Epidemiological studies in humans 
as well as in vitro
and in vivo
studies in rodents 
, have been conducted to determine if iAs exposure is a risk factor for T2DM. Analyses of human samples from subjects exposed to iAs are lacking, especially studies of the effects of the metalloid in tissues such as pancreas and liver. Therefore, pilot studies such as this one are very valuable, even though the number of subjects involved was small. Although evidence shows that high iAs exposure and T2DM are significantly associated, additional research is needed to determine whether the relationship is causal as proposed by The National Institute of Environmental Health Sciences (NIEHS), Division of the National Toxicology Program (NTP) this past year 
. Moreover, evidence showing that iAs exposure is associated with an increased risk for T2DM in humans is still limited 
, and until now, studies that try to explain the mechanism of how arsenic acts as a risk factor for T2DM have been carried out in rodents or in in vitro
studies but never in humans; therefore, this pilot study is relevant. The data obtained from this work support the previous findings from studies carried out in non-human subjects, especially those focusing on beta-cell dysfunction.
In this pilot study, we measured the interaction of iAs on glucose metabolism in humans using non-invasive methods. We also analyzed the association between this environmental metalloid and genetic factors for the disease. Our results show that iAs indeed does affect the pathogenesis of T2DM, particularly in disturbing beta-cell function, and this in turn is also associated with SNPs found in CAPN-10
, a susceptibility gene for T2DM in certain populations, including Mexicans 
It was expected that daily arsenic consumption and levels of arsenic excreted in the urine would be correlated. Nevertheless, non-diabetic subjects consumed less iAs but had greater concentrations of arsenic in the urine than subjects with T2DM. This phenomenon could be due to differences in arsenic metabolism or, to a less extent, to an inaccuracy in self-reporting of water consumption habits.
The relationship between the concentration of arsenic in urine and the function of beta-cells in diabetic and non-diabetic subjects show the same trend, but the association was statistically significant only in the diabetic subjects. These data suggest that iAs exposure is a risk factor for T2DM rather than a direct inducer of the disease. Failure of beta-cell function is necessary for progression from insulin resistance to T2DM; nevertheless, it is not the only event that is required because many other risk factors are also needed for the development of the diabetic state 
. In this work, we noticed that, in subjects without T2DM, iAs exposure was associated with a decrease in the function of beta-cells, but this was not sufficient for the development of T2DM. Other risk factors would need to exert their actions, indicating a multifactorial metabolic disruption in the organism. Another possibility may be that the beta-cells of diabetic subjects are extremely sensitive to iAs exposure; however, in this study it was not possible to determine this as a cause (i.e., beta-cells are impaired directly by iAs) or a consequence (i.e., the function of beta-cells from diabetic subjects is deficient and thus cells are more susceptible to the effects of iAs). However, in vitro
studies have shown that iAs impairs insulin secretion, leading to beta-cell insensitive to glucose 
. It could also be possible that by increasing the sample size, a clearer association could be determined between iAs exposure and beta-cell function in non-diabetic subjects.
T2DM is a complex disease, and an interaction between environmental factors and genetic components occurs in the pathogenesis of the disease. In this work we evaluated the influence of SNPs in CAPN-10
on beta-cell dysfunction. Other genes have been associated with T2DM such as TCF7L2
, KCNQ1 and MTNR1B
, as determined through genome-wide association studies (GWAS) 
. However, this study analyzed SNPs in CAPN-10
because these have been associated with T2DM in the Mexican population 
and calpain-10 participates in both glucose-stimulated insulin secretion in beta-cells 
and in insulin-stimulated GLUT4 translocation to the plasma membrane in adipocytes 
. Moreover, we previously showed that iAs can reduce insulin secretion in beta-cells through a decrease in the proteolysis of SNAP-25 by calpain-10 
SNP-43 in CAPN-10
is involved in many of the phenotypes associated with T2DM, such as insulin resistance, lipogenesis, insulin secretion and microvascular function 
, while SNP-44 has been associated with increase risk for T2DM in Mexican population 
and with other populations as evaluated through meta-analysis 
. SNP-44 has also been associated with alterations in the pattern of the oral glucose tolerance curve 
and is in linkage disequilibrium with the missense variant T504A (SNP-110), which unlike the other SNPs, is located in the open reading frame of CAPN-10 
Beta-cell function was lower in subjects without T2DM that carried the at-risk genotypes G/G at SNP-43 and T/C at SNP-44. In subjects with T2DM, the influence of the genotype on beta-cell function was not evident because the level of function of these pancreatic cells is already altered due to the disease per se
and to all the factors that contributed to the onset of it; therefore, the influence of the genotype has been lost. Once the disease has been established, which not only involves the dysfunction of beta-cells but also other events 
, it is likely that at-risk variants in CAPN-10
are not more associated with the level of beta-cell function, even when iAs exposure occurs. Instead, other mechanisms, such epigenetic modifications, may be taking place, as has been revealed in a genome wide site-specific DNA methylation study conducted in peripheral blood lymphocyte DNA of 16 individuals, half with established elevated levels of iAs exposure and showing signs of arsenicosis (skin lesions). The investigators in this study found that epigenetic changes in genes were associated with arsenic-induced diseases, including T2DM 
From the analysis of beta-cell function stratified by CAPN-10 genotypes through multivariate analysis, it can be postulated that the at-risk heterozygote allele T/C in SNP-44 is associated with decreased beta-cell function independently of arsenic exposure or other risk variables for T2DM, such as age, sex and BMI. On the contrary, the risk for the development of T2DM with the G/G genotype at SNP-43 depends upon intrinsic and environmental factors, including iAs exposure.
As mentioned above, for T2DM to occur, other components, such as insulin resistance, are also needed. In contrast to in vitro
findings in the literature in which arsenic induces insulin resistance in adipocytes 
, in this pilot study insulin sensitivity was not associated with iAs exposure or with at-risk alleles in CAPN-10
. This lack of correlation may be due to the relationship between glucose and insulin in the basal state (fasting) in the HOMA analysis, which reflects the balance between hepatic glucose output and insulin secretion that is maintained by a feedback loop between the liver and the pancreas 
, and less with muscle or adipose tissue insulin sensitivity, which are the organs where glucose uptake occurs in an insulin-dependent manner. In fact, the studies of in vitro
arsenic effects were conducted mostly in adipocytes and not in hepatic cells. Additionally, it cannot be ruled out that the lack of correlation was influenced by metformin treatment in diabetic subjects (50%) or by the small sample size.
In a recent cross-sectional study conducted in Zimapán, México, in addition to the prevalence of diabetes being positively associated with iAs in drinking water and with the concentration of its metabolite dimethylarsinous acid in urine, an inverse association (p
<0.01) between HOMA-insulin resistance and iAs exposure was also shown, contrary to our results and to in vitro
. Nonetheless, more studies in humans are needed to understand more deeply this phenomenon.
Taken together, these results provide evidence in humans that support the idea that iAs is a risk factor for T2DM through the disturbance of beta-cell function. As is known, individuals with beta-cells unable to sustain increased insulin secretion to compensate for insulin resistance will develop T2DM, and thus inadequate beta-cell function is essential to the course of the disease 
. Here we show that both iAs exposure, as well as presence of the at-risk alleles of SNP-43 and SNP-44 in CAPN-10
, a protein that plays an active role in insulin secretion as part of the machinery of the secretion complex 
, have an adverse effect on beta-cell function. It is possible that beta-cell failure may occur earlier and/or in an exacerbated manner in individuals with the at-risk genotypes in SNP-44 (T/C) and/or in subjects with the at-risk genotypes in SNP-43 (G/G) and exposure to iAs.
Epidemiological associations between chemical exposure and disorders of glucose metabolism are increasing every day. According to The Environmental Protection Agency (EPA), an endocrine disrupting chemical (EDC) is defined as an exogenous agent that interferes with the production, release, transport, metabolism, binding, action, or elimination of natural hormones in the body responsible for the maintenance of homeostasis, reproduction, development and/or behavior 
. iAs as well as other pollutants, such as polychlorinated biphenyls, organochlorine pesticides, dioxins, and bisphenol A, among others 
, need to be seriously taken into account as environmental factors that increase the risk for T2DM.
Finally, we believe this study improves the current understanding of T2DM, particularly by adding evidence of iAs acting as a diabetogen in humans and contributing to the fact that environmental factors other than diet and exercise, together with genetic influences, are involved in the development of the disease. In additional to epidemiological studies, it is very important to conduct biological analyses in humans and to find the way to eliminate efficiently iAs exposure.