Cellular pathologies and genotoxic effects arising secondary to heavy metal exposure: A review

Introduction

Heavy metals or metalloids are natural elements that have a density greater than 5 g/cm³. ¹ Heavy metals are ubiquitous, and they show a persistent profile in the environment. Their specific abilities and potential uses allow them to be used in the wide variety of industrial applications. Consequently, to increasing heavy metal usage, geogenic, industrial, agricultural, pharmaceutical, domestic effluents and atmospheric sources continuously release heavy metals to the environment, inducing perpetual concern on ecological and global public health effects. Environmental pollution is significant and oftentimes hazardous in the areas, where mining, foundries and smelters and other metallurgical industrial operations are located. ² Heavy metals, namely cadmium (Cd), lead (Pb), mercury (Hg), chromium (Cr) and arsenic (As) are all major threats to human health. These metals are known toxic agents and millions of people are exposed to these compounds either occupationally or environmentally. ³

Pb is one of the most abundant metals in Earth’s crust. Estimated annual Pb production is 15,000 million tonnes followed by Cd (22,000 tonnes) and Hg (2000 tonnes). Pb was suspected of 143,000 deaths and 0.6% of the global burden of diseases in 2004. ⁴ According to the research conducted by International Agency for Research on Cancer (IARC) and National Toxicology Programme, approximately 270,000 people were occupationally exposed to Cd compounds and 70,000 workers to Hg in the 1990s. ^5,6 Systematic biomedical research on the chronic effects of metals has been started during the past century; nevertheless, it is evident that even today, there are large gaps in knowledge with regard to the assessment of the health effects caused by environmental and occupational exposures to these metals. Furthermore, humans are not the only organisms affected by the exposure to heavy metals. A magnitude of species which differ substantially in regard to their physicochemical and biological properties are also affected by exposure to heavy metals. ⁷

One of the major characteristics of heavy metals is the induction of DNA damage and cancer via various modes of action. ^8,9 Heavy metals are known genotoxins that can lead to single- or double-strand DNA breaks causing damage in DNA. ¹⁰ Exposure to heavy metals causes genotoxicity through different pathways (Figure 1). They induce the production of reactive oxygen species (ROS) causing oxidative stress, make several cellular changes, which have an effect on repair mechanisms, and alter the reparation of double-strand breaks. They favour the ‘false’ repairing of DSBs, propagate DNA mutations and increase the risk of carcinogenesis. ¹¹ Thus, this study aims is to summarize the modes of actions of heavy metals by which they exert their genotoxicity.

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Figure 1.

Exposure to heavy metals causes genotoxicity through different pathways. ROS: reactive oxygen species; DSB: double-strand break.

Heavy metals can induce genetic damage via multiple pathways. Such pathways include the creation of micronuclei, chromosomal aberrations (CAs), and other anomalies and molecular-level DNA damage. The type of which heavy metals can induce which forms of DNA damage is dependent on their chemical identity, therefore mechanism of action. These effects can be identified and quantitated by the application of a battery of genotoxicity assays, which typically include the likes of cytokinesis-blocked micronucleus assay, CA assay, sister chromatid exchange (SCE) assay and alkaline comet assay (Table 1).

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Table 1.

Studies conducted on heavy metals and their genotoxic effects.

	Observed genotoxic effects
Metal/metalloid	Nuclear levela	Chromosome levelb	Molecular levelc
Cr	Yes 12	N/A	Yes 13
Cd	No 14	Yes 15,16	Yes 15
As	Yes 17	Yes 17	Yes 17 –19
Pb	Yes 20	N/A	Yes 21
Hg	Yes 22	Yes 22	Yes 22

^aMicronucleus test of either variant.

^bChromosomal aberration assay or sister chromatid exchange assay.

^cAlkaline comet assay.

This article summarizes and reviews the available data in scientific literature concerning the emergence of genotoxic effects secondary to heavy metal exposure.

Materials and methods

A detailed literature search was performed using the MedLine/PubMed database (National Library of Medicine, http://www.ncbi.nlm.nih.gov/PubMed), SCOPUS (Elsevier, https://www-scopus-com.web.bisu.edu.cn), Thomson ISI’s Web of Science (Thomson Reuters Corporation, http://apps.webofknowledge.com) and Google Scholar (Google, http://scholar.google.com). The keywords and the combinations of search terms were used as follows: metals (lead, mercury, cadmium, arsenic, chromium) + toxicity; metals + genotoxic; metals + mutagenic; metals + carcinogenic; metals + exposure; genotoxicity; metal-induced genotoxicity. All articles obtained were evaluated for further references.

Uses of heavy metals and human exposure

As is a naturally occurring metalloid. It is well-known for its ability to contaminate water and Pb to important adverse health effects including carcinogenesis upon chronic exposure. ²³ As is ubiquitous in nature, occurring in igneous and sedimentary rocks, as well as due to anthropogenic activities. In the environment, As is found in four different oxidation states as follows: arsenate (+5), arsenite (+3), elemental As (0) and arsenide (−3). Depending on the oxidation state, As’s physicochemical properties are subject to change. Elemental As is insoluble in water, but As salts show different solubility properties according to environmental pH. As is most abundant in its arsenite state, and it shows stability in oxygenated aquatic environments as arsenate. Groundwater mostly contains inorganic forms of As, which are thought to be more toxic than the organic forms. ²⁴ As is a highly toxic substance, and it is listed as the first in the list of priority substances compiled by the United States Department of Health and Human Services, Public Health Service. Thus, As in drinking water is strictly regulated to be lower than 10 μg/L. ²⁴

In industry, As-containing compounds are manufactured and are used to produce agricultural products. Insecticides, herbicides, fungicides, sheep dips, wood preservatives and dyestuffs are good examples of As-containing industrial products. Veterinary medicines that are used for tapeworm eradication in sheep and cattle also contain As. ²⁵ As has also medical applications, namely the treatment of some diseases such as syphilis, yaws, and trypanosomiasis in the past, and it is still being used in the treatment of amoebic dysentery and African sleeping sickness. ^25,26 As is known to induce programmed cell death (apoptosis) in leukaemia cells. Thus, FDA approved an As compound, arsenic trioxide as an anticancer drug in the treatment of acute promyelocytic leukaemia. ²⁷

Cd is a heavy metal characterized by its soft, ductile, bluish-silvery white constitution, shimmering and electropositive properties. It is tasteless and odourless. It can form different complex of organic amines, sulphur complexes, chlorocomplexes and chelates. In addition, Cd ions form salts of carbonates, arsenates, phosphates, and ferrocyanide, all of which are soluble. ²⁸ Cd attracts attention as an environmental and occupational concern. The level of Cd in the Earth’s crust is high with an average concentration of approximately 0.1 mg/kg. ²⁹ Cd is used industrially during the production of alloys, pigments and batteries. ³⁰ The use of Cd batteries has shown an increasing trend. However, industrial use of Cd has become restricted because of environmental concerns. In response to restrictions, in the United States, the daily Cd intake is about 0.4 μg/kg/day, less than half of the US EPA’s oral reference dose. ⁶ The primary sources of Cd exposure are smoking, inhalation and ingestion. Transdermal absorption is relatively rare. Occupational exposure is high for people who work in metal industries. Mining and smelting activities, battery manufacture, pigments, stabilizers and alloys increase Cd emissions into the environment. ^{31 –33} Low amounts of Cd are present in foodstuffs, such as potatoes, grains and seeds, liver, kidney, and leafy vegetables. ³⁴ Consuming Cd-containing foods can highly increase the risk of Cd accumulation in the human body. ³⁵ During the past century, due to the continuing use of industrial applications of Cd, there exists a dramatic increase of environmental pollution and human exposure to Cd. ³⁶

Cr is found in the Earth’s crust naturally with valence states ranging from Cr (II) to Cr (IV). ³⁷ Natural and anthropogenic activities allow Cr to enter air, water and soil. Metal processing, tannery facilities, chromate production, stainless steel welding, as well as ferrochrome and chrome pigment production are the most suspected industries for Cr release to the environment. Environmental concentrations of Cr thus show a trend of increase, and Cr acts as an environmental pollutant in many environmental systems. ³⁸ Commercial uses of Cr are industrial welding, chrome plating, dyes and pigments, leather tanning, wood preservation, and as an anticorrosive in cooking systems and boilers. ^39,40 As it is widely used in industrial applications, Cr [VI] compounds which are mostly water soluble are important industrial wastes. Cr [VI] is listed as a human carcinogen and occupational exposure may lead to nasal, oesophageal and lung cancers. ⁴¹ It is estimated that more than 300,000 individuals are occupationally exposed to Cr worldwide. ⁴² Annual Cr release is estimated to be 33 tonnes. ⁴³ Average atmospheric levels of Cr range from 1 to 100 ng/cm³. However, these levels might become higher in areas, where Cr is being processed. ⁴⁴ Cr can be ingested via Cr continued food and water. ⁴⁵ Generally, most of the fresh foods contain low levels of Cr. Although non-occupational Cr exposure is not negligible, people who are occupationally exposed to Cr get into contact with at least twice as much Cr as the average. ⁴³ Inhalation is the primary route of human exposure in terms of occupational exposure. Thus, the lung is the most affected organ. Despite this fact, dermal contact with Cr is not insignificant. ^46,47

Hg is a ubiquitous element. It is thus environmentally present as mercury vapour (Hg⁰), inorganic mercurous (Hg⁺¹) and mercuric (Hg⁺²) as well as inorganic Hg compounds. This property of Hg makes it available for exposure on all living organisms at some level. ^48,49 In addition to its natural occurrence, Hg is released by many anthropogenic activities. In the electrical industry, dentistry, nuclear, wood and pharmaceutical industries, Hg is widely used. ⁵⁰ Human exposure to Hg can be amplified in some circumstances, such as accidents, environmental pollution, consumption of contaminated food, dental care, preventative medical practices and industrial, agricultural and other occupational activities. ⁵¹ The primary sources of long-term, low-level exposure to Hg are dental amalgams and consumption of contaminated fish. Hg enters the water naturally as it is found it the Earth’s crust. It can also enter through industrial activities. ⁵² Methylation of Hg is carried out by algae and bacteria in the water. Then, methyl mercury (MeHg) enters the food chain through fish and shellfish and bioaccumulates. ⁵³ The Hg⁰ and MeHg are the most readily absorbed forms of Hg. Hg⁰ is present in more than 50% of all dental amalgams. ⁴⁹ As the elemental vapour has highly lipophilic properties, it is readily absorbed by the lungs and epithelial lining of the mouth. Furthermore, Hg⁰ in the blood can pass through cell membranes and important defensive barriers including blood–brain and the placental barriers. ⁵⁴ Inside the cell, Hg⁰ rapidly undergoes oxidation and becomes highly reactive Hg⁺². On the other hand, MeHg that enters the body via ingestion is absorbed in the gastrointestinal tract and like Hg⁰, it can pass through blood–brain and the placental barriers. Unfortunately, the excretion rate of the Hg is very low. It accumulates especially in the kidneys, nervous tissue, and the liver. Regardless of its form, Hg is a highly toxic element, and it shows its effect as enterotoxic, neurotoxic and nephrotoxic agent. ⁵⁰

Pb is a naturally occurring bluish-grey metallic element. Despite its natural occurrence, combustion of fossil fuel, mining and manufacturing are the human activities yielding an increase in the release of Pb to the environment at high concentrations. Pb is being used industrially, agriculturally and domestically. The most well-known uses of Pb can be listed as the production of Pb-acid batteries, ammunition, metal products and X-ray shielding. Production of Pb-acid batteries is the most liable source of Pb release to the environment, which accounts for approximately 83%. ^55,56 The majority of Pb exposure occurs as a result of Pb-contaminated dust particles or aerosols and ingestion of Pb-contaminated food, water, and paints. ^57,58 Depending on several factors including age and physiological condition, the effectiveness of absorption of Pb is subject to change. In human subjects, kidneys take up the greatest percentage of Pb, followed by the liver, heart, brain and the skeleton. ⁵⁹ Pb poisoning greatly affects the nervous system with symptoms, such as headaches, poor attention span, irritability, loss of memory and dullness. ^58,60 Apart from being a nervous system toxicant, Pb also affects several organs and systems, including the kidneys, liver, haematopoietic, endocrine and reproductive systems. ⁵⁸ In general, Pb exposure occurs due to Pb-contaminated household paints, Pb-containing crystals, and ceramics, several Pb-containing medicines and cosmetics. Occupational Pb exposure is also widespread. ⁵⁷ Exposure to Pb during pregnancy is one of the major concerns in gestation. Pb can directly penetrate foetal tissues and may cause a decrease in birthweight, early delivery and neurodevelopmental deficiencies. ^{61 –63}

Heavy metal-induced genotoxicity

Depending on the mechanism of action, arsenicals can act as genotoxins, non-genotoxin agents and also carcinogens. As compounds induce genotoxicity, and it has been shown that they cause DNA damage repair inhibition, induction of CAs, SCEs and formation of micronuclei as well as molecular-level DNA damage. ^17,64,65 Arsenic trioxide also induces DNA damage. ⁶⁶ Furthermore, As compounds cause the induction of gene amplification, cellular arrests and induction of c-fos gene expression and oxidative stress. ^65,67 It has been shown that As compounds are highly cytotoxic, and they induce the transcription of stress genes. ⁵⁰ Specifically, inorganic As⁺³ (iAsIII) and its methylated metabolites (monomethyl arsenite (MMAIII) and dimethyl arsenite (DMAIII)) are suspected to inhibit DNA repair. Furthermore, iAsIII can bind to the zinc finger motifs in recombinant proteins that contain three or more cysteine residues leading to alteration in the function of DNA repair mechanisms. To compare, methylated trivalent As metabolites interact with high affinity with cysteine containing repair proteins involved in nucleotide excision repair (NER). Additionally, xeroderma pigmentosum group A protein that has a significance in NER mechanism is affected by As compounds. ⁶⁸ It must be noted that although many As-bearing species have been demonstrated to have genome-level and chromosome-level DNA disorganizing properties, the question of whether As can cause molecular-level DNA damage has been a topic of contention, with both negative and positive results, although there is a growing body of scientific literature supporting a causal relationship between As exposure and the development of molecular-level DNA damage as can be detected via the use of the comet assay. ¹⁷

Cd compounds are considered as human carcinogens. ³² Cd can bind to proteins, decrease DNA damage repair, activate protein degradation, up-regulate cytokines and proto-oncogenes (c-fos, c-jun and c-myc), induce the expression of metallothionein, haeme-oxygenases, glutathione (GSH) transferases, heat-shock proteins, acute-phase reactants, and DNA polymerase β at lower concentrations. ^{69 –72} Cd has different influences on cell proliferation, differentiation and apoptosis. These activities lead to interaction with DNA repair mechanisms, induction of ROS as well as apoptosis. At low concentrations, Cd interacts with mitochondria and inhibits both cellular respiration and oxidative phosphorylation. This may cause CAs, SCE, DNA strand breaks, and DNA–protein cross-links in cell lines. Moreover, Cd may cause mutations and potential chromosomal deletions. Its toxicity relies on the depletion of reduced GSH, binding sulphhydryl/thiol groups within proteins and inducing the generation of ROS, including superoxide (O₂ ⁻), hydrogen peroxide (O₂ ⁻²) and hydroxyl (•OH) radicals. Some enzymatic activities, for instance, manganese superoxide dismutase (SOD) and copper/zinc dismutase are inhibited by Cd. Cd sensitivity depends on metallothionein, which is a zinc-containing protein with 33% cysteine. It acts as a free-radical scavenger and binds specifically to hydroxyl and superoxide radicals. In general, the cells that contain metallothioneins show resistance to Cd toxicity, and others show sensitivity to Cd intoxication. Furthermore, Cd can indirectly activate the apoptotic processes by modulating the cellular Ca⁺² levels and activation of caspases and mitogen-activated protein kinases in the cells. ²⁸ All the Cd-treated male rats showed pathological testicular alterations and liver and kidney damage after chronic exposure. The lung is the most susceptible organ for human carcinogenesis caused by Cd exposure. ³² Occupational and environmental exposure to Cd may increase the development of certain cancer types, such as cancers of prostate, kidney, liver, hematopoietic system and stomach. ^73,74 It is also known that chronic exposure of Cd may lead to pathological testicular alterations. ⁷⁵

Toxicity of Cr compounds depends on the valence state and solubility. Cr (VI) compounds are known to be powerful oxidizing agents, therefore act as irritating and corrosive agents. In comparison, Cr (VI) compounds show more toxicity than Cr (III) compounds. ^76,77 The reason why Cr (VI) is more toxic is that Cr (VI) can pass through cell membranes and be reduced to reactive intermediates. On the other hand, Cr (III) is poorly absorbed via any route of administration. Cr (VI) can be absorbed by the lung, gastrointestinal tract and skin. Reduction of Cr (VI) is either a detoxification process or a toxification process depending on the site. Reduction processes taking place away from the target site is considered detoxification. In contrast, reduction of Cr (VI) shows toxic effects if it takes place in or near cell nucleus of target organs. ⁷⁸ Reduction of Cr (VI) relies on several enzymes depending on the type of the cell that it penetrates. Hydrogen peroxide, GSH reductase, ascorbic acid and GSH are the key factors during this process. As a result of reduction reactions, Cr (V), Cr (IV), thionyl radicals, hydroxyl radicals and Cr (III) are the reactive intermediates produced. The major mechanism of Cr-induced DNA damage is the generation of oxidative stress through the production of reactive intermediates. These reactive intermediates can disrupt cellular integrity and functions by attacking DNA, proteins and membrane lipids ^79,80 Cr (VI) induces DNA intrastrand cross-links, DNA adducts and DNA single-strand breaks. DNA double-strand breaks are also created in Cr (VI) exposure. Thus, Cr (VI) exposure leads to genotoxicity indirectly as it cannot directly interact with DNA. Furthermore, Cr (VI) exposure can activate cell cycle checkpoints and apoptotic cell response. ⁴¹

The main pathway in which Hg exerts its toxicity is the induction of oxidative stress by producing ROS. ⁸¹ Hg has mechanisms related to sulphhydryl/thiol reactivity. Inside the cell, MeHg and Hg⁺² covalently bind the cysteine residues of proteins and cause the depletion of cellular antioxidants. Antioxidant enzymes act as a defence mechanism against Hg compounds. ⁸² Inorganic Hg damages oxidative phosphorylation and electron transport pathways at the ubiquinone–cytochrome b5 locus and, thus, induces ROS production ⁸³ Moreover, Hg accelerates the rate of electron transfer in the electron transfer chain in the mitochondria. This, in turn, causes the premature release of an electron to molecular oxygen and generation of ROS. ⁸⁴ According to studies conducted by Leaner et al. and Zalups et al., Hg has negative effects on cellular organelles and their biological functions. ^85,86 There is a controversy surrounding exposure to Hg and carcinogenesis. Some studies have indicated that Hg is genotoxic. On the other hand, others show no correlation between Hg exposure and genotoxicity. ⁸¹ In studies that support the induction of genotoxicity by Hg, it is shown that Hg increases the ROS production that damages DNA in cells and may result in the induction of carcinogenesis. ^82,87 ROS may cause conformational changes in DNA, mitotic spindle and chromosomal segregation proteins. ⁸²

Pb has the ability to inhibit or mimic calcium and interact with proteins. ⁵⁸ Pb behaves similarly to calcium within the skeleton. There are certain mechanisms by which Pb exerts its actions. By interacting with sulphhydryl/thiol and amide groups of enzymes, Pb diminishes enzymatic activity. It also exerts its action via competing with essential elements for their binding sites, inhibiting the activity of the enzymes and altering the transport of essential cations. ⁸⁸ According to the studies, Pb induces the formation of ROS and, thus, causes cellular damage. ⁸⁹ People who are exposed occupationally to Pb are shown to have increased antioxidant enzyme activities, especially SOD and GSH peroxidase in their erythrocytes. ⁹⁰ Various research studies demonstrated that Pb interferes with calcium-dependent processes interrelated to intracellular signal transduction and neuronal signalling. Pb disrupts intracellular calcium cycling and alters the release of calcium from endoplasmic reticulum and mitochondria. ^91,92 Pb can also inhibit calcium-dependent release of neurotransmitters and also receptor-coupled ionophores in glutamatergic neurons. ⁹³ In contrast, it may augment protein kinase C and calmodulin activity. ^91,94 Furthermore, exposure to Pb increases the likelihood of gene mutations and SCEs. ^95,96

Conclusion and discussion

Heavy metals are capable of inducing the generation of ROS and leading to oxidative stress. As they act through different mechanisms of action, the outcome of metal-induced oxidative stress is subject to change. It must be noted that the mechanisms of action of heavy metals are not fully understood. Thus, further studies should ideally pay particular attention to improvements in the understanding of the mechanisms of actions of heavy metals.

Environmental heavy metal pollution is one of the major problems facing many countries around the globe. Abandoned mining areas and industrial wastes they create persist in the environment. These specific sites are in need of urgent remediation with maximum efficiency and minimum cost. This will increase the need for methodologies employing green and sustainable remediation. Phytoremediation is important in that it is widely used as an in situ remediation technique for heavy metals. ¹ Advantages of phytoremediation can be listed as follows: the use of solar energy to power the process, cost-effectiveness, easy operation, reduction in secondary air and water contaminants, and the use of biomass for biofuel production and low-cost adsorbents. ^97,98 Several disadvantages have also been documented: selective metal uptake by plants, slow growth, minimal biomass, long clean-up periods and treatment/disposal of metal-loaded plant biomass. ^99,100 With sufficiently high-volume remediation efforts, the recovery of heavy metals as raw material may become economically viable.

Countries with relatively advanced levels of socio-economical and industrial development tend to have in place laws and regulations limiting the contamination of the environment with sources containing heavy metals, therefore, such toxicants are disproportionately more likely to affect underdeveloped countries, which are also typically characterized by a relatively underdeveloped healthcare system. This exposes a large number of people to extensive heavy metal exposure with bleak hopes of treatment, management or prevention of such environmental hazards, resulting in widely prevalent heavy metal-induced maladies, which, however, are difficult to diagnose and quantitate due to the aforementioned scarcity in high-quality medical care.

As the potential of heavy metals to cause carcinogenesis in humans has been established in scientific literature, ^101,102 exposure to heavy metals is strongly associated with cancers of various systems. In 2015, cancer.org ¹⁰³ estimates that the cost of treating cancer in the United States alone was 80.2 billion USD, divided over some 14 million people, averaging around 5700 USD per person per year. Therefore, cancer has a quite tangible burden on the economy of a country as well, especially small countries with widespread contamination and limited economic means. Therefore, decontamination and remediation efforts may not only allow for the economically viable recovery of valuable industrial resources but may also reduce the prevalence of economically costly diseases such as cancer by a significant margin.

The test results can indicate the type of damage occurring due to heavy metal exposure. Cd, for instance, yields positive results for genetic damage in all assays concerned except micronuclei, indicating that the genetic damage it causes is affected on multiple levels upon the genome and results in multiple forms of genetic instabilities both at the molecular level and at the supramolecular level. Similarly, Hg yields positive results on all four major assays, indicating the presence of detrimental genotoxic effects on all levels of the genome. As, on the other hand, shows marked development of genetic abnormalities only in micronuclei testing, indicating that although it causes genetic damage, it is at DNA organization level and not due to molecular insult to DNA (Table 1).