Use of monoclonal antibodies to characterize the induction response of the cytochrome P-450-dependent mixed function oxidase system to nitrofluoranthenes.
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 greater than 3-NF greater than 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 P-450 (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 (approximately 80%) AHH activity induced by 3-NF and NFs. On the other hand FL-induced AHH activity was only moderately (approximately 30%) inhibited by MAb 1-7-1 whereas higher inhibition (approximately 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.
Khan
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WA
Department of Dermatology, University Hospitals of Cleveland, Case Western Reserve University, OH.
Asokan
P
P
Park
S S
SS
Gelboin
H V
HV
Bickers
D R
DR
Mukhtar
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis; D001564:Benzo(a)pyrene / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D005449:Fluorenes / Q000494:pharmacology*; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010105:Oxygenases / Q000096:biosynthesis*; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D002273:Carcinogens; D005449:Fluorenes; D001564:Benzo(a)pyrene; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C033643:3-nitrofluoranthene