Monoclonal antibodies (MAbs) are superior reagents for determining the role of specific P450s in endo- and xenobiotic metabolism and activating procarcinogens. In a situation where different P450 isozymes can metabolize the same substrates, MAbs inhibiting the enzymatic activity of specific P450 are uniquely suited for defining the role of an individual P450 in the metabolism of xenobiotics in human tissues which contain multiple P450s. Two panels of inhibitory and non-inhibitory MAbs against h2E1 and h3A4 were prepared and tested for inhibition of metabolism of typical substrates by h2E1 and h3A3/4/5, and for binding activity with various human and rodent P450 isforms. MAb 1-73-18 specific to h2E1 inhibited h2E1-catalyzed metabolism of 5 typical substrates. The inhibition of metabolism by vaccina expressed h2E1 ranged from 70 to 90%. The contribution of P450 h2E1 to total metabolism of 5 substrates in human liver ranged from 25 to 75%. MAb 3-29-9 specific against h3A4 inhibited the P450 h3A3, 3A4, 3A5-catalyzed metabolism of 6 typical substrates. Strong inhibition of metabolism of 5 substrates (73-97%) was observed. The contribution of P450h3A to total metabolism in human liver ranged from 27 to 90%. Both MAbs did not inhibit metabolism of the tested substrates by rat 2E1 and 3A1, respectively. All 17 MAbs against h2E1 and 2 MAbs against h3A4 were antigen specific and did not cross-react with the tested human and rodent P450 isoforms of the heterologous families in ELISA and IB. Selected MAbs against h2E1 bound 2E1 of microsomal fraction of human and acetone- treated rat livers in IB. One MAb against h3A4 cross-reacted in IB with h3A3 but not with h3A5 and thus distinguished between h3A3/4 and h3A5. The results demonstrate that MAbs generated against baculo-expressed P450 h2E1 or h3A4 can be successfully used for the studies on P450 enzymes in human tissues by metabolic inhibition and immunoprecipitation assays.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01CP005711-04
Application #
5201553
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Division of Cancer Epidemiology and Genetics
Department
Type
DUNS #
City
State
Country
United States
Zip Code