Mutagenicity of arsenic in mammalian cells: role of reactive oxygen species

Abstract

Arsenite, the trivalent form of arsenic present in the environment, is a known human carcinogen that lacked mutagenic activity in bacterial and standard mammalian cell mutation assays. We show herein that when evaluated in an assay (AL cell assay), in which both intragenic and multilocus mutations are detectable, that arsenite is in fact a strong dose-dependent mutagen and that it induces mostly large deletion mutations. Cotreatment of cells with the oxygen radical scavenger dimethyl sulfoxide significantly reduces the mutagenicity of arsenite. Thus, the carcinogenicity of arsenite can be explained at least in part by it being a mutagen that depends on reactive oxygen species for its activity.

Figure 1

Survival curve for A_L cells exposed to graded doses of sodium arsenite. Exponentially growing A_L cells were treated with the drug for either 1 or 5 days. Each data point represents an average of four experiments. The D_o values of the curves were 1.7 and 1.3 μg/ml, respectively. Error bars represent ±SEM.

Figure 2

Mutation induction in A_L cells by graded doses of sodium arsenite, expressed as number of induced mutants per 10⁵ clonogenic survivors, at the S1 and HPRT loci after either 1- or 5-day treatment. Induced mutant fractions are the total mutant yield minus background. Each point represents data pooled from three to five experiments. Error bars represent ±SEM.

Figure 3

Diagram of human chromosome 11 showing the M1C1 gene used in defining the S1⁻ phenotype and the relative positions of other markers used in the multiplex PCR analysis to determine the extent of the S1 mutations. The M1C1 gene maps to 11p13.5. The two nearest markers flanking M1C1, CAT and WT, are separated by approximately 3.6 megabase pairs (Mbp) so that the S1⁻ mutants that retained these neighboring markers could result from a base change to deletions as large as 3.6 Mbp.

Figure 4

Deletion spectra of S1⁻ mutants of spontaneous origin or from cells exposed to graded doses of sodium arsenite for either a 1- or a 5-day treatment period. Each line depicts the spectrum of a single independent mutant. The absence or presence of markers among the mutants was determined by multiplex PCR using DNA from S1⁻ mutants as templates and primers for parathyroid hormone (PTH), Wilms’ tumor (WT), catalase (CAT), apolipoprotein A1 (APO-A1), and RAS. Blank space shows missing markers.

Figure 5

Effects of the free radical scavenger DMSO (0.1%) on induced mutant yield in A_L cells treated with sodium arsenite (2 μg/ml) for 24 hr. The survival fraction of the various treatment group is shown above each bar. Data were pooled from two or three experiments. Error bars represent ±SEM.