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Different mechanisms of reversion of HPRT-deficient V79 Chinese hamster cells
M Fox1, B J Rossiter, J Brennand
1Department of Biochemical Genetics, Paterson Institute for Cancer Research, Manchester, UK.
Abstract:
The revertibility of three spontaneous hypoxanthine phosphoribosyl transferase (HPRT)-deficient V79 cell lines has been determined after exposure to a number of alkylating agents. TG11 and 19 reverted at frequencies ranging from 1 X 10(-5) to 1 X 10(-4) after exposure to doses of ethylmethane sulphonate (EMS) N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) resulting in surviving fractions between 1.0 and 0.1. Reversion frequencies in TG15 ranged from 10(-7) to 5 x 10(-6) over a similar dose range. The relative efficiencies of different monofunctional alkylating agents in causing reversion of TG11 at equitoxic doses were ENU greater than EMS greater than N-ethyl-N-nitroso-guanidine greater than MNU greater than N-methyl-N-nitrosoguanidine greater than methylmethane sulphonate. Revertant frequencies for all three cell lines were maximal immediately after treatment and declined thereafter at a rate inversely proportional to dose. Such kinetics are explicable if reversion is due to miscoding opposite alkylated guanines. Reversion frequencies after N-butyl-N-nitrosourea exposure were 100-fold lower than after MNU and kinetics of expression of revertant colonies differed. Frequencies were low immediately after treatment, increased between 0 and 24 h then remained at a plateau. Similar kinetics were observed after chlorozotocin and bis-chloroethylnitrosourea exposure. This difference in expression kinetics suggests that reversion in this case is not the result of direct miscoding but of errors in excision repair. TG11, 15 and 19 had low spontaneous mutant frequencies which were either unaffected or only marginally increased by treatment with 5-azacytidine.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
This study investigated how alkylating agents affect hypoxanthine phosphoribosyl transferase (HPRT)-deficient V79 cells. Different agents and exposure times revealed distinct mechanisms of genetic reversion, involving miscoding and DNA repair errors.
Area of Science:
- Molecular Biology
- Genetics
- Toxicology
Background:
- Hypoxanthine phosphoribosyl transferase (HPRT)-deficient V79 cell lines are utilized to study genetic mutations.
- Alkylating agents are known mutagens that can induce DNA damage and subsequent cellular responses.
- Understanding the mechanisms of genetic reversion is crucial for assessing the genotoxicity of chemical compounds.
Purpose of the Study:
- To determine the revertibility of three spontaneous HPRT-deficient V79 cell lines after exposure to various alkylating agents.
- To investigate the kinetics of reversion and elucidate the underlying molecular mechanisms (miscoding vs. excision repair errors).
- To compare the relative efficiencies of different monofunctional alkylating agents in inducing genetic reversion.
Main Methods:
- Exposure of HPRT-deficient V79 cell lines (TG11, TG15, TG19) to a range of alkylating agents including ethylmethane sulphonate (EMS), N-methyl-N-nitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU).
- Quantification of reversion frequencies at different time points post-treatment.
- Analysis of dose-dependent effects and comparison of relative efficiencies of various alkylating agents.
Main Results:
- TG11 and TG19 cell lines showed higher reversion frequencies (1 x 10^-5 to 1 x 10^-4) compared to TG15 (10^-7 to 5 x 10^-6) after exposure to EMS, MNU, and ENU.
- The relative efficiencies of monofunctional alkylating agents on TG11 were ranked: ENU > EMS > N-ethyl-N-nitroso-guanidine > MNU > N-methyl-N-nitrosoguanidine > methylmethane sulphonate.
- Distinct reversion kinetics were observed: immediate maximal frequencies declining with dose (suggesting miscoding) for some agents, versus delayed increases and plateaus (suggesting excision repair errors) for others like N-butyl-N-nitrosourea, chlorozotocin, and bis-chloroethylnitrosourea.
Conclusions:
- The study differentiates between two primary mechanisms of genetic reversion in V79 cells induced by alkylating agents: direct miscoding and errors in DNA excision repair.
- The kinetics of revertant expression are indicative of the underlying mutagenic mechanism.
- The findings contribute to a better understanding of genotoxicity assessment and DNA repair pathways.