Measurement of unscheduled DNA synthesis in rat kidney cells following in vivo treatment with genotoxic agents

Publisher Website
Google Scholar
Abstract
The kidney is a key target tissue in animal and human carcinogenesis, yet there are no established short-term tests for studying the genotoxicity of chemicals in the kidney. We have developed an assay for the measurement of chemically induced DNA repair as unscheduled DNA synthesis (UDS) in isolated rat kidney cells following in vivo treatment. Male Fischer-344 rats were injected intraperitoneally with chemicals dissolved in saline or corn oil. After various treatment times, the kidneys were perfused with a collagenase/trypsin solution (CTS), minced into small pieces, and stirred in CTS at 37°C for 1 hr to dissociate cells. Cultures contain a high proportion of epithelial cells from the proximal and distal tubules. Cultures were incubated for 16–18 hr with 3H-thymidine in Williams' Medium E supplemented with 20% fetal bovine serum. UDS was measured by quantitative autoradiography as net grains/nucleus (NG). The percentage of cells in repair (% IR) was defined as the percentage of cells with ⩾ 3 NG. Saline- or corn oil-injected controls consistently produced −3 to −5 NG with < 1 % IR. The time course of DNA repair following treatment with the direct-acting mutagen methylmethane sulfonate (MMS) or the renal carcinogen azaserine showed a peak response at 2 hr after treatment. Azaserine showed a rapid decline in UDS at 12 and 24 hr, whereas MMS exhibited a relatively high UDS level at 24 hr. The renal carcinogens methylazoxymethanol acetate, N-methyl-N-nitrosourea, and streptozotocin all yielded strong positive UDS responses. The liver and intestinal carcinogen 1, 1-dimethylhydrazine at doses up to 50 mg/kg was cytotoxic to kidney cells, but induced < 0 NG. Treatment with 1,2-dimethylhydrazine, which induces kidney tumors in mice but not rats, also induced < 0 NG. Treatment with o-anisidine, a weak renal carcinogen, did not induce UDS in the kidney, suggesting that it may be acting as a tumor promotor. These results demonstrate the usefulness of this assay for the detection and study of a variety of genotoxic kidney carcinogens.