In vitro metabolism and covalent binding of ethylbenzene to microsomal protein as a possible mechanism of ethylbenzene-induced mouse lung tumorigenesis

This study was conducted to determine species differences in covalent binding of the reactive metabolites of ethylbenzene (EB) formed in the liver and lung microsomes of mouse, rat and human in the presence of NADPH. These data further the understanding of the mechanism by which EB causes mouse specific lung toxicity and a follow-up to our earlier report of the selective elevation, although minor, of the ring-oxidized reactive metabolites in mouse lung microsomes (Saghir et al., 2009). Binding assays were also conducted with or without 5-phenyl-1-pentyne (5P1P), an inhibitor of CYP 2F2, and diethyldithiocarbamate (DDTC), an inhibitor of CYP 2E1 to evaluate their role in the formation of the related reactive metabolites. Liver and lung microsomes were incubated with 14C-EB (0.22 mM) in the presence of 1 mM NADPH under physiological conditions for 60 min. In lung microsomes, binding activity was in the order of mouse (812.4 ± 102.2 pmol/mg protein) >> rat (57.0 ± 3.2 pmol/mg protein). Human lung microsomes had little binding activity (15.7 ± 1.4 pmol/mg protein), which was comparable to the no-NADPH control (9.9-16.7 pmol/mg protein). In liver microsomes, mouse had the highest activity (469.0 ± 38.5 pmol/mg protein) followed by rat (148.3 ± 14.7 pmol/mg protein) and human (89.8 ± 3.0 pmol/mg protein). Presence of 5P1P or DDTC decreased binding across species and tissues. However, much higher inhibition was observed in mouse (86% [DDTC] and 89% [5P1P]) than rat (56% [DDTC] and 59% [5P1P]) lung microsomes. DDTC showed ∼2-fold higher inhibition of binding in mouse and human liver microsomes than 5P1P (mouse = 85% vs. 40%; human = 59% vs. 36%). Inhibition in binding by DDTC was much higher (10-fold) than 5P1P (72% vs. 7%) in rat liver microsomes. These results show species, tissue and enzyme differences in the formation of reactive metabolites of EB. In rat and mouse lung microsomes, both CYP2E1 and CYP2F2 appear to contribute in the formation of reactive metabolites of EB. In contrast, CYP2E1 appears to be the primary CYP isozyme responsible for the reactive metabolites of EB in the liver.