Sister-chromatid exchange (SCE) among individuals chronically exposed to arsenic in drinking water

EnvironmentalMutagenesis

ELSEVIER

Mutation Research 312 (1994) 111-120

Sister-chromatid exchange (SCE) among individuals chronically exposed to arsenic in drinking water D. Lerda

*

Bromatology, Hygiene and Environmental Health Direction, 2580 Marcos Judrez, Argentina

(Received 14 February 1993) (Revision received 1 November 1993) (Accepted 19 November 1993)

Abstract A study was carried out on human subjects of various ages and backgrounds who had been drinking water containing more than 0.13 mg/l (0.13 ppm) arsenic for a period of at least 20 years. The main aim was not only to correlate the frequency of sister-chromatid exchanges in the lymphocytes with the amount of arsenic in water and urine but also to correlate the frequency of SCE with sex and age. In addition, family background regarding skin alterations or other arsenic-related symptoms was explored, so that individual health conditions could be assessed. External factors such as exposure to other chemical or contaminating agents (pesticides, battery manufacturing plants, foundries) were also taken into consideration. The data on sister-chromatid exchanges (282 exposed and 155 control individuals) showed that arsenic at concentrations used by our population (0.13 mg/1) induced a significantly elevated response. Other health effects of arsenic at these concentrations were found, e.g., hyperkeratosis, melanosis, actinic keratosis, basal cell carcinoma. Key words." Arsenic; Sister-chromatid exchange; Skin alterations; Drinking water

I. Introduction Arsenic and its compounds in water have repeatedly been observed worldwide. Arsenic may appear in water either due to natural causes or as a byproduct of human activities. Natural causes include: soil erosion, surface rocks and volcanic areas formed during the pre-Andes pre-Rocky Mountains period (as far as South and North America are concerned), a fact wich explains why

* Corresponding author.

the same As-related problem has been detected in Argentina, Chile (Antofagasta) (Ugarte and Donoso, 1978), Oregon (Morton, 1976), and Alaska (Harrington, 1978). ' M a n - m a d e ' sources basically include industrial effluents from As compound manufacturing plants and non-ferrous metal foundries. It should be noted that, even though the latter source of As is quite important in some countries, this is not the case in Argentina. A positive correlation between exposure to higher As concentrations and cancer or skin diseases has been documented (Bencko, 1977; Tsuchiya, 1977; Pershagen, 1981).

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The first observation of the relationship between As and skin cancer was by Ayrton Paris, in Cornwall (1820) (Bishop and Kipling, 1978). In Argentina, the first observations were performed by Goyenechea (1917) and Pusso (1918). The present study is aimed at determining the incidence of cytogenetic injury in individuals consuming drinking water with a high As content.

The arsenic content in urine was determined also by the Sedivec and Vasak colorimetric method using SDDC.

2.4. Sister-chromatid exchange

Drinking water samples from each subject's home were obtained from wells ranging in depth from 20 to 200 m or more. The arsenic content in water was determined by the Sedivec and Vasak colorimetric method (1952), using silver diethyldithiocarbamate (SDDC).

For each subject, two replicate cultures were carried out by incubating 0.3 ml of whole blood in 4 ml of culture medium Ham F10 (Gibco) with fetal calf serum (15%), L-glutamine (2 mM), penicillin (100 units/ml), streptomycin (100 /xg/ml) and phytohemagglutinin (PHA M; 1%). The medium was supplemented with 5'bromodeoxyuridine (BrdUrd) at 100/xM (Sigma). Lymphocytes were cultured in the dark for 72-74 h and metaphases were blocked during the last 4 h with 0.1/xg/ml of colcemid (Gibco). Cells were treated with up to 15 ml of hypotonic KC1 (75 mM) at room temperature for about 8 rain (range 4-10 min), then fixed with freshly prepared, room temperature, methanol:glacial acetic acid (3:1, v/v). After two washes in fixative, the cell suspensions were usually stored in fixative at about 4°C overnight or longer before preparation of air-dried slides. For detection of SCEs, slides were stained by a modified 'fluorescence plus Giemsa' technique (Perry and Wolff, 1974). Slides were stained for 10 min in Hoechst 33258 (5 /zg/ml; American Hoechst) in phosphate buffer (M/15, pH 6.8; Baker), mounted in the same buffer and exposed at 55-65°C on a hotplate to 'black light' from two 15-W tubes for the amount of time required for differentiation between chromatids (5-20 min). Finally, slides were stained with 5% Giemsa (Biolabor) in water, for 5-20 min. Since the concentration of BrdUrd and the number of cells in a culture have both been shown to affect the frequency of SCEs in cultured lymphocytes, an attempt was made to reduce variability by using a high concentration of BrdUrd (100 /~M) (Carrano et al., 1980). For each subject, SCEs in 55 second-division metaphases with more than 44 chromosomes were scored blind on coded slides.

2.3. Urinalysis

2.5. Statistical analysis

During 24 h samples, kept in plastic flasks were collected from each subject in both groups.

SCE/cell mean was analyzed for each individual in both groups. Student's t-test was used to

2. Material and methods

2.1. Samples The arsenic-exposed population included 282 volunteers, non-smokers living in the town of Marcos Jufirez (Province of C6rdoba, Argentine Republic). This population has been consuming drinking water containing arsenic at a concentration of 0.13 m g / l or more, for more than 20 years' time. The control group included 155 volunteers living in the town of Tortugas, from the neighboring Province of Santa Fe (Tortugas is 37 Km East of Marcos Jufirez). All control group volunteers have been drinking water containing less than 0.02 mg/1 of arsenic for more than 20 years' time. External factors such as exposure to other chemical or contaminating agents (pesticides, battery manufacturing plants, foundries) were taken into account. Furthermore, the family background of each volunteer regarding skin alterations or other arsenic-related symptoms was investigated, so that individual health conditions could be assessed.

2.2. Water

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Table 1 Mean, standard deviation and range of age, arsenic in water and urine of the control and exposed groups Population group

n

Age Mean + SD Range

Arsenic in water (rag/l) Mean + SD Range

Arsenic in urine (mg/1) Mean _+ SD Range

Exposed

282

Control

155

56.71 ± 8.42 27.00-82.00 38.90 +_ 3.11 29.00-51.00 p < 0.001

0.13 _+0.09 0.01-0.66 0.02 _+0.02 0.00-0.07 p < 0.001

0.16 _+0.08 0.06-0.41 0.07 ± 0.04 0.01-0.60 p < 0.001

Significance (Student's t)

compare mean contents of arsenic in water, arsenic in urine and SCE between exposed individuals and controls. Pearson's correlation coefficient was calculated among arsenic-related parameters, frequencies of SCE, sex, age and other external parameters such as exposure to chemical or contaminating agents.

3. Results and discussion

The range of arsenic content in well water for the control group was 0.00-0.07 mg/1 while the range for the exposed group was 0.01-0.66 mg/1 (Table 1). The mean As content in water for the control group was 0.02 +_ 0.02 mg/1 while the mean for the exposed group was 0.13 + 0.09 mg/1. Data show a marked difference as regards exposure to As in both groups (p < 0.001). High As levels (0.9-3.4 mg/l) have been detected in villages and towns around Marcos Jufirez (Arguello et al., 1983). The mean age of the control group (38.9 years) was significantly (p < 0.001) lower than that of the exposed group (56.7 years). Exposure level was determined by As content in urine, where control group subjects ranged from 0.01 m g / l to 0.60 mg/1 while exposed sub-

jects ranged from 0.06 mg/1 to 0.41 mg/1 (Table 1). The control group's mean arsenic content in urine was 0.07 m g / l + 0.04 while the exposed individual's mean was 0.16 mg/1 + 0.08. As regards normal As levels in urine, values found in the literature cover a wide margin indeed, probably because of the influence of dietary sources, and differences in analytical methods. Nevertheless the present As level in urine correlated with As content in well water. Harrington et al. (1978) conducted research among a population who drank well water with a high As content. The authors found a group of individuals consuming water whose As content exceeded 0.1 mg/1 (a mean of 0.401 mg/l, with a daily consumption estimated in As of 0.324 mg/l). Their As content in urine was 0.178 mg/1. Another group who drank water with a 0.031 mg/1 As concentration (with an estimated daily consumption of 0.046 mg/1) showed 0.042 m g / l As in urine. In the present study cell cultures for SCE were performed among 437 individuals (282 exposed and 155 controls). The SCE/cell mean is presented in Table 2. The SCE range among the exposed population in this study was 7.23-14.90, a value that is high compared to the usual range of 6-11 SCEs for normal, healthy individuals whose lymphocytes are treated with 100 /~M Br-

Table 2 Mean SCE/cell frequencies in lymphocytes of the control and exposed groups Population group

n

Cells scored

Mean SCE/cell ± SD

Range

Significance (Student's t)

Exposed Control

282 155

15620 8525

10.46 _+ 1.02 7.49 _+0,97

7.23-14.90 5.17-10.87

p < 0.001

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dUrd. Other authors (Lee et al., 1985; Let et al., 1986; Jan et al., 1986; Gomez Arroyo et al., 1988) have also reported a increase in SCE in As-treated plants and animals. The relationships between SCE and age and SCE and sex were studied in order to determine the probable factors that could affect the frequency of SCE. To homogenize the age of the exposed group subjects older than 50 were excluded. In individuals younger than 50 no correla*

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tion between SCE and sex and SCE and age was found. Moreover, the correlation between arsenic in well water and SCE, arsenic in urine and SCE and arsenic in well water and arsenic in urine in individuals of both sexes was studied. Non-predictive positive relations were found (see Figs. 1-6). In the present study, when Pearson's correlation coefficient was applied to calculate arsenic I

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parameters against other external parameters (such as exposure to other chemical or polluting agents), no correlation was found. A most important finding has been our detecting among individuals exposed to the above mentioned As concentrations many chronic arsenicism features, such as hyperkeratosis, melanosis, actinic keratosis and basal cells carcinoma. Moreover, the datas of the C6rdoba Ministry of Public Health (1990) about the causes of death of the populations of

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Marcos Ju~trez in 1970-1988 report a significant increase in deaths due to lung tumor. These results suggest that lung cancer could be associated with ingested arsenic.

4. Conclusions

This is the first cytogenetic study performed in an area next to the city of Bell Ville (Province of

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C6rdoba, Argentina), a city where the first cases of chronic arsenicism have been detected (Astolfi et al., 1981). A higher As mean in water as well as in urine could be detected in the exposed group than in the control group (p < 0.001). The subjects' estimated daily arsenical water consumption was 0.13 mg/l, with a urinary excretion of 0.16 mg/1. In the Province of C6rdoba several towns and vii-

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lages have drinking water with a higher As content ranging from 1.6 mg/1 to 4.5 mg/1, a fact also to be found in other Argentine provinces such as Chaco, Buenos Aires, Salta, Formosa, La Pampa, San Luis, Santa Cruz, Santiago del Estero, and Tucuman with As quantities in water ranging from 0.5 to more that 1 mg/1 (Castro, 1982). Our data regarding individuals that have been exposed to As in their drinking water for

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Fig. 3. Relationship between arsenic in urine and SCE for 60 females. * controls; • exposed; - -

prediction.

D. Lerda /Mutation Research 312 (1994) 111-120

more than 20 years' time suggest that further assessments of the potential effect of As in drinking water on human health are in order, so that realistic standards can be set up. As regards the cytogenetic aspect, according to our findings, an increase in the incidence of genetic harm (as measured by SCE) can be observed. Sex and age did not affect the frequency of SCE.

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Petres et al. (1977) and Nordenson et al. (1978) have reported similar findings (measured as chromosomal aberrations) in exposed individuals. Thus there exists sufficient evidence that As at relatively higher doses favors both mutagenesis and carcinogenesis (Bencko, 1977; Pershagen, 1981). As regards the present study, besides findings

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D. Lerda /Mutation Research 312 (1994) 111-120

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related to a higher As concentration in water and urine, coupled to a SCE increase, emphasis should be placed on skin alterations that have been detected among exposed subjects since evidence about As mutagenic action in humans includes: (a) observations on a higher frequency of some As-related clinical consequences, and (b) direct observations of abnormalities at the genetic ma-

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terial level. In summary, from studies performed in Taiwan, Germany, Chile and Argentine, it can safely be said that a regular consumption of drinking water with an As content higher than 0.1 ppm allows the development of cytogenetic injuries, and, in some cases, skin cancer, besides evidencing clear poisoning. In this research, a carcinogenic effect of ingested arsenic on lung

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D. Lerda / Mutation Research 312 (1994) 111-120 I

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and skin was suggested. Skin lesions, cytogenetic injuries, and cancer depend on: (a) arsenic concentration in drinking water; (b) subjects using exclusively water containing As; (c) duration of exposure; (d) the susceptibility of the individual. In this connection, it is the author's contention that every effort should be made to reduce the

prediction.

possibility of As-contaminated water being consumed.

5. Acknowledgements The author wishes to thank Jorge Alasia, MD (Cintra, Argentine), Rogelio Azcona, MD (Los

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Surgentes, Argentine) and Nobuyuki Hotta, MD (Kumamoto, Japan) for their help in the completion of this work. I am very grateful to Beatriz Massiero of the INTA-EEA, Marcos Ju~irez, Argentine for statistical help.

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Jan, K.Y., R.V. Huang and T.C. Lee (1986) Different modes of action of sodium arsenite, 3-aminobenzamide and caffeine on the enhancement of ethyl methane sulfonate clastogenicity, Cytogenet. Cell Genet., 41, 2-8. Lee, T,C., M. Oshimura and J.C. Barrett (1985) Comparison of arsenic induced cell transformation, cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture, Carcinogenesis, 6, 1421-1426. Let, T.C., K.C. Lee, Y.J. Tzeng, R.Y. Huang and K.Y. Jan (1986) Sodium arsenite potentiates the clastogenicity and mutagenicity of DNA cross linking agents, Environ. Mutagen., 8, 119-128. Morton, W., G. Starr, D. Pohl, J. Stoner, S. Wagner and P. Weswig (1976) Skin cancer and water arsenic in Lane County, Oregon, Cancer, 37, 2523-2532. Norderson, I., G. Beckman, L. Beckman and S. Nordstrom (1978) Occupational and environmental risks in and around a smelter in Northern Sweden. II, Chromosomal aberrations in workers exposed to arsenic, Hereditas, 88, 47-50. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature, 251, 156-158. Pershagen, G. (1981) The carcinogenicity of arsenic, Environ. Health Perspect., 40, 93-100. Petres, J., D. Baron and M. Hagedorn (1977) Effects of arsenic on cell metabolism and cell proliferation: cytogenetic and biochemical studies, Environ. Health Perspect., 19, 223-227. Pusso, A. (in Aymi, M.J.) (1918) La intoxicaci6n cr6nica en nuestro pats, Thesis, C6rdoba, No. 514, 32. Tsuchiya, K. (1977) Various effects of arsenic in Japan depending on type of exposure, Environ. Health Perspect., 19, 35-42. Ugarte, G., and S. Donoso (1978) Primary hepatic tumors in Chile, in: H. Remmer, H.M. Bolt, P. Bamasch and H. Popper (Eds.), Primary Liver Tumors, MTP Press, Lancaster, pp. 165-169. Vasak, V., and V. Sedivec (1952) The colorimetric determination of arsenic, Chem. Listy, 46, 341-344.