Molecular and epigenetic markers as promising tools to quantify the effect of occupational exposures and the risk of developing non-communicable diseases.

Non-communicable diseases (NCDs) are chronic diseases that are by far the leading cause of death in the world. Many occupational hazards, together with social, economic and demographic factors, have been associated to NCDs development. Genetic susceptibility or environmental exposures alone are not usually sufficient to explain the pathogenesis of NCDs, but can be integrated in a more complex scenario that can result in pathological phenotypes. Epigenetics is a crucial component of this scenario, as its changes are related to specific exposures, therefore potentially able to display the effects of environment on the genome, filling the gap between genetic asset and environment in explaining disease development. To date, the most promising biomarkers have been assessed in occupational cohorts as well as in case/control studies and include DNA methylation, histone modifications, microRNA expression, extracellular vesicles, telomere length, and mitochondrial alterations.

Occupational and lifestyle factors role in non-communicable disease development

Non-communicable diseases (NCDs) are chronic diseases that are by far the leading cause of death in the world, representing 71% of all annual deaths (WHO, 2018; https://www.who.int/news-room/fact-sheets/detail/noncommunicable-diseases). The four main types of NCDs are cardiovascular diseases (like heart attacks and stroke), cancer, chronic respiratory diseases (such as chronic obstructed pulmonary disease and asthma) and diabetes. The risk of NCDs is increased by modifiable behaviors, such as tobacco use, physical inactivity, and unhealthy diet. Many occupational hazards, together with social, economic and demographic factors, have also been associated to NCDs development, in particular with chronic respiratory diseases and cardiovascular diseases. Moreover, high levels of occupational stress, isolated living conditions, worksite food catering services, nocturnal work shifts and other related factors have been shown to affect NCD development (13).

Same exposure, different risk: theimportance of detecting the hypersusceptible subjects

Occupational risks have been identified by observations of increased morbidity or mortality among specific populations of workers. Hypersusceptibility is defined as a condition characterized by a set of risk factors that make some people more susceptible than others to certain exposures (37, 72, 107). Risk factors include distinct categories, as genetic asset and lifestyle/occupational factors. Genetic susceptibility variants are inherited traits at the bases of biological variability, and contribute to determine the individual molecular response to environmental insults. Lifestyle/occupational factors include all non-genetic forces experienced during life, such as nutritional status, drug use, socioeconomic status, behavioral traits, occupational and environmental exposures (37, 107). Both these components are crucial in the onset of several diseases, such as NCDs, which are often associated with older age groups, although evidences show that 15 million of all deaths attributed to NCDs occur between the ages of 30 and 69 years. Of these “early” deaths, over 85% are estimated to occur in low- and middle-income countries, where several environmental exposures reach high levels and occupational conditions are often life threatening.

As an example, to investigate a population of workers, we cannot avoid considering concurrent risk factors that are well represented in the general population, such as obesity. Obesity is a condition of increased chronic low-grade inflammation, which results in an increased susceptibility to cardiovascular risk associated with particulate exposure. For this reason, the inclusion of several hypersusceptibility factors must be taken into account to obtain a comprehensive understanding of individual risk of developing NCDs.

Epigenetics at the crossroads between genetics, environmental exposures and disease

Genetic susceptibility or environmental exposures alone are not usually sufficient to explain the pathogenesis of NCDs, but should be integrated in a more complex scenario that can result in pathological phenotypes. Epigenetics is a crucial component of this scenario, as its changes are related to specific exposures, therefore potentially able to display the effects of environment on the genome, filling the gap between genetic asset and environment, in explaining disease development. As the epigenome is a plastic entity modified by the environment and some modifications are detectable in accessible tissues (e.g. peripheral blood, urine, buccal cells, etc.) and show long-term stability, epigenetic pattern might be considered as a memory of occupational/environmental exposures experienced throughout life (57, 77).

In this last decade, the Genome Wide Association Studies (GWAS) have clearly established that high-penetrance disease-associated genetic variants cause defined functional consequences (e.g. nonsynonymous amino acid changes or truncated proteins). On the other hand, a large portion of low-penetrance variants are difficult to interpret, as they do not cause downstream functional changes able to determine per se a pathogenic effect. Epigenetics might offer an opportunity to disentangle how these variants impact the effects of occupational/environmental exposures, clarifying their role as risk factors in NCD onset (58). The effects of a particular genetic variant can be modulated by epigenetic modifications, associated for instance with occupational/environmental exposures, able to limit the expression of the corresponding allele (111, 114).

Altered epigenetic states have been shown to be associated with a wide range of NCDs including cancer (94, 112, 119), cardiovascular diseases (1), neurodegeneration and aging (44, 71, 103), psychiatric (75, 104, 120) and autoimmune diseases (30, 68, 74), all of which are known to be influenced also by occupational/environmental exposures (57).

Besides epigenomics, “Omics” includes genomics, transcriptomics, miRNomics, proteomics, metabolomics, and microbiomics (figure 1). The emerging field of omics – i.e. large-scale data-rich biological measurements- provides new opportunities to advance our knowledge in the field. A deep analysis of this topic goes beyond the purpose of the present review, but a useful introduction can be found in the review by Karczewski and Snyder (53).

Figure 1

The emerging field of “omics” includes a variety of large-scale data-rich biological measurements and provides new opportunities to better understand how occupational exposure, lifestyle factors and susceptibility factors can modulate disease risk

Novel biomarkers

While genetic mutations are relatively rare events and, once acquired, cannot be reverted, epigenetic changes are dynamic and can allow the cells to adapt their functioning in response to every stimulus. At the same time, as epigenetic profiles can reflect several diseases, they can help to explain the molecular mechanisms underlying the disease risks, and can also be considered as suitable clinical biomarkers (both diagnostic and prognostic) of several NCDs, among which the most consolidated is cancer (39, 78).

In order to be able to detect the epigenetic changes induced by the external environment, high sensitive and specific biomarkers are needed. Moreover, the ideal biomarker should be easy to measure, not only in a research laboratory setting.

DNA methylation, histone modifications, microRNA expression, extracellular vesicles, telomere length, and mitochondrial alterations are promising biomarkers to be used in occupational settings.

DNA methylation

DNA methylation represents the best-characterized epigenetic modification. It implies the addition of a methyl group to the fifth carbon of the cytosine DNA base, giving rise to a 5-methyl-cytosine (figure 2). In differentiated cells, only cytosine followed by a guanine can be efficiently methylated by specific enzymes, called DNA methyltransferase. All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor (76). Patterns of DNA methylation are established early during development, with two critical waves of methylation and demethylation occurring during embryogenesis. On the one hand, when DNA methylation pattern is established (in utero), it is then maintained into subsequent cellular generations, being a mechanism through which each cell lineage becomes highly specialized. On the other hand, any stimulus reaching a particular cell through life is potentially able to modulate its DNA methylation (3).

Figure 2

DNA methylation implies the addition of a methyl group to the fifth carbon of the cytosine DNA base, giving rise to a 5-methyl-cytosine. In differentiated cells, only cytosine followed by a guanine can be efficiently methylated by specific enzymes, called DNA methyltransferase. All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor. DNA methylation occurring in gene promoters can directly alter gene expression, by inhibiting the access to DNA of the transcriptional machinery function. This methylation is often referred as “gene-specific methylation”. Methylation taking place in repetitive transposable elements (e.g. Alu, LINE-1, HERVs, etc.) can compact the chromatin structure and therefore increase DNA resistance to toxicants

DNA methylation occurring in gene promoters can directly alter gene expression, by inhibiting the access to DNA of the transcriptional machinery function. This methylation is often referred as “gene-specific methylation” (79). Methylation taking place in repetitive transposable elements (e.g. Alu, LINE-1, HERVs, etc.) has a different function, as it can compact the chromatin structure and therefore increase DNA resistance to toxicants (116).

DNA methylation is the first player that allows the body to shape gene expression in response to environmental stimuli. This first adaptive reaction might later evolve in a stable change potentially contributing to the development of disease.

One of the most fascinating open points on DNA methylation is the possibility for transgenerational inheritance of methylation changes, implying both an impact on the offspring through direct effects of exposure of the gametes or embryos, or rather a true transgenerational effect on subsequent generations that did not directly experience the exposure.

Bisulfite treatment is the method of choice for the analysis of DNA methylation in complex genomes as it is a simple method to convert an epigenetic mark into a genetic difference, which can be further analyzed by standard molecular biology techniques, such as pyrosequencing. Several additional methods have been developed in the last few years for DNA methylation analysis, however bisulfite treatment and pyrosequencing remain the gold standard to evaluate the effects of environmental exposure, due to its sensitivity that allows the detection even of very slight modifications (14).

Several types of exposures experienced during occupational life have been shown to modulate both global and gene-specific DNA methylation.

An increased global methylation was reported in coal workers and in hairdresser exposed to formaldehyde (10, 28). A reduced global methylation was reported for workers in petro-chemical factories, and workers exposed to low-dose benzene, such as petrol-station workers (16, 34, 95, 102). Global methylation studies conducted on farmers exposed to different pesticides were not conclusive. Most of the studies reported a decrease of global methylation, while Benedetti and colleagues recently reported genomic hypermethylation in soybean farmers exposed to complex mixture of pesticides (12, 27, 51, 52, 97).

To evaluate the effects of occupational exposure, methylation levels have also been measured on promoters of several genes, mainly involved in cancer risk, and epigenetic alterations may be early indicators of genotoxic and non-genotoxic carcinogen exposure (99).

In hairdressers, gene specific methylation analysis of selected cancer-related genes allowed the identification of higher methylation of the CDKN2A gene, a master regulator of cell cycle (66). Increased methylation levels of CDKN2A were also reported in nurses and midwives (19), together with BRCA1 and BRCA2 tumor-suppressor genes (88). Increased methylation levels of tumor-suppressor genes were also confirmed in lead-exposed workers (125).

Low-dose benzene exposure was associated with aberrant promoter methylation of genes involved in the inflammation process, in nitrosative stress and in xenobiotic metabolism (50). In petrochemical workers, the tumor-suppressor p15 altered expression was reported (16, 102), and cancer-related NRF2 and KEAP1 genes altered promoter methylation was reported in men occupationally exposed to arsenic (47). DNA methylation of the cancer-related genes F2RL3 and AHRR was associated with occupational exposure to polycyclic aromatic hydrocarbons (PAHs) (4). Occupational exposure to vinyl chloride monomer (VCM)-occupational exposure was associated with altered methylation of tumor-suppressor p16 (117), and cancer-related MGMT and MLH1 genes (122).

Exposure to pesticides was associated with several altered gene specific-methylation levels: p16 (52); CDH1, GSTp1, and MGMT (97); BRCA1, and genes involved in p70S6K signaling, and PI3K signaling in B lymphocytes (33, 42).

Methylation levels of NOS3 and EDN1 genes, involved in coagulation, were altered in steel workers (108).

Goodrich and colleagues reported that dental workers exposure to mercury (Hg) was associated with reduced methylation of SEPP1 gene, which is involved in the counterbalance of Hg toxic effects in buccal epithelium (38).

Interestingly, long-term exposure to shift work in cohorts of nurses and midwives, was associated with altered methylation of CLOCK genes, which are involved in the control of circadian rhythm and whose alterations have been linked to increased cancer risk (18, 93).

The increasing experimental and epidemiological studies of DNA methylation changes caused by occupational exposures suggest that both global and gene specific methylation are strongly impacted, and that epigenetic marks may have the potential to be future biomarkers for occupational risk assessment.

Histone modifications

Each human cell contains approximately 2 meters of DNA, while the average nucleus is 0.006 mm in diameter. This simple observation underlines two opposing requirements: DNA needs to be compacted, but on the other hand it needs to maintain accessibility in order to be transcribed, replicated, and repaired.

Human DNA is therefore compacted by being wound around octamers of core histone proteins that form nucleosomes. Nucleosomes comprise 147 base pairs of DNA wrapped approximately twice around the protein core that contains two copies of each histone, H2A, H2B, H3, and H4 (69). The interaction between DNA and histones is critical to regulating transcription, replication, and repair of the genome and is regulated by more than 100 distinct histone modifications, such as phosphorylation, methylation, ubiquitination, and acetylation (127). Histone code hypothesis states that gene regulation is partly dependent on histone modifications that primarily occur on histone tails (48). These modifications modify the net charge of the core histone, modulating the affinity between the DNA (negatively charged) and the octamer: adding positive charges would result in a closure of the nucleosome, while adding negative charges would result in an opening of the nucleosome (figure 3).

Figure 3

Human DNA is compacted by being wound around octamers of core histone proteins that form nucleosomes. Nucleosomes comprise 147 base pairs of DNA wrapped approximately twice around the protein core that contains two copies of each histone, H2A, H2B, H3, and H4. The interaction between DNA and histones is critical to regulating transcription, replication, and repair of the genome and is regulated by more than 100 distinct histone modifications, such as phosphorylation, methylation, ubiquitination, and acetylation. Histone code hypothesis states that gene regulation is partly dependent on histone modifications that primarily occur on histone tails. These modifications modify the net charge of the core histone, modulating the affinity between the DNA (negatively charged) and the octamer: adding positive charges would result in a closure of the nucleosome, while adding negative charges would result in an opening of the nucleosome

Histone acetylation/deacetylation, probably the most investigated modification, is conducted by histone acetyltransferases (HATs) and histone deacetylases (HDACs). For example, histone acetylation is associated with transcriptionally active genes, while deacetylation is associated with inactive genes.

Several environmental conditions (e.g. metals, air pollutants, dietary conditions) can induce histone modifications, possibly altering both specific gene expression and global chromatin conformation (7, 20, 56, 106). Interestingly, the influences of these toxicants can endure even after the toxicant is no longer present.

The standard method for histone modification analysis is the enzyme-linked immunosorbent assay (ELISA). However, many efforts have been made in order to develop high-throughput methods by combining chromatine immunoprecipitation (ChIP) and DNA-microarray analysis (chip) techniques, abbreviated as “ChIP-on-chip” (65, 91, 96).

Histone 3 lysine 4 dimethylation (H3K4me2) and histone 3 lysine 9 acetylation (H3K9ac) are two histone modifications associated with open chromatin states and considered as main markers of exposures (9). H3K4me2 and H3K9ac were associated with several occupational exposures, including different metals such as nickel, arsenic, and iron, and with polycyclic aromatic hydrocarbons (PAH) and benzene (7, 20, 21, 55, 64, 70, 126). While DNA methylation seems to be modified in a few days, histone modifications are probably a suitable epigenetic biomarker induced by long-term exposures. However, as the comprehensive analysis of histone modifications is almost impossible to be obtained, and even specific modification analyses are quite time consuming, histone modifications are still largely unexplored in large epidemiological cohorts.

microRNA expression

MicroRNAs (miRNAs) are short RNAs of approximately 22 nucleotides in length. miRNAs post-transcriptionally regulate gene expression by silencing protein expression through cleavage and degradation of the mRNA transcript or inhibiting translation (23) (figure 4). A single miRNA can bind multiple mRNA targets (more than 100), while several miRNAs may regulate a single mRNA target (35).

Figure 4

MicroRNAs are short RNAs of approximately 22 nucleotides in length. miRNAs post-transcriptionally regulate gene expression by silencing protein expression through cleavage and degradation of the mRNA transcript or inhibiting translation. A single miRNA can bind multiple mRNA targets (more than 100), while several miRNAs may regulate a single mRNA target

The relative expression of miRNAs can be studied by various techniques, such as hybridisation-based methods (e.g. microarrays) used as an initial approach to find the potential candidate miRNAs, or quantitative real-time PCR (qPCR) used to validate highly dysregulated miRNAs. qPCR, due to its high precision and accuracy, remains the gold standard for miRNA quantification. In addition, miRNAs can be sequenced and quantified by using next-generation sequencing (NGS) platforms (2).

Thanks to their stability, miRNAs have been extensively investigated in peripheral blood and urine as potential biomarkers of exposure. Lower expression of miR-24-3p, miR-27a-3p, miR-142-5p, and miR-28-5p in blood was associated with urinary monohydroxy-PAHs and plasma benzo[a]pyrene-r-7,t-8,c-10-tetrahydrotetrol-albumin (BPDE–Alb) adducts in coke oven workers (29). miR-6819-5p and miR-6778-5p were observed to be upregulated in blood in workers exposed to organic solvents, such as ethylbenzene, toluene, and xylene, while the up-regulation of miR-92a and miR-486 was reported in plasma samples in association with chronic mercury occupational exposure (31, 105). Decreased expression of miR-548h-5p, miR-145-5p, miR-4516, miR-331-3p, miR-181a-5p, and miR-1260a, all involved in tumor suppressor activity, was observed in incumbent firefighters while the expression of miR- 374a-5p and miR-486-3p, involved in cancer survival, was increased (49).

miRNA levels were evaluated also as biomarkers of pesticide exposure in urine. Interestingly, miR-223, -518d-3p, -597, -517b, -133b, and -28-5p were observed to be positively associated with farmworkers status during the post-harvest season, and a positive dose response relationship with organophosphate pesticide metabolites was reported by Weldon and colleagues (118).

Although miRNAs have the potential of being sensitive biomarkers of different occupational exposures, further studies in larger populations are needed to empower the associations identified between biomarkers and exposures.

Extracellular vesicles

Extracellular vesicles (EVs) are a heterogeneous group of membrane vesicles, which are released from cells under both physiological and pathological conditions (124). EVs play a central role in cell-to-cell communication, as they are able to transfer between cells biological active molecules, such as proteins and nucleic acids (113). Of particular interest is the fact that EVs contain microRNAs, thus being potentially able to modulate cell expression “at a distance” (124). As EVs can transmit information from one cell to another, they represent a potential mechanism to help explaining how several environmental exposures interact with the molecular machinery of the body (figure 5). EVs have been detected in most body fluids, including blood, urine, saliva, cerebrospinal fluid, bronchoalveolar lavage fluid, amniotic fluid, seminal plasma, and breast milk.

Figure 5

Extracellular vesicles are a heterogeneous group of membrane vesicles, including microvesicles and exosomes, which are released from cells under both physiological and pathological conditions. Extracellular vesicles play a central role in cell-to-cell communication, as they are able to transfer between cells biological active molecules, such as proteins and nucleic acids. Extracellular vesicles contain microRNAs, being able to modulate cell expression “at a distance”, and they have been detected in most body fluids, including blood, urine, saliva, cerebrospinal fluid, bronchoalveolar lavage fluid, amniotic fluid, seminal plasma, and breast milk

The study of EVs is quite a new research field, and the standardization of methods for their isolation and characterization is still debated. The current guidelines for EVs processing are reported in the Minimal information for studies of extracellular vesicles 2018 (MISEV2018) (109).

In a recent work, we isolated plasma EVs from fifty-five healthy male workers employed for at least 1 year in a steel production plant, before and after workplace PM exposure, and evaluated the expression of 88 EV-associated miRNAs by quantitative qPCR. miR-128 and miR-302c expression was increased after 3 days of workplace PM exposure. Moreover, in vitro experiments conducted on the A549 alveolar epithelium derived-cell line confirmed a dose-dependent expression of miR-128 in EVs released after 6 h of PM treatment. These findings suggest that PM exposure induces the release of specific miRNAs within EVs generated from alveolar or other lung cells, which can be detected in plasma (17). Moreover, in the same cohort, the increased expression of 17 EV-associated miRNAs was associated with individual level of PM and metal exposure, suggesting the involvement of different molecular mechanisms such as insulin biosynthesis, inflammation and coagulation in response to those specific exposures (82).

Telomere length

All the cells have a biological clock in telomeres, which are DNA sequences located at the ends of chromosomes (figure 6) involved in maintaining genomic stability and regulating cellular proliferation (67). Telomeres are DNA repeat sequences (TTAGGG) that, together with associated proteins, form a sheltering complex that caps chromosomal ends and protects their integrity (25). Chromosomal stability is gradually lost as telomeres shorten with each round of cell division. Telomere length can be measured in peripheral blood mononuclear cells (Leukocyte Telomere Length, LTL) as marker of biological aging (73). In fact, in proliferating tissues, LTL is longer at birth and shortens progressively as individuals’ age (25).

Figure 6

Telomeres are DNA repeat sequences (TTAGGG) that, together with associated proteins, form a sheltering complex that caps chromosomal ends and protects their integrity. Chromosomal stability is gradually lost as telomeres shorten with each round of cell division. Telomere length can be measured in leukocytes as marker of biological aging. In proliferating tissues, leukocyte telomere length is generally longer at birth and shortens progressively as individuals’ age. Most of the mammalian cells contain hundreds to more than a thousand mitochondria each. The mitochondrion works as a factory for ATP (adenosine triphosphate) and metabolite supplies for cell survival and releases cytochrome c to initiate cell death. Each organelle harbors 2–10 copies of mitochondrial DNA (mtDNA). mtDNA is known to be more sensitive to oxidative damage than nuclear DNA due to its lack of protective histones, introns, and an efficient DNA repair mechanisms. mtDNA copy number may serve as a promising biomarker for oxidative stress–related health outcomes

Retrospective and prospective epidemiological studies have shown that LTL shortening is a risk factor for age-related NCDs (40, 83, 85). Oxidative stress and inflammation, the two major intermediate mechanisms for such diseases, are also risk factors for LTL shortening (84, 86). Telomere erosion can however be further accelerated by external stressors. Telomeres, in fact, as triple-guanine-containing sequences, are highly sensitive to physical and chemical genotoxic exposures. Occupational exposure to genotoxic compounds, directly, by damaging DNA, and indirectly, by favoring the cumulative effect of oxidative damages and the onset of chronic inflammation, might accelerate the physiological process of telomere erosion and facilitate the onset of NCDs (15).

LTL are measured by using qPCR methods described by Cawthon (24). This assay measures relative LTL i.e., T/S ratio, by determining the ratio of telomere repeat copy number (T) to single nuclear copy gene (S), in a given sample relative to that of a reference DNA. The single-copy gene used in this study is usually human ß-globin (HBB). This technique can be applied to a high-throughput format and therefore this method is widely used in large population studies (46).

Our research groups published the first evidences of LTL shortening in association with occupational exposures: LTL decreased in traffic workers exposed to benzene (43) and in coke-oven workers exposed to high levels of polycyclic aromatic hydrocarbons (80). After this initial observation, Fu and coworkers confirmed LTL reduction in Chinese coke-oven workers, longitudinally studied (36). Exposure to high levels of vinyl chloride monomer was also associated with LTL shortening (128, 129), as well as N-nitrosamines in rubber industries and polychlorinated biphenyls exposure in a transformer cycling company (62, 130). In hairdressers, aromatic amine exposure, evaluated by hemoglobin adducts, reduced LTL (66). Studies evaluating particle matter (PM)-related exposures at work coherently report modifications of LTL in association with long-term exposure (43, 63, 121). LTL reduction was reported also in smelters exposed to Pb (87).

Several studies associate exposure to pesticides to LTL but the results are not conclusive. A decrease of LTL in blood was found in different type of exposures (51, 52, 98). Similar results were reported also for TL measured in DNA from saliva (41). On the other hand, increased LTL was also reported in association with exposure to different pesticides (6, 32, 115).

The association of LTL with exposure to ionizing radiations presents contrasting results. Shorter LTL was found in Chernobyl nuclear power plant (CNPP) clean-up workers exposed to low levels of radiation (45). On the contrary, longer telomere were found in Chernobyl CNPP clean-up workers who worked during 1986, in those undertaking ‘dirty’ tasks (digging and deactivation) (92) and in workers of a plutonium production plant (100). Authors suggest that longer telomeres, revealed in people more heavily exposed to ionizing radiation probably indicate a later activation of telomerase as a chromosome healing mechanism following damage, and reflect defects in telomerase regulation that could potentiate carcinogenesis. LTL shortening was reported also in workers cardiac catheterization laboratory staff exposed to long-term low-dose ionizing radiation (5).

Telomere length may be an underlying mechanism by which a wide range of occupational exposures may influence ageing and may relate to an early risk of NCD development and mortality.

Mitochondrial DNA copy number

Most of the mammalian cells contain hundreds to more than a thousand mitochondria each. A mitochondrion is an essential organelle of eukaryotic cells, with many functions that ensure differentiation, survival and control of cell death (59). The mitochondrion works as a factory for ATP (adenosine triphosphate) and metabolite supplies for cell survival and releases cytochrome c to initiate cell death. Each organelle harbors 2–10 copies of mitochondrial DNA (mtDNA). mtDNA is known to be more sensitive to oxidative damage than nuclear DNA due to its lack of protective histones, introns, and an efficient DNA repair mechanisms (90). Increased oxidative stress may contribute to alterations in the copy number mtDNAcn and integrity of mtDNA in human cells (60, 61). Epidemiologic studies have linked reduced leukocyte mtDNA copy number (mtDNAcn) with a range of adverse clinical outcomes, including all-cause mortality, coronary heart disease and sudden cardiac death, metabolic syndrome, and chronic kidney disease (8, 26, 110). Therefore, mtDNAcn may serve as a promising biomarker for oxidative stress–related health outcomes. Progressive loss of mitochondrial function is a common feature of ageing being influenced by the life-long production of ROS, as by-products of oxidative metabolism (11, 54).

mtDNAcn is measured using a qPCR method similar to the one used for TL analysis. The assay measures relative mtDNAcn by determining the ratio of mitochondrial (MT) DNA copy number to single copy gene (S) copy number in experimental samples relative to the MT/S ratio of a reference-pooled sample (22, 81).

Since mtDNA are highly prone to damage, some studies explored the effect of occupational pollutants on it. Reports found that alteration of mtDNAcn in peripheral blood can be related to toxic exposures, such as PAHs (81, 123) and benzene (22).

It has been suggested that the oxidative stress, as a consequence of exposure to PAHs, has a dual influence on mitochondrial DNA content. Environmental low-dose exposure to PAHs can stimulate mitochondrial DNA production to fulfill the respiratory needs of the cell in order to allow the cell survival (89). On the other hand, excessive oxidative stress generated by tobacco smoke that, containing many toxic, carcinogenic and mutagenic chemicals, as well as stable and unstable free radicals and reactive oxygen species (ROS), with the potential for oxidative DNA damage, might result in decreased or no synthesis of mtDNA, eventually leading to cell senescence or cell death. This hypothesis may also explain the different results obtained in terms of mtDNAcn in association with occupational exposures.

A considerable amount of research is still needed to understand the influence of the exposure to occupational toxicants on mtDNAcn.

Markers of exposure or markers of effect?

The biomarkers we have described are modified by many factors and probably every stimulus that our body receives, is later summarized as “epigenetic memory”.

For this reason, epigenetic biomarkers cannot be considered as markers of single exposures, as they are not specific for one particular occupational toxicant and are not useful to quantify the individual dose to which each worker was exposed. Nonetheless, these markers have the potential for becoming important markers of early effect, as they might assist the occupational physician in understanding who are the workers at higher risk of developing specific diseases in a given occupational setting.

The concept of precision medicine postulates that medicine should make individual prevention and treatment by taking into account each patient’s genomics, pre-existing conditions, exposures, and a variety of potential risk factors unique to that individual. Epigenetics might integrate this concept, shedding light on individual susceptibility to toxicants and giving irreplaceable tools for measuring such susceptibility in a quantitative manner. Epigenetics data would therefore potentiate several parameters such as toxicodynamics, toxicokinetics and the mechanism of action toxicants within the body, thus eventually contributing to the risk assessment processes (101).

However, these markers have not been standardized yet, so the next step in order to translate the findings from basic research to medicine will have to go in this direction.

The challenge that occupational physicians and scientists are facing in the (near) future is big, as they need to interpret these markers not only as markers of occupational risk but also, in addition, as markers of risk related to modifiable lifestyle factors. If the entire professional and scientific community of occupational medicine and health accept this challenge, this might represent a huge step forward in the comprehension of occupational risks and towards workers’ health protection and well-being promotion.

No potential conflict of interest relevant to this article was reported by the authors