Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity

Abstract

Although arsenic is a well-established human carcinogen, the mechanisms by which it induces cancer remain poorly understood. We previously showed arsenite to be a potent mutagen in human-hamster hybrid (A(L)) cells, and that it induces predominantly multilocus deletions. We show here by confocal scanning microscopy with the fluorescent probe 5',6'-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate that arsenite induces, within 5 min after treatment, a dose-dependent increase of up to 3-fold in intracellular oxyradical production. Concurrent treatment of cells with arsenite and the radical scavenger DMSO reduced the fluorescent intensity to control levels. ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOL-H) as a probe in conjunction with superoxide dismutase and catalase to quench superoxide anions and hydrogen peroxide, respectively, indicates that arsenite increases the levels of superoxide-driven hydroxyl radicals in these cells. Furthermore, reducing the intracellular levels of nonprotein sulfhydryls (mainly glutathione) in A(L) cells with buthionine S-R-sulfoximine increases the mutagenic potential of arsenite by more than 5-fold. The data are consistent with our previous results with the radical scavenger DMSO, which reduced the mutagenicity of arsenic in these cells, and provide convincing evidence that reactive oxygen species, particularly hydroxyl radicals, play an important causal role in the genotoxicity of arsenical compounds in mammalian cells.

Figure 1

Representative images of fluorescent signals generated from composite images obtained by confocal microscopy of A_L cells pretreated with the fluorescent probe CM-H₂DCFDA for 40 min with or without subsequent arsenite treatment. (A) A_L cells treated with only the fluorescent probe; (B) five minutes after the addition of sodium arsenite at a dose of 1 μg/ml (7.7 μM); (C) five minutes after treatment with 2 μg/ml (15.4 μM) sodium arsenite; (D) treatment as in C, but with concurrent 0.1% DMSO.

Figure 2

Average fluorescent intensity in A_L cells treated with 2 μg/ml (15.4 μM) of sodium arsenite alone or with DMSO. The intensity of the fluorescent signals was obtained from the composite images generated by image analysis software. The relative intensities are expressed in arbitrary units. Data from four independent experiments were pooled. Error bars represent ± SD.

Figure 3

ESR spectra generated by (a) 25 mM TEMPOL-H in PBS buffer containing 3 × 10⁶ A_L cells; (b) condition a + arsenite, 4 μg/ml (30.8 μM); (c) condition b + catalase, 5,000 units/ml; and (d) 25 mM TEMPOL-H solution in PBS alone as control. Cells were incubated with the chemicals for 1 h at 37°C and then cooled to room temperature for the ESR measurements.

Figure 4

Surviving fractions of A_L cells exposed to graded doses of sodium arsenite with or without pretreatment of BSO at either 10 or 25 μM. BSO pretreatment reduced the nonprotein sulfhydryls levels of A_L cells to less than 5% of control levels. Data from three experiments were pooled. Error bars represent ± SD.

Figure 5

Induction of CD59⁻ mutants in A_L cells treated with graded doses of arsenite with or without pretreatment with BSO. Induced mutant fractions are the total mutant yield minus background, which amounts to 46 ± 10 mutants per 10⁵ clonogenic survivors among the A_L cells used in these studies. Data from three experiments were pooled. Error bars represent ± SD.