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Introduction

A huge number of studies have been performed on well known toxicants such as asbestos, pesticides and benzene. Even though there is plenty of evidence toward the toxicity of these compounds, recent findings are pointing out some novelties like new pathogenetic mechanisms, or previously unsuspected work-related pathologies (1).

In occupational settings, it is very important to keep an eye on these toxicants because of their great distribution: pesticides are globally used to enhance agricultural production and to control pests; benzene is still used in petrochemical reactions and its exposure involves many human activities (e.g., gas station attendants); asbestos-like fibers [e.g., carbon nanotubes (CNTs)] are raising great concern in the scientific community because of their increasing use in industry despite their potential, and yet partially known, toxicity (2). The present review summarizes some of the most recent and supported findings on these toxicants.

Data collection methods

In the present study, a computerized search on PubMed was performed up to November 2016. Search terms were combinations of the following keywords and their abbreviations: ‘carbon nanotubes’ and ‘toxicity’ or ‘exposure’. ‘Pesticides’ and ‘chronic diseases’ or ‘Parkinson’ or ‘cancer’ or ‘epigenetics’. ‘Benzene’ and ‘low dose exposure’ or ‘chronic exposure’ or ‘epigenetics’ or ‘exposure’ or ‘assessment’. References in reports were also reviewed. The attention was focused on recent findings on these toxicants, which could be of particular interest towards occupational exposure. For this reason, in vivo studies were generally preferred to in vitro studies.

Asbestos-like fibers

Fiber-induced pulmonary toxicity was widely debated in the 1990s because of the emerging harmful effects of asbestos, which led to the banning of this material from commerce. Later on, it was demonstrated that the high aspect ratio of asbestos fibers was the main pathogenetic factor, and that other fibers could have the same pathogenicity. This is the case in other natural fibers like fluoro-edenite and in silicon carbides (SiC) (1–6).

Recently, some studies have linked CNT exposure with the same pathologies produced by asbestos exposure, such as pleural plaques, fibrosis, and pleural mesothelioma (7,8). In 2014, the International Agency for Research on Cancer published its conclusions on these ‘asbestos-like’ fiber (1), assessing fluoro-edenite fibrous amphibole as carcinogenic to human (Group 1). Furthermore, occupational exposure associated with the Acheson process (of which SiC fibres are unwanted byproducts) was also classified as carcinogenic to humans (Group 1), while fibrous SiC was included in Group 2b, as possible carcinogen to humans. Finally, multi-walled CNT-7 (MWCNT-7) was classified as possible carcinogen to humans (Group 2b), while single-walled CNTs (SWCNT) and other forms of MWCNT were recorded as not classifiable for their carcinogenicity to humans (Group 3), for lack of sufficient evidence (1).

CNTs toxicity

A recent review examined the detrimental health effects of asbestos-like fibers, including CNTs (2). The authors found a general consensus about the toxicity of these particles, and their potential toxicity towards the serosal cavities like the pleura and the peritoneum in animal models. In particular, a study in 2008 reported a high incidence of mesothelioma after intraperitoneal injection of MWCNT in mice, and, furthermore, MWCNT injection produced the highest mortality compared to fullerene and crocidolite (9). Another experiment aimed to discover if the presence of structural defects in MWCNTs could be a pathogenetic factor. After intraperitoneal injection in mice of structurally imperfect nanotubes (MWCNT+) and nanotubes without structural defects (MWCNT-) and comparison with a similar dose of crocidolite, the authors found that only crocidolite was capable of inducing mesothelioma (10). The hypothesis that a particular group of MWCNT (with structural defects) could have been more toxic than other types was of particular interest in occupational settings, and the authors of the study concluded that the negative results obtained were attributable to the short length of the MWCNT used for the experiment. According to this hypothesis, later studies confirmed that length is a key parameter in fibrous nanoparticle toxicology (11).

Recently, another review examined the toxicity of carbon nanoparticles and CNTs (CNPs/CNTs) on the respiratory system, especially in occupational settings. The authors found that the majority of studies indicate that CNPs/CNTs induce respiratory system pathologies such as hypersensitivity, inflammation, fibrosis, and cancer and that these nanomaterials are released in the air during their manufacturing and handling (12).

Guseva Canu et al reviewed the current knowledge on occupational exposure to CNTs and nanofilaments (13). The authors found that the most exposed subjects are: academic and private laboratory workers, which engineer and elaborate new materials, workers employed at facilities which produce nanomaterials for selling them to the industry (primary workers) and workers employed in industries or laboratories which use the previously produced nanomaterials for the development of final products (secondary workers). The authors concluded that exposure to CNTs/CNFs is possible during the whole life-cycle, even if a real assessment of exposure has been done only for the early stages of production, while information on exposure at final stages of production is lacking. Furthermore, since MWCNTs appear to be the most hazardous material because of its toxicity (1), the majority of studies focus on the exposure/toxicity of this kind of nanotubes. Actually, exposure to SWCNTs and, even more, to carbon nanofilaments (CNFs) is quantitatively even more relevant than to MWCNTs and should be further investigated. On the other hand, consumer exposure seems to be limited.

To date, there is no conclusive data about the toxicity of these molecules on humans, and, furthermore, animal models can only partially indicate the potential toxicity of these materials because of the numerous variants of CNTs and CNFs produced in the facilities. For these reasons, the National Institute for Occupational Safety and Health (NIOSH) recommends maximum caution for any occupational exposure to these materials, which levels have to be maintained below recommended exposure limit. At this time, given the available literature, NIOSH advises not to exceed 1 µg/m3 of respirable elemental carbon as 8 h time weighted average for CNT and CNF. Furthermore, these regulations are recommended for any type of CNT and CNF, until scientific evidence will definitively shed light on their actual toxicity.

Pesticides

Because of their undisputed utility, pesticides are today an essential tool in agriculture, and many efforts have been made by the scientific community to identify the possible biological and pathological consequences of environmental and professional exposure to pesticides. Assessing professional exposure, though, is far from simple. A previous study of our group summarized the latest findings on the relation between pesticide exposure and development of chronic diseases (14). Even though the link between pesticide exposure and the development of neurological diseases (15,16), reproductive disorders (17,18) and cancer (19–21) seems clear and almost obvious in some cases [like Parkinson's disease (PD)], it is still difficult to correlate these pathologies to exposure to a narrow spectrum of pesticides. Many studies, infact, evaluate exposure to pesticide in general. In particular, epidemiological studies suffer of inaccuracy, because pesticides are almost invariably used as mixtures, so that it is difficult for the worker to remember the exact name of the pesticides he used throughout the years, and for the researcher to determine the specific pesticide which could be responsible for any specific alteration. Furthermore, pesticides are usually applied in open fields, and variables like humidity, wind, heat and correct use of personal protective equipment are almost impossible to be precisely evaluated over a long period of working activity. The use of questionnaires and expert interviews has been used as a tool to provide a more precise assessment of previous exposure of individuals, but the results are still controversial (22,23).

Pesticide tasks

Individuals who are involved in pesticide application processes in agricultural and non-agricultural settings or spend their activity nearby the site of application, are potentially exposed to these toxicants: i) mixer/loaders, individuals perform tasks in preparation for an application. For example, prior to application, mixer/loaders would mix a liquid pesticide concentrate with water and load it into the holding tank; ii) applicators, individuals operate application equipment during the release of a pesticide product onto its target; iii) mixer/loader/applicators, individuals who perform all aspects of the pesticide application process; iv) flaggers, individuals that guide aerial applicators during the release of a pesticide product onto its target.

Furthermore, other categories of workers can be exposed to pesticides like farmers, greenhouse workers, plants manufacturers, workers responsible of re-entry activities in vineyards or other crops.

Pesticide toxicity

Pesticides, in general, have been linked with a really wide range of pathologies. Cancer at various sites of the body is one of the most discussed themes, as well as neurological diseases. Other pathologies such as diabetes mellitus (24–26), reproductive disorders (27,18) and cardiovascular diseases (28,29) have also been linked to pesticide exposure.

The most consistent findings for the correlation between pesticides and chronic diseases development are with cancer and with Parkinsons disease (PD).

PD is a multifactorial, neurodegenerative disease chiefly characterized by tremor, bradykinesia, rigidity and postural instability as clinical symptoms (30). The main pathogenetic event behind this clinical presentation is the death of dopaminergic cells in the substantia nigra pars compacta, and progressive degeneration of its projections to the basal ganglia. There are numerous studies pointing towards the conclusion that pesticides could be a risk factor for the development of PD, even if the majority of studies did not find a specific bond between a single or a group of pesticides and the disease. The link appeared very likely for pesticides in general, even if it is a heterogeneous group of compounds. In this regard, a meta-analysis performed by Hernández et al serves perfectly as an example, since the authors could not draw any specific conclusion (15).

Similar results were attained by Freire and Koifman, who reviewed epidemiological studies between 2000 and 2011 about the connection between organophosphate exposure and PD (31). The authors found several studies reporting a positive correlation between pesticide exposure and PD: in particular, 13 case-control studies reported a significant association. Other studies reported an increase in the risk of developing PD for those subjects who have genetic polymorphism like paraoxonase enzyme 1 and nitric oxide synthase, both involved in detoxification of pesticides, as confirmed by other studies (18). Furthermore, a strong association was reported in particular for clorpyrifos (organophosphate), organochlorines, and for maneb and paraquat, especially when combined.

Another meta-analysis (32) examined 12 cohort studies and concluded that occupational exposure to pesticides is a possible risk factor for the development of PD, even if, once again, they were not able to isolate specific compounds causing the disease.

As evidenced before, methods for a proper assessment of exposure are still under discussion. Nevertheless, some interesting pathogenetic mechanisms are emerging regarding the role of pesticides in the development of chronic diseases, and, in particular, of cancer development.

Pesticides, in fact, have been proved to have immunotoxic properties, through the alteration of cytokine profiles (33). The role of immune system is central in cancer development, as demonstrated by recent investigations: the recognition and destruction of tumor cells is indeed a mechanism of protection for the body, but the development of a chronic inflammatory microenvironment could, on the other hand, support tumor progression (34). So, the immune system can, in turn, promote or contrast cancer progression. This characteristic is known as cancer immunoediting and summarily consists of three phases: i) elimination (immunosurveillance), ii) equilibrium (control of tumor growth) and iii) escape (alteration of tumor cells which elude the immune system or induce immunosuppressive mechanisms, which ultimately lead to tumor growth) (35). The role of pesticides in cancer development could likely include one or more of these mechanisms.

A study by Cassidy et al was performed regarding the correlation between exposure to pesticides and breast cancer development. Biopsies of breast lesions were examined and the authors found a positive link between heptachlor epoxide exposure and prevalence of cancer (36).

A study by Lee et al demonstrated a link between pesticide exposure and the incidence of lung cancer in pesticide applicators, even after adjusting the results for cigarette smoke and other confounding factors (37). Later on, another study from the same group analyzed a very large cohort of agricultural workers (56,813) - called the agricultural health study cohort - to assess the incidence of colorectal cancer. Given the already known issues regarding occupational exposure assessment in agricultural settings, this study tried to surpass those issues by administering an enrolment questionnaire, followed by a more detailed take-home questionnaire. The questionnaires included information regarding previous exposure up to 50 pesticides and working habits like use of personal protective equipment, equipment status and application methods. Questions regarded also lifestyle habits like physical activity and fruit consumption. The results elucidated a significant connection between the use of some pesticides (chlorpyrifos and aldicarb) and colorectal cancer among these workers, even if this result has to be contextualised as an epidemiological observation which was not supported by pathogenetic hypothesis prior to the study. Besides, the study results could be questioned given the large population analysed, the large number of pesticides included in the questionnaires, and the incidence of colorectal cancer among the general population, even if the design of the study tried to minimize the bias (38). Several studies have been performed on the agricultural health study cohort, and their results have been summarised in a review by Weichenthal et al (39). The authors concluded that most of the pesticides examined were not strongly correlated with the incidence of cancer, even if, in some cases, an increased relative risk/odds ratio was registered. Despite these results, further studies are needed to assess the role of pesticides in cancer development.

Interesting results have been attained regarding the exposure to organochlorines and hepatocellular cancer (HCC). A study conducted in California showed a positive association between exposed workers and HCC development in agricultural areas. Similar results were attained in another study, which highlighted an increased prevalence of HCC in areas contaminated by widespread pesticide use, if compared to the median population values (40).

Benzene

Benzene toxicity has been assessed for a long time. Even if the use of this molecule has been limited, it remains necessary in many industrial processes, like the production of styrene, cyclohexane, cumene, inks, solvents, pesticides, lubricants. As a consequence, occupational exposure is still possible (41). Alongside this professional routes of exposure, benzene is also an environmental contaminant mainly because of industrial emissions, automobile service stations, car exhaust gas, and, more importantly, cigarette smoking habits.

It has been demonstrated that benzene exposure is linked with the risk of developing hematological diseases like aplastic anemia, myelodysplastic syndrome, acute and chronic lymphocytic leukemia and cancers like multiple myeloma and non-Hodgkin's lymphoma (42,43). Furthermore, benzene toxicity has been linked to the development of diseases at other sites of the human body, like the respiratory system (44), the immune system (45,46), nervous (47) and reproductive systems (48–50,41). Studies indicate that benzene could be a risk factor for the development of breast cancer (51–53), prostate cancer, stomach cancer, colon cancer, pharyngeal cancer and malignant melanoma (54–57).

Several mechanisms of toxicity have been proposed for benzene, like production of chromosomal aberrations, sister chromatid exchange, and micronuclei (58). Recently, some studies have evidenced that some of the harmful effects of benzene could be caused by the induction of oxidative stress. A recent review (59) simulated chronic exposure to benzene on mice, with the aim to evaluate the mechanism of toxicity of benzene itself and of its metabolite hydroquinone. After the evaluation of plasmatic values for insulin and glucose and the examination of liver and pancreatic histological samples, the authors concluded that repeated administration of benzene could be capable of determining oxidative stress-related damage to the liver and the pancreas (the latter determined by hydroquinone, a benzene metabolite). These results suggest the potential role of benzene in the development of glucose metabolism dysregulations. Even if the study of Bahadar et al (59) examined the molecular effects of benzene at relatively high doses, similar pathways may be involved in the pathogenesis of chronic cellular damage at other sites (60), even at low degrees of exposure. In this regard, a study of our group enrolled 91 male gas station attendants and compared them to a control population of 63 male office workers (61). The aim was to evaluate the effects of low dose, chronic exposure to benzene on NF-κB, STAT3, p38-MAPK and stress-activated protein kinase/Jun aminoterminal kinase signal transduction pathways in peripheral blood mononuclear cells of gas station workers. Urine samples were collected at the end of the work shift to evaluate the concentration of trans, trans-muconic acid (t,t-MA), and oxidative stress biomarkers were determined on blood samples. The results showed a significant increase of t,t-MA in the urine of gasoline station attendants, compared with those of the control group. NF-κB and phospho-IκB-α proteins were also higher in the exposed population, while phosphorylated STAT3 was significantly decreased in the benzene exposed group, compared to controls. These results indicate the action of benzene on some oxidative stress-related signal transduction pathways, even at low doses of occupational exposure (61,62).

A similar study conducted by Xia et al involved 144 workers, of which 96 were gasoline filling station attendants (with no history of exposure to heavy metals and organic solvents) and 48 were cashiers, the latter used as controls. Aim of the study was to assess the oxidative stress status of workers exposed to benzene, toluene, ethylbenzene, xylene (BTEX) and manganese (Mn) (63). Mn levels were investigated since methylcyclopentadienyl manganese tricarbonyl (MMT), an organic derivative, is a widely used additive for gasoline. The authors combined environmental monitoring with biological monitoring for the assessment of exposure. After that, the authors calculated the cumulative exposure index from 8 h of BTEX and Mn exposure time-weighted average and multiplied this value for the years of exposure. The authors assessed oxidative stress biomarker levels, such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and Hsp70. Given that the airborne concentration of BTEX were significantly higher in gas station attendants working environment than for office workers, blood levels of SOD and GSH-Px were significantly lower in the exposed group than in the control group, and, conversely, MDA levels (a marker of oxidative stress) were significantly higher in the exposed group, especially for those who had been working in gas stations for a longer time (>10 years). Hsp70 hematic concentration was also found higher in gas station attendants who worked for >10 years in the field, compared to the control group. The authors concluded that MMT-containing gasoline may diminish the antioxidant capabilities of the body and enhance lipid peroxidation levels in occupationally exposed workers. Hsp70 could be an interesting biomarker in this regard.

Conclusions and future perspectives

The pollution of living environment and workplaces by a large number of toxic agents, makes it essential to continue investigating the links between exposure to toxicant and development of certain pathologies, basing on epidemiological evidence and laboratory experiments.

Occupational and environmental exposure to CNTs and to nanoparticles in general is particularly important in this regard, as this scenario reminds us of what happened with asbestos some decades ago. Although this comparison may sound exaggerated, the studies presented above show that, even if some international agencies are recommending limits for occupational exposure, occupational and environmental exposure still continues even if the possible consequences are not yet fully clear.

Pesticides exposure shows similar issues. Even after years of investigations, scientific evidence is still conflicting with the fact that a proper assessment of occupational exposure is very difficult to obtain and this corrupts the reliability of a large number of epidemiological and retrospective studies. Recently, the use of algorithms and tools like job exposure matrices has been proposed to surpass this issue and establish the correct links between exposure to specific pesticides and the eventual development of diseases.

Lastly, the examination of some of the latest evidence regarding benzene toxicity points out another very interesting argument. In fact, the evidence that benzene exposure could be related to immunotoxic and epigenetic effects opens new scenarios about individual susceptibility to this molecule (as well as others), and probably leads towards a novel conception of occupational safety. Personal characteristics such as genetic polymorphisms and eventual epigenetic changes should, in fact, be considered for a real assessment of risk in the future.

References