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The role of metabolic activation of chemical precarcinogens in the induction of DNA damage and repair in mammalian cells

University of British Columbia

It has been proposed that DNA alterations may be involved in the "early" stages of chemical carcinogenesis. The relationship between metabolic activation of chemical precarcinogens, DNA damage induced by reactive carcinogenic metabolites, and repair of the damaged DNA was therefore investigated in cultured mammalian cells and in the intact animal. Attention was also focused on the relationship of these events to organ specificity of tumor induction by chemical precarcinogens. Synthetically prepared metabolites of the precarcinogen 2-acetyl aminofluorene (AAF) - the proximate carcinogen N-hydroxy-2-acetyl aminofluorene (N-hydroxy-AAF), and the ultimate carcinogen N-acetoxy-2-acetyl aminofluorene (N-acetoxy-AAF) - were used to investigate the relationship between the stage of metabolic activation and the extent of DNA damage induced in cultured human fibroblasts. As estimated by the alkaline sucrose gradient technique, the extent of DNA damage induced by equimolar doses of AAF and its metabolites, followed the order: N-acetoxy-AAF > N-hydroxy-AAF > AAF. This parallelled the order of the levels of DNA repair synthesis, estimated by the unscheduled incorporation of tritiated thymidine (³H-TdR). Unscheduled incorporation of ³H-TdR may, therefore, be a reflection of DNA damage, the amount of which, in turn, may be determined by the reactivity of the carcinogenic metabolite to which the cells are exposed. A technique of in vitro simulation of in vivo metabolism of precarcinogens provided a workable system in which the role of metabolic activation of precarcinogens in the induction of DNA damage and repair, chromosome aberrations, and cell lethality could be studied with cultures of human "receptor" cells. When the precarcinogens dimethylnitrosamine (DMN), aflatoxin Bl, and sterigmatocystin were "activated", by combination with NADPH-dependent activation systems containing the post-mitochondrial supernatant (9S) fraction of mouse liver homogenate, their ability to induce high levels of DNA repair synthesis, chromosome aberrations, and cell lethality was increased. The results of alkaline sucrose gradient studies indicated that DNA damage induced by reactive metabolites may account for these biological responses. Different subcellular fractions of mouse, rat, and duck livers were found capable of potentiating the ability of aflatoxin Bl to induce high levels of DNA repair synthesis in cultured human cells. High levels of DNA repair synthesis were also induced by aflatoxin Bl or sterigmatocystin in combination with the 9S fraction from liver, kidney, or lung of several animal species. This implies that circulating aflatoxin Bl or sterigmatocystin in vivo may be converted to DNA-damaging metabolites in lung, kidney, and liver of these animals. It is proposed, therefore, that the "hepatocarcinogens" aflatoxin Bl and sterigmatocystin should have the capacity to induce tumors in organs other than the liver. Mycotoxins that contain an isolated vinyl ether double bond (aflatoxins Bl, Gl, aflatoxicol, and sterigmatocystin), when "activated", induced higher levels of DNA repair synthesis than mycotoxins lacking this molecular feature (aflatoxins B2 and G2). The activity of "activated" aflatoxins Bl, Gl, B2, and G2 in this in vitro system paralleled their activity as carcinogens. Repair-deficient xeroderma pigmentosum (XP) cells respond with lower levels of unscheduled incorporation of ³H-TdR, as compared with normal cells, following exposure to UV-radiation and numerous chemical carcinogens. Alkaline sucrose gradient studies, in combination with studies of the unscheduled incorporation of ³H-TdR by human cells exposed to 4-nitroquinoline l-oxide (4NQ0), demonstrated that the repair of 4NQ0-induced DNA damage may entail the "resynthesis" of excised DNA strand segments and the "rejoining" of small DNA strands. A comparatively slow return of the sedimentation profiles of 4NQ0-damaged XP-DNA towards the position of control XP-DNA in alkaline sucrose gradients, may reflect slower rates of repair of 4NQ0-induced damage in XP cells than in normal cells. Treatment with "activated" DMN and aflatoxin Bl resulted in the induction of lower levels of DNA repair synthesis, higher frequencies of chromosome aberrations, and higher levels of cell lethality in XP cells than in similarly treated normal cells. The results obtained with XP cells indicate that defective or incomplete DNA repair synthesis may play a role in carcinogenesis induced by these precarcinogens. The possible role of metabolic activation and DNA damage and repair in organ-specific tumor induction was investigated in vivo. The extent of DNA damage and patterns of repair in lung, kidney, and liver of mice, after single subcutaneous injections of 4NQ0 or DMN, were demonstrated by an alkaline sucrose gradient technique. The extent of DNA damage appeared maximal at 4 h post-injection. At this time, the extent of DNA damage from 4NQ0 followed the order: lung > kidney > liver, while that from DMN followed the order: liver > kidney and lung. The levels of DNA damage may reflect differences in the distribution and metabolic activation of the precarcinogens in these organs. Repair of induced DNA damage was evident at later sampling times and was not complete in the tissues which had sustained the highest levels of DNA damage. This organ specificity of DNA damage seems to correlate with levels of DNA repair synthesis, estimated by the incorporations of ³H-TdR, and with sites of tumor formation. Tumor induction by chemical precarcinogens may be influenced by multiple factors that control "early" stages of chemical carcinogenesis such as metabolic activation of pre-carcinogens, binding to DNA, DNA damage, and DNA repair. The central hypothesis is that high levels of DNA damage induced by reactive metabolites of precarcinogens in vivo, require high levels of DNA repair synthesis and result in the possible occurrence of defective or incomplete DNA repair. This may lead to neoplastic transformation through alterations of the informational content of DNA.

Text

2010-02-05

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http://hdl.handle.net/2429/19625

Genetics

University of British Columbia

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