Arsenic represents a major threat to global health, with millions of people exposed through contaminated food and water, particularly in areas of the world where arsenic is naturally occurring in the environment (IARC, 2012). In Bangladesh, an estimated 45 million people are exposed to arsenic concentrations in drinking water greater than the World Health Organization (WHO) guideline value (10 µg/L) and 20 million people are exposed to concentrations greater than the Bangladeshi national standard (50 µg/L) (Flanagan et al., 2012). A recognized toxicant (IARC, 2012) and carcinogen (IARC, 2012), arsenic has been related to increased risk of cardiovascular, lung, kidney, bladder, skin, prostate, and liver disease (IARC, 2012; Liu and Waalkes, 2008; Wu et al., 2014). Arsenic has also been linked to increased systemic inflammation via oxidative stress (Ahmed et al., 2011) and chronic metabolic disorders including diabetes (Navas-Acien et al., 2008). Appreciation of the immunotoxic effects of arsenic and its potential to alter immune function is emerging (Dangleben et al., 2013). Laboratory studies reveal that arsenic inhibits antigen-presentation of macrophages (Banerjee et al., 2009) and T cell proliferation (Soto-Pena and Vega, 2008), decreases CD4+ T cell numbers in the spleen (Sikorski et al., 1989), and suppresses contact hypersensitivity responses (Patterson et al., 2004). The ability of arsenic to alter susceptibility to infection by affecting immune response is becoming increasingly recognized (Bailey et al., 2013; Farzan et al., 2013b; Kozul et al., 2009; Rager et al., 2014).

Pregnancy represents a unique period of susceptibility to infection (Kourtis et al., 2014) in part because of a shift toward an anti-inflammatory immune response (Kraus et al., 2012) due to changes in estrogen and progesterone levels (Shelly et al., 2012). No studies, to our knowledge, have investigated the dynamics of specific objectively measured infections among pregnant women exposed to arsenic. One study among 140 pregnant women in Matlab, Bangladesh, showed that increasing urinary arsenic concentrations were associated with increasing frequency of self-reported days of fever and diarrhea during pregnancy (Raqib et al., 2009). Though this study lacked objective measures of specific infections, its findings suggest a possible involvement of arsenic exposure in susceptibility to infection during pregnancy. Most attention has focused on the relation of in-utero arsenic exposure with the fetal immune repertoire (Nadeau et al., 2014) and infectious outcomes during early childhood (Farzan et al., 2013a,, 2013b; Rahman et al., 2011). Studies have also shown that in-utero and childhood arsenic exposure can alter vaccine immune responses in children (Ahmed et al., 2014; Saha et al., 2013). This emerging literature reflects a growing understanding of the immunomodulatory properties of arsenic; however, whether (and how) this affects susceptibility to specific infections in humans is not well understood.

Hepatitis E virus (HEV) is a non-enveloped positive-strand RNA virus, transmitted primarily via the fecal-oral route (e.g., contaminated surface water). HEV infection can be measured objectively via assessment of anti-HEV IgG seroconversion (Innis et al., 2002). Among individuals who seroconvert, HEV infection can be either asymptomatic or cause disease (hepatitis E) which is usually self-limited. However, some individuals with hepatitis E experience severe disease, known as acute fulminant hepatitis. HEV is a leading global cause of acute viral hepatitis (Mahtab et al., 2009; Rein et al., 2012) and causes significant morbidity and mortality during pregnancy (Labrique et al., 2012), particularly in South Asia where seasonal floods and poor sanitation have led to widespread contamination of surface water supplies with HEV and large epidemics of HEV infection (Gurley et al., 2014; Labrique et al., 2010). For reasons that are unclear, the case-fatality rate is markedly elevated (10–40%) in some pregnant women infected with HEV in South Asia, compared to the general population (3%) (Hamid et al., 1996; Tsega et al., 1992).

Although the liver is a known target of arsenic carcinogenesis (Liu and Waalkes, 2008) and emerging data support arsenic immunotoxicity (Stone, 1969), whether arsenic exposure during pregnancy can enhance susceptibility to objectively measured HEV infection during pregnancy has not been investigated. The aims of this nested case-control study were to assess arsenic exposure during pregnancy and evaluate its association with incident HEV seroconversion and changes in cytokine concentrations.

Results of this study suggest that elevated urinary arsenic levels during pregnancy may be related to HEV seroconversion. In the 1st TM, prior to HEV seroconversion, urinary ∑As levels were similar between women who later went on to seroconvert to HEV and those who did not. By the 3rd TM, however, urinary ∑As levels decreased significantly among non-seroconverters, while urinary ∑As levels did not decline among HEV seroconverters. Higher urinary arsenic levels during early pregnancy and on average across pregnancy were associated with incident HEV seroconversion, particularly after adjustment for socioeconomic factors and nutritional biomarkers. Although the sample size was relatively small, strengths of the study include the prospective design, high quality exposure and outcome assessment, adjustment for relevant confounders and the evaluation of immune markers.

The biological basis of enhanced susceptibility to HEV seroconversion among women with high urinary inorganic arsenic levels during pregnancy is supported by knowledge of arsenic hepato- and immunotoxicity. The liver is a primary target site of arsenic metabolism, which involves biomethylation through one-carbon metabolism into monomethylated and dimethylated arsenic species (Vahter, 2009). As one-carbon metabolism increases during the course of pregnancy, the methylation of inorganic arsenic into dimethylated arsenic species typically increases, (Gardner et al., 2011) resulting in increased proportion of DMA in urine. (Vahter, 2009) DMA is considered a less toxic metabolite than MMA and we found that HEV seroconversion was associated with higher % MMA and lower % DMA in urine, especially during the 1st TM of pregnancy, although the model estimates were imprecise, likely due to the small sample size. The association of higher % MMA and lower % DMA with increased risk of disease has been shown for cardiovascular disease and cancer in other studies (IARC, 2012; Wu et al., 2014). These results could identify an adverse profile of arsenic-related metabolism that is associated with increased risk of HEV seroconversion among pregnant women.

Arsenic is known to alter key functions of the innate and adaptive immune system. Arsenic has been shown to disturb innate immune responses, including the inhibition of macrophage function (Banerjee et al., 2009). Such disturbance of innate immune responses is believed to be one potential contributor to the increased frequency of symptoms of respiratory tract infections, diarrhea, and fever among arsenic-exposed pregnant women (Kile et al., 2014; Raqib et al., 2009). During pregnancy the immune response is associated with a shift to an anti-inflammatory or Th2-type response (Chaouat et al., 1997). While beneficial for the outcome of pregnancy, reduced inflammatory processes may enhance women's susceptibility to certain infections that require Th1-type immunity for clearance. Th1- and Th2-type responses transcriptionally down-regulate each other – a strong Th2-type response should reduce a Th1-type response and vice-versa (Lee et al., 2006; Spilianakis and Flavell, 2007). Research suggests that arsenic exposure may exacerbate anti-inflammatory immune responses during pregnancy (Cho et al., 2012), which could further enhance susceptibility to infection, particularly viral infections that require Th1-type immune responses for successful clearance. It is possible that arsenic exposure during pregnancy may alter Th1-type immune responses resulting in susceptibility to seroconversion and viral infection at a lower challenge dose of virus than in the absence of arsenic exposure. A compromised immune response, increasing morbidity, and higher pulmonary viral titers were observed following a sub-lethal influenza virus challenge dose in mice exposed to arsenic relative to arsenic unexposed mice (Kozul et al., 2009), but human population-based studies of arsenic immunotoxicity and infection among pregnant women are lacking. We could not investigate whether arsenic-induced immune alteration is related to symptomatic HEV disease because pregnant women in our study did not experience symptomatic HEV disease (as reflected by anti-HEV IgG seroconversion without reports of acute viral hepatitis symptoms).

Arsenic is known to impair adaptive immunity. It is recognized that arsenic delays or inhibits adaptive T-cell immune response. One mechanism that has been described in detail is the delay of T cell proliferation (Soto-Pena and Vega, 2008), and promotion of T cell anergy, senescence or tolerance (Soto-Pena et al., 2006) after arsenic exposure. Impairment of T cell proliferation has been shown to have adverse effects when an infectious agent (such as HEV) is encountered because a delayed or ineffective T-cell response (Galicia et al., 2003), combined with ineffective innate immune response, could enhance susceptibility to infection. Among the cytokines examined in this study, IL-2, an indicator of immune activation, were higher in HEV seroconverters and its levels showed a significant positive association with urinary arsenic concentration only among pregnant women who seroconverted to HEV, potentially indicating there was activation of the immune response by arsenic. IL-2 plays a critical role in initiating the adaptive immune response and T cell proliferation. Laboratory studies suggest that after inducing an initial delay in IL-2 production, arsenic may subsequently induce a “ramping-up” of IL-2 production to achieve an effective adaptive immune response (Soto-Pena et al., 2006; Soto-Pena and Vega, 2008). This is supportive of a possible role of arsenic exposure in the dynamics of HEV seroconversion, and future studies involving functional measures of immune response (e.g., immune cell populations, immunoglobulin isotypes) could improve understanding of this exposure–response relationship.

Limitations of the study include a small sample size and a lack of assessment of arsenic in drinking water and food. However, we measured urine arsenic, which is a well-established biomarker that integrates all sources of arsenic exposure (including drinking water and food). The observed differences in urinary arsenic levels were unlikely due to changes to an alternative water supply because sources of drinking water in the study area remained constant, nor was it likely due to differences in hydration status since all analyses were adjusted for urine dilution via specific gravity. Future studies should measure arsenic and pathogens in water and food and assess urinary arsenic levels during the postpartum period to address these limitations.

Despite the small sample size, important strengths of the study include use of a prospective design, nested within a large cohort, objective measures of arsenic exposure in urine, objective assessment of outcome (incident seroconversion) for a pathogen that causes a significant burden of morbidity and mortality in pregnancy (HEV), measurement of pro- and anti-inflammatory cytokines in plasma, the inclusion of objectively measured micronutrients as confounders, and the availability of repeated measurements during pregnancy and postpartum. These strengths represent an improvement on previous studies of arsenic exposure and infection during pregnancy because most involved classification of exposures or outcomes via participant self-reports of symptoms (Kile et al., 2014; Raqib et al., 2009). Anti-HEV IgG seroconversion constitutes incident HEV infection; however, pregnant women who seroconverted to HEV in our study did not report symptoms of acute HEV disease – e.g., none reported icterus, fever, clay-colored stools during the follow-up period. Our findings may therefore provide important insights into the dynamics of arsenic exposure with early sentinel immune responses and asymptomatic infection and could inform novel hypotheses for future research of symptomatic HEV infection during pregnancy in population-based and clinical settings.

The possibility of reverse causation should be considered. Pregnant women who seroconverted to HEV may have experienced a cascade of events related to viral seroconversion that could have led to reduced metabolism of arsenic in the liver and thus resulted in higher unmethylated arsenic, which has a longer half-life than methylated arsenic species, and then higher urinary ∑As levels. To address this question, future studies should study the relation of arsenic exposure among pregnant women with symptomatic HEV disease because this may reflect dynamics of more severe metabolic stress on the liver than among asymptomatic HEV infections observed in our study. The small sample size precluded a more extensive analysis of confounding and/or effect measure modification. Micronutrients included in our analysis were those known to influence one-carbon metabolism and arsenic metabolism (folate) (Gamble et al., 2006) and those hypothesized to be associated with immune response (zinc, vitamin D) (Bartley,2010; Mahalanabis et al., 2004). However, given that all women in this nested case-control study received antenatal multiple micronutrients at a daily requirement level or the same level of iron-folic acid alone as part of an intervention trial, future studies should be conducted among women not receiving micronutrient supplements in order to assess the potential impact of micronutrient deficiency status on arsenic exposure, infection, and immune response.

Our findings contribute to understanding arsenic immunotoxic effects during pregnancy and postpartum. In Bangladesh, a large proportion of the population relies on water from tube wells that are contaminated with arsenic (Karagas, 2010). In Bangladesh and other areas where genotype 1 HEV is endemic, transmission occurs via the fecal-oral route principally via fecally-contaminated surface water. During pregnancy, it may be important to consider the context of exposure to arsenic and HEV (as well as other pathogens related to water, hygiene, and sanitation conditions). In rural Bangladesh, near the location of the present study, HEV seroprevalence estimates range between 20% and 23% (Labrique et al., 2010) and have been observed as high as 40–80% in other areas of Bangladesh (Mahtab et al., 2009). With such high HEV endemicity and co-occurring arsenic exposure, if the associations we observed are causal, the population at risk could be large. A more complete understanding of the effects of arsenic on immune alterations and other infectious diseases during pregnancy could inform interventions to reduce morbidity and mortality where exposure to pathogenic and immunotoxic agents co-exists.