Urgent - Help - Info about Aflatoxin M1

miller rellim at MAILHOST.TCS.TULANE.EDU
Wed Jan 13 17:20:08 EST 1999

Here are a few references. For more on obtaining toxicological information,
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J Food Prot 1998 Jun;61(6):738-41
Survey of the occurrence of aflatoxin M1 in dairy products marketed in Italy.
Galvano F, Galofaro V, de Angelis A, Galvano M, Bognanno M, Galvano G
Dipartimento di Scienze e Technologie Agroforestali ed Ambientali,
Universita di Reggio Calabria.

During 1995, 159 samples of milk, 97 samples of dry milk for infant
formula, and 114 samples of yogurt were randomly collected in supermarkets
and drug stores in four large Italian cities and checked for aflatoxin M1
(AFM1) by immunoaffinity column extraction and HPLC. AFM1 was detected in
136 (86%) of the milk samples (in amounts ranging from < 1 ng/liter to
108.5 ng/liter; mean level: 10.19 ng/liter), in 81 (84%) of the dry milk
samples (in amounts ranging from < 1 ng/liter to 101.3 ng/kg; mean level:
21.77 ng/kg), and in 91 (80%) of the yogurt samples (in amounts ranging
from < 1 ng/liter to 496.5 ng/liter; mean level: 18.08 ng/liter).
Altogether, only two samples of milk, two samples of yogurt, and one sample
of dry milk had levels of AFM1 exceeding the Swiss legal limits, which are
the most restrictive in the world. AFM1 contamination levels in milk and
yogurt samples collected in the period of November to April were ca. four
times as high as those in samples collected in the period of May to
October. It is concluded that during 1995, despite the widespread
occurrence of AFM1, the mean contamination levels in dairy products sold in
Italy were not a serious human health hazard.
PMID: 9709261, UI: 98374923

Toxicol Appl Pharmacol 1998 Jul;151(1):152-8
Metabolism and toxicity of aflatoxins M1 and B1 in human-derived in vitro
Neal GE, Eaton DL, Judah DJ, Verma A
Medical Research Council Toxicology Unit, University of Leicester,
Leicester, United Kingdom.

Aflatoxin M1 (AFM1) is the principal hydroxylated aflatoxin metabolite
present in the milk of dairy cows fed a diet contaminated with aflatoxin
B1, (AFB1) and the metabolite is also present in the milk of human nursing
mothers consuming foodstuffs containing the toxin. AFM1 is usually
considered to be a detoxification product of AFB1 and this appears
warranted if the biological endpoints involved are carcinogenicity and
mutagenicity. However, it may not be a valid conclusion in the case of
cytotoxicity. The metabolism of AFM1 and AFB1 have been studied in vitro
using human liver microsomes. Formation of primary metabolites associated
with metabolic activation to the respective epoxides reflected the
differences between the carcinogenic potentials of the two toxins and,
similar to AFB1, the conjugation of AFM1 epoxide with reduced GSH was
catalyzed by mouse, but not human liver cytosol. Although the majority of
the binding of [3H]AFB1 to microsomal protein was dependent on metabolic
activation, a high level of retention of [3H]AFM1 by microsomes,
nonextractable in methanol and unrelated to metabolic activation, was
observed. It appears possible that this property is related to the high
cytotoxicity of AFM1. Experiments using human cell line cells either
expressing or not expressing human cytochrome P450 enzymes in assays of
acute toxicity (MTT assays) have demonstrated a directly toxic potential of
AFM1 in the absence of metabolic activation, in contrast to AFB1. Caution
therefore needs to be exercised in designating the formation of AFM1 as
essentially detoxification when considering a biological response in which
cytotoxicity may play a significant role, e.g.,  immunotoxicity. Copyright
1998 Academic Press.
PMID: 9705898, UI: 98372937

Anal Chem 1998 Jun 1;70(11):2366-71
Flow injection monitoring of aflatoxin M1 in milk and milk
preparations using filter-supported bilayer lipid membranes.
Andreou VG, Nikolelis DP
Department of Chemistry, University of Athens, Greece.

This work describes a technique for the rapid and sensitive electrochemical
flow injection monitoring of aflatoxin M1 (AFM1) using stabilized systems
of filter-supported bilayer lipid membranes (BLMs). Injections of AFM1 were
made into flowing streams of a carrier electrolyte solution, and a
transient current signal with a duration of seconds reproducibly appeared
less than 10 s after exposure of the lipid membranes to the toxin. The
magnitude of this signal was linearly related to the concentration of AFM1,
with detection limits at the subnanomolar level. The mechanism of signal
generation was investigated by differential scanning calorimetric
experiments. The technique was applied for the rapid flow injection
determination of AFM1 in milk and milk preparations. The effect of potent
interferences such as proteins and lipids was investigated, and the results
show that interferences from these milk constituents can be eliminated by
modulation of the flow rate of the carrier solution so as not to allow
adsorption of these compounds in BLMs. AFM1 could be determined in
continuous flowing systems with a rate of at least 4 samples min-1.
Repetitive cycles of injection of AFM1 showed no signal degradation during
each cycle.

Food Addit Contam 1997 Jul;14(5):451-6
Occurrence of aflatoxin M1 in commercial pasteurized milk
determined with ELISA and HPLC.
Markaki P, Melissari E
Department of Food Chemistry, University of Athens, Greece.

Eighty-one samples of commercial pasteurized milk from Athens market were
analysed for the presence of aflatoxin M1 (AFM1). A combination of a
commercial ELISA kit and a modified HPLC method was applied for the rapid
and reliable determination of AFM1. AFM1 concentrations in milk extracts
were initially estimated by ELISA. Samples found to contain more than 5
ng/l were further quantitated by HPLC. Determination was performed after
derivatization of AFM1 to its hydroxylated product AFM2a. The recovery of
the HPLC method used was found to be close to 100%. Thirty-two samples
contained aflatoxin M1 at levels of 2.5-5 ng/l, none contained more than 5
ng/l, while 31 contained only traces of aflatoxin (0.5-1 ng/l). In nine
samples no AFM1 was detected. There was no seasonal influence on the
aflatoxin content of the milk samples analysed.
PMID: 9328529, UI: 97469057

Rev Saude Publica 1996 Dec;30(6):542-8
Evaluation of enzyme-linked immunosorbent assay (ELISA) in milk
powder contaminated with known concentrations of aflatoxin M1.
[Article in Portugese]
de Oliveira CA, Germano PM

Departamento de Producao Animal da Faculdade de Medicina Veterinaria e
Zootecnia da Universidade de Sao Paulo, Brasil.

The evaluation of commercially available test systems of competitive
enzyme-linked immunosorbent assay (ELISA), for Aflatoxin M1 (AFM1) was
performed in experimental conditions, through repeated analysis, in samples
of milk powder contaminated with known concentrations of the toxin.
Recoveries of AFM1 added to milk at levels of 0.10, 0.20, 0.50 and 1.00
ng/ml were 83.0%, 87.5%, 103.0% and 111.8% respectively. Relative standard
deviations for the above mentioned concentrations were 65.5%, 31.8%, 10.9%
and 13.6%, respectively (n = 10, per spiking level). According to these
results the ELISA is appropriate for AFM1 research and/or surveying, mainly
for concentrations between 0.20-1.00 ng/ml.
PMID: 9302824, UI: 97448416

Cancer Epidemiol Biomarkers Prev 1997 Jul;6(7):523-9
Use of an improved method for analysis of urinary aflatoxin M1 in a
survey of mainland China and Taiwan.
Cheng Z, Root M, Pan W, Chen J, Campbell TC
Division of Nutritional Sciences, Comell University, Ithaca, New York
14853, USA.

An improved monoclonal antibody immunoaffinity chromatography/high-pressure
liquid chromatography/ fluorescence detection method was developed to
measure aflatoxin (AF) exposure by quantifying AFM1 in human and rat urine
samples. Analysis of different amounts of various AF metabolites showed
that the immunoaffinity resin was highly selective for aflatoxin B1 (AFB1),
AFB2, and AFM1. Recovery of added AFs increased with the amount of
immunoaffinity resin and was virtually complete within the range of 0.01-10
ng of AFM1 by using 7 ml of resin. The detection limit of this method is
0.5 pg/ml urine. Rats dosed with tritiated AFB1 excreted in their urine
tritiated AFM1, among other AF metabolites, as indicated by chemical
derivative confirmation and cochromatography with authentic AFM1 and
agreement of radioactivity and fluorescence quantitation. A linear
dose-response relationship was found over the range of 0.05-50
micrograms/kg of body weight/day. Two humans dosed with 1.0 microgram of
pure AFB1 excreted 6-7% of the dose as urinary AFM1 over 5-7 days. Pooled
urine samples from 30 men from each of 69 rural counties in mainland China
and 16 survey areas in Taiwan, with two villages per county or area, were
analyzed with this improved method (170 villages total). The correlation
coefficient of urinary excretion of AFM1 compared between villages within
all 85 survey areas was 0.50 (P < 0.001). Sixty-five % of the samples
contained detectable concentrations of AFM1 with an average excretion of
3.1 ng/12 h. Assuming an excretion rate of 2-6%, this AFM1 excretion
corresponds to a very low average daily AF consumption of 0.1-0.3
microgram/day (possible range, 0-11 micrograms/day). Patterns of urinary
excretion of AFM1 were similar in mainland China and Taiwan.
PMID: 9232340, UI: 97376168

Food Addit Contam 1997 May-Jun;14(4):341-4
Aflatoxin M1 occurrence in samples of Grana Padano cheese.
Peitri A, Bertuzzi T, Bertuzzi P, Piva G
Istituto di Scienze degli Alimenti e della Nutrizione, Facolta di Agraria
U.C.S.C., Piacenza, Italy.

A total of 223 samples of Grana Padano cheese manufactured in 4 years
(1991-94) by dairies in 11 provinces of the Po valley were checked for
aflatoxin M1. Grated cheese was extracted with chloroform and the defatted
extract was purified by an immunoaffinity column; aflatoxin M1 was
determined by HPLC using a fluorescence detector. From the analysis of the
data it has emerged that only one sample exceeded the maximum tolerated
level in cheese in some European countries (250 ng/kg). Most samples (91%)
were in the range 5-100 ng/kg and only 15 (6.7%) in the range 100-250
ng/kg. notwithstanding a diffuse microcontamination, the situation
regarding the AFM1 levels can be considered fairly satisfactory. Mean
contamination levels of 1992 and 1994 were significantly higher (P < 0.05)
than those of 1993 and1991. No significant difference was observed among
provinces or dairies of origin.
PMID: 9205562, UI: 97349641

Dr. Charles A. Miller III,   rellim at mailhost.tcs.tulane.edu
Dept. Environmental Health Sciences, SL29
Tulane-Xavier Center for Bioenvironmental Research and
Tulane Univ. School of Public Health and Tropical Medicine
1430 Tulane Ave.
New Orleans, LA 70112
(504)585-6942, fax (504)584-1726
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