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Platinum-induced mutations to 8-azaguanine resistance in Chinese hamster ovary cells
Abstract:
6 platinum (Pt) compounds were compared in suspension cultured Chinese hamster ovary (CHO-S) cells with respect to their inhibition of growth, their reduction of cloning efficiency, and their induction of mutants resistant to 200 microM (30 micrograms/ml) 8-azaguanine (8-AG) and 3 mM ouabain (OUA), respectively. The toxicity of these compounds can be ranked by the medium concentrations which decrease suspension growth/or cloning efficiency by 50%: cis-Pt(NH3)2-Cl2 (0.9/1.5 microM) greater than Pt(SO4)2 + methylcobalamin (MeB-12) methylation product (20/10 microM) greater than K2PtCl4 (32/50 microM) = K2PtCl6 (34/50 microM) = MePtCl2-3 (60/50 microM) greater than Pt(SO4)2 (66/105 microM). Following 20 h exposures to concentrations which resulted in relative survivals of 80-2%, none of the foregoing compounds increased consistently the frequency of OUA(R) mutants above the spontaneous frequency (6.0 x 10(-6)). Parallel treatments with 800 microM (100 micrograms/ml) ethyl methanesulfonate (EMS) increased the OUA(R) mutant frequency 10--12-fold. Using 8-AG for mutant selection, dose-dependent increases of 5--7-fold above the spontaneous frequency (3--8 x 10(-5) were obtained with cis-Pt(NH3)2Cl2, Pt(S04)2, and the product from Pt(SO4)2 + MeB-12. Identical 20 h exposures to varying amounts of K2PtCl4, K2PtCl6, and MePtCl2-3 did not induce 8-AG(R) mutants. Optimal detection of Pt-induced 8-AG(R) mutants required 7 post-treatments, expression doublings in suspension culture. Under our selection conditions 8/8 spontaneous and 24/24 Pt-induced 8-AG(R) variants contained reduced hypoxanthine-guanine phosphoribosyl transferase (HGPRT) specific activities (means ranging from 3 to 11% of the parental CHO-S cells). When compared from linear plots of the 8-Ag(r) frequency against the initial medium concentration, cis-Pt(NH3)2Cl2 is 134 times and Pt(SO4)2 si 3.5 times more mutagenic than EMS. However, on a cell-survival basis EMS is 8--10-fold more mutagenic than these two Pt-compounds. 6-Thioguanine (10 microM) can be substituted for 8-AG to assay mutant induction by cis-Pt(NH3)2Cl2 and Pt(SO4)2 in CHO-S cells. The sensitivity of the CHO-S HGPRT locus for detecting mutagenesis by Pt complexes can be increased several fold by continuous subculture in the presence of these agents for 10--25 population doublings. By this procedure K2PtCl6 is seen to be weakly mutagenic and 20 microM Pt(SO4)2 produces 8-AG(R) mutants at frequencies requiring 7--8-fold higher concentrations when a fixed 20 h exposure is used.
Insights
Six platinum compounds were evaluated for their toxicity and mutagenicity in Chinese hamster ovary cells. Cisplatin and a platinum sulfate derivative showed mutagenic potential, while others did not induce significant mutations.
Area of Science:
- Toxicology
- Genetics
- Biochemistry
Background:
- Platinum compounds are widely used in chemotherapy.
- Understanding their genotoxic effects is crucial for risk assessment.
- Chinese hamster ovary (CHO-S) cells are a standard model for genotoxicity testing.
Purpose of the Study:
- To compare the toxicity and mutagenicity of six platinum compounds in CHO-S cells.
- To evaluate their potential to induce mutations resistant to 8-azaguanine (8-AG) and ouabain (OUA).
- To assess the mutagenic potency relative to ethyl methanesulfonate (EMS).
Main Methods:
- Culturing CHO-S cells in suspension.
- Exposing cells to varying concentrations of six platinum compounds for 20 hours.
- Assessing inhibition of growth and cloning efficiency.
- Measuring the frequency of 8-AG-resistant (8-AG(R)) and OUA-resistant (OUA(R)) mutants.
- Determining hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in resistant mutants.
Main Results:
- Toxicity ranking: cis-Pt(NH3)2Cl2 > Pt(SO4)2 + MeB-12 product > K2PtCl4 = K2PtCl6 = MePtCl2-3 > Pt(SO4)2.
- No platinum compounds consistently increased OUA(R) mutant frequency above spontaneous levels.
- cis-Pt(NH3)2Cl2 and Pt(SO4)2 dose-dependently increased 8-AG(R) mutant frequency (5-7 fold).
- K2PtCl4, K2PtCl6, and MePtCl2-3 did not induce 8-AG(R) mutants.
- cis-Pt(NH3)2Cl2 was 134 times, and Pt(SO4)2 was 3.5 times more mutagenic than EMS per concentration.
- On a cell-survival basis, EMS was 8-10 fold more mutagenic than cis-Pt(NH3)2Cl2 and Pt(SO4)2.
- All tested 8-AG(R) mutants exhibited reduced HGPRT activity.
Conclusions:
- cis-Diamminedichloroplatinum(II) (cisplatin) and platinum(IV) sulfate exhibit significant mutagenic potential in CHO-S cells.
- Other tested platinum compounds showed minimal or no mutagenic activity under the study conditions.
- The HGPRT locus is a sensitive target for platinum-induced mutagenesis.
- Continuous culture can enhance the detection of mutagenesis by platinum complexes.
