Use of monoclonal antibodies to characterize the induction response of the cytochrome P-450-dependent mixed function oxidase system to nitrofluoranthenes

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Abstract
In prior studies with neonatal rats we have suggested that nitrated polycyclic aromatic hydrocarbons (NPAH) are 3-methylcholanthrene (3-MC) type of inducers of cytochrome P-450. These observations have been extended by studying the effect of fluoranthene (FL) and its nitrated derivative, 3-nitrofluoranthene (3-NF) and a mixture of nitrated fluoranthenes (NFs) on the induction of hepatic and pulmonary monooxygenase activities in adult rats. We have characterized the effect of these compounds on hepatic cytochrome P-450 isozyme(s) using immunoblot analysis. The administration of 3-NF and NFs to rats resulted in highly significant induction (1.9- to 5.8-fold) of hepatic and pulmonary aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin-O-deethylase (ERD) activities. FL was either ineffective or much less effective in inducing these enzyme activities. The enzyme induction response to these compounds occurred in the following order: NFs > 3-NF > FL. SDS-PAGE of hepatic microsomes prepared from FL-, 3-NF- and NFs-treated animals revealed a higher content of protein migrating in the P-450 region. Characterization of isozymes of P-450 was carried out by Western blot analysis with highly specific monoclonal antibodies (MAb) raised against 3-MC-specific P-450 (MAb 1-7-1) and phenobarbital-specific P450 (MAb-2-66-3) isozymes. Hepatic microsomes prepared from 3-NF- and NFs-treated rats showed two distinct immunoprecipitin bands with MAb 1-7-1 whereas microsomes prepared from FL-treated animals showed a sharp band with MAb 2-66-3. Mab 1-7-1 significantly inhibited (.apprx. 80%) AHH activity induced by 3-NF and NFs. On the other hand FL-induced AHH activity was only moderately (.apprx.30%) inhibited by MAb 1-7-1 whereas higher inhibition (.apprx.60%) was observed with MAb 2-66-3. Analysis of BP metabolites by h.p.l.c. revealed enhanced production of metabolites by liver microsomes from 3-NF- and NFs-treated animals. The formation of BP 7,8-diol was 1.8- to 2.4-fold increased following treatment of animals with 3-NF and NFs respectively. Addition of MAb 1-7-1 to a microsomal mixture from 3-NF- and NFs-treated rats inhibited the formation of BP phenols (60-75%) and BP 7,8 diol (52-60%). These inhibitory effects were not observed with microsomes prepared from FL-treated rats. These studies suggest that NFs induce specific monooxygenases in liver and that they are inducers of P-450 isozymes c and d.