Production of oxidative DNA damage during the metabolic activation of benzo[a]pyrene in human mammary epithelial cells correlates with cell killing.

Abstract

We have studied the generation of reactive oxygen species during the metabolism of a carcinogen, benzo [a]pyrene, by human mammary epithelial cells. We have quantitated the production of one type of oxidative DNA damage, thymine glycols, by using a monoclonal antibody specific to this base modification. Thymine glycols were produced in DNA in a dose-dependent manner after exposure of human mammary epithelial cells to benzo[alpyrene. The number of thymine glycols formed in the DNA was similar to that of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ajpy-rene covalently bound to the DNA. Exposure of cells to the carcinogen in the presence of superoxide dismutase, which reduces superoxide anions, inhibited the production of thymine glycols and increased cell survival but had little effect on adduct formation. At equitoxic doses, =10-fold more thymine glycols were formed after exposure to benzo[alpyrene than to yirradiation. Thymine glycols, produced by either agent, were efficiently removed from the DNA of the cells. Since thymine glycols represent only a portion of the oxidative damage possibly produced, our results indicate that the total amount of oxidative damage induced during the exposure of human mammary epithelial cells to benzo [a]pyrene greatly exceeds the amount produced by direct adduct formation and that this indirect damage plays an important role in the cytotoxicity ofgens, it is important to identify the primary DNA lesions produced directly by adduct formation and indirectly by free radicals.One product that is formed in DNA as a consequence of chemical oxidation or ionizing radiation is thymine glycol (13,14). A sensitive immunoassay for thymine glycols has been developed with a monoclonal antibody to this product in DNA (15). By using this methodology, thymine glycols have been detected in DNA after treatment of cultured human fibroblasts with ionizing radiation, 254-nm ultraviolet light, and an active metabolite of the carcinogen 2-naphthylamine (16,17). We have used this method to determine whether reactive oxygen species are being produced during the metabolism of B[a]P by HMEC and what role these reactive oxygen species play in cellular toxicity.Our results show that: (i) thymine glycols are produced in DNA in a dose-dependent manner at levels comparable to the amount of B[a]P diol epoxide covalently bound to DNA adducts after exposure of HMEC to B[a]P; (ii) the production of thymine glycols, but not that of B[a]P adducts, is reduced if superoxide dismutase (SOD) is present at the time of treatment; (iii) survival is increased if SOD is present during treatment with B[a]P; (iv) at equitoxic doses, 10-fold more thymine glycols are produced after B[a]P treatment than after ionizing radiation.There is increasing evidence implicating the involvement of free radicals, particularly those derived from molecular oxygen, in many stages of chemical carcinogenesis (1, 2). Active oxygens and the ensuing lipid peroxidations could affect carcinogenic processes in at least two ways: (i) by causi...

Evidence for the aldo-keto reductase pathway of polycyclic aromatic trans -dihydrodiol activation in human lung A549 cells

“…PAHs have previously been reported to cause oxidative DNA damage in vitro and in vivo when using methods of varying sensitivity and specificity (32,33). Base modifications such as thymine glycol, etheno adducts, and 8-oxo-dGuo all have been detected upon PAH exposure (34).…”

Formation of 8-Oxo-7,8-dihydro-2‘-deoxyguanosine (8-Oxo-dGuo) by PAH o-Quinones: Involvement of Reactive Oxygen Species and Copper(II)/Copper(I) Redox Cycling

“…The types of DNA damage detected upon PAH exposure were 8-oxo-dGuo, thymine glycol and etheno adducts (23)(24)(25)(26). Irrespective of the final lesion the mechanism by which PAH cause this oxidative damage is uncertain.…”

“…Parent PAH and PAH trans-dihydrodiols have been shown to cause oxidative DNA damage in vitro and in vivo (23)(24)(25)(26)(27)(28). The AKR pathway of PAH activation provides a route to ROS generation and ROS-mediated DNA lesions seen with PAH (Scheme 1).…”

Oxidative Stress Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin. An Application of Oxidative Stress Markers to Cancer Risk Assessment of Dioxins.

“…Paraquat is metabolized by NADPH cytochrome P-450 reductase to a reduced form, which is a paraquat radical. 57) . CYP1A1 and 1A2 show their highest activity on conversion of polycyclic aromatic hydrocarbons, such as B[a]P, to diol metabolites 53) .…”