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Induction of ouabain-resistant mutants by chemical carcinogens in rat prostate epithelial cells
Abstract:
We determined optimal conditions to quantitatively select ouabain-resistant (Ouar) mutants induced by chemical carcinogens in a rat prostate epithelial cell line (RPYK). These conditions included selection of Ouar mutants in 3 mM ouabain, an expression time of two days following a two-day treatment with carcinogens, and a reseeding density of 2 X 10(5) mutagenized cells per 100 mm dish to select mutants in ouabain. Ouar mutants induced by N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) remained stably Ouar when passaged in nonselective medium. Hemicyst formation, a characteristic of epithelial cells, was reversibly inhibited by ouabain in wild-type cells and was resistant to ouabain in Ouar cells. The direct-acting carcinogens MNNG and methylazoxymethanol-acetate (MAMA) and the environmentally widespread procarcinogens aflatoxin B1 and benzo(a)pyrene increased the frequency of Ouar mutants in RPYK cells. The procedures developed here now make it possible to detect some environmental carcinogens likely to cause prostate cancer by virtue of their ability to mutate cultured prostate epithelial RPYK cells. The sensitivity of the RPYK cell line to aflatoxin-induced cytotoxicity and mutagenesis also makes it a useful cell system in which to study enzymes governing the conversion of aflatoxin to genotoxic metabolites.
Insights
Researchers developed a method to detect prostate cancer-causing carcinogens using rat prostate epithelial cells. This technique identifies mutations induced by chemicals like aflatoxin B1 and benzo(a)pyrene.
Area of Science:
- Toxicology
- Cell Biology
- Cancer Research
Background:
- Prostate cancer is a significant health concern.
- Identifying environmental carcinogens is crucial for prevention.
- Developing sensitive cell-based assays for carcinogen detection is needed.
Purpose of the Study:
- To establish optimal conditions for selecting ouabain-resistant (Ouar) mutants in a rat prostate epithelial cell line (RPYK).
- To validate the use of RPYK cells for detecting environmental carcinogens that may cause prostate cancer.
- To assess the utility of RPYK cells in studying aflatoxin metabolism.
Main Methods:
- Optimized selection conditions for ouabain-resistant mutants: 3 mM ouabain, 2-day expression time post-carcinogen treatment, and reseeding at 2 x 10(5) cells/100 mm dish.
- Induced mutations using chemical carcinogens: N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG), methylazoxymethanol-acetate (MAMA), aflatoxin B1, and benzo(a)pyrene.
- Assessed stability of ouabain resistance and ouabain's effect on hemicyst formation in wild-type and mutant cells.
Main Results:
- Established reproducible conditions for selecting ouabain-resistant mutants in RPYK cells.
- Demonstrated stable ouabain resistance in induced mutants.
- Showed that MNNG, MAMA, aflatoxin B1, and benzo(a)pyrene increase Ouar mutant frequency in RPYK cells.
- Observed reversible inhibition of hemicyst formation by ouabain in wild-type cells, with resistance in Ouar cells.
Conclusions:
- The developed RPYK cell-based assay can detect environmental carcinogens that induce mutations, potentially linked to prostate cancer.
- RPYK cells are sensitive to aflatoxin-induced cytotoxicity and mutagenesis, making them valuable for studying enzymes involved in aflatoxin activation.
- This method provides a new tool for identifying carcinogens that target prostate epithelial cells.