Demographic characteristics of the study participants

The summary demographic characteristics and exposure status of the entire study population based in rural West Bengal, India, are shown in Table 1. All the study groups (A–F), selected on the basis of equally spaced classification boundaries with respect to cooked rice arsenic, are similar with respect to their age and gender distribution, body weight and also their tobacco usage. For all groups, the mean arsenic content of drinking water was between 3 and 6 μg/L and the mean drinking water intake between 2.9 and 3.8 L/day. Combined with mean cooked rice intakes of between 540 and 600 g/day, this means that arsenic exposure from drinking water contributed no more than 20% of total dietary exposure for any group and less than 12% of total dietary exposure for the study population taken as a whole.

Table 1 Demographic characteristics and arsenic exposure status in the study population Full size table

Urinary arsenic and exposure from cooked rice

There is a strong correlation (r2 = 0.81) between grouped urinary arsenic and cooked rice arsenic data (Table 1, Fig. 1) confirming the overwhelming importance of rice as the major dietary exposure route in the study population. This relationship was also found to varying degrees of fits to all of: males, females, tobacco-users, non-tobacco users and participants from each of the 3 study areas.

Figure 1 Cross-plot of mean urinary arsenic vs mean cooked rice arsenic. The linear best-fit trendline is indicative only. Error bars represent ± 1 standard deviation for each parameter for each exposure group (A–F). Full size image

Genetic damage status as measured by micronucleus assay

For the whole cohort, MN ranged from 0.50 to 4.98, with a median of 1.91 and an inter-quartile range from 1.56 to 2.56. The whole cohort mean MN was 2.12 ± 0.89 (SD, n = 417). On a grouped basis, MN increased monotonically with mean cooked rice arsenic content from 1.85 ± 0.63 (SD, n = 113) for the lowest cooked rice arsenic group (A) up to 3.23 ± 0.93 (SD, n = 37) for the highest cooked rice arsenic group (F). Preliminary linear regression analysis, using the cooked rice arsenic groups as categorical variables and with adjustment for gender, body weight, tobacco usage and drinking water arsenic concentration, showed that groups with mean cooked rice arsenic >200 μg/kg (D, E & F) each showed significantly higher (p < 0.001) micronuclei frequencies than the lowest exposure group (A), with the coefficients for the predicted increased in MN for each group relative to the reference group (A) as follows: Group B: 0.05 (95% CI −0.15 to 0.25; p = 0.631; Group C: 0.11 (95% CI −0.11 to 0.33; p = 0.338); Group D: 0.66 (95% CI 0.35 to 0.96; p < 0.001); Group E: 0.83 (95% CI 0.50 to 1.15; p < 0.001) and Group F: 1.38 (95% CI 1.09 to 1.68; p < 0.001). Further preliminary statistical analysis (One-way ANOVA with Tukey-Kramer Multiple Pairwise Comparisons Test modified to account for unequal group sizes and variance) shows that groups with mean cooked rice arsenic >200 μg/kg (D, E & F) each have significantly higher (p < 0.05) induction of genetic damage compared to each of the groups with mean cooked rice arsenic < = 200 μg/kg (A, B & C), although a relatively low p(>~0.005) was required by the w/s test invoked in order to fully comply with the requirement of this test for within group normal distributions of MN. A more robust non-parametric analysis (Kruskal-Wallis test followed by Wilcoxon Rank Sum Test with continuity correction) confirmed the result that all the groups with mean cooked rice arsenic >200 μg/kg (D, E & F) showed significantly higher (p < 0.05) micronuclei frequencies than the lower exposure groups (A, B & C) (Kruskal-Wallis Rank Sum test (χ2 = 83.9113; df = 5; p < 2.2e-16); Wilcoxon Rank Sum tests for: groups A & D: W = 951; p = 3.654e-07; groups A & E: W = 695.5; p = 7.293e-07; groups A&F: W = 512.5; p = 6.077e-12; groups B & D: W = 1127; p = 4.651e-05; groups B & E: W = 862; p = 1.499e-05; groups B & F: W = 664.5; p = 1.864e-10; groups C & D: W = 840.5; p = 0.0002438; groups C & E: W = 586; p = 1.462e-05; groups C & F: W = 500.5; p = 3.138e-09) (Fig. 2).

Figure 2 Urothelial cell genetic damage, as measured by frequency (per 1000 cells) of induction of micronuclei (MN), as a function of total arsenic concentration (CR-As) in consumed cooked rice, grouped as indicated. For this rural West Bengal population consuming rice as a staple, high arsenic in cooked rice is associated with elevated genotoxic effects. All groups with a mean As in cooked rice >200 μg/kg (D, E, F) have mean micronuclei frequencies (MN/1000 cells) significantly higher (p < 0.05) than those of the lower exposure groups (A, B, C). Numbers in each group as in Table 1. Full size image

No significant differences (p < 0.05) were found by either the modified Tukey-Kramer test or the more robust non-parametric Wilcoxon Rank Sum test between the group mean MN for any pairs of groups with cooked rice arsenic < = 200 μg/kg (A, B & C), but because of the small sample size, we are unable to determine if this reflects that there is no relationship between MN and cooked rice arsenic at these lower cooked rice arsenic concentrations or merely that our study had insufficient power to detect such a relationship. Similarly, the relatively small number of samples (n = 5) for which cooked rice arsenic exceeded 600 μg/kg means that we are unable to determine whether or not the relationship between cooked rice arsenic and MN for our study was more linear or sub-linear for these very high concentrations.

Notwithstanding these limitations, the highest rice arsenic content which has not been observed to be unequivocally associated with significantly increased genetic damage (Group C, Fig. 1) is 200 μg/kg, equivalent to 112 μg of arsenic solely from rice sources each day and, given the mean body weight of the study participants of 50.8 kg (Table 1), equivalent to a dosage of 2.2 μg As/kg-bw/day. Our results clearly demonstrate for this study population consuming around 500 g of cooked rice per day, that a cooked rice arsenic content above 200 μg/kg is - on its own - sufficient to give rise to significant amounts of genetic damage, even when there is little exposure through drinking water (Fig. 2).

Genetic damage association with high arsenic rice is not confounded here by other factors

Age, gender and tobacco-usage are often the major confounding factors in a genetic toxicity study, but the similar distribution of these factors throughout the groups suggests that it is unlikely for them to have substantially confounded the results. We note that the same positive relationship between micronuclei frequency and arsenic content of cooked rice is found in our study for both men and for women (Fig. 3a), for both tobacco-users and for tobacco-non-users (Fig. 3b) and for each of the 3 study areas (Fig. 3c). Questionnaire-based data shows that almost all of these individuals seldom travelled outside their local area and almost always used the same water source, thus suggesting that other sources of water are not a significant confounder (Table 1). Comparable body weight distributions in each study group act as a proxy variable that shows that the mean rice intakes of the different study groups were also comparable and, as such, the observed differences in genetic damage status in groups with >200 μg/kg in their consumed rice (Groups D, E, F) are, from a preliminary inspection, not likely due to differences in the level of rice intake (Table 1).

Figure 3 Cross-plot of urothelial micronuclei frequency (MN/1000 cells) and arsenic content in cooked rice for grouped data. (a) males (squares) and females (circles); (b) tobacco-users (squares) and tobacco-non-users (circles); (c) groups from each of the 3 study areas, viz. Murshidabad (squares), Nadia (circles) and East Midnapur (triangles). The linear best-fit trendlines are indicative only. Error bars represent ± 1 standard deviation for each parameter for each exposure group (A–F). Full size image

Following this preliminary inspection, in order to more robustly test whether or not key measured covariates, including age, gender, tobacco usage, bodyweight (as a proxy for cooked rice intake), drinking water arsenic, drinking water intake and study sub-area, are significant confounders to the association between MN and cooked rice arsenic, a stepwise (forwards/backwards) regression model was constructed utilising the data for the whole study population (i.e. without considering groups A–F). Distribution characteristics, including the scaling units, for these categorical and continuous covariates are listed in Table 2. The first step of this analysis (Table 3) revealed that the only covariates of significance in predicting MN, expressed as (MN/1000 cells)1/2, were, most significantly, cooked rice arsenic (p < 2.2e-16) and, to a much lower level of significance, tobacco usage (p = 0.022) with all of the following covariates tested not statistically significant (p > 0.05) confounders, viz. age (p = 0.648), body weight (p = 0.411), drinking water arsenic (p = 0.561), drinking water intake (p = 0.910), area (p = 0.126). The final model (Table 4) includes linear and quadratic cooked rice arsenic terms, tobacco usage and gender as covariates and indicates that gender is not a significant confounder (p = 0.704), tobacco usage is a weak confounder (p = 0.0478), whereas the overwhelming most important covariates in determining MN are the cooked rice arsenic linear (p = 4.50e-16) and quadratic (p = 1.43e-05) terms.

Table 2 Whole groupa summary of categorical and continuous covariates used in linear regression model of micronuclei frequency (MN/1000 cells) in human volunteers living in the rural West Bengal study areas and consuming rice as a staple Full size table

Table 3 Summary of first stagea of stepwise selection of covariates to include in model of micronuclei frequency, (MN/1000 cells)1/2, for rural West Bengal study group consuming rice as a staple. None of the covariates – area, body weight, drinking water arsenic, age or drinking water intake are significant confounders for the association between MN and cooked rice arsenic Full size table

Table 4 Summary of modela of micronuclei frequency, (MN/1000 cells)1/2, for rural West Bengal study group consuming rice as a staple Full size table

The elevated toxicity of inorganic arsenic species compared to organic arsenic moieties, such as arsenobetaine, arsenocholine, arsenolipids and arsenosugars, is well known5,9, but although we did not determine arsenic speciation in every cooked rice sample collected, the percentage inorganic arsenic (% i-As) content of a large sub-set of samples was relatively uniform and high (mean 88 ± 14%; SD; n = 92) in agreement with previous studies in the region9,10,16 and with only a weak (r2 = 0.24) correlation between % i-As and total arsenic, thus systematic variations in the percentage of inorganic arsenic in rice may also be eliminated as a significant confounding factor in this study.

Non-rice dietary sources of arsenic, for example from vegetables, fruit and seafood, may also be readily eliminated as significant confounders in this study. Drinking water intake of arsenic has already been shown to be low for the selected study group as a result of the study design. Halder et al.17 have shown that (i) the mean contribution to arsenic intake in rural West Bengal from vegetables is less than 0.4 μg/kg-bw/day, corresponding to less than 20% of total dietary exposure; and (ii) more importantly in relation to the present study, the inter-quartile range of such intake is less than 0.1 μg/kg-bw/day. Rowchowdhury et al.18 has previously shown that rice contributes over 90% of non-drinking water dietary exposure to arsenic in West Bengal.

Lastly, historical exposure is another potential confounding factor for other assays, but the employment of MN assay ensures that the results we obtained were only due to current exposure, since MN is not an inheritable property of a cell, rather each cell acquires MN during its short lifetime of being exposed to arsenic.