[Zbrafish] Re: Cyp activity

Tyrone via zbrafish%40net.bio.net (by tgenade from gmail.com)
Fri Jul 4 02:06:55 EST 2008


Hello,

On Jul 2, 8:55 pm, "Ana Schneider" <schneide... from gmail.com> wrote:

> I am looking for a method to evaluate CYP 450 in liver of Zebrafish. Does
> anyone know any work about this?

P450 enzymes, such as CYP3A4, were subject of my masters thesis
(though I worked on steroid synthesizing P450s of the adrenal) which
is accesible at http://tgenade.freeshell.org (you will have to explore
a bit). My thesis includes a protocol for the isolation of microsomal
P450s as well as protocol for P450 concentration estimation and
microsomal P450 enzyme assays in microL quantities.

You will probably need to isolate the enzyme which means you will have
to liquidize large numebers of zebrafish fry. It is possible that the
protocol can be downscaled but much optimization will be needed. You
may want to look into dissecting out embryonic livers (good luck...
you will have to find someone else to help with that, I have no idea
if it is even possible) and then culturing them up, but I do not know
if you will get representative data. I gather you are trying look at
induction of the enzyme under physiological conditions in response to
some environmental insult, suggesting that enzyme isolation may be the
way to go.

You may want to pick the brains of my ex-professor: Piet Swart. His
email is (was?) pswart from sun.ac.za.

The isolation protocol is rather generic, I think your biggest problem
will be scaling the P450 concentration and enzyme assays to meet your
requirments. I used about half a kg of sheep adrenals to get a couple
of mg of P450. I cannot even begin to speculate on how many zebrafish
fry you will need!

Kind regards

Tyrone



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