The human gut microbiome mediates thousands of interactions between their hosts and xenobiotic compounds daily (1
). For example, gut microbes can metabolize xenobiotics, modulate the absorption and dissemination of toxicants (2
), and alter bioavailability or activity of pharmaceuticals (3
). As microbiome composition is highly personalized, the magnitude of these effects may differ across individuals (4
). Environmental factors that alter the composition or function of the microbiome, such as dietary variation (6
), may then affect individual variation in response to xenobiotic exposure (5
). These exposures could also alter the sensitivity of the microbiome to subsequent xenobiotic exposures, potentially driving the microbiome into a dysbiotic state. Given the well-described associations between microbiome variation and disease (7–11
), it is important to understand the extent to which environmental parameters may influence the microbiome’s susceptibility to xenobiotics or its ability to mediate or modify the effects thereof. Yet, no study has explored how dietary variation and environmental chemical exposure interact to affect the microbiome.
Nearly 2 billion people worldwide consume insufficient zinc (12
). While the impact of various macronutrients (e.g., fat and protein) on the gut microbiome is well described (6
), less is known about how dietary micronutrient variation, such as zinc deficiency, impacts the microbiome and how these impacts may influence host health. The studies that have explored the impact of zinc deficiency on the microbiome find that this single dietary micronutrient can significantly affect the microbiome’s composition (15
), which follows from the fact that zinc is an essential nutrient for microbial cells. However, these studies tend to focus on extreme zinc deficiencies (i.e., complete lack of dietary zinc [16
]); little is known about how moderate insufficiencies, such as the marginal zinc deficiencies that typically arise from inadequate dietary intake of zinc, impact the microbiome.
Many individuals who consume inadequate amounts of zinc also live in regions where the risk of exposure to toxicants, such as arsenic, is high (18–20
). Hundreds of millions of people worldwide (21
) routinely consume inorganic and organic forms of arsenic in drinking water and food (22
). The concentrations of dietary exposure to the more toxic inorganic forms of arsenic vary widely and frequently exceed safety thresholds (10 µg/liter [19
]). Chronic exposure to high arsenic concentrations increases the risk of cancer, cardiovascular disorders, and neuropathies (25
). At low concentrations—even those nearing safety thresholds—exposure can also negatively impact health, but the severity of these effects varies (26
). This interindividual variation may result from personalized susceptibility to arsenic exposure (21
). Several genetic and dietary factors affect arsenic susceptibility (21
). Among these factors are micronutrient deficiencies, including zinc deficiency. Zinc and arsenic interact with common proteins (29
), and zinc deficiency and arsenic exposure yield similar pathologies (31–33
). Moreover, restriction of dietary zinc alters the host’s sensitivity to toxicant exposures, including arsenic (28
). Despite these observations, it remains unclear how zinc restriction modulates the physiological effects of arsenic exposure.
While we know that dietary zinc can impact arsenic toxicity in the host (28
), we do not understand how marginal zinc deficiency affects the microbiome’s response to subsequent arsenic exposure or how any such combinatorial effects on the microbiome relate to host physiology. For example, consumption of a zinc-deficient diet may enrich for microbes lacking traits required to metabolically detoxify or resist arsenic (36
). Consequently, gut bacteria may suffer increased sensitivity to arsenic upon exposure, which may magnify the effects of arsenic on the microbiome. It is important to define how zinc and arsenic interact to affect the gut microbiome because such interactions could contribute to the physiological response of dual exposure in the host. For example, if a zinc-deficient gut microbiome is more sensitive to arsenic, then it is possible that its contribution to homeostasis is more likely to break down upon arsenic exposure.
To determine if multifactorial interactions between zinc, arsenic, and the gut microbiome exist, we examined the impact of physiologically relevant levels of zinc deficiency, through marginal zinc deficiency (37
), on the response of the microbiome to environmentally relevant levels of arsenic exposure. Given the independent effects of zinc and arsenic on the gut microbiome, we hypothesized that zinc deficiency and exposure to arsenic yield combinatorial effects on the gut microbiome. We found that arsenic exposure had a modest effect on the microbiome of animals fed zinc-adequate (ZA) diets; however, mice fed marginally zinc-deficient (MZD) diets experienced significant shifts in microbiome composition in response to arsenic exposure. Changes in microbial relative abundance were also associated with host physiological responses to zinc restriction and arsenic exposure. Our data indicate that zinc restriction alters the microbiome’s sensitivity to arsenic exposure and that gut microbes are linked to the physiological changes associated with arsenic exposure and dietary zinc deficiency. Considering the associations between the gut microbiome and health, moderate micronutrient deficiencies may have broader health risks than previously appreciated.
Our study finds that both marginal zinc deficiency and arsenic exposure yield moderate impacts on the composition of the microbiome. However, when zinc deficiency occurs in conjunction with arsenic exposure, these effects on the microbiome magnify. We also observe associations between microbial abundance and indicators of host DNA damage and oxidative stress. Collectively, these results indicate that marginal zinc deficiency sensitizes the microbiome to the impact of environmentally relevant concentrations of arsenic and that these changes to the microbiome are linked to host physiological changes that occur during zinc restriction and arsenic exposure. Moreover, these results highlight that dietary micronutrient status and environmental chemical exposure manifest synergistic effects on the gut microbiome. The finding that dietary micronutrient exposure influences the microbiome’s response to subsequent exposures has implications in almost every field of biomedical research and may help explain some of the variation in microbiome studies in human populations.
Despite the importance of arsenic toxicity and the growing awareness of the microbiome as an agent of health, we understand relatively little about the effects of arsenic exposure on the gut microbiome. Prior research found that exposure to high concentrations of arsenic alters microbial community structure and operation (41
). Here, we found that environmentally relevant concentrations of arsenic elicit moderate impacts on microbiome diversity. This finding complements a recent study that also showed that concentrations of arsenic similar to those used here (100 ppb) disrupt microbiome composition and function (42
). However, this prior study observed larger effects of arsenic exposure on the microbiome. Chi and colleagues leveraged a longer exposure period in their study (13 weeks), which could account for this variation in effect size. Moreover, facility, strain, or background diet effects could contribute to the differences in magnitude that we observed (43
). For example, if the initial microbiome of mice in our facility were enriched for taxa that rapidly detoxified arsenic, then any effects of arsenic exposure in our mice may have been mitigated. Despite the differences in magnitude, both studies highlight the impact of short-term arsenic exposure on the composition of the microbiome. Further study is warranted to determine the minimum exposure length and dose that are sufficient to disrupt the microbiome.
While micronutrient deficiencies are well studied in terms of their effects on health (12
), we are only beginning to understand their impact on the gut microbiome. In the case of zinc, prior research demonstrated that severe depletion of zinc (0 to 2.5 µg/g) alters the composition and operation of the gut microbiome (15–17
). Our study extends this prior work by demonstrating that marginal dietary zinc deficiency similarly results in a modest, yet significant, restructuring of the gut microbiome. In bacteria, zinc starvation inhibits growth (44
), disrupts enzyme activity (45
), and increases the expression of zinc transporters (46
). Although we did not measure the metabolic outputs of the microbiome during zinc deficiency, it is possible that altered microbial metabolism in MZD animals played a role in the microbiome’s heightened sensitivity to arsenic exposure.
Combining marginal zinc deficiency and arsenic exposure amplified their effects on the gut microbiome. This observation is consistent with the intermediate disturbance hypothesis, which postulates that community diversity maximizes at intermediate levels of ecological disturbance (47
). Under this framework, large disturbances, such as frequent antibiotic exposure, reduce diversity as the disturbance selects for a relatively small set of organisms. It is possible that both marginal zinc deficiency and arsenic exposure constitute moderate microbiome-perturbing agents, and therefore their administration results in increased microbiome diversity. However, when applied in combination, their synergistic effect induces a perturbation of far greater magnitude and results in decreasing diversity. If this were the case, then we would expect that micronutrient deficiency would increase susceptibility to many other microbiome-perturbing agents. Moreover, this would also suggest that these deficiencies might lower the exposure threshold needed to perturb the gut microbiome.
Both zinc deficiency and arsenic exposure modulate oxidative stress, inflammation, DNA repair and metabolism (12
). Correspondingly, our study finds that marginal zinc deficiency and arsenic exposure both independently increased DNA damage and decreased plasma zinc. Cellular DNA damage positively associated with the abundance of the phylum Tenericutes
, which prior work links to perturbed gut microbiomes, such as those subject to pathogenic infection (52
). Zinc restriction also decreased plasma adiponectin. The family Neisseriaceae
, which is depleted in inflamed guts (53
), positively associates with adiponectin. These associations suggest that the microbiome may contribute to some of the physiological effects associated with zinc restriction and arsenic exposure.
Collectively, the results of this study bolster the hypothesis that the gut microbiome affects an individual’s physiological response to zinc deficiency and arsenic exposure. A thorough test of this hypothesis ultimately requires demonstrating that the exposure-induced perturbations to the gut microbiome contribute to the physiological responses to those perturbations. Though our study design cannot disentangle cause-and-effect relationships, our results point to potential mechanisms through which zinc deficiency and arsenic exposure may impact the gut microbiome to disrupt physiology. For example, zinc deficiency and arsenic exposure can yield gastrointestinal dysfunctions and elevated intestinal inflammation (54
). Our results imply that these exposures could deplete members of the families Rikenellaceae
to consequently contribute to these dysfunctions. Rikenellaceae
depletion is associated with impaired mucosal immune function and increased gut inflammation (55
). Similarly, Lachnospiraceae
contains taxa that produce butyrate, which reduces oxidative stress and inflammation (56
), and prior work links their depletion to inflammatory disorders such as Crohn’s disease (57
). Correspondingly, MZD animals exposed to arsenic experienced elevated levels of Akkermansia
, which contain taxa that induce proinflammatory immune responses in human peripheral blood mononuclear cells (58
has also been shown to exacerbate inflammation during infection with intestinal pathogens (59
). These observations specifically point to inflammation as a potential means through which the microbiome mediates the effects of zinc deficiency and arsenic exposure. However, it is unclear if the microbiome plays a role in the proinflammatory immune environment during zinc restriction and arsenic exposure or if the altered microbial abundance was due to altered host physiology in response to marginal zinc deficiency or arsenic. Zinc restriction may also reduce both the host’s and microbiome’s ability to detoxify arsenic exposure. For example, zinc restriction may alter the expression or activity of enzymes involved in the excretion or methylation of arsenic, such as highly conserved arsenic(III) methyltransferase (60
). This would increase or prolong the exposure to arsenic, potential enhancing its effects on microbiome and host physiology. It is worth disentangling these relationships in future studies because if the microbiome contributes to the physiological effects of micronutrient deficiency and arsenic exposure, then it may be used as a therapeutic intervention to mitigate these effects. Alternatively, the gut microbiome may be a useful diagnostic for assessing arsenic exposure, though any such diagnostics would need to account for the interacting effects of alternative exposures as documented here.
If the microbiome is important in detoxification of xenobiotics, then any stimuli that alter microbiome composition, function, resistance to perturbation, or resilience may, in turn, alter an individual’s response to subsequent xenobiotic exposures. Thus, based on our prior work and the results of this study, we hypothesize that micronutrient status may have a significant impact on an individual’s response to toxicant exposure such as arsenic. This study demonstrates that coupling zinc deficiency with environmentally relevant exposures to arsenic yields combinatorial effects on the gut microbiome. Moreover, these exposure-induced effects on the gut microbiome correlate with specific changes to physiology. Future work should seek to identify the mechanisms that dictate the microbiome’s sensitivity to zinc deficiency and arsenic exposure as well as the underlying causes of the amplified diversification of the microbiome upon multifactorial exposure. Additional studies are also needed to measure the specific physiological consequences of this diversification and to determine if similar outcomes are observed in humans. Ultimately, the gut microbiome may be proven to be a factor that defines personalized exposure effects, which can help advance microbiome-based preventative therapeutics of exposure.