Concepts and determination of reference values for human biomonitoring of environmental contaminants

Human biomonitoring (HBM) of environmental contaminants plays an important role in estimating exposure and evaluating risk, and thus it has been increasingly applied in the environmental field. The results of HBM must be compared with reference values ( RV). The term ""reference values"" has always been related to the interpretation of clinical laboratory tests. For physicians, RV indicate ""normal values"" or ""limits of normal""; in turn, toxicologists prefer the terms ""background values"" or ""baseline values"" to refer to the presence of contaminants in biological fluids. This discrepancy leads to the discussion concerning which should be the population selected to determine RV. Whereas clinical chemistry employs an altered health state as the main exclusion criterion to select a reference population ( that is, a ""healthy"" population would be selected), in environmental toxicology the exclusion criterion is the abnormal exposure to xenobiotics. Therefore, the choice of population to determine RV is based on the very purpose of the RV to be determined. The present paper discusses the concepts and methodology used to determine RV for biomarkers of chemical environmental contaminants.

Application of alkaline comet assay in human biomonitoring for genotoxicity: a study on occupational exposure to cytostatics

The use of cytostatics drugs in anticancer therapy is increasing. Health care workers can be occupationally exposed to these drugs classified as carcinogenic, mutagenic or teratogenic. Cytostatics drugs are a heterogeneous group of chemicals widely used in the treatment of cancer, nevertheless have been proved to be also mutagens, carcinogens and teratogens. Workers may be exposed to this drug, being in the hospital settings the main focus dwelled upon the pharmacy, and nursing personnel. Alkaline comet assay is one of the most promising short-term genotoxicity assays for human risk assessment, being recommended to monitor populations chronically exposed to genotoxic agents. DNA glycosylase (OGG1) represents the main mechanism of protecting the integrity of the human DNA with respect to 8-OHdG, the most well studied biomarker of oxidative damage.

Selenium role in an human biomonitoring study applied to occupational health

Selenium functions as a co-factor for the reduction of antioxidant enzymes and is an important component of antioxidant enzymes. Dietary selenium significantly inhibits the induction of skin, liver, colon, and mammary tumours in experimental animals by a number of different carcinogens, as well as the induction of mammary tumours by viruses. Selenium shows a “U” shaped curve for functionality, whereby too little is as damaging as too much. At optimal levels, selenium may protect against the formation of DNA adducts, DNA or chromosome breakage, chromosome gain or loss, mitochondrial DNA, and telomere length and function. Aim of study: Investigate the relation between selenium and genotoxic effects in a human biomonitoring study applied to occupational health.

Human biomonitoring: biomarkers, susceptibility, and nutrigenetics

The methods of molecular biology applied in epidemiological research lead us to the realm of molecular epidemiology, where there is immense potential for the establishment of associations between cancer and exposure to risk factors in lifestyle, profession, or pollution. Human biomonitoring consists, on the one hand, in research and identification of hazardous environmental conditions and, on the other hand, in the assessment of cancer risk following exposure to such conditions. Since carcinogenesis is a lengthy process, the biomarkers used to recognize biological abnormalities are selected and developed in the realm of molecular epidemiology. Such biomarkers are quantifiable and allow for the recognition of progression from normal to abnormal biological conditions at the molecular level. They can be categorized in biomarkers of exposure, effect, and genetic susceptibility. Genotoxicity biomarkers are a particular subset of effect biomarkers and are used to assess genomic instability caused by environmental or occupational exposure, being considered useful carcinogenesis predictors.

Micronutrients intake associated with DNA damage assessed by in a human biomonitoring study

Nutrition science has evolved into a multidisciplinary field that applies molecular biology and integrates individual health with the epidemiologic investigation of population health. Nutritional genomics studies the functional interaction of food and its components, macro and micronutrients, with the genome at the molecular, cellular, and systemic level. Diet can influence cancer development in several ways, namely direct action of carcinogens in food that can damage DNA, diet components (macro or micronutrients) that can block or induce enzymes involved in activation or deactivation of carcinogenic substances. Moreover, inadequate intake of some molecules involved in DNA synthesis, repair or methylation can influence mutation rate or changes in gene expression. Several studies support the idea that diet can influence the risk of cancer; however information concerning the precise dietary factor that determines human cancer is an ongoing debate. A lot of epidemiological studies, involving food frequency questionnaires, have been developed providing important information concerning diet and cancer, however, diet is a complex composite of various nutrients (macro and micronutrients) and non-nutritive food constituents that makes the search for specific factors almost limitless.

First steps toward harmonized human biomonitoring in Europe : demonstration project to perform human biomonitoring on a European scale

'Reproduced with permission from Environmental Health Perspectives'

Comet assay as a human biomonitoring tool: application in occupational exposure to antineoplastic drugs

Antineoplastic drugs are a heterogeneous group of chemicals used in the treatment of cancer, and have been proved by IARC to be mutagens, carcinogens and teratogens agents. In general, chemicals that interact directly with DNA by biding covalently or by intercalating, or indirectly by interfering with DNA synthesis, were among the first chemotherapeutics developed. Also, these drugs can induce reactive oxygen species that can lead to DNA damage and, consequently, mutations. These drugs are often used in combination to achieve synergistic effects on tumour cells resulting from their differing modes of action. However, most if not all of these chemical agents are generally nonselective and, along with tumour cells, normal cells may undergo cytotoxic/genotoxic damage. The in vivo exposure to antineoplastic drugs has been shown to induce different types of lesions in DNA, depending on the particular stage of cell cycle at the time of treatment. Besides the patients that use these drugs as a treatment, workers that handle and/or administer these drugs can be exposed to these substances; namely pharmacy, and nursing personnel in hospital context.

Contribute of the MYCOMIX project and future perspectives for Human Biomonitoring and health risk assessment in Portugal

There is a growing concern within public health about mycotoxin involvement in human diseases, namely those related to children. The MycoMix project (2012-2015), funded by the Portuguese Foundation for Science and Technology, gathered a multidisciplinary team aiming at answering several questions: 1) Are Portuguese children exposed daily to one or several mycotoxins through food? 2) Can this co-exposure affect children´s health? and 3) Are there interaction effect between mycotoxins? Mycomix results revealed that Portuguese children (< 3 years old, n=103) are exposed to multiple mycotoxins through food consumption. Cumulative risk assessment results revealed a potential health concern for the high percentiles of intake, specially for aflatoxins which are carcinogenic compounds. This fact assumes particular importance considering the interactive effects found in in vitro bioassays. These results highlight the need for a more accurate approach to assess the human exposure to mycotoxins6. Within the Mycomix project the assessment of mycotoxin exposure was based on calculations combining mycotoxin data in food with population data on food consumption. This approach does not consider some aspects as the inter-individual metabolism variation, the exposure through sources other than food and the heterogeneous distribution of mycotoxins in food. Exposure assessment of mycotoxins in Portuguese population through biomarkers is still missing and further studies are urgent to be developed. The European Human Biomonitoring Initiative (EHBMI), a proposal within the European Joint Programme, aims to advance the understanding of the extent of exposure to environmental chemicals across Europe and the impact on human health, by gathering national expertise in human biomonitoring domain. At national level Mycomix project uncovered the potential health risk of exposure of Portuguese children to multiple mycotoxins. The risk assessment expertise acquired within Mycomix, namely in analysis and toxicology of chemical mixtures, will be brought together as a contribute to EHBMI objectives.

Viable human buccal mucosa cells do not yield typical nucleoids: Impacts on the single-cell gel electrophoresis/comet assay

Buccal mucosa (BM) cells have been used in human biomonitoring studies for detecting DNA adducts and chromosomal damage in an epithelial cell population. In the present study, we have investigated if human BM cells are suitable for use in the single-cell gel electrophoresis (SCGE)/Comet assay as an approach for estimating the exposure of epithelial cells to DNA-damaging agents. Our results indicate that only a few cells from BM cell samples yield comets that can be analyzed by current methods, and that the yield of cells with comets is independent of the percentage of viable BM cells in the sample. Data generated after enzymatic enrichment of viable cells and immunomagnetic separation of epithelial cells suggest that most of the BM cells that do form comets are probably leukocytes. Moreover, by reevaluating specific cells after running the Comet assay, we found that viable epithelial BM cells give rise to atypical comets that are not included in the analysis. Comparing DNA migration patterns between small groups of smokers and nonsmokers indicated that long-term smoking had no effect on the subpopulation of cells that yield typical comets. Our results indicate that the SCGE assay, as it is commonly performed, may not be useful for genotoxicity monitoring in human epithelial BM cells.

Relation between DNA damage measured by comet assay and OGG1 Ser326Cys polymorphism in antineoplastic drugs biomonitoring

Antineoplastic drugs are hazardous chemical agents used mostly in the treatment of patients with cancer, however health professionals that handle and administer these drugs can become exposed and develop DNA damage. Comet assay is a standard method for assessing DNA damage in human biomonitoring and, combined with formamidopyrimidine DNA glycosylase (FPG) enzyme, it specifically detects DNA oxidative damage. The aim of this study was to investigate genotoxic effects in workers occupationally exposed to cytostatics (n = 46), as compared to a control group with no exposure (n = 46) at two Portuguese hospitals, by means of the alkaline comet assay. The potential of the OGG1 Ser326Cys polymorphism as a susceptibility biomarker was also investigated. Exposure was evaluated by investigating the contamination of surfaces and genotoxic assessment was done by alkaline comet assay in peripheral blood lymphocytes. OGG1 Ser326Cys (rs1052133) polymorphism was studied by Real Time PCR. As for exposure assessment, there were 121 (37%) positive samples out of a total of 327 samples analysed from both hospitals. No statistically significant differences (Mann-Whitney test, p > 0.05) were found between subjects with and without exposure, regarding DNA damage and oxidative DNA damage, nevertheless the exposed group exhibited higher values. Moreover, there was no consistent trend regarding the variation of both biomarkers as assessed by comet assay with OGG1 polymorphism. Our study was not statistically significant regarding occupational exposure to antineoplastic drugs and genetic damage assessed by comet assay. However, health professionals should be monitored for risk behaviour, in order to ensure that safety measures are applied and protection devices are used correctly.

The influence of genetic polymorphisms in XRCC3 and ADH5 genes on the frequency of genotoxicity biomarkers in workers exposed to formaldehyde

The International Agency for Research on Cancer classified formaldehyde as carcinogenic to humans because there is “sufficient epidemiological evidence that it causes nasopharyngeal cancer in humans”. Genes involved in DNA repair and maintenance of genome integrity are critically involved in protecting against mutations that lead to cancer and/or inherited genetic disease. Association studies have recently provided evidence for a link between DNA repair polymorphisms and micronucleus (MN) induction. We used the cytokinesis-block micronucleus (CBMN assay) in peripheral lymphocytes and MN test in buccal cells to investigate the effects of XRCC3 Thr241Met, ADH5 Val309Ile, and Asp353Glu polymorphisms on the frequency of genotoxicity biomarkers in individuals occupationally exposed to formaldehyde (n = 54) and unexposed workers (n = 82). XRCC3 participates in DNA double-strand break/recombination repair, while ADH5 is an important component of cellular metabolism for the elimination of formaldehyde. Exposed workers had significantly higher frequencies (P < 0.01) than controls for all genotoxicity biomarkers evaluated in this study. Moreover, there were significant associations between XRCC3 genotypes and nuclear buds, namely XRCC3 Met/Met (OR = 3.975, CI 1.053–14.998, P = 0.042) and XRCC3 Thr/Met (OR = 5.632, CI 1.673–18.961, P = 0.005) in comparison with XRCC3 Thr/Thr. ADH5 polymorphisms did not show significant effects. This study highlights the importance of integrating genotoxicity biomarkers and genetic polymorphisms in human biomonitoring studies.

Re-evaluation of a reported increased micronucleus frequency in lymphocytes of workers occupationally exposed to formaldehyde

A replicate evaluation of increased micronucleus (MN) frequencies in peripheral lymphocytes of workers occupationally exposed to formaldehyde (FA) was undertaken to verify the observed effect and to determine scoring variability. May–Grünwald–Giemsa-stained slides were obtained from a previously performed cytokinesis-block micronucleus test (CBMNT) with 56 workers in anatomy and pathology laboratories and 85 controls. The first evaluation by one scorer (scorer 1) had led to a highly significant difference between workers and controls (3.96 vs 0.81 MN per 1000 cells). The slides were coded before re-evaluation and the code was broken after the complete re-evaluation of the study. A total of 1000 binucleated cells (BNC) were analysed per subject and the frequency of MN (in ‰) was determined. Slides were distributed equally and randomly between two scorers, so that the scorers had no knowledge of the exposure status. Scorer 2 (32 exposed, 36 controls) measured increased MN frequencies in exposed workers (9.88 vs 6.81). Statistical analysis with the two-sample Wilcoxon test indicated that this difference was not significant (p = 0.17). Scorer 3 (20 exposed, 46 controls) obtained a similar result, but slightly higher values for the comparison of exposed and controls (19.0 vs 12.89; p = 0.089). Combining the results of the two scorers (13.38 vs 10.22), a significant difference between exposed and controls (p = 0.028) was obtained when the stratified Wilcoxon test with the scorers as strata was applied. Interestingly, the re-evaluation of the slides led to clearly higher MN frequencies for exposed and controls compared with the first evaluation. Bland–Altman plots indicated that the agreement between the measurements of the different scorers was very poor, as shown by mean differences of 5.9 between scorer 1 and scorer 2 and 13.0 between scorer 1 and scorer 3. Calculation of the intra-class correlation coefficient (ICC) revealed that all scorer comparisons in this study were far from acceptable for the reliability of this assay. Possible implications for the use of the CBMNT in human biomonitoring studies are discussed.

Assessment of genotoxicity in oral epithelial cells of health professionals occupationally exposed to ionizing radiations

Dissertação de Mestrado em Ambiente, Saúde e Segurança.

Formaldehyde: exposure and genotoxicity assessments and potential health effects

Worldwide formaldehyde is manipulated with diverse usage properties, since industrial purposes to health laboratory objectives, representing the economic importance of this chemical agent. Therefore, many people are exposed to formaldehyde environmentally and/or occupationally. Considering the latter, there was recommended occupational exposure limits based on threshold mechanisms, limit values and indoor guidelines. Formaldehyde is classified by the International Agency for Cancer Research (IARC) as carcinogenic to humans (group 1), since a wide range of epidemiological studies in occupational exposure settings have suggested possible links between the concentration and duration of exposure and elevated risks of nasopharyngeal cancer, and others cancers, and more recently, with leukemia. Although there are different classifications, such as U.S. EPA that classified formaldehyde as a B1 compound, probable human carcinogen under the conditions of unusually high or prolonged exposure, on basis of limited evidence in humans but with sufficient evidence in animals. Formaldehyde genotoxicity is well-known, being a direct-acting genotoxic compound positively associated for almost all genetic endpoints evaluated in bacteria, yeast, fungi, plants, insects, nematodes, and cultured mammalian cells. There are many human biomonitoring studies that associate formaldehyde occupational exposure to genomic instability, and consequently possible health effects. Besides the link with cancer, also other pathologies and symptoms are associated with formaldehyde exposure, namely respiratory disorders such as asthma, and allergic contact dermatitis. Nowadays, there are efforts to reduce formaldehyde exposure, namely indoor. Europe and United States developed more strict regulation regarding formaldehyde emissions from materials containing this agent. Despite the regulations and restrictions, formaldehyde still continues to be difficult to eliminate or substitute, being biomonitoring an important tool to control possible future health effects.

Biological monitoring of chemical exposure : toxicokinetic differences due to age and sex

Human biomonitoring is a widely used method in the assessment of occupational exposure to chemical substances and recommended biological limits are published periodically for interpretation and decision-making. However, it is increasingly recognized that a large variability is associated with biological monitoring, making interpretation less efficient than assumed. In order to improve the applicability of biological monitoring, specific factors responsible for this variability should be identified and their contribution quantified. Among these factors, age and sex are easily identifiable, and present knowledge about pharmaceutical chemicals suggests that they play an important role on the toxicokinetics of occupational chemical agents, and therefore on the biological monitoring results.The aim of the present research project was to assess the influence of age and sex on biological indicators corresponding to organic solvents. This has been done experimentally and by toxicokinetic computer simulation. Another purpose was to explore the effect of selected CYP2E1 polymorphisms on the toxicokinetic profile.Age differences were identified by numerical simulations using a general toxicokinetic model from a previous study which was applied to 14 chemicals, representing 21 specific biological entities, with, among others, toluene, phenol, lead and mercury. These models were runn with the modified parameters, indicating in some cases important differences due to age. The expected changes are mostly of the order of 10-20 %, but differences up to 50 % were observed in some cases. These differences appear to depend on the chemical and on the biological entity considered.Sex differences were quantified by controlled human exposures, which were carried out in a 12 m3 exposure chamber for three organic solvents separately: methyl ethyl ketone, 1-methoxy-2-propanol and 1,1,1-trichloroethane. The human volunteer groups were composed 12 of ten young men and fifteen young women, the latter subdivided into those with and without hormonal contraceptive. They were exposed during six hours at rest and at half of the threshold limit value. The kinetics of the parent compounds (organic volatiles) and their metabolite(s) were followed in blood, urine and expired air over time. Analyses of the solvent and their metabolites were performed by using headspace gas chromatography, CYP2E1 genotypes by using PCR-based RFLP methods. Experimental data were used to calibrate the toxicokinetic models developed for the three solvents. The results obtained for the different biomarkers of exposure mainly showed an effect on the urinary levels of several biomarkers among women due to the use of hormonal contraceptive, with an increase of about 50 % in the metabolism rate. The results also showed a difference due to the genotype CYP2E1*6, when exposed to methyl ethyl ketone, with a tendency to increase CYP2E1 activity when volunteers were carriers of the mutant allele. Simulations showed that it is possible to use simple toxicokinetic tools in order to predict internal exposure when exposed to organic solvents. Our study suggests that not only physiological differences but also exogenous sex hormones could influence CYP2E1 enzyme activity. The variability among the urinary biological indicators levels gives evidence of an interindividual susceptibility, an aspect that should have its place in the approaches for setting limits of occupational exposure.