title: Retinal pigment epithelial cell DNA is damaged by exposure to benzo[a]pyrene, a constituent of cigarette smoke.
abstract: This study examined the effect of exogenous benzo[ a ]pyrene (BaP), an important constituent of cigarette smoke, on cultured bovine retinal pigment epithelial (RPE) cells. Evidence is presented for its metabolic conversion into benzo[ a ]pyrene diol epoxide (BPDE) and the consequent formation of potentially cytotoxic nucleobase adducts in DNA. Cultured RPE cells were treated with BaP at concentrations in the range of 0-100 microM. The presence of BaP was found to cause inhibition of cell growth and replication. BaP induced the expression of a phase I drug metabolizing enzyme which was identified as cytochrome P450 1A1 (CYP 1A1) by RT-PCR and by Western blotting. Coincident with the increased expression of CYP 1A1, covalent adducts between the mutagenic metabolite BPDE and DNA could be detected within RPE cells by immunocytochemical staining. Additional support for their formation was afforded by nuclease P1 enhanced (32)P-postlabelling assays on cellular DNA. Single-cell gel electrophoresis (comet) assays showed that exposure of RPE cells to BaP rendered them markedly more susceptible to DNA damage induced by broad band UVB or blue light laser irradiation. In the case of UVB, this is consistent with the photosensitization of DNA cleavage by nucleobase adducts of BPDE. Collectively, these findings imply that BaP has a significant impact on RPE cell pathophysiology and suggest mechanisms whereby exposure to cigarette smoke might cause RPE dysfunction and cell death, thus possibly contributing to degenerative disorders of the retina.
authors.affiliation: Department of Ophthalmology and Vision Science, Queen's University of Belfast, Northern Ireland, UK.
authors.forename: William P
authors.initials: WP
authors.lastname: Patton
authors.forename: Michael N
authors.initials: MN
authors.lastname: Routledge
authors.forename: George D
authors.initials: GD
authors.lastname: Jones
authors.forename: Sheena E M
authors.initials: SE
authors.lastname: Lewis
authors.forename: Desmond B
authors.initials: DB
authors.lastname: Archer
authors.forename: R Jeremy H
authors.initials: RJ
authors.lastname: Davies
authors.forename: Usha
authors.initials: U
authors.lastname: Chakravarthy
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000494:pharmacology*; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism; D018736:DNA Adducts / Q000187:drug effects; D004249:DNA Damage; D008027:Light; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics; D012906:Smoke; D014026:Nicotiana; D014466:Ultraviolet Rays
chemical_list: D018736:DNA Adducts; D012333:RNA, Messenger; D012906:Smoke; D001564:Benzo(a)pyrene; D019363:Cytochrome P-450 CYP1A1