Use of monoclonal antibodies to identify cytochrome P450 isozymes in rat liver microsomes that hydroxylate N-nitrosomethylamylamine at each of six positions.
Inhibition of enzyme activity by monoclonal antibodies (MAbs) was used to indicate which cytochrome P450 isozymes in Sprague-Dawley rat liver microsomes catalyse hydroxylation of the oesophageal carcinogen N-nitrosomethyl-n-amylamine (NMAA) to give 2- to 5-hydroxy-NMAA (HO-NMAA), formaldehyde and pentaldehyde. Liver microsomes (0.3-0.6 mg protein) were incubated (15 min, 23 degrees C) with 0.4 mg MAb and, after adding NMAA to 6 mM, incubated for 20 min at 37 degrees C. Mixtures were analysed for HO-NMAAs by gas chromatography-thermal energy analysis and for aldehydes by high-performance liquid chromatography of their 2,4-dinitrophenylhydrazones. The percentage inhibition by each MAb indicates the percentage metabolism by the corresponding P450 isozyme(s). These results indicate that the MAb to P450 IIB1 cross-reacts with P450 IIE1 and that the MAb to male-specific constitutive IIC11 cross-reacts with female-specific IIC12. Taking this into account, the main results were as follows. With uninduced male microsomes, 4-hydroxylation was catalysed mainly by IIC11 and demethylation by IIC11 and IIE1. With uninduced female microsomes, P450s reacting with the MAb to IIC11 (probably mainly IIC12) were responsible for most of the 4-hydroxylation and demethylation. With 3-methylcholanthrene-induced male microsomes, most 3-hydroxylation and some depentylation were due to IA1 or IA2. With phenobarbital-induced microsomes, all six reactions, but especially 4-hydroxylation and depentylation, were largely due to IIB1. With Aroclor-induced microsomes, all six reactions were catalysed by IIB1 and IA1 or IA2. The role of P450 IIC11 in 4-(omega-1)-hydroxylation was striking.
Mirvish
S S
SS
Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha.
Ji
C
C
Huang
Q
Q
Wang
S
S
Park
S S
SS
Gelboin
H V
HV
D000818:Animals; D000911:Antibodies, Monoclonal; D002273:Carcinogens / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000276:immunology; D004790:Enzyme Induction; D005260:Female; D006900:Hydroxylation; D007527:Isoenzymes / Q000032:analysis* / Q000276:immunology; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009602:Nitrosamines / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D002273:Carcinogens; D007527:Isoenzymes; D009602:Nitrosamines; C025033:N-amyl-N-methylnitrosamine; D003577:Cytochrome P-450 Enzyme System