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I am currently working with induced rat liver microsomes and I have
difficulties to get a linear relationship when it is incubated with
I am combining this activation system with fish embryos. I want to
after 48h CPA teratogenic effects and lethality on the fish embryos.
I don't know if my incubation conditions are correct!
I am using a "special" metabolic activation system because standard
concentrations & incubation conditions for microsomes activity are too
toxic for the normal development of the fish eggs.
Here it is:
In my test tubes, I have:
1-Tris-HCl buffer 25mM
Preincubation time 5 min with shaking at 125rpm at 32*C
4-Input of the fish embryos
5-Input of 1mM NADPH (final concentration)
6-Incubation time 90 min at 32*C/125rpm
7-Transfer of the fish embryos in fish medium at 26*C for 48h
8- Observation of the malformations
I assumed that my problem comes from the metabolic activation system and
not from the diffusion of the activated metabolites in the embryos. I
increased the shaking, the preincubation time or even changed the vessel
(petri dishes) but it had only an impact on the teratogenicity
nothing on the linearity..I was thinking maybe to replace the NADPH
NADPH regenerating system??
Toxikologie Institute, U9/2111
Frankfurter Str. 250
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First of all, what kind of linearity do you expect from this kind of
If you seek the linear relationship between time and product
formation, then you should determine first if your system performs
under linear conditions without the fish embrios.
1) If it does, then you know that the embrios might "spoil" your
conditions either by uptake of the product, substrate, change in pH,
cofactors etc. Then you should probably change your microsome
incubation volume / fish egg ratio.
2) If your system does not function under linear conditions alone,
then you should try to modify your incubating conditions: try with
shorter incubation time, use NADPH regenerating system [NaHCO3, NADP
+, Glucose-6P, Glucose-6P-dehydrogenase], try carbonate buffer
instead of Tris, as the latter can be toxic to embrios by itself,
maybe try lower microsome concentration etc.
Hope this helps,
Faculty of pharmacy
University in Ljubljana, Slovenia
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