Role of cytochrome P-450IA1 in the metabolism and binding to DNA of benzo(a)pyrene

Jeanmarie Kern Eberhart, Purdue University

Abstract

The proportion of BaP converted to a highly carcinogenic metabolite, (+)-anti-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxide-7,8,9,$ 10$,-tetrahydrobenzo(a)pyrene ((+)-anti-BaPDE) increases with time of exposure in cell cultures from a number of species. These studies were designed to examine the role of cytochrome P450IA1 in formation of (+)-anti-BaPDE in hepatocyte cultures. Pretreatment of primary rat hepatocytes in culture or a human hepatoma cell line (Hep G2) with BaP increased the amounts of ($\sp3$H) BaP metabolized to water-soluble metabolites bound to DNA, and the (+)-anti-BaPDE bound to deoxyguanosine. To examine the role of specific P450 isozymes in activation of BaP to DNA binding metabolites, inhibitory monoclonal antibodies against certain P450 isozymes were incubated with microsomes from acetone- and BaP-treated primary rat hepatocytes or Hep G2 cells in culture. The amount of BaP metabolized to water-soluble and DNA-binding metabolites was increased four-fold and twenty-eight-fold in microsomes from BaP-treated primary rat hepatocytes or Hep G2 cells respectively, compared to microsomes from acetone-treated cells. BaP metabolism, BaP DNA-binding and the amount of (+)-anti-BaPDE bound to DNA were decreased to the control level in the presence of a cytochrome P450IA1-inhibitory monoclonal antibody. Western blot analysis demonstrated the presence of cytochrome P450IA1 in microsomes from cells pretreated with BaP but not in those from acetone-pretreated cells. To selectively inhibit BaP metabolism in intact cells, attempts were made to insert the cytochrome P450IA1-inhibitory monoclonal antibody directly into cells using two techniques: the osmotic lysis of pinocytic vesicles and electroporation. Neither approach was successful. In primary rat hepatocytes in culture and Hep G2 cell cultures, treatment with BaP induces cytochrome P450IA1 which increases the metabolism of BaP and metabolizes a higher proportion of the BaP to (+)-anti-BaPDE. These studies demonstrate that the time-dependent increase in the proportion of BaP bound to DNA through the (+)-anti-BaPDE results from induction of P450IA1 by the BaP.

Degree

Ph.D.

Advisors

Baird, Purdue University.

Subject Area

Biochemistry|Molecular biology

COinS