Genetic toxicology of a paradoxical human carcinogen, arsenic: a review

Mutation Research 488 (2001) 171–194

Review

Genetic toxicology of a paradoxical human carcinogen, arsenic: a review A. Basu, J. Mahata, S. Gupta, A.K. Giri∗ Division of Human Genetics and Genomics, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta 700032, India Received 11 October 2000; received in revised form 6 February 2001; accepted 11 February 2001

Abstract Arsenic is widely distributed in nature in air, water and soil in the form of either metalloids or chemical compounds. It is used commercially, as pesticide, wood preservative, in the manufacture of glass, paper and semiconductors. Epidemiological and clinical studies indicate that arsenic is a paradoxical human carcinogen that does not easily induce cancer in animal models. It is one of the toxic compounds known in the environment. Intermittent incidents of arsenic contamination in ground water have been reported from several parts of the world. Arsenic containing drinking water has been associated with a variety of skin and internal organ cancers. The wide human exposure to this compound through drinking water throughout the world causes great concern for human health. In the present review, we have attempted to evaluate and update the mutagenic and genotoxic effects of arsenic and its compounds based on available literature. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Mutagenicity; Genotoxicity; DNA damage; Arsenic; Review

1. Introduction Arsenic is a naturally occurring metalloid that exists in inorganic as well as organic forms. The major inorganic forms of arsenic include the trivalent meta arsenite and the pentavalent arsenate. The majority of arsenic in surface water exists as As5+ and in ground water in deep anoxic wells as As3+ . The trivalent arsenic is more toxic than the pentavalent form. The organic forms are the methylated metabolitesmonomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and trimethylarsine oxide (TMAO). The source of arsenic is geological. Arsenic is released ∗ Corresponding author. Tel.: +91-33-473-0492; fax: +91-33-473-5197/0284. E-mail address: [email protected] (A.K. Giri).

to the environment through natural weathering of arsenic-rich geological forms, pesticide use, mining, manufacturing, burning of fossil fuels and incineration. The majority of humans are chronically exposed to low levels of arsenic, principally through ingestion of food and water and to some extent due to inhalation of arsenic in the ambient air. Incidents of arsenic contamination in the ground water have been reported from widespread areas such as Taiwan, Mexico, Chile, Argentina, Thailand, Bangladesh and India. Minor cases of chronic arsenic toxicity have occurred in Poland, USA (Minnesota and California), Canada (Ontario), Hungary and Japan. It has been reported that earth arsenic concentration increases with depths of less than 22 m and decreases at depths of over 22 m [1]. According to WHO, the preferred level of arsenic in water is ≤10 ␮g/l and the

1383-5742/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 ( 0 1 ) 0 0 0 5 6 - 4

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maximum permissible limit accepted by the WHO is 50 ␮g/l. Arsenic has been found to be above the permissible limit in seven districts of West Bengal, India, with concentrations ranging from 200–600 ␮g/l according to latest reports. It has been regarded as the biggest arsenic calamity in the world [2]. In contrast to most other carcinogens, vast human epidemiological studies on arsenic poisoning are available. Treatment with arsenic alone, however, does not easily induce cancer in animals. Chronic ingestion of high levels of inorganic arsenic in drinking water is associated with increased incidence of human cancer at various sites such as skin, lung, bladder and other internal organs [3]. In the six arsenic affected districts of West Bengal, India about 175,000 people are showing arsenical skin lesions that are the late stages of manifestation of arsenic toxicity [2]. The clinical manifestations of chronic arsenic poisoning are many but the most commonly observed symptoms include arsenical skin lesions, melanosis, conjunctivities, keratosis and hyperkeratosis. Cases of gangrene in limbs and malignant neoplasms have been observed [2]. We have been working on the mutagenic and genotoxic effects of different environmental chemicals and drugs [4–9]. There is no report on the genetic damage induced by arsenic through drinking water from West Bengal, India. Very recently we started working on the assessment of the cytogenetic damage in the patients showing arsenical skin lesions from the populations exposed to arsenic through drinking water in West Bengal, India. Considering the widespread reports of carcinogenicity in human beings, we recognised the need to review and update the mutagenic and genotoxic effects of arsenic based on available literature. This review takes into account in vivo and in vitro experimental studies as well as human observational studies.

2. Mutagenicity assays Table 1 represents the summary of results of the short-term mutagenicity assays of arsenic and its compounds available so far in the literature. As far as mutagenicity of arsenicals is concerned it appears to be nonmutagenic in bacterial and standard mammalian cell mutation assays which measure mutation at single gene loci [10]. However, inorganic arsenic has

been found to potentiate the mutagenic action of UV and a number of other mutagenic agents [11]. In general, arsenic does not appear to cause point mutations [12–15]. Arsenic compounds were found to be negative in the Ames assay [16,17]. Arsenobetaine, the predominant form of arsenic occurring in marine fishery products, was found to be nonmutagenic using the Salmonella typhimurium strains TA97, TA98 and TA100 without activation or after addition of a liver-enzyme fraction or gut-flora extract by Jongen et al. [18]. The compound was also negative in the forward mutation assay of the HGPRT gene in the V79 Chinese hamster cells. Kharab and Singh [19] inferred that sodium arsenite was virtually ineffective as a gene conversion agent but gave a positive result for reverse mutation in Saccharomyces cerevisiae. In reversion assays with Escherichia coli a positive result [20–22] was reported for sodium arsenite however, sodium arsenate, octyl ammonium methyl arsonate and dodecyl ammonium methyl arsonate gave negative results [23,24]. Absence of arsenite mutagenicity was also reported in E. coli and Chinese hamster cells by Rossman et al. [25]. In the E. coli system, Trp+ revertants were not induced by arsenite in a variety of experimental protocols including spot test, treat and plate protocols, and fluctuation tests. Moreover, arsenite did not cause induction of lambda prophage. In Chinese hamster cells, arsenite did not induce ouabain-resistant mutants or thioguanine-resistant mutants. When added to the plating medium, arsenite can act as an anti-mutagen by inhibiting the induction of the error-prone SOS repair system in E. coli [25]. Enhancement of UV-mutagenesis by low concentrations of arsenite in E. coli WP2 strain was also reported by Rossman [26]. The optimum enhancing (comutagenic) effect was seen at 0.2 mM concentration. At higher concentrations, the comutagenic action of arsenite decreases. No comutagenic effect was observed with arsenate [26]. Mutagenicity of dimethylated metabolites of inorganic arsenicals was investigated by Yamanaka et al. [27] using E. coli B tester strains. When H/r30R (wild type; Exc+ Rec+ ) and Hs30R (uvrA− ; Exc− Rec+ ) cells were incubated with DMA(V) for 3 h, many more revertants appeared in sealed tubes than in the control, but this was not the case in unsealed tubes, suggesting that the reaction product(s) between

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dimethylarsine and molecular oxygen is responsible for the mutagenesis. Okui and Fujiwara [28] analysed the comutagenic effects of inorganic arsenic in V79 Chinese hamster cells. When the cells were treated with small doses of As2 O3 (0.5 ␮g/ml) and Na2 HAsO4 (5 ␮g/ml), the compounds enhanced the UV induction of 6-thioguanine-resistant mutations of V79 cells thus exhibiting comutagenic effect. Comutagenesis of sodium arsenite with UV radiation in V79 cells was also studied by Li and Rossman [14]. Irradiation of Chinese hamster V79 cells with UVA (360 nm), UVB (310 nm) and UVC (254 nm) caused a fluence-dependent increase in mutations at the HPRT locus. Non toxic concentrations of arsenite increased the toxicity of UVA, UVB and UVC. Arsenite acted as a comutagen at the three wavelengths. Treatment of G12 cells for 24 h with sodium arsenite resulted in mutagenesis at the transgenic gpt locus, but the increase was not significant at P < 0.05. Arsenite was found to enhance the mutagenesis of UVC and MNU [29]. Walker and Bradley [30] and Ahmed and Walker [31] reported that sodium arsenate exerts some effect on cross over frequencies characterised by increases in single exchange classes in Drosophila. An experiment was conducted by Rasmuson [32] to study the mutagenic effects of sodium arsenite in a somatic eye-colour test system in Drosophila melanogaster. It was observed that arsenite caused a reduction of the MMS-induced mutation frequency of red somatic spots in the eyes of adult males. The Drosophila wing spot test (SMART) was used by de la Rosa et al. [11] to study the modulating action of inorganic arsenic on the recombinogenic and mutagenic effects of the alkylating agents ethylnitrosourea (ENU), methyl methane sulphonate (MMS), and ethylene oxide (EO) as well as gamma rays. It was found that arsenic exerted an inhibitory effect on mitotic recombination induced by alkylating agents and gamma irradiation. Mutagenicity of sodium arsenite and sodium arsenate was tested in germinal and somatic cells of Drosophila by Ramos-Morales and Rodriguez-Arnaiz [33]. In the sex-linked recessive lethal test (SLRLT) both the compounds were found to be positive, sodium arsenite was an order of magnitude more mutagenic and toxic than sodium arsenate. Tripathy et al. [34] tested sodium arsenite and sodium arsenate at low levels and obtained negative results in the Drosophila wing spot test.

Sram and Beneko [35] using the dominant lethal test (DLT) in mice evaluated the genetic risk of exposure of mammalian germ cells to arsenic after an acute oral administration of 250 mg/kg and a chronic administration during four generations. Arsenic trioxide does not induce dominant lethals in male germ cells at the dosage range of 1/40 to 1/4 LD50 in hybrid mice (CBA X C57B1/6J)F1 [36]. Treatment with effective dose of thioTEPA followed by injection of As2 O3 inhibited the mutagenic activity of thioTEPA. Deknudt et al. [37] also obtained negative results in the dominant lethality assay on male germ cells in vivo in mice using sodium meta arsenite (NaAsO2 ). Wiencke et al. [38] reported that sodium arsenite produces gene mutations (large scale rearrangements, frameshifts and base pair substitutions) in the supF gene using pZ189 shuttle vector system in DNA repair proficient GM637 human fibroblast. They also observed a synergistic effect in supF mutation induction with arsenic and UV light. Arsenite is a dose dependent mutagen, and it induces large deletion mutations. Co-treatment of cells with DMSO significantly reduce the mutagenicity of arsenite in AL cell assay [10]. A pilot biomarker study was conducted by Harrington-Brock et al. [39] to investigate the effects of arsenic exposure on the frequency of HPRT-mutant lymphocytes in a population of copper roasters in Chile. Individuals were classified into three potential exposure groups: high, medium and low. An increased mutant frequency was observed in the highly exposed groups. However, conclusive information regarding the arsenic level typically present in the environment was not available from this study.

3. DNA damage assay A number of publication are available on the effect of arsenic on DNA damage in different test systems. Table 2 represents the summary of results of DNA damage of arsenic and its compunds available so far in literature. Arsenic trichloride, sodium arsenite, sodium arsenate and dimethldithiocarbamate cause damage to DNA as shown by the rec assay [21,22,40,41] but negative results were obtained with sodium methylarsonate and calcium methylarsonate [41]. Rossman et al. [42] have shown that arsenite

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inhibited post-replication repair pathway in E. coli. Strains of E. coli, differing from each other in one or more repair functions were exposed to UV light and then plated in the presence or absence of arsenite. Survival after irradiation of wild type E. coli (WP2 ) was significantly decreased [42]. The modifying effects of arsenic on UV-induced cytotoxicity for Hs30R and NG30 cells (E. coli) was examined by Okada et al. [43]. Their results indicate that arsenite and arsenate enhance error-free excision repair of UV-damaged DNA by retarding the DNA replication and by prolonging the period for excision repair. Two reports are available on DMA(V)-induced DNA damage in mouse and rat lung cells by Yamanaka et al. [44,45]. Induction of DNA damage was found to be due to both active oxygen and dimethylarsenic peroxyl radical produced in DMA(V) metabolism. Preferential increase of heterochromatin clumping was noted by Nakano et al. [46] in the endothelial nuclei of alveolar wall capillaries of lung in mice after administration of DMA(V). This was thought to be the result of damage of RNA transcription due to protein binding to DNA. Yamanaka et al. [47] reported crosslink formation between DNA and nuclear proteins by in vivo and in vitro exposure of cells to dimethylarsinic acid(V). Sodium arsenite did not induce DNA damage in rat lung cells. Yamanaka and Okada [48] observed the induction of lung specific DNA damage by methylated arsenics via the production of free radicals. Brown and Kitchin [49] suggested that sodium arsenite is more likely to be a nongenotoxic carcinogen than a genotoxic carcinogen. DNA strand breaks in bovine aortic endothelial cells were investigated by Liu and Jan [50] by a 4 h treatment with sodium arsenite at sublethal concentrations. Treatment with arsenite increased nitrite production suggesting that arsenic increased NO may react with O2 ∗ – to produce peroxynitrite and cause DNA damage. The effect of arsenite on the DNA repair of UV irradiated Chinese hamster ovary cells was studied by Lee-Chen et al. [51,52]. They suggested that arsenite may exert its cogenotoxic effect by inhibiting DNA repair. On exposure to UV or MMS, DNA strand breaks were induced in CHO cells. When the cells were allowed to recover in a drug free medium the strand breaks gradually disappeared. Post-treatment with sodium arsenite retarded the disappearance

of UV-induced DNA strand breaks and inhibited postreplication repair in UV irradiated cells. Li and Rossman [53] reported that arsenite can inhibit the DNA ligase II activity in vitro in V79 cells. Both the constitutive and MNU inducible levels of DNA ligase II activity were inhibited. They concluded that DNA ligase II was more sensitive to arsenite-induced inhibition than DNA ligase I [53]. Gebel et al. [54] using V79 Chinese hamster cells in comet assay showed that As (III) mediated generation of DNA protein crosslinks and DNA strand lesions. Piperakis and Mc Lennan [55] treated HeLa cells with ethanol and sodium arsenite before infection with UV irradiated adenovirus-2. They reported enhanced reactivation of the damaged virus due to inhibition of DNA synthesis caused by these agents. The presence of As2 O3 for 24 h after UV irradiation inhibited the excision of thymine dimers from the DNA of normal and XP variant cells. This result provided the first evidence that arsenic inhibited the excision of pyrimidine dimers [28]. The effect of trivalent arsenic on DNA synthesis and induction of unscheduled DNA synthesis (UDS) were evaluated in cultured human lung fibroblasts by Chang [56]. It was found to inhibit DNA synthesis when the cells were treated for 2 h. Dong and Luo [57] observed that sodium arsenite-induced UDS in human fetal lung fibroblasts. Sodium arsenite at concentrations of 1, 5 and 10 ␮M increased UDS value, indicating that arsenic directly damaged DNA but did not inhibit DNA repair. A series of experiments were carried out by Yamanaka et al. [58–60] using human embryonic cell line type II alveolar epithelial cells to evaluate the induction of DNA damage by DMA(V). In 1990 they treated the cells with dimethyl arsine and found presence of single strand breaks (SSBs) of DNA. Similarly in 1995 they observed SSBs after 9 and 12 h of 10 mM DMA(V) treatment. The occurrence of DNA–protein cross-linking in the cultured cells was also observed. In 1997, Yamanaka et al. [60] assayed arsenite, MMA(V) and DMA(V) for the induction of DNA damage by determining DNA repair synthesis using polymerisation inhibitors. DNA SSBs resulting from inhibition of repair polymerisation were produced by exposure to DMA(V) at 5–100 ␮M, while not by that to arsenite and MMA(V). Human alveolar type II (L-132) cells were exposed to 10 mM DMA(V) for 10 h by Tezuka et al. [61] to determine

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DNA SSBs. This suggested the possibility of DNA adduct formation. Kato et al. [62] incubated L132 human alveolar cells with 5, 7.5 and 10 mM DMA(V) at 37◦ C for up to 12 h and observed concentration dependent DNA strand breaks. Effects of arsenic on the persistence of mutagen-induced DNA lesions in human cells were observed by Hartmann and Speit [63]. Human blood and SV40 transformed fibroblasts were treated with MMS for 2 h followed by incubation with sodium arsenite for 2 h. MMS-induced concentration related DNA damage in fibroblasts. When incubated without arsenic the effects were reduced in the cells. Post treatment with arsenite inhibited the repair process. Hartwig et al. [64] investigated the effect of arsenic (III) on nucleotide excision repair (NER) after UV irradiaton in human fibroblasts. The results show that two steps of NER are affected. Furthermore, both the global genome repair pathway and the transcription-coupled repair pathway are affected by arsenite. Arsenite induces DNA–protein crosslinks and cytokeratin expression in the WRL-68 human hepatic cell line [65]. By single cell gel (SCG) assay Hartmann and Speit [66] detected more DNA damage in cells treated with sodium arsenite (in concentrations ranging from 200–1500 ␮M) than the control culture expressed as increasing length of DNA migration. Schaumloffel and Gebel [67] also observed a significant induction of DNA damage with 0.01 ␮M As (III) by SCG assay. As (III) proved to be a potent inducer of DNA–protein crosslinks. Hu et al. [68] examined several different types of human cells in culture (HOS cells, AG06 SV40-transformed keratinocytes, and W138 fibroblasts) and observed that arsenic-induced DNA repair inhibition is not the result of direct enzyme inhibition. Arsenic inhibits only a few enzymes involved in DNA repair at physiologically relevant concentrations (micromolar or less). The authors further reported that out of eight enzymes tested only pyruvate dehydrogenase, an enzyme not involved in DNA repair, is inhibited by low micromolar concentrations of As (III), but not As (V). Lynn et al. [69] used single cell alkaline electrophoresis to detect DNA strand breaks in human vascular smooth muscle cells treated with arsenite at a concentration above 1 ␮M/l for 4 h. According to them, NADH oxidase is activated by arsenite to produce superoxide which then causes oxidative DNA damage.

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4. Cytogenetic assays Table 3 summarizes the cytogenetic assay results as measured by chromosomal aberrations (CA), sister chromatid exchanges (SCE) and micronucleus formation (MN). Arsenic compounds have been shown to induce CA or abnormal cell divisions in animal and plant cells [70]. Mitotic indices and CA were observed in maize root tips [71] and in Allium cepa [72]. Genotoxic evaluation of arsenic trioxide using Tradescantia micronucleus (Trad-MN) assay in soil and aqueous media was done by Gill and Sandhu [73]. Arsenic trioxide yielded clastogenic responses in both the media. SCE in Vicia faba root tips were also used to examine well water containing high levels of arsenic [74]. Cytogenetic effects of arsenic have been studied in different rodents using CA, MN, and SCE assays. Pashin et al. [36] observed a significant increase in MN in erythrocytes of bone marrow cells of hybrid mice, following i.p. injection of As2 O3 . In vitro test systems were employed by Muller et al. [75] to study MN in mouse embryo by sodium arsenite. A minimum dose of 0.7 ␮M of sodium arsenite was effective in inducing MN formation. Deknudt et al. [37] observed that sodium arsenite given by i.p. injection produced a clear dose response relationship in micronuclei in bone marrow polychromatic erythrocytes of mice from 0.5 up to 5 mg/kg which is about half of the reported oral LD50 value of As3+ in mice and rats [76]. Tinwell et al. [77] studied the effects of sodium arsenite, potassium arsenite, and Fowler’s solution on induction of MN. The authors reported that all the three compounds are equally active in mouse bone marrow MN assay. The effect of hepatic methyl donor status in male B6C3F1 mice treated with sodium arsenite was studied by Tice et al. [78]. Bone marrow erythrocyte micronucleus assay was used to assess chromosomal damage. Treatment with sodium arsenite once or four times in hepatic methyl donor sufficient and deficient mice induced a significant increase in the frequency of micronucleated polychromatic erythrocytes. Synergistic effects could not be demonstrated by Poma et al. [79] in somatic and germ cells of mice given a combined treatment of arsenic trioxide solution (12 mg/kg body weight) with EMS (200 mg/kg body weight). Moore et al. [80] tested sodium arsenite, sodium arsenate, pentavalent monomethylarsonic acid (MMA) and pentavalent dimethylarsinic acid

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Table 3 Cytogenetic assays induced by arsenica Test systems

Specific forms of arsenic

Lowest effective dose

End points

Effects

References

Zea mais

p-Methyl-phenyl-arsonic acid p-Chlorophenyl-arsonic acid p-Nitrophenyl-arsonic acid Methl chlorophenyl arsonic acid Arsenic trioxide Arsenic Arsenic trioxide Alkyl arsonic acid Dialkyl arsinic acid Alkylaminotrimethyl arsonic acid Arsenic trioxide Arsenite Arsenate DMA(V) MMA(V) Arsenobetaine Arsenite Arsenate Arsenite Arsenite Arsenite Arsenite Arsenite Arsenite Arsenite Arsenite Arsenite Arsenite Arsenite Arsenite Arsenate Arsenite Arsenite Arsenite Arsenite Arsenite Arsenite Arsenite DMA(V) DMA(V) Arsenite Arsenite Arsenate Arsenite Arsenite Arsenite Arsenic trioxide dissolved in potassium bicarbonate Arsenic trioxide Arsenite Arsenic trioxide Arsenite

NC NC NC NC 4 ␮g/ml 0.267 mg/l NA NC NC NC 28.5 mg/m3 2.0 ␮g/ml 14.0 ␮g/ml 10,000 ␮g/ml 4500 ␮g/ml 2 mg/cm3 NC NC 5 ␮M 10 ␮M 10 ␮M NC NC NC NC 10 ␮M NC NC 50 ␮M 10−5 M 10−5 M NC 20 ␮M 40 ␮M NC 40 ␮M 10 ␮M NC NC NC NC NC NC NC 1.0 mg/kg 1.0 mg/kg 1.0 mg/kg

CA CA CA CA MN SCE CA CA CA CA CA CA, MN CA, MN CA, MN CA, MN CA, SCE CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA MN SCE, MN MN MN MN CA, SCE, MN MN CA CA CA, SCE CA, SCE CA, SCE MN MN MN MN

+

[71] [71] [71] [72] [73] [74] [79] [161] [161] [161] [81] [80] [80] [80] [80] [126] [91] [91] [12] [93] [93] [100] [101] [99] [97] [98] [96] [103] [104] [106] [106] [105] [107] [108] [102] [110] [111] [109] [112] [113] [12] [114] [114] [37] [77] [77] [77]

1/40 LD50 2.5 mg/kg 250 mg/l 2.5 mg/kg

MN MN CA CA

Allium cepa Tradescantia Vicia faba root tips Mouse germ and somatic cells Mouse fibroblast cells

Mouse fetal cells, in vivo Mouse lymphoma cell, in vitro

Mouse BALB/c 3T3 cells CHO cells

CHO-XRS-5 cells CHO-K1 cells

CHO (XRS5, XRS6 and CHO-K1) V79 cells SHE cells

Mouse bone marrow cells, in vivo

+ + + − + + +

+ +

+ + + + + + + + + +

+ +

[36] [78] [79] [83]

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179

Table 3 (Continued) Test systems

Mouse embryos Sheep Human lymphocytes, in vitro

Human lymphocytes, in vitro

Human lymphocytes, in vitro Human skin fibroblasts Human fibroblasts

Human exfoliated bladder cells, in vivo

Specific forms of arsenic

Lowest effective dose

End points

Arsenite DMA(V) Arsenite

0.1 mg/kg 300 mg/kg 2.5 mg/kg 2.5 mg/kg 100 ␮M NA 3 × 10−5 g m/ml NC 0.05 ppm 0.05 ppm 0.05 ppm 0.05 ppm 0.2 ppm NA NA NC NC 0.5 ␮M 5 ␮M 10−10 ␮M 0.001 ␮M 10−6 M 2 × 10−6 M NC NC NC 0.1 ␮g/ml 2.5 ␮g/ml NC NC 0.5 ␮M 5 ␮M 2 × 10−4 M 5 × 10−7 M 5 × 10−7 cM 5 × 10−7 M NC NC NC 0.5 ␮M 0.5 ␮M NC NC NC NC 0.8 × 10−5 M 1.6 × 10−5 M 0.7 × 10−4 M 1.4 × 10−5 M 3.7 × 10−2 M 1.25 ␮M NA NA

CA CA CA CA MN SCE CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA SCE SCE SCE SCE SCE SCE SCE SCE SCE SCE SCE SCE SCE SCE SCE SCE SCE SCE SCE MN CA CA CA CA CA CA CA CA CA MN MN MN

Arsenite Inorganic arsenic Arsenate Arsenite Arsenic trichloride Arsenic trioxide Arsenic pentaoxide Arsenic acid Disodium hydrogen arsenate Arsenic in the atmosphere Arsenate; arsenite Arsenic Arsenite Arsenite Arsenite Arsenite Arsenite Arsenate Arsenite Arsenate Arsenite Arsenite; Arsenate Inorganic arsenic Arsenite Arsenite Arsenite Arsenite Arsenite Arsenate DMA(V) MMA(V) DMA(V) TMAO Arsenite Arsenic (III) Arsenite Arsenite Arsenite Arsenite Arsenite Arsenate DMA(V) MMA(V) TMAO Arsenite Inorganic arsenic Inorganic arsenic

Effects

+ − +

+

+ + +

+ +

References [84,85,86] [82] [87,88] [89] [75] [90] [132] [133] [131] [131] [131] [131] [131] [135] [120] [163] [101] [115] [116] [117] [118] [119] [119] [120] [120] [122] [121] [121] [163] [123] [115] [116] [66] [125] [125] [125] [124] [124] [124] [147] [67] [93] [127] [101] [128] [129] [129] [129] [129] [129] [130] [151] [152]

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Table 3 (Continued) Test systems

Human lymphocytes, in vivo

Human lymphocytes, in vivo

Human lymphocytes, in vivo

Specific forms of arsenic

Lowest effective dose

End points

Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Arsenic trioxide Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic Inorganic arsenic

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

MN MN CA CA CA CA CA CA CA CA SCE SCE SCE SCE SCE SCE SCE MN MN MN MN

Effects

+

− +

− +

References [154] [80] [133] [134] [136] [137] [139] [140] [142] [138,143] [144] [149] [145] [146] [147] [143] [148] [137] [150] [148] [139]

a (+): positive effects; (−): negative effects; CA: chromosome aberrations; SCE: sister chromatid exchanges; MN: micronuclei formation; NC: not consulted in original; NA: not applicable.

(DMA) for their relative mutagenic and clastogenic potential, using mouse lymphoma assay. Sodium arsenate and MMA(V), but not sodium arsenite and DMA(V)-induced micronuclei formation. Sodium arsenite, sodium arsenate and MMA(V) are clastogenic. DMA(V) induced a slight increase in gross aberration frequency. Both the inorganic arsenicals induced polyploidy and endoreduplication. The organic arsenicals were much less potent as clastogenic agents than the inorganic arsenicals. An increase in CA in the fetal chromosome of mouse due to exposure of 28.3 mg/m3 As2 O3 was observed by Nagymajtenyi et al. [81]. Kashiwada et al. [82] observed that DMA(V) increased mitotic indices significantly and induced aneuploids. They suggested that DMA(V) may cause mitotic arrest in vivo as well as in vitro. Several reports are available on the genotoxic effects of arsenic in vivo in mice as well as the reduction of the toxic effects by some natural products and also with some other compounds [83–89]. In all the reports treatment with arsenic induced a significant increase in CA in vivo in Swiss albino mice. Four reports are available showing reduced clastogenic

effect of arsenic by garlic extract [83–86]. Similarly, Biswas et al. [87] observed protection against the cytotoxic effects of arsenic by dietary supplementation with crude extract of Emblica officinalis fruit in Swiss albino mice. They also reported reduction in arsenic genotoxicity through dietary intervention of selenium in vivo in Swiss albino mice [88]. Recently, Poddar et al. [89] observed that ferrous sulfate, given together with or before exposure to sodium arsenite, significantly reduced the effects of the latter in vivo in mice. Assessment of a possible genotoxic environmemtal risk in sheep bred on grounds with strongly elevated contents of arsenic in Germany was done by Gebel et al. [90]. In the SCE analysis there was no significant increase in the different sheep collectives. This indicates that the transfer rate of genotoxic compounds of arsenic from the environment is too low to register effects with SCE. A number of authors have reported significant increase in CA and SCE in CHO cells in vitro. Wan et al. [91] observed the cytotoxic and cytogenetic effects of sodium arsenite and sodium arsenate. CA was induced by both the arsenic compounds. They

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further concluded that trivalent arsenic was more clastogenic than pentavalent arsenic. SCE was also increased by sodium arsenite. Lee et al. [92] carried out a series of experiments to study the potentiating effects of sodium arsenite. Cytotoxicity and chromosomal aberrations were synergistically increased by incubating the UV irradiated CHO cells in medium containing arsenite. The effect of arsenite on CAs varied with cell-harvesting time and it decreased with increasing time intervals between UV and arsenite treatments. They further observed that pretreatment with sodium arsenite overcomes the inhibition of mitosis and cell proliferation but has no apparent effect on the clastogenicity in MMS treated CHO cells. Thus, pretreatment does no harm and may even benefit the MMS treated cells. Post-treatment of sodium arsenite was found to be cogenotoxic [93]. To see if arsenite enhances the clastogenicity of DNA crosslinking agents, CHO cells were exposed to cis-diamminedichoroplatinum(II) plus UV light and then to sodium arsenite. The results indicate that the clastogenicity of cis-Pt (II) plus UV are enhanced by the post treatment [94]. Post-treatment with sodium arsenite during G2 phase enhanced the frequency of CAs induced by S-dependent clastogens such as 4-nitroquinoline 1-oxide in CHO cells [95]. Jan et al. [96] observed that CAs induced by EMS in CHO cells were potentiated by subsequent exposure to sodium arsenite. This coclastogenicity was most effective when arsenite was applied 3 or 6 h immediately after removal of EMS. The authors further studied sodium arsenite mediated synergistic increase in chromosome aberrations induced by alkylating agents such as methyl-nitrosourea (MNU), methyl-methanesulfonate (MMS), ethyl-methanesulfonate (EMS), ethyl-nitrosourea (ENU) [97]. Similar potentiating effect by sodium arsenite at a dose of 10 ␮M was reported by Lin and Tseng [98]. The frequencies of CAs scored in the form of chromatid break, chromatid exchange, chromosome breaks and chromosome rings, induced by direct acting mutagen N-nitroso-2-acetylaminofluorene (N-NO-AF) was significantly increased in the presence of sodium arsenite. Cytotoxicity of UV-light in CHO cells was found to be potentiated by sodium arsenite [99]. Positive result of coclastogenic effect of sodium arsenite in increasing CA induced by EMS was also investigated by Huang et al. [100,101]. In CHO-K1 cells chromosomal aberrations induced by

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UV radiation was enhanced by post treatment with sodium arsenite after 24 h [102]. In 1993, Huang et al. [103] used glutathione (GSH) as cellular defence against arsenite toxicity in cultured CHO cells. A 2 h pretreatment of CHO cells with GSH reduced the clastogenicity and cytotoxicity of arsenite. The enhancing effects of arsenite on CAs and cell destruction induced by UV were also reduced. GSH given after arsenite treatment decreased the cellular arsenic content and increased the cell survival, but did not reduce the clastogenicity of arsenite. The authors further established that cells in the transition stages from late G1 to early S phase are most vulnerable to the coclastogenic effect of treatment with UV radiation and arsenite. A significant dose dependent induction of CA in the form of chromatid breaks and tetraploidy were observed when CHO cells were incubated with 0–100 ␮m arsenite during the G2 phase. Arsenite also retarted reentry of mitotic cells into interphase [104]. Wang and Huang [105] while analysing the induction of micronuclei by arsenite in CHO-XRS-5 cell line, reported that XRS-5 cells are more sensitive to arsenite in terms of MN formation than the parental CHO-K1 cells and concluded that MN is induced via the overproduction of H2 O2 by arsenite. Sodium salts of trivalent and pentavalent arsenic were tested by Kochhar et al. [106] for their effect in inducing CAs and SCEs in CHO cells. It was discovered that arsenite (As3+ ) produced excessive endoreduplication of the chromosome at higher levels. The potential protective effect of squalene on sodium arsenite-induced MN in CHO-K1 cells was studied by Fan et al. [107]. The CHO-K1 cells treated with sodium arsenite induced a dose-dependent increase in MN frequency. However, cotreatment with 80 ␮M of squalene significantly inhibited the formation of MN. Involvement of calcium dependent protein kinase C in arsenite-induced genotoxicity in CHO-K1 cells was investigated by Liu and Huang [108]. The authors found that the calcium channel blocker, Verapamil can potentiate arsenite-induced MN. When extracellular calcium was depleted during arsenite treatment, the induced MN formation was significantly suppressed. These data indicated that the calcium ion plays an essential role in arsenite-induced genotoxicity. Recently, Wang et al. [109] studied the role of catalase and glutathione peroxidase in the cellular defence mechanism against the genotoxicity of arsenite in different CHO cell lines

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(XRS5, XRS6, and CHO-K1). Cells were treated with 0–160 ␮M arsenite for 4 h and counted after 72 h. MN frequencies of 10.8, 4.5, and 2.1 micronuclei per 1000 binucleated cells per ␮M arsenite respectively was observed. The authors concluded that the elevated glutathione peroxidase activity in CHO-K1 cells was associated with decreased arsenite toxicity. According to Lynn et al. [110], who observed MN formation in CHO-K1 cells by arsenite treatment, arsenite stimulated poly ADP-ribosylation by generation of nitic oxide and exerted its toxicity by generating reactive oxygen species. Similarly, Gurr et al. [111] reported that calcium-dependent NO generation is involved in arsenite-induced MN in CHO-K1 cells. Mechanism of mitotic arrest and tetraploidy induced by nine organic and three inorganic arsenic compounds was studied by Endo et al. [112] in cultured Chinese hamster V79 cells. They incubated V79 cells with 50 or 100 ␮l of various concentrations of the test compounds for 8 h at 37◦ C. Mitotic indices were scored by counting metaphases in 500 cells. They observed that DMA(V)-induced tetraploids and mitotic arrest. The authors further concluded that DMA(V) directly inhibited cell division and DMA-induced tetraploid may be an important part of arsenic genotoxicity. The cytotoxic effect of DMA(V)-induced tetraploidy and the inhibitory role of selenium was reported by Ueda et al. [113] in V79 cells. Two in vitro reports are available on effect of arsenic in inducing cell transformation, cytogenicity and cytotoxicity in Syrian Hamster Embryo (SHE) cells. Lee et al. [99] evaluated the genetic effects of two arsenicals (tri and penta) at same doses. They observed that cytogenetic effects like endoreduplication, CA and SCE were formed with similar dose response. They further concluded that trivalent arsenicals were more clastogenic. Exactly similar findings were obtained by Barrett et al. [114]. Specificity of arsenite in potentiating cytogenetic damage induced by DNA crosslinking agent diepoxybutane (DEB) was measured by Wiencke and Yager [115]. DEB treatments alone increased SCE frequencies and CAs. The yields of chromatid deletions and exchanges in lymphocytes exposed to both arsenite and DEB were markedly increased above the levels expected if the effects of the two agents had been simply additive. Genotoxic effects of sodium arsenite in human lymphocyte culture were also studied by Jha et al. [116] both alone and in combination with X-rays.

It was found that sodium arsenite induced chromatid type aberrations as a function of concentration. Synergistic enhancement of X-ray-induced chromosomal damage was also observed. Vega et al. [117] carried out investigations on aneugenic effect of sodium arsenite in human lymphocytes. The blood cultures were incubated for 72 h and treated with various concentrations of sodium arsenite for the last 24 h. The number of chromosome in 200 metaphases of first and second division cells were scored. They concluded that sodium arsenite has an aneuploidogenic and mitotic arrestant effect. The mechanism of induction of aneuploid cells by disruption of microtubule assembly and spindle formation by sodium arsenite was evaluated by Ramirez et al. [118] in cultured human lymphocytes. Sister chromatid exchange in human lymphocytes in vitro was evaluated by several authors. Zanjoni and Jung [119] stated that the addition of Na2 HAsO4 induced a significant dose dependent increase in SCE. The effects of arsenite and arsenate compounds on SCE were investigated by Nordenson et al. [120] and Crossen [121]. The rate of SCEs was found to be increased by the authors after cultured human lymphocytes were exposed to trivalent arsenic. Further, Crossen [121] has concluded that although arsenic can induce SCEs in human lymphocytes, there is considerable variation in SCE response among individuals. Sodium arsenite-induced SCE was investigated by Wen et al. [122] in cultured lymphocytes of 13 Blackfoot disease patients and observed a significant increase of baseline SCE among the exposed individuals compared with controls. Sister chromatid exchanges (SCE) has also been examined by Sahu et al. [123] in human lymphocytes following in vitro treatment with sodium arsenite. It produced significant increase in SCE frequencies over the levels for untreated lymphocytes. A dose dependent response in SCE rate was reported by Hartmann and Speit [66]. Treatment of cells with NaAsO2 for 2 or 24 h beginning 48 h after the start of the blood culture in concentrations from 2 × 10−4 to 1.5 × 10−3 M caused a small but significant SCE induction at the highest concentration. Iwami et al. [124] examined the genotoxic effects of organic arsenic compounds by assessing induction of SCE. Human lymphocyte cultures were exposed to methylarsonic acid(V), dimethyl-arsinic acid(V) and trimethyl-arsine oxide. Mitotic arrest and aneuploidy were found to be significantly increased.

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A study was undertaken by Rasmussen and Menzel [125] to compare the genotoxic effects of arsenite in cultured human lymphocytes and lymphoblastoid cell lines from a group of normal humans. Cultures were exposed for 40 h to sodium arsenite. SCE assays revealed a dose dependent increase over the concentration range of 10−7 to 10−5 M. Comparison of SCE frequency showed substantial variation in sensitivity to arsenite. When pooled data from the primary lymphocytes were compared to that obtained from the lymphoblastoid cells, the slopes of the dose response curves for arsenite-induced SCEs were similar. It appears that arsenic can significantly induce CA, SCE and MN in various mammalian cells both in vivo and in vitro. Kaise et al. [126] used the mammalian cell culture technique to investigate the cytotoxicological aspects of organic arsenic compounds present in marine products. These compounds exhibited a low ability to induce CAs at a concentration range of 2–10 mg cm−3 and no SCE was observed at a concentration of 1.0 mg cm−3 . The CAs caused by arsenobetaine at a concentration of 10 mg cm−3 consisted mainly of chromatid gaps and chromatid breaks. Like all other studies on human cell lines, fibroblast cells also showed positive clastogenic effects with arsenic. Earlier, Happle and Hoehn [127] noted CA in fibroblast culture established from tumor tissue. A study on the effect of sodium arsenite as an enhancer of clastogenicity and CA induction by DNA crosslinking agent cis-diammino-dichloroplatinium II plus long wave UV in human fibroblasts (HFW) was conducted by Lee et al. [99]. A significant increase in chromosome breaks and exchanges were noted. Moreover, Huang et al. [101] have stated that posttreatment with sodium arsenite has synergistic effect on CA induced by ethylmethanesulphonate (EMS) in log phase only but not in stationary phase. Later, in 1995, Huang et al. [128] arrested HFW cells in the S-phase and then allowed these to synchronously progress into G2 phase. The G2 enriched HFW were treated with sodium arsenite at a dose of 0–200 ␮M. They observed that sodium arsenite was capable of producing cell cycle arrest at G2 phase, interference with mitotic division, inhibition of spindle assembly and chromosome endoreduplication in the second mitosis. Clastogenicity of variety of arsenic compounds on cultured human fibroblast were examined by Oya-Ohta et al. [129]. Among all the compounds,

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DMA(V) was found to be very potent and produced chromosome pulverizations. All arsenicals produced gaps and breaks. The authors have ranked the different compounds in terms of clastogenic potency as arsenite > arsenate > DMA(V) > MMA(V) > TMAO. Effects of exposure protocols on induction of kinetochore-plus and -minus micronuclei by arsenite in diploid human fibroblasts was observed by Yih and Lee [130]. HFW were treated with 1.25–10 ␮M arsenite for 24 h (low dose and long exposure) and 5–80 ␮M for 4 h (high dose and short exposure). Arsenite induced mainly kinetochore-positive MN by low dose exposure whereas kinetochore-negative MN was induced by high dose exposure. A comparative study of CA induced by tri and pentavalent arsenic in cultured human leucocytes was reported by Nakamuro and Sayato [131]. The chromosome breaking activity was much higher for the compounds with trivalent (NaAsO2 , AsCl3 and As2 O3 ) than with pentavalent arsenic (Na2 HAsO4 , H3 AsO4 and As2 O5 ). Petres et al. [132,133] conducted chromosome analysis of lymphocytes from patients who had been exposed to arsenic. In another study the occurrence of CAs was studied in lymphocytes from nine workers exposed to arsenic at the Ronnskar smelter in Sweden [134]. Nordenson et al. [135,136] studied CA patients previously treated with arsenic in comparison with patients with no such previous treatment. In all the above cases, a significant increase in CA was observed in exposed patients when compared with the control. Hu [137] also reported an increase in CA frequency among smelter workers in Yunnan Tin Corporation, China. A pilot study was carried out by Ostrosky-Wegman et al. [138] on individuals exposed to arsenic in Mexico; 11 of them were chronically exposed and 13 had lower exposure to the metal. The percentages of CAs were similar in both the populations, although complex aberrations were more frequent in the highly exposed group. Gonsebatt et al. [139] have shown the difference in cytogenetic effects of arsenic exposure among populations with different degrees of exposure. A group of inhabitants of Santa Ana (408.17 ␮g/l of As in drinking water) were considered the exposed individuals and some inhabitants of Nazareno (29.88 ␮g/l) were cosidered as controls. The frequencies and types of CAs were studied in lymphocytes while the presence of MN were studied in exfoliated epithelial cells obtained

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from oral mucosa and urine samples. Exposed individuals showed an increase in the frequency of chromatid deletions in lymphocytes and of MN in oral and urinary epithelial cells. Males were more affected than females. Similarly, increased frequency of CAs was also reported by Maki-Paakkanen et al. [140] in a study on 42 individuals exposed to arsenic through well waters in Finland. The median concentration of arsenic in the wells was 410 ␮g/l. Increased arsenic exposure, indicated best by increased concentrations of arsenic in urine, was associated with increased CAs. Black foot disease is associated with the consumption of arsenic containing well water [141]. A cytogenetic survey was conducted in a blackfoot endemic area in Taiwan by Liou et al. [142]. A cohort of 686 residents was recruited from three villages. During a 4-year follow-up period, 31 residents developed cancer. Among those who did not develop cancer, 22 subjects were selected as control. The frequencies of total chromosome-type aberrations, were significantly higher than those in the control group. The only negative result in chromosome aberration study was reported by Vig et al. [143]. They carried out a 2-year survey on human subjects who had been drinking water containing more than 0.05 mg/l arsenic for a period of at least 5 years. Burgdorf et al. [144] reported an elevated SCE rate in the lymphocytes of six patients treated with arsenic. The arsenic exposed patients had a mean of 14.00 SCE/cell while 44 normal controls had a mean of 5.8 SCE/cell. Lerda [145] conducted a study of cytogenetic abnormalities in persons drinking arsenic contaminated water. They observed that people who drink water containing arsenic at the concentrations of 0.13 mg/l or more exhibit an increased frequency of SCEs in their lymphocytes. In another study Hantson et al. [146] noted mean SCE frequency and the population of high frequency cells (HFC) in the peripheral blood lymphocytes of four patients who ingested high doses of arsenicals (150 mg KAsO2 , 1, 10 and 20 g As2 O3 ). The mean frequency of SCEs/cell was affected only after the highest dose. The clastogenic effects of arsenic were studied in lymphocyte cultures of 15 Bowen’s disease patients from arseniasis-hyperendemic villages in Taiwan [147]. Blood samples from 34 healthy subjects served as matched control. Notably higher spontaneous SCEs and HFCs were found in patients compared to controls.

Although the majority of the studies show positive clastogenic effects in populations exposed to arsenic, a few authors have reported negative results. Vig et al. [143] concluded that arsenic at concentrations (>0.05 mg/l) used by the sample population, in a 2-year study, showed no effects on SCE frequency. In another investigation on native Andean women and children from Northwestern Argentina, exposed to high levels of arsenic (0.2 mg/l) in drinking water, Dulout et al. [148] did not find any notable effect on the frequencies of SCEs. Several lines of evidence indicate that arsenic acts indirectly with other agents to enhance specific genotoxic effects that may ultimately lead to carcinogenesis. However, Yager and Wiencke [149] stated that arsenite does not effect the induction of SCE induced by a DNA cross-linking agent 1,3-butadiene diepoxide. Three reports are available on the increased incidence of MN in human lymphocytes in vivo. Hu [137] reported an increased MN frequency in cultured lymphocytes among smelter workers in Yunnan Tin Corporation. Nilsson et al. [150] carried out MN study on a population exposed to arsenic in Srednogorie (Bulgaria). The third report was provided by Dulout et al. [148] who observed an increase in MN in the lymphocytes among native children and women of northwestern Argentina exposed to high level of arsenic via drinking water (0.2 mg/l) in contrast to controls. Warner et al. [151] compared the frequency of micronucleated cells in exfoliated bladder and buccal cells between a group of 18 individuals in Nevada who chronically ingested high levels of inorganic arsenic from their well-water (1312 ␮g/l) and an individually matched control group with low exposure to arsenic (16 ␮g/l). A 1.8-fold increase was observed in the mean frequency of micronucleated bladder cells in the exposed group (2.79/1000 cells) compared with the unexposed group (1.57/1000 cells). Moreover, the frequency of micronucleated bladder cells was positively associated with the urinary concentration of arsenic. In the same study, exfoliated cell micronucleus assay using fluorescent in situ hybridisation (FISH) with a centromeric probe was utilised by Moore et al. [152] to detect the presence or absence of centromeric DNA in MN induced by chronic arsenic ingestion. Frequencies of micronuclei containing acentric fragments and those containing whole chromosomes both increased. In 1997, Moore et al. [153] investigated

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the relationship between arsenic ingestion and genetic damage to the urothelium in two cross-sectional biomarker studies, one in Nevada and one in Chile. In both studies they found that increased levels of micronucleated cells in exfoliated bladder cells were associated with elevated concentrations of arsenic in drinking water. Biggs et al. [154] conducted a cytogenetic survey to determine the relationship of urinary arsenic to intake estimates using bladder cell micronuclei as a biomarker of effect. The study group consisted of 232 persons living in northern Chile whose drinking water supply were contaminated with arsenic. Among them 124 persons drank water containing arsenic at a concentration of 670 ␮g/l while 108 persons consumed arsenic at a concentration of 15 ␮g/l. Urine samples were analysed for total arsenic and arsenicals. Exfoliated bladder cells were analysed for micronuclei. It was found that the proportion of micronucleated cells increased with increased exposure index.

5. Discussion As far as mutagenicity of arsenic is concerned it appears to be largely non-mutagenic in bacterial test systems that measure mutation at single gene loci. Arsenic showed no mutagenic effects in Salmonella both with and without metabolic activation [16–18]. Contradictory reports are available on the mutagenicity in other bacterial species (E. coli). It was found to be negative [25] in one case and positive in another [27]. In yeasts, arsenic gave a positive result for reverse mutation even though it was ineffective as gene convertogen [19]. Arsenic was demonstrated to be comutagenic with UV radiation in V79 cells [14,28] but it lacked this potentiating effect in Drosophila [11,32]. Reports on mutagenicity in mouse were also negative [36,37]. However, in human fibroblasts and lymphocytes arsenic was found to induce supF and HPRT mutations [38,39]. Sodium arsenite at relatively non-toxic concentrations (5 ␮M for 24 h or 10 ␮M for 3 h) was also comutagenic with N-methyl-N-nitrosourea (MNU) at the hprt locus in V79 cells [155]. Arsenic and its metabolites can induce DNA damage in multiple test systems both in vivo and in vitro [45,47,48,50,57,62,66,67,156]. Several reports on DNA SSBs indicate that arsenic and its compounds are capable of inducing DNA strand breaks

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[57–59,61,107]. Arsenic also leads to DNA synthesis inhibition [55,56], DNA repair inhibition [28,52,54,63] and retardation of DNA replication [43]. Thus, the positive results reported in the in vitro and in vivo assays indicate that arsenicals can interact with DNA indirectly leading to CA, SCE and MN in populations exposed to arsenic. Several studies show that inorganic arsenic induce structural [157] and numerical [92,118,128] chromosome changes, cell transformation [92,128] and gene amplification [158] in mammalian cells in culture. However, arsenite was unable to induce amplification in SV40 transformed human keratinocytes in vitro [159]. Some metabolites of arsenic have been observed to cause numerical chromosome changes in mammalian cells in vitro [160]. King and Lunford [161] studying the effect of different arsenicals on mouse fibroblasts, observed CA and spindle aberrations. It has been suggested that arsenite causes endoreduplication by inhibition of the protein phosphatase activity [128] and hyperdiploidy by disruption of microtubule function [118]. The coclastogenic effects of arsenicals have been highlighted in various in vitro experiments using CHO cells where CA induced by UV or other alkylating agents was synergistically increased [94,97,102]. The genotoxicity of arsenic has been proved in V79 cells [112] and SHE cells [114] also. Mass [162] in his review clearly indicated that arsenic is a genotoxic agent that induces CA, MN and SCE in mammalian cells as well as neoplastically transformed SHE cells. Almost all the results of in vitro assays of CA, SCE and MN induced by arsenic in various mammalian cells showed positive clastogenic effects [116–125,132,133,163]. Cytogenetic assays in mice gave positive results in most cases, demonstrating the aneuploidigenic and mitotic arrestant effect of different arsenicals. Genotoxic effects of sodium arsenite were also carried out in human lymphocytes. All arsenicals produced gaps and breaks [129]. The inorganic arsenicals were more potent clastogens, the trivalent arsenic being more potent than the pentavalent form [131]. Leonard and Lauwerys [164] in their review stated that most of the studies performed on Zea mais, Allium cepa and Drosophila as well as observations on the ability of arsenic to induce, in vitro and in vivo, chromosomal aberrations in mammalian cells provided positive results.

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Studies on cytogenetic assays in populations with arsenic exposure clearly indicate positive clastogenic effects on human lymphocytes in vivo [134,142,145,150]. The majority of exposed populations who had a history of relatively long periods of exposure showed higher incidences of CA [134,136,138], SCE [144,147] and MN [137,148]. Significantly increased rate of CA and MN was also found in human fibroblasts [99,130] and human exfoliated bladder cells [151,154]. In most of the cytogenetic assays of arsenic exposed populations the effects on MN were more pronounced than either CA or SCE. Gonsebatt et al. [139] reported a higher incidence of MN in exfoliated urothelial and buccal cells than CA in lymphocytes of exposed individuals. This was attributed to the fact that the exfoliated cells are in direct contact with arsenic. They also observed that exposed males had higher frequencies of CA and MN than females. This is because men worked at the fields and hence drank more water than women. Implications of arsenic genotoxicity for the dose response of carcinogenic effects were thoroughly reviewed by Rudel et al. [165]. Epidemiological data relating arsenic ingestion and skin and internal cancers strongly suggest a sublinear or threshold relationship. They have evaluated the molecular basis for sublinearity in light of new data and hypotheses regarding arsenic genotoxicity and chemical carcinogenesis. Except SCE, sublinear dose-response relationships for arsenic-induced CAs were observed repeatedly in different mammalian and human cell systems. They concluded that arsenic indirectly induces genetic damage, thus providing a biological basis for a sublinear dose-response relationship for human cancer. Very recently, we have started working on the assessment of genetic damage in the populations exposed to arsenic through drinking water in West Bengal, India [166]. We have studied the cytogenetic effects in patients showing arsenical skin lesions as measured by micronuclei (MN) formation in buccal mucosa cells, urothelial cells, lymphocytes and CA and SCE in whole blood lymphocyte cultures. Exposed individuals with arsenical skin lesions showed a statistically significant increase in the frequency of MN in oral mucosa, urothelial cells and lymphocyte when compared with control. There was a definite correlation between the arsenic content particularly in the nail and hair and the incidence of MN formation in the exposed populations. Results of CA and SCE are

under progress [166]. Arsenic is believed to be a progressor facilitating the transition from benign to malignant tumours [160,163]. As is characteristic with progression, chromosomal instability has been induced as a result of arsenic exposure. Analyses on humans occupationally exposed to arsenic indicate that arsenic appear to be active late in the carcinogenic process [167]. Only some studies on the genotoxicity of arsenic in humans are contradictory. Some studies did not find cytogenetic effects in individuals exposed to arsenic via drinking water [140,144]. The negative result of Vig et al. [144] may be due to low exposure level (0.05 mg/l) and shorter duration of study. Similarly, the negative result in the SCE study by Dulout et al. [148] may be due to the very small sample size (n = 22). The reason why Ostrosky-Wegman et al. [138] obtained negative result may be attributed to the fact that actual intake of arsenic for several of the studied individuals could be lower than was previously assumed [149]. Chen et al. [168] reported that arsenic trioxide can be effectively applied to the treatment of acute promyelocytic leukemia (APL). Their results showed that arsenic trioxide triggers relatively specific NB4 cell apoptosis at micromolar concentration. This apoptotic effect was selective because neither cell growth inhibition nor cell death increase was noted in HL-60 and U937 cells. It does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. Arsenic trioxide also induced a significant modulation of the PML (Promyelocytic leukemia) staining pattern (indirect fluorescence staining of PML using antiserum specific to the N-terminal region of PML) in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR␣ in NB4 cells disappear after treatment with arsenic trioxide, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. A low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after treatment of arsenic trioxide. A similar PML staining pattern is observed in apoptotic cells. All-trans retinoic acid (ATRA) pretreatment does not influence arsenic trioxide-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of arsenic trioxide [168].

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Arsenic compounds are the only compounds that the IARC considers to have sufficient evidence for human carcinogenicity, but inadequate evidence for animal carcinogenicity [76]. Several attempts to induce carcinomas in animals by arsenic either failed or provided very limited evidence for carcinogenesis [169]. Although transgenic strains of mice hold some promise, but in general there is no good animal model for arsenic-induced tumorigenesis [170]. The failure to demonstrate the relationship between arsenic exposure and rodent cancer may result from inappropriate design of rodent carcinogenicity bioassay [153]. Humans may be more sensitive to arsenic than other species because arsenic methylation in humans is less efficient than in other species [171]. Very high doses of arsenic compounds are required for tumour production in rodents. A few reports of successful arsenic carcinogenesis exist in rats and mice [169]. Wei et al. [172] demonstrated that long-term p.o. administration of high dose of DMA(V)-induced urinary bladder carcinomas in male F344 rats. Therefore, the results indicate that DMA is carcinogenic for the rat urinary bladder, which may be related to the human carcinogenicity of arsenicals [172]. However, there is no analog to this exposure in humans. Arsenite and DMA were detected to be skin carcinogens, a tissue relevant to humans, in K6/ODC transgenic mice [173]. Germolec et al. [174] studied the influence of arsenic in humans with arsenic skin disease and on mouse skin tumour development in transgenic mice. After low dose application of tetradecanoyl phorbol acetate (TPA) a marked increase in the number of skin papillomas occurred in Tg. AC mice, which carry the v-Ha-ras oncogene, that received arsenic in the drinking water as compared with control drinking water, whereas no papillomas developed in arsenic-treated transgenic mice that did not receive TPA or arsenic/TPA-treated wild-type FVB/N mice. They concluded that arsenic enhanced skin neoplasia development via the chronic stimulation of keratinocyte-derived growth factors. Thus, arsenic acts a co-promoter in chemical carcinogenesis [174]. Since most of the animal experiments provided negative results and some even reported a decrease in tumour induction by arsenic [169] it seems reasonable to suggest that arsenic might act as a cocarcinogen, or tumour promoter rather than an initiating direct carcinogen with a no-threshold effect [169].

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The mechanism by which arsenic compounds cause human cancers are not yet known [169]. Arsenic research in the past has shown that the arsenic does not directly react with DNA or cause gene mutations, except to a small extent at high doses. However, it does cause gene amplification and chromosome damage at lower doses and can enhance mutagenesis by other agents apparently by inhibiting DNA repair [175]. Li and Rossman [53] stated that inhibition of DNA repair is due to the inhibitory effect of arsenite on DNA ligase II and to a lesser extent on DNA ligase I enzymes which is a possible mechanism of its comutagenesis [53]. Hu et al. [68] observed that purified DNA repair enzymes-DNA polymerase ␣, DNA ligase I and II were insensitive to arsenic. They concluded that arsenic-induced DNA repair inhibition is probably not the result of direct enzyme inhibition. It maybe an indirect effect caused by arsenic-induced changes in cellular redox levels or alterations in signal transduction pathways and consequent changes in gene expression. In a new cell transformation system using rat liver epithelial cell line TRL 1215, it was found that chronic exposure to arsenite-induced malignant transformation [176]. This was associated with global DNA hypomethylation, decreased DNA methyl transferase activity, and activation (overexpression) of protooncogene c-myc. These authors suggested that arsenic may act as a carcinogen by causing DNA hypomethylation leading to aberrant gene expression [176]. Mass and Wang [177] studied DNA methylation by arsenite in a small region on the p53 promoter in human lung A549 carcinoma cells. They hypothesized that arsenic-induced carcinogenesis could involve alterations of MTase/SAM-dependent DNA methylation of a tumor suppressor gene. They found that chronic exposure to arsenite caused a progressive increase in CpG methylation within the p53 promoter, which was expected to block transcription of the p53 gene [177]. The p53 tumour suppressor gene, whose protein product plays an important role in cell-cycle control, apoptosis and control of DNA repair, was found to have an altered expression after arsenite treatment [178]. Cells with mutant p53 are more likely to continue to divide, and fail to undergo apoptosis, in spite of DNA damage to their chromosomes [177]. Such cells show greatly elevated rates of CA such as deletions, translocations, amplifications and aneuploidy

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[179,180], exactly the classes of genotoxic events induced by arsenite; p53 protein is normally degraded through ubiquitin-dependent proteolysis [181], a process inhibited by arsenite [182]. The increase in p53 protein after arsenite treatment probably causes continuous over-expression of p53 protein. In the long term, it results in shutting down its expression by hypermethylation of its promoter. Trivalent arsenite may activate nuclear oncogene, perhaps via its ability to bind to vicinal dithiols within a protein, or to bridge two thiols between two proteins [169]. Tumour suppressor fau gene product or some other aspect of ubiquitin system maybe a target for arsenic toxicity and that disruption of the ubiquitin system may contribute to the genotoxicity and carcinogenicity of arsenite [183].

keratotic, premalignant and malignant tissues from arsenic exposed populations. (iv) To establish suitable animal models for arsenic-induced carcinogenesis which will facilitate the study of the mechanism of carcinogenesis in human beings. (v) Specific epidemiological studies are recommended that consider the following elements-characterization of dose-response relationships; characterization of duration response relationships; linkage to mechanistic studies and evaluation of population variability. (vi) To identify individuals with greater genetic sensitivity to arsenic toxicity and carcinogenicity and possibly genotyping of specific genes that may be found to be relevant to arsenic susceptibility. References

6. Conclusion Arsenic is genotoxic with a carcinogenic potential in humans. Arsenic showed rare mutagenic effects in bacterial systems either with or without metabolic activation. From the results of the DNA damage induced by arsenic both in vivo and in vitro it appears that, it induces DNA damage indirectly by inhibiting DNA repair. But the exact mechanism of DNA repair inhibition is still unknown. Results of cytogenetic assays both in vivo and in vitro clearly reflect its behaviour as a highly genotoxic agent. Almost all the results of cytogenetic assays showed positive clastogenic effects. The results of DNA damage and the genotoxicity data indicates that arsenic can induce genetic damage indirectly. Arsenic is believed to be a progressor, facilitating the transition from benign to malignant tumours in humans. This is still a theory that is hotly debated. Further studies are required on the mechanism of action in multiple test systems so that the exact mechanism of action of arsenic genotoxicity and carcinogenicity can be understood. This work is necessary for the monitoring of the harmful effects of arsenic and to find out suitable preventive measures. A few recommendations for arsenic research in future are: (i) Detailed study of the mechanisms of arsenic resistance in mammalian systems. (ii) Effects of arsenic on signal transduction for specific gene expression. (iii) Mutational analysis of tumour suppressor genes like p53 and p16 from the biopsies of

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