In vitro cytotoxic and genotoxic effects of diphenylarsinic acid, a degradation product of chemical warfare agents

Toxicology and Applied Pharmacology 200 (2004) 64 – 72 www.elsevier.com/locate/ytaap

In vitro cytotoxic and genotoxic effects of diphenylarsinic acid, a degradation product of chemical warfare agents Takafumi Ochi, a,* Toshihide Suzuki, a Hideo Isono, a and Toshikazu Kaise b a

Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan b Tokyo University of Pharmacy and Life Sciences, Hachioji, 192-0392, Japan Received 30 January 2004; accepted 18 March 2004 Available online 18 May 2004

Abstract Diphenylarsinic acid [DPAs(V)], a degradation product of diphenylcyanoarsine or diphenylchloroarsine, both of which were developed as chemical warfare agents, was investigated in terms of its capacity to induce cytotoxic effects, numerical and structural changes of chromosomes, and abnormalities of centrosome integrity and spindle organizations in conjunction with the effects of glutathione (GSH) depletion. DPAs(V) had toxic effects on cultured human hepatocarcinoma HepG2 cells at concentrations more than 0.5 mM. Depletion of GSH reduced the toxic effects of DPAs(V) as well as dimethylarsinic acid [DMAs(V)] toxicity, while toxicity by arsenite [iAs(III)] was enhanced. Exogenously added sulfhydryl (SH) compounds, such as dimercapropropane sulfonate (DMPS), GSH, and dithiothreitol (DTT), enhanced the toxic effects of DPAs(V) while they suppressed iAs(III) toxicity. DPAs(V) caused an increase in the mitotic index, and also structural and numerical changes in chromosomes in V79 Chinese hamster cells. Abnormality of centrosome integrity in mitotic V79 cells and multipolar spindles was also induced by DPAs(V) in a time- and concentration-dependent manner. These results suggested that highly toxic chemicals were generated by the interaction of DPAs(V) with SH compounds. Moreover, enhancements of toxicity by a combination of DPAs(V) and SH compounds suggested a risk in the use of SH compounds as a remedy for intoxication by diphenylarsenic compounds. Investigations on the effects of SH compounds on animals intoxicated with DPAs(V) are warranted. D 2004 Elsevier Inc. All rights reserved. Keywords: Diphenylarsinic acid; Centrosome abnormality; Multipolar spindles; Chromosome aberrations; SH compounds

Introduction A variety of chemical warfare agents have been developed to this day and used in modern warfare (Hu et al., 1989; Orient, 1989). Chlorine and phosgene as suffocation gas (Lung, 1915), Yperit (sulfur mustard) and lewisite as sore gas (Hobbs, 1944; Dacre and Goldman, 1996; Marshall, 1919; Sinclair, 1948), and nerve agents (Dunn and Sidell, 1989; Gunderson et al., 1992; Nozaki et al., 1995), such as tabun, soman, sarin, and VX, are well-known and some were used practically in World War I and the Iraq – Iran War. Sternutatory or vomiting agents, such as diphenylchloroarsine and diphenylcyanoarsine (Henriksson et al., * Corresponding author. Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Suarashi 1091-1, Sagamikomachi, Tsukui-gun, Kanagawa-ken, 199-0195, Japan. Fax: +81-426-853751. E-mail address: [email protected] (T. Ochi). 0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2004.03.014

1996; Pitten et al., 1999), were also developed to decrease the soldier’s will to fight. On the other hand, a poisonous incident occurred in Kamisu-machi, Ibaraki Prefecture, Japan, on May, 2003 (Ishii et al., 2003). Arsenicals contaminating ground water were considered to be the cause, because the arsenicals were detected at a concentration of 4.5 mg/l, which is 450 times higher than the safety standard for drinking water in Japan. Thereafter, analysis by gas chromatography – mass spectrometry of the arsenicals demonstrated that diphenylarsinic acid [DPAs(V)] was the main contaminant in the ground water (Hanaoka et al., 2003; Ishizaki et al., 2003). However, DPAs(V) is not a natural compound, and it was, therefore, possible that DPAs(V) may have been a degradation product of diphenylcyanoarsine or diphenylchloroarsine abandoned by Japanese troops after the end of World War II. Indeed, it became known that the military installations were in Kamisu-machi. Fig. 1 depicts degradation in nature of diphenylchloroarsine and diphenylcyanoarsine. In addition

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Fig. 1. Degradation of diphenylcyanoarsine and diphenylchloroarsine.

to DPAs(V), bis(diphenylarsine)oxide, an intermediate degradation product, was also detected in the ground water in Kamisu-machi. Most patients who suffered arsenic poisoning in Kamisumachi showed dysfunction of the central nervous system, such as trembling, cerebellar ataxia, brainstem ataxia including double vision, etc., and these were very similar to the case of chronic poisoning by organic mercuric compounds, methylbromide and organic chlorinated compounds. Hereafter, a follow-up survey must be conducted not only on the central nervous system, but also on other clinical symptoms. Thus far, the biological effects of human-made organic arsenicals have not been investigated extensively in either experimental animals or cultured mammalian cells. No information is available on the toxicity of DPAs(V). Accordingly, in contrast to the case of inorganic arsenic poisoning, no remedy specific for the poisoning by DPAs(V) has been established. In the present study, cytotoxicity and genotoxicity of DPAs(V) in cultured human cells or animal cells were investigated in conjunction with the effects of glutathione (GSH) depletion. The effects of DPAs(V) on centrosome integrity and microtubule organization were also investigated in terms of the mechanism of abnormality of cell division. In addition, the effects of sulfhydryl (SH) compounds on the cytotoxicity of DPAs(V) were investigated to gain some insights into the development of treatment for poisoning by arsenicals.

Japan). Cell counting Kit-8 and 5-sulfosalicylic acid dihydrate (SSA) were obtained from Wako (Osaka, Japan). LButhionine-SR-sulfoximine (BSO), 4,6-diamidino-2-phenylindol (DAPI), dithiothreitol (DTT), 2, 3dimercapto-1-propane sulfonic acid (DMPS), glutathione (GSH, reduced form), glutathione reductase (GSSG-Rx), monoclonal anti-h-tubulin antibody (TUB 2.1), NADPH ,and rabbit polyclonal anti-g-tubulin antibody were obtained from Sigma (St. Louis, MO, USA). Skim milk was obtained from Difco Laboratories (Detroit, MI, USA), FITC-labeled sheep anti-mouse IgG1 antibody from Serotec Ltd. (Oxford, UK), and TRITC-labeled goat anti-rabbit IgG antibody from EY Laboratories, Inc, (San Mateo, CA, USA).

Materials and methods

Determination of cell glutathione. HepG2 cells, seeded at a cell density of 2  104 cells/cm2 in 3-cm Petri dish (IWAKI, Japan) and preincubated for 24 h, were incubated with various concentrations of BSO for 6 h, and then incubated in control medium for 24 h. The levels of intracellular glutathione were determined by a DTNBGSSG reductase recycling assay as described previously (Ochi, 1997). Proteins were quantitated by the coomassie assay (Coomassie Plus Protein Assay Reagent Kit, PIERCE, IL, USA) with bovine serum albumin as the standard.

Chemicals. Arsenite [iAs(III)] and dimethylarsinic acid [DMAs(V)] were obtained from Tori Chemical Corp. (Uenohara, Japan). Diphenylarsinic acid [DPAs(V)] was prepared by a modification of the method of Baker et al. (1949). In short, aqueous arsenic acid (60%) and phenylhydrazin were mixed at 75 jC in the presence of catalysts, cuprous oxide, and then the mixture was extracted with carbon tetrachloride. After evaporation, bis (diphenylarsine) oxide was obtained and the crude crystals were oxidized with hydrogen peroxide. DPAs(V) was recrystallized with double-distilled water 3 times. 5,5V-Dithiobis(2-nitrobenzoic acid) (DTNB) was from Nakarai Chemicals, Ltd., (Kyoto,

Cell culture. HepG2 cells derived from human hepatocarcinoma were grown in a monolayer in Dulbecco’s modified Eagle’s medium and supplemented with 10% fetal bovine serum. V79 cells derived from the lung fibroblasts of a male Chinese hamster were grown in a monolayer in Eagle’s MEM medium and supplemented with 10% fetal bovine serum. These cells were cultured in an incubator in an atmosphere of 5% CO2 in humidified air. Treatment of cells with the compounds. Diphenylarsinic acid [DPAs(V)] was dissolved in dimethylsulfoxide (DMSO) as a stock solution of 0.25 M, then diluted with culture medium immediately before use. DMSO controls were prepared according to the final concentrations of DMSO in each DPAs(V)-treated group.

Cytotoxicity. HepG2 cells, seeded at a density of 1  104 cells/well in 96-well plates (IWAKI) and incubated for 24 h, were incubated in medium with or without 1.0 mM BSO for

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times. After incubation, the cells were fixed with 3.7% formaldehyde in PBS for 20 min at room temperature, then soaked in methanol at 20 jC for 5 min. The chamber slides were incubated with 2% skim milk in PBS for 30 min at room temperature, then immunofluorescence analysis of g- and h-tubulin was performed as described previously (Ochi et al., 2003). Cells double stained with anti-h-tubulin and anti-g-tubulin were observed and photographed using a Nikon BIOPHOTO fluorescence microscope. The incidence of mitotic cells with abnormalities in the g-tubulin signals and spindle morphology was measured in more than 100 mitotic cells.

Fig. 2. Depletion of glutathione (GSH) in cultured HepG2 cells by Lbuthionine-SR-sulfoximine (BSO). Cells, seeded at a density of 2  104 cells/cm2 in a 3-cm Petri dish and preincubated for 24 h, were incubated with 0.5 – 2.0 mM BSO for 6 h. The cells were then placed in control medium and further incubated for 24 h. After washing with PBS, the cells were scraped and acid-soluble materials were extracted with ice-cold 2.5% sulfosalicylic acid. GSH was quantitated by the DTNB-GSSG reductase recycling assay as described in Materials and methods. Data are represented as means F SEM (n = 3).

6 h. The cells were then placed in medium that contained test chemicals and incubated for specified times. Cytotoxicity was assayed by the method of WST-8 (Cell Counting Kit-8, Wako), in which WST-8 was converted to soluble formazan by the action of mitochondrial dehydrogenase in viable cells. The cultures in 96-well plates were placed in 100 Al of medium that contained WST-8 and incubated for 1 h at 37 jC. The absorbance at 450 nm was determined by a multi-plate reader. Determination of structural and numerical changes of chromosomes and mitotic index. V79 cells, seeded at a density of 1.8 – 2  104 cells/cm2 in 6-cm dish and preincubated for 24 h, were placed in fresh medium that contained test chemicals and then incubated for specified times. After incubation, the cells were trypsinized and treated with 0.075 M KCl solution at room temperature for 10 min, fixed with Carnoy’s solution (methanol/acetic acid, 3:1), and spread on glass slides by the air-drying method. Cells on the slides were stained with 3% Giemsa solution for 30 min at room temperature. Fifty metaphase cells were scored to determine the distribution of the chromosome number and 100 metaphase cells to detect chromosome structural aberrations. To determine the mitotic index, more than 500 cells were counted in each group. Immunofluorescence analysis of tubulins. V79 cells, seeded at a density of 1  104 cells/well in eight-chamber slides (NUNC) and preincubated for 24 h, were placed in fresh medium that contained test chemicals for the specified

Fig. 3. Cytotoxic effects of arsenic compounds in cultured HepG2 cells depleted and not depleted of glutathione. Cells, seeded at a density of 1  104 cells/well in 96-well plates and incubated for 24 h, were incubated in medium with and without 1.0 mM BSO for 6 h. The cells were then placed in medium that contained arsenic compounds and incubated for specified times. The cytotoxic effects were assessed by WST-8 assay as described in Materials and methods. Data are given as percent of the untreated control and represented as means F SEM (n = 3). (A) Diphenylarsinic acid [DPAs(V)]; (B) dimethylarsinic acid [DMAs(V)]; (C) arsenite [iAs(III)].

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Results Cytotoxicity of arsenic compounds in HepG2 cells depleted and not depleted of glutathione (GSH) Cytotoxicities of DPAs(V), iAs(III), and DMAs(V) were evaluated in cultured HepG2 cells that were depleted and not depleted of GSH by incubation with a specific inhibitor of GSH synthesis, L-buthionine-SR-sulfoximine (BSO) (Griffith and Meister, 1979). As shown in Fig. 2, when the cells were incubated with BSO for 6 h, then incubated in control medium for 24 h, GSH levels decreased to 25% of the control level with 0.5 mM, 14% with 1 mM, and 13% with 2 mM. As a result, 1 mM BSO was chosen for the depletion of GSH in HepG2 cells. As shown in Fig. 3A, when cells depleted and not depleted of GSH were incubated for 24 h with DPAs(V), the cytotoxic effects appeared at concentrations of more than 0.5 mM and GSH-depleted cells were somewhat less sensitive to DPAs(V) toxicity than cells not depleted of

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GSH. Prolonged incubation by 48 h enhanced the toxicity. Fig. 3B shows the cytotoxic effects of DMAs(V) and GSH depletion reduced the cytotoxicities. By contrast, as shown in Fig. 3C, toxic effects of iAs(III) were enhanced substantially by depletion of GSH. Effects of exogenously added DMPS, DTT, and GSH on the cytotoxic effects of DPAs(V) and iAs(III) in HepG2 cells depleted and not depleted of GSH Effects of sulfhydryl (SH) compounds on the cytotoxic effects of arsenic compounds were investigated in cells depleted and not depleted of GSH. As shown in Fig. 4A, none of 0.5 mM DMPS, 0.75 mM DMPS, 0.5 mM DTT, and 5 mM GSH when incubated for 24 h were toxic to cells depleted and not depleted of GSH. By contrast, as shown in Fig. 4B, when 1.25 mM DPAs(V), which by itself was not cytotoxic, was incubated in combination with 0.5 or 0.75 mM DMPS for 24 h, cytotoxicity was enhanced markedly and almost all cells died. Combinations with 0.5 mM DTT

Fig. 4. Effects of sulfhydryl (SH) compounds on the cytotoxic effects of DPAs(V) and iAs(III) in cultured HepG2 cells depleted and not depleted of glutathione. Cells, seeded at a density of 1  104 cells/well in 96-well plates and incubated for 24 h, were incubated in medium with or without 1.0 mM BSO for 6 h. The cells were placed in medium that contained arsenic compounds in the presence or absence of SH compounds, then incubated for 24 h. The cytotoxic effects were assessed by WST-8 assay as described in Materials and methods. Data are given as percent of untreated control and represented as means F SEM (n = 3).

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Fig. 5. Structural changes in chromosomes in V79 cells exposed to DPAs(V). Cells, seeded at a density of 2  104 cells/cm2 in 6-cm dish and preincubated for 24 h, were placed in fresh medium that contained 2.5 mM DPAs(V) and then incubated for 24 h. After incubation, chromosome preparations were made as described in Materials and methods.

and 5 mM GSH also caused enhancement of the toxicity by 1.25 mM DPAs(V). In addition, Fig. 4C showed enhancement of the cytotoxic effects of 2 mM DPAs(V), which by itself was only slightly toxic to the cells, by combinations with DMPS, DTT, and GSH. By contrast, as shown in Fig. 4D, depletion of GSH caused marked enhancement of the cytotoxic effects by 20 AM iAs(III). However, when iAs(III) was combined with 0.75 mM DMPS, the enhanced cytotoxic effects in GSH-depleted cells were inhibited completely. Likewise, the presence of DTT and GSH partly reduced the enhanced toxicity caused by iAs(III) in GSH-depleted cells. Structural and numerical changes of chromosome in cultured V79 cells exposed to DPAs(V) DPAs(V) was not found to be mutagenic in the Ames test using Salmonella typhimurium (our unpublished data). Therefore, in the present study, inducibility of chromosome structural aberrations was investigated to determine whether DPAs(V) has clastogenic potential on V79 cells. In addition,

numerical changes in the chromosome were investigated to obtain evidence on the inducibility of aneuploidy and polyploidy. V79 cells were used instead of HepG2 cells as the number of chromosome has remained stable at 21 or 22 during long-term culture and it was therefore easy to evaluate structural and numerical changes. Fig. 5 shows the types of chromosome aberrations caused by DPAs(V). Chromatid gaps and chromatid breaks were induced by incubation with 2.5 mM DPAs(V) for 24 h. C-mitosis that was often observed when cells were incubated with microtubule drugs, such as colchicine, was also induced with a high incidence. Table 1 demonstrates the time and concentration dependence of the induction of chromosome structural aberrations. When incubated for 24 h, the structural aberrations, consisting mainly of chromatid gaps and chromatid breaks, were induced in a concentration-dependent manner. By contrast, the incidence of aberrations decreased when the duration of incubation was prolonged for 48 h. In addition, DPAs(V) caused an increase in the mitotic index. When incubated for 24 h, the incidence reached a maximum

Table 1 Induction of chromosome structural aberrations and mitotic arrest in V79 cells exposed to DPAs(V) Treatment with DPAs(V)

Types of aberrations (%) CG

CB

24 h Control 0.5 mM 1.0 mM 1.5 mM 2.0 mM 2.5 mM

1 0.7 1.7 2.3 9.7 12.0

F F F F F F

0 0.6 0.6 0.6 1.5 2

0.3 0.3 0.3 1.7 3.3 3.7

F F F F F F

48 h Control 0.5 mM 1.0 mM 1.5 mM 2.0 mM 2.5 mM

0.7 0.3 1.7 2.3 3.7 6.0

F F F F F F

0.6 0.6 0.6 0.6 0.6 2.6

0.3 0.3 0.3 1.7 0 1

F F F F

Percentage of aberrant metaphase

Mitotic index (%)

1.7 7.3 16.4 20.3 16.1 9.5

F F F F F F

0.5 0.6* 1.7* 2.7* 0.8* 2.2*

2.1 13.3 9.8 9.7 6.1 2.3

F F F F F F

0.8 2.2* 1.4* 2.0* 0.8* 0.7

Pulv

ERD

0.6 0.6 0.6 0.6 1.5 1.2

0 0 0 0 0 0.3 F 0.6

0 0.3 F 0.6 0 0 0.3 F 0.6 1.3 F 1.5

1.3 1 2 4 12.3 16.7

0.6 0.6 0.6 0.6

0 0 0 0 0.7 F 0.6 0.3 F 0.6

0 0.3 0 0.3 0.3 0.3

1 0.7 1.7 4 4.7 7.7

F1

F 0.6 F 0.6 F 0.6 F 0.6

F F F F F F

F F F F F F

0.6 0 1 1 2.5* 2.5*

1 0.6 0.6 2 0.6* 2.5*

Cells, seeded at a cell density of 2  104 cells/cm2 in 6-cm Petri dish and preincubated for 24 h, were placed in fresh medium that contained DPAs(V) for specified times. After incubation, chromosome preparations were made as described in Materials and methods. One hundred metaphase cells were scored for the determination of chromosome structural aberrations. The data shown are means F SEM (n = 3). CG: chromatid gaps; CB: chromatid breaks; Pulv: pulverizations; ERD: endoreduplications. * Significance of difference between the treated groups and control ( P value < 0.05, Student’s t test).

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Fig. 6. Numerical changes in chromosomes in V79 cells exposed to DPAs(V). Cells, seeded at a density of 2  104 cells/cm2 in 6-cm dish and preincubated for 24 h, were placed in fresh medium that contained DPAs(V) and then incubated for specified times. After incubation, chromosome preparations were made as described in Materials and methods. Fifty metaphase cells were scored to determine the distribution of the chromosome number. Data shown are representative of results from two separate experiments.

at 1.5 mM DPAs(V), then decreased. After 48-h incubation, the increased incidences were lower than those at 24 h. Fig. 6 shows numerical changes of chromosomes resulting from exposure of V79 cells to DPAs(V). No losses and

gains of chromosomes were observed when incubated for 24 h. However, when incubated at 1.0 –2.0 mM for 48 h, some hyper-diploid, hypo-tetraploid, and tetraploid cells appeared.

Fig. 7. Induction of mitotic centrosome abnormality and multipolar spindles in V79 cells exposed to DPAs(V). Cells, seeded at a cell density of 2  104 cells/ cm2 in eight-chamber slides (Nunc) and preincubated for 24 h, were placed in fresh medium that contained DPAs(V) and then incubated for 24 h. The cells on the slides were stained for h-tubulin (microtubules), g-tubulin (centrosomes), and chromosomal DNA as described in Materials and methods.

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Table 2 Induction of abnormalities of centrosome integrity and spindle organization in cultured V79 cells exposed to diphenylarsinic acid (DPAs) Treatment with DPAs (mM) 6h Control 0.5 0.75 1.0

24 h Control 0.5 0.75 1.0 1.5 2.0

Percentage of mitotic cells with various number of g-tubulin signals 1

2

3

0.3 F 0.6 7.3 F 2.7 10.3 F 1.3 Microtubule disruption

99.0 F 1.0 86.3 F 3.2 72.7 F 3.2

0.7 F 0.6 1.8 F 1.3 12.7 F 2.1 12.5 F 1.7 9.9 F 1.1 Microtubule disruption

98.7 72.9 66.5 66.0 47.3

F F F F F

1.2 1.9 7.2 1.7 4.1

0.7 F 0.6 2.7 F 1.1 9.0 F 1.7

0.7 14.1 10.8 5.9 17.0

F F F F F

0.6 3.6 3.2 1.2 1.5

Percentage of mitotic cells with multiple signals of g-tubulin

4

>5

0 3.7 F 1.4 3.7 F 1.9

0 0 4.3 F 0.8

1.0 F 1.0 13.7 F 3.2* 27.3 F 3.2*

0 8.4 6.1 8.5 8.1

0

1.3 27.1 33.5 34.0 52.7

F F F F

2.6 2.4 1.5 1.2

2.9 4.2 7.1 17.6

F F F F

1.0 1.9 1.5 5.5

F F F F F

1.2 1.9* 7.2* 1.7* 4.1*

Cells, seeded at a cell density of 2  104 cells/cm2 in eight-chamber slides (Nunc) and preincubated for 24 h, were placed in fresh medium that contained DPAs(V) and then incubated for specified times. The cells on the slides were double stained for h-tubulin and g-tubulin as described in Materials and methods. One hundred mitotic cells were scored for determination of the number of tubulin-tubulin signals. The data are given as the percent of mitotic cells with a centrosome abnormality and represented as means F SEM (n = 3). * Significance of differences between the treated groups and control (P value < 0.05, Student’s t test).

Abnormalities of centrosome integrity and spindle organization in V79 cells exposed to DPAs(V) Increase in mitotic index by DPAs(V), as demonstrated in Table 1, led us to predict that the arrested cells may have an abnormality of centrosome integrity and a resultant abnormality of mitotic spindles. Therefore, centrosome integrity was evaluated by immunofluorescence of g-tubulin, the protein component of microtubule organizing centers (MTOCs) in centrosomes, and h-tubulin of the spindles. Fig. 7 shows g-tubulin signals, h-tubulin signals, and chromosomal DNA in cells arrested in the mitotic phase by incubation with 0.5 mM DPAs(V) for 24 h. The arsenical caused formation of tripolar or quadripolar spindles colocalized with multiple g-tubulin signals, without microtubule disruption. Table 2 showed time and concentration dependence of the induction of abnormality in the mitotic centrosome by DPAs(V). When incubated for 6 h, the abnormality increased by 0.5 and 0.75 mM DPAs(V) in a concentrationdependent manner. However, the abnormality was not observed at 1.0 mM as the microtubules (spindles) were disrupted. By contrast, when the duration of incubation was prolonged for 24 h, the incidences of abnormalities of centrosome and spindles further increased by 0.5 – 1.5 mM DPAs(V) in a concentration-dependent manner. No disruption of the microtubules by DPAs(V) was observed at the concentration of 1.5 mM.

Discussion Diphenylarsinic acid [DPAs(V)], a stable pentavalent oxidation product of diphenylcyanoarsine or diphenyl-

chloroarsine, was found in abnormally high concentration in ground water in an area of Kamisu-machi. DPAs(V) is a diphenylated analogue of pentavalent dimethylarsinic acid [DMAs(V)], a methylated metabolite of inorganic arsenics. It was therefore considered that the biological effects of DPAs(V) may be similar to those of DMAs(V). In earlier studies, it was shown that DMAs(V) caused cytotoxicity (Ochi et al., 1994), genotoxicity (Oya-Ohta et al., 1996), apoptosis (Ochi et al., 1996), and abnormalities of mitotic centrosome integrity and spindle organization (Ochi et al., 1999) in cultured mammalian cells. However, depletion of cell GSH reduced the activities of DMAs(V), suggesting that GSH was needed for the induction of a variety of toxic effects by DMAs(V), and that highly toxic chemicals were generated by interaction of DMAs(V) with GSH (Ochi et al., 1994, 1996; Oya-Ohta et al., 1996). Studies have also shown that DMAs(III), a reduced form of DMAs(V), was 10 000 times more potent than DMAs(V) in causing the toxic effects (Ochi et al., 2003). Taking these findings into consideration, the cytotoxic effects of DPAs(V) were investigated in conjunction with the effects of GSH depletion. As shown in Fig. 3, depletion of GSH by BSO markedly enhanced the cytotoxic effects of iAs(III) on cultured human HepG2 cells, suggesting that cell GSH plays a role in defense against iAs toxicity. By contrast, the depletion of GSH significantly reduced the cytotoxic effects of DPAs(V) and DMAs(V). The results suggest that GSH is necessary for the expression of toxic effects caused by both arsenicals, and also that highly toxic chemicals were generated by the interaction of the arsenicals with GSH. In addition, as shown in Fig. 4, the fact that the combination of treatments for DPAs(V) and SH compounds was highly toxic to cells,

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although each of these compounds alone was not toxic to cells, strongly supported the hypothesis that highly reactive chemicals were generated by the interaction of DPAs(V) with SH compounds, such as GSH, DTT, and DPMS. By contrast, the cytotoxicity of iAs(III), which had been enhanced substantially by GSH depletion, was completely suppressed by the presence of DMPS, and partly by the presence of DTT and GSH. Among SH compounds, only DMPS has chelating activity owing to the formation of a ring mercaptide by two adjacent sulfhydryl groups. In contrast, it was considered that GSH and DTT suppressed iAs(III) toxicity by forming metal-mercaptide. When we consider the remedy for arsenic intoxication, especially intoxication by aryl- and alkylarsenic compounds such as DPAs(V) and DMAs(V), we must keep in mind the fact that arsenic toxicity is enhanced remarkably by combination with chemicals that have been used as effective remedies for the treatment of inorganic arsenic poisoning. Hereafter, in vivo studies must be conducted on the effects of SH compounds on experimental animals intoxicated with DPAs(V). The mechanism for enhancement by SH compounds of the cytotoxic effects of DPAs(V) is unclear. However, when we considered that trivalent DMAs was 10 000 times more potent than pentavalent DMAs in causing cytotoxic effects, structural and numerical changes of chromosomes, and abnormalities of centrosomes and spindles (Ochi et al., 2003), reduction may be important in association with the activation of DPAs(V). Indeed, most GSH was oxidized to glutathione disulfide (GSSG) by in vitro incubation of DPAs(V) and GSH (data not shown). An approach to identify highly reactive chemicals that can be generated by the interaction of DPAs(V) with GSH or other SH compounds is under investigation using the techniques of high-performance liquid chromatograph (HPLC)-mass spectrometry (MS) and HPLC-inductively coupled plasma (ICP) MS. At the present stage, the toxic principle has not been specified, but chemical analysis of the substances formed by the incubation of DPAs(V) with GSH at various ratios is under investigation in parallel with the toxic effects. DPAs(V), not only by itself, but also in combination with SH compounds, such as GSH and DMPS, was not mutagenic in the Ames test using S. typhimurium. The results suggest that DPAs(V) is not an effective inducer of gene mutation. By contrast, DPAs(V) caused chromosome structural aberrations. However, the effects were limited to 2.0 – 2.5 mM of DPAs(V), which were highly toxic concentrations, and moreover, the incidence of aberrations was not high relative to the potency of DMAs(III). In addition, DPAs(V) caused losses or gains of chromosomes in low incidences when incubated for 48 h, suggesting that DPAs(V) is a weak clastogen and aneuploidogen. Both increases in mitotic index and aneuploidy suggest the induction of non-disjunction of mitotic chromosomes by DPAs(V). However, the mechanism is unclear. On the other hand, centrosomes play a pivotal role as microtubule orga-

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nizing centers (MTOCs) in the maintenance of various microtubule functions in interphase cells and also an equal segregation of chromosomes via spindle function in the mitotic phase (Brinkley, 1985; Kellog et al., 1994). Moreover, centrosomes have gained attention recently as a command center in the regulation of progress of the cell cycle (Doxsey, 2001). Accordingly, abnormality of centrosome integrity caused by DPAs(V) and the resultant induction of multipolar spindles may be responsible for the induction of aneuploidy and polyploidy via abnormality of cell division.

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