Rapidly growing primary cultures of normal human mammary epithelial cells (HMEC) were exposed to 1 jig of benzo [a]pyrene (B[a]P) per ml for two or three 24-hr periods. The B[a]P-treated populations consistently contained cells displaying a longer period of active growth in culture compared to the untreated control cells. Widespread heterogeneity in morphology and growth patterns was evidenced in these "extended life" (EL) cultures, with multiple sequential changes in these parameters occurring during the course of their life in culture. Two apparently immortal continuous cell lines have thus far emerged from these EL cultures. These lines have been characterized to be of human mammary epithelial origin and derived from the originally treated HMEC specimen. The continuous lines do not appear to be malignantly transformed as they do not cause tumor formation in nude mice and show little or no anchorage-independent growth. Nonetheless, they have acquired several properties characteristic of tumor-derived HMEC, which distinguish them from their normal progenitors. These cell lines, as well as the EL strains, may provide useful substrates for studies to determine what agents can induce further transforming events. Additionally, analysis of the multiple steps occurring in the EL cultures, as well as in the emergence of the continuous cell lines, could potentially elucidate the processes occurring during human epithelial cell carcinogenesis and escape from senescence. Establishment of methods

No 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...

No abstract