Differential Effects of Pre- and Posttreatment of Sodium Arsenite on the Genotoxicity of Methyl Methanesulfonate in Chinese Hamster Ovary Cells1

Pretreatment of sodium arsenite reduces hypoxanthine-guanine phosphoribosyltransferase mutagenicity and overcomes the inhibition of mi tosis and cell proliferation but has no apparent effect on the cytotoxicity and clastogenicity in methyl methanesulfonate (MMS)-lreaU'd Chinese hamster ovary cells. Posttreatment of sodium arsenite drastically in creases the cytotoxicity, clastogenicity, hypoxanthine-guanine phosphoribosyltransferase mutagenicity, and inhibition of mitosis and cell prolif eration induced by MMS. Sodium arsenite either preor posttreatment has no apparent effect on the MMS-induced sister chromatid exchanges. The present results indicate that pretreatment of sodium arsenite not only does no harm but may even benefit the MMS-treated cells. On the contrary, posttreatment of sodium arsenite is cogenotoxic. INTRODUCTION Arsenic is the 20th most abundant of the earth's crustal elements and most of it is in the form of highly insoluble metal arsenides. Unfortunately, the insoluble arsenides have been steadily transferred to the soluble forms of arsenale (pentavalent) and arsenite (trivalent) by industrialization and metal smelting. Ingested inorganic arsenic, which is readily absorbed from the gastrointestinal tract by humans, is excreted primarily via the kidneys (1). Recently, the presence of pentavalent, trivalent, and methylated arsenic in urine of exposed humans has been reported (2). Trivalent arsenic is generally considered to be more toxic than pentavalent arsenic (3); however, they may to a certain extent be mutually transformed into each other by oxidation and reduction (4). From epidemiolÃ3gica! investigation, arsenic exposure is strongly correlated with the increased incidences of human skin and lung cancers, but there is no reliable evidence showing arsenic carcinogenicity in experimental animals (5, 6). There fore, arsenicals have been considered as comutagens and/or cocarcinogens. Arsenic can apparently affect cancer develop ment through several indirect or direct mechanisms, although little is understood regarding the mechanism of action of arsenic at the cellular and molecular levels (7). We have previously demonstrated that AS3 posttreatment synergistically increases the UV-induced cytotoxicity, clastogenicity, and 6-thioguanineresistant mutagenicity, but not the rate of SCE in CHO cells (8). The comutagenicity of AS has also been demonstrated in UV-irradiated Escherìchiacoli (9). For a better understanding of the cogenotoxicity of arsenic, we have studied the effects of postas well as pretreatment of AS on MMS-induced genetic damages in CHO cells. Cytotoxicity, CA, SCE, and mutation at the HGPRT locus were the end points used to monitor the genetic damages in this study. Received 5/28/85; revised 9/20/85; accepted 11/6/85. 1This study was supported by a grant from the National Science Council, Republic of China. 2To whom requests for reprints should be addressed. 3The abbreviations used are: AS, sodium arsenite; BrdUrd, 5-bromodeoxyuridine; CA, chromosome aberrations; CHO, Chinese hamster ovary; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; MMS, methyl methanesulfon ate; SCE, sister chromatid exchanges. MATERIALS AND METHODS All chemicals for cell culture were obtained from GIBCO (Grand Island, NY). MMS was purchased from Sigma Chemical Co. (St. Louis, MO). AS was purchased from Merck (Darmstadt, Federal Republic of Germany). CHO cells used for all the experiments were maintained in McCoy's Medium 5A supplemented with 10% heat-inactivated fetal calf serum and antibiotics as described previously (8). Cells were plated at desired density 2 to 3 h before the experiments. The log phase growing cells were then subjected to chemical treatment. A general scheme of treatment with AS and MMS is shown in Fig. 1. MMS and AS were dissolved in double distilled water immediately before use. Control cultures were treated with 25 //I of double distilled water per 5 ml of medium. Cytotoxicity and the mutation frequency at the HGPRT locus were determined according to the procedures described by Gupta and Singh (10). One million cells were plated in 10(1-mm Petri dish and treated with AS and MMS (see Fig. 1). The duration of AS preor posttreat ment was 24 h. At the end of chemical treatment, the cells were trypsinized, replated at 200 cells per 60-mm Petri dish in triplicate, and incubated in normal medium for 7 days to determine survival. The colony numbers were determined after fixation with absolute methanol and staining with 10% Giemsa solution. The remaining cells were plated at a density of 1 x 10* cells/100-mm Petri dish and subcultured every other day for the expression of the 6-thioguanine-resistant mu tants. The mutants were selected by splitting 1 x 10*cells into five 100mm dishes and feeding with medium containing 6-thioguanine (11 ng/ ml) on the eighth day. Along with plating in selective medium, an aliquot of 200 cells was also plated in the normal medium to determine the plating efficiency of the cells. The dishes were incubated for 7 days at 37°Cwithout change of medium and then fixed and stained as described above. The mutation frequency was calculated from the number of cells plated, the number of mutant colonies observed, and the plating efficiency of cells as described by Gupta and Singh (10). For the analyses of SCE and cell proliferation, BrdUrd (20 MM)was added immediately after MMS was removed (see Fig. 1). Colcemid (0.2 Mg/ml) was added at 2 h before harvesting. Metaphase cells were collected by a shake-off technique and chromosomes were prepared by the air-dry method. Sister chromatid differential staining was per formed as described by Jan et al. (l 1). Thirty 2nd-division metaphases in coded slides were scored for the SCE value of each treatment. Cell cycle kinetics was determined by the BrdUrd differential chromatid labeling technique as described by Schneider <•/ al. (12). One hundred metaphase cells were examined for each harvesting point. The replica tion index was calculated by the formula (1 x % of first cycle metaphases) + (2 x % of second cycle metaphases) + (3 x % of third cycle metaphases) 100 Mitotic index was determined from cultures without Colcemid. Cells were harvested by trypsinization, and at least 1000 cells were scored to determine the percentage of cells in metaphase. For the analysis of CA, metaphase cells were harvested from cultures without BrdUrd. For each treatment 100 metaphases in coded slides were examined. CAs were identified by following the criteria described by Buckton and Evans (13). Gaps were not included in calculating the percentages of aberrant metaphases. The method of calculating the expected values was adopted from Morgan and Cleaver (14), i.e., summation of the values of two single treatments then minus the value of no treatment.