[Neuroscience] faults in 1999 July EPA 468-page formaldehyde profile: Elzbieta Skrzydlewska PhD, Assc. Prof., Medical U. of Bialystok, Poland, abstracts -- ethanol, methanol, formaldehyde, formic acid, acetaldehyde, lipid peroxidation: Murray 2004.08.08 2005.07.11

Rich Murray rmforall at att.net
Mon Jul 11 20:52:54 EST 2005


*************************************************************

http://groups.yahoo.com/group/aspartameNM/message/1108
faults in 1999 July EPA 468-page formaldehyde profile:
Elzbieta Skrzydlewska PhD, Assc. Prof., Medical U. of Bialystok, Poland,
abstracts -- ethanol, methanol, formaldehyde, formic acid, acetaldehyde,
lipid peroxidation, green tea, aging: Murray 2004.08.08 2005.07.11

Rich Murray, MA    Room For All    rmforall at comcast.net
1943 Otowi Road, Santa Fe, New Mexico 87505 USA  505-501-2298

Herein I offer abstracts and three full texts of dozens of studies by a
world-class biochemist and her associates, mostly experiments with rats, on
ethanol toxicity since 1984 and methanol toxicity since 1993.  Enough
details are provided to show the competency and credibility of E.
Skrzydlewska and her colleagues over two decades, and to make access to
their literature more convenient for professionals.

For instance, anyone can click on this post at the above URL, and in Outlook
Express, use Control F to search the text for any word.  Yahoo Groups also
includes a fine search function.

A conscientious, responsible review of any reseach that affects the
interests of vast commercial vested interests has to provide justified
criticism of dubious studies, reviews, and conclusions, that are
characteristically the main sources for private and professional
information.   My experience since I first started investigating toxicity
issues in 1999 is, count on it, wolves guard the sheep.

To be effective, this criticism has to be calm, civil, detailed, specific,
reasonable, founded on evidence, focused on issues and not on persons, and
based on easily accessed public sources, so that anyone interested in basing
their conclusions on facts can start the laborous process of deciding for
themselves.

http://groups.yahoo.com/group/aspartameNM/message/667
25 rules of disinformation , Sweeney 1997: Murray 2001.07.04 rmforall

As a medical layman, age 62, earning my living the last 16 years as a home
health care giver, my volunteer public service for toxicity issues on the
Net since January 1999 depends entirely on earning credibility by deserving 
it:

http://groups.yahoo.com/group/aspartameNM/messages
186 members,  1,182 posts in a public searchable archive

I regret that I almost never receive negative feedback based on any
specifics of this work,  for this would enable me, in the finest tradition
of science, to either reverse, amend, or clarify my positions, to the
benefit of humanity, as well as deepening my own satisfaction and confidence
in a long-term effort.

However, it is indubitable that the best disinformation strategy is to
simply ignor any inconvenient researchers and their work, or, if that
becomes problematic due to the quality of the worker and his work, to firmly
repeat the exact opposite of truth, for example, "Aspartame is the most
tested food additive in history," while using ad hominen statements to
dismiss, ridicule, and marginalize the opposition.  Fanning a "flame war" of
escalating incivility is a virtually infallible strategy for muddying the
waters, preventing any real examination of actual facts.

I should also here assert that, so far, I have not ever received a
penny of support for my toxicity service, save for about $ 300 of free
books, from both sides of the debate.  Furthermore, when the happy day
ensues when I receive payment of any sort, I will immediately and forever
make this clearly and completely known on the Net, along with full details
for contacting the sources, who must agree to repond responsibly,
immediately, and publicly to all inquiries.  All my financial information
about this work will be immediately, clearly, and forever public.  I cannot
be bought, bent, or borrowed.   I don't deal in secret.

A persistent, consistent campaign that provides facts about an actual toxin
can only succeed.

Fact is, the 11% methanol component of aspartame, readily released into the
G.I. tract, is within hours converted into potent amounts of formaldehyde
and formic acid, in amounts scores of times higher than that allowed by the
EPA for daily drinking water.

I have recently summarized mainstream evidence that the similar amounts of
methanol impurity, about one part in ten thousand, in dark wines and
liquors, are largely responsible for the famed "morning after" hangover:

http://groups.yahoo.com/group/aspartameNM/message/1106
hangover research relevant to toxicity of 11% methanol in aspartame
(formaldehyde, formic acid): Calder I (full text): Jones AW:
Murray 2004.08.06 rmforall

Similar potent levels of methanol, and its inevitable products in the human
body, formaldehyde and formic acid, can also ensue from fermentation of
fruits by certain yeast and bacteria in the colon:

http://groups.yahoo.com/group/aspartameNM/message/1110
methanol (formaldehyde and formic acid) from fermentation of fruit in the
colon: Lindinger W, 1997 Aug: Murray 2004.08.10

Alcohol Clin Exp Res. 1997 Aug; 21(5): 939-43.
Endogenous production of methanol after the consumption of fruit.
Lindinger W, Taucher J, Jordan A, Hansel A, Vogel W.
Institut fur Ionenphysik, Leopold Franzens Universitat Innsbruck, Austria.

After the consumption of fruit, the concentration of methanol in the human
body increases by as much as an order of magnitude.
This is due to the degradation of natural pectin (which is esterified with
methyl alcohol) in the human colon.
In vivo tests performed by means of proton-transfer-reaction mass
spectrometry show that consumed pectin in either a pure form (10 to 15 g) or
a natural form (in 1 kg of apples) induces a significant increase of
methanol in the breath (and by inference in the blood) of humans.
The amount generated from pectin (0.4 to 1.4 g) is approximately equivalent
to the total daily endogenous production (measured to be 0.3 to 0.6 g/day)
or that obtained from 0.3 liters of 80-proof brandy (calculated to be 0.5
g).
This dietary pectin may contribute to the development of nonalcoholic
cirrhosis of the liver.  PMID: 9267548

Alcohol Clin Exp Res. 1995 Oct; 19(5): 1147-50.
Methanol in human breath.
Taucher J, Lagg A, Hansel A, Vogel W, Lindinger W.
Institut fur Ionenphysik, Universitat Innsbruck, Austria.

Using proton transfer reaction-mass spectrometry for trace gas analysis of
the human breath, the concentrations of methanol and ethanol have been
measured for various test persons consuming alcoholic beverages and various
amounts of fruits, respectively.
The methanol concentrations increased from a natural (physiological) level
of approximately 0.4 ppm up to approximately 2 ppm a few hours after eating
about 1/2 kg of fruits, and about the same concentration was reached after
drinking of 100 ml brandy containing 24% volume of ethanol and 0.19% volume
of methanol.  PMID: 8561283

[ Corrected 2005.07.11: 24 ml means 19 g ethanol, and 0.19 ml means 0.15 g =
150 mg  methanol.
One L diet soda has 61.5 mg methanol in the aspartame molecule, so 100 ml
diet soda has 6.15 mg methanol, so the brandy has 24.4 times more methanol
than diet soda.  ]

These three potent dietary sources of methanol, formaldehyde, and formic
acid, which impact many people, and cause the same symptoms in vulnerable
and sensitized people, are ignored in the following prestigious, official
source:

http://groups.yahoo.com/group/aspartameNM/message/1110
Toxicological Profile for Formaldehyde 1/4 plain text, start to 111 of 468
pages USA DHHS PHS ATSDR 1999 July: Murray 2004.08.10 rmforall

[ Text unaltered, except for spacing for enhanced clarity.  My comments are
in square brackets.  The four sections have URLs    /1110,  /1111,  /1112 ,
/1113   ]

http://www.atsdr.cdc.gov/toxprofiles/tp111.pdf

TOXICOLOGICAL PROFILE FOR FORMALDEHYDE
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
Public Health Service  Agency for Toxic Substances and Disease Registry
July 1999

FORMALDEHYDE page 338  Table 7-1   REGULATIONS AND ADVISORIES

Table 7-1. Regulations and Guidelines Applicable to Formaldehyde (continued)
Agency   Description   Information   References
NATIONAL (cont.)....b. Water:...
Lifetime Health Advisory (adult)-draft 1 mg/L...   [ 1 ppm drinking water ]


FORMALDEHYDE page 339  Table 7-1  REGULATIONS AND ADVISORIES

Table 7-1. Regulations and Guidelines Applicable to Formaldehyde (continued)
Agency Description Information References
STATE (cont.)...
b. Water
Water Quality Criteria: Human Health...    [ compared to federal standard ]
CA Drinking water (guideline) 30 µg/L FSTRAC 1995  [  three times less ]
MD Drinking water (guideline) 10 µg/L                  [  ten times less ]
ME Drinking water (guideline) 30 µg/L                [  three times less ]


http://groups.yahoo.com/group/aspartameNM/message/1109
faults in 1999 July EPA 468-page formaldehyde profile, extracts &  full
references:  Murray 2004.08.09 rmforall

[ Extracts ]  [ My comments are in square brackets. ]

[  http://www.atsdr.cdc.gov   atsdric at cdc.gov  888-42-ATSDR
404-498-0110   fax  404-498-0093

Agency for Toxic Substances and Disease Registry  Division of Toxicology
1600 Clifton Road NE, Mailstop E-29  Atlanta, GA 30333
* Information line and technical assistance  Phone: (800) 447-1544  Fax:
(404) 639-6359

* To order toxicological profiles, contact
National Technical Information Service  5285 Port Royal Road  Springfield,
VA 22161    Phone: (800) 553-6847 or (703) 487-4650

The National Center for Environmental Health (NCEH) focuses on preventing or
controlling disease, injury, and disability related to the interactions
between people and their environment outside the workplace.
http://www.cdc.gov/nceh/   888-232-6789
http://www2.cdc.gov/nceh/contactnceh/frmSubmit.asp  email contact
Contact: NCEH, Mailstop F-29, 4770 Buford Highway, NE, Atlanta, GA
30341-3724        . Phone: 770-488-7000 . FAX: 770-488-7015.

The National Institute for Occupational Safety and Health (NIOSH) conducts
research on occupational diseases and injuries, responds to requests for
assistance by investigating problems of health and safety in the workplace,
recommends standards to the Occupational Safety and Health Administration
(OSHA) and the Mine Safety and Health Administration (MSHA), and trains
professionals in occupational safety and health.
http://www.cdc.gov/niosh/homepage.html  eidtechinfo at cdc.gov
Contact: NIOSH, 200 Independence Avenue, SW, Washington, DC 20201 . Phone:
513-533-8328  800-356-4674 or NIOSH Technical Information Branch, Robert A. 
Taft Laboratory, Mailstop C-19, 4676 Columbia Parkway, Cincinnati, OH 
45226-1998   Phone: 800-35-NIOSH fax 513-533-8573

The National Institute of Environmental Health Sciences (NIEHS) is the
principal federal agency for biomedical research on the effects of chemical,
physical, and biologic environmental agents on human health and well-being.
http://www.niehs.nih.gov/
Contact: NIEHS, PO Box 12233, 104 T.W. Alexander Drive,
Research Triangle Park, NC 27709 . Phone: 919-541-3212.
Office of Communications  919-541-3345      TTY  919-541-0731


Referrals
The Association of Occupational and Environmental Clinics (AOEC) has
developed a network of clinics in the United States to provide expertise in
occupational and environmental issues. Contact:
AOEC, 1010 Vermont Avenue, NW, #513, Washington, DC 20005
Phone: 202-347-4976    FAX: 202-347-4950
Web Page: http://www.aoec.org/   e-mail: AOEC at AOEC.ORG

AOEC Clinic Director:  http://occ-envmed.mc.duke.edu/oem/aoec.htm.

The American College of Occupational and Environmental Medicine
(ACOEM) is an association of physicians and other health care providers
specializing in the field of occupational and environmental medicine.
http://www.acoem.org/     http://www.acoem.org/feedback/   email contact
Contact: ACOEM, 55 West Seegers Road, Arlington Heights, IL 60005
Phone: 847-818-1800   FAX: 847-818-9266.  ]

FORMALDEHYDE  page iii

UPDATE STATEMENT
Toxicological profiles are revised and republished as necessary, but no less
than once every three years.  [ I could not locate any more recent updates
than July 1999 via Google. ]

FORMALDEHYDE ix

CONTRIBUTORS
CHEMICAL MANAGER(S)/AUTHORS(S):
Sharon Wilbur, M.A.  [ Not a PhD level degree ]
[ Environmental Health Scientist ]
ATSDR, Division of Toxicology, Atlanta, GA

M. Olivia Harris, M.A.   [ Not a PhD level degree ]
ATSDR, Division of Toxicology, Atlanta, GA
[  Environmental Health Scientist
1600 Clifton Road NE, E29  Atlanta, GA 30333
P: 404-639-5091  F: 404-639-6315   oxh0 at cdc.gov ]

Peter R. McClure, Ph.D., DABT [ Veterinarian ]
Syracuse Research Corporation, North Syracuse, NY
[   Syracuse Research Corporation  Environmental Science Center
301 Plainfield Road Suite 350  Syracuse, New York 13212  (315) 452 8420
mcclure at syrres.com   ]

Wayne Spoo, DVM, DABT, DABVT [ Veterinarian ]
Research Triangle Institute, Research Triangle Park, NC
[ Jerry Wayne Spoo  Operations Director, Life Sciences and Toxicology
919-541-6000     jwspoo at rti.org   http://www.rti.org    http://www.abvt.org/

CPT Spoo, HHC, USACAPOC, AOCP-MS, 910-432-2209.
jerry.w.spoo at us.army.mil  ]

FORMALDEHYDE xi

PEER REVIEW
A peer review panel was assembled for formaldehyde. The panel consisted of
the following members:
1. Carson Conaway, Research Scientist, American Health Foundation, Valhalla,
New York 10595;
[  http://www.ahf.org/contact/  914-789-7210    914-789-7243
1 Dana Road  Valhalla, NY 10595
300 E. 42nd. Street  New York, NY 10017

 http://www.ifcp.us/Scientists-Scientists-Carson_Conaway.cfm
Carson Clifford Conaway, Ph. D., DABT [ Veterinarian ]
Research Scientist  phone: (914) 789-7210  email: cconaway at ifcp.us
Institute for Cancer Prevention
In addition to his research work, Dr. Conaway is an Adjunct Associate
Professor in the Department of Pharmacology, New York Medical College.  In
that capacity, he is called upon to present lectures in toxicology to
graduate students in the College of Basic Medical Sciences and in the School
of Public Health.

2. John Egle, Jr., Professor, Department of Pharmacology and Toxicology,
Medical College of Virginia, Smith Bldg., Room 656, Richmond, VA 23219; and
[  http://www.medschool.vcu.edu/   John L. Egle, Jr no longer listed.  Last
PubMed study in 1995   .  Studies on formaldehyde, 2 in 1974, 1 in 1972, no
PubMed abstracts for these. ]

3. Vincent Garry, Director, Environmental Medicine, University of Minnesota,
421 29th Ave.,  SE Minneapolis, MN 55414.

[
http://www.iatp.org/foodandhealth/library/admin/uploadedfiles/Vincent_Garry_Bio.pdf
Vincent F Garry  Title: Professor
Department: Lab Medicine/Pathology (office: Lab Med/Pathology Department)
Dept Campus: UMN Twin Cities
E-mail Address: garry001 at umn.edu
Office Address: Lab Med/Pathology Department
225 Mayo  8609   420 Delaware St SE  Minneapolis, MN 55455
Campus Mail: Lab Medicine and Pathology
MMC 609 Mayo 8609  420 Delaware St SE  Minneapolis, MN 55455
Office Phone:+1 612-626-3354  Fax:+1 612-626-3380
Address: 4829 Girard Ave So  Minneapolis, MN 55409
Phone:+1 612-827-7316

Toxicol Appl Pharmacol. 2004 Jul 15; 198(2): 152-63.
Pesticides and children.
Garry VF.
Department of Laboratory Medicine and Pathology and Program in Toxicology,
University of Minnesota School of Medicine, Minneapolis, MN 55455, USA.

Prevention and control of damage to health, crops, and property by insects,
fungi, and noxious weeds are the major goals of pesticide applications.
As with use of any biologically active agent, pesticides have unwanted
side-effects. In this review, we will examine the thesis that adverse
pesticide effects are more likely to occur in children who are at special
developmental and behavioral risk. Children's exposures to pesticides in the
rural and urban settings and differences in their exposure patterns are
discussed.
The relative frequency of pesticide poisoning in children is examined.
In this connection, most reported acute pesticide poisonings occur in
children younger than age 5.
The possible epidemiological relationships between parental pesticide use or
exposure and the risk of adverse reproductive outcomes and childhood cancer
are discussed.
The level of consensus among these studies is examined.
Current concerns regarding neurobehavioral toxicity and endocrine disruption
in juxtaposition to the relative paucity of toxicant mechanism-based studies
of children are explored.  PMID: 15236951 ]

These experts collectively have knowledge of formaldehyde's physical and
chemical properties, toxicokinetics, key health end points, mechanisms of
action, human and animal exposure, and quantification of risk to humans.

All reviewers were selected in conformity with the conditions for peer
review specified in Section 104(I)(13) of the Comprehensive Environmental
Response, Compensation, and Liability Act, as amended.

Scientists from the Agency for Toxic Substances and Disease Registry (ATSDR)
have reviewed the peer reviewers' comments and determined which comments
will be included in the profile.

A listing of the peer reviewers' comments not incorporated in the profile,
with a brief explanation of the rationale for their exclusion, exists as
part of the administrative record for this compound. [ Not easily accessible
by public ]

A list of databases reviewed and a list of unpublished documents cited are
also included in the administrative record. [ Not easily accessible by
public ]

The citation of the peer review panel should not be understood to imply its
approval of the profile's final content. The responsibility for the content
of this profile lies with the ATSDR....  [ Apparently, the peer review
panel's opinions carry little weight. ]

1. PUBLIC HEALTH STATEMENT
This public health statement tells you about formaldehyde and the effects of
exposure....

1.2 WHAT HAPPENS TO FORMALDEHYDE WHEN IT ENTERS THE
ENVIRONMENT?

Most of the formaldehyde you are exposed to in the environment is in the
air....  [ A very misleading statement, as already pointed out above ]

There is usually more formaldehyde present indoors than outdoors.
[ Ignors the issue of dietary sources ]

Formaldehyde is released to the air from many home products and you may
breath in formaldehyde while using these products.

Latex paint, fingernail hardener, and fingernail polish release a large
amount of formaldehyde to the air.  [ Bound to be much less than the potent
amounts in dietary sources, which have immediate strong effects on sensitive
and sensitivized persons and other vulnerable groups ]

Plywood and particle board, as well as furniture and cabinets made from
them, fiberglass products, new carpets, decorative laminates, and some
permanent press fabrics give off a moderate amount of formaldehyde.
[ This shows why new buildings, and especially mobile homes and RVs are
toxic for many people. ]

Some paper products, such as grocery bags and paper towels, give off small
amounts of formaldehyde.

Because these products contain formaldehyde, you may also be exposed on the
skin by touching or coming in direct contact with them.

You may also be exposed to small amounts of formaldehyde in the food you
eat.  [ The amounts in dietary sources are the most potent sources for most
people. ]

You are not likely to be exposed to formaldehyde in the water you drink
because it does not last a long time in water.  [ This ignores the fact that
methanol, always turned into formaldehyde and formic acid in humans, does
indeed last a very long time in water and dietary sources. ]

Many other home products contain and give off formaldehyde although the
amount has not been carefully measured. [ Potent dietary sources have been
systematically ignored for decades. ]

These products include household cleaners, carpet cleaners, disinfectants,
cosmetics, medicines, fabric softeners, glues, lacquers, and antiseptics.
[ Notice "cosmetics", "medicines", "disinfectants", "antiseptics" -- to this
list of direct skin contact items, we can add hair care products and shoe
leather. ]

You may also breath formaldehyde if you use unvented gas or kerosene heaters
indoors or if you or someone else smokes a cigar, cigarette, or pipe
indoors.

The amount of formaldehyde in mobile homes is usually higher than it is in
conventional homes because of their lower air turnover. [ Evades the issue
that particleboard and other materials common in mobile homes are strong
formaldehyde sources, which this study showed caused symptoms and immune
system signs:

http://www.drthrasher.org/formaldehyde_1990.html  full text   Jack Dwayne
Thrasher, Alan Broughton, Roberta Madison. Immune activation and
autoantibodies in humans with long-term inhalation exposure to formaldehyde.
Archives of Environmental Health. 1990; 45: 217-223.  "Immune activation,
autoantibodies, and anti-HCHO-HSA antibodies are associated with long-term
formaldehyde inhalation."  PMID: 2400243  toxicology at drthrasher.org ]

People who work at or near chemical plants that make or use formaldehyde can
be exposed to higher than normal amounts of formaldehyde.

Doctors, nurses, dentists, veterinarians, pathologists, embalmers, workers
in the clothing industry or in furniture factories, and teachers and
students who handle preserved specimens in laboratories also might be
exposed to higher amounts of formaldehyde. The National

FORMALDEHYDE 4 1. PUBLIC HEALTH STATEMENT

Institute for Occupational Safety and Health (NIOSH) estimates that
1,329,332 individuals in the United States have had the potential for
occupational exposure to formaldehyde.  [ Common sense suggests that health
professionals who have been exposed to formaldehyde and have become
sensitized and symptomatic naturally will have a prejudice against
discovering the real extent of the danger. ]

1.4 HOW CAN FORMALDEHYDE ENTER AND LEAVE MY BODY?

Formaldehyde can enter your body after you breath it in, drink or eat it, or
when it comes in contact with your skin....

Once absorbed, formaldehyde is very quickly broken down. [ Notice the phrase
"very quickly broken down", which skirts the issue that potent levels of
formaldehyde and formic acid are inevitably produced in humans, and retained
in complex, unresearched amounts. ]

Almost every tissue in the body has the ability to break down formaldehyde.
[ Again the dangers are waved away by definition.  The correct way to say
this is that formaldehyde and formic acid toxicity affects every tissue in
the body. ]

It is usually converted to a non-toxic chemical called formate, which is
excreted in the urine. [  This is an astonishing, brazen deceit, defining
formic acid as "non-toxic".   Notice the qualification "usually". ]

Formaldehyde can also be converted to carbon dioxide and breathed out of the
body. [ Notice the qualification "can" . ]

It can also be broken down so the body can use it to make larger molecules
needed in your tissues, or it can attach to deoxyribonucleic acid (DNA) or
to protein in your body.... [ In one sentence, formaldehyde is portrayed as
a useful food, while the very serious and complex issue of formaldehyde
adducts to DNA and proteins in all tissues and cells is minimalized:

C. Trocho (1998 July 26): [ Not cited in this lengthy tome. ]
"In all, the rats retained, 6 hours after administration, about 5% of the
label, half of it in the liver."

They used a very low level of aspartame ingestion, 10 mg/kg, for rats, which
have a much greater tolerance for aspartame than humans.
So, the corresponding level for humans would be about 1 or 2 mg/kg.
[ 60 to 120 mg aspartame for a 60-kg person, of which 11% is methanol,
6.6 to 13.2 mg ]
Many headache studies in humans used doses of about 30 mg/kg daily.
[ 1800 mg aspartame for a 60-kg person, of which 11% is methanol, 198 mg ]

http://groups.yahoo.com/group/aspartameNM/message/925
aspartame puts formaldehyde adducts into tissues, Part 1/2
full text, Trocho & Alemany 1998.06.26: Murray 2002.12.22 rmforall

http://ww.presidiotex.com/barcelona/index.html  full text
Formaldehyde derived from dietary aspartame binds to tissue components in
vivo.
Life Sci June 26 1998; 63(5): 337-49.
Departament de Bioquimica i Biologia Molecular,
Facultat de Biologia, Universitat de Barcelona, Spain.
http://www.bq.ub.es/cindex.html    Línies de Recerca: Toxicitat de
l'aspartame     http://www.bq.ub.es/grupno/grup-no.html
Sra. Carme Trocho, Sra. Rosario Pardo, Dra. Immaculada Rafecas,
Sr. Jordi Virgili, Dr. Xavier Remesar,  Dr. Jose Antonio
Fernandez-Lopez, Dr. Marià Alemany [male]
Fac. Biologia Tel.: (93)4021521, FAX: (93)4021559
Sra. Carme Trocho "Trok-ho"  Fac. Biologia Tel.:   (93)4021544,
FAX: (93)4021559  alemany at porthos.bio.ub.es ; bioq at sun.bq.ub.es

Abstract:
Adult male rats were given an oral dose of 10 mg/kg aspartame,
14C-labeled in the methanol carbon.
At timed intervals of up to 6 hours, the radioactivity in plasma and several
organs was investigated.
Most of the radioactivity found (>98% in plasma, >75% in liver) was bound to
protein.
Label present in liver, plasma and kidney was in the range of 1-2% of total
radioactivity administered per g or mL, changing little with time.
Other organs (brown and white adipose tissues, muscle, brain, cornea and
retina) contained levels of label in the range of 1/12th to 1/10th of that
of liver.
In all, the rats retained, 6 hours after administration, about 5% of the
label, half of it in the liver.

The specific radioactivity of tissue protein, RNA and DNA was quite uniform.
The protein label was concentrated in amino acids, different from
methionine, and largely coincident with the result of protein exposure to
labeled formaldehyde.
DNA radioactivity was essentially in a single different adduct base,
different from the normal bases present in DNA.
The nature of the tissue label accumulated was, thus, a direct consequence
of formaldehyde binding to tissue structures.

The administration of labeled aspartame to a group of cirrhotic rats
resulted in comparable label retention by tissue components, which suggests
that liver function (or its defect) has little effect on formaldehyde
formation from aspartame and binding to biological components.

The chronic treatment of a series of rats with 200 mg/kg of non-labeled
aspartame during 10 days results in the accumulation of even more label when
given the radioactive bolus, suggesting that the amount of formaldehyde
adducts coming from aspartame in tissue proteins and nucleic acids may be
cumulative.

It is concluded that aspartame consumption may constitute a hazard because
of its contribution to the formation of formaldehyde adducts.  PMID: 9714421

[ Extracts ]
"The high label presence in plasma and liver is in agreement with the
carriage of the label from the intestine to the liver via the portal vein.
The high label levels in kidney and, to a minor extent, in brown adipose
tissue and brain are probably a consequence of their high blood flows (45).
Even in white adipose tissue, the levels of radioactivity found 6 hours
after oral administration were 1/25th those of liver.
Cornea and retina, both tissues known to metabolize actively methanol
(21,28) showed low levels of retained label.
In any case, the binding of methanol-derived carbon to tissue proteins was
widespread, affecting all systems, fully reaching even sensitive targets
such as the brain and retina....

The amount of label recovered in tissue components was quite high in all the
groups, but especially in the NA rats.
In them, the liver alone retained, for a long time, more than 2 % of the
methanol carbon given in a single oral dose of aspartame, and the rest of
the body stored an additional 2 % or more.
These are indeed extremely high levels for adducts of formaldehyde, a
substance responsible of chronic deleterious effects (33), that has also
been considered carcinogenic (34,47).
The repeated occurrence of claims that aspartame produces headache and other
neurological and psychological secondary effects-- more often than not
challenged by careful analysis--  (5, 9, 10, 15, 48)  may eventually find at
least a partial explanation in the permanence of the formaldehyde label,
since formaldehyde intoxication can induce similar effects (49).

The cumulative effects derived from the incorporation of label in the
chronic administration model suggests that regular intake of aspartame may
result in the progressive accumulation of formaldehyde adducts.

It may be further speculated that the formation of adducts can help to
explain the chronic effects aspartame consumption may induce on sensitive
tissues such as brain (6, 9, 19, 50).

In any case, the possible negative effects that the accumulation of
formaldehyde adducts can induce is, obviously, long-term.

The alteration of protein integrity and function may needs some time to
induce substantial effects.

The damage to nucleic acids, mainly to DNA, may eventually induce cell death
and/or mutations.

The results presented suggest that the conversion of aspartame methanol into
formaldehyde adducts in significant amounts in vivo should to be taken into
account because of the widespread utilization of this sweetener.

Further epidemiological and long-term studies are needed to determine the
extent of the hazard that aspartame consumption poses for  humans." ]

Some people are more sensitive to the effects of formaldehyde than
others....
[ Again a very significant, complex, and problematic issue is mentioned and
minimalized in one sentence-- notice the phrase "some people". ]

The Department of Health and Human Services (DHHS) has determined that
formaldehyde may reasonably be anticipated to be a human carcinogen (NTP).

The International Agency for Research on Cancer (IARC) has determined that
formaldehyde is probably carcinogenic to humans.

This determination was based on specific judgements that there is limited
evidence in humans and sufficient evidence in laboratory animals that
formaldehyde can cause cancer.

The Environmental Protection Agency (EPA) has determined that formaldehyde
is a probable human carcinogen based on limited evidence in humans and
sufficient evidence in laboratory animals.... [ These extremely alarming
admissions ought to be emphasized and used to support calls for urgent
research, action, and public warning. ]

The most common way for children to be exposed to formaldehyde is by
breathing it. [ Again, kids are kept at risk with this policy of denial of
the potent role of dietary sources. ]

Children may also be exposed by wearing some types of new clothes or
cosmetics.

A small number of studies have looked at the health effects of formaldehyde
in children.  [ Notice the term "small number", which serves to both mention
and minimalize the problem of a dire shortage of adaquate research. ]

It is very likely that breathing formaldehyde will result in nose and eye
irritation (burning feeling, itchy, tearing, and sore throat). [ The focus
is placed on the most unimportant symptoms. ]

We do not know if the irritation would occur at lower concentrations in
children than in adults. [ Research that could threaten vested interests
somehow just doesn't get funded. ]

Studies in animals suggest that formaldehyde will not cause birth defects in
humans.  [ Notice the qualification "suggest". ]

Inhaled formaldehyde or formaldehyde applied to the skin is not likely to be
transferred from mother to child in breast milk or to reach the developing
fetus....  [ Notice the qualification "not likely".

ttp://groups.yahoo.com/group/aspartameNM/message/915
formaldehyde toxicity:  Thrasher & Kilburn: Shaham: EPA: Gold:
Wilson: CIIN: Murray 2002.12.12 rmforall

Thrasher (2001): "The major difference is that the Japanese demonstrated
the incorporation of FA and its metabolites into the placenta and fetus.
The quantity of radioactivity remaining in maternal and fetal tissues
at 48 hours was 26.9% of the administered dose." [ Ref. 14-16 ]

Arch Environ Health 2001 Jul-Aug; 56(4): 300-11.
Embryo toxicity and teratogenicity of formaldehyde. [100 references]
Thrasher JD, Kilburn KH.  toxicology at drthrasher.org
Sam-1 Trust, Alto, New Mexico, USA.
http://www.drthrasher.org/formaldehyde_embryo_toxicity.html   full text  ]

Formaldehyde is usually found in the air.

Formaldehyde levels are also higher indoors than outdoors.

Opening windows or using a fan to bring in fresh air is the easiest way to
lower formaldehyde levels in the home and reduce the risk of exposure to
your family. [ This reassuring, simple advice is dangerous, since the potent
dietary sources are ignored. ]

Removing formaldehyde sources from the house will also reduce the risk of
exposure.

Since formaldehyde is found in tobacco smoke, not smoking or smoking outside
will reduce exposure to formaldehyde.

Unvented heaters, such as portable kerosene heaters, also produce
formaldehyde. If you do not use these heaters in your home or shop, you help
to prevent the build up of formaldehyde indoors.

Formaldehyde is found in small amounts in many consumer products including
antiseptics, medicines, dish-washing liquids, fabric softeners, shoe-care
agents, carpet cleaners, glues, adhesives, and lacquers.

If you or a member of your family uses these products, providing fresh
outdoor air when you use them, this will reduce your exposure to
formaldehyde.

Some cosmetics, such as nail hardeners, have very high levels of
formaldehyde.

If you do not use these products in a small room, or if you have plenty of
ventilation when you use them, you will reduce your exposure to
formaldehyde.

If your children are not in the room when you use these products, you will
also reduce their exposure to formaldehyde.

Formaldehyde is emitted from some wood products such as plywood and particle
board, especially when they are new.

The amount of formaldehyde released from them decreases slowly over a few
months. [ Notice "slowly over a few months". ]

If you put these materials in your house, or buy furniture or cabinets made
from them, opening a window will lower formaldehyde in the house. [ What
happens in winter? ]

The amount of formaldehyde emitted to the house will be less if the wood
product is covered with plastic laminate or coated on all sides.

If it is not, sealing the unfinished sides will help to lower the amount of
formaldehyde that is given off.

Some permanent press fabrics emit formaldehyde. [ Why aren't there warning
labels? ]

Washing these new clothes before use will usually lower the amount of
formaldehyde and reduce your family's risk of exposure.

FORMALDEHYDE 7 1. PUBLIC HEALTH STATEMENT

1.8 IS THERE A MEDICAL TEST TO DETERMINE WHETHER I HAVE BEEN EXPOSED TO
FORMALDEHYDE?

We have no reliable test to determine how much formaldehyde you have been
exposed to or whether you will experience any harmful health effects....
[ Another casual mention of an alarming reality ]

1.9 WHAT RECOMMENDATIONS HAS THE FEDERAL GOVERNMENT MADE TO PROTECT HUMAN
HEALTH?

The federal government develops regulations and recommendations to protect
public health.

Regulations can be enforced by law.

Federal agencies that develop regulations for toxic substances include the
EPA, the Occupational Safety and Health Administration (OSHA), and the Food
and Drug Administration (FDA).

Recommendations provide valuable guidelines to protect public health but
cannot be enforced by law. [ Not reassuring... ]

Federal organizations that develop recommendations for toxic substances
include the Agency for Toxic Substances and Disease Registry (ATSDR) and the
NIOSH.  [ ATSDR and NIOSH cannot enforce their recommendations. ]

Regulations and recommendations can be expressed in not-to-exceed levels in
air, water, soil, or food that are usually based on levels that affect
animals, then they are adjusted to help protect people.  [ This bypasses the
issue that the reseach on humans is very inadequate to determine the actual,
complex toxicity of methanol, formaldehyde, and formic acid. ]

Sometimes these not-to-exceed levels differ among federal organizations
because of different exposure times (an 8-hour workday or a 24-hour day),
the use of different animal studies, or other factors....
[ An outstanding example of disharmony in the EPA is the fact that the
1998.05.05 EPA IRIS level for oral methanol in humans (Oral Rfd)  is 0.5
mg/kg/day, or 30 mg oral methanol daily for a 60 kg human.   The animal
study used was:

U.S. EPA. 1986. Rat oral subchronic toxicity study with methanol. Office of
Solid Waste, Washington, DC.

I have not found on the Net any information as to the authors,  institution,
abstract, or full text of this study.

But the EPA ATSDR limit for formaldehyde in drinking water is
1 ppm, or 2 mg daily for a typical daily consumption of 2 L of water:

http://groups.yahoo.com/group/aspartameNM/message/835
ATSDR: EPA limit 1 ppm formaldehyde in drinking water July 1999:
Murray 2002.05.30 rmforall

http://www.atsdr.cdc.gov/tfacts111.html
[excerpts]

Agency for Toxic Substances and Disease Registry    Division of Toxicology
1600 Clifton Road NE, Mailstop E-29Atlanta,   GA 30333    888-422-8737 FAX:
(404)498-0057 ATSDRIC at cdc.gov  http://www.atsdr.cdc.gov/contacts.html

"The EPA recommends that an adult should not drink water containing more
than 1 milligram of formaldehyde per liter of water (1 mg/L) for a
lifetime exposure, and a child should not drink water
containing more than 10 mg/L for 1 day or 5 mg/L for 10 days."

This stringent limit means that if over 13% of the oral methanol limit
results in production of formaldehyde in the human body by the liver, then
the formaldehyde limit would be exceeded.  This is cutting things pretty
close.

http://www.epa.gov/iris/subst/0305.htm
also  http://www.china-pops.net/enwww/IRIS-Mirror/subst/0305.htm  1998.05.05

USA Environmental Protection Agency  EPA
Integrated Risk Information System   IRIS

This site explains that the harmful rat dose of 500 mg/kg body weight per
day was divided
by 10 for "interspecies extrapolation" (the higher vulnerability of
humans than rats),
by 10 for "range of sensitivity" (the variation of individual human
vulnerability), and
by 10 for "subchronic to chronic exposure" (the increased danger from
lifetime as compared to the 3 month exposure in the rat test),
giving a total reduction of 10x10x10 = 1000 for the UF = Uncertainty Factor.

The human Oral RfD is the rat Oral RfD divided by 1000, so
500 mg/kg/day  is reduced to  0.5 mg/kg/day , so that the allowed dose for a
60 kg human is 30 mg oral methanol daily.


Moreover, a recent study found that after 4 months of moderate oral
aspartame, rats took four times longer to finish a simple, one-turn maze-- 
an alarming level of neurotoxicity:

http://groups.yahoo.com/group/aspartameNM/message/1088
Murray, full plain text & critique:
chronic aspartame in rats affects memory, brain cholinergic receptors, and
brain chemistry, Christian B, McConnaughey M et al, 2004 May:
2004.06.05 rmforall

"Control and treated rats were trained in a T-maze to a particular side and
then periodically tested to see how well they retained the learned response.

Rats that had received aspartame (250 mg/kg/day) in the drinking water
for 3 or 4 months showed a significant increase in time to reach the reward
in the T-maze,  suggesting a possible effect on memory due to the artificial
sweetener."

The 11% methanol component of aspartame is immediately released in the GI
tract, so these rats were being exposed to only 27.5 mg/kg/day methanol.

The EPA IRIS on 1998.05.05 used a 1986 90 day rat study to find a
No-Observed-Effect Level (NOEL) value of 500 mg/kg/day, which, divided by
1000, became their human long-term safe methanol level of 0.5 mg per kg body
weight per day, which for a 60 kg average person is 30 mg methanol daily,
for oral exposure.

However, the rat level is 18 times greater than that for the level of
dramatic memory loss and clear-cut brain changes found by McConnaughey M,
May 2004.

This suggests reducing the human long-term safe level twenty times to
.025 mg/kg/day = 25 micrograms per kg body weight per day,
which for a 60 kg average person is 1.5 mg oral methanol per day.


It is certain that high levels of aspartame use, above 2 liters daily for
months and years, must lead to chronic formaldehyde-formic acid toxicity.

Fully 11% of aspartame is methanol--  1,120 mg aspartame  in 2 L diet soda,
almost six 12-oz cans,  gives 123 mg methanol (wood alcohol), about 22 mg
methanol per can.

If only 10% of the methanol accumulates daily as formaldehyde, that would
give 12 mg daily formaldehyde accumulation-- about 60 times more than the
0.2 mg from 10% retention of the 2 mg EPA daily limit for formaldehyde in
drinking water.

If about 30% of oral methanol is retained as formaldehyde and formic acid,
then this EPA ATSDR formaldehyde limit of 2 mg daily for 2 L drinking water
suggests a corresponding methanol limit of 6.7 mg daily, about 4.5 times the
safe limit based on the McConnaughey data.  This is much closer than the
1998 EPA IRIS limit of 30 mg daily oral methanol, which is 20 times the
McConnaughey data limit.  ]
**************************************************************



Returning to the voluminous work of Elzbieta Skrzydlewska, it is important
that many of her studies suggest that many safe substances may prevent or
treat toxicity from methanol and its inevitable toxic human body products,
formaldehyde and formic acid:

N-acetylcysteine (2000); U-83836E containing a trolox ring (1997);
green tea (2004); vitamins E, C, A, and beta-carotene (2004);
glutathione (2001); N-Acetylcysteine (NAC) (2001); melatonin (2001);
low and medium levels of cysteine (1990).



"Methanol, when introduced into all mammals, is oxidized into formaldehyde
and then into formate, mainly in the liver.

Such metabolism is accompanied by the formation of free radicals....

The consequences of methanol metabolism and toxicity distinguish the human
and monkey from lower animals.

Formic acid is likely to be the cause of the metabolic acidosis and ocular
toxicity in humans and monkeys,
which is not observed in most lower animals.

Nevertheless, chemically reactive formaldehyde and free radicals may damage
most of the components of the cells of all animal species, mainly proteins
and lipids...."

http://taylorandfrancis.metapress.com/openurl.asp?genre=article&eissn=1537-6524&volume=13&issue=4&spage=277

Toxicology Mechanisms and Methods
 Publisher:  Taylor & Francis Health Sciences, part of the Taylor & Francis
Group   Issue:  Volume 13, Number 4 / Oct-Dec 2003   Pages:  277 - 293

Toxicological and Metabolic Consequences of Methanol Poisoning
Elzbieta Skrzydlewska, Assoc. Professor, MSc, PhD, Deputy Dean of Faculty of 
Pharmacy,  Head of Department of Analytical Chemistry, Medical University of 
Bialystok,  Mickiewicza 2A   15-230 Bialystok 8, P.O. Box 14, Poland
skrzydle at amb.ac.bialystok.pl
http://www.amb.edu.pl/en/sites/university.html  dzss at amb.edu.pl
Kilinskiego 1   15-089  Bialystok, Poland     fax (48 85)7485408

Abstract:
Methanol, when introduced into all mammals, is oxidized into formaldehyde
and then into formate, mainly in the liver.

Such metabolism is accompanied by the formation of free radicals.

In all animals, methanol oxidation, which is relatively slow, proceeds via
the same intermediary stages, usually in the liver,
and various metabolic systems are involved in the process, depending on the
animal species.

In nonprimates, methanol is oxidized by the catalase-peroxidase system,
whereas in primates, the alcohol dehydrogenase system takes the main role in
methanol oxidation.

The first metabolite (formaldehyde is rapidly oxidized by formaldehyde
dehydrogenase) is the reduced glutathione (GSH)-dependent enzyme.

Generated formic acid is metabolized into carbon dioxide with the
participation of H4folate and two enzymes, 10-formyl H4folate synthetase and
dehydrogenase,
whereas nonprimates oxidize formate efficiently.

Humans and monkeys possess low hepatic H4folate and 10-formyl H4folate
dehydrogenase levels
and are characterized by the accumulation of formate after methanol
intoxication.

The consequences of methanol metabolism and toxicity distinguish the human
and monkey from lower animals.

Formic acid is likely to be the cause of the metabolic acidosis and ocular
toxicity in humans and monkeys,
which is not observed in most lower animals.

Nevertheless, chemically reactive formaldehyde and free radicals may damage
most of the components of the cells of all animal species, mainly proteins
and lipids.

The modification of cell components results in changes in their functions.

Methanol intoxication provokes a decrease in the activity and concentration
of antioxidant enzymatic as well as nonenzymatic parameters,
causing enhanced membrane peroxidation of phospholipids.

The modification of protein structure by formaldehyde as well as by free
radicals results changes in their functions,
especially in the activity of proteolytic enzymes and their inhibitors,
which causes disturbances in the proteolytic-antiproteolytic balance toward
the proteolytics and
enhances the generation of free radicals.

Such a situation can lead to destructive processes because components of the
proteolytic-antiproteolytic system during enhanced membrane lipid
peroxidation may penetrate from blood into extracellular space, and an
uncontrolled proteolysis can occur.

This applies particularly to extracellular matrix proteins.

Keywords:
Free Radicals, Methanol Metabolism, Methanol Poisoning, Proteases, Protease
Inhibitors

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subscription sales from this site.    The price for this article is $25.00.

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Taylor & Francis Group
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J Pharm Pharmacol. 2000 May; 52(5): 547-52.
Protective effect of N-acetylcysteine on rat liver cell membrane during
methanol intoxication.    kasacka at amb.edu.pl
Dobrzynska I, Skrzydlewska E, Kasacka I, Figaszewski Z.
Institute of Chemistry, University in Bialystok, Poland.

Methanol is oxidized in vivo to formaldehyde and then to formate, and these
processes are accompanied by the generation of free radicals.
We have studied the effect of N-acetylcysteine on liver cell membrane from
rats intoxicated with methanol (3.0 g kg(-1)).
Evaluation of the effect was achieved by several methods.
Lipid peroxidation and surface charge density were measured.
An ultrastructural study of the liver cells was undertaken.
The concentration of marker enzymes of liver damage (alanine
aminotransferase and aspartate aminotransferase) in blood serum was
measured.
Methanol administration caused an increase in lipid peroxidation products
(approximately 30%) as well as in surface charge density (approximately
60%).
This might have resulted in the membrane liver cell damage visible under
electron microscopy and a leak of alanine aminotransferase and aspartate
aminotransferase into the blood (increase of approximately 70 and 50%,
respectively).
Ingestion of N-acetylcysteine with methanol partially prevented these
methanol-induced changes.
Compared with the control group, lipid peroxidation was increased by
approximately 3% and surface charge density by approximately 30%.
Alanine aminotransferase and aspartate aminotransferase activity increased
by 9 and 8%, respectively, compared with the control group.
The results suggested that N-acetylcysteine was an effective antioxidant in
methanol intoxication.
It may have efficacy in protecting free radical damage to liver cells
following methanol intoxication.  PMID: 10864143



"Changes in protein structure resulted both from free radical action and
formaldehyde generation during methanol intoxication."

J Appl Toxicol. 2000 May-Jun; 20(3): 239-43.
Effect of methanol intoxication on free-radical induced protein oxidation.
Skrzydlewska E, Elas M, Farbiszewski R, Roszkowska A.
Department of Analytical Chemistry, Medical University, 15-230 Bialystok 8,
Poland.

Oxygen free radicals are generated during methanol-induced liver injury, as
was shown for ethanol.
The effect of methanol intoxication (6 g kg(-1) body wt.) on protein
modification in the liver of rats was investigated.
Electron spin resonance determination indicated an increase in the free
radical signal 6 and 12 h after intoxication.
After 7 days of treatment, the contents of malondialdehyde and carbonyl
groups in proteins were significantly increased.
The level of amino groups and sulphydryl groups and the amount of tryptophan
in proteins were decreased,
whereas the amount of bi-tyrosine was increased significantly.
Changes in protein structure resulted both from free radical action and
formaldehyde generation during methanol intoxication.
Copyright 2000 John  Wiley & Sons, Ltd.    PMID: 10797478



Toxicology. 2000 Dec 7; 156(1): 47-55.
N-acetylcysteine or trolox derivative mitigate the toxic effects of methanol
on the antioxidant system of rat brain.
Farbiszewski R, Witek A, Skrzydlewska E.
Department of Analytical Chemistry, Bialystok Medical Academy, Mickiewicza
Str 2, P.O. Box 14, 15-230 Bialystok 8, Poland.

The effect of two compounds: N-acetylcysteine (NAC) and trolox derivative
(U-83836E) on the methanol induced impairment of the antioxidant system of
the rat brain was studied in male Wistar rats (approx. 250 g body weight).
The animals were divided into six main groups:
control group (0.5 ml of physiological saline intragastrically),
NAC group (150 mg/kg intraperitoneally-i.p),
U-83836E group (10 mg/kg i.p.),
methanol group (3 g/kg intragastrically),
NAC+methanol and U-83836E+methanol groups.
In these particular groups the changes in antioxidant parameters were
observed for 6,12,24,48 h and 5 and 7 days.
The results proved that the use of methanol and N-acetylcysteine increased
the activities of Cu,Zn-superoxide dismutase, glutathione peroxidase and
glutathione reductase by about 15,15 and 41%, respectively, in comparison to
the group of rats receiving methanol alone.
Similarly, the level of GSH increased by about 17%, the concentration of
ascorbate by 20%, while the thiobarbituric acid-reactive substances (TBA-rs)
diminished to the values as in control group.
The use of new antioxidant U8383E and methanol showed less beneficial effect
in the measured parameters however,
it serves as a better protector for the methanol induced decrease in
GSH-content. These data suggest that NAC and U-83836E mitigate the toxic
effects of methanol on the antioxidant system of the rat brain.  PMID: 
11162875




Rocz Akad Med Bialymst. 1999; 44: 89-101.
Morphological changes in the liver of rats intoxicated with methanol.
Kasacka I, Skrzydlewska E.
Department of Histology and Embryology, Medical Academy of Bialystok.

On the basis of morphological examinations in light and electron microscope,
the evaluation of methanol influence on the liver of rats was conducted.
The examination was carried out in the group of 36 rats that were given a
single dose of methanol (1.5 g/kg b.w.) into the stomach through a gastric
tube.
The liver was taken from rats under the ether anaesthesia after 6, 12, and
24 hours as well as after 2, 5, and 7 days of methanol administration.
Results showed that methanol intoxication caused visible changes in the
examined organ.
Only 6 h after intoxication, lobular peripheral hepatocytes presented
characteristic features of vacuolar degradation persisting up to 48 h.
Since the second day of intoxication, many cells with double nuclei were
found more frequently than in controls.
Single hepatocytes or small hepatocytic clusters with the features of
deliquescent necrosis could be seen after 5 and 7 days of examination.
All animals intoxicated with methanol showed distinct weakness of glycogen
reaction.
The loss of glycogen resources was highest at 24 h after methanol
administration.
The results indicate, that methanol causes morphological changes in the rat
liver and that intensification of these changes depends on the time after
intoxication.  PMID: 10697423



Rocz Akad Med Bialymst. 1999; 44: 76-88.
Activity of lysosomal proteases in the liver and in the plasma from rats
intoxicated with methanol.
Skrzydlewska E.
Department of Analytical Chemistry, Medical Academy of Bialystok.

The activity of lysosomal proteolytic enzymes (cathepsin A, B, C, D and E)
in cytosol and in the whole homogenate of the liver and in the blood plasma
from rats intoxicated with 1.5, 3.0 and 6.0 g methanol/kg b.w. was measured
6, 12 and 24 h and 2, 5 and 7 days after the intoxication.
The activity of all proteases was increased in the cytosol from 12 h to 5
days of intoxication, whereas the activity of these enzymes was decreased in
the whole liver homogenate during the same time.
The magnitude of the decrease in proteolytic activity in the whole
homogenate of the liver depended on the amino acid active center of the
enzyme.
The greatest decrease was observed for sulfhydryl and hydroxyl proteases and
smaller one for carboxyl proteases.
The proteases activity in the plasma was increased from 12 h to 5 days after
methanol intoxication.
These results suggest that during methanol intoxication the cellular and
lysosomal membranes are impaired and proteases are translocated into the
blood. However, changes in proteases activities and proteases distribution
within the hepatocytes may lead to disturbances in the catabolism of cell 
proteins
and to destruction of liver cells.  PMID: 10697422



"The primary metabolic appropriation of methanol is oxidation to
formaldehyde and then to formate.
These processes are accompanied by formation of superoxide anion and
hydrogen peroxide....
Methanol administration,[ by ] increasing concentration of the lipid
peroxidation products, decreased the liver glutathione-peroxidase and
glutathione reductase (GSSG-R) activities, GSH concentration and total
antioxidant status (TAS)."

Drug Alcohol Depend. 1999 Nov 1; 57(1): 61-7.
Protective effect of N-acetylcysteine on reduced glutathione, reduced
glutathione-related enzymes and lipid peroxidation in methanol intoxication.
Skrzydlewska E, Farbiszewski R.   skrzydle at amb.ac.bialystok.pl
Department of Analytical Chemistry, Bialystok Medical University, Poland.

The primary metabolic appropriation of methanol is oxidation to formaldehyde
and then to formate.
These processes are accompanied by formation of superoxide anion and
hydrogen peroxide.
This paper reports data on the effect of N-acetylcysteine (NAC) on reduced
glutathione (GSH) and on activity of some GSH-metabolising enzymes in the
liver, erythrocytes and serum of rats intoxicated with methanol (3 g/kg
b.w.) during 7 days after intoxication.
Methanol administration,[ by ] increasing concentration of the lipid
peroxidation products, decreased the liver glutathione-peroxidase and
glutathione reductase (GSSG-R) activities, GSH concentration and total
antioxidant status (TAS).
The use of NAC after methanol ingestion apparently diminished lipid
peroxidation, elevated the GSH level in the liver and erythrocytes, and
increased activity of GSH-related enzymes in the serum, erythrocytes and in
the liver.
These results suggest that NAC exerts its protective effect by acting as a
precursor for glutathione, the main low molecular antioxidant and as a free
radical scavenger.  PMID: 10617314



"Methanol ingestion in humans caused changes in activities of proteases and
their inhibitors with similar direction as in rats.
These changes in activity of proteases and their inhibitors produce signific
ant disturbances in proteolytic-antiproteolytic balance after methanol
administration."

J Toxicol Environ Health A. 1999 Jul 23; 57(6): 431-42.
Activity of cathepsin G, elastase, and their inhibitors in plasma during
methanol intoxication.
Skrzydlewska E, Szmitkowski M, Farbiszewski R.
Department of Analytical Chemistry, Medical University, Bialystok, Poland.
skrzydle at amb.ac.bialystok.pl

Methanol oxidation in the liver is accompanied by formation of formaldehyde
and free radicals.
These compounds can react with biologically active proteins, including
proteolytic enzymes and their inhibitors.
The activity of cathepsin G and elastase and their inhibitors such as
alpha-1-antitrypsin and alpha-2-macroglobulin in plasma of rats given
methanol orally in doses of 1.5, 3, and 6 g/kg was investigated for 7 days.
The activity of cathepsin G and elastase was increased from 12 h to 5 d,
proportionally to methanol dose.
At the same time, activity of their inhibitors was reduced.
Methanol ingestion in humans caused changes in activities of proteases and
their inhibitors with similar direction as in rats.
These changes in activity of proteases and their inhibitors produce signific
ant disturbances in proteolytic-antiproteolytic balance after methanol
administration.  PMID: 10478824


Folia Histochem Cytobiol. 1999; 37(2): 111-2.
Parenchymal cell mitochondria in the liver of rats after methanol
intoxication.
Kasacka I, Skrzydlewska E.
Department of Histology, Medical University, Bialystok, Poland.
PMID: 10352983


"Our findings indicate decreased antioxidative potential both in the brain
and in the liver of rats after methanol ingestion."

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1998 Aug; 120(2):
289-94.
The comparison of the antioxidant defense potential of brain to liver of
rats after methanol ingestion.
Skrzydlewska E, Witek A, Farbiszewski R.
Department of Instrumental Analysis, Bialystok Medical University, Poland.

The antioxidant enzymatic and nonenzymatic potential in the brain of rats
given methanol orally was investigated for 7 days consecutively and compared
to that one in the liver.
Glutathione (GSH) and the activities of superoxide dismutase (Cu, Zn-SOD),
glutathione peroxidase (GSH-Px) and glutathione reductase (GSSG-R) were
reduced in the brain after the first 24 h, whereas
in the liver these parameters were diminished after 6 h.
The brain catalase (CAT) activity was very low and constant in contrast to
high and changeable CAT in the liver.
At the beginning of intoxication, the activities of Cu, Zn-SOD and CAT in
the liver were increased;
after 5 days they were restored to normal values
while Cu, Zn-SOD diminished gradually in the brain.
An early change that occurred 6 h after intoxication was a decrease of
ascorbate in the brain and in the liver.
The increase in thiobarbituric acid-reactive substances (TBA-rs) in the
liver was preceded by their increase in the brain.
Our findings indicate decreased antioxidative potential both in the brain
and in the liver of rats after methanol ingestion.
The regulatory mechanisms of the antioxidant enzymes in the brain of
intoxicated rats differ from those ones in the liver.  PMID: 9827043



Acta Biol Hung. 1998; 49(2-4): 345-52.
Formaldehyde-induced modification of hemoglobin in vitro.
Farbiszewski R, Skrzydlewska E, Roszkowska A.
Department of Analytical Chemistry, Medical University, Bialystok, Poland.

Formaldehyde is known to react with proteins.
The purpose of our experiments was to analyse in vitro the effect of
formaldehyde on the physicochemical and biological properties of hemoglobin
molecules.
The effect of formaldehyde concentration, reaction time, pH and temperature
on hemoglobin free amino groups was estimated.
The modified hemoglobin was analysed using electrophoretic, potentiometric
and spectrophotometric techniques.
Reaction between formaldehyde and hemoglobin was accelerated by increasing
concentration of formaldehyde and higher temperature.
This reaction was most intensive during the first few hours at pH 7.4 so the
amount of free amino groups of hemoglobin was significantly diminished by
directly mixing formaldehyde with hemoglobin.
The modified protein was characterized by the increase in electrophoretic
mobility and the decrease in maximum absorption derived from porphyrin
rings. Formaldehyde modified hemoglobin was less susceptible to the action
of
cathepsin D.  PMID: 10526979



"These results indicate that methanol intoxication in rats leads to an
increase in the lipid peroxidation and impairment in the antioxidant
mechanisms in liver, erythrocytes, and blood serum."

J Toxicol Environ Health A. 1998 Apr 24; 53(8): 637-49.
Lipid peroxidation and antioxidant status in the liver, erythrocytes, and
serum of rats after methanol intoxication.
Skrzydlewska E, Farbiszewski R.
Department of Instrumental Analysis, Medical Academy, Bialystok, Poland.

Lipid peroxidation products measured as a malondialdehyde and activities of
superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione
reductase (GSSG-R), and concentrations of ascorbic acid, alpha-tocopherol,
and glutathione (GSH) were measured in the liver, erythrocytes, and serum of
rats 6, 14, and 24 h and 2, 5, and 7 d after treatment with 3 g methanol/kg.
GSH-Px and GSSG-R activities, GSH level, and ascorbate concentration in the
liver, erythrocytes, and blood serum were significantly decreased.
In addition, SOD and alpha-tocopherol in erythrocytes were diminished, while
malondialdehyde (MDA) in liver, erythrocytes, and serum were elevated.
Further, erythrocyte counts, hemoglobin levels, hematocrit, and mean
corpuscular volume (MCV) were reduced.
These results indicate that methanol intoxication in rats leads to an
increase in the lipid peroxidation and impairment in the antioxidant
mechanisms in liver, erythrocytes, and blood serum.  PMID: 9572161



Rocz Akad Med Bialymst. 1997; 42 Suppl 2: 47-55.
Ultrastructural evaluation of lysosomes and biochemical changes in cathepsin
D distribution in hepatocytes in methanol intoxication.
Skrzydlewska E, Szynaka B.
Department of Instrumental Analysis, Medical Academy of Bialystok.

Methanol oxidation is accompanied by free radicals and formaldehyde
formation.
It is likely to cause damage of lysosomal membranes.
Lysosomal ultrastructure under transmission electron microscope and
biochemical
localization of cathepsin D were estimated after rats intoxication with
methanol.
The examination was carried out 6, 12 and 24 h and 2.5 and 7 days after
intoxication.
Ultrastructural examination showed that methanol causes
extension of Golgi apparatus cisterns and an increase in a number of
lysosomes.
>From 12 h to 2 days lysosomes were characterized by damage of structure of
membrane enclosing lysosomes.
During the first days of intoxication activity of cathepsin D decreased in
lysosomes and increased in cytosol.
These changes may lead to uncontrolled extralysosomal proteolysis in the
liver cells and to the onset of liver tissue destruction.  PMID: 9646682



Rocz Akad Med Bialymst. 1997; 42 Suppl 2: 39-46.
Ultrastructural evaluation of hepatocytes membranes and changes in cytosolic
enzymes distribution in methanol intoxication.
Skrzydlewska E, Szynaka B.
Department of Instrumental Analysis, Medical Academy of Bialystok.

Acute methanol intoxication causes metabolic and structural disturbances of
liver cells.
The aim of this paper, therefore, was to evaluate the
ultrastructure of liver cells membrane and the amount of lipid peroxidation
products, as well as the concentration of marker enzymes of liver damage
(ALT and AST) in blood serum.
The experiment was done on Wistar rats which once received intragastrically
6.0 g methanol/kg b.w. as a 50% solution.
The animals were decapitated 6, 12 and 24 h and 2, 5 and 7 days after the
methanol administration.
The liver was evaluated under transmission electron microscope and lipid
peroxidation products were determined in the liver homogenate.
The serum ALT and AST activity were also assayed.
The biochemical results indicate the increase in lipid peroxidation
products.
The consequence of this is probably the membrane liver cell damage visible
in the electron microscope.   PMID: 9646681




"The primary metabolic fate of methanol is oxidation to formaldehyde and
then to formate by enzymes of the liver....

Changes due to methanol ingestion may indicate impaired antioxidant defense
mechanisms in the liver tissue. "

Free Radic Res. 1997 Oct; 27(4): 369-75.
Decreased antioxidant defense mechanisms in rat liver after methanol
intoxication.
Skrzydlewska E, Farbiszewski R.
Department of Instrumental Analysis, Medical Academy, Poland.

The primary metabolic fate of methanol is oxidation to formaldehyde and then
to formate by enzymes of the liver.
Cytochrome P-450 and a role for the hydroxyl radical have been implicated in
this process.
The aim of the paper was to study the liver antioxidant defense system in
methanol intoxication, in doses of 1.5, 3.0 and 6.0 g/kg b.w., after
methanol administration to rats.
In liver homogenates, the activities of Cu,Zn-superoxide dismutase and
catalase were significantly increased after 6 h following methanol ingestion
in doses of 3.0 and 6.0 g/kg b.w. and persisted up to 2-5 days,
accompanied by significant decrease of glutathione reductase and glutathione
peroxidase activities.
The content of GSH was significantly decreased during 6 hours to 5 days.
The liver ascorbate level was significantly diminished, too, while MDA
levels were considerably increased after 1.5, 3.0 and 6.0 g/kg b.w. methanol
intoxication.
Changes due to methanol ingestion may indicate impaired antioxidant defense
mechanisms in the liver tissue.  PMID: 9416465



Arch Toxicol. 1997; 71(12): 741-5.
Glutathione consumption and inactivation of glutathione-related enzymes in
liver, erythrocytes and serum of rats after methanol intoxication.
Skrzydlewska E, Farbiszewski R.
Department of Instrumental Analysis, Medical Academy, Bialystok, Poland.

The primary metabolic fate of methanol is oxidation to formaldehyde and then
to formate.
These processes are accompanied by formation of superoxide anion
and further hydrogen peroxide.
Glutathione plays a unique role in the cellular defense system against
xenobiotics.
The glutathione (GSH) content and glutathione peroxidase (GSH-Px) and
glutathione reductase (GSSG-R) activities were measured in liver,
erythrocytes and serum of rats.
Rats were intoxicated with 3.0 and 6.0 g methanol/kg body wt. and
measurements taken after 6, 12 and 24 h and 2, 5 and 7 days of intoxication.
The decrease in GSH content and in GSH-related enzyme activity was observed
during the whole time-course of the intoxication.
The most significant changes were observed in the erythrocytes.
The results obtained show that the protection against oxidative damage due
to methanol intoxication in rats seems to be less efficient than in control
rats.  PMID: 9388006



Acta Biochim Pol. 1997; 44(2): 339-42.
Activity of liver proteases in experimental methanol intoxication.
Skrzydlewska E, Skrzydlewski Z, Worowski K.
Department of Instrumental Analysis, Medical Academy, Bialystok, Poland.

Intoxication of rats with methanol (1.5 and 3.0 g/kg body weight) led to a
significant, time- and dose-dependent decrease in the activities of
cathepsins A, B and C, while the activity of cathepsin D was unaffected.
The decrease was associated with a different partial release of individual
cathepsins to the post-lysosomal fraction.  PMID: 9360724



Fundam Clin Pharmacol. 1997; 11(5): 460-5.
Trolox-derivative antioxidant protects against methanol-induced damage.
Skrzydlewska E, Farbiszewski R.
Department of Instrumental Analysis, Medical Academy, Bialystok, Poland.

This paper reports data on the effect of a new antioxidant, U-83836E, on the
lipid peroxidation and antioxidant status of liver, red blood cells (RBCs)
and blood serum of rats intoxicated with methanol (3.0 g/kg body weight).
Methanol administration slightly increased the levels of peroxidation
products in the liver, and markedly increased them in RBCs and serum.
In contrast, glutathione-peroxidase, glutathione-reductase activity, reduced
glutathione concentration and total antioxidant status were decreased.
The use of U-83836E, containing a trolox ring, appeared to be beneficial in
reducing lipid peroxidation products and in partially in preventing the
decrease in glutathione and antioxidant enzymes induced by methanol in liver
and serum.
These results show that antioxidant U-83836E may partially prevent methanol
toxicity.  PMID: 9342600

"These results indicate that methanol in rats leads to the impairment of
antioxidant mechanisms in the liver, erythrocytes, and blood serum. "

Alcohol. 1997 Sep-Oct; 14(5): 431-7.
Antioxidant status of liver, erythrocytes, and blood serum of rats in acute
methanol intoxication.
Skrzydlewska E, Farbiszewski R.
Department of Analytical Chemistry, Medical Academy, Poland.

SOD, CAT, GSH-Px, GSSG-R, ascorbic acid, alpha-tocopherol, nonprotein- and
protein-bound sulfhydryl compounds, and TBA-rs content
in the liver, erythrocytes, and blood serum of rats treated with methanol
after 6, 12, and 24 h and 2, 5, and 7 days were investigated.
Furthermore, hematological parameters of erythrocytes were analysed.
GSH-Px, GSSG-R, sulfhydryl compounds, and ascorbic acid in the liver,
erythrocytes, and in blood serum were significantly decreased.
In addition, Cu,Zn-SOD and tocopherol in erythrocytes were diminished,
whereas TBA-rs in the three biological materials was enhanced.
Simultaneously, erythrocytes amount, hemoglobin level, hematocrit, and MCV
were reduced.
These results indicate that methanol in rats leads to the impairment of
antioxidant mechanisms in the liver, erythrocytes, and blood serum.  PMID:
9305457



Alcohol. 1997 May-Jun; 14(3): 295-9.
Influence of methanol and its metabolites on the activity of
alpha1-antitrypsin.
Skrzydlewska E, Mielczarska J.
Department of Instrumental Analysis, Medical Academy, Bialystok, Poland.

Among methanol and its metabolites, formaldehyde was found to have the
strongest inactivating effect on the activity of alpha1-antitrypsin
preparation and inhibitor existing in blood serum.
The influence of formaldehyde on the activity of serum alpha1-antitrypsin is
lower in comparison with purified inhibitor.
alpha1-Antitrypsin modified by formaldehyde inactivates the trypsin in its
action on the BAPA to a smaller degree than on the hemoglobin.
The effective formaldehyde concentration in the case of the BAPA is about 64
mM and in the case of the hemoglobin is about 256 mM.
The significant inhibitory effect of methanol on alpha1-antitrypsin appears
only at a high concentration of this compound.
Formate does not decrease alpha1-antitrypsin activity.
In people intoxicated with methanol, alpha1-antitrypsin activity decreases,
whereas the content of this inhibitor does not change.  PMID: 9160807



Acta Biochim Pol. 1997; 44(1): 139-45.
Liver and serum antioxidant status after methanol intoxication in rats.
Skrzydlewska E, Farbiszewski R.
Department of Instrumental Analysis, Medical Academy, Bialystok, Poland.

Activities of superoxide dismutase (SOD), catalase, glutathione peroxidase
(GSH-Px) and glutathione reductase (GSSG-R) and concentration of ascorbate,
alpha-tocopherol, non-protein and protein-bound sulfhydryl compounds and
thiobarbituric acid-reactive substances (TBA-rs) were measured in liver and
serum of rats 6, 12 and 24 h and 2, 5 and 7 days after intoxication with
1.5 g or 3.0 g methanol/kg b.w.
Liver GSH-Px and GSSG-R activities and SH-groups and ascorbate content were
significantly diminished at 6 and 24 h,
while TBA-rs were increased.
Serum SOD, GSH-Px and GSSG-R activities and SH-groups concentration were
reduced, while TBA-rs were elevated.
The changes were more intensive after application of the higher dose of
methanol.
It is concluded that methanol impairs the liver and blood serum antioxidant
mechanisms in rats.   PMID: 9241366




Vet Hum Toxicol. 1996 Dec; 38(6): 429-33.
Diminished antioxidant defense potential of liver, erythrocytes and serum
from rats with subacute methanol intoxication.
Skrzydlewska E, Farbiszewski R.
Department of Analytical Chemistry, Medical Academy, Bialystok, Poland.

The activities of superoxide dismutase (SOD), catalase glutathione
peroxidase (GSH-Px) and glutathione reductase (GSSG-R) and the concentration
of ascorbate, alpha-tocopherol, non-protein and protein-bound sulfhydryl
compounds, and thiobarbituric acid-reactive substances (TBA-rs)
in liver, erythrocytes and serum of rats dosed with 1.5 g methanol/kg bw
were measured after 6, 12 and 24 h and 2, 5 and 7 d.
Hematological erythrocyte parameters were also determined.
Liver GSH-Px and GSSG-R activities, SH-groups and ascorbate were
significantly diminished at 12 and 24 h, while TBA-rs increased.
Blood SOD, GSH-Px and GSSG-R activities and sulfhydryl-group concentrations
were reduced while TBA-rs were elevated.
Methanol given to rats impaired liver, erythrocyte and blood serum
antioxidant mechanisms.  PMID: 8948074



Rocz Akad Med Bialymst. 1996; 41(2): 397-404.
Decreased antioxidant status and increased lipid peroxidation in rats after
methanol intoxication.
Skrzydlewska E.
Department of Instrumental Analysis, Medical Academy of Bialystok.

The liver is the main metabolic place where the methanol is oxidized to
formaldehyde and to formate.
The aim of this paper was to study the liver antioxidant system in acute
methanol intoxication, after 6, 12, 24 hours and 2, 5 and 7 days of alcohol
administration into rats.
In liver homogenates the superoxide dismutase, catalase, peroxidase and
reductase glutathione activity and content of malondialydehyde (MDA),
SH-compounds in protein and non-protein fraction and ascorbate were
estimated.
Activity of superoxide dismutase and catalase was significantly increased
after 6 hours following methanol ingestion and persisted up to 2-5 days of
intoxication.
It was accompanied by significant decreased of reductase and peroxidase
glutathione activities.
The protein and non-protein SH-groups were significantly decreased during 6
hours to 5 days following methanol ingestion.
The liver MDA content was considerably increased.
After 2 days since methanol intoxication the liver vitamin C content was
significantly decreased in comparison with the control group.
The obtain results demonstrated that during methanol induced liver injury
there are increase of lipid peroxidation and impairment of proantioxidant
equilibrium in favour to prooxidant.   PMID: 9020552



Pol Tyg Lek. 1993 May 3-10; 48(18-19): 433-6.
[Metabolism and toxic effects of methanol]
[Article in Polish]
Skrzydlewska E.
Zakladu Analizy Instrumentalnej AM, Bialymstoku.
Publication Types: Review Review, Tutorial  PMID: 8309827



Cell Mol Biol Lett. 2003; 8(2): 391-413.
DNA damage caused by lipid peroxidation products.
Luczaj W, Skrzydlewska E.
Department of Analytical Chemistry, Medical Academy of Bialystok,
Mickiewicza 2A, P.O. Box 14, 15-230 Bialystok 8, Poland.

Lipid peroxidation is a process involving the oxidation of polyunsaturated
fatty acids (PUFAs), which are basic components of biological membranes.
Reactive electrophilic compounds are formed during lipid peroxidation,
mainly alpha, beta-unsaturated aldehydes.
These compounds yield a number of adducts with DNA.
Among them, propeno and substituted propano adducts of deoxyguanosine with
malondialdehyde (MDA), acrolein, crotonaldehyde and etheno adducts,
resulting from the reactions of DNA bases with epoxy aldehydes, are a very
important group of adducts.
The epoxy aldehydes are more reactive towards DNA than the parent
unsaturated aldehydes.
The compounds resulting from lipid peroxidation mostly react with DNA
showing both genotoxic and mutagenic action;
among them, 4-hydroxynonenal is the most genotoxic, while MDA is the most
mutagenic.
DNA damage caused by the adducts of lipid peroxidation products
with DNA can be removed by the repairing action of glycosylases.
The formed adducts have been hitherto analyzed using the IPPA
(Imunopurification-(32)P-postlabelling assay) method and via gas
chromatography/electron capture negtive chemical ionization/mass
spectrometry (GC/EC NCI/MS).
A combination of liquid chromatography with electrospray tandem mass
spectrometry (LC/ES-MSMS) with labelled inner standard has mainly been used
in recent years.  PMID: 12813574



Hepatogastroenterology. 2003 Jan-Feb; 50(49): 126-31.
Antioxidant potential in esophageal, stomach and colorectal cancers.
Skrzydlewska E, Kozuszko B, Sulkowska M, Bogdan Z, Kozlowski M,
Snarska J, Puchalski Z, Sulkowski S, Skrzydlewski Z.
skrzydle at amb.ac.bialystok.pl
Department of Analytical Chemistry Medical Academy, 15-230 Bialystok 8, P.O.
Box 14, Poland.

BACKGROUND/AIMS: The gastrointestinal tract is particularly susceptible to
reactive oxygen species attack which lead to carcinogenesis.
An important role in defense strategy against reactive oxygen species is
played by antioxidants.
The present study aims at examining antioxidant parameters and
malondialdehyde-- the product of lipid peroxidation as well as the marker of
cancer progression-- and cancer procoagulant in esophageal, gastric and
colorectal cancer patients.
METHODOLOGY: The activity of superoxide dismutase, catalase, glutathione
peroxidase and reductase and the level of glutathione, vitamin C,
malondialdehyde and cancer procoagulant were determined in tumors and normal
mucous from 18 patients with esophageal cancer, 18 patients with stomach
tumor and 62 patients with colorectal cancer.
RESULTS: In esophageal tumor the activity of all enzymes has been increased
compared with normal mucous.
Stomach tumor has been also characterized by an increase in antioxidant
enzymes activity except glutathione peroxidase and reductase whose
activities have been decreased.
However in colorectal tumor the activity of enzymes has been increased apart
from catalase.
In all cases the glutathione level has been increased while the vitamin C
content has been significantly decreased.
Tumor malondialdehyde level was significantly increased, too.
The level of cancer procoagulant also increased in cancer tissues as well as
in the serum.
CONCLUSIONS: Antioxidant potential in all cases of
gastrointestinal tract cancer has been unbalanced which has lead to increase
in reactive oxygen species action and enhancement of lipid peroxidation and
cancer procoagulant generation.  PMID: 12630007



J Toxicol Environ Health A. 2004 Apr 9; 67(7): 595-606.
Green tea protection against age-dependent ethanol-induced oxidative stress.
Luczaj W, Waszkiewicz E, Skrzydlewska E, Roszkowska-Jakimiec W.
Department of Analytical Chemistry, Medical University of Bialystok,
Bialystok, Poland.

Ethanol intoxication leads to oxidative stress, which may be additionally
enhanced by aging.
The aim of this study was to investigate the influence of green tea as a
source of water-soluble antioxidants on the ability to prevent oxidative
stress in aged rats sub-chronically intoxicated with ethanol.
Two-, 12-, and 24-mo-old male Wistar rats were divided into 4 experimental
groups: (1) control, (2) green tea, (3) ethanol, and (4) ethanol and green
tea. Ethanol intoxication produced age-dependent decrease in the activity of
serum superoxide dismutase, glutathione peroxidase, and reductase and in
levels of glutathione (GSH), vitamins C, E, and A, and beta-carotene.
Changes in the serum antioxidative ability were accompanied by enhanced
oxidative modification of lipid (increase in lipid hydroperoxides,
malondiadehyde, and 4-hydroxynonenal levels) and protein (rise in carbonyl
group levels).
Green tea partially protected against changes in antioxidant enzymatic as
well as nonenzymatic parameters produced by ethanol and enhanced by aging.
Administration of green tea significantly protects cellular components such
as lipids and proteins against oxidative modification.
Results indicate that green tea effectively protects blood serum against
oxidative stress produced by ethanol as well as aging.  PMID: 15129554



Postepy Hig Med Dosw (Online). 2004 Mar 30; 58: 194-201.
[Antioxidative abilities during aging]  [Article in Polish]
Augustyniak A, Skrzydlewska E.
Zaklad Chemii Nieorganicznej i Analitycznej Akademii Medycznej w
Bialymstoku.

Biological aging is associated with increased cellular levels of reactive
oxygen species (ROS) as well as the formation and accumulation of oxidized
biomolecules.
During evolution, organisms developed a highly-efficient and adaptive
antioxidant defense system.
Antioxidants can generally be divided into two categories: enzymatic and
non-enzymatic. During the aging process the activity of antioxidant enzymes,
e.g. SOD, CAT, GSH-Px, and GSSG-R, depends on factors such as race, gender,
tissue and subcellular localization of enzymes.
The age-dependent decrease in antioxidant enzyme activity may be attributed
to oxidative modifications of enzymes.
During the aging process, ROS may also lead to the induction of some enzyme
activity which is explained as an adaptive phenomenon.
The decrease in GSH concentration with age can be explained by decreased GSH
synthesis and/or increased GSH consumption in the removal of peroxides and
xenobiotics.
In plasma albumin, ferritin, transferrin, and caeruloplasmin exert
protective action.
Plasma proteins can inhibit ROS generation and lipid peroxidation by
chelating free transition metals.
Plasma protein concentrations changes with age.
The major exogenous antioxidants, mostly derived from the diet, are vitamin
E, C, A, and beta-carotene.
During the aging process the level of vitamins may decrease or increase,
depending on such factors as diet, and diseases.  PMID: 15077054



Folia Morphol (Warsz). 2004 Feb; 63(1): 123-6.
Green tea as an antioxidant which protects against alcohol induced injury in
rats -- a histopathological examination.
Baltaziak M, Skrzydlewska E, Sulik A, Famulski W, Koda M.
Department of General Pathology, Medical University, Bialystok, Poland.
drbal at poczta.onet.pl

Our study with animal models was designed to test the hypothesis that green
tea protects against chronic (over 4 weeks) alcohol induced liver injury in
rats.
The research was conducted on Wistar male rats divided into 4 research
groups:
I - received the Libera-De Carli control diet (L-DC),
II - received (L-DC) and green tea,
III - received (L-DC) and ethanol and
IV - received (L-DC), green tea and ethanol.
When comparing groups I and II we saw less intensive steatosis in group II
than in group I, which can suggest that green tea may affect the
accumulation of fat in the hepatocytes and protect them against steatosis
and disruption.
In III, the ethanol group, the steatosis of the liver increased considerably
and
the green tea which was given with ethanol in group IV did not halt this,
as in group IV we also observed intensive steatosis in the liver.
>From this data we conclude that green tea has an important, although not
fully understood role in preventing liver injury.  PMID: 15039917



Alcohol. 2004 Jan; 32(1): 25-32.
Green tea protects against ethanol-induced lipid peroxidation in rat organs.
Ostrowska J, Luczaj W, Kasacka I, Rozanski A, Skrzydlewska E.
Department of Analytical Chemistry, Medical Academy of Bialystok,
PO Box 14, 15-230 Bialystok, Poland.

Ethanol metabolism is accompanied by generation of free radicals, which
stimulates lipid peroxidation.
Natural antioxidants are particularly useful in such a situation.
The current study was designed to investigate the efficacy of green tea, as
a source of water-soluble antioxidants (catechins), on lipid peroxidation in
liver, brain, and blood induced by chronic (4 weeks) ethanol intoxication in
rats. Feeding of ethanol led to a significant increase in lipid
peroxidation, as
measured by increased concentrations of lipid hydroperoxides,
4-hydroxynonenal, and malondialdehyde.
Feeding of ethanol also changed the glutathione-dependent lipid
hydroperoxide decomposition system, resulting in a decrease in both reduced
glutathione concentration and activity of glutathione peroxidase.
Observed changes were statistically significant in all examined tissues.
Enhancement in lipid peroxidation was associated with disruption of
hepatocyte cell membranes, as observed through electron microscopic
evaluation.
Green tea protects phospholipids from enhanced peroxidation and prevents
changes in biochemical parameters and morphologic changes observed after
ethanol consumption.
These results support the suggestion that green tea protects membranes from
peroxidation of lipids associated with ethanol consumption.  PMID: 15066700




Journal of Toxicology and Environmental Health Part A
Publisher:  Taylor & Francis Health Sciences, part of the Taylor & Francis
Group         Issue:  Volume 67, Number 7 / April 9, 2003  Pages:  595 - 606

Green Tea Protection Against Age-Dependent Ethanol-Induced Oxidative Stress
Wojciech  Luczaj  A1, Ewa Waszkiewicz  A1, Elzbieta  Skrzydlewska  A1,
Wiesawa Roszkowska-Jakimiec  A2

A1  Department of Analytical Chemistry, Medical University of Bialystok,
Bialystok, Poland
A2  Department of Instrumental Analysis, Medical University of Bialystok,
Bialystok, Poland

Abstract:
Ethanol intoxication leads to oxidative stress, which may be additionally
enhanced by aging.
The aim of this study was to investigate the influence of green tea as a
source of water-soluble antioxidants on the ability to prevent oxidative
stress in aged rats sub-chronically intoxicated with ethanol.
Two-, 12-, and 24-mo-old male Wistar rats were divided into 4 experimental
groups: (1) control, (2) green tea, (3) ethanol, and (4) ethanol and green
tea. Ethanol intoxication produced age-dependent decrease in the activity of
serum superoxide dismutase, glutathione peroxidase, and reductase and in
levels of glutathione (GSH), vitamins C, E, and A, and g-carotene.
Changes in the serum antioxidative ability were accompanied by enhanced
oxidative modification of lipid (increase in lipid hydroperoxides,
malondiadehyde, and 4-hydroxynonenal levels) and protein (rise in carbonyl
group levels).
Green tea partially protected against changes in antioxidant enzymatic as
well as nonenzymatic parameters produced by ethanol and enhanced by aging.
Administration of green tea significantly protects cellular components such
as lipids and proteins against oxidative modification.
Results indicate that green tea effectively protects blood serum against
oxidative stress produced by ethanol as well as aging.




Addict Biol. 2002 Jul; 7(3): 307-14.
Green tea as a potent antioxidant in alcohol intoxication.
Skrzydlewska E, Ostrowska J, Stankiewicz A, Farbiszewski R.
Department of Analytical Chemistry, Medical Academy of Bialystok, Bialystok,
Poland. skrzydle at amb.ac.bialystok.pl

Ethanol oxidation to acetaldehyde and next to acetate is accompanied by free
radical generation.
Free radicals can affect cell integrity when antioxidant mechanisms are no
longer able to cope with the free radical generation observed in ethanol
intoxication.
Natural antioxidants are particularly useful in such a situation.
The present study was designed to investigate the efficacy of green tea as a
source of water-soluble antioxidants (catechins) on the liver and blood
serum antioxidative potential of rats chronically (28 days) intoxicated with
ethanol.
Alcohol caused a decrease in liver superoxide dismutase, glutathione
peroxidase and catalase activities and an increase in activity of
glutathione reductase.
Moreover, a decrease in the level of reduced glutathione, ascorbic acid,
vitamins A and E and beta-carotene were observed.
The activity of serum glutathione peroxidase decreased while glutathione
reductase activity increased.
The level of serum non-enzymatic antioxidants was also decreased in the
liver.
Alcohol administration caused an increase in the liver and serum lipid
peroxidation products, measured as thiobarbituric acid-reactive substances.
However, green tea prevents the changes observed after ethanol intoxication.
Green tea also protects membrane phospholipids from enhanced peroxidation.
These results indicate a beneficial effect of green tea in alcohol
intoxication.  PMID: 12126490



Phytomedicine. 2002 Apr; 9(3): 232-8.
Protective effect of green tea against lipid peroxidation in the rat liver,
blood serum and the brain.
Skrzydlewska E, Ostrowska J, Farbiszewski R, Michalak K.
Department of Analytical Chemistry, Medical Academy of Bialystok, Poland.
skrzydle at solar.amb.edu.pl     michalak at biofiz.am.wroc.pl

This paper reports data on the effect of green tea on the lipid peroxidation
products formation and parameters of antioxidative system of the liver,
blood serum and central nervous tissue of healthy young rats drinking green
tea for five weeks.
The rats were permitted free access to solubilized extract of green tea.
Bioactive ingredients of green tea extract caused in the liver an increase
in the activity of glutathione peroxidase and glutathione reductase and in
the content of reduced glutathione as well as marked decrease in lipid
hydroperoxides (LOOH), 4-hydroksynonenal (4-HNE) and malondialdehyde (MDA).
The concentration of vitamin A increased by about 40%.
Minor changes in the measured parameters were observed in the blood serum.
GSH content increased slightly, whereas the index of the total antioxidant
status increased significantly.
In contrast, the lipid peroxidation products, particularly MDA was
significantly diminished.
In the central nervous tissue the activity of superoxide dismutase and
glutathione peroxidase decreased while the
activity of glutathione reductase and catalase increased after drinking
green tea.
Moreover the level of LOOH, 4-HNE and MDA significantly decreased.
The use of green tea extract appeared to be beneficial to rats in reducing
lipid peroxidation products.
These results support and substantiate traditional consumption of green tea
as protection against lipid peroxidation in the liver, blood serum, and
central nervous tissue. PMID: 12046864



Rocz Akad Med Bialymst. 2001; 46: 240-50.
The influence of green tea on the activity of proteases and their inhibitors
in plasma of rats after ethanol treatment.
Skrzydlewska E, Roszkowska A, Makiela M, Skrzydlewski Z.
Department of Analytical Chemistry, Medical Academy of Bialystok, Bialystok,
Poland.

Ethanol oxidation in the liver is accompanied by formation of acetaldehyde
and free radicals.
These compounds can react with biologically active proteins, including
proteolytic enzymes and their inhibitors.
The aim of this paper was to determine the influence of green tea on the
activity of cathepsin G and elastase and their inhibitors such as
alpha-1-antitrypsin and alpha-2-macroglobulin, total antioxidant status and
lipid peroxidation in plasma of young rats chronically intoxication with
ethanol.
The activity of cathepsin G and elastase was increased, while the activity
of their inhibitors was reduced after ethanol treatment.
At the same time, the total antioxidant status was significantly decreased
while lipid peroxidation measured as malondialdehyde and 4-hydroxynonenal
was significantly increased.
Giving green tea to rats did not change the proteases and their inhibitors
activity, but significantly increased total antioxidant status and decreased
lipid peroxidation.
Drinking green tea with ethanol partially prevents the changes observed
after ethanol intoxication.  PMID: 11780568



J Toxicol Environ Health A. 2001 Oct 12; 64(3): 213-22.
Antioxidant status and lipid peroxidation in colorectal cancer.
Skrzydlewska E, Stankiewicz A, Sulkowska M, Sulkowski S, Kasacka I.
Department of Analytical Chemistry, Medical Academy of Bialystok, Poland.
skrzydle at amb.ac.bialystok.pl

Colon carcinogenesis is a multistep process where oxygen radicals were found
to enhance carcinogenesis at all stages: initiation, promotion, and
progression.
Since insufficient capacity of protective antioxidant system can result in
cancer, the aim of this study was to examine the activity of antioxidant
enzymes (superoxide dismutase, catalase, glutathione peroxidase, and
glutathione reductase) and the levels of reduced glutathione, vitamin C, and
vitamin E.
The lipid peroxidation products were also determined by measuring
malondialdehyde and 4-hydroxynonenal levels in colorectal cancer tissue
collected from 55 patients.
In these cases the activity of superoxide dismutase, glutathione peroxidase,
and glutathione reductase was significantly increased
while the activity of catalase was significantly decreased in cancer tissue.
However, the level of nonenzymatic antioxidant parameters (glutathione,
vitamin C, and vitamin E) was significantly decreased in cancer tissue.
Further lipid peroxidation was enhanced during cancer development,
manifested by a significant increase in malondialdehyde and 4-hydroxynonenal
levels.
The obtained results indicate significant changes in antioxidant capacity of
colorectal cancer tissues, which lead to enhanced action of oxygen radicals,
resulting in lipid peroxidation.  PMID: 11594700



Postepy Hig Med Dosw. 2001; 55(6): 871-89.
[Melatonin as an antioxidant]  [Article in Polish]
Skrzydlewska E.
Zaklad Chemii Nieorganicznej i Analitycznej Akademii Medycznej w
Bialymstoku.

This review describes the structure and properties of melatonin.
The interaction of melatonin with reactive oxygen species and its protective
action in relation to DNA, lipids and proteins are presented.
The effect of melatonin on antioxidant and prooxidant enzymes is discussed,
too.       Publication Types: Review  Review, Tutorial  PMID: 11875783



Rocz Akad Med Bialymst. 2001; 46: 133-44.
Ethanol and N-acetylcysteine influence on the development of liver changes
in experimental methanol intoxication.
Kasacka I, Skrzydlewska E.
Departments of Histology & Embriology, Medical Academy of Bialystok,
Bialystok, Poland.

The evaluation of ethanol and N-Acetylcysteine (NAC) influence on
histopathological changes in rat liver intoxicated with 3 g of methanol/kg
b.w. was conducted, based on morphological examinations in light and
electron microscope.
The rats received intragastrically 3.0 g of methanol/kg b.w. as a 50%
solution, 10% ethanol for 24 hours before methanol and next 48 hours after
methanol ingestion and NAC (150 mg/kg b.w.) after 15 min. methanol
administrated.
The results indicate that methanol intoxication causes pronounced
morphological changes in the examined organ.
Ethanol administered to methanol-intoxicated rats caused intensification of
certain parameters of hepatocytes morphological damage.
A simultaneous administration of methanol and NAC resulted in a lower degree
of parenchymal damage.  PMID: 11780556




Psychiatr Pol. 1992 Sep-Oct; 26(5): 421-9.
[The influence of disulfiram on the metabolism of ethanol]  [Article in
Polish]
Skrzydlewska E.
Zakladu Analizy Instrumentalnej AM, Bialymstoku.

The author discusses the metabolism of disulfiram and the enzymes which
metabolize ethanol.
The restraining of the activity of ALDH in the liver by disulfiram causes an
accumulation of acetaldehyde which in their turn cause a series of
psychophysical symptoms which are unpleasant and in some instances dangerous
for the patient.
Thus, it is important to monitor changes in the activity of ALDH after
administration of disulfiram.
Publication Types:  Review Review, Tutorial  PMID: 1302340



Postepy Hig Med Dosw. 1992; 46(1): 117-30.
[Metabolism of liver proteins in ethanol poisoning]  [Article in Polish]
Skrzydlewska E, Worowski K, Roszkowska-Jakimiec W.
Zaklad Analizy Instrumentalnej AM, Bialymstoku.

The present paper reviews the literature on influence of ethanol and
acetaldehyde on synthesis, export and degradation of liver proteins.
Direction and intensification changes caused by ethanol and acetaldehyde
depend on concentration, time of activity and the way of administration of
these compounds, and the way of feeding.
Publication Types:  Review Review Literature PMID: 1641374



Postepy Hig Med Dosw. 1992; 46(2): 159-72.
[Proteolytic enzymes of the digestive tract in ethanol poisoning] [Article
in Polish]
Skrzydlewska E, Worowski K, Roszkowska-Jakimiec W.
Zaklad Analizy Instrumentalnej Akademii Medycznej, Bialymstoku.

The present paper reviews the literature on changes of proteolytic enzymes
activity, disorders of protein digestion and absorption of protein
degradation products from digestive tract in ethanol intoxication.
Magnitude of the change depends on concentration, dose and time of ethanol
consumption.
Acute ethanol intoxication causes increase in gastric and pancreatic
proteolytic enzymes secretion and reduces amino acids and peptides
absorption.
Chronic ethanol consumption results in reduced synthesis and secretion of
gastric and pancreatic proteinases.
Publication Types:  Review Review Literature PMID: 1470579



Postepy Hig Med Dosw. 1992; 46(4): 417-30.
[Interaction of acetaldehyde and proteins] [Article in Polish]
Skrzydlewska E, Roszkowska-Jakimiec W.
Zaklad Analizy Instrumentalnej Akademii Medycznej, Bialymstoku.

A review of literature dealing with acetaldehyde-proteins reactions in vitro
and in vivo was done.
The changes in proteins structure and functions resulting from acetaldehyde
binding were discussed.
Publication Types:  Review  Review, Academic  PMID: 1293589


Rocz Akad Med Bialymst. 1990-91; 35-36: 119-27.
[Histopathological evaluation of protective effect of L-cysteine in
ethanol-induced liver damage in rats]  [Article in Polish]
Worowski K, Chyczewski L, Skrzydlewska E.
Zakladu Analizy Instrumentalnej, Akademii Medycznej Bialymstoku.

Rats fed standard diet were intoxicated during 4 weeks with ethanol at the
dose of 0.6 g/100 g of the body weight.
This poisoning causes vacuolar degeneration, disappearance of glycogen
granules, steatosis of hepatocytes and focal necrosis changes in the liver.
The intake of food with cysteine at the dose of 0.012 and 0.024 g/100 g/24
hrs markedly prevents histopathological changes in the liver of rats
intoxicated with ethanol.
Larger amounts of cysteine (0.044 g/100 g/24 hrs) intensify
histopathological changes caused by ethanol in the liver of rats. PMID:
2136542



Rocz Akad Med Bialymst. 1990-91; 35-36: 129-41.
[Effect of cysteine on protein metabolism in the liver of rats with
ethanol-induced liver damage]  [Article in Polish]
Skrzydlewska E, Worowski K, Chyczewski L.
Zakladu Analizy Instrumentalnej, Akademii Medycznej, Bialymstoku.

Rats intoxicated with ethanol at the dose of 0.6 g/100 g of the body weight
during 4 weeks were fed on standard diet and the one containing 0.125, 0.25
and 0.5% L-cysteine.
 Intoxication of rats fed standard food causes an increase in the activity
of cathepsin D and gamma-glutamyl-transpeptidase in the liver and an
increase in the activity of alanine aminotransferase and
gamma-glutamyl-transpeptidase in the blood serum.
Consuming by rats food containing small and medium quantity of cysteine
causes normalization of the activity of all enzymes,
whereas consuming food containing large quantity of cysteine does not give
such effect.  PMID: 1983788



Rocz Akad Med Bialymst. 1990-91; 35-36: 163-75.
[Effect of immunomodulating drugs on the release and activities of lysosomal
proteinases of the liver of rats with ethanol poisoning]  [Article in
Polish]
Skrzydlewska E, Chyczewski L, Worowski K.
Zakladu Analizy Instrumentalnej, Akademii Medycznej, Bialymstoku.

Increased activity of cathepsin A and D in the cytosol fraction and
homogenate of the liver of rats intoxicated for 4 weeks with ethanol (0.6
g/100 g of the body weight) was found.
The cytosol cathepsin A and D activities were unaffected under the influence
of Levamisole and isoprinosine++.
Encorton reduced the activity of both cathepsins in the cytosol fraction
while it did not diminish their activities in the liver homogenates.
Encorton, and to a markedly lesser degree, Levamisole and isoprinosine++
caused a regression of vacuolar degeneration and of necrotic lesions and
an increase in the number of glycogen granules in the livers of
ethanol-intoxicated rats.  PMID: 1726679



Mater Med Pol. 1989 Jul-Sep; 21(3): 225-7.
Inhibitory effect of ethanol and acetaldehyde on the amidolytic activity of
trypsin and chymotrypsin.
Skrzydlewska E, Worowski K, Zakrzewska I, Prokopowicz J, Puchalski Z,
Piotrowski Z.
Department of Instrumental Analysis, Bialystok, Poland.

Ethanol and in higher degree acetaldehyde displayed inhibitory effect
directed against amidolytic activity of trypsin and chymotrypsin.
The decrease of the activity of both enzymes is related to the concentration
of these compounds.
The rate of inhibition of amidolytic activity of chymotrypsin with both
reagents is more evident in comparison to trypsin.  PMID: 2491274



Acta Med Pol. 1988; 29(1-2): 41-5.
Effect of ethanol and acetaldehyde on the enzymatic activity of human
pancreatic juice in vitro. I. Inhibition of alpha-amylase and lipase
activity. Zakrzewska I, Worowski K, Skrzydlewska E, Prokopowicz J, Puchalski
Z,
Piotrowski Z.    PMID: 3267162



Acta Med Pol. 1988; 29(1-2): 47-52.
Effect of ethanol and acetaldehyde on the enzymatic activity of human
pancreatic juice in vitro. II. Inhibition of the activity of proteolytic
enzymes. Skrzydlewska E, Worowski K, Zakrzewska I, Prokopowicz J, Puchalski
Z,
Piotrowski Z. PMID: 3076740



Folia Med Cracov. 1987; 28(1-2): 205-14.
[Effect of ethanol and acetaldehyde on the release and activity of proteases
and protein degradation in the rat liver (in vitro and in vivo studies)]
[Article in Polish]   Skrzydlewska E.  PMID: 3623321



Rocz Akad Med Im Juliana Marchlewskiego Bialymst. 1986-87; 31-32: 3-18.
[Effects of ethanol and acetaldehyde on proteolytic enzyme activity of the
stomach]  [Article in Polish]  Skrzydlewska E.   PMID: 3152147



Rocz Akad Med Im Juliana Marchlewskiego Bialymst. 1986-87; 31-32: 19-28.
[Effects of ethanol and acetaldehyde on proteolytic enzyme activity of the
small intestine and pancreas]  [Article in Polish]
Skrzydlewska E.  PMID: 3152145



Acta Biochim Pol. 1985; 32(3): 271-7.
Release of acid proteolytic activity from lysosomes and degradation of
protein in organs of rats intoxicated with ethanol and acetaldehyde.
Skrzydlewski Z, Worowski K, Skrzydlewska E.

Intoxication with ethanol and acetaldehyde resulted in a marked increase of
the acid proteolytic activity in the post-lysosomal supernatant of rat
kidney, lung, and liver, while the content of protein and acid-soluble
tyrosine remained practically unchanged.
Proteins of the post-lysosomal supernatant were degraded in vitro by the
endogenous proteinase(s) of lysosomal origin at pH 3.5 and 5.5 but not at pH
7.0.    PMID: 3911694



Rocz Akad Med Im Juliana Marchlewskiego Bialymst. 1984-85; 29-30: 59-76.
[Effect of ethanol and acetaldehyde on the activity and release of
cathepsins from lysosomes of the canine liver (studies in vitro)]
[Article in Polish]     Skrzydlewska E, Worowski K.  PMID: 6443826



Rocz Akad Med Im Juliana Marchlewskiego Bialymst. 1984-85; 29-30: 153-62.
Influence of ethanol and acetaldehyde on blood coagulation (examinations in
vitro).      Skrzydlewska E, Worowski K.  PMID: 6443814



Rocz Akad Med Im Juliana Marchlewskiego Bialymst. 1984-85; 29-30: 163-73.
Influence of ethanol and acetaldehyde on fibrinolytic system (examinations
in vitro).      Skrzydlewska E, Worowski K.  PMID: 6242841
********************************************************************

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Postepy Higieny I Medycyny Doswiadczalnej     http://www.phmd.pl

Postepy Hig Med Dosw (Online). 2004 Mar 30; 58: 194-201.
[Antioxidative abilities during aging]
[Article in Polish]  [ Abstract and 80 References, in English, are given
here. ]
Augustyniak A, Skrzydlewska E.
Zaklad Chemii Nieorganicznej i Analitycznej Akademii Medycznej w
Bialymstoku.

Biological aging is associated with increased cellular levels of reactive
oxygen species (ROS) as well as the formation and accumulation of oxidized
biomolecules.
During evolution, organisms developed a highly-efficient and adaptive
antioxidant defense system.
Antioxidants can generally be divided into two categories: enzymatic and
non-enzymatic. During the aging process the activity of antioxidant enzymes,
e.g. SOD, CAT, GSH-Px, and GSSG-R, depends on factors such as race, gender,
tissue and subcellular localization of enzymes.
The age-dependent decrease in antioxidant enzyme activity may be attributed
to oxidative modifications of enzymes.
During the aging process, ROS may also lead to the induction of some enzyme
activity which is explained as an adaptive phenomenon.
The decrease in GSH concentration with age can be explained by decreased GSH
synthesis and/or increased GSH consumption in the removal of peroxides and
xenobiotics.
In plasma albumin, ferritin, transferrin, and caeruloplasmin exert
protective action. Plasma proteins can inhibit ROS generation and
lipid peroxidation by chelating free transition metals.
Plasma protein concentrations changes with age.
The major exogenous antioxidants, mostly derived from the diet, are vitamin
E, C, A, and beta-carotene.
During the aging process the level of vitamins may decrease or increase,
depending on such factors as diet, and diseases.  PMID: 15077054

www.phmd.pl
Review
Postepy Hig Med Dosw (online), 2004; 58: 194-201

page 194

Zdolnosci antyoksydacyjne w starzejacym
sie organizmie
Antioxidative abilities during aging
Agnieszka Augustyniak, Elzbieta Skrzydlewska
Zaklad Chemii Nieorganicznej i Analitycznej Akademii Medycznej w Bialymstoku
Streszczenie
Received: 2003.04.22
Accepted: 2003.08.08
Published: 2004.03.30
Key words: aging . antioxidant enzymes . non-enzymatic antioxidants
Full-text PDF: http://www.phmd.pl/pub/phmd/vol_58/5343.pdf
Word count: 3953
Tables: -
Figures: 1
References: 80
Source of support: Praca finansowana z grantu KBN 6PO5F01720.
Adres autorki: dr hab. Elzbieta Skrzydlewsdka, Zaklad Chemii Nieorganicznej
i Analitycznej AM, ul. Mickiewicza 2a,
15-230 Bialystok, e-mail: skrzydle at amb.edu.pl

page 196

Postepy Hig Med Dosw (online), 2004; tom 58: 194-201  References

[1] Adachi T., Wang J., Wang X.L.:
Age-related change of plasma extracellular superoxide dismutase.
Clin. Chim. Acta, 2000; 290: 169-178

[2] Alejendro D.B., Martha S.B., Nestor O.B.:
Superoxide dismutase, catalase and glutathione peroxidase activities in
human blood: influence of sex, age, and cigarette smoking.
Clin. Biochem., 1997; 30: 449-454

[3] Ames B.N., Shigenaga M.K., Hagen T.M.:
Oxidants, antioxidants and the degenerative diseases of aging.
Proc. Natl. Acad. Sci. USA, 1993; 90: 7915-7922

[4] Andersen H.R., Nielsen Y.B., Nielsen F., Grandjean P.:
Antioxidative enzyme activities in human erytrocytes.
Clin. Chem., 1997; 43: 562-568

[5] Bajra G.:  Rate of generation of oxidative stress-related damage and
animal longevity. Free Radic. Biol. Med., 2002; 33: 1167-1172

[6] Beckman K., Ames B.N.:  The free radical theory of aging matures.
Physiol. Rev., 1998; 78: 547-581

[7] Bettger W.J., Fish T.J., O'dell B.L.:
Effects of dietary copper and zinc on erytrocyte stability and superoxide
dismutase activity.
Proc. Soc. Exp. Biol. Med., 1978 June; 158(2): 279-282 PMID: 674233

[8] Blanchard J., Conrad K. A, Garry P. J.:
Effects of age and intake on vitamin C disposition in females.
Eur. Clin. Nutr., 1990; 44: 447-460

[9] Blot W., Li J.-Y., Taylor P.R., Guo W., Dawsey S., Wang G.Q., Yang
C.S., Zheng S.F., Gail M., Li G.Y.:
Nutrition intervention trials in Linxian, China: supplementation with
specific vitamin-mineral combinations, cancer incidence and disease-specific
mortality in the general population
J. Natl. Cancer Inst., 1993; 85: 1483-1492

[10] Bunker V.W.:
Free radicals, antioxidant and ageing. Med. Lab. Sci., 1992; 49: 299-312

[11] Burch R.E., Sullivan J.F., Jetton M.M., Hahn H.K.:
The effect of aging on trace element content of various rat tissues:
I. Early stages of aging. Age, 1979; 2: 103-107

[12] Camougrand N., Rigoulet M.:
Aging and oxidative stress: studies of some genes involved both in aging and
in response to oxidative stress.  Respir. Physiol., 2001; 128: 393-401

[13] Campbell D., Bunker V.W., Thomas A.J., Clayton B.E.:
Selenium and vitamin E status of healthy and institutionalized elderly
subjects: analysis of plasma, erythrocytes and platelets.
Br. J. Nutr., 1989; 62: 221-227

[14] Chance B., Oshino N.:
Kinetics and mechanisms of catalase in peroxisomes of the mitochondrial
fraction. Biochem. J., 1971; 122: 225-233

[15] Chow C.K., Ibrahim W., Wei Z., Chan A.C.:
Vitamin E regulates mitochondrial hydrogen peroxide generation
Free Radic. Biol. Med.. 1999; 27: 580-587

[16] Darley-Usmar V., Starke-Reed P.E.:
Antioxidants: strategies for interventions in aging and age-related
diseases.
A workshop sponsored by the National Institute on Aging and by the Office of
Dietary Supplements.
Antioxidant Redox Signal, 2000; 2: 375-377

[17] De A.K., Darad R.:
Age-associated changes in antioxidants and antioxidative enzymes in rats.
Mech. Aging Dev., 1991; 59: 123-128

[18] Devasagayam T.P.A.:
Senescence-associated decrease of NADPH induced lipid peroxidation in rat
liver microsomes.  FEBS Lett., 1986; 205: 246-250

[19] Gale C.R., Hall N.F., Phillips D.I.W., Martyn C.N.:
Plasma antioxidant vitamins and carotenoids and age related cataract
Ophthalmology, 2001; 108: 1992-1998

[20] Garry P.J., Vanderjagt D.J., Hunt W.C.:
Ascorbic acid intakes and plasma levels in healthy elderly.
Ann. N. Y. Acad. Sci., 1987; 498: 90-99

[21] Ghezzo-Schoneich E., Esch S.W., Sharov V.S., Schoneich C.:
Biological aging does not lead to the accumulation of oxidized Cu,
Zn-superoxide dismutase in the liver of F344 rats.
Free Radic. Biol. Med., 2001 April 15; 30(8): 858-864

[22] Grimble R.F., Hughes R.E.:
The glutathione: dehydroascorbate oxidoreductase activity of guinea pigs
from two different age groups.  Life Sci., 1968; 7: 383-386

[23] Halliwell B., Gutteridge J.M.:
Free Radical in Biology and Medicine.
III edition. Oxford University Press, Inc. New York, 1999

[24] Handler J.A., Genell C.A., Goldstein R.S.:
Hepatobiliary function in senescence male Sprague-Dawley rats.
Hepatology, 1994; 19: 1496-1503

[25] Harman D.:  Aging: A theory based on free radical and radiation
chemistry.
J. Gerontol., 1956; 11: 298-300

[26] Hasselgren P.O., Pedersen P., Sax H.C., Warner B.W., Fischer J.E.:
Methods for studying protein synthesis and degradation in liver and skeletal
muscle.  J. Surg. Res., 1988; 45: 389-415

[27] Hernanz A., Fernandez-Vivanocos E., Montiel C., Vazquez J.J.,
Arnalich F.:
Changes in the intracellular homocysteine and glutathione content associated
with aging.  Life Sci., 2000; 67: 1317-1324

[28] Hollander D., Dadufalza V.:
Lymphatic and portal absorption of vitamin E in aging rats.
Dig. Dis. Sci., 1989; 34: 768-772

[29] Inal M.E., Kanbak G., Sunal E.:
Antioxidant enzyme activities and malondialdehyde levels related to aging.
Clin. Chim. Acta, 2001; 305: 75-80

[30] Inal M.E., Sunal E., Kanbak G.:
Age-related changes in the glutathione redox system
Cell. Biochem. Funct., 2002; 20: 61-66

[31] Ishikawa T.:  The ATP-dependent glutathione S-conjugate export pump.
TIBS, 1992; 17: 463-468

[32] Jiang Q., Christen S., Shigenaga M.K., Ames B.N.:
Gamma-tocopherol, the major form of vitamin E in the US diet, deserves more
attention.  Am. J. Clin. Nutr., 2001 Dec; 74(6): 714-722

[33] Jones D.P., Mody V.C., Carlson J.L., Lynn M.J., Sternberg P. Jr.:
Redox analysis of human plasma allows separation of pro-oxidant
events of aging from decline in antioxidant defences.
Free Radic. Biol. Med., 2002; 33: 1290-1300

page 200

Postepy Hig Med Dosw (online), 2004; tom 58: 194-201

page 201

Augustyniak A. i Skrzydlewska E. - Zdolnosci antyoksydacyjne.

[34] Kushi L.H., Folsom A.R., Prineas R.J., Mink P.J., Wu Y., Bostik R.M.:
Dietary antioxidant vitamins and death from coronary hearth disease in
postmenopausal women.  N. Engl. J. Med., 1996; 34: 1156-1162

[35] Laila G., Yues A., Bernard H., Claude J., Gerard C., Gerard S.:
Biological variability of superoxide dismutase, glutathione peroxidase and
catalase in blood.  Clin. Chem., 1991; 37: 1932-1937

[36] Lee J., Hunt J.A., Groves J.T.:
Manganese porphyrins as redox-coupled peroxynitrite reductases.
J. Am. Chem. Soc., 1998; 120: 6053-6061

[37] Leutner S., Eckert A., M?ler W.E.:
ROS generation, lipid peroxidation and antioxidant enzyme activities in the
aging brain.  J. Neural. Transm., 2001; 108: 955-967

[38] Liu R.M., Choi J.:
Age-associated decline in gamma-glutamylcysteine synthetase gene expression
in rats.  Free Radic. Biol. Med., 2000; 28: 566-574

[39] Masaaki K., Masatoshi S., Nihal S.A.:
Antioxidant system and erytrocyte life-span in mammals.
Comp. Biochem. Physiol., 1993; 106B: 477-487

[40] Matsuo M., Gomi F., Dooley M.M.:
Age-related alterations in antioxidant capacity and lipid peroxidation in
brain, liver, and lung homogenates of normal and vitamin E-deficient rats.
Mech. Aging Dev., 1992; 64: 273-292

[41] Mecocci P., Polidori M.C., Troiano L., Cherubini A., Cecchetti R.,
Pini G., Straatman M., Monti D., Stahl W., Sies H., Franceschi C.,
Senin U.:  Plasma antioxidants and longevity: a study on healthy
centenarians.
Free Radic. Biol. Med., 2000; 28: 1243-1248

[42] Melov S., Schneider J.A., Day B.J., Hinerfeld D., Coskun P., Mirra
S.S., Crapo J.D., Wallace D.C.:
A novel neurological phenotype in mice lacking mitochondrial manganese
superoxide dismutase.  Nat. Genet., 1998; 18: 159-163

[43] Meydani M.:  Vitamin E.  Lancet., 1995; 345: 170-175

[44] Meydani S.N., Meydani M., Blumberg J.B., Leka L.S., Siber G., Loszewski
R., Thompson C., Pedrosa M.C., Diamond R.D., Stollar B.D.:
Vitamin E supplementation and in vivo immune response in healthy elderly
subjects: a randomised trial.
JAMA, 1997; 277: 1380-1386

[45] Misra D.P., Loudon J.M., Staddon G.E.:
Albumin metabolism in elderly patients.  J. Gerontol., 1975; 30: 304-306

[46] Nakata K., Kawase M., Ogino S., Kinoshita C., Murata H., Sakaue
T., Ogata K., Ohmori S.:
Effects of age on levels of cysteine, glutathione and related enzyme
activities in livers of mice and rats and an attempt to replenish hepatic
glutathione level of mouse with cysteine derivatives.
Mech. Ageing Dev., 1996; 90: 195-207

[47] Oreopoulos D.G., Lindeman R.D., Vanderjagt D.G., Tzamaloukas
A.H., Bhagavan H.N., Garry P.D.:
Renal excretion of ascorbic acid: Effect of age and sex.
Am. J. Clin. Nutr., 1993; 12: 537-542

[48] Pallast E.G., Schouten E.G., de Waart F.G., Fonk H.C., Doekes G.,
von Blomberg B.M., Kok F.J.:
Effect of 50- and 100-mg vitamin E supplements on cellular immune function
in non institutionalised elderly persons.
Am. J. Clin. Nutr., 1999; 69: 1273-1281

[49] Reiss U., Gershon D.:
Comparison of cytoplasmic superoxide dismutase in liver heart and brain of
aging rats and mice.  Biochem. Biophys. Res. Commun., 1976; 73: 255-262

[50] Rikans L.E., Hornbrook K.R.:
Lipid peroxidation, antioxidant protection and aging.
Biochim. Biophys. Acta., 1997; 1363: 116-127

[51] Rikans L.E., Moore D.R.:
Effect of aging on aqueous-phase antioxidants in tissues of male Fischer
rats.
Biochim. Biophys. Acta, 1988; 966: 269-275

[52] Rikans L.E., Moore D.R., Snowden C.D.:
Sex dependent differences in the effects of aging on antioxidant defence
mechanisms of rat liver.  Biochim. Biophys. Acta, 1991; 1074: 195-200

[53] Rikans L.E., Snowden C.D., Moore D.R.:
Effect of aging on enzymatic antioxidant defences in rat liver mitochondria.
Gerontology, 1992; 38: 133-138

[54] Ritchie J.P., Lang C.A.:
A decrease in cysteine levels causes the glutathione deficiency of the aging
mosquito. Proc. Soc. Exp. Biol. Med., 1988; 187: 235-240

[55] Salo D.C, Pacifini R.E, Davies K.N.J.:
Superoxide dismutase is preferentially degraded by a proteolytic system from
red blood cells following oxidative modification by hydrogen peroxide.
Free Radic. Biol. Med., 1988; 5: 335-339

[56] Samiec P.S., Drews-Botsch C., Flagg E, Kurtz J.C., Sternberg P. Jr.,
Reed R.L., Jones D.P.:
Glutathione in human plasma. Decline in association with aging, age related
macular degeneration and diabetes.
Free Radic. Biol. Med., 1998; 24: 699-704

[57] Sanz N., Diez-Fernandez C., Andres D., Cascales M.:
Hepatotoxicity and aging: endogenous antioxidant systems in hepatocytes
from 2-, 6-, 18-, 30-month-old rats following a necrogenic dose
thioacetamide. Biochim. Biophys. Acta, 2002; 1587: 12-20

[58] Sastre J., Pallardo F.V., Vina J.:
Glutathione, oxidative stress and aging.  Age, 1996; 19: 129-139

[59] Semsei I., Rao G., Richardson A.:
Changes in the expression of superoxide dismutase and catalase as a function
of age and dietary restriction.
Biochem. Biophys. Res. Commun., 1989; 164: 620-625

[60] Shindo Y., Akiyama J., Yamazaki Y., Saito K., Takase Y.:
Changes in enzyme activities in skin fibroblasts from persons of various
ages.
Exp. Gerontol., 1991; 26: 29-35

[61] Sies H., Stahl W., Sundquist A.R.:
Antioxidant functions of vitamins. Vitamins E and C, beta-carotene and other
carotenoids.  Ann. N.Y. Acad. Sci., 1992; 669: 7-20

[62] Sohal R.S.:
Role of oxidative stress and protein oxidation in the aging process.
Free Radic. Biol. Med., 2002; 33: 37-44

[63] Sohal R.S., Svensson I., Brunk U.T.:
Hydrogen peroxide production by liver mitochondria in different species.
Mech. Ageing Dev., 1990; 53: 209-215

[64] Sohal R.S., Svensson I., Sohal B.H., Brunk U.T.:
Superoxide anion radical production in different animal species.
Mech. Ageing Dev., 1989; 49: 129-135

[65] Sohal R.S., Mockett R.J., Orr R.C.:
Mechanisms of aging: an appraisal of the oxidative stress hypothesis.
Free Radic. Biol. Med., 2002; 33: 575-586

[66] Somville M., Houben A., Raes M., Houbion A., Henin V., Remacle J.:
Alteration of enzymes in ageing human fibroblasts in culture.
III. Modification of superoxide dismutase as an environmental
and reversibleprocess. Mech. Ageing Dev., 1985; 29: 35-51

[67] Storz G., Tartaglia L.A., Ames B.N.:
Transcriptional regulator of oxidative stress inducible genes: direct
activation by oxidation.  Science, 1990; 248: 189-194

[68] Tian L., Cai Q., Wei H.:
Alterations of antioxidant enzymes and oxidative damage to macromolecules in
different organs of rats during aging.
Free Radic. Biol. Med., 1998; 24: 1477-1484

[69] Tolmasoff J.M., Ono T., Cutler R.G.:
Superoxide dismutase: correlation with life span and specific metabolic rate
in primate species.  Proc. Natl. Acad. Sci. USA, 1980; 77: 2777-2781

[70]  van der Loo B., Labugger R., Aebischer C.P., Skepper J.N., Bachschmid
M., Spitzer V., Kilo J., Altwegg L., Ullrich V., Luscher TF.:
Cardiovascular aging is associated with vitamin E increase.
Circulation, 2002 April 9; 105(14): 1635-1638

[71] van der Loo B., Labugger R., Skepper J.N.:
Enhanced peroxynitrite formation is associated with vascular aging.
J. Exp. Med., 2000; 192: 1731-1743

[72] Vina J., Sastre J., Anton V., Bruseghini L., Esteras A., Asensi M.:
Effect of aging on glutathione metabolism. Protection by antioxidants.
In: Free Radical and Aging, ed.: Emerit I., Chance B.
Birkhauser Verlag, Basel, 1992

[73] Wasowics W,., Kantorski J., Perek D.:
Concentration of zinc and zinc-cooper superoxide dismutase activity in red
blood cells in normal and children with cancer.
J. Clin. Chem. Clin. Biochem., 1989; 27: 413-418

[74] Wickens A.P.:
Ageing and the free radical theory.  Respir. Physiol., 2001; 128: 379-391

[75] Yan H., Harding J.J.:
Glycation-induced inactivation and loss of antigenicity of catalase and
superoxide dismutase.  Biochem J., 1997; 328: 599-605

[76] Yanagawa K., Takeda H., Egashira T., Sakai K., Takasaki M.,
Matsumiya T.:
Age related changes in alpha-tocopherol dynamics with relation to lipid
hydroperoxide content and fluidity of rat erythrocyte membrane.
J. Gerontol. Biol. Sci., 1999 Sep; 54(9): B379-B383

[77] Yip R., Johnson C., Dallman R.:
Age related changes in laboratory values used in the diagnosis of anaemia
and iron deficiency.  Am. J. Clin. Nutr., 1984; 39: 427-436

[78] Yu B.P., Laganiere S., Kim J.W.:
Influence of life-prolonging food restriction on membrane lipoperoxidation
and antioxidant status.
In: Oxygen Radicals in Biology and Medicine, ed.: Simic M.G., Taylor
K.A., Ward J.F., Von Sonntag C., Plenum Press, New York, 1989

[79] Yunice A.A., Lindeman R.D., Czerwinski A.W., Clark M.:
Influence of age and sex on serum copper and ceruloplasmin.
J. Gerontol., 1974 May; 29(3): 277-281

[80] Zarling E.J., Mobarhan S., Bowen P., Kamath S.:
Pulmonary pentane excretion increases with age in healthy subjects.
Mech. Aging Dev., 1993; 67: 141-147
*************************************************************

"CONCLUSION
There is much strong evidence that the background of etheno-DNA and
propano-DNA detected in tissues from unexposed humans and rodents arises
from endogenous lipid peroxidation products, such as MDA.

With the advent of ultrasensitive, specific detection methods for this kind
of DNA damage in human tissues and cells, new insights can be gained into
the mechanisms involved in human cancers.

Moreover, etheno-DNA adducts can now be used as biomarkers to investigate
the potential role of lipid peroxidation in human cancers."

Cell Mol Biol Lett. 2003; 8(2): 391-413.
DNA damage caused by lipid peroxidation products.
Luczaj W, Skrzydlewska E.
Department of Analytical Chemistry, Medical Academy of Bialystok,
Mickiewicza 2A, P.O. Box 14, 15-230 Bialystok 8, Poland.

Lipid peroxidation is a process involving the oxidation of polyunsaturated
fatty acids (PUFAs), which are basic components of biological membranes.
Reactive electrophilic compounds are formed during lipid peroxidation,
mainly alpha, beta-unsaturated aldehydes.
These compounds yield a number of adducts with DNA.
Among them, propeno and substituted propano adducts of deoxyguanosine with
malondialdehyde (MDA), acrolein, crotonaldehyde and etheno adducts,
resulting from the reactions of DNA bases with epoxy aldehydes,
are a very important group of adducts.
The epoxy aldehydes are more reactive towards DNA than the parent
unsaturated aldehydes.
The compounds resulting from lipid peroxidation mostly react with DNA
showing both genotoxic and mutagenic action;
among them, 4-hydroxynonenal is the most genotoxic,
while MDA is the most mutagenic.
DNA damage caused by the adducts of lipid peroxidation products
with DNA can be removed by the repairing action of glycosylases.
The formed adducts have been hitherto analyzed using the IPPA
(Imunopurification-(32)P-postlabelling assay) method and via gas
chromatography/electron capture negtive chemical ionization/mass
spectrometry (GC/EC NCI/MS).
A combination of liquid chromatography with electrospray tandem mass
spectrometry (LC/ES-MSMS) with labelled inner standard has mainly been used
in recent years.  PMID: 12813574

http://www.cmbl.org.pl/vol8/V8Page391.pdf

CELLULAR & MOLECULAR BIOLOGY LETTERS
Volume 8, (2003) pp 391 - 413
http://www.cmbl.org.pl
Received 6 January 2003
Accepted 16 April 2003

Abbreviations used: eA - 1,N6-ethenoadenine;
eC - 3,N4-ethenocytosine; edA - 1,N6-
ethenodeoxyadenosine; edC - 3,N4-ethenodeoxycytidine;
AdG - acrolein-derived 1,N2-propanodeoxyguanosine;
CdG - crotonaldehyde-derived 1,N2-propanodeoxyguanosine;
EH - 2,3-epoxy-4-hydroxynonanal;
GC/EC NCI/MS - gas chromatography with mass spectrometry with electron
capture negative chemical ionization detection;
GSH - gluthatione; HNE - trans-4-hydroxy-2-nonenal;
IPPA - Imunopurification-32Ppostlabelling assay;
LC/ES-MSMS - liquid chromatography with electrospray tandem mass
spectrometry;
M1 C - N4-(3-oxo-propenyl)deoxycytidine;
M1 G - pirymido[1,2a]purin-10(3H)-one); M1A -
N6-(3-oxo-propenyl)deoxyadenosine; MDA - malondialdehyde; PUFAs -
polyunsaturated fatty acids.

DNA DAMAGE CAUSED BY LIPID PEROXIDATION PRODUCTS

WOJCIECH LUCZAJ and  ELZIBIETA SKRZYDLEWSKA
Department of Analytical Chemistry, Medical Academy of Biaystok,
Mickiewicza 2A, P.O. Box 14, 15-230 Biaystok 8, Poland

Abstract: Lipid peroxidation is a process involving the oxidation of
polyunsaturated fatty acids (PUFAs), which are basic components of
biological membranes. Reactive electrophilic compounds are formed during
lipid
peroxidation, mainly a,b-unsaturated aldehydes. These compounds yield a
number of adducts with DNA. Among them, propeno and substituted propano
adducts of deoxyguanosine with malondialdehyde (MDA), acrolein,
crotonaldehyde and etheno adducts, resulting from the reactions of DNA bases
with epoxy aldehydes, are a very important group of adducts. The epoxy
aldehydes are more reactive towards DNA than the parent unsaturated
aldehydes. The compounds resulting from lipid peroxidation mostly react with
DNA showing both genotoxic and mutagenic action; among them, 4-
hydroxynonenal is the most genotoxic, while MDA is the most mutagenic. DNA
damage caused by the adducts of lipid peroxidation products with DNA can be
removed by the repairing action of glycosylases. The formed adducts have
been hitherto analyzed using the IPPA (Imunopurification-32P-postlabelling
assay) method and via gas chromatography/electron capture negtive chemical
ionization/mass spectrometry (GC/EC NCI/MS).
A combination of liquid chromatography with electrospray tandem mass
spectrometry (LC/ES-MSMS) with labelled inner standard has mainly been used
in recent years.

Key Words: Lipid Peroxidation, Etheno Adducts, Propano Adducts, á,â-
Unsaturated Aldehydes

CELL. MOL. BIOL. LETT. Vol. 8. No. 2. 2003

page 392

INTRODUCTION
Carcinogenesis was induced by chronic infections caused by viruses, bacteria
or parasites and the inflammation accompanying them. A hypothesis explaining
carcinogenesis mechanisms suggested that endogenic compounds causing DNA
damage are formed by the organism in the inflammation state [1]. The main
species/compounds involved in them are oxygen and nitrogen (species). The
production of these species markedly increases in such conditions, and they
can induce enhanced lipid peroxidation in direct reactions and/or cause DNA
damage [1].

Various reactive electrophilic compounds can be formed by lipid
peroxidation and some of them, showing mutagenic and genotoxic properties,
reaily react with proteins and DNA. This is particularly true for trans-4-
hydroxynonenal, malondialdehyde, and crotonaldehyde, which form adducts
with DNA [7]. The formation of these compounds was described as early as in
the eighties, but their identification and quantitative determination in
animal organisms under physiological conditions has only been possible in
recent studies, in which ultrasensitive analytical methods such as IPPA
(Imunopurification-32P-postlabelling assay) and gas chromatography/electron
capture negtive chemical ionization/mass spectrometry (GC/EC NCI/MS) were
used [86-89]. This also created chances to obtain evidence of the toxic and
genetic consequences of such DNA damage [1].

LIPID PEROXIDATION
Lipid peroxidation occurs in physiological conditions. It involves the
oxidation of polyunsaturated fatty acids (PUFAs), which are basic biological
membrane components. Many unsaturated components, mainly aldehydes, are then
formed [2]. Fundamental reactions occurring during peroxidation are showing
in Fig. 1 [2].

Polyunsaturated fatty acids contain active methylene groups situated between
cis double bonds. Such groups readily react with oxidizing agents and their
hydrogen atoms are removed to form carbon-centered radicals (compound 1,
Fig. 1) [2]. These radicals react with molecular oxygen-yielding peroxyl
radicals, which are the initial products of polyunsaturated fatty acid
oxidation [3].

Further transformations of peroxyl radicals depend on their position in
the carbon chain of the fatty acids [3]. If the peroxyl radical exists at
the
end of a double bond system (compound 2, Fig. 1) then it can be reduced to a
hydroperoxide. Conjugated diene hydroperoxides formed in this way (compound
4, Fig. 1) are relatively stable lipid peroxidation products in the absence
of transition metal ions [2]. Peroxyl radicals can be also reduced to
hydroperoxides by other fatty acid molecules or by vitamin E [4].

The reduction of a peroxyl radical by another fatty acid molecule results in
the formation of a new carbon centered radical which propagates the fatty
acid oxidation. In this way, an oxidized molecule can induce the oxidation
of many other fatty acid molecules. Approximately 60 linoleic acid molecules
and 200 molecules of arachidonic acid

CELLULAR & MOLECULAR BIOLOGY LETTERS  393

are oxidized as the result of transformations initiated by one free-radical
reaction [4]. The length of the free-radical reaction chain depends on many
factors.

The main factor determining the free-radical reaction chain length in vivo
is
vitamin E concentration in the lipid bilayer [5]. This vitamin reduces
peroxyl
radicals to hydroperoxides, thereby breaking the reaction chain and slowing
the rate of lipid peroxidation [6].
However, vitamin E can initiate other free-radical chain reactions if
present in very small concentrations [6].

Fig. 1. The pathways of lipid peroxidation [3].

If the peroxyl radical is located within a fatty acid chain
(compound 3, Figure 1) then it can undergo cyclization owing to a
neighbouring double bond [7],
yielding a cyclic peroxide located in vicinity of a carbon-centered radical
(compound 5, Fig. 1). This radical can undergo further transformations. It
can bind an oxygen molecule, yielding a peroxyl radical which can be reduced
to
hydroperoxide (compound 6, Fig. 1), as described earlier, or it can undergo
a new cyclization, yielding a bicyclic peroxide. This can bind another
oxygen
molecule and be reduced to a compound (compound 7, Fig. 1) structurally
analogous to prostaglandin endoperoxide but lacking a stereochemical control
[7]. The chemical conversion of the bicyclic peroxide group of this compound
gives malondialdehyde (MDA) and isoprostanes [8] and 17-carbon fatty acids
are simultaneously formed as side products (compound 8, Fig. 1) [9].

There is a growing belief that the most valuable biomarkers of lipid
peroxidation in the human body are the isoprostanes [10-14]. Highly
sensitive and accurate
spectrophotometric methods have hitherto been used to determine isoprostane

CELL. MOL. BIOL. LETT. Vol. 8. No. 2. 2003  394

concentration in blood serum [13, 15, 16]. Isoprostates undergo relatively
rapid metabolic transformations. Therefore, lipid peroxydation in various
organs
can be readily monitored by determining the level of their decomposition
products in urine [13, 17].

It is known that transition metal ions initiate free-radical reactions
including lipid peroxidation because they participate in generating reactive
oxygen species
(O2-*, *OH). At the same time, they contribute to the propagation of the
process by reducing lipid hydroperoxides [18, 19]. Compounds of such ions as
Cd(II), Co(II), Cu(II), Hg(II), Ni(II), Pb(II), Sn(II), V(V), Fe(II), and
Fe(III)
provoke enhanced lipid peroxidation under both in vitro and in vivo
conditions
[20-25].

All the hydroperoxides presented in Fig. 1, as well as their regio- and
stereoisomers, can be reduced to their akoxyl radicals by transition metal
cations and then they can undergo b-cleavage yielding many products [26,
27].

This results in a number of epoxy compounds (e.g. 2,3-epoxybutanal or
2,3-epoxy-4-hydroxynonanal), hydroperoxides (e.g. compounds 4, 6, 7, Fig. 1)
 and saturated and á,â-unsaturated aldehydes (e.g. acrolein, crotonaldehyde,
4-hydroxynonenal, 2,4-nonadienal, 2,4-decadienal) [28]. These compounds are
formed from fatty acids in various amounts depending on their stucture and
oxidation conditions.

Recently, a new product of lipid peroxidation, 4-oxo-2-nonenal, was reported
to be generated by the decomposition of linoleic acid hydroperoxide [13(S)-
hydroperoxy-(Z,E)-9, 11-octadecadienoic acid] [29].

All lipid peroxidation pathways leading to product formation are not known
in sufficient detail. Nevertheless, it has been found that crotonaldehyde is
mainly generated from a-linoliec acid and linoleate.

Crotonaldehyde is also formed
in small amounts by the peroxidation of arachidonate and such acids as cis-
5,8,11,14,17-eicosapentaenoic and cis-4,7,10,13,16,19-docosahexaenoic [30].
It is interesting that arachidonate and w-3-polyunsaturated fatty acids were
earlier stated to be primary compounds of acrolein generation [31].

It was also found that hexanal and 4-hydroxynonenal (HNE) were formed from
lipids containing
w-6-polyunsaturated fatty acids (18:2, 20:4), whereas 4-hydroxyhexenal and
propanal are formed from w-3-polyunsaturated fatty acids (22:6) [32]. It was
shown that hexanal is formed as a result of the â-cleavage of
15-hydroperoxyarachidonic acid or 13-hydroperoxy-linoleic acid [32, 33].

However, it was suggested that HNE could be formed by the transformation of
w-6-polyunsaturated fatty acids like 11-hydroperoxy-arachidonic acid (or 9-
hydroperoxy-linoleic acid) or from isomers of 15-hydroperoxy-arachidonic
acid and 13-hydroperoxy-linoleic acid [34]. On the other hand, lipids
containing
polyunsaturated fatty acids with three or more double bonds separated by
methylene groups, mainly arachidonic acid (20:4) and docosahexaenoic acid
(22:6), yield malondialdehyde (MDA) [32, 33]. It is believed that oleic and
linoleic acid to a small degree take part in MDA formation [32, 35].
Bicyclic peroxides with an oxygen bridge located inside the molecule are
involved as intermediates in the pathway of MDA formation; at higher
temperatures or in an acid medium, they undergo decomposition to free MDA
[7]. Two other

CELLULAR & MOLECULAR BIOLOGY LETTERS  395

pathways to MDA formation from polyunsaturated fatty acids were proposed
[36]. The main stages of MDA formation by the oxidation of arachidonic acid
are presented in Fig. 2: â-cleavage of the fatty acid giving
hydroperoxyaldehyde, which further yields MDA or the acrolein radical; the
latter undergoes transformation to the enol form of MDA in reaction with a
hydroxyl radical [2].

ADDUCTS OF LIPID PEROXIDATION PRODUCTS WITH DNA
There are many known adducts formed by low molecular weight compounds
resulting from lipid peroxidation with DNA bases, but the compounds formed
by a,b-unsaturated aldehydes have been studied the most thoroughly [37, 38].
Among them, there are etheno and substituted ethano adducts of
deoxyadenosine, deoxyguanosine, and deoxycytidine, propeno and substituted
propano adducts of deoxyguanosine, and bicyclic adducts with deoxyguanosine.

A particularly important group of these compounds are propeno and
substituted
propano adducts of deoxyguanosine with malondialdehyde, acrolein, or
crotonaldehyde. Their content in human liver DNA is from 1500 to 5000
adducts per cell [86, 39].

Fig. 2. Possible mechanisms of the formation of MDA through the autoxidation
of intermediate polyunsturated aldehydes derived from â-cleavage of
5-hydrperoxy or 15-hydrperoxy arachidonic acid [7].

It has been proved in numerous studies carried out in various laboratories
that propano adducts are mainly formed in reactions of small-molecule a,b-
unsaturated aldehydes with deoxyguanosine [1, 40-42, 48]. The mechanism of

CELL. MOL. BIOL. LETT. Vol. 8. No. 2. 2003  396

the nucleophilic Michael addition of the nitrogen atom of deoxyguanosine
amino group to the aldehyde double bond followed
by cyclization in position N-1 of
deoxyguanosine is presented in Fig. 3a. Reactivity of enals with
deoxyguanosine
during the formation of propano adducts varies with increasing alkyl chain
length: acrolein > crotonaldehyde > HNE [41, 59]. Accordingly, adduct
formation rate in the case of HNE is considerably lower than in the case of
shorter chain unsaturated aldehydes [48]. In addition, the modification
degree of nucleic acid is lower in the case of double-stranded DNA than in
the case of single-stranded DNA,
because two nitrogen atoms of the bases must then be
bonded to form a cyclic adduct, which are normally bonded by a hydrogen bond
in the DNA a-helix [59].

Fig. 3. The mechanism of the reaction of 4-hydroxyalkenals with
deoxyguanosine.
(a) The reaction of the parent unsaturated aldehyde;
(b) the reaction of epoxyaldehyde [48].

Acrolein is the most electrophilic of the á,â-unsaturated aldehydes. This is
reflected by its great ability to react with thiol and amino groups [43,
44].
It was found, for example,
that acrolein reacts with GSH 110-150 times more rapidly
than HNE or crotonaldehyde [45]. Acrolein yields cyclic adducts with
deoxyguanosine, deoxyadenosine and deoxycytidine, but it is its compounds
with deoxyguanosine that have been studied the most extensively [46]. Three
stereoisomeric adducts are formed in the reaction with deoxyguanosine:
AdG 1, AdG 2, and AdG 3 (Fig. 4).
AdG 1 and AdG 2 are a pair of diastereoisomers and
AdG 3 is a regio-isomer formed by ring closure in the opposite direction; it
is supposedly composed of AdG 1 and AdG 2 stereoisomers. With these
compounds, the formation mechanism is based on the Michael addition of
nitrogen N-1 of the deoxyguanosine amino group to the third acrolein carbon
atom, followed by ring closure between deoxyguanosine nitrogen N-2 and the
carbon C-1 of acrolein [41].

Crotonaldehyde, a homolog of acrolein, reacts with deoxyguanosine like
acrolein, mainly yielding 1,N2-propano adducts: CdG 1 and CdG 2 (Fig. 4)
[36].

CELLULAR & MOLECULAR BIOLOGY LETTERS  397

However, the number of guanosine modifications caused by crotonaldehyde is
considerably smaller than in the case of acrolein [46]. The Michael addition
between the nitrogen N-1 and the double-bond carbon atom is not possible in
this case because of steric hindrance generated by the methyl group and the
oxygen atom at the C-6 carbon atom. As a result, only one diastereoisomer
pair is formed. The trans configuration was assigned to the CdG isomers on
the
ground of NMR magnetic coupling constants [47].

Fig. 4. The reaction of acrolein (1) and crotonaldehyde (2) with
deoxyguanosine [47].
4-Hydroxyalkenals, including HNE, have three exceptionally reactive groups:
the aldehyde group, the double carbon-carbon bond, and the hydroxyl group.

All of them can react with other molecules, either independently from each
other
of or in cooperation. In a neutral medium, 4-hydroxyalkenals readily react
with
the sulfhydryl groups (SH) of thiols such as glutathione, or they react with
protein or nucleic acid base amino groups, mainly of guanine, adenine, and
cytosine [36].

As in the case of acrolein and crotonaldehyde, Michael addition of the
nitrogen deoxcyguanosine amino group to the HNE double bond followed by
cyclization in the N-1 position of deoxyguanosine are involved in the
mechanism of the HNE reaction with deoxyguanosine [48]. It results in the
formation of two
pairs of 1,N2-propano-dG (HNE-dG 1,2 and 3,4) diastereomeric adducts [48,
49]. The formation of four different adducts is a consequence of the trans
configuration of the hydroxy group and
the side alkyl chain with a chiral carbon atom [50].
An HNE adduct with deoxyguanosine is presented in Fig. 5.

MDA also reacts with DNA bases yielding various compounds (Fig. 6). The
problem of MDA interaction with DNA is somewhat complicated by the ability
of MDA to oligomerize to dimers and trimers which are also able to react
with DNA.
However, MDA oligomerization is relatively slow in a neutral medium,
and compounds of the monomers presented in Fig. 6 are considered to be the
main products formed in physiological conditions [51, 52]. The in vitro
experiments suggest that the adducts M1G (pirymido[1,2a]purin-10(3H)-one)
and M1A (N6-(3-oxo-propenyl)deoxyadenosine)
are the main products of MDA reaction with DNA [57, 1].
The amount of M1G was found to be approximately
five times greater than that of M1A [57]. Trace amounts of N4-(3-oxo-

CELL. MOL. BIOL. LETT. Vol. 8. No. 2. 2003  398

propenyl)deoxycytidine (M1C) were also formed [53]. It should be stressed
that the main adduct of malondialdehyde
with dG is not a malondialdehyde-specific lesion.
Dedon et al. demonstrated that base propenals as structural analogs
of MDA derived from oxidative DNA damage are capable of forming the M1G.
Adenine propenal was predicted to be more reactive than malondialdehyde in
the formation of M1G [58].

Fig. 5. The formation of 1,N2-propano-dG adduct in a reaction of HNE with
deoxyguanosine [50].

Fig. 6. The formation of MDA-DNA adducts [3].

It was demonstrated that unsaturated aldehydes could be converted to
epoxyaldehydes (aldehydes with an oxygen bridge in the molecule) in the
presence of H2O2 or fatty acid hydroperoxides or in conditions in which
these oxidants are formed [54-56]. Epoxyaldehydes are more reactive towards
DNA than parent unsaturated aldehydes, particularly those formed from
long-chained enals [54-56]. Reactions of epoxy aldehydes with DNA bases
yield etheno or ethano adducts [1], mainly 1,N2-ethenoguanosine (or
1,N2-ethanoguanosine), 1,N6-ethenoadenosine, and 3,N4-ethenocytidine (Fig.
7). Acrolein epoxide (glycidaldehyde) reacts with guanine yielding
substituted 1,N2-ethano and 1,N2-

CELLULAR & MOLECULAR BIOLOGY LETTERS  399

Fig. 7. The formation of various etheno adducts from reactions of
deoxyrybonucleosides with epoxyaldehydes. The substituted etheno adducts are
formed by a loss of H 2O from the intermediate ethano adducts and the
unsubstituted etheno adducts are formed by base-catalyzed losses of R'CHO
and H2O [47].

etheno adducts and an unsubstituted 1,N2-etheno adduct [57] while 2,3-
epoxybutanal obtained from crotonaldehyde yields 1,N2-ethenoguanosine, 1,N6-
ethenoadenosine, and 3,N4-ethenocytidine [54]. The relative generation
yields of substituted and unsubstituted etheno adducts depend on
epoxyaldehyde and on the pH of the reaction. 2,3-epoxy-4-hydroxynonanal (EH)
is a mixture of two diastereomers, I and II;
it is formed in vitro in the reaction of HNE with
t-butyl hydroperoxide. It reacts with deoxyguanosine yielding a unique
tetracyclic
1,N2 ethano adduct as two pairs of diastereomers [55, 56].
Adducts formed at pH 10 and 37ºC readily undergo conversion to
1,N2-ethenodeoxyguanosine (1,N2-edG)

CELL. MOL. BIOL. LETT. Vol. 8. No. 2. 2003  400

- compound 9 in Fig. 3b [55]. 2,3-Epoxy-4-hydroxynonanal reacting with
deoxyadenosine yields edA adduct (1,N6-ethenoadenosine) and the pair of its
substituted isomers. It was also demonstrated that both 2,3-epoxy-4-
hydroxynonanal isomers (I and II) react stereoselectively with
deoxyadenosine
and deoxyguanosine, yielding etheno adducts; isomer I preferentially
modifies adenine over guanine, while isomer II exhibits similar rectivity to
both
purine bases [59].
Isomer II of 2,3-epoxy-4-hydroxynonanal is more mutagenic than
isomer I because of its higher reactivity towards nucleic acids. These
results
suggest that epoxidation is possibly an important transition stage
activating
relatively unreactive long-chain enals. Unfortunately, there is no evidence
that epoxidation of 4-hydroxy-2-nonenal can occur in vivo, as the compound
is a
convenient substrate of glutathione S-transferases and aldo-keto reductases
[60].

Recent studies have also furnished us with information on DNA damage caused
by 2,4-decadienal (DDE), a highly toxic lipid peroxidation product [61, 62].
Initially, the formation of 1,N6-etheno-2'-deoxyadenosine
and of two etheno adducts:
1-[3-(2-deoxy-beta-D-erythro-pentafuranosyl)-3H-imidazo[2,1-i]purin-
7-yl]-1,2,3-octanetriol (adduct 1) and
1-[3-(2-deoxy-beta-D-erythropentafuranosyl)-
3H-imidazo[2,1-i]purin-7-yl]-1,2-heptanediol (adduct 2) are
generated in the reaction of the aldehyde with deoxyadenosine [61, 62]. On
the other hand, Loureiro et al. proved that the interaction of DDE with
deoxyguanosine yielded the adduct which is a tautomer of 1,N2-ethene-2'-
deoxyguanosine (the commonly known reaction product of epoxy aldehydes
with dG) and, in addition, two new diastereomeric products [63]. The
formation mechanism that was
proposed for these compounds involved double DDE
epoxidation, nucleophilic addition of the dG amino group nitrogen atom to
the C1 carbon atom of the aldehyde, followed by cyclization as the result of
nucleophilic attack of the N1 nitrogen atom on the C2 carbon atom of the
epoxy group, and dehydratation [63].

Similarly, 4-oxo-2-nonenal, a recently discovered lipid peroxidation
product, forms adducts with the DNA of the structure corresponding to that
of
substituted etheno adducts [64].
The three initially formed ethano adducts arose from
the highly regioseletive nucleophilic addition of the nitrogen N-2 of dG to
the
C-1 aldehyde of 4-oxo-2-nonenal, followed by the reaction of N-1 at the C-2
of
the resulting á,â-unsaturated ketone. All three ethano adducts then
dehydrated
to a single heptanone etheno-dG adduct
(3-(2-deoxy-beta-D-erythropentafuranosyl)
imidazo-7-(heptane-2-one)-9-hydroxy[1,2-alpha]purine) [64]. Nucleophilic
addition of the N-6 of dA to the C-1 aldehyde of 4-oxo-2-nonenal followed by
the reaction of N-1 at the C-2 of the resulting á,â-unsaturated ketone
results in the generation of a mixture of
two ethano adducts that could inter-convert
to each other. The ethano adducts are subsequently dehydrated, yielding a
single heptanone etheno-dA adduct
(1''-[3-(2'-deoxy-beta-D-erythropentafuranosyl)-3H-imidazo[2,1-i]purin-7-yl]
heptane-2''-one) [65, 66].

CELLULAR & MOLECULAR BIOLOGY LETTERS  401

THE GENOTOXICITY AND MUTAGENEITY OF LIPID
PEROXIDATION PRODUCTS AND OF THEIR ADDUCTS WITH DNA
Most compounds formed by lipid peroxidation react with DNA and proteins.
These processes have genotoxic and mutagenic effects. The most genotoxic
lipid peroxidation product is 4-hydroxynonenal [70]. HNE and other 4-
hydroxyalkenals provoke genotoxic phenomena in various animal tissues as
well as in the Salomonella typhimurium bacteria cell [71]. A similar action
was
observed in the case of 2-nonenal, whereas nonanal shows no genotoxic action
even at high non-physiological concentration (100 mM) [70]. On the other
hand, HNE was found to provoke chromosomal aberrations and sister chromatid
exchange even at 0.1 mM [70]. It should be taken into account that HNE
concentration in the liver is in the range of about 0.1-0.5 mM and, for this
reason, the aldehydes generated by lipid peroxidation
are a constant source of DNA mutagenic DNA damage [36].

Mutagenic properties, both towards eukaryotic and bacterial cells, are
characteristic for acrolein, crotonaldehyde, and MDA [67-69]. MDA appears to
be the most mutagenic product of lipid peroxidation [70]. MDA shows
mutagenic activity towards Salomonella typhimurium bacteria culture [72] and
towards eukaryotic cells and is carcinogenic in rodents. The mutagenic
abilities of MDA-DNA adducts were based on MDA reaction (in a neutral
medium) with the single-stranded M13 vector
containing the lacZá gene giving rise to
mutation during the formation of the lacZá- phenotype [76]. The mutation was
found to be related to the increase in the
amount of M1G-major MDA-doexyguanosine
adduct. Most sequence changes induced by MDA were base pair substitutions.
43% of these substitutions were transversions (most of which were G.T) and
57% were C.T and A.G transitions [76]. The ability of MDA to induce base
pair substitution mutation at dG, dA, and dC is due to the possibility of
forming adducts with these deoxynucleosides. No mutation was detected in the
case of dT, which is concurrent with the fact
that MDA does not form adducts with
this deoxynucleoside.
An experiment in which M1G was positioned at a defined site
in a duplex M13 genome was used to determine the mutagenic potential of the
lesion. M1G-modified and unmodified genomes were transoformed into E. coli
strains [77]. A 500-fold increase in mutations was observed compared to the
unadducted genome. The recent work of Lloyd and Harris shows that cyclic
adducts can be reactive species that participate in intra- and interchain
crosslinking reactions in the genome. They demonstrated that the primary
acrolein adduct of deoxyguanosine,
3-(2-deoxy-beta-D-erythro-pentofuranosyl)-
5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purin-10(3H)-one (2), can
spontaneously but reversibly be forme as an interchain cross-link with the
exocyclic amino group of deoxyguanosine in the opposing chain [78].

The carcinogenic action of MDA was known of as early as 1972 [73]. This
compound, administered locally to mice, provokes a rapidly propagating skin

CELL. MOL. BIOL. LETT. Vol. 8. No. 2. 2003  402

tumour without showing characteristics which are typical for a mouse skin
assay [74]. In addition, MDA was found to induce thyroid tumours in rats
[75].
DNA damage caused by the formation of adducts with lipid peroxidation
products can be repaired.
This is mainly mediated by the action of
glycosylase, which results in apurinic/apyrimidinic sites [79].
Human cells contain a
protein signalling the ranges of damaged DNA owing to its high binding
affinity to
the etheno adducts of DNA, and glycosylase acts there as the repairing
enzyme
[80].

This protein is able to detect nucleic acid damage in the position of åA
(1,N6-ethenoadenine) coupled either
with thymine or with cytosine [80], while the
range where eA is bonded to adenine is poorly detectable and the adduct of
eA with guanine is undetectable. It is worth noting that the molecular
weight
of the discussed protein is
similar to those of many animal DNA glycosylases [81].
Since it was found that 3-methyladenine-DNA glycosylase was able to remove
eA and 3-methyladenine cooperating with a protein having affinity to eA, the
repairing action to eA in the human organism was assigned to
3-methyladenine-DNA glycosylase [79]. In further studies, it was found that
all four etheno adducts:
eA, 3,N4-ethenocytosine (eC), N2,3-ethenoguanine (N2,3-eG) and
1,N2-ethenoguanine (1N2-eG) can be removed both
by human glycosylase and by
Escherichia coli expressing cloned human 3-methyladenine-DNA glycosylase
[82]. It is also supposed that etheno adducts of adenine and cytosine are
removed much more effectively than the guanine adducts [83]. It was recently
reported that human alkylpurine-DNA N-glycosylase (APNG) is able to remove
both 1,N6-ethenoadenine(eA) and 1,N6-ethanoadenine (EA);
unlike eA, EA has a
single bond in its five-membered ring instead of double bond [84]. It was
also found experimentally that the ability of this enzyme to remove EA is 65
times lower than its ability to remove eA [84].
It should be stressed that the possibility to remove etheno adducts of DNA
by ubiquitous and non-specific DNA
glycosylase suggests an endogenous origin of these adducts.

METHODS OF IDENTIFICATION AND QUANTITATIVE
DETERMINATION OF THE ADDUCTS OF LIPID PEROXIDATION
PRODUCTS WITH DNA
Etheno and propano DNA adducts are formed in small amounts in vivo. For this
reason, sensitive separation and detection methods are needed in their
analysis to
monitor their formation and to determine their role in pathological states
including carcinogenesis.

The first method applied in the quantitative analysis of the etheno adducts
of dA and of dC occurring in the lung and liver DNA
of rats after a short exposure
to vinyl chloride involved the resolution of DNA hydrolysis products by HPLC
and the determination of the level of the resulting adducts via the
radioimmunoassay (RIA) technique using monoclonal antibodies [85].
Parallelly, a sensitive method was developed to determine the level of
etheno dG adducts based on electrophore labelling followed by negative ion
chemical ionization [86].
The

CELLULAR & MOLECULAR BIOLOGY LETTERS  403

detection limit, depending on the sensitivity of the methods, was three
adducts (edA or edC) per 108 parent bases and six edG adducts per 107 parent
bases, respectively.
Unfortunately, several mg of DNA were needed to carry out the
determination using these methods.

A method combining gas chromatography with mass spectrometry with electron
capture negative chemical ionization detection (GC/EC NCI/MS) used in
quantitative determination of etheno adducts, mainly MDA with dG, proved to
be more sensitive [86, 88]. The detection limit of this method is about 1
M1G
adduct per 108 parent bases, and the required DNA amount is 1 mg [87, 88].
In this method, deoxyribonucleosides are separated using monoclonal
antibodies
against M1G-deoxyribose-albumine and M1G-deoxyribose conjugates.
The M1G base is converted in its derivative using pentafluorobenzyl bromide
(PFB) and the derivative is analysed using the GC/EC NCI/MS method.

The ultrasensitive IPPA method (Imunopurification-32P-postlabelling assay)
involves the isolation of modified DNA bases using immunoaffinity followed
by labelling e-deoxyribonucleoside 3'-monophosphates
with 32P phosphorus [89-91].
This was the basic method in determining the level of etheno adducts of
deoxyadenosine and deoxycytidine (edA and edC, respectively) isolated from
normal nucleotides using monoclonal antibodies against those adducts. The
attainable detection limit of this method is about 1 adduct per 107-109
parent DNA bases, and the required DNA amount is only 10-50 mg. Thus, it is
applicable to determinations in human tissues, where the expected
concentration of etheno adducts of DNA is close to one per 108 parent bases
[77, 79].
The use of large amounts of an energetic radioisotope and a great amount of
work were needed in the sensitive 32P-postlabelling method and, in addition,
this method was very time-consuming.
For this reason, another method has recently
been applied - liquid chromatography and electrospray tandem mass
spectrometry (LC/ES-MSMS) with the use of a labelled inner standard.
It was applied in quantitative determination of the etheno adducts of dA
with
guanine formed in the placenta [92, 93] and of etheno adducts of guanine in
the
human liver [94]. The resulting detection limit was about 1 adduct per
106-107 parent DNA bases. The LC/ES-MSMS method proved to be more sensitive
than the 32P-postlabelling method,
but 1 mg DNA was needed to carry out the analysis.
For this reason Roberts et al. have recently taken advantage of the unique
properties of graphitized carbon to attain maximal efficiency of
chromatographic
separation [95]. Such a modification of the method made it possible to
determine the level of the etheno adducts of dC of the order of 5 adducts
per 108
parent bases using an amount of DNA less than 100 mg.

Examples of the occurrence of etheno adducts in various tissues and the
techniques used for their identification and quantitative determination are
presented in Tab. 1.

CELL. MOL. BIOL. LETT. Vol. 8. No. 2. 2003  404

Tab. 1. Etheno adducts in various tissues and the methods of their
determination.
Adducts Tissue Technique Reference
M1G liver 32P [96, 97]
M1G kidney 32P [98]
M1G white cells 32P [99, 100]
M1G breats 32P [99, 101]
M1G liver GC/MS [87]
M1G leukocyte DNA GC/EC NCI/MS [88]
M1G pancreas GC/EC NCI/MS [102]
M1G liver GC/EC NCI/MS [103]
edA liver 32P [90, 104, 105, 108]
edC liver 32P [90, 104, 105, 108]
edA spleen 32P [106]
edC spleen 32P [106]
edA white cells 32P [107]
edC white cells 32P [107]
1,N2-propano-dG liver 32P [50]
1,N2-propano-dG colon 32P [50]
edA placenta GC/ECNCI/MS [92]
edA placenta LC/MS/MS [93]
edA liver LC/MS/MS [93]
edC liver LC/ES-MS/MS [93, 94]
1,N2-propano-dG brain 32P [109]

CONCLUSION
There is much strong evidence that the background of etheno-DNA and
propano-DNA detected in tissues from unexposed humans and rodents arises
from endogenous lipid peroxidation products, such as MDA.
With the advent of ultrasensitive, specific detection methods for this kind
of DNA damage in human tissues and cells, new insights can be gained into
the mechanisms involved in human cancers.
Moreover, etheno-DNA adducts can now be used as biomarkers to investigate
the potential role of lipid peroxidation in human cancers.

Acknowledgements. This study was supported by the Polish State Committee
for Resesarch, grant No. 3P05B07922.

REFERENCES
1. Marnett, L.J. Oxyradicals and DNA damage. Carcinogenesis 21 (2000)
361-370.

2. Porter, N.A. Mechanisms for the autoxidation of polyunsaturated lipids.
Acc. Chem. Res. 19 (1986) 262-268.

CELLULAR & MOLECULAR BIOLOGY LETTERS  405

3. Marnett, L.J. Lipid peroxidation - DNA damage by malondialdehyde. Mut.
Res. 424 (1999) 83-95.

4. Pryor, W.A. Oxy-radicals and related species: their formation, lifetimes,
and reactions. Annu. Rev. Physiol. 48 (1986) 657-667.

5. Barclay, L.R.C., Locke, S.J., MacNeil, J.M., VanKessel, J., Burton, G.W.
and Ingold, K.U. Autoxidation of micelles and model membranes.
Quantitative kinetic measurements can be made by using either watersoluble
or lipid-soluble chainbreaking antioxidants. J. Am. Chem. Soc.
106 (1984) 2479-2481.

6. Waldeck, A.R. and Stocker, R. Radical-initiated lipid peroxidation in low
density lipoproteins: insights obtained from kinetic modeling. Chem. Res.
Toxicol. 9 (1996) 954-964.

7. Pryor, W.A. and Stanley, J.P. A suggested mechanism for the production of
malondialdehyde during the autoxidation of polyunsaturated fatty acids.
Non-enzymatic production of prostaglandin endoperoxides during
autoxidation. J. Org. Chem. 40 (1975) 3615-3617.

8. Morrow, J.D. and Roberts, L.J. The isoprostanes-current knowledge and
directions for future research. Biochem. Pharmacol. 51 (1996) 1-9.

9. Hamberg, M. and Samuelsson, B. On the mechanism of the biosynthesis of
prostaglandins E1 and F1á. J. Biol. Chem. 242 (1967) 5336-5343.

10. Praticò, G., Iuliano, L., Mauriello A., Spagnoli, L., Lawson, J.A.,
Maclouf, J., Violi, F. and FitzGerald, G.A. Localization of distinct
F2-isoprostanes in human atherosclerotic lesions. J. Clin. Invest. 100
(1997), 2028-2034.

11. Mallat, Z., Philip, I., Lebret M., Chatel, D., Maclouf, J. and Tedgui,
A.  Elevated levels of 8-iso-prostaglandin F2á in pericardial fluid of
patients with heart failure. Circulation 97 (1998) 1536-1539.

12. Richelle, M., Turini, M. E., Guidoux R., Tavazzi, I., Metairon, S. and
Fay, L.B. Urinary isoprostane excrection is not confounded by the lipid
content of the diet. FEBS Lett. 459 (1999) 259-262.

13. Lawson, J.A., Rokach, J. and FitzGerald, G.A. Isoprostanes: formation,
analysis and use as indices of lipid peroxidation in vivo.
J. Biol. Chem. 247 (1999) 24441-24444.

14. Davi, G., Ciabattoni, G., Consoli A., Mezzetti, A., Falco, A.,
Santarone, S., Pennese, E., Vitacolonna, E., Bucciarelli, T., Costantini,
F., Capani, F. and Patrono, C. In vivo formation of 8-iso-PGF2á and platelet
activation in diabetes mellitus: effects of improved metabolic control and
vitamin E supplementation. Circulation 99 (1999) 224-229.

15. Praticò, D. F2-isoprostanes: sensitive and specific non-invasive indices
of lipid peroxidation in vivo. Atherosclerosis 147 (1999) 1-10.

16. Li, H., Lawson, J.A., Reilly M., Adiyaman, M., Hwang, S.-W., Rokach, J.
and FitzGerald, G.A. Quantitative high performance liquid chromatography/
tandem mass spectrometric analysis of the four classes of F2-isoprostanes in
human urine. Proc. Natl. Acad. Sci. USA 96 (1999) 13381-13386.

CELL. MOL. BIOL. LETT. Vol. 8. No. 2. 2003  406

17. Basu, S. Metabolism of 8-iso-prostaglandin PGF2á. FEBS Lett. 428 (1998)
32-36.

18. Buettner, G.R. The pecking order of free radicals and antioxidants:
Lipid peroxidation, á-tocopherol, and ascorbate. Arch. Biochem. Biophys. 300
(1993) 535-543.

19. Halliwell, B. Oxidation of low-density lipoproteins: Questions of
initiation,
propagation, and the effect of antioxidants. Am. J. Clinical Nutrition 61
(1995) 670-677.

20. Solé, J., Huguet, J., Arola, L. and Romeu, A. In vivo effects of nickel
and cadmium in rats on lipid peroxidation and ceruloplasmin activity. Bull.
Environ. Contam. Toxicol. 44 (1990) 686-691.

21. Sarkar, S., Yadav, P., Trivedi, R., Bansal A.K. and Bhatnagar, D.
Cadmium-induced lipid peroxidation and the status of the antioxidant
system in rat tissues. J. Trace Elem. Med. Biol. 9 (1995) 144-149.

22. Xie, J., Funakoshi, T., Shimada, H. and Kojima, S. Effects of chelating
agents on testicular toxicity in mice caused by acute exposure to nickel.
Toxicology 103 (1995) 147-155.

23. Hartwig, A., Klyszcz-Nasko, H., Schlepegrell, R. and Beyersmann, D.
Cellular damage by ferric nitrilotriacetate and ferric citrate in V79 cells:
Interrelationship between lipid peroxidation, DNA strand breaks and sister
chromatid exchanges. Carcinogenesis 14 (1993) 107-112.

24. Minotti, G. Sources and role of iron in lipid peroxidation. Chem. Res.
Toxicol. 6 (1996) 134-146.

25. Glass, G.A. and Stark, A.A. Promotion of glutathione-ã-glutamyl
transpeptidase-dependent lipid peroxidation by copper and ceruloplasmin:
The requirement for iron and the effects of antioxidants and antioxidant
enzymes. Environ. Mol. Mutagen. 29 (1997) 73-80.

26. Dix, T.A. and Aikens, J. Mechanisms and biological relevance of lipid
peroxidation initiation. Chem. Res. Toxicol. 6 (1993) 2-18.

27. Esterbauer, H. Lipid peroxidation products: formation, chemical
properties and biological activities, in: Free Radicals in Liver Injury,
(Pli, G.,
Cheeseman, K.H., Dianzani, M.U., Slater T.F., Eds.), IRL Press, Oxford,
1985, 29-47.

28. Kaneko, T., Honda, S., Nakano, S.I. and Matsuo, M. Lethal effects of a
linoleic acid hydroperoxide and its autoxidation products, unsaturated
aliphatic aldehydes, on human dipoloid fibroblasts. Chem. Biol. Interact.
63 (1987) 127-137.

29. Lee, S.H. and Blair, I.A. Characterization of 4-oxo-2-nonenal as a novel
product of lipid peroxidation. Chem. Res. Toxicol. 13 (2000) 698-702.

30. Ichihashi, K., Osawa, T., Toyokuni, S. and Uchida, K. Endogenous
Formation of Protein Adducts with Carcinogenic Aldehydes. J. Biol.
Chem. 276 (2001) 23903-23913.

31. Uchida, K., Kanematsu, M., Morimitsu, Y., Osawa, T., Noguchi, N., Niki
and E.J. Acrolein is a product of lipid peroxidation reaction. Formation of

CELLULAR & MOLECULAR BIOLOGY LETTERS  407

free acrolein and its conjugate with lysine residues in oxidized low density
lipoproteins. J. Biol. Chem. 273 (1998) 16058-16066.

32. Esterbauer, H., Zollner, H. and Schaur, R.J. Aldehydes formed by lipid
peroxidation; mechanisms of formation, occurrence and determination. In:
Membrane lipid oxidation, (Vigo-Pelfrey, C., Ed.), vol. I, Boca Raton,
FL:CRC Press, 1990, 239-283.

33. Grosch, W. Reactions of hydroperoxides - products of low molecular
weight. in: Autoxidation of unsaturated lipids (Chan, H. W. S., Ed.),
New York: Academic Press , 1987, 95-139.

34. Segall, H.J., Wilson, D.W., Dallas, J.L. and Haddon, W.F. Trans-4-
hydroxy-2-hexenal. A reactive metabolite from the macrocyclic
pyrrolizidine alkaloid senecionine. Science 229 (1985) 472-475.

35. Esterbauer, H. and Cheeseman, K.H. Determination of aldehydic lipid
peroxidation products: malonaldehyde and 4-hydroxynonenal. Meth.
Enzymol. 186 (1990) 407-421.

36. Esterbauer, H., Schaur, R.J. and Zollner, H. Chemistry and biochemistry
of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic.
Biol. Med. 11 (1991) 81-128.

37. The role of cyclic nucleic acid adducts in carcinogenesis and
mutagenesis, (Singer, B., Bartsch, H., Eds), IARC Sci. Publ. no. 70,
International Agency for Research on Cancer, Lyon, France, 1986.

38. Marnett L.J., in: DNA Adducts: Identification and Biological
Significance, (Hemminki, K., Dipple, A., Shuker, D.E.G., Kadlubar, F.F.,
Segerbäck, D., Bartsch, H., Eds.), IARC Sci. Publ. no. 125, International
Agency for Research on Cancer, Lyon, France, 1994, 151-163.

39. Nath, R.G. and Chung, F.-L. Detection of exocyclic1,N2-
propanodeoxyguanosine adducts as a common DNA lesions in rodents and
humans. Proc. Natl. Acad. Sci. USA 91 (1994) 7491-7495.

40. Chung, F. -L. and Hecht, S.S. Formation of cyclic 1,N2-adducts by
reaction of deoxyguanosine with á-acetoxy-N-nitrosopyrrolidine, 4-
(carbethoxynitrosamino) butanal, and crotonaldehyde. Cancer Res. 43
(1983) 1230-1235.

41. Chung, F.-L., Young, R. and Hecht, S.S. Formation of cyclic 1,N2-
propanodeoxyguanosine adducts in DNA upon reaction with acrolein or
crotonaldehyde. Cancer Res. 44 (1984) 990-995.

42. Burcham, P.C. Genotoxic lipid peroxidation products: their DNA damaging
properties and role in formation of endogenous DNA adducts. Mutagenesis
13 (1998) 287-305.

43. Schauenstein, E., Esterbauer, H. and Zollner, H. Aldehydes in biological
system: Their natural occurence, and biological activities. London: Pion
Ltd., 1977.

44. Witz, G. Biological interactions of alpha, beta-unsaturated aldehydes.
Free Radic. Biol. Med. 7 (1989) 333-349.

CELL. MOL. BIOL. LETT. Vol. 8. No. 2. 2003  408

45. Esterbauer, H., Zollner, H. and Scholz, N. Reaction of glutathione with
conjugated carbonyls. Z. Naturforsch 30 (1975) 466-473.

46. Sodum, R.S. and Shapiro, R. Reaction of acrolein with cytosine and
adenine deratives. Bioorg. Chem. 16 (1988) 272-282.

47. Chung, F.-L., Chen, H-J. C. and Nath, R.G. Lipid peroxidation as
apotential endogenous source for the formation of exocyclic DNA adducts.
Carcinogenesis 17 (1996) 2105-2111.

48. Winter, C.K., Segall, H.J. and Haddon, W.F. Formation of cyclic adducts
of deoxyguanosine with the aldehydes trans-4-hydroxy-2-hexenal and
trans-4-hydroxy-2-nonenal in vitro. Cancer Res. 46 (1986) 5682-5686.

49. Yi, P., Zhan, D.J., Samokyszyn, V.M., Doerge, D.R. and Fu, P.P.
Synthesis and 32P-postlabelling/high-performance liquid chromatography
separation of daistereomeric, N2-(1,3-propano)-2'-deoxyguanosine 3'-
phosphate adducts formed from 4-hydroxy-2-nonenal. Chem. Res.
Toxicol. 10 (1997) 1259-1265.

50. Chung, F.-L., Nath, R.G., Ocando, J., Nishikawa, A. and Zhang, L.
Deoxyguanosine Adducts of t-4-Hydroxy-2-nonenal Are Endogenous DNA
Lesions in Rodents and Humans: Detection and Potential Sources. Cancer
Res. 60 (2000) 1507-1511.

51. Seto, H., Okuda, T., Takesue, T. and Ikemura, T. Reaction of
malondialdehyde with nucleic acid: I. Formatin of fluorescent pyrimido[1,2
-a]purin-10(3H)-one nucleosides. Bull. Chem. Soc. Jpn. 56 (1983) 1799-
1802.

52. Marnett, L.J., Basu, A.K., O'Hara, S.M., Weller, P.E., Rahman, A.F.M.M.
and Oliver, J.P. Reaction of malondialdehyde with guanine nucleosides:
formation of adducts containing oxadiazabicyclononene residues in the
base-pairing region. J. Am. Chem. Soc. 108 (1986) 1348-1350.

53. Chaudhary, A.K., Reddy, G.R., Blair, I.A. and Marnett, L.J.
Characterization of an N6-oxo-propenyl-2'-deoxyadenosine adduct in
malondialdehyde-modified DNA using liquid chromatography electrospray
ionization tandem mass spectrometry. Carcinogenesis 17 (1996)1167-
1170.

54. Nair, V. and Offerman, R.J. Ring-extended products from the reaction of
epoxy carbonyl compounds and nucleic acid bases. J. Org. Chem. 50
(1985) 5627-5631.

55. Sodum, R.S. and Chung, F.-L. Structural characterization of adducts
formed in the reaction of 2,3-epoxy-4-hydroxynonanal with
deoxyguanosine. Chem. Res. Toxicol. 2 (1989) 23-28.

56. Chen, H.-J. C. and Chung, F.-L. Epoxidation of trans-4-hydroxy-2-nonenal
by fatty acid hydroperoxides and hydrogen peroxide. Chem. Res. Toxicol.
9 (1996) 306-312.

57. Golding, B.T., Slaich, P.K., Kennedy, G., Bleasdale, C. and Watson, W.P.
Mechanism of formation of adducts from reaction of glycidaldehyde with
2'-deoxyguanosine and/or guanosine. Chem. Res. Toxicol. 9 (1996) 147-
157.

CELLULAR & MOLECULAR BIOLOGY LETTERS  409

58. Dedon, P.C., Plastaras, J.P., Rouzer, C.A. and Marnett, L.J. Indirect
mutagenesis by oxidative DNA damage: Formation of the
pyrimidopurinone adduct of deoxyguanosine by base propenal. Proc. Natl.
Acad. Sci. USA 95 (1998) 11113-11116.

59. Sodum, R.S. and Chung, F.-L. Stereoselective formation of in vitro
nucleic acid adducts by 2,3-epoxy-4-hydroxynonanal. Cancer Res. 51 (1991)
137- 143.

60. Burczynski, M.E., Sridhar, G.R., Palackal, N.T. and Penning, T.M. The
reactive oxygen species and Michael acceptor-inducible human aldo-keto
reductase AKR1C1 reduces the á,â-unsaturated aldehyde 4-hydroxy-2-
nonenal to 1,4-dihydroxy-2-nonene. J. Biol. Chem. 276 (2001) 2890-2897.

61. Cadet, J., Carvalho, V.M., Onuki, J., Douki, T., Medeiros, H.M., Di
Mascio, P.D., in: Exocyclic DNA Adducts in Mutagenesis and
Carcinogenesis, (Singer, B., Bartsch, H., Eds.), IARC Sci. Publ. no. 150,
International Agency for Research on Cancer, Lyon, France, 1999, 103- 113.

62. Carvalho, V.M., Asahara, F., Di Mascio, P., de Arruda Campos, I.P.,
Cadet, J. and Medeiros, H.M. Novel 1,N(6)-etheno-2'-deoxyadenosine adducts
from lipid peroxidation products. Chem. Res. Toxicol. 13 (2000) 397-405.

63. Loureiro, A.P., Di Mascio, P., Gomes, O.F. and Medeiros, M.H. Trans,
trans-2,4-decadienal-induced 1,N(2)-etheno-2'-deoxyguanosine adduct
formation. Chem. Res. Toxicol. 13 (2000) 601-609.

64. Rindgen, D., Nakajima, M., Wehrli, S., Xu, K. and Blair, I.A. Covalent
modifications to 2'-deoxyguanosine by 4-oxo-2-nonenal a novel product of
lipid peroxidation. Chem. Res. Toxicol. 12 (1999) 1195-1204.

65. Rindgen, D., Lee, S.H., Nakajima, M. and Blair, I.A. Formation of a
substituted 1,N6-etheno2'-deoxyadenosine adduct by lipid
hydroperoxidemediated generation of 4-oxo-2-nonenal. Chem. Res. Toxicol. 13
(2000) 846-852.

66. Lee, S.H., Rindgen, D., Bible, R.A., Hajdu, E. and Blair, I.A.
Characterization of 2'-deoxyadenosine adducts derived from 4-oxo-2-
nonenal, a novel product of lipid peroxidation. Chem. Res. Toxicol. 13
(2000) 565-574.

67. Foiles, P.G., Akerkar, S.A. and Chung, F.-L. Application of an
immunoassay for cyclic acrolein deoxyguanosine adducts to assess their
formation in DNA of salmonella typhimurium under conditions of mutation
induction by acrolein. Carcinogenesis 10 (1989) 87-90.

68. Chung, F.-L., Young, R. and Hecht, S.S. Detection of cyclic 1,N2-
propanodeoxyguanosine adducts in DNA of rats treated with
Nnitrosopyrrolidine and mice treated with crotonaldehyde. Carcinogenesis
10 (1989) 1291-1297.

69. Chung, F.-L., Wang, M. and Hecht, S.S. Detection of exocyclic guanine
adducts in hydrolysates of hepatic DNA of rats treated with N-

CELL. MOL. BIOL. LETT. Vol. 8. No. 2. 2003  410

nitrosopyrrolidine and in calf thymus DNA reacted with
á-acetoxy-Nnitrosopyrrolidine. Cancer Res. 49 (1989) 2034-2041.

70. Esterbauer, H., Eckl, P. and Ortner, A. Possible mutagens derived from
lipids and lipid precursors. Mutat. Res. Rev. Genet. Toxicol. 238 (1990)
223-233.

71. Eckl, P. and Esterbauer, H. Genotoxic effects of 4-hydroxynonenals. Adv.
Biosci. 76 (1989) 141-157.

72. Mukai, F.H. and Goldstein, B.D. Mutagenicity of malondialdehyde, a
decomposition product of peroxidized polyunsaturated fatty acids. Science
191 (1976) 868-869.

73. Shamberger, R.J., Andreone, T L. and Willis, C.E. Antioxidants and
cancer: IV. Initiating activity of malonaldehyde as a carcinogen. J. Natl.
Cancer Inst. 53 (1974) 1771-1773.

74. Fischer, S.M., Olge, S., Marnett, L.J., Nesnow, S. and Slaga, T.J. The
lack of initiating and/or promoting activity of sodium malondialdehyde on
Sencar mouse skin. Cancer Lett. 19 (1983) 61-66.

75. Spalding, J.W. Toxicology and carcinogenesis studies of malondialdehyde
sodium salt (3-hydroxy-2-propenal, sodium salt) in F344/N rats and
B6C3F1 mice. NTP Technical Report 331 (1988) 5-13.

76. Benamira, M., Johnson, K., Chaudhary, A., Bruner, K., Tibbetts, C. and
Marnett, L.J. Induction of mutations by replication of
malondialdehydemodified
M13 DNA in Escherichia coli: determination of the extent of DNA
modification, genetic requirements for mutagenesis, and types of mutations
induced. Carcinogenesis 16 (1995) 93-99.

77. Fink, S.P., Reddy, G.R. and Marnett, L.J. Mutagenicity in Escherichia
coli of the major DNA adduct derived from the endogenous mutagen
malondialdehyde. Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 8652-8657.

78. Kozekov, I. D., Nechev, L. V., Sanchez, A., Harris, C. M. Lloyd, R. S.,
Harris, T. M. Interchain cross-linking of DNA mediated by the principal
adduct of acrolein. Chem. Res. Toxicol. 14 (2001) 1482-1485.

79. Oesch, F., Adler, S., Rettelbach, R. and Doerjer, G. in: The role of
Cyclic and Nucleic Acid Adducts in Carcinogenesis and Mutagenesis, (Singer,
B., Bartsch, H., Eds.), IARC Sci. Publ. no. 70, International Agency for
Research on Cancer, Lyon, France, 1986, 373-379.

80. Rydberg, B., Qui, Z.-H., Dosanjh, M.K. and Singer, B. Partial
purification of a human DNA glycosylase acting on the cyclic carcinogen
adduct 1,N6-ethenodeoxyadenosine. Cancer Res. 52 (1992) 1377-1379.

81. Vollberg, T.M., Siegler, K.M., Cool, B.L. and Sirover, M.A. Isolation
and characterization of the human uracil DNA glycosylase gene. Proc. Natl.
Acad. Sci. USA 86 (1989) 8693-8697.

82. Singer, B., Antoccia, A., Basu, A.K., Dosanjh, M.K., Fraenkel-Conrat,
H., Gallagher, P.E., Kusmierek, J.T., Qiu Z.-H. and Rydberg, B. Both
purified
human 1,N6-ethenoadenine-binding protein and purified human 3-
methyladenine-DNA glycosylase act on 1,N6-ethenoadenine and 3-
metyladenine. Proc. Natl. Acad. Sci. USA 89 (1992) 93-97.

CELLULAR & MOLECULAR BIOLOGY LETTERS  411

83. Dosanjh, M.K., Chenna, A., Kim, E., Fraenkel-Conrat, H., Samson, L. and
Singer, B. All four known cyclic adducts formed in DNA by the vinyl
chloride metabolite chloroacetaldehyde are released by a human DNA
glycosylase. Proc. Natl. Acad. Sci. USA 91 (1994) 1024-1028.

84. Guliaev, A.B., Hang, B. and Singer, B. Structural insights by molecular
dynamics simulations into differential repair efficiency for ethano-A versus
etheno-A adducts by the human alkylpurine-DNA N-glycosylase. Nucleic
Acids Res. 30 (2002) 3778-3787.

85. Eberle, G., Barbin, A., Laib, R.J., Ciroussel, F., Thomale, J., Bartsch,
H. and Rajewsky, M.F. 1,N6-etheno-2'-deoxyadenosine and 3,N4-etheno-2'-
deoxycytidine detected by monoclonal antibodies in lung and liver DNA of
rats exposed to vinyl chloride. Carcinogenesis 10 (1989) 209-212.

86. Fedtke, N., Boucheron, J.A., Walker, V.E. and Swenberg, J.A. Vinyl
chloride-induced DNA adducts. II: Formation and persistence of 7-(2'-
oxoethyl)guanine and N2,3-ethenoguanine in rat tissue DNA.
Carcinogenesis 11 (1990) 1287-1292.

87. Chaudhary, A.K., Nokubo, M., Reddy, G.R., Yeola, S.N., Morrow, J.D.,
Blair, I.A. and Marnett, L.J. Detection of endogenous
malondialdehydedeoxyguanosine adducts in human liver. Science 256 (1994)
1580-1582.

88. Rouzer, C.A., Chaudhary, A.K., Nokubo, M., Ferguson, D.M., Reddy,
G.R., Blair, I.A. and Marnett, L.J. Analysis of the malondialdehyde-2'-
deoxyguanosine adduct, pyrimidopurinone, in human leukocyte DNA by
gas chromatography/electron capture negtive chemical ionization/mass
spectrometry. Chem. Res. Toxicol. 10 (1997) 181-188.

89. Guichard, Y., Nair, J., Barbin, A. and Bartsch, H. Immunoaffinity
clean-up combined with 32P-postlabelling analysis of 1,N6-ethenoadenine and
and 3,N4-ethenocytosine in DNA, in: Postlabelling Methods for Detection of
DNA Adducts (Phillips, D.H., Castegnaro, M., Bartsch, H. Eds.), vol. 124,
IARC Sci. Publ. IARC, Lyon, 1993, 263-269.

90. Nair, J., Barbin, A., Guichard, Y. and Bartsch, H. 1,N6-
ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine in liver DNA from
humans and untreated rodents detected by immunoaffinity/32Ppostlabelling.
Carcinogenesis 16 (1995) 613-617.

91. Leuratti, C., Singh, R., Deag, E.J., Griech, E., Hughes, R., Bingham,
S.A., Plastaras, J.P., Marnett, L. J. and Shuker D.E.G., in: Exocyclic DNA
Adducts in Mutagenesis and Carcinogenesis, (Singer, B., Bartsch, H.,
Eds.), IARC Sci. Publ. no. 150, International Agency for Research on
Cancer, Lyon, France, 1999, 197-203.

92. Chen, H.-J.C., Chiang, L.-C., Tseng, M.C., Zhang, L.L., Ni, J. and
Chung, F.-L. Detection and quantification of 1,N6-ethenoadenine in human
placental DNA by mass spectrometry. Chem. Res. Toxicol 12 (1999)
1119-1126.

93. Doerge, D.R., Churchwell, M.I., Fang, J-L. and Beland, F.A.
Quantification of etheno-DNA addducts using liquid chromatography, on-line
sample

CELL. MOL. BIOL. LETT. Vol. 8. No. 2. 2003  412

processing, and electrospray tandem mass spectrometry. Chem. Res.
Toxicol 13 (2000) 1259-1264.

94. Yen, T.Y., Christova-Gueoguieva, N.I., Scheller, N., Holt, S., Swenberg,
J. A. and Charles, J.M. Quantitative analysis of the DNA adduct N2,
3-ethenoguanine using LC-ESI/MS. J. Mass. Spectrom. 31 (1996) 1271-
1276.

95. Roberts, D.W., Churchwell, M.I., Beland, F.A., Fang, J-L. and Doerge,
D.R. Quantitative analysis of etheno-2'-deoxycytidine DNA adducts using
on-line immunoaffinity chromatography coupled with LC/ES-MS/MS
detection. Anal. Chem. 73 (2001) 303-309.

96. Vaca, C.E., Vodicka, P. and Hemminki, K. Determination of
malonaldehyde-modified 2'-deoxyguanosine-3'-monophosphate and DNA
by 32P -postlabelling. Carcinogenesis 13 (1992) 593-599.

97. Kautiainen, A., Vaca, C.E. and Granath, F. Studies on the relationship
between hemoglobin and DNA adducts of malonaldehyde and their
stability in vivo. Carcinogenesis 14 (1993) 705-708.

98. Wang, M.-Y. and Liehr, J.G. Induction by estrogens of lipid peroxidation
and lipid peroxide-derived malonaldehyde -DNA adducts in male Syrian
hamsters: Role of lipid peoxidation in estrogen-induced kidney
carcinogenesis. Carcinogenesis 16 (1995) 1941-1945.

99. Vaca, C.E., Fang, J.-L., Mutanen, M. and Valsta, L. 32P -Postlabelling
determination of DNA adducts of malonaldehyde in humans: total white
blood cells and breast tissue. Carcinogenesis 16 (1995) 1847-1851.

100. Fang, J.L., Vaca, C.E., Valsta, L.M. and Mutanen, M. Determination of
DNA adducts of malonaldehyde in humans: effects of dietary fatty acid
composition. Carcinogenesis 17 (1996) 1035-1040.

101. Wang, M., Dhingra, K., Hittelman, W.N., Liehr, J.G., de Andrade, M. and
Li, D. Lipid peroxidation-induced putative malondialdehyde-DNA adducts
in human breast tissue. Cancer Epidemiol. Biomarkers Prev. 5 (1996)
705-710.

102. Kadlubar, F., Anderson, K., Lang, N., Thompson, P., MacLeod, S.,
Mikhailova, M., Chou, M., Plastaras, J., Marnett, L., Haussermann, S. and
Bartsch, H. Comparison of endogenous DNA adducts levels in human
pancreas. Proc. Am. Assoc. Cancer Res. 39 (1998) 286, Abstract.

103. Chaudhary, A.K., Nokubo, M, Oglesby, T.D., Marnett, L.J. and Blair,
I.A. Characterization of endogenous DNA adducts by liquid
chromatography/electrospray ionization/tandem mass spectrometry. J.
Mass Spectrom. 30 (1995) 1157-1166.

104. Nair, J., Sone, H., Nagao, M., Barbin, A. and Bartsch, H.
Copperdependent formation of miscoding etheno-DNA aducts in the liver of
Long Evans Cinnamon (LEC) rats developing hereditary hepatitis and
hepatocellular carcinoma. Cancer Res. 56 (1996) 1267-1271.

105. Nair, J., Carmichael, P.L., Fernando, R.C., Phillips, D.H., Strain,
A.J. and Bartsch, H. Lipid peroxidation-induced etheno-DNA adducts in liver
of patients with the genetic metal storage disorders Wilson's disease and

CELLULAR & MOLECULAR BIOLOGY LETTERS  413

primary hemochromatosis. Cancer Epidemiol. Biomarkers Prev. 7
(1998) 435 - 440.

106. Nair, J., Gal, A., Tamir, S., Tannenbaum, S.R., Wogan, G.N. and
Bartsch, H. Etheno adducts in spleen DNA of SJL mice stimulated to
overproduce nitric oxide. Carcinogenesis 19 (1998) 2081-2084.

107. Nair, J., Vaca, C.E., Velic, I., Mutanen, M., Valsta, L.M. and Bartsch,
H. High dietary w-6 polyunsaturated fatty acids drastically increase the
formation of etheno-DNA base adducts in white blood cells of female
subjects. Cancer Epidemiol. Biomarkers Prev. 6 (1997) 597-601.

108. Nair, J., Barbin, A., Velic, I. and Bartsch, H. Etheno DNA-base adducts
from endogenous reactive species. Mutat. Res. 424 (1999) 59-69.

109. Götz, M.E., Wacker, M., Luckhaus, C., Wanek, P., Tatschner, T.,
Jellinger, K., Leblhuber, F., Ransmayr, G., Riederer, P. and Eder, E.
Unaltered brain levels of 1,N2-propanodeoxyguanosine adducts of trans-4-hydr
oxy-2-
nonenal in Alzheimer's disease. Neurosci. Lett. 324 (2002) 49-52.
*************************************************************

Med Sci Monit. 2001 Nov-Dec; 7(6): 1230-5.
 Role of reactive oxygen species (ROS) in patients with erythema migrans, an
early manifestation of Lyme borreliosis.
Pancewicz SA, Skrzydlewska E, Hermanowska-Szpakowicz T, Zajkowska JM,
Kondrusik M.
Department of Infection Diseases and Neuroinfections, Medical University,
Bialystok, Poland.

BACKGROUND: Lyme borreliosis is a tick-transmitted, chronic, zoogenous
disease caused by Borrelia burgdorferi spirochete.
The clinical picture of Lyme disease is characterized by the variety of
tissue and organ involvement and differing severity of symptoms.
One of the pathogenic symptoms of early Lyme disease is a skin lesion called
erythema migrans.
MATERIAL AND METHODS: The purpose of our research was to estimate the
parameters of the antioxidant system and the concentration of lipid
peroxidation products in the plasma of patients with erythema migrans (EM).
The parameters measured included the activity levels of superoxide dismutase
(SOD) according to Sykes,
gluthatione reductase (GSSG-R) according to Mize and Langdon,
glutathione peroxidase (GSH-Px) according to Paglia and Valentine;
the concentrations of malondialdehyde (MDA)
were examined by means of a Bioxytech LPO-586 kit.
The total sulphydryl groups (-SH) according to Ellman and
reduced glutathione (GSH)
were measured using a Bioxytech GSH-400 test
in plasma samples collected from 20 patients with EM aged from 19 to 50,
taken before (examination 1) and after (examination 2) therapy with
amoxycycline.
The control group consisted of 8 healthy people.
RESULTS: The results of our examinations prove that beta-lactamase
antibiotic therapy brings non-enzymatic antioxidant parameters to control
values,
though the treatment causes no change in enzymatic antioxidant parameters,
resulting in the further activation of free radicals.
CONCLUSIONS: In patients with Erythema migrans, the decreased capability to
reduce lipid superoxidants leads to maintaining a high concentration of
membrane lipid peroxidation products.   PMID: 11687735

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Clinical Research

page 1230

Med Sci Monit, 2001; 7(6): 1230-1235
Role of reactive oxygen species (ROS) in patients with erythema
migrans, an early manifestation of Lyme borreliosis
Slawomir A. Pancewicz1, Elzbieta Skrzydlewska2, Teresa
Hermanowska-Szpakowicz1,
Joanna Zajkowska1, Maciej Kondrusik1
1 Department of Infection Diseases and Neuroinfections, Medical University,
Bialystok, Poland
2 Department of Analytical Chemistry, Medical University, Bialystok, Poland
key words: Lyme borreliosis, erythema migrans, reactive oxygen species,
antioxidants

SUMMARY
Background: Lyme borreliosis is a tick-transmitted, chronic, zoogenous
disease caused by Borrelia burgdorferi
spirochete. The clinical picture of Lyme disease is characterized by the
variety of tissue and organ involvement
and differing severity of symptoms. One of the pathogenic symptoms of early
Lyme disease is a skin lesion called erythema migrans.
Material and methods: The purpose of our research was to estimate the
parameters of the antioxidant system
and the concentration of lipid peroxidation products in the plasma of
patients with erythema migrans (EM).
The parameters measured included the activity levels of superoxide dismutase
(SOD) according to Sykes, gluthatione
reductase (GSSG-R) according to Mize and Langdon, glutathione peroxidase
(GSH-Px) according to
Paglia and Valentine; the concentrations of malondialdehyde (MDA) were
examined by means of a Bioxytech
LPO-586 kit. The total sulphydryl groups (-SH) according to Ellman and
reduced glutathione (GSH) were measured
using a Bioxytech GSH-400 test in plasma samples collected from 20 patients
with EM aged from 19 to
50, taken before (examination 1) and after (examination 2) therapy with
amoxycycline. The control group consisted
of 8 healthy people.
Results: The results of our examinations prove that beta-lactamase
antibiotic therapy brings non-enzymatic antioxidant
parameters to control values, though the treatment causes no change in
enzymatic antioxidant parameters,
resulting in the further activation of free radicals.
Conclusions: In patients with Erythema migrans, the decreased capability to
reduce lipid superoxidants leads to
maintaining a high concentration of membrane lipid peroxidation products.

Received: 2000.08.28
Accepted: 2001.09.28

Correspondence address: S.awomir A. Pancewicz, Department of Infection
Diseases and Neuroinfections, Medical University,
ul. °urawia 14, 15-540 Biaystok, Poland

BACKGROUND
Lyme borreliosis (tick spirochaetosis, Lyme disease)
is a tick-transmitted, chronic, multisystem, zoogenous
disease of worldwide distribution, caused by
Borrelia burgdorferi spirochete, and in Europe also
by Borrelia garini and Borrelia afzeli. Ticks of the
Ixodes type are main carriers of B. burgdorferi. In
Europe, I. ricinus and I. persulcatus are main carriers
of B. burgdorferi, whereas in the Unites States
I. dammini is present in the North-eastern states
and I. pacificus in the Western states. I. neotomae,
I. spinipalpis, I. hexagonus, and I. granulatus ticks
are of less importance in the transmission of B.
burgdorferi [1-4].

Research done in Poland by Sifski et al. revealed
B. burgdorferi infection in 3.5% to 58.33% of ticks,
depending on the region of Poland [3]. Wegner et
al. documented infection in 11.5% of the I. ricinus
ticks in the region of Olsztyn, and in 4.0% to
15.6% in the region of Bia.ystok [5].

1231

Pancewicz SA et al - Role of reactive oxygen species (ROS) in patients.
The tests performed by many authors, both in Poland
and elsewhere, show that B. burgdorferi is
commonly present in high risk groups, especially
among forestry employees and farmers. Pancewicz
et al. in 1993-1997 examined 1466 forestry employees
from the northeastern region of Poland,
and found B. burgdorferi antibodies in 23.81% of
their subjects [6]. Hulsse C. von Stenglin found B.
burgdoferi infection in 7.8% of the ticks in the
Mecklenbug-Vorpmmern region of Germany, and
detected B. burgdorferi antibodies in 31.4% of forestry
employees [7]. According to Rath et al, 53%
of forestry workers reported tick bites, but erythema
migrans appeared after the bite only in 8% of
these persons. IgG antibodies to B. burgdorferi (immunoblot
assay) were found in 18% of the persons
examined [8]. Burek et al. discovered IgG antibodies
to B. burgdorferi in 9.7% of their total research
population, but in the group coming from
a high risk region the figure rose to 44%, as against
8% in the group from a low risk region; moreover,
antibodies were found in 42.9% of forestry workers [9].

The pathomechanisms controlling the course of
Lyme borreliosis have not yet been fully explained.
It is known, however, that the accompanying
inflammatory states are characterized by the
increased stimulation of phagocytes, which leads
to the increased generation of reactive oxygen
species (ROS) [10,11]. The presence of ROS causes
modifications to occur in low- and macromolecular,
endo- and exogenous compounds [10]. It
has been proved that B. burgdorferi induces O2 generation,
and that ROS derived from macrophages,
mainly nitrous oxide (NO), take part in the inactivation
of B. burgdorferi spirochete [12-15].
The balance between free radicals and antioxidants
is the main factor in the defense against
harmful processes at the cellular and tissue levels.
According to current findings, excessive ROS production
in the organism and imbalance between
the concentrations of ROS and defense antioxidants
may be related to such pathologic processes
as intestinal inflammation, joint inflammation, local
ischemic processes, cardiovascular diseases,
multiple sclerosis, or central and peripheral nervous
system diseases [16-26].

The prevalence of Lyme borreliosis among the population
in the northeastern region of Poland and
difficulties in the treatment and prognosis of the
course of the disease have prompted us to evaluate
selected parameters of the antioxidant system and
lipid peroxidation products in patients with erythema
migrans, an early manifestation of Lyme borreliosis.

MATERIAL AND METHODS
Twenty patients aged from 21 to 66 years (x=41.9)
treated for Lyme borreliosis in the form of erythema
migrans (EM) were enrolled in the study. The
diagnosis of the disease was based on the patients'
epidemiological history and the characteristic clinical
picture. The examinations were carried out
from July to September of 1998. All the patients reported
for treatment within 2 to 4 weeks after
a tick bite. Skin changes of 5 to 30 cm in diameter
and characteristic of EM were found on the patients'
skin, primarily on the extremities. Four weeks
of therapy with Amoxycycline at a dosage of
2.0 g daily resulted in complete remission of pathological
skin changes.

IgM class antibodies to B. burgdorferi were measured
in serum by means of the ELISA method, using
Borrelia recombinant IgG and IgM High Sensitivity
kits from Biomedica (Austria) within 4 to 6 weeks
after treatment.

The control group consisted of 8 healthy individuals
(2 women and 6 men) aged 21 to 45 years (x= 31 years).

Enzyme activity and the concentration of micromolecular
substances were assessed twice: before
Amoxycycline treatment (examination 1) and after
4 weeks of therapy (examination 2).

The activity of Cu, Zn-SOD (EC. 1.15.1.1) was determined
by the method of Misra and Fridovich
[27] as modified by Sykes et al. [24]. A standard
curve for SOD activity was made by using SOD
from bovine erythrocytes (Sigma Biochemicals, St.
Louis, Missouri, USA). One unit of SOD was defined
as the amount of the enzyme needed to inhibit
epinephrine oxidation to adrenochrome by
50%. Enzyme activity was expressed in units per
mg of protein for liver and serum, or per mg of hemoglobin
for erythrocytes.

Glutathione peroxidase (EC. 1.11.1.6) activity was
measured in the liver, erythrocytes and serum specrophotometrically
using a technique based on Paglia
and Valentine [28], whereas GSH formation was
assayed by measuring the conversion of NADPH to
NADP. Enzyme activity was expressed as micromoles
of NADP/min per mg of protein for the liver and
per mg of hemoglobin for erythrocytes.
The activity of glutathione reductase (EC. 1.6.4.2)
was measured by the method of Mize and Langdon,
which involves monitoring the oxidation of nicotinamide
adenine dinucleotide phosphate
(NADPH) at 340 nm [29].

Sulfhydryl compounds were estimated according to
Ellman using 5,5'-dithiobis (nitrobenzoic acid,
DTNB) [30].

The glutathione (GSH) concentration was measured
using a Bioxytech GSH-400 test. This method
proceeds in two steps. The first step leads to the
formation of substitution products between a patented
reagent and all the mercaptans (GSH) present
in the sample. The second step transforms
specifically the substitution product obtained with
GSH into a chromophoric thione whose maximum
absorbency wavelength is 400 nm.
Lipid peroxidation in liver, erythrocytes and serum
was assayed using a Bioxytech LPO-586 kit that
measures MDA together with 4-hydroxyalkanals or
MDA alone. The colorimetric assay uses a chromogenic
reagent which reacts with the products mentioned
above, generating a stable chromophore
which is measured spectrophotometrically at 586
nm. This technique requires sample incubation at
45°C, thus avoiding undesirable artifacts.

Statistical analysis
The results of our examinations were analyzed statistically
by calculating the arithmetic mean and
standard deviation for each variable. The evaluation
of statistical significance was performed by
means of the Wilcoxon test for two related and
unrelated trials, since the variables were not subjected
to normal distribution. The value p<0.05
was taken as the limit of statistical significance.

RESULTS
SOD activity in healthy controls was 3.02 u/ml,
whereas in patients with erythema migrans, it was
lower before treatment than in the controls, and
decreased insignificantly after treatment, to 2.77
u/ml (Tables 1 and 2).

GSSG-R activity was insignificantly higher than in
the controls, amounting to 26.46 u/ml in examination
1, before treatment. After 4 weeks' treatment
with Amoxycycline this figure increased to 29.57
u/ml (Tables 1 and 2).

GSH-Px activity was insignificantly lower in both
examination 1 and 2 in comparison with controls:
93.15% in examination 1, decreasing after treatment
to 91.74% of the values found in controls
(Tables 1 and 2).

The total concentration of SH-groups was significantly
lower (76.71%) in examination 1 when
compared to the concentration in the controls.
After treatment, an insignificant increase to 183.3
nmol/l was observed in the total concentration of
SH-groups, which again was significantly lower in
comparison to the controls (92.19%) (Tables 1
and 2).

1232

Clinical Research
Enzymes examined p= p<
SOD
GSSG-R
GSH-Px
-SH
GSH
MDA
* statistical significance p< 0.05
Test 1 - Test 2
Test 1 - controls
Test 2 - controls
Test 1 - Test 2
Test 1 - controls
Test 2 - controls
Test 1 - Test 2
Test 1 - controls
Test 2 - controls
Test 1 - Test 2
Test 1 - controls
Test 2 - controls
Test 1 - Test 2
Test 1 - controls
Test 2 - controls
Test 1 - Test 2
Test 1 - controls
Test 2 - controls
0.72
0.0932
0.627
0.66
0.6437
0.381
0.75
0.0776
0.0336
0.02
0.0119
0.3393
0.05
0.0076
0.1675
0.21
0.00001
0.00001
0.8
0.100
0.700
0.7
0.700
0.400
0.8
0.080
0.05*
0.05*
0.02*
0.400
0.06
0.008*
0.200
0.3
0.0001*
0.0001*

Table 2. Comparison of enzyme and micromolecular substance
activity in patients with Erythema migrans before and after
treatment in comparison with controls.
Enzymes examined Examination 1 Examination 2 Controls
SOD (u/ml)
GSSG-R (u/ml)
GSH-Px (u/ml)
-SH (nmol/l)
GSH (nmol/l)
MDA (nmol/l)
2.93±1.14
26.46±11.88
132.0±7.21
152.52±19.58
15.66±3.66
15.66±4.21
2.77±1.14
29.57±5.28
130.00±9.11
183.30±53.22
18.14±2.39
14.21±3.99
3.02±2.50
24.87±10.73
141.71±12.52
198.83±65.02
21.23±5.89
4.76±3.13

Table 1. Activity of antioxidation parameters and peroxidation products
(MDA) in patients with Erythema migrans before and after
treatment and in healthy controls.

1233

Pancewicz SA et al - Role of reactive oxygen species (ROS) in patients.
The GSH concentration was 26.24% lower in comparison
to controls in examination 1. After treatment,
it increased insignificantly to 18.14 nmol/ml,
and did not differ significantly when compared to
healthy controls (Tables 1 and 2).

The mean MDA concentration was significantly higher
in both examinations 1 and 2 than in the controls.
In examination 1, before treatment, it was
328.99% of control value; after treatment it decreased
insignificantly, but was still significantly higher
than in the controls (298.53%).

DISCUSSION
The skin lesion called erythema migrans is an early
pathogenic symptom of Lyme disease, appearing
from 10 days to several weeks after infection. Erythema
migrans is present as a ring-like or homogeneous
erythema at the site of B. burgdorferi spirochete
penetration. Afterwards, it spreads concentrically
in the intercellular matrix, which explains why
erythema migrans is later so dispersed [4,31]

Patients with erythema migrans present with nonspecific
symptoms, such as malaise, fatigue, fever,
cephalalgia, atralgia and myalgia.
Neutrophils and macrophages belong to the most
important elements of the host's defense system
against the bacterial infection. The activation of
neutrophils and macrophages greatly intensifies
their metabolism, causing an increase in oxygen
absorption, and in glucose, lipid, and protein metabolism,
the production of proinflammatory cytokin,
and the release of proteolytic enzymes from
these cells, such as collagenase and elastase. Neutrophils
are capable of producing active oxygen
metabolites, which act as bactericidal agents, but
when produced excessively they may cause harmful
reactions [32].

Suhonen et al. examined oxidative burst, calcium
mobilization and phagocytosis induced by B. burgdoferi
proper, B. afzeli and B. garini. They proved
that each genotype of B. burgdorferi induces all
neutrophil functions, depending on the complement.
The CR3 (CD11b) integrin was shown to have
a role in the oxidative burst and calcium mobilization
induced by B. burgdorferi [15].

Modolell et al. examined B. burgdorferi's interaction
with murine macrophages derived from bone
marrow. They found that this interaction caused
microorganized phagocytosis, the induction of nitric
oxide (NO) and oxygen free radicals, and the
elimination of spirochete. The phagocytosis of B.
burgdorferi by macrophages and the generation of
NO and free oxygen radicals was intensified through
spirochete opsonization with monoclonal antibodies.

The addition of NO-synthesis specific inhibitors
to macrophage and spirochete cultures,
together or separately, caused only a partial reduction
in the effector cell-killing potential. The
data they obtained suggest that NO and oxygen
free radicals are essential but insufficient for the
complete elimination of B. burgdorferi by macrophages.
The authors concluded that the defense
against B. burgdorferi infection is associated with
the humoral response, and that specific antibodies
play an important role in the spirochete control
mediated by macrophages [14].

Harter L. et al. stated that mRNA regulation of nitric
oxide (NO) synthetasis, one of the free radicals,
was part of the host's immune response to B. burgdorferi
infection. They suggested that NO may play
a role in arthritis in dogs [33].

Georgili et al. have proved that all B. burgdorferi
strains have a different susceptibility to elimination
through phagocytosis; however, all of them provoked
oxidative burst [13].

Active oxygen metabolites, especially the hydroxylic
radical, exhibit a high level of chemical activity,
and in they organism they interact with proteins, lipids,
carbohydrates and nucleinic acids, which
causes changes in cell function and structure. However,
the excessive release of these compounds
by the cell may cause tissue damage. An excess of
reactive oxygen species activates the mechanisms
which immobilize them by means of enzymatic superoxide
dismutase, catalase and glutathione peroxidation.
The activity of oxygen reactive species is
harmful when the antioxidant system is disturbed
or when it is unable to eliminate the results of ROS
activity [10,11,18].

The excess of ROS together with the inefficiency
of the antioxidant mechanisms intensifies lipid peroxidation,
causing a decrease in cell membrane
flow, damage to the nucleinic acid structure, and
enzyme inactivation. Lipid peroxidation is an avalanche
process, continually supplying free radicals
that initiate further peroxidation reactions. The
products of lipid peroxidation and their reactions
with other cell components cause changes in cell
membrane properties, resulting in cell homeostasis
disorder, which leads to cell death [10,11,18,32].

1234

Clinical Research
The results obtained in our studies indicate that there
are changes in the serum antioxidant system of
patients with Lyme borreliosis presenting in the
form of erythema migrans. The activity of antioxidant
enzymes (SOD and GSH-Px) in patients before
treatment was significantly lower than in the controls.
After treatment, despite complete remission of
pathological changes, a further decrease was observed
in the activity of these enzymes. The concentration
of SH- and GSH- group micromolecular substances,
which was significantly lower before treatment
when compared to controls, increased after
treatment, and did not differ significantly from the
values of healthy individuals. The MDA concentration
was significantly higher both before and after
treatment than the values found in the controls.
These results indicate that beta-lactamase antibiotic
therapy brings non-enzymatic antioxidant parameters
to control values, though no change is observed
in the enzymatic antioxidant parameters,
resulting in the further activation of free radicals.
This may suggest an ongoing asymptomatic pathological
process, leading to the appearance of late
borreliosis symptoms.

A constant decrease in the activation of glutathion
peroxidase indicates a decreased capability to reduce
lipid superoxidants, thus maintaining a high concentration
of membrane lipid peroxidation products.

CONCLUSIONS
1. In Lyme borreliosis presenting as erythema migrans,
changes in the antioxidant system are observed in the plasma of patients,
i.e. a decrease in the activity of SOD and GSH-Px antioxidant enzymes,
 a decrease in the concentration of antioxidant -SH and GSH non-enzymatic
parameters, and an increase in MDA concentration.

2. Beta-lactamase antibiotic therapy brings non-enzymatic antioxidant
parameters to control values,
though it has no influence on enzymatic antioxidant parameters.

3. In erythema migrans, the decreased ability to reduce lipid superoxidants
leads to maintaining a high concentration of membrane lipid peroxidation
products.

REFERENCES:
1. Burgdorfer W: Lyme disease - a tick-borne spirochetosis. Science,
1982; 216: 1317-1318

2. Gustafson R: Epidemiological studies on Lyme borreliosis and tick-
-borne encephalitis. Scand J Inf Dis, 1994; Sup. 92: 8-21

3. Sifski E, Karbowiak G, Siuda K et al: Zaka½enie kleszczy Borrelia
burgdorferi w wybranych rejonach Polski. Przeg Epidemiol, 1994; 4: 385

4. Stanek G, O'Connell S, Cimmino M et al: European Union concerted
action on risk assessment in Lyme borreliosis: clinical case definition
for Lyme borreliosis. Wien Klin Wochenschr, 1996; 108/23: 741-747

5. Wegner Z, Stafczak J, Racewicz N et al: Wyst´powanie kr´tków Borrelia
burgdorferi w kleszczach Ixodes ricinus na terenie województwa bia-
.ostockiego. Mat Mi´dz Symp Borelioza z Lyme. Bia.owie½a 1995, 12

6. Pancewicz SA, Zajkowska JM, Kondrusik M et al: WykrywalnoÊç
przeciwcia. przeciwko B. burgdorferi wÊród pracowników leÊnictwa
w pó.nocno-wschodnim regionie Polski. Med Prac, 1998; 49(3): 253

7. Hulsse C, von Stenglin M: Incidence of Lyme borreliosis in Mecklenburg-
Vorpommern. Gesundheitswesen, 1995; 57(1): 21-4

8. Rath PM, Obershoff B, Mohnhaupt A et al: Seroprevalence of Lyme
borreliosis in forestry workers from Brandenburg, Germany. Eur
J Clin, Microbiol Infect Dis, 1996; 15(5): 372 -7

9. Burek V, Misic-Mayerus L, Maretic T: Antibodies to Borrelia burgdorferi
in various population groups in Croatia. Scan. J Infect Dis, 1992; 24(5):
683-4

10. Conner EM, Grisham MB: Inflammation, free radicals and antioxidants.
Nutrition, 1996; 12: 274-277

11. Kehrer JP: Free radicals as mediators of tissue injury and disease, Crit
Rev Toxicol, 1993; 23: 21-48

12. Garcia-Monco JC, Benach JL: Mechanism of injury in Lyme
neuroborreliosis, Semin Neurol, 1997; 171: 67

13. Georgilis K, Steere AC, Klempner MS: Infectivity of Borrelia burgdorferi
correlates with resistance to elimination by phagocytic cells. J Infect
Dis, 1991; 163: 150-155

14. Modolell M, Schaible UE, Ritting M, Simon MM: Killing of Borrelia
burgdorferi by macrophages is dependent on oxygen radicals and nitric
oxide and can be enhanced by antibodies to outer surface proteins of
the spirochete. Immunol Lett, 1994; 40(2): 139-146

15. Suhonen J, Hartiala K, Tuominen-Gustafsson H, Viljanen MT: Borrelia
burgdorferi-induced oxidative burst, calcium mobilization and phagocytosis
of human neutrophils are complement dependent. J Infectious
Dis, 2000; 181: 195-202

16. Bruckdorfer KR, Hillary JB, Bunce T et al: Increased susceptibility to
oxidation of low-density lipoproteins isolated from patients with systemic
sclerosis. Arthritis Rheum, 1995; 38(8): 1060-1067

17. De Deyn PP, Hiramatsu M, Borggreve F et al: Superoxide dismutase
activity in cerebrospinal fluid of patients with dementia and some other
neurological disorders. Alzheimer Dis Assoc Disord, 1998; 12(1): 26-32

18. Eapen CE, Madesh M, Balasubramanian KA et al: Mucosal mitochondrial
function and antioxidant defences in patients with gastric
carcinoma. Scand J Gastroenterol, 1998; 33(9): 975-981

19. Hall ND, Maslen CL, Blake DR: The oxidation of serum sulphhydryl
groups by hydrogen peroxide secreted by stimulated phagocytic cells in
rheumatoid arthritis, Rheumatol Int, 1984; 4: 35-38

20. Honkkanen VEA: The factors affecting plasma glutathione peroxidase
and selenium in rheumatoid arthritis: a multiple linear regression analysis.
Scan J Rheumatol, 1991; 20: 385-391

21. Kiziltunc A, Cogalgil S, Cerrahoglu L: Carinitine and antioxidants
levels in patients with Rheumatoid Arthritis, Scand J Rheumatol, 1998;
27: 441-445

1235

Pancewicz SA et al - Role of reactive oxygen species (ROS) in patients.
22. Maeda H, Akaike T: Nitric oxide and oxygen radicals in infection,
inflammation and cancer. Biochemistry (Mosc) 1998; 63(7): 854-865

23. Stoessl AJ: Etiology of Parkinson's disease. Can J Neurol Sci, 1999;
26(suppl 2): 5-12

24. Sykes JA, McCormac FX, O'Brien TJ: Preliminary study of the superoxide
dismutase content of some human tumours. Cancer Res, 1978,
38, 2759-2762

25. Tarp U, Hansen JC, Overvad K et al: Glutathione peroxidase activity in
patients with rheumatoid arthritis and in normal subjects: effects of long-
-term selenium supplementation. Arthritis Rheum, 1987; 30: 1162-1165

26. Tarp U, Stengaard Pedersen K, Hansen JC, Thorling EB: Glutathione
redox cycle enzymes and selenium in severe rheumatoid arthritis: lack
of antioxidative response to selenium supplementation in polymorphonuclear
leucocytes. Ann. Rheum. Dis, 1992; 51: 1044-1049

27. Blum J, Fridovich I: Inactivation of glutathione peroxidase by
superoxide radical. Arch Biochem Biophys, 1985; 240: 500-508.

28. Paglia DE, Valentine WN: Glutathione peroxidase and selenoprotein
activity in various tissue. Biol Chem, 1967; 145: 233

29. Mize CE, Langdon. RG: Hepatic glutathione reductase. Purification
and general kinetic properties. J Biol Chem, 1962; 237: 1589

30. Ellman GL: SH groups determination in biological fluids. Anal Biochem,
1970; 46: 237

31. Sigal LH: Lyme disease: a review of aspects of its immunology and
immunopathogenesis. Annu Rev Immunol, 1997; 15: 63-92

32. Zeman K: Rola neutrofilów w procesach zapalnych. W: Zapalenie.
Patofizjologia i klinika, red. H. Tchórzewski. Medpress Warszawa,
1998; 76.

33. Harter L, Straubinger RK, Summers BA et al: Up-regulation of inducible
nitric oxide synthase mRNA in dogs experimentally with Borrelia burgdorferi.
Vet Immunol Immunopathol, 1999; 22, 67(3): 271-284
 *************************************************************

http://groups.yahoo.com/group/aspartameNM/message/1106
hangover research relevant to toxicity of 11% methanol in aspartame
(formaldehyde, formic acid): Calder I (full text): Jones AW:
Murray 2004.08.05 rmforall

Rich Murray, MA    Room For All    rmforall at comcast.net
1943 Otowi Road, Santa Fe, New Mexico 87505 USA  505-501-2298

Since no adaquate data has ever been published on the exact disposition  of
toxic metabolites in specific tissues in humans of the 11% methanol
component of aspartame, the many studies on morning-after hangover from the
methanol impurity in alcohol drinks are the main available resource to date.

Jones AW (1987) found next-morning hangover from red wine with
100 to 150 mg methanol
(9.5% w/v ethanol, 100 mg/l methanol, 0.01%, one part in ten thousand).
Fully 11% of aspartame is methanol --  1,120 mg aspartame  in 2 L diet soda,
almost six 12-oz cans,  gives 123 mg methanol (wood alcohol) -- the same
amount that produces hangover from red wine.

The expert review by Monte WC (1984) states:  "An alcoholic consuming 1500
calories a day from alcoholic sources alone may consume between 0 and 600 mg
of methanol each day depending on his choice of beverages (Table 1)...."
Table 1 lists red wine as having 128 mg/l methanol, about one part in ten
thousand.

http://groups.yahoo.com/group/aspartameNM/message/870
Aspartame: Methanol and the Public Interest 1984: Monte:
Murray 2002.09.23 rmforall

Dr. Woodrow C. Monte  Aspartame: methanol, and the public health.
Journal of Applied Nutrition 1984;  36 (1):  42-54.
(62 references)   Professsor of Food Science [retired 1992]
Arizona State University,  Tempe, Arizona 85287  woodymonte at xtra.co.nz
The methanol from 2 L of diet soda, 5.6 12-oz cans, 20 mg/can, is
112 mg, 10% of the aspartame.
The EPA limit for water is 7.8 mg daily for methanol (wood alcohol), a
deadly cumulative poison.
Many users drink 1-2 L daily.
The reported symptoms are entirely consistent with chronic methanol
toxicity. (Fresh orange juice has 34 mg/L, but, like all juices, has 16
times more ethanol, which strongly protects against methanol.)

"The greater toxicity of methanol to man is deeply rooted in the limited
biochemical pathways available to humans for detoxification.
The loss of uricase (EC 1.7.3.3.),
formyl-tetrahydrofolate synthetase (EC 6.3.4.3.) (42)
and other enzymes (18) during evolution sets man apart from all
laboratory animals including the monkey (42).

There is no generally accepted animal model for methanol toxicity (42, 59).

Humans suffer "toxic syndrome" (54) at a minimum lethal dose
of  <1 gm/kg, much less than that of monkeys, 3-6 g/kg (42, 59).

The minimum lethal dose of methanol
in the rat, rabbit, and dog is 9.5, 7.0 , and 8.0 g/kg, respectively (43);
ethyl alcohol is more toxic than methanol to these test animals (43)."

"Fruit and vegetables contain pectin with variable methyl ester content.
However, the human has no digestive enzymes for pectin (6, 25) particularly
the pectin esterase required for its hydrolysis to methanol (26).

Fermentation in the gut may cause disappearance of pectin (6) but the
production of free methanol is not guaranteed by fermentation (3).
In fact, bacteria in the colon probably reduce methanol directly to formic
acid or carbon dioxide (6)  (aspartame is completely absorbed before
reaching the colon).
Heating of pectins has been shown to cause virtually no demethoxylation;
even temperatures of 120 deg C produced only traces of methanol (3).
Methanol evolved during cooking of high pectin foods (7) has been accounted
for in the volatile fraction during boiling and is quickly lost to the
atmosphere (49).
Entrapment of these volatiles probably accounts for the elevation in
methanol levels of certain fruits and vegetable products during canning (31, 
33)."
************************************************************* 




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