PharmPK Discussion - Rat liver microsomes

• On 18 Apr 2007 at 10:08:58, francois.busquet.aaa.merck.de sent the message
  

  
  The following message was posted to: PharmPK
  
  Dear all,
  I am currently working with induced rat liver microsomes and I have
  difficulties to get a linear relationship when it is incubated with
  cyclophosphamide (CPA).
  I am combining this activation system with fish embryos. I want to
  detect
  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
  2-microsomes (0.35mg/ml)
  3-CPA
  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
  severity and
  nothing on the linearity..I was thinking maybe to replace the NADPH
  by an
  NADPH regenerating system??
  Greetings,
  francois busquet
  
  --
  Francois BUSQUET
  Merck Serono
  Toxikologie Institute, U9/2111
  Frankfurter Str. 250
  D-64271 Darmstadt
  
  

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• On 18 Apr 2007 at 18:22:53, "Trontelj Jurij" (Jurij.Trontelj.at.ffa.uni-lj.si) sent the message
  

  
  Dear Francois,
  
  First of all, what kind of linearity do you expect from this kind of
  experiment ?
  
  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,
  Kind regards,
  
  Jurij Trontelj
  Faculty of pharmacy
  University in Ljubljana, Slovenia
  
  

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