Toxicogenomics of Arsenic, Lead and Mercury: The Toxic Triad

Abstract

The metalloid arsenic (As) and the metals lead (Pb) and mercury (Hg), which together we call the “Toxic Triad”, are among the pollutants of greatest global concern, harming the health of millions of people and contributing to biodiversity loss. The widespread distribution of As, Pb and Hg facilitates the exposure of humans and other species to these elements simultaneously, potentially amplifying their individual toxic effects. While As, Pb and Hg are well established as toxic elements, the mechanisms by which they interact with genetic material and impact the health of various species remain incompletely understood. This is particularly true regarding the combined effects of these three elements. In this context, the objective of this work was to perform a toxicogenomic analysis of As, Pb and Hg to highlight multiple aspects of element-gene interactions, in addition to revisiting information on the genotoxicity and carcinogenicity of the Toxic Triad. By using The Comparative Toxicogenomics Database, it was possible to identify that As interacts with 7666 genes across various species, while Pb influences 3525 genes, and Hg affects 692 genes. Removing duplicate gene names, the three elements interact with 9763 genes across multiple species. Considering the top-20 As/Pb/Hg-interacting genes, catalase (CAT), NFE2 like bZIP transcription factor 2 (NFE2L2), caspase 3 (CASP3), heme oxygenase (HMOX1), tumor necrosis factor (TNF), NAD(P)H quinone dehydrogenase 1 (NQO1) and interleukin 6 (IL6) were the most frequently observed. In total, 172 genes have the potential to interact with the three elements. Gene ontology analysis based on those genes evidenced that the Toxic Triad affects several cellular compartments and molecular functions, highlighting its effect on stimulation of toxic stress mechanisms. These 172 genes are also associated with various diseases, especially those of the urogenital tract, as well as being related to biological pathways involved in infectious diseases caused by viruses, bacteria and parasites. Arsenic was the element with the best-substantiated genotoxic and carcinogenic activity. This article details, through a toxicogenomic approach, the genetic bases that underlie the toxic effects of As, Pb and Hg.

1. Introduction

The global shift toward renewable energy and the widespread adoption of electronic devices are notable realities, especially in middle- and high-income countries. Nevertheless, the continuous rise in consumption and use of materials lead to worrying environmental and health consequences. Demand for metals in the production of personal electronic devices, electric vehicle batteries, rockets and other technologies, along with the accumulation of global material stocks and the jewelry industry, is collectively increasing mining activities worldwide. Excessive and poorly managed mining leads to metal pollution in the atmosphere, as well as in terrestrial and aquatic ecosystems, including in critical high-biodiversity areas [1,2,3].

Mining often leads to pollution from unintended elements, as seen in gold mining in the Amazon and Orinoco basins. This process involves large quantities of mercury (Hg), which contaminates the atmosphere, soil and rivers, posing serious risks to both human and animal life [4,5,6]. In addition, other anthropogenic factors also contribute to metal pollution, such as attempts at artisanal recycling of electronic devices (e-waste), that commonly contaminate the environment with various metals, including nickel (Ni), copper (Cu), cadmium (Cd), and lead (Pb) [7,8]. Fossil fuel combustion, cement production and poor waste management contribute to metal pollution globally [9]. Agrochemical production and the inappropriate use of pesticides and fertilizers also pollute ecosystems and food with toxic metals [10,11]. Combined, these different sources of pollution remobilize and disseminate a variety of metals into the biosphere. However, some specific elements are of particular concern.

The metalloid arsenic (As), and the metals Pb and Hg, are among the major toxic elements of global concern [12,13]. Arsenic is widely distributed around the world due to its natural occurrence combined with environmental contamination from anthropogenic sources. Exposure to As occurs mainly through contaminated water and, in humans, this metalloid is associated with the development of different types of cancer, skin disorders, cognitive impairment, among other conditions [14,15]. Multiple anthropogenic sources increase concentrations of Pb in the environment, such as the use of Pb-based paints, battery recycling, and combustion of fuels containing Pb. Exposure to Pb harms brain tissues, blood, bones, kidneys, heart, eyes, among other organs [16,17], being a particularly important problem in low- and middle-income countries [18]. Mercury is also toxic to several body systems, particularly affecting neurodevelopment and the locomotor function. Anthropogenic emissions (e.g., artisanal gold mining, fossil fuel combustion) associated with natural emissions (e.g., biomass burning) contribute to Hg pollution globally [5,9]. Pollution by As, Hg and Pb may negatively affect plants’ genomic stability, growth and survival, and can lead to the bioaccumulation of these elements in plant tissues such as leaves, flowers, and stems. This, in turn, poses a risk to herbivorous animals, including pollinators, which are essential for maintaining ecological balance [19,20]. Moreover, elevated metal concentrations can impair reproduction, behavior and other vital traits necessary for the survival of wildlife species. Thus, metal pollution not only threatens human health but also exerts a detrimental impact on biodiversity [5]. Given the high toxicity and the widespread environmental distribution of As, Pb and Hg, we propose the term “Toxic Triad” to refer to these elements together.

It has been estimated that up to 1.4 billion people live in areas at high risk of metal pollution [21]. Although the individual toxic effects of As, Pb and Hg are well known, in the “real world” humans and animals are usually exposed to complex mixtures of pollutants. The toxic effects of pollutants are often synergistic and cumulative, making exposure to multiple pollutants of high concern [22,23,24]. Furthermore, exposure to mixtures of several toxic elements, even within “safety standards” for each element, can have significant detrimental impacts on exposed organisms [23]. Thus, understanding the toxic effects of elements in combination is the next frontier of ecotoxicological studies. Toxicogenomic approaches are useful in this task, as they allow investigating the integrated effects of different pollutants on thousands of genes and biological systems of various species, bringing robustness to the results and conclusions [25,26,27,28]. Toxicogenomics also aids in hazard identification, distinguishes between genotoxic and non-genotoxic carcinogens, supports dose-response analysis, uncovers chemical-induced mRNA and protein expression patterns, and contributes to human health risk assessment [29,30]. In this context, this study aimed to perform a toxicogenomic analysis of As, Pb and Hg, investigating the main genes and biological systems jointly affected by the Toxic Triad, providing an update on the genotoxic and health effects of these elements based on gene-chemical interaction data from multiple species.

2. Materials and Methods

2.1. Target Elements

The toxicogenomic analyses of this study were focused on As, Pb and Hg, the first being classified as a “metalloid” and the other two as “metals” by the Royal Society of Chemistry [31]. From here on, these three elements will be collectively referred to as “metals” for convenience. This is a hypothesis-free article with the intention of analyzing the available toxicogenomic information on As, Pb and Hg.

Arsenic, Pb and Hg are listed by the World Health Organization among the ten chemicals of public health concern [32]. Recently, Marti et al. [13] used a structured expert judgement approach to select the most relevant pollutants in terms of health impacts. Among the five metals (As, chromium (Cr), Cd, Pb and Hg) identified by Marti et al. [13] as part of the sixteen most prevalent global pollutants, As, Pb and Hg account for the highest number of premature deaths annually (considering performance-weighted decision maker’s 50% percentile). More precisely, As had an estimated value of 136,000 premature deaths associated with its exposure, while Pb and Hg had, respectively, 1,660,000 and 79,800 deaths. These three elements also led the criterion Disability-Adjusted Life Years (DALYs) annually (As: 987,000 lost DALYs; Pb: 40,500,000 lost DALYs; Hg: 2,950,000 lost DALYs) [13].

Arsenic, Pb and Hg were also previously considered the metals of concern by Mamtani et al. [12]. However, we highlight that the choice to focus our study on As, Pb and Hg does not put in question the fact that other metallic pollutants are also highly relevant to public health and biodiversity in terms of toxicity, especially Cd and Cr [13,32,33,34].

2.2. Metal–Gene Interactions

Data on metal-gene interactions were obtained from The Comparative Toxicogenomics Database—CTD in March 2025, comprising CTD Revision 17685M [35]. The CTD is a database that has been publicly available for over 20 years, integrating robust, human-curated data on gene-chemical interactions, based on information derived from studies conducted in humans and other species [36]. The toxicogenomic approach used in this study was based on previous works by our group [37,38,39].

At CTD, chemical searches were performed for “arsenic” (Chemical Abstracts Service (CAS) registry number 7440-38-2), “lead” (CAS registry number 7439-92-1), and “mercury” (CAS registry number 7439-97-6). For these metals, the raw data (Excel files) concerning “genes” were downloaded and then we collected (I) gene names, (II) number of genes, (III) number of interactions (with the metals) and (IV) number of organisms on which these interactions were based. Of note, although the genes annotated in the CTD are from varied species, including humans and animal models, in this article we will standardize the writing of gene names in capital letters and italics.

Finally, we emphasize that the pooled analysis of toxicogenomic data from multiple species is relevant because it allows identifying potential health risks for different species, independently or even jointly. Furthermore, conclusions drawn from data derived from the integration of studies carried out with different organisms and under varied experimental conditions can be very robust, especially for pointing out biological trends and health risks. On the other hand, this approach can disregard some species particularities in relation to dose response, effects related to time of exposure to the chemical agent, and specific detoxification mechanisms acquired during the particular evolutionary processes of each species.

2.3. Analyses of Gene–Metal Interactions

First, the top-20 genes that interact with each metal were ranked based on the number of gene-chemical interactions (first ranking criterion) and number of organisms (second ranking criterion). The genes that were repeated in the top-20 rankings of the three metals were identified and highlighted. Next, using Microsoft Excel (Microsoft 365), the gene lists for the three metals were combined, duplicates were removed, and the total number of unique gene names was obtained.

Using VennViewer tool [40], Venn diagrams were made to obtain the genes that interact with As, Pb and Hg (considering commonalities between exact terms and any descendant terms), aiming to know which genes are most commonly affected by these three metals. Only curated chemical interactions were considered. First, Venn diagrams were generated considering (I) all chemical–gene interaction types combined (i.e., “increases”, “decreases”, “affect (degree unspecified)”), (II) “increases” chemical–gene interaction type, (III) “decreases” chemical–gene interaction type, and (IV) “affect (degree unspecified)” chemical–gene interaction type. Second, Venn diagrams were generated considering (I) “increased expression” chemical–gene interaction type and (II) “decreased expression” chemical–gene interaction type.

2.4. Gene Ontology, Enriched Diseases, Enriched Pathways, and Gene–Gene Interaction Network

Using the list of genes that interact with all three metals (obtained with the Venn diagram considering all chemical–gene interaction types combined, as detailed in the previous section), a gene ontology (GO) analysis (enriched GO functional annotations) was performed with the aim of knowing which “biological processes”, “molecular functions” and “cellular components” are regulated by these genes. Subsequently, also using the list of genes that interact with the three metals, enriched diseases and enriched pathways were obtained [41]. According to CTD, enriched diseases analysis “displays the diseases that are statistically enriched among your input genes/proteins” and enriched pathways analysis “displays the pathways that are statistically enriched among your input genes/proteins” [42]. The p-value of 0.001 was considered as the threshold for the GO analyses, enriched diseases and enriched pathways [41]. As described by CTD, “the significance of enrichment is calculated by the hypergeometric distribution and adjusted for multiple testing using the Bonferroni method” [42]. The top-20 results of the GO analyses, enriched diseases and enriched pathways were ranked based on corrected p-values (from lowest to highest).

Finally, a gene–gene interaction network was generated based on the list of genes that interact with all three metals [41]. Although the network is generated (input) with the total list of genes that interact with the Toxic Triad, n = 172, the obtained network (output) may contain a smaller number of genes/proteins, depending on the availability of information in BioGRID [43,44], the database that is used by CTD to generate the interaction networks.

2.5. Genotoxic and Carcinogenic Activity

The genotoxic and carcinogenic potential of “arsenic” (DSSTox substance identifier (DTXSID): DTXSID4023886), “lead” (DTXSID2024161) and “mercury” (DTXSID1024172) was also assessed in April 2025 through the CompTox Chemicals Dashboard v2.5.2 [45,46]. This is a database provided by the U.S. Environmental Protection Agency [46] that aggregates a range of toxicological data and information for several groups of chemicals.

3. Results

3.1. Metal–Gene Interactions

Arsenic interacts with a total of 7666 genes from multiple species. Lead interacts with 3525 genes, and Hg interacts with 692 genes. Combining the gene lists for the three metals (n = 11,883 gene names) and removing duplicates names, the three metals interact with 9763 genes across multiple species.

Table 1. Top-20 metal-interacting genes.

Arsenic (n = 7666 Interacting Genes)			Lead (n = 3525 Interacting Genes)			Mercury (n = 692 Interacting Genes)
Top-20 Gene Names	Number of Metal–Gene Interactions	Organism Number	Top-20 Gene Names	Number of Metal–Gene Interactions	Organism Number	Top-20 Gene Names	Number of Metal–Gene Interactions	Organism Number
CXCL8	101	1	TNF	61	4	CYP1A1	41	2
CAT	87	8	CYP1A1	57	3	HMOX1	37	4
NFE2L2	77	4	CAT	56	7	NQO1	25	2
CASP3	68	5	MT1	54	1	TNF	21	4
AS3MT	67	5	HMOX1	53	5	IL6	19	4
HMOX1	62	4	ALAD	49	4	ABCC2	17	5
MAPK1	62	6	CASP3	49	4	NFE2L2	16	4
MAPK3	59	6	MT2	48	2	MT2	15	6
TNF	53	3	NFE2L2	42	4	MT1	14	5
GSR	42	8	APP	41	3	CAT	13	4
VIM	42	2	NQO1	37	4	GSTP1	13	3
CDH1	39	1	PTGS2	36	4	IFNG	13	2
APOE	38	2	IL6	28	4	GSTA1	12	2
ERBB2	37	1	IL1B	26	5	ALB	11	3
NQO1	36	4	SOD1	26	9	MT3	11	3
TP53	36	6	HSPA5	25	4	CASP3	9	2
IL6	35	3	BCL2	24	4	CRYZ	9	1
SNAI1	35	1	MAPK3	24	3	GSTA2	9	3
SQSTM1	31	4	BAX	23	4	RELA	8	4
ATF3	31	2	RELA	22	4	SOD1, MT1A, MT2A *	8	3

Data from The Comparative Toxicogenomics Database. Number of metal–gene interactions: first ranking criteria. Organism number: second ranking criteria. Gene names in bold are those observed in the top-20 lists of the three metals. * Genes in the last position with the same numbers of metal–gene interactions and organisms.

details the top-20 metal-interacting genes. The genes CAT, NFE2L2, CASP3, HMOX1, TNF, NQO1 and IL6 were observed in the top-20 lists of As, Pb and Hg.

3.2. Profile of Metal–Gene Interactions

Figure 1 shows the profile of metal–gene interactions. In total, 172 genes (Figure 2; Supplementary List S1) have the potential to interact with As, Pb and Hg, considering all types of interactions combined (Figure 1, panel (a)). Notably, as evidenced by the gene–gene interaction network, many of these genes (Figure 2) are highly functionally connected (Figure 3). The types of interactions classified as “increases”, “decreases” and “affect” are shown in panels (b), (c) and (d), respectively (Figure 1).

Finally, we specifically filtered the number of genes that had increased or decreased expression by As, Pb and Hg. Figure 4 shows only the “increased expression” (panel (a)) and “decreases expression” (panel (b)) interaction types. Although there is a greater number of total metal–gene interactions of the type “decreases expression” (n = 4007; sum of the three metals individually) than “increases expression” (n = 3494; sum of the three metals individually), it is evident that together the three metals tend to increase gene expression (n = 45; Supplementary List S2) rather than decrease (n = 25; Supplementary List S3) (Figure 4).

3.3. Gene Ontology, Enriched Diseases and Enriched Pathways

Table 2. Gene ontology (top-20 results) based on genes (n = 172) that interact with all three metals.

Biological Processes		Molecular Functions		Cellular Components
Gene Ontology Term	Corrected p-Value	Gene Ontology Term	Corrected p-Value	Gene Ontology Term	Corrected p-Value
cellular response to chemical stimulus	1.14 × 10−78	Binding	3.04 × 10−53	cytoplasm	7.07 × 10−48
response to chemical	1.28 × 10−74	protein binding	1.69 × 10−52	cellular anatomical entity	7.91 × 10−47
response to stress	1.28 × 10−70	identical protein binding	6.64 × 10−41	intracellular anatomical structure	2.45 × 10−39
response to stimulus	1.16 × 10−68	enzyme binding	1.41 × 10−29	membrane-bounded organelle	6.35 × 10−37
response to organic substance	2.67 × 10−62	catalytic activity	4.73 × 10−21	intracellular membrane-bounded organelle	2.30 × 10−36
response to external stimulus	1.14 × 10−59	heterocyclic compound binding	3.14 × 10−20	cytosol	2.35 × 10−34
cellular response to stimulus	2.09 × 10−59	organic cyclic compound binding	7.01 × 10−20	organelle	1.80 × 10−33
positive regulation of biological process	2.89 × 10−59	molecular function regulator activity	2.16 × 10−19	intracellular organelle	3.84 × 10−33
negative regulation of biological process	7.67 × 10−56	antioxidant activity	1.58 × 10−18	extracellular region	3.77 × 10−29
biological regulation	1.95 × 10−55	protein dimerization activity	3.77 × 10−18	endomembrane system	4.78 × 10−27
regulation of biological process	3.15 × 10−55	protein-containing complex binding	6.43 × 10−17	intracellular organelle lumen	9.04 × 10−26
response to oxygen-containing compound	5.97 × 10−55	ion binding	9.93 × 10−17	membrane-enclosed lumen	9.04 × 10−26
positive regulation of cellular process	1.43 × 10−53	protein homodimerization activity	3.96 × 10−15	organelle lumen	9.04 × 10−26
negative regulation of cellular process	2.84 × 10−53	oxidoreductase activity	6.02 × 10−15	extracellular space	1.22 × 10−25
cellular process	4.04 × 10−53	receptor ligand activity	2.42 × 10−14	cell periphery	2.19 × 10−24
metabolic process	1.79 × 10−50	signaling receptor activator activity	3.52 × 10−14	plasma membrane	5.20 × 10−22
cellular metabolic process	8.36 × 10−50	molecular function activator activity	6.47 × 10−14	membrane	1.14 × 10−20
apoptotic process	1.03 × 10−49	signaling receptor regulator activity	1.13 × 10−13	nucleus	2.22 × 10−19
cellular response to organic substance	1.64 × 10−49	signaling receptor binding	1.89 × 10−13	vesicle	1.02 × 10−18
programmed cell death	1.47 × 10−48	ubiquitin protein ligase binding	9.62 × 10−12	cytoplasmic vesicle	1.43 × 10−16

Data from The Comparative Toxicogenomics Database.

details the top-20 results of the gene ontology analysis based on genes (n = 172) that interact with As, Pb and Hg. The terms of gene ontology show that the Toxic Triad affects numerous biological processes (n = 967 gene ontology terms), cellular compartments (n = 64 gene ontology terms) and molecular functions (n = 69 gene ontology terms), highlighting the effects of As, Pb and Hg on the stimulation of toxic stress mechanisms, as expected (Table 2).

Table 3. Enriched diseases (top-20 results) based on genes (n = 172) that interact with the Toxic Triad (As, Pb and Hg).

Disease	Corrected p-Value
Pathologic processes	5.72 × 10−102
Pathological conditions, signs and symptoms	1.15 × 10−97
Digestive system diseases	1.23 × 10−94
Liver diseases	9.37 × 10−92
Cardiovascular diseases	1.86 × 10−87
Urologic diseases	1.22 × 10−81
Neoplasms by site	2.00 × 10−81
Vascular diseases	3.42 × 10−81
Chemically-induced disorders	1.72 × 10−79
Neoplasms	4.76 × 10−79
Kidney diseases	3.91 × 10−77
Male urogenital diseases	7.47 × 10−77
Nutritional and metabolic diseases	4.56 × 10−76
Metabolic diseases	8.84 × 10−76
Female urogenital diseases	3.62 × 10−74
Female urogenital diseases and pregnancy complications	2.07 × 10−73
Neoplasms by histologic type	2.79 × 10−72
Neoplasms, glandular and epithelial	1.28 × 10−71
Urogenital diseases	1.11 × 10−69
Nervous system diseases	3.04 × 10−69

Urogenital diseases are highlighted in bold. Data from The Comparative Toxicogenomics Database.

details the top-20 enriched diseases based on genes (n = 172) that interact with As, Pb and Hg. Although these genes are involved in a large number of diseases, including varied cancer types and metabolic diseases (n = 512), urogenital diseases are prominent in the top-20 ranking (Table 3).

Finally, the top-20 enriched pathways based on genes (n = 172) that interact with the Toxic Triad are shown in

Table 4. Enriched pathways (top-20 results) based on genes (n = 172) that interact with the Toxic Triad (As, Pb and Hg).

Pathways	Corrected p-Value
Immune system	9.40 × 10−41
Fluid shear stress and atherosclerosis	1.39 × 10−31
Innate immune system	1.62 × 10−29
Chagas disease (American trypanosomiasis)	9.88 × 10−28
Signaling by interleukins	2.98 × 10−27
Cytokine signaling in immune system	5.26 × 10−27
Apoptosis	1.64 × 10−26
AGE-RAGE signaling pathway in diabetic complications	3.93 × 10−26
Hepatitis B	2.38 × 10−24
Influenza A	3.09 × 10−24
Metabolism	1.39 × 10−23
Interleukin-4 and 13 signaling	5.24 × 10−23
Pathways in cancer	7.23 × 10−23
Pertussis	9.62 × 10−23
TNF signaling pathway	1.08 × 10−21
IL-17 signaling pathway	4.11 × 10−21
Tuberculosis	9.85 × 10−21
MAPK signaling pathway	1.92 × 10−20
Salmonella infection	6.85 × 10−20
Toxoplasmosis	1.41 × 10−19

The terms are those defined in the CTD, but do not reflect a total independence between the pathways (for example, “innate immune system” can be considered as part of the “immune system”). Infectious/parasitic diseases are highlighted in bold. Data from The Comparative Toxicogenomics Database. AGE: advanced glycation end-products. RAGE: receptor for advanced glycation end-products. TNF: tumor necrosis factor. IL-17: interleukin-17. MAPK: mitogen-activated protein kinase.

. The presence of various infectious and parasitic diseases in the top-20 ranking was noteworthy. Thus, the complete list of pathways (n = 208; Supplementary List S4) was analyzed more carefully (manually) and a total of 21 (10.1%) pathways related to infectious diseases caused by viruses, bacteria or parasites were found among the pathways. Of note, prion disease was also included among infectious diseases as it can assume an infectious character.

3.4. Genotoxic and Carcinogenic Activity

Table 5. Genotoxic and carcinogenic activity of the Toxic Triad.

Metal	Genotoxic Activity: Summary Report	Carcinogenic Activity: Summary Report
Arsenic	Positive (based on assay performed on human peripheral blood lymphocytes)	Positive (considered carcinogenic to humans by multiple health agencies)
Lead	Positive (assays: sperm morphology, mammalian sperm morphology test (in vivo))	Probable/possibly/reasonably anticipated to be carcinogenic to humans by different sources
	Negative (assays: human (in vitro) and non-human (in vivo) chromosome aberration tests)
Mercury	No information available	Mixed results (not classifiable as to human carcinogenicity by multiple health sources; classified as possible human carcinogen by one source)

Summary reports synthesized by the authors based on data from CompTox Chemicals Dashboard v2.5.2.

summarizes the information on the genotoxic and carcinogenic activity of As, Pb and Hg. With the exception of Hg, there is evidence of the genotoxic action of the other two metals evaluated in this study. A robust body of evidence indicated the As is a human carcinogen. Lead is considered a probable human carcinogen. Regarding the carcinogenic activity of Hg, the results are mixed, but there is a possibility that this element has carcinogenic action. It is important to highlight that these results are based solely on the sources that feed the CompTox Chemicals Dashboard v2.5.2 [46], without considering other sources in literature.

4. Discussion

This study suggests that As, Pb and Hg can interact with 9763 genes across multiple species. This finding shows, through a toxicogenomic approach, that the Toxic Triad affects a wide variety of organs and biological systems, as well as are associated with several groups of diseases in humans. Furthermore, the interaction of these metals with such a large number of genes, coupled with the fact that the toxicogenomic information accessed in this study comes from multiple species, makes it likely that the Toxic Triad is responsible for harm to non-human species living in natural environments, as suggested elsewhere [5,20]. The effects of As, Pb and Hg on biodiversity are possibly underestimated since most researchers focus their studies on the effects of metals on human health.

We observed that As, Pb and Hg share the ability to interact with 172 genes from different species. As expected, due to the varied metal-related toxic mechanisms [47], gene ontology analyses showed that the Toxic Triad is related to chemical stress response pathways and effects in multiple cellular components. Gene ontology analyses and enriched diseases and pathways related to these 172 genes showed that they are involved in different groups of diseases, with emphasis on urogenital tract diseases. The kidney’s participation in the excretion of metals [48] may help explain, at least partially, why the Toxic Triad is related to urogenital tract diseases. Of note, individuals with urothelial carcinoma showed higher urinary levels of metals, including As and Pb, compared to controls [49]. The ability of toxic metals to influence the expression of genes involved in inflammation and cancer progression pathways [50,51] may also help explain the association observed between the Toxic Triad and diseases of the urogenital tract.

The robust carcinogenic activity of As and the potential carcinogenic activity of Pb evidenced by the U.S. Environmental Protection Agency [46] are supported by other sources of evidence [52,53], as well as the genotoxic activity of these two elements [52,53,54]. Contamination of water and food with As is a global problem, posing risks to food production and the health of animals and humans [55,56]. A recent systematic review based on 35 years of evidence supported an association between As exposure via drinking water and increased lung cancer risk [57]. The health effects of As can be attributed to its multiple genotoxic and epigenomic impacts, which may influence cancer signaling pathways [58]. Similarly, recent systematic reviews identified an association between Pb exposure and increased risk of human respiratory diseases [59], as well as negative health effects (ranging from subclinical to fatality) of Pb exposure in 57 mammal species [60].

Although data from the CompTox Chemicals Dashboard [46] indicate a lack of genotoxic information on Hg and mixed results on its carcinogenic activity, other sources indicate that this element has genotoxic activity [61,62] and increase the risk of cancer in humans [63]. Mercury has the ability to participate in different processes associated with DNA damage and carcinogenesis, such as production of free radicals and oxidative stress, direct interaction with DNA and microtubules, and disruption of DNA repair. Differences in the doses of exposure to this metal and its various compounds (e.g., methylmercury, dimethylmercury, inorganic mercury, phenylmercury acetate, thimerosal) help to explain the mixed results observed in relation to the toxic effects of Hg [64].

In this context, it is important to emphasize that this study analyzed different chemical forms of metals in an integrated manner, which may lead to a simplification or generalization of the specific toxicological properties of each metal species. For instance, in As biogeochemistry, speciation plays a critical role in determining mobility, reactivity, ability to interact with cells and other biological systems, and toxicity. Inorganic arsenic species, such as arsenite (As(III)) and arsenate (As(V)), differ significantly from organic methylated forms in terms of bioavailability and health effects. Furthermore, particularities in terms of environmental exposure (e.g., through food, water, atmosphere, soil) to different chemical forms of As significantly modify their potential toxic effects on multiple organisms [65,66]. Similarly, in the case of Hg, methylmercury, an organic form of Hg, is the primary neurotoxic form of concern considering environmental and food exposure (e.g., consumption of fish contaminated with methylmercury from artisanal gold mining activities), while other Hg species may exert distinct toxic effects [5,67]. Thus, metal speciation is a key factor influencing toxicity, and this should be considered when interpreting our results.

We observed that catalase (CAT), NFE2 like bZIP transcription factor 2 (NFE2L2), caspase 3 (CASP3), heme oxygenase (HMOX1), tumor necrosis factor (TNF), NAD(P)H quinone dehydrogenase 1 (NQO1) and interleukin 6 (IL6) are the main genes affected by As, Pb and Hg, all of them being in the top-20 lists of the Toxic Triad. The HMOX1 can be induced by chemical stimuli [68]. The gene CAT encodes the catalase, a major antioxidant enzyme that plays a crucial role in defending cells against oxidative stress [69]. The NQO1 also protects cells against oxidative stress [70]. The NFE2L2 participates in antioxidant and inflammatory responses, promoting protection from xenobiotics and proteotoxicity [71]. Both TNF and IL6 are major components of pro-inflammatory reactions [72]. Finally, the gene CASP3 participates in cell apoptosis [73]. With the exception of the CAT, all other genes had increased expression by As, Pb and Hg. Together, these gene functions suggest that the Toxic Triad triggers stress responses in exposed organisms, encompassing both inflammation and apoptosis, while also activating antioxidant defenses.

Through a data search of orthologs retrieved on 10 June 2025, on NCBI Orthologs [74], considering “vertebrates” or “tetrapods” and focused on the main genes affected by As, Pb and Hg, we found that such genes are observed in a large number of species (number of orthologs shown in parentheses): CAT—reference gene ID: 847 (662 orthologs), NFE2L2—reference gene ID: 4780 (681 orthologs), CASP3—reference gene ID: 836 (535 orthologs), HMOX1—reference gene ID: 3162 (665 orthologs), TNF—reference gene ID: 7124 (273 orthologs), NQO1—reference gene ID: 1728 (313 orthologs), and IL6—reference gene ID: 3569 (445 orthologs). These findings show that these genes occur across a wide range of species, supporting the cautious extrapolation of our results to multiple organisms.

The 172 genes affected by As, Pb and Hg are also highly related to biological pathways involved in infectious diseases caused by different classes of pathogens. At first glance, this result may seem intriguing, since the toxic effects of metals are classically associated with chronic and degenerative diseases. However, this result is in line with a growing body of evidence that has linked metal exposure to infectious diseases [75,76]. This is potentially due to the immunosuppressive capacity of metals that predispose hosts to pathogens [75,77]. Our results also suggest that metals may exacerbate infectious disease pathogenesis by interacting with genes involved in pathogen-related immune response. These results are highly relevant because although pollution is associated with infectious diseases [76], the mechanisms behind this association still need to be investigated. However, we stress that the large number of infectious diseases observed in our study may also be the result of indirect associations caused by the ability of metals to affect genes related to physiological stress, immune responses, and inflammation, which are common events in infectious processes. In this sense, we emphasize that the associations between genes, metals and infectious diseases observed in our study are correlative and should be interpreted as suggestive and hypothesis-generating only, rather than mechanistic or definitive.

Several limitations of this study warrant consideration. First, the datasets available on databases used in our analyses are influenced by research frequency (i.e., the number of publications per chemical or gene), potential reporting biases, and variability in experimental design across studies. For example, the high number of gene interactions reported for As may reflect its more extensive investigation compared to Hg, rather than a true greater biological significance as compared to other elements. Second, the identification of overlapping gene interactions does not necessarily indicate synergistic effects of the metals on specific genes. Third, our analyses grouped different metal compounds, potentially overlooking compound-specific effects on gene and protein interactions. These limitations should be carefully considered when interpreting our findings.

An additional limitation of this study is that toxicogenomic data from multiple species were analyzed in a pooled manner. This multi-species analysis may generalize the results obtained and disregard particularities of each species regarding dose-response, detoxification mechanisms, and evolutionary differences regarding responses to different chemical agents and environmental stresses. Toxicogenomic studies may also have difficulty in separating which genes are orthologous across species [78]. On the other hand, many genes included in toxicogenomic databases are well conserved across species, allowing the extrapolation of results to different organisms [39]. This is the case for the main genes evaluated in this study, as previously discussed. Furthermore, this cross-species approach allows identifying environmental risks relevant to both humans and non-human species, which is relevant in a One Health approach.

5. Conclusions

Our toxicogenomic approach suggested that As, Pb and Hg, the Toxic Triad, have the ability to affect multiple genes in combination, potentiating their individual toxic effects and explaining why these elements are associated with such a wide variety of diseases. Our results obtained with metal–gene interaction data from multiple species indicate that the Toxic Triad affects both humans and non-human species, advancing the knowledge of the toxic effects of metals on biodiversity.

Unlike studies that explore in detail the effects of one metal or a limited set of metals/metal compounds on a given organism, our article, using broad and robust toxicogenomic data, showed the global effects of As, Pb and Hg on different species. Despite the limitations of this approach, this article brings a key message: the metals that make up the Toxic Triad represent a health risk for both humans and other organisms due to their ability to interfere with multiple genetic and protein pathways. In this sense, our study contributes to strengthening the body of evidence indicating the need to limit environmental exposure to these metals. In addition, this work highlights the fact that different metals can affect common biological pathways, potentially exacerbating metal-related toxic effects.

This study also showed an interesting association of As, Pb and Hg with infectious and parasitic diseases, which is worrying considering the increase in pollution and dissemination of pathogens currently observed around the world. Finally, in addition to highlighting additional genotoxic basis of the Toxic Triad, this study emphasizes the urgency of controlling metal pollution, especially at a time of escalating mining activities in many countries.