Genotoxicity of pesticides: a review of human biomonitoring studies

Pesticides constitute a heterogeneous category of chemicals specifically designed for the control of pests, weeds or plant diseases. Pesticides have been considered potential chemical mutagens: experimental data revealed that various agrochemical ingredients possess mutagenic properties inducing mutations, chromosomal alterations or DNA damage. Biological monitoring provides a useful tool to estimate the genetic risk deriving from an integrated exposure to a complex mixture of chemicals. Studies available in scientific literature have essentially focused on cytogenetic end-points to evaluate the potential genotoxicity of pesticides in occupationally exposed populations, including pesticide manufacturing workers, pesticide applicators, floriculturists and farm workers. A positive association between occupational exposure to complex pesticide mixtures and the presence of chromosomal aberrations (CA), sister-chromatid exchanges (SCE) and micronuclei (MN) has been detected in the majority of the studies, although a number of these failed to detect cytogenetic damage. Conflicting results from cytogenetic studies reflect the heterogeneity of the groups studied with regard to chemicals used and exposure conditions. Genetic damage associated with pesticides occurs in human populations subject to high exposure levels due to intensive use, misuse or failure of control measures. The majority of studies on cytogenetic biomarkers in pesticide-exposed workers have indicated some dose-dependent effects, with increasing duration or intensity of exposure.Chromosomal damage induced by pesticides appears to have been transient in acute or discontinuous exposure, but cumulative in continuous exposure to complex agrochemical mixtures.Data available at present on the effect of genetic polymorphism on susceptibility to pesticides does not allow any conclusion.     

Cytogenetic analysis and occupational health in the Czech Republic

This paper summarizes the experiences of the Czech Hygiene Service since the middle 1970s until the present time, using cytogenetic analysis as a biomarker of exposure. We have included a review of all the available scientific literature in Czech as well as in English, and have described the most important milestones in the history of the use of cytogenetic analysis in the Czech Republic. Details on the levels of occupational exposure and the corresponding observed frequencies of aberrant cells are provided. Furthermore, we discuss the interpretation of the cytogenetic findings in the context of occupational health and give several examples when the results of cytogenetic analysis provided the chief argument used to enforce improved working conditions and to establish safer maximum allowable concentrations.     

The characteristics of the realization of cytogenetic damage in mammalian and plant cells exposed to low doses of radiation]

A complicated character of the cytogenetic injury dependence upon radiation dose was revealed after low-level gamma irradiation of Vicia faba seedlings and Chinese hamster fibroblasts. The dependence was linear with low-level secondary exposure to 70 GeV protons. The authors discuss a threshold nature of induction of the cytogenetic damage repair responsible for a high outcome of damages under the effect of low-level gamma radiation.     

Security considerations in blinded exposure experiments using electromagnetic waves

Whether exposure to electromagnetic fields well below accepted exposure limits has a cytogenetic effect on human cells has long been debated. It is widely published and generally accepted that the exposure unit invariably used in these experiments is capable of providing blinded exposure conditions. The following short report illustrates, however, that exposure conditions might not always be as effectively masked as is generally assumed.     

Increased risk of cancer in radon-exposed miners with elevated frequency of chromosomal aberrations

In spite of the extensive use of cytogenetic analysis of human peripheral blood lymphocytes in the biomonitoring of exposure to various mutagens and carcinogens, the long-term effects of an increased frequency of chromosomal aberrations in individuals are still uncertain. Few epidemiologic studies have addressed this issue, and a moderate risk of cancer in individuals with an elevated frequency of chromosomal aberrations has been observed.In the present study, we analyzed data on 1323 cytogenetic assays and 225 subjects examined because of occupational exposures to radon (range of exposure from 1.7 to 662.3 working level month (WLM)). Seventy-five subjects were non-smokers. We found 36 cases of cancer in this cohort.Chromatid breaks were the most frequently observed type of aberrations (mean frequency 1.2 per 100 cells), which statistically significantly correlated with radon exposure (Spearman's correlation coefficient R=0.22, P<0.001). Also, the frequency of aberrant cells (median of 2.5%) correlated with radon exposure (Spearman's correlation coefficient R=0.16, P<0.02). Smoking and silicosis were not associated with results of cytogenetic analyses.The Cox regression models, which accounted for the age at time of first cytogenetic assay, radon exposure, and smoking showed strong and statistically significant associations between cancer incidence and frequency of chromatid breaks and frequency of aberrant cells, respectively. A 1% increase in the frequency of aberrant cells was paralleled by a 62% increase in risk of cancer (P<0.000). An increase in frequency of chromatid breaks by 1 per 100 cells was followed by a 99% increase in risk of cancer (P<0.000). We obtained similar results when we analyzed the incidence of lung cancer and the incidence other than lung cancer separately.Contrary to frequency of chromatid breaks and frequency of aberrant cells, the frequency of chromatid exchanges, and chromosome-type aberrations were not predictive of cancer.     

The cytogenetic effect of bleomycin on human peripheral lymphocytes in vitro and in vivo.

The cytogenetic effect of bleomycin (BLM) in human lymphocytes was studied after exposure to different doses during the G0 and G2 phases. BLM produced a marked specific effect on the cell cycle. The main aberration types after exposure in tg0 were dicentrics and deletions; and after exposure in G2, open chromatid breaks. A linear dose--response was calculated for all these aberration types as well as for the number of aberrant cells. In the G2 experiments, partially and totally pulverized cells also increased linearly with dose. The intercellular distributions of the most frequent aberration types after exposure in G0 and G2--the dicentrics and chromatid breaks, respectively--showed over-dispersion. These results show that the cytogenetic effect of BLM may be compared with that of densely ionizing irradiation. Preliminary results of chromosome analysis of three cancer patients in the course of BLM therapy showed effects similar to those in the G0 experiments.     

Review of the genotoxicity of ozone.

Ozone is a powerful oxidant, reactive to biomolecules. In aqueous solution it decomposes to give hydrogen peroxide, superoxide and hydroxy radicals which can take part in secondary reactions. Ozone is a disinfectant that inactivates both viruses and bacteria. Although other reactions are primarily responsible for the inactivation, cellular DNA is also damaged. Ozone is genotoxic to microorganisms, plants and cell cultures in vitro. The results from in vivo cytogenetic studies with laboratory animals after inhalation exposure are contradictory. Chromosome aberrations in lymphocytes, but not SCEs, have been demonstrated in Chinese hamsters but not in mice. Chromatid deletions were induced in pulmonary macrophages in rats. No cytogenetic effects have been reported for bone marrow cells or spermatocytes. The few experimental and epidemiological studies with human subjects do not allow a conclusion on the cytogenetic effects of ozone in lymphocytes in humans. No life-long cancer studies have been performed with ozone. However, after 4 and 6 months of inhalation exposure, lung adenomas were induced in strain A/J mice, but not in Swiss-Webster mice.     

A systematic review of cytogenetic studies conducted in human populations exposed to cadmium compounds

BACKGROUND: Exposure to cadmium fumes or dusts has been associated with an increased risk of lung cancer and the characterisation of the genotoxic potential of cadmium compounds is, among other possible mechanisms, an important element in the assessment of the carcinogenic hazard of the element. While there is some evidence that in experimental systems, cadmium compounds may exert genotoxic effects, the results of the epidemiological studies having examined cytogenetic endpoints in humans exposed to cadmium appear conflicting. Therefore, a systematic review was undertaken to assess whether a cytogenetic effect of cadmium exposure is supported by the studies with the strongest design. METHODS: The relevant literature was identified through several databases and assessed with a check-list by two reviewers. Causes of heterogeneity between studies were looked for. Results were extracted and the strength of the evidence was evaluated with causality criteria. RESULTS: No studies met the criteria for being considered as very convincing. Several factors were identified that could explain contradictory findings (small sample size, selection bias, insufficient characterisation of exposure, lack of consideration of confounders) but their actual impact could not be conclusively assessed with the published information. Importantly, it should be recognised that the absence of a clear mechanism for the cytogenetic action of cadmium compounds did not allow to select the most appropriate endpoint to be examined. CONCLUSIONS: No clear association between cadmium exposure and cytogenetic endpoint appeared but no definite conclusion can be drawn from the existing studies in humans. Future research efforts should mainly focus on experimental studies to understand how cadmium compounds could produce genotoxic/carcinogenic effects, in order to target the most relevant endpoint to be examined in humans.     

The effect of the spontaneous level of the chromosome aberration rate on its alterations after a single local irradiation

With X-ray examinations of the stomach and duodenum, changes in chromosomal aberration frequencies in peripheral blood lymphocytes were shown to depend on the spontaneous level of these aberrations in patients exposed to a single local irradiation by 25 R. The cytogenetic efficiency of radiation exposure was found to be local in patients with the lower frequency of spontaneous chromosomal aberrations. A genetic genesis of the revealed dependence is supposed. The effect of irradiation on the correlation between radiosensitivity and radioresistance of the cytogenetic process is regarded as a possible mechanism of the appearance of the observed dependence.     

Micronuclei in peripheral blood lymphocytes and buccal epithelial cells of Polish farmers exposed to pesticides.

In this biomonitoring study, we investigated whether an occupational exposure to a complex mixture of chemical pesticides produced a significant increase of micronuclei (MN) in both peripheral blood lymphocytes and buccal cells. Forty-nine male workers exposed to pesticides, from an agricultural area of Malopolska Region in Southern Poland, together with 50 men from the same area without indication of exposure to pesticides that served as controls, were used in this investigation.No statistically significant differences in the frequencies of cytogenetic damage were detected between exposed and control individuals, for either type of cells. The multiple linear regression analysis in the case of lymphocytes indicated that the studied cytogenetic endpoints were inversely influenced by alcohol; whilst a negative binomial regression, in the case of buccal cells, indicated that the MN values were directly influenced by the ingestion of red meat. An inverse negative relationship between the cytokinesis-block proliferation index and age, and a significant increase of miscarriages due to the exposure to pesticides were also observed.     

Chromosomal aberrations in lymphocytes of employees in transformer and generator production exposed to electromagnetic fields and mineral oil

The objective was to study the risk of cytogenetic damage among high voltage laboratory workers exposed to electromagnetic fields and mineral oil. This is a cross sectional study of 24 exposed and 24 matched controls in a Norwegian transformer factory. The exposure group included employees in the high voltage laboratory and in the generator soldering department. Electric and magnetic fields and oil mist and vapor were measured. Blood samples were analyzed for chromosomal aberrations in cultured lymphocytes. In addition to conventional cultures, the lymphocytes were also treated with hydroxyurea and caffeine. This procedure inhibits DNA synthesis and repair in vitro, revealing in vivo genotoxic lesions that are repaired during conventional culturing. In conventional cultures, the exposure group and the controls showed similar values for all cytogenetic parameters. In the DNA synthesis- and repair-inhibited cultures, generator welders showed no differences compared to controls. Among high voltage laboratory testers, compared to the controls, the median number of chromatid breaks was doubled (5 vs. 2.5 per 50 cells; P<0.05) the median number of chromosome breaks was 2 vs. 0.5 (P>0.05) and the median number of aberrant cells was 5 vs. 3.5 (P<0.05). Further analysis of the inhibited culture data from this and a previous study indicated that years of exposure and smoking increase the risk of aberrations. We conclude that there was no increase in cytogenetic damage among exposed workers compared to controls in the conventional lymphocyte assay. In inhibited cultures, however, there were indications that electromagnetic fields in combination with mineral oil exposure may produce chromosomal aberrations.     

Cytogenetic monitoring of industrial populations potentially exposed to genotoxic chemicals and of control populations

Currently the most applied technique for monitoring biological effects of exposure to genotoxic chemicals in industrial workers is the measurement of chromosome aberrations in peripheral blood lymphocytes. In the Shell petrochemical complex in The Netherlands cytogenetic monitoring studies have been carried out from 1976 till 1981 inclusive, in workers potentially exposed to a variety of genotoxic chemicals, i.e. vinyl chloride, ethylene oxide, benzene, epichlorohydrin, epoxy resins. Average exposure levels to these chemicals were well below the occupational exposure limits. Results of these studies indicate that no biologically significant increase in the frequencies of chromosome aberrations in the exposed populations occurred compared with control populations. Our experience with this methodology has shown that the results of chromosome analyses are difficult to interpret, due to the variable and high background levels of chromosome aberrations in control populations and in individuals. It is concluded that the method is not sufficiently sensitive for routine monitoring of cytogenetic effects in workers exposed to the low levels of genotoxic compounds.     

Tabulation and assessment of 113 human surveillance cytogenetic studies conducted between 1965 and 1984

There is an increasing tendency to monitor human exposure to genotoxic chemicals by the assessment of chromosomal aberrations or sister-chromatid exchanges (SCEs) in peripheral blood lymphocytes. In order to assess the sensitivity of these techniques, and to discern minimal criteria for their conduct, a survey of 113 human lymphocyte cytogenetic surveillance studies conducted between 1965 and 1984 has been undertaken. The present survey indicates the urgent need for standardization of study protocols. It is suggested that a common method of reporting chromosomal aberrations should be adopted, and that this should be based on the system described by Scott et al. It is also suggested that a minimum acceptable size of control and exposed populations should be agreed, and that potentially important factors such as the gender, the period and extent of exposure and individual smoking habits be defined in advance of the commission of future surveillance studies. As general awareness of the possible hazard presented by exposure of man to genotoxic chemicals increases, so appropriate preventative industrial hygiene measures will be instituted. This implies that future human cytogenetic surveillance studies may yield either weakly positive or negative data. This emphasizes the current need for agreement on appropriate study protocols. The formation of a central repository for control databases, and its subsequent updating and use by those involved in human cytogenetic surveillance studies, is recommended. Minimal experimental criteria for the design of future studies are also outlined.     

Induction of biotransformation enzymes by the carcinogenic air-pollutant 3-nitrobenzanthrone in liver, kidney and lung, after intra-tracheal instillation in rats

We investigated the effect of the seasonal variability of environmental air pollutants on oxidative stress and cytogenetic biomarkers in a group of 59 city policemen working in Prague, Czech Republic. The studied group was monitored in February and May 2007. The exposure to environmental pollutants (carcinogenic polycyclic aromatic hydrocarbons, c-PAHs, including benzo[a]pyrene, B[a]P, and particulate matter of aerodynamic diameter<2.5μm, PM2.5) was measured by personal and/or stationary monitors. Levels of c-PAHs were significantly higher in winter than spring, while exposure to PM2.5 was higher in May than in February 2007. We did not observe any significant difference between the two seasons for any biomarker of oxidative stress (8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxodG, 15-F(2t)-isoprostane, 15-F(2t)-IsoP, protein carbonyl levels) or any cytogenetic parameter, including the genomic frequency of translocations (F(G)/100), the percentage of aberrant cells (%AB.C.) or the number of acentric fragments (ace). Analyses of associations between oxidative stress biomarkers and cytogenetic parameters showed a negative relationship between protein oxidation and F(G)/100, as well as protein oxidation and ace. We further analyzed the effect of air pollution on all subjects regardless of the season. Data from stationary monitors showed that 8-oxodG levels were significantly increased by exposure to PM2.5 over a 2-day period before sampling and by exposure to B[a]P over a 28-day period, days 57-84 before sampling. 15-F(2t)-IsoP levels were increased after exposure to B[a]P over both 2-day and 3-day periods preceding sample collection and after exposure to c-PAHs over a 2-day period before sampling. %AB.C. was significantly affected by exposure to B[a]P over a 14-day period, days 57-70 before sampling. In summary, our results indicate that the exposure to environmental pollutants affects urinary excretion of 8-oxodG, lipid peroxidation and the frequency of chromosomal aberrations.     

Frequency of stable chromosomal aberrations determined by FISH in 49 Chernobyl nuclear accident liquidators exposed to various doses of radiation

FISH technique was tested on the 49 liquidators of the Chernobyl Accident. Groups were formed according to average group exposure doses and/or individual ones determined by the method of EPR. The essential variability of spontaneous and irradiation-induced frequencies of reciprocal translocations was observed. In groups with the lowest EPR-doses (below 25 cGy) the average cytogenetic dose was considerably higher then doses determined by EPR-method. The best coincidences between average in-group EPR- and FISH-doses were in the group with 25-50 cGy doses for "acute irradiation" model and in the group with 50-100 cGy and higher for "chronic irradiation" model.     

Induction of micronuclei in human lymphocytes exposed in vitro to microwave radiation

Increasing applications of electromagnetic fields are of great concern with regard to public health. Several in vitro studies have been conducted to detect effects of microwave exposure on the genetic material leading to negative or questionable results. The micronucleus (MN) assay which is proved to be a useful tool for the detection of radiation exposure-induced cytogenetic damage was used in the present study to investigate the genotoxic effect of microwaves in human peripheral blood lymphocytes in vitro exposed in G(0) to electromagnetic fields with different frequencies (2.45 and 7.7GHz) and power density (10, 20 and 30mW/cm(2)) for three times (15, 30 and 60min). The results showed for both radiation frequencies an induction of micronuclei as compared to the control cultures at a power density of 30mW/cm(2) and after an exposure of 30 and 60min. Our study would indicate that microwaves are able to cause cytogenetic damage in human lymphocytes mainly for both high power density and long exposure time.     

Effect of interferon on the number of cytogenetic disorders occurring in human cultured lymphocytes treated with thio-TEPA and fotrin

The paper deals with the modifying effects of natural (leukocytic) and synthesized (recombinant) interferons on the number of cytogenetic injuries in the cultured lymphocytes of human peripheric blood after exposure to alkylating chemicals--thio-TEPA and fotrin. The analysis of chromosomal aberration levels is suggestive of a significant protective effect exerted by interferons. The addition of the recombinant interferon increased the number of sister chromatid exchanged frequency in mutagen treated variants. The application of the test system that involves two types of interferon made it possible to reveal differences in their cytogenetic effect during the protector-sensitive period of cultivation.     

The genotoxic risk of underground coal miners from Turkey

A cytogenetic monitoring study was carried out on a group of workers from a bituminous coal mine in Zonguldak province of Turkey, to investigate the genotoxic risk of occupational exposure to coal mine dust. Cytogenetic analysis, namely sister chromatid exchanges (SCEs), chromosomal aberrations (CAs) and micronucleus (MN) tests were performed on a strictly selected group of 39 workers and compared to 34 controls matched for gender, age, and habit. Smoking and age were considered as modulating factors. Both SCE and CA frequencies in coal miners appeared significantly higher than in controls. Similarly, there was a significant increase in the frequency of total micronuclei in exposed group as compared to control group. The effect of smoking on the level of SCE and MN was significant in the control group. A positive correlation between the age and the level of SCE was also found in controls. The frequencies of both SCE and CA were significantly enhanced with the years of exposure. The results of this study demonstrated that occupational exposure to coal mine dust leads to a significant induction of cytogenetic damage in peripheral lymphocytes of workers engaged in underground coal mining.     

The genotoxic risk of underground coal miners from Turkey

A cytogenetic monitoring study was carried out on a group of workers from a bituminous coal mine in Zonguldak province of Turkey, to investigate the genotoxic risk of occupational exposure to coal mine dust. Cytogenetic analysis, namely sister chromatid exchanges (SCEs), chromosomal aberrations (CAs) and micronucleus (MN) tests were performed on a strictly selected group of 39 workers and compared to 34 controls matched for gender, age, and habit. Smoking and age were considered as modulating factors. Both SCE and CA frequencies in coal miners appeared significantly higher than in controls. Similarly, there was a significant increase in the frequency of total micronuclei in exposed group as compared to control group. The effect of smoking on the level of SCE and MN was significant in the control group. A positive correlation between the age and the level of SCE was also found in controls. The frequencies of both SCE and CA were significantly enhanced with the years of exposure. The results of this study demonstrated that occupational exposure to coal mine dust leads to a significant induction of cytogenetic damage in peripheral lymphocytes of workers engaged in underground coal mining.