The role of cytochrome b5 and P450 oxidoreductase in the cytochrome P450 mediated activation of the carcinogenic compounds benzo[a]pyrene and ellipticine

Abstract

Benzo[a]pyrene (BaP), an environmental pollutant, and ellipticine, an anticancer drug, are carcinogens that exert genotoxic effects after metabolic activation by cytochrome P450 enzymes. In the present study two mouse models, the Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) and the cancer cell lines Hepa-1c1c7 and HepG2 have been used to study the role of P450-mediated metabolism and disposition of BaP and ellipticine. In HRN mice cytochrome P450 oxidoreductase (POR), the electron donor to P450 enzymes, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b5 (Cyb5), which can also act as an electron donor from cytochrome b5 reductase to P450 enzymes, is also deleted in the liver. HRN and HBRN mice were treated once with BaP or ellipticine for 24 hours. DNA adduct levels were measured by ³²P-postlabelling analysis. Mice treated with ellipticine had significantly fewer DNA-adducts in the livers on HRN and HBRN mice compared to WT, with no significant difference between HRN and HBRN. In the mice treated with BaP, however, there were significantly higher levels of DNA adducts in the livers of HRN mice than wild-type (WT) mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Hepatic microsomal fractions were also incubated with BaP/ellipticine and DNA in the presence of NAD(P)H in vitro which showed as electron donors were lost, adduct formation decreased. This correlated with incubations carried out with BaP or ellipticine which measured formation of metabolites which decreased as electron donors were lost. The results from the incubations also correlated with measured enzyme activity. In order to model the paradoxical phenomenon seen in the HRN mice, Hepa-1c1c7 and HepG2 cells that do not express POR were exposed to BaP. In the Hepa-1c1c7 cells, despite the absence of POR expression, both formation of metabolites, DNA damage (comet assay) and adduct formation was increased compared to WT cells. The HepG2 cell line showed decreased levels of DNA damage (comet assay) in the absence of POR compared to WT cells. Whilst formation of metabolite and adduct formation was reduced compared to WT HepG2 cells 24 hours after treatment with BaP, metabolite and adduct formation was higher in POR KO HepG2 cells compared to WT cells after 48 hours.

Date of Award	1 Apr 2019
Original language	English
Awarding Institution	King's College London
Supervisor	Volker Arlt (Supervisor) & David Phillips (Supervisor)