Phenotyping of cytochromes P -450 in human tissues with monoclonal antibodies

Abstract

Cytochrome P450 (P450)-dependent aryl hydrocarbon hydroxylase (AHHase) and 7-ethoxycoumarin deethylase (ECDEtase) in human tissues were differentially inhibited by monoclonal antibodies (MAbs) that were prepared to inhibit and completely inhibited the activity of3-methylcholanthrene-induced rat liver P-450. The AHHase and ECDEtase of placentas from individual women who smoked were inhibited by the MAbs by 83-90% and by 34-74%, respectively. Benz [a]anthracene (BaA)-induced AHHase and ECDEtase in lymphocytes were inhibited 18-65% and 30-78%, respectively. The enzymes in both control and BaA-induced human cells in culture were inhibited to different extents. Both the AHHase and ECDEtase in control and BaAinduced monocytes and in normal liver were largely unaffected by the MAb. Thus, we have with the MAbs: (i) identified P-450s with a common antigenic site in placenta, lymphocytes, and human cells in culture; (ii) identified two forms of hydrocarbon-induced P450s in lymphocytes, at least one of which is common with the induced P450s of placenta and with a P-450 form present in uninduced lymphocytes; (iii) identified two forms of P-450 responsible for smoking-induced ECDEtase activity in placenta, one of which is also responsible for AHHase activity; (iv) shown that the P-450s of liver, basal, and BaA-induced monocytes are different from the MAb-sensitive P-450s of placenta and lymphocytes; (v) quantitated in several human tissues the percentages of control and inducible AHHase and ECDEtase that are dependent on the MAb-sensitive P-450; and (vi) defined by HPLC the contribution of the MAb-sensitive P-450 to the formation of specific benzo[a]-pyrene metabolites. The results demonstrate the value of MAbs for defining antigenic site relatedness for different enzymatic functions ofP450s and for identifying and quantifying the amount of a specific enzyme activity in a tissue dependent on specific P450s. This study may be a prototype for the use of MAbs for phenotyping and mapping of P-450s responsible for specific metabolic reactions and, thus, may be useful in determining the relationship of P450 phenotype to individual differences in drug metabolism and carcinogen susceptibility.The mixed-function oxidases containing cytochromes P450 metabolize a variety ofxenobiotics and endogenous compounds, including carcinogens, drugs, and steroids (1, 2). The P-450s govern reactions that lead to detoxified metabolites or to toxic, mutagenic, and carcinogenic compounds (1, 2). The P-450s convert benzo[a]pyrene (BaP) to an active form that is covalently bound to DNA (3). The latter system activates xenobiotics to their mutagenic forms in the Ames assay (4). Xenobiotic activation and detoxification may occur through alternative metabolic pathways regulated by different P-450s with overlapping specificities. Thus, BaP is converted by P-450s and linked enzymes, to >40 metabolites (1). BaP metabolism resulting in activation involves P450-catalyzed epoxidation, followed by formation of (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[...

Coumarin 7‐hydroxylase activity in human liver microsomes. Properties of the enzyme and interspecies comparisons.

“…Another explanation is that there are more or less marked differences in isozyme composition between man and rodents. There are immunochemical similarities between some rodent and human P-450 isozymes (Fujino et al, 1982), but at least thus far it has been very difficult to find really analogous isozymes in human and rodent livers (Wang et al, 1983). On this basis one is easily led to a conclusion that qualitative and quantitative interspecies differences make it very difficult to extrapolate from animal species to man.…”

The immunocytochemical localisation and distribution of cytochrome P‐ 450 in normal human hepatic and extrahepatic tissues with a monoclonal antibody to human cytochrome P‐450.

“…Cytochrome P-450 has previously been identified and partially purified in human polymorphonuclear leucocytes (Mungikar & Gothoskar, 1986;Shak & Goldstein, 1984. Cytochrome P-450 has not previously been identified in human bone marrow, although cytochrome P-450 associated monooxygenase activity has been demonstrated in cultured human sternal marrow cells (Fujino et al, 1982). Cytochrome P-450 recognised by MAb-HL3 was not present in platelets, monocytes or lymphocytes, although cytochrome P-450 or cytochrome P-450 associated activity has been described in these human cell types Figure 4 Immunostaining of a group of myeloid cells with MAb-HL3 showing strong cytoplasmic staining (arrow identifies a myeloid cell).…”

“…(x 340, scale bar represents 25 ,um.) (Cinti & Feinstein, 1976;Haurand & Ullrich, 1985;McLemore et al, 1978;Burke et al, 1977;Fujino et al, 1982).…”

The effect of cigarette smoking on 7‐ethoxyresorufin O‐deethylase and other monooxygenase activities in human liver: analyses with monoclonal antibodies.

“…As a control, an ascites fluid (NBS 1-48-5) containing non-specific IgG was used. The preparation, characterization and properties of the monoclonal antibodies have been previously described Fujino et al, 1982;. The MAb 1-7-1 inhibits the AHH and ECDE activities of the purified MC-induced cytochrome P-450 by more than 90%, while not inhibiting the same activities of the purified PB-induced cytochrome P-450 .…”