Lindey, Susannah; (1997) Isolation, characterization and induction studies of human cytochromes P450. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Animal models, particularly rodents, are used for biotransformation studies. Marmosets may provide a better model as they are more closely evolutionarily linked to man. A human CYP2A6 and three marmoset CYP2A cDNA clones were isolated from their respective liver cDNA libraries. The DNA sequences of the three marmoset CYP2As were 92% identical to the human CYP2A6 DNA sequence whereas the predicted protein sequences were 88% identical. In experimental animals anti-epileptic drugs, such as phenobarbital and valproic acid, lead to increased expression of CYPs. It has been shown using western blot analysis with polyclonal antibodies to CYP2As, CYP2B6, CYP2Cs, CYP3As and NADPH-cytochrome P450-reductase that both these drugs induce the expression of the above enzymes in cultured human hepatocytes derived from two different individuals. The anti-epileptic agents induced the expression of these enzymes after four and seven days (three and six days of treatment with the drugs, respectively) of culture. The antibodies used in this study recognise all isoforms of each subfamily and were mainly raised to non-human CYPs. The major aim of this thesis was to produce monoclonal antibodies to two human CYPs, CYP2A6 and CYP3A4, using an adaptation of the recombinant phage antibody system. These CYPs were purified from insect cell microsomes and injected separately into mice. Antibody ScFv cDNA libraries were constructed from mice spleen mRNA using an RT-PCR based cloning strategy. The antibodies were expressed in E. coli using a flexible phagemid vector that permitted either phage-displayed or soluble antibody expression. Soluble antibodies that recognised a CYP3A4 antigen preparation were isolated from the CYP3A4 ScFv antibody library by a two-membrane immunodetection screen.

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