Epoxide hydratase: sex specific expression and rate-limiting role in DMBA metabolism

The rate of generation of the proximate carcinogen 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol (DMBA 3,4-diol) from DMBA, was 3-fold lower in rat liver microsomes (RLM) from female, compared to male Sprague-Dawley rats. However, the sum of products that potentially derive from the common intermediate DMBA 3,4-oxide namely, DMBA 3,4-diol, 3- and 4-hydroxy DMBA, was comparable between the two sexes (18 versus 20 pmol/mg/min). Addition of purified microsomal epoxide hydratase (EHm) (150 nM) to female RLM increased the rate of DMBA 3,4-diol formation to a level comparable to that obtained in male RLM. This activity was not increased when equivalent amounts of EHm were added to male RLM. Female RLM contained 2-fold lower levels of EHm protein compared to male RLM, based on Western blot analyses, and exhibited correspondingly lower hydrating activities towards both benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) and DMBA 5,6-oxide. The rate of DMBA 3,4-diol formation was also limited by the availability of EHm in lung and adrenal microsomes from male rats. Addition of exogenous EHm to both tissues stimulated DMBA 3,4-diol formation by 2 and 6-fold respectively. EHm activity was still more deficient in the formation of the K-region, DMBA 5,6-diol, and this limitation was seen in both female and male-RLM as well as in microsomes from extrahepatic tissues. These limiting effects were enhanced by 3-methylcholanthrene treatment, through increases in DMBA monooxygenase but not EHm activities, but were diminished by phenobarbital treatment where EHm was also induced. Manipulation of EHm activity (through addition of pure EHm or its inhibitor trichloropropylene oxide) demonstrated a direct relationship between EHm activity and total DMBA metabolism. A deficiency in EHm apparently caused a selective loss of products resulting from initial DMBA 5,6- and 8,9-monooxygenation. Substantial reduction of DMBA 5,6-oxide to DMBA was measured in RLM under anaerobic conditions. This reduction was inhibitable by ambient oxygen levels. These data indicate that insufficient cellular EHm levels result in the reversion of microsomally generated DMBA 5,6-oxide (and 8,9-oxide) back to DMBA and also lead to reduced activation of DMBA via the carcinogenic bay region diol epoxide.