Biochemical and Molecular Characterization of Beagle Dog CYP4A Public Deposited

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  • March 20, 2019
  • Graham, Richard Alan
    • Affiliation: Eshelman School of Pharmacy
  • The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely -naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n = 3) with -naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with -naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9- fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase (CYP2E1) activity in vitro. Rifampin caused a 13-fold induction of testosterone 6 - hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results 40 suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats. (Abstract from Chapter 2) Compared to other species, little information is available on the xenobiotic-induced regulation of cytochrome P450 enzymes in the beagle dog. Dogs are widely used in the pharmaceutical industry for many study types, including those that will impact decisions on compound progression. The purpose of this study was 1) to determine the temporal kinetics of drug-induced changes in canine CYP1A, CYP2B, and CYP3A mRNA and enzymatic activity, and 2) to characterize concentration-response relationships for CYP1A2, 2B11 and 3A12 using primary cultures of canine hepatocytes treated with -naphthoflavone (BNF), Phenobarbital (PB) and Rifampin (RIF), respectively. CYP1A1 and CYP1A2 mRNA exhibited maximal expression (12,700-fold and 206-fold, respectively) after 36 hr of treatment with BNF. PB treatment, but not RIF treatment, caused maximal induction of CYP2B11 mRNA (149-fold) after 48 hr of treatment. CYP3A12 and CYP3A26 mRNA levels were increased maximally after 72 hr of treatment with PB and RIF (CYP3A12, 35- fold and 18-fold, CYP3A26, 72-fold and 22-fold with PB and RIF treatment, respectively). Concentration-response relationships for BNF induced 7-ethoxyresorufin O-dealkylation (EROD) (EC50=7.8 4.2 mu-M), PB induced 7-benzyloxyresorufin O-dealkylation (BROD) (EC50=123 30 mu-M) and PB and RIF induced testosterone 6 -hydroxylation (EC50=132 28 ?M and EC50=0.98 0.16 ?M) resembled the relationship for human CYP induction compared to that of rodent. Interestingly, RIF had no effect on CYP2B11 expression, which represents a species difference overlooked in previous investigations. Overall, the induction of dog CYP1A, CYP2B and CYP3A exhibits characteristics that are intermediate to those of rodent and human. (Abstract from Chapter 3) In addition to its function as a fatty acid hydroxylase the peroxisome proliferatoractivated receptor (PPAR ) target gene, CYP4A, has been shown to be important in conversion of arachidonic acid to the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE), suggesting a role for this enzyme in mediating vascular tone. In the present study the cDNA sequence of beagle dog CYP4A37, CYP4A38 and CYP4A39 from liver was determined. Open reading frame analysis predicted that CYP4A37, CYP4A38 and CYP4A39 are each comprised of 510 amino acids with ~90% sequence homology, and ~71% and ~78% homology to rat CYP4A1 and human CYP4A11, respectively. PCR analysis revealed that the three dog CYP4A isoforms are expressed in kidney>liver>>lung>>intestine>skeletal muscle>heart. Treatment of primary dog hepatocytes with the PPAR agonists GW7647X and clofibric acid resulted in an increase in CYP4A37, CYP4A38 and CYP4A39 mRNA expression (up to 4-fold), whereas HMG-CoA synthase mRNA expression was increased to a greater extent (up to 10-fold). These results suggest that dog CYP4A37, CYP4A38 and CYP4A39 are expressed in a tissue dependent manner and that beagle dog CYP4A is not highly inducible by PPAR agonists, similar to the human CYP4A11 gene. (Abstract from Chapter 4)
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  • LeCluyse, Edward
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