Zinc as a Mechanism-Based Strategy for Mitigation of Metals Toxicity

Abstract   

Purpose of Review

Zinc is an essential micronutrient with a myriad of key roles in human health. This review summarizes mechanistic data supporting the protective effects of zinc on metal toxicity and discusses the framework for an interventional clinical trial of zinc supplementation within a metal exposed Native American community.

Recent Findings

Many metals have common underlying mechanisms of toxicity that contribute to adverse human health effects. Studies demonstrate that multiple aspects of metal toxicity can be attributed to disruption of essential zinc-dependent functions. Multiple lines of evidence suggest that zinc may confer protection against metal toxicity in human populations with mixed-metal exposures. Thinking Zinc is a mechanism-informed intervention study of zinc supplementation to test the potential benefits of zinc while maintaining a culturally responsive research approach.

Summary

The current knowledge of diverse metal and zinc interactions, coupled with strong mechanistic evidence for zinc benefits in the context of toxic metal exposures, supports the hypothesis that zinc supplementation may mitigate the impact of toxic metals exposures in populations with chronic mixed metal exposures and in populations with low zinc status.

Introduction

Numerous heavy metals and metalloids with established human toxicity and carcinogenic potential are present in the environment. The International Agency for Research on Cancer (IARC) has classified arsenic, beryllium, cadmium, chromium and nickel as Group 1 carcinogens [1]. Distribution of specific metals and human exposure differs by location and local geology or anthropogenic causes [1–6]. The United States Environmental Protection Agency (USEPA) established drinking water maximum contaminant levels (MCL) for ten metals: antimony, arsenic, barium, beryllium, cadmium, chromium, mercury, selenium, thallium, and uranium. Despite regulatory guidelines, community water systems may exceed one or more MCLs and elevated metal levels have been documented in unregulated private wells [3, 5, 6]. The scope of metal exposures is highlighted by data obtained from the National Health and Nutrition Examination Survey (NHANES). Based on measures of urinary or blood metal levels, the study found that nearly 50% of the United States population was exposed to a combination of three or more metals [7]. In a recent meta-analysis, exposure to certain metals such as cadmium, lead, and arsenic were reported to be associated with mortality from all causes, cardiovascular disease and cancer [8] and metals exposures are linked to cardiovascular disease and other adverse human health effects [1, 9–11].

Although metals differ in specific toxic profiles, they share a number of underlying mechanisms of toxicity that contribute to adverse human health effects. Generation of reactive oxygen species (ROS) is a common response to metal exposure and excessive ROS is damaging to cellular components such as lipids, proteins and DNA [2, 11–17]. Metal binding to proteins may modify signaling pathways, cellular functions and genomic integrity through direct and indirect mechanisms [1, 2, 11, 13, 15]. A number of metals compete with essential metal nutrients (e.g. iron, copper, zinc) for specific binding sites in proteins or metal transporters that regulate essential and non-essential metal absorption and excretion [1, 4, 10, 11, 17–19]. Understanding the mechanistic underpinnings of metal toxicity has prompted exploration of strategies to reduce the human impact of metal exposure. For example, a number of nutritional approaches to mitigate arsenic toxicity have been tested in experimental models and a limited number of human population studies. These include, but are not limited to, supplementation with folates or glutathione to modify arsenic metabolism and enhance excretion, treatment with antioxidants to counteract arsenic-induced oxidative damage and supplementation with essential metal micronutrients such as zinc or selenium [20, 21].

This review will discuss the potential for zinc to mitigate the adverse effects of toxic metals. Zinc is the second most abundant metal micronutrient after iron and it is estimated that nearly 3000 human proteins bind zinc [22–26]. Zinc plays catalytic and structural functions, and is critical for proper immune function, suppression of inflammation, growth and development, wound repair, sensory and neurological functions and general antioxidant defense among other physiologic roles that support human health [22, 25, 27–30]. As will be discussed in more detail in subsequent sections, zinc is an attractive strategy for nutritional intervention in metal-exposed human populations. This prediction is based on abundant experimental evidence that zinc counteracts multiple mechanisms of metal toxicity and nutritional supplementation is a tractable approach for a community-level intervention. In terms of mechanistic considerations, we will focus on the available evidence of metal and zinc interaction. This is not meant to suggest that potential benefits of zinc are limited to these specific examples–a mechanism of toxicity shared amongst metals is likely to be responsive to zinc. We will discuss a case study for the design and early implementation phases of a zinc intervention clinical trial in Navajo communities exposed to legacy uranium mine wastes. The review will conclude with perspectives regarding development of clinical trials involving zinc supplementation to reduce human health burdens related to toxic metal exposures.

Zinc and Metal-Induced Mechanisms of Toxicity

Oxidative Stress Response

Oxidative stress is a common molecular mechanism underlying metal-induced toxicity [2, 11–15, 17, 19]. Exposure to many metals leads to the excess production of reactive oxygen and nitrogen species (ROS/RNS) through both direct and indirect mechanisms [13]. This imbalance of ROS/RNS promotes cellular damage with consequences that may include mitochondrial dysfunction, endoplasmic reticulum stress, disruption of DNA repair, genotoxicity, apoptosis, inflammation, and other adverse effects. Oxidative stress, inflammation and resulting cellular damage contributes to the pathology of numerous chronic diseases such as cardiovascular and pulmonary disease, diabetes, renal dysfunction, neurological disorders, cancer and aging [1, 2, 11–13, 15–17, 25]. In vivo studies indicate protective activity of molecules with antioxidant properties against metal toxicity [11, 15, 31, 32]. Biomarkers of oxidative stress such as measurement of F2-isoprostanes, oxidized lipids, 8-OHdG and total antioxidant status have been used in human studies including populations exposed to environmental metals [10, 16, 33–36].

While zinc is often described as an antioxidant it does not formally function in a direct antioxidant capacity. Multiple mechanisms such as protection of protein sulfhydryl groups, competition with redox-active metals, actions as a cofactor for antioxidant enzymes such as Cu/Zn superoxide dismutase and induction of the antioxidant system response underlie zinc’s role in antioxidant defense [22, 26, 28, 30, 32]. The critical role of zinc in regulating oxidative stress response is illustrated by clinical and experimental studies demonstrating that zinc deficiency is associated with increased biomarkers of oxidative stress, DNA damage, and inflammatory cytokines that can be alleviated by zinc supplementation [22, 25, 25, 27–29, 37]. For these reasons, zinc has been considered as a candidate to protect against metal-induced toxicity resulting from oxidative stress [11, 32, 38].

Metal Displacement of Zinc in Zinc-Binding Proteins

Zinc binding proteins are highly represented in the proteome and are critically important to broad aspects of biology [23, 25, 26]. Although there are many zinc binding motifs, the zinc finger domains are present in approximately 5% of human proteins and facilitate binding to DNA, RNA, proteins, lipids and protein post-translational modifications [23, 39]. Zinc finger proteins are represented in a vast array of cellular functions and are characterized by coordination of zinc by four histidine and/or cysteine residues [Fig. 1]. A number of toxic metals, including cadmium, cobalt, lead, arsenic and uranium, interfere with zinc finger proteins by displacing zinc or causing oxidation of zinc coordinating cysteine residues in this key structure [1, 11, 17, 39–41]. The consequences for metal interference with zinc finger proteins depends on the protein function. Using DNA repair as an example, arsenic binding and zinc finger motif disruption has been studied at the peptide, protein, cellular and organismal level. This arsenic and zinc interaction results in inhibition of DNA repair protein activity and elevated DNA damage [11, 17, 19, 42]. There are differences noted between metals; arsenic as arsenite displays binding preference for zinc finger motifs containing ≥ three cysteine residues, whereas uranium [40] and cadmium [1] bind effectively to zinc finger structures with two cysteine and two histidine residues. Other zinc binding motifs such as RING domains are also vulnerable to metals with impact on genomic integrity, RNA splicing and mitosis [43–46]. Investigation on metal interference with zinc-dependent protein functions is a fertile area for further study.

Fig. 1.

A Schematic of Arsenic (As)/Uranium (U)/Cadmium (Cd) interactions at protein zinc finger (ZF) motifs demonstrating that metals can displace zinc resulting in protein disruption. Replacement of zinc in the binding pocket restores function. B Native artist Mallery Quetawki (Zuni Pueblo) rendering of zinc (turquoise jewelry) binding as beautiful and healthy versus metal binding (green circles) causing damage and dysfunction

Importantly, in vitro studies demonstrate that adequate or supplemental zinc counteracts the effects of toxic metal binding to zinc finger motifs and/or protein function [11, 19, 41, 42, 45, 46]. In the context of arsenic inhibition of DNA repair, protein zinc content and DNA repair capacity is largely rescued by supplemental zinc in cell models and in vivo [11, 19, 42]. The importance of the balance of zinc and toxic metals in cells and tissues is further highlighted in studies that find that metal toxicity is exacerbated under zinc deficient conditions [20, 47–52]. Overall, research findings indicate that zinc may overcome the adverse effects of metal interaction with zinc-binding proteins.

Metallothionein

Metallothioneins are a family of proteins that bind both essential and toxic metals with different affinities. They are dynamically regulated by metal exposure and play a particularly key role in zinc and copper homeostasis [4, 22, 26, 53, 54]. The binding of toxic metals to metallothionein sequesters these metals and this property is generally thought to reduce metal toxicity [4, 26, 38, 54]. Metallothioneins are regulated in response to essential and non-essential metals through gene expression and at the protein level [54]. Zinc induction of metallothionein expression may be a moderating factor for metal interaction with intracellular molecular targets.

Another function of metallothioneins is scavenging reactive oxygen species through the oxidation of its abundant cysteine residues [26, 38, 53]. This mechanism can provide protection even for toxic metals with low binding affinities for metallothioneins such as chromium and arsenic [51, 55, 56]. Reduction of cellular metallothionein under zinc deficient conditions enhances chromium toxicity [51]. Conversely, zinc administration leading to induction of metallothionein expression and restoration of its antioxidant capacity reduces arsenic-stimulated oxidative stress and toxicity [57]. Thus, zinc offers protection against toxic metals via metallothioneins through both metallothionein protein induction and redox homeostasis [38].

Metal Transporters

There are numerous proteins responsible for carrying zinc throughout the body [24]. Distribution of metals into cells and tissues and elimination of metals from the body depends largely on the expression and activity of transporters. Metal-induced organ-specific toxicity or carcinogenesis is often associated with greater accumulation of metal in a particular target tissue and transporters play an important role in reducing metal burden in the body along excretory routes. Many transporters are metal selective, but not metal specific. One example is ZIP8 which was originally identified as a zinc transporter, but is now well known to transport cadmium and manganese [58, 59]. Based on high expression of ZIP8 in renal proximal tubules, it may be involved in reabsorption of cadmium [59]. Metal transport may be influenced by transporter expression or activity in response to metal exposure. Overexpression of ZIP8 increased lung cadmium burden and lung tissue loss following prolonged smoke exposure [52], and exposure of cultured urothelial cells to cadmium decreased expression of a subset of zinc transporter genes including ZIP10 [60]. In a study of chronic zinc and arsenic co-exposure, zinc reduced the amount of arsenic detected in all tissues tested and arsenic significantly affected expression of two zinc transporters in liver and kidney tissue [61]. Significant interactions between arsenic and zinc were evident for two transporters associated with arsenic uptake and efflux. The complexity of potential metal and zinc interactions at the level of transporters is not fully understood, but the evidence suggests that for at least some metals, zinc may decrease metal toxicity by reducing tissue burden of toxic metals based on transport mechanisms.

In vivo evidence for zinc protection against metal toxicity

Animal Models of Metal and Zinc Interactions

Experimental evidence from rodent models provides examples to support the mechanisms of zinc and metal interaction discussed above. Zinc supplementation before or during metal exposures is protective against toxicity from exposures to depleted uranium [62], lead [63], cadmium [38, 64–67] and arsenic [11, 42, 47, 49, 61]. The mechanisms of zinc protection include reduced oxidative stress and damage, restoration of zinc-dependent protein function, and decreased accumulation or increased excretion of toxic metals in target organs. Zinc also proved beneficial in the presence of metal mixtures [68, 69]. In the case of cadmium, zinc treatment ameliorated bone defects, toxicity in the testis, kidney, liver, blood vessels, oxidative stress and oxidative damage to lipids and proteins in the brain [52, 65–67, 70–72]. Zinc administration reduced lead levels in the blood and testis and counteracted the effects of lead on sperm health and renal function, histology and oxidative stress [63, 73, 74]. Zinc decreased arsenic-induced hepatic toxicity and restored antioxidant parameters, offset arsenic teratogenicity and reduced arsenic-augmented DNA damage [42, 57, 75–77]. Zinc was also effective at preventing acute toxicity following exposure to depleted uranium [62]. Often, zinc supplementation is associated with reduced tissue burden of toxic metals including uranium [62], cadmium [67] and arsenic [61, 75].

Additional evidence for the importance of metal and zinc interactions is provided by studies of metal exposures in zinc deficient animals. Arsenic-induced oxidative stress and inflammatory markers are exacerbated in zinc deficient mice [49] and synergistic changes are observed in the gut microbiome diversity upon arsenic exposure and zinc deficiency [50]. Marginal zinc deficiency enhanced cadmium accumulation in multiple organs including lung, liver and kidney and enhanced cadmium toxicity and carcinogenesis [52, 78, 79]. These observations may be particularly important for human populations with inadequate zinc intake.

Human Population Studies

There are a number of studies that report an inverse relationship between serum zinc status or dietary zinc intake and metal toxicity. There is a negative association between zinc and cadmium in blood or urine, suggesting that zinc modifies the absorption, excretion and/or accumulation of cadmium in the body [80–83]. For cadmium exposures, higher zinc level is associated with a decreased risk of renal toxicity, cardiovascular disease, prostate cancer, overall cancer mortality and mortality from all causes [4, 83–85]. Interactions between zinc and cadmium for epigenetic markers of ageing have been reported with differences between populations [86, 87]. Elevated lead levels were associated with low blood zinc and an inverse effect of zinc on DNA damage was noted in an occupationally exposed population. The authors suggest that lead absorption is modulated by zinc, thereby explaining the protective effects of zinc on lead exposure toxicity [88, 89]. In children, inverse correlations were noted between zinc and lead for neurodevelopment including autism spectrum disorders [90, 91] and a negative association between lead and stature was more pronounced and statistically significant in children identified as zinc deficient [92]. For arsenic, skin lesions linked to arsenic exposure were more common in people low in certain nutrients including, but not restricted to, zinc [20]. A few human studies suggest that zinc may modulate arsenic metabolism leading a less toxic form of arsenic that is readily excreted [20].

Human studies in metal-exposed populations do not uniformly report zinc benefits. Serum zinc did not have any causal mediation of the effects of other metals for oxidative stress in a Navajo population [33]. In a Spanish population, zinc was related to increased biomarkers of oxidative stress [34] in contrast to other studies suggesting zinc benefits for reducing oxidative stress. More work is needed in human populations to better understand the impact of zinc on metal toxicities.

Although the evidence for protective effects of zinc against metal exposures is limited in humans, the mechanistic underpinnings of metal and zinc interaction, coupled with abundant data from cell and animal experimental models, supports the hypothesis that supplemental zinc could provide benefits in metal exposed human populations. Zinc as a mechanism-based intervention is attractive because it is likely to target multiple pathways contributing to metal toxicities which may be particularly beneficial for populations that experience chronic exposures to mixtures of metals that interact with multiple zinc-dependent processes.

Thinking Zinc: A Zinc Intervention Clinical Trial in Partnership with the Navajo Nation

With more than 500 abandoned uranium mines, some of which are in close proximity to inhabited areas, Navajo communities have raised concerns regarding potential health consequences related to metal exposures [93]. Toxic metals including uranium and arsenic co-occur with other metals with exceedances of EPA’s MCL as much as 100-fold in unregulated water sources [5, 94–99], of relevance to the 30% of the Navajo Nation population who lack access to a regulated water source [5, 100]. Modeling of multiple contaminant exposure pathways classifies greater than 20% of the Navajo Nation as high potential for contamination from mine and mine waste sites [95], including often overlooked soil and dust contributions [101].

Biomonitoring of blood and urine samples as part of ongoing health studies confirms ongoing exposures to environmental metals with urine concentrations of uranium, manganese, cadmium and lead above levels reported in NHANES and identification of participant subgroups with distinct exposure profiles [94, 102]. Health effects associated with living in proximity to mines and mine sites or metal exposures identified through biomonitoring include increased risk for kidney disease, hypertension, diabetes, autoimmunity, and other immune disruptions in adults [93, 103–106], elevated risk of preterm birth associated with metal mixture profiles [102], developmental delays in children [107] and alterations in systemic immune responses in mother and infant pairs [108].

The compelling mechanistic data of zinc and toxic metal interactions provided the framework for development and implementation of a clinical trial on Navajo Nation. A scientifically sound and culturally acceptable study design was conceptualized through a strong community partnership. Zinc as a metal from Mother Earth to restore balance from unhealthy metal exposures is consistent with an indigenous view of wellness. Contributions from Navajo community leaders and community liaisons knowledgeable in language, culture, environmental and health conditions were instrumental for all aspects of the Thinking Zinc clinical trial. Community input led to the study name, expansion of the age inclusion criteria allowing older people to participate, and the overall trial design. Informational materials were developed in both English and the Navajo (Diné) language, then vetted by Navajo community members. Artwork was specifically created as a science communication tool to illustrate the concepts of metals competition with zinc-binding proteins (see Fig. 1), DNA damage, and DNA repair in a culturally resonant manner (Fig. 2). Presentations about Thinking Zinc include information about zinc-rich foods overall and in the Navajo diet. Blue corn mush is a common Navajo dish that is high in zinc when prepared in the traditional manner. Community liaisons prepare and serve blue corn mush to highlight a culturally relevant zinc-rich food source.

Fig. 2.

A Native artist Mallery Quetawki (Zuni Pueblo) rendering of DNA damage caused by uranium, reactive oxygen species, and other environmental chemicals and toxicants acting as “wrecking-balls” to a strand of DNA. The painted designs are taken from Pendleton blankets that are used both in gift giving, trade and ceremony. The designs used are from a collection of tribes throughout the Southwest and signify connectedness. B Native artist Mallery Quetawki (Zuni Pueblo) rendering of DNA repair depicted as re-stringing beautiful native beadwork. The flower design is a symbol of regrowth (Crow Nation)

Thinking Zinc (NCT03908736, Clinical Trials.gov) has a longitudinal study design to assess individual exposures and responses to zinc supplementation over time. The two baseline and two post-zinc supplementation collections are scheduled 3 months apart (Fig. 3). The study includes biomonitoring of metals in urine and blood samples. Other study endpoints are informed by mechanisms of metal toxicities, established effects of toxic metal exposures and endpoints reported to responsive to zinc. Endpoints include biomarkers of oxidative stress, inflammation, DNA damage using the single cell gel electrophoresis (Comet) assay, Poly (ADP-ribose) polymerase (PARP) activity and immune parameters including cytokines and lymphocyte profiles. The one-armed cohort intervention is an important aspect of Thinking Zinc clinical trial strongly favored by Navajo community advisors because risks or benefits are shared equally across all participants. This design bypasses research practices that contributed to mistrust in the communities [109, 110] and strikes the appropriate balance between ensuring respect for the Navajo culture and values while maintaining strong scientific validity. Scientifically, the longitudinal one-armed cohort design can account for substantial participant heterogeneity in both metal exposures and health status that have significant impact on the measured biomarkers such as cytokines and markers of inflammation. We have observed striking variability in metal concentrations and metal mixture profiles within individuals over time, between individuals, and between the participating communities. Individual responses pre- and post-zinc supplementation allows evaluation of changes within individuals (intra-individual) that might be obscured when looking across two groups (inter-individual). Quantitative comparisons of one-arm vs two -arm designs have shown that both designs are warranted under certain situations. The one-arm design normally requires less sample size, is appropriate when randomization to a placebo is not appropriate or ethical, and is more favorable when there is minimal standard of care activity or in the presence of a positive historical bias [111, 112]. Although current findings from Thinking Zinc are preliminary, the work has been well received within partnering communities [113].

Fig. 3.

Longitudinal one-arm study design. Each participant has two pre-zinc and two post-zinc samples taken approximately 3 months apart. Blood and urine samples are analyzed for metals, including zinc and biomarkers (DNA damage/repair, inflammation, oxidative stress). Graphic by Mallery Quetawki (Zuni Pueblo)

Perspectives

There is sound mechanistic evidence to support dietary zinc supplementation in metal-exposed human populations. Based on the diverse nature of metal and zinc interactions, zinc may be beneficial in situations of chronic mixed metal exposures and in populations with low zinc status where metal effects may be more pronounced. Low zinc status is widespread in many parts of the world [22, 25, 27, 28] and due to changing diet patterns, a decrease in zinc intake below recommended levels has been observed in the United States in recent years [114, 115]. Specifically, 15% of the U.S. population is estimated to have inadequate zinc intake [116]. Given the evidence for an inverse relationship between zinc and metal toxicity, a better understanding of potential vulnerabilities associated with marginal zinc status is needed. Zinc supplementation is an approach to mitigate metal toxicity that is not reliant on exposure reduction in the face of barriers to waste remediation and clean up. Zinc sufficiency can be achieved either through supplementation or increased consumption of zinc-rich foods. However, many foods with high zinc content, such as oysters, seafood, and red meats are not commonly consumed in all communities and alternate sources of zinc, such as a low-cost dietary supplement may be most effective to counteract metals-toxicity.

Although zinc is generally recognized as safe, there are a number of considerations for development of zinc intervention trials. Zinc dose and formulations are both important factors for zinc supplementation studies. Over the counter zinc supplements are readily available, but formulations of zinc (e.g. zinc oxide, zinc gluconate, zinc picolinate) differ significantly in their bioavailability so comparable oral dose may not yield equivalent results [22, 53, 117]. Zinc has a favorable safety profile, but excess zinc may lead to adverse health effects. Gastrointestinal symptoms have been reported and at high doses over extended periods, zinc may interfere with copper absorption, alter immune function and disrupt blood lipids [30, 117, 118]. Study participants and personnel should be well informed on appropriate zinc intake, zinc sources in the diet and as necessary, dietary sources relevant to the participating community.

Standardization of zinc measurement methods and assessment of biologic response to zinc are challenges for research studies. Serum zinc levels remain valuable because there are commonly accepted definitions of zinc sufficiency and insufficiency in human populations; the World Health Organization defines insufficiency as ≤ 70 µg/dL. Species and sex differences in animal models has not been adequately explored or reported. However, there are sex differences in humans with serum zinc significantly lower in women compared to men [120, 121]. No significant differences across the adult age span were detected in either women (P = 0.45) or men (P = 0.02) [121]. Unfortunately, numerous physiological variables such as sex, age, inflammation and time of blood draw in addition to sample handling and processing parameters affect serum zinc measurements [27, 119–122]. Zinc supplementation does not reliably result in detectable changes in serum zinc [27, 37, 121, 123] and although measurement of exchangeable zinc pool is reportedly responsive to zinc supplementation [124], the reliance on zinc isotopes is not amenable for studies in rural or remote locations. A number of functional biomarkers for zinc response are under consideration such as expression of zinc-sensitive proteins or genes including metallothionein or zinc transporters or biomarkers related to oxidative stress or DNA damage [27, 37, 125]. A double blind, randomized clinical trial of zinc supplementation found no difference in serum zinc between groups after 17 days, but significant decreases in DNA damage as measured by the comet assay [37]. This issue of zinc assessment and intervention response is an ongoing barrier to the field.

Conclusion

The current status of research findings in experimental models and population studies provide strong rationale and support for interventional clinical trials of zinc supplementation in communities chronically exposed to mixed metals. Given the ongoing barriers to waste remediation, a feasible and mechanism-based approach to combat multiple pathways contributing to metal toxicity provides an alternative to offset health risks for these populations. Moreover, ensuring zinc sufficiency is a low risk intervention that may provide additional health effects based on the established role of zinc to improve immune function, decrease oxidative damage and reduce inflammation all of which are known to be contributors to chronic diseases.

Author Contributions

L.H. and D.M. conceptualized the manuscript. L.H. wrote the main manuscript text with significant contributions from D.M. E. D-T. contributed to review of current literature utilized in the manuscript. All authors critically reviewed the manuscript.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

All reported studies/experiments with human or animal subjects performed by the authors have been previously published and complied with all applicable ethical standards (including the Helsinki declaration and its amendments, institutional/national research committee standards, and international/national/institutional guidelines).The Thinking Zinc clinical trial (NCT03908736) has been approved by University of New Mexico Institutional Human Research Protections Office (HRPO 18–380) and the Navajo Nation Human Research Review Board (NNR 18–330).

Competing Interests

The authors declare no competing interests.