Induction of Cyp1a-1 and Cyp1a-2 gene expression by a reconstituted mixture of polynuclear aromatic hydrocarbons in B6C3F1 mice

The potential non-additive interactions of polynuclear aromatic hydrocarbon (PAH) mixtures as inducers of Cyp1a-1 and Cyp1a-2 gene expression were investigated in B6C3F1 mice using a reconstituted PAH mixture. The chemical composition (% by weight) of the reconstituted PAH mixture was: 2-ring PAHs - indan (0.22), naphthalene (23.8), 2-methylnaphthalene (23.2) and 1-methylnaphthalene (13.3); 3-ring PAHs - acenaphthylene (7.7), acenaphthene (0.6), dibenzofuran (0.7), fluorene (4.3), phenanthrene (10.5) and anthracene (3.4); ≥4-ring PAHs - fluoranthene (2.4), pyrene (4.3), benz[a]anthracene (1.4), chrysene (1.5), benzo[b]fluoranthene (0.8), benzo[k]fluoranthene (0.9) and benzo[a]pyrene (0.9). The composition of the 2-, 3- and ≥4-ring PAH fractions were based on the relative concentration of individual PAHs as noted above. The ≥4-ring PAH fraction and reconstituted mixture induced hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity and Cyp1a-1 mRNA levels, whereas the 2- and 3-ring PAHs were only weakly active. Direct comparison of the potencies of the reconstituted mixture and ≥4-ring PAHs showed that the Cyp1a-1 induction activity of the reconstituted mixture was due to the ≥4-ring PAHs. The reconstituted PAH mixture and ≥4-ring PAHs also induced Cyp1a-2 hepatic mRNA levels and microsomal methoxyresorufin O-deethylase (MROD) activity; however, their dose-response curves indicated that the reconstituted PAH mixture was more potent as a Cyp1a-2 inducer than the ≥4-ring PAHs. The differences in potency were due to 3-ring PAHs which were found to be strong inducers of hepatic Cyp1a-2 mRNA levels and microsomal MROD activity at the lowest dose administered (37 mg/kg). The 3-ring mixture caused a maximal 29-fold increase in hepatic MROD activity at a dose of 292 mg/kg, but only 28% of maximal induction of EROD activity. Northern analysis of liver mRNA from mice treated with 3-ring PAHs showed that there was minimal induction of Cyp1a-1 mRNA levels. The 3-ring PAHs did not competitively bind to the mouse hepatic cytosolic aryl hydrocarbon (Ah) receptor suggesting that 3-ring PAHs are a new class of Cyp1a-2 inducers which do not act through the Ah receptor.