Role of biomarkers in monitoring exposures to chemicals: present position,future prospects

Biomarkers are becoming increasingly important in toxicology and human health. Many research groups are carrying out studies to develop biomarkers of exposure to chemicals and apply these for human monitoring. There is considerable interest in the use and application of biomarkers to identify the nature and amounts of chemical exposures in occupational and environmental situations. Major research goals are to develop and validate biomarkers that reflect specific exposures and permit the prediction of the risk of disease in individuals and groups. One important objective is to prevent human cancer. This review presents a commentary and consensus views about the major developments on biomarkers for monitoring human exposure to chemicals. A particular emphasis is on monitoring exposures to carcinogens. Significant developments in the areas of new and existing biomarkers, analytical methodologies, validation studies and field trials together with auditing and quality assessment of data are discussed. New developments in the relatively young field of toxicogenomics possibly leading to the identification of individual susceptibility to both cancer and non-cancer endpoints are also considered. The construction and development of reliable databases that integrate information from genomic and proteomic research programmes should offer a promising future for the application of these technologies in the prediction of risks and prevention of diseases related to chemical exposures. Currently adducts of chemicals with macromolecules are important and useful biomarkers especially for certain individual chemicals where there are incidences of occupational exposure. For monitoring exposure to genotoxic compounds protein adducts, such as those formed with haemoglobin, are considered effective biomarkers for determining individual exposure doses of reactive chemicals. For other organic chemicals, the excreted urinary metabolites can also give a useful and complementary indication of exposure for acute exposures. These methods have revealed ‘backgrounds’ in people not knowingly exposed to chemicals and the sources and significance of these need to be determined, particularly in the context of their contribution to background health risks.     

Mercapturic acids as biomarkers of exposure to electrophilic chemicals:applications to environmental and industrial chemicals

The use of mercapturic acids (N-acetyl-L-cysteine S-conjugates, MAs) in the biological monitoring of human exposure to environmental and industrial chemicals is receiving more and more attention. Mercapturic acids (MAs) are formed from glutathione (GSH) S-conjugates via the MA-pathway. Although this pathway can lead to different end-products, the formation of MAs is the predominant route in most species, including man. Two GSH S-transferases (GSTs) show genetic polymorphisms in humans and this can have major consequences for individual susceptibility to toxic effects and for MA formation. In occupational toxicology, adducts to biomacromolecules are also used as biomarkers. DNA adducts are a measure for the effective dose, while protein adducts are related to the dose at critical site. Both type of adducts are normally determined in blood, while MAs are determined in urine. Most MAs are excreted with relatively short half-lifes, allowing a direct evaluation of the occupational circumstances. For many compounds similar (linear) dose-dependency was found for MA excretion, formation of macromolecular adducts, and for various biomarkers of toxic effects. These relations together with fact that MAs relate to the electrophilic character of compounds, allows for the conclusion that MAs are biomarkers of toxicologically relevant internal doses of chemicals or their metabolites. An overview will be given here of the use of MAs in the assessment of internal human exposure to electrophilic environmental and industrial chemicals. Additionally, the formation of GSH S-conjugates, their catabolism to MAs and several of the frequently used analytical approaches are discussed. When appropriate, the influence of genetic polymorphisms on formation of MAs and on susceptibility to toxicity will be discussed for different chemicals as well.     

Mercapturic acids as biomarkers of exposure to electrophilic chemicals:applications to environmental and industrial chemicals

The use of mercapturic acids (N-acetyl-L-cysteine S-conjugates, MAs) in the biological monitoring of human exposure to environmental and industrial chemicals is receiving more and more attention. Mercapturic acids (MAs) are formed from glutathione (GSH) S-conjugates via the MA-pathway. Although this pathway can lead to different end-products, the formation of MAs is the predominant route in most species, including man. Two GSH S-transferases (GSTs) show genetic polymorphisms in humans and this can have major consequences for individual susceptibility to toxic effects and for MA formation. In occupational toxicology, adducts to biomacromolecules are also used as biomarkers. DNA adducts are a measure for the effective dose, while protein adducts are related to the dose at critical site. Both type of adducts are normally determined in blood, while MAs are determined in urine. Most MAs are excreted with relatively short half-lifes, allowing a direct evaluation of the occupational circumstances. For many compounds similar (linear) dose-dependency was found for MA excretion, formation of macromolecular adducts, and for various biomarkers of toxic effects. These relations together with fact that MAs relate to the electrophilic character of compounds, allows for the conclusion that MAs are biomarkers of toxicologically relevant internal doses of chemicals or their metabolites. An overview will be given here of the use of MAs in the assessment of internal human exposure to electrophilic environmental and industrial chemicals. Additionally, the formation of GSH S-conjugates, their catabolism to MAs and several of the frequently used analytical approaches are discussed. When appropriate, the influence of genetic polymorphisms on formation of MAs and on susceptibility to toxicity will be discussed for different chemicals as well.     

Degree of alkylation of macromolecules in vivo from variable exposure.

Measurements of adducts formed with blood proteins, particularly haemoglobin, are increasingly being used to monitor human exposures to genotoxic chemicals. Information about the relationships between levels of genotoxic chemicals in the environment, e.g., concentration in the air, and levels of protein adducts in the blood is particularly important in setting safety standards and assessing risks. This paper describes the relationships between level of exposure to alkylating agents and level of haemoglobin adducts, considering the zero-order kinetics of the disappearance of these adducts. For comparison the corresponding relationship for adducts to macromolecules subjected to turnover, with first-order kinetics of disappearance, is described. For chemically stable and unstable adducts different exposure situations are considered: acute, chronic, intermittent and varying exposure levels. It is shown how an optimum solution of the problem of establishing the relationship between long-term exposure at varying levels (e.g., in work environments) and adduct level can be reached. Through mathematical derivations, which are given, expressions applicable to various exposure patterns are obtained and presented.     

Future directions in butadiene risk assessment and the role of cross-species internal dosimetry

The 2005 International Symposium on the evaluation of butadiene and chloroprene health risks provided the opportunity to consider the past, present and future state of research issues for 1,3-butadiene. Considerable advancements have been made in our knowledge of exposure, metabolism, biomarkers of exposure and effect, and epidemiology. Despite this, uncertainties remain which will impact the human health risk assessment for current worker and environmental exposures. This paper reviews key aspects of recent studies and the role that biomarkers of internal dosimetry can play in addressing low to high exposure, gender, and cross-species differences in butadiene toxicity and metabolism. Considerable information is now available on the detection and quantification of protein adducts formed from the mono-, di- and dihydroxy-epoxide metabolites of butadiene. The diepoxide metabolite appears to play a key role in mutagenesis. Species differences in production of this critical metabolite are reflected by the diepoxybutane-specific hemoglobin adduct, pry-Val. To date, the pry-Val adduct has not been quantifiable in human blood samples from workers with cumulative occupational exposures up to 6.3 ppm-weeks; whereas, the pry-Val was quantifiable in the blood of mice and rats with similar cumulative exposures. Levels in mice were much higher than in rats. Further improvements in analytical sensitivity for the pyr-Val adduct are on the horizon. Epidemiology studies are also described and ongoing efforts promise to help bridge our understanding of past and future risks.     

Duration of exposure to environmental carcinogens affects DNA-adduct level in human lymphocytes

Background and objective: An important issue in human biomonitoring is determining how exposure duration affects the kinetics of molecular biomarkers. In this study we compare the influence of exposure variables on DNA adducts.

Methods: DNA adducts were analysed by 32P-postlabelling in lympho/monocytes of 677 Caucasian subjects.

Results: After correction for other variables, DNA adducts increased depending on the length of occupational and smoke exposures. Higher DNA adducts were detected in workers with more than 14 years of exposure than in workers with shorter exposures (RR?=?1.19, p?=?0.049) and in smokers with more than 10 years of exposure than in smokers with shorter exposure (RR?=?1.21, p <0.001).

Conclusions: Exposure length is the primary factor affecting DNA-adduct level in lympho/monocytes both in smokers and in occupationally exposed subjects.     

Biomonitoring of human genotoxicity induced by complex occupational exposures

Sensitivity, specificity and correlations among several biomarkers for monitoring occupational exposure to complex mixtures of genotoxic agents were assessed in occupational environments in Hungarian study populations. The studies have been focused on DNA adduct formation, urinary metabolites, mutations and micronuclei induced by exposures to complex organic mixtures. In two Hungarian aluminium plants, increased DNA adduct and 1-hydroxypyrene (1-OH-PY) levels were observed in workers as compared to controls. However, no association between the biomarker levels was evident on an individual basis. In Hungarian garage mechanics, DNA adduct determinations did not show increased genotoxic exposure as compared to the controls. However, ambient air measurements, significantly enhanced 1-OH-PY levels, and slightly enhanced frequency of micronuclei indicated increased polycyclic aromatic hydrocarbon (PAH) exposure in the garages, as compared to the general environment. In a Hungarian vulcanizing plant, DNA adduct determinations and 1-OH-PY did not show significantly elevated exposure levels as compared to controls. The glycophorin A (GPA) somatic mutation assay was also negative for this occupational exposure. The results support previous observations of a lack of correlation between DNA adducts detectable by 32P-postlabelling and those measured by the PAH-DNA immunoassay in the same DNA sample. These studies also demonstrate a lack of close correlation between levels of DNA adducts and urinary 1-OH-PY in the same individual.     

Chemical safety in animal care,use, and research

Chemical safety is an essential element of an effective occupational health and safety program. Controlling exposures to chemical agents requires a careful process of hazard recognition, risk assessment, development of control measures, communication of the risks and control measures, and training to ensure that the indicated controls will be utilized. Managing chemical safety in animal care and use presents a unique challenge, in part because research is frequently conducted in two very different environments--the research laboratory and the animal care facility. The chemical agents specific to each of these environments are typically well understood by the employees working there; however, the extent of understanding may not be adequate when these individuals, or chemicals, cross over into the other environment. In addition, many chemicals utilized in animal research are not typically used in the research laboratory, and therefore the level of employee knowledge and proficiency may be less compared with more routinely used materials. Finally, the research protocol may involve the exposure of laboratory animals to either toxic chemicals or chemicals with unknown hazards. Such animal protocols require careful review to minimize the potential for unanticipated exposures of the research staff or animal care personnel. Numerous guidelines and regulations are cited, which define the standard of practice for the safe use of chemicals. Key chemical safety issues relevant to personnel involved in the care and use of research animals are discussed.     

1,3-Butadiene: Biomarkers and application to risk assessment

1,3-Butadiene (BD) is a known rodent and human carcinogen that is metabolized mainly by P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB) and 1,2-epoxy-3,4-butanediol (EB-diol). The individual epoxides vary up to 200-fold in their mutagenic potency, with DEB being the most mutagenic metabolite. It is important to understand the internal formation of the individual epoxides to assign the relative risk for each metabolite and to understand the molecular mechanisms responsible for major species differences in carcinogenicity. We have conducted extensive exposure-biomarker studies on mice, rats and humans. Using low exposures that range from current occupational levels to human exposures from tobacco smoke has provided evidence that mice are very different from humans, with mice forming ～200 times more DEB than humans at exposures of 0.1-1.5ppm BD. While no gender differences have been noted in mice and rats for globin adducts or N-7 guanine adducts, female rats and mice had 2-3-fold higher Hprt mutations and DNA-DNA cross-links, suggesting a gender difference in DNA repair. Numerous molecular epidemiology studies have evaluated globin adducts and Hprt mutations, SCEs and chromosomal abnormalities. None of the blinded studies have shown evidence of human genotoxicity at current occupational exposures and studies of globin adducts have shown similar or lower formation of adducts in females than males. If one calculates the EB dose-equivalents for the three species, mice clearly differ from rats and humans, being ～44 and 174 times greater than rats and humans, respectively. These data provide a scientific basis for improved risk assessment of BD.     

Biomarkers in styrene-exposed boatbuilders.

14 fiberglass-reinforced plastics (FRP) boatbuilders were compared with 9 unexposed controls with respect to several chemical specific and nonspecific biomarkers measured in peripheral blood. Biomarkers included styrene-hemoglobin adducts (styrene-Hb), sister-chromatid exchanges (SCEs), micronuclei (MN), single-strand breaks (SSBs) and N-acetoxy-2-acetylaminofluorene-induced DNA binding (NA-AAF binding) as a measure of susceptibility to DNA damage. Workers' exposures averaged 11 ppm (8-h TWA; geometric mean) and ranged from 0.6 to 44 p.p.m. Mandelic acid levels were measured in end-of-shift urine samples and reflected an average styrene exposure equivalent to 15 p.p.m. There was a large though not significant difference in levels of styrene-Hb adducts among exposed workers and controls, largely the consequence of a single heavily-exposed individual with an extremely high level of adducts. Significant differences between biomarker levels in exposed workers and controls were observed with MN, SSBs and NA-AAF binding. No significant differences were seen in mean levels of SCEs nor in the incidence of cells with a high frequency of SCEs. The data suggest that exposure to levels of styrene in occupational settings near or below the current OSHA standard (50 p.p.m.) can induce damage at the cellular/molecular level. Appropriately-selected panels of biomarkers can be useful in identifying potentially harmful exposures.     

Polycyclic aromatic hydrocarbons and PAH-related DNA adducts

Investigations on the impact of chemicals on the environment and human health have led to the development of an exposome concept. The exposome refers to the totality of exposures received by a person during life, including exposures to life-style factors, from the prenatal period to death. The exposure to genotoxic chemicals and their reactive metabolites can induce chemical modifications of DNA, such as, for example, DNA adducts, which have been extensively studied and which play a key role in chemically induced carcinogenesis. Development of different methods for the identification of DNA adducts has led to adopting DNA adductomic approaches. The ability to simultaneously detect multiple PAH-derived DNA adducts may allow for the improved assessment of exposure, and offer a mechanistic insight into the carcinogenic process following exposure to PAH mixtures. The major advantage of measuring chemical-specific DNA adducts is the assessment of a biologically effective dose. This review provides information about the occurrence of the polycyclic aromatic hydrocarbons (PAHs) and their influence on human exposure and biological effects, including PAH-derived DNA adduct formation and repair processes. Selected methods used for determination of DNA adducts have been presented.     

The role of DNA adducts in smoking-related carcinogenesis

Epidemiological studies indicate a close association between smoking and cancer. Biological activity of many chemical carcinogens and of their metabolites is induced by covalent binding of their reactive derivatives to DNA, which consequently causes mutations in critical genes. Carcinogen-DNA adducts formed by exposure to tobacco smoke have a key role in the initiation of various types of cancer including lung cancer. Presence of tobacco smoke-related carcinogen-DNA adducts in various tissues of smokers proves the DNA damaging effect of smoking. DNA adducts are important biomarkers for the biomonitoring of human genotoxic exposures to tobacco smoke. The paper gives a short overview on the role of smoking-related DNA adducts in carcinogenesis.     

The Use of Epidemiology in Risk Assessment: Challenges and Opportunities

The assessment of risk from environmental and occupational exposures incorporates and synthesizes data from a variety of scientific disciplines including toxicology and epidemiology. Epidemiological data have offered valuable contributions to the identification of human health hazards, estimation of human exposures, quantification of the exposure–response relation, and characterization of risks to specific target populations including sensitive populations. As with any scientific discipline, there are some uncertainties inherent in these data; however, the best human health risk assessments utilize all available information, characterizing strengths and limitations as appropriate. Human health risk assessors evaluating environmental and occupational exposures have raised concerns about the validity of using epidemiological data for risk assessment due to actual or perceived study limitations. This article highlights three concerns commonly raised during the development of human health risk assessments of environmental and occupational exposures: (a) error in the measurement of exposure, (b) potential confounding, and (c) the interpretation of non-linear or non-monotonic exposure–response data. These issues are often the content of scientific disagreement and debate among the human health risk assessment community, and we explore how these concerns may be contextualized, addressed, and often ameliorated.     

The endogenous exposome

The concept of the Exposome is a compilation of diseases and one's lifetime exposure to chemicals, whether the exposure comes from environmental, dietary, or occupational exposures; or endogenous chemicals that are formed from normal metabolism, inflammation, oxidative stress, lipid peroxidation, infections, and other natural metabolic processes such as alteration of the gut microbiome. In this review, we have focused on the endogenous exposome, the DNA damage that arises from the production of endogenous electrophilic molecules in our cells. It provides quantitative data on endogenous DNA damage and its relationship to mutagenesis, with emphasis on when exogenous chemical exposures that produce identical DNA adducts to those arising from normal metabolism cause significant increases in total identical DNA adducts. We have utilized stable isotope labeled chemical exposures of animals and cells, so that accurate relationships between endogenous and exogenous exposures can be determined. Advances in mass spectrometry have vastly increased both the sensitivity and accuracy of such studies. Furthermore, we have clear evidence of which sources of exposure drive low dose biology that results in mutations and disease. These data provide much needed information to impact quantitative risk assessments, in the hope of moving towards the use of science, rather than default assumptions.     

Molecular epidemiology studies of carcinogenic environmental pollutants. Effects of polycyclic aromatic hydrocarbons (PAHs) in environmental pollution on exogenous and oxidative DNA damage

Exposure to high levels of environmental air pollution is known to be associated with an increased carcinogenic risk. The individual contribution to this risk derived from specific carcinogenic chemicals within the complex mixture of air pollution is less certain, but may be explored by the use of molecular epidemiological techniques. Measurements of biomarkers of exposure, of effect and of susceptibility provide information of potential benefit for epidemiological and cancer risk assessment. The application of such techniques has been mostly concerned in the past with the carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) that are associated with particulate matter in air pollution, and has showed clear evidence of genotoxic effects, such as DNA adducts, chromosome aberrations (CA) and ras oncogene overexpression, in environmentally exposed Czech and Polish populations. We are currently extending these studies by an investigation of populations exposed to environmental pollution in three European countries, Czech Republic, Slovak Republic and Bulgaria. This pays particular attention to PAHs, but also investigates the extent of radically induced (oxidative) DNA damage in the exposed populations. Policemen, bus drivers and controls, who carried personal monitors to determine their exposures to PAHs have been studied, and blood and urine were collected. Antioxidant and dietary status were assessed in these populations. Stationary monitors were also used for ambient air monitoring. Amongst the parameters studied in the biological samples were: (a) exposure biomarkers, such as PAH adducts with DNA, p53 and p21(WAF1) protein levels, (b) oxidative DNA damage, (c) the biological effect of the exposure by measurement of chromosome damage by fluorescence in situ hybridisation (FISH) or conventional methods, and (d) polymorphisms in carcinogen metabolising and DNA repair enzymes. Repair ability was also measured by the Comet assay. In vitro systems are being evaluated to characterise the genotoxicity of the organic compounds adsorbed to air particles.     

Methodologies for bulky DNA adduct analysis and biomonitoring of environmental and occupational exposures

It is undisputed that DNA adduct formation is one of the key processes in early carcinogenesis. Therefore, analysis of DNA adduct levels may be one of the best tools available to characterize exposure to complex mixtures of genotoxic chemicals as occurring in different environmental and occupational exposure settings. However, from an analytical point of view the detection and quantification of DNA adducts is a challenging enterprise as extremely high sensitivity and selectivity are required. The entire spectrum of chromatographic techniques, including thin-layer chromatography (TLC), gas and liquid chromatography as well as capillary electrophoresis has been used in combination with different detection systems, all with their own specific characteristics. Among the various combinations of techniques, the TLC-(32)P-postlabeling combination appears to meet best with criteria of sensitivity and requirements of minimal amounts of material. Recent developments in the application of capillary electrophoresis in combination with either immunochemical or mass spectrometric detection techniques may offer new and promising approaches, with higher selectivity as compared to TLC-(32)P postlabeling. The applicability of these new techniques in biomonitoring studies aiming at the exposure and risk assessment of low and chronic exposures remains to be determined. In this paper we compare and discuss the advantages and limitations of different techniques used in DNA adduct analysis, with specific emphasis on those adducts formed by the polycyclic aromatic hydrocarbons and heterocyclic aromatic amines.     

Polycyclic nitroarenes (nitro-PAHs) as biomarkers of exposure to diesel exhaust

Diesel exhaust contains numerous genotoxic carcinogens. It is essentially unknown to which extent this source contributes to the total load of these chemicals in humans. One possible approach to the problem is to find suitable biomarkers. To this end five polycyclic mononitroarenes (nitro-PAH) were selected and methods developed to determine the sulfinic acid-type hemoglobin adducts they form in vivo. The nitro-PAHs are: 1-nitropyrene, 2-nitrofluorene, 3-nitrofluoranthene, 9-nitrophenanthrene, and 6-nitrochrysene. Hydrolysis of the hemoglobin adducts yields the respective arylamines which were analyzed by gas chromatography/mass spectrometry. The detection limit was 0.01-0.08 pmol/g Hb. Blood samples were analyzed from 29 bus garage workers, occupationally exposed to diesel exhaust, and from 20 urban hospital workers and 14 rural council workers as controls. Hb adducts above the detection limit were found in most blood samples. The most abundant cleavage products were 1-aminopyrene and 2-aminofluorene with levels ranging from 0.01 to 0.68 pmol/g Hb. However, there was no significant difference between the groups for 1-nitropyrene and 2-nitrofluorene supporting the conclusion that both are widespread environmental contaminants resulting in significant background exposures. A significant difference on a group from individuals from urban and rural areas was found only if all five adducts were added, this may indicate an additional exposure from traffic. The new specific nitro-PAH Hb adducts are proposed to be used as biomarkers to trace the sources and to identify above-background exposures.     

Prospects for incorporation of epigenetic biomarkers in human health and environmental risk assessment of chemicals

Epigenetic mechanisms have gained relevance in human health and environmental studies, due to their pivotal role in disease, gene × environment interactions and adaptation to environmental change and/or contamination. Epigenetic mechanisms are highly responsive to external stimuli and a wide range of chemicals has been shown to determine specific epigenetic patterns in several organisms. Furthermore, the mitotic/meiotic inheritance of such epigenetic marks as well as the resulting changes in gene expression and cell/organismal phenotypes has now been demonstrated. Therefore, epigenetic signatures are interesting candidates for linking environmental exposures to disease as well as informing on past exposures to stressors. Accordingly, epigenetic biomarkers could be useful tools in both prospective and retrospective risk assessment but epigenetic endpoints are currently not yet incorporated into risk assessments. Achieving a better understanding on this apparent impasse, as well as identifying routes to promote the application of epigenetic biomarkers within environmental risk assessment frameworks are the objectives of this review. We first compile evidence from human health studies supporting the use of epigenetic exposure‐associated changes as reliable biomarkers of exposure. Then, specifically focusing on environmental science, we examine the potential and challenges of developing epigenetic biomarkers for environmental fields, and discuss useful organisms and appropriate sequencing techniques to foster their development in this context. Finally, we discuss the practical incorporation of epigenetic biomarkers in the environmental risk assessment of chemicals, highlighting critical data gaps and making key recommendations for future research within a regulatory context.     

Evaluation of long-term occupational exposure to styrene vapor on olfactory function

The primary sensory neurons of the olfactory system are chronically exposed to the ambient environment and may therefore be susceptible to damage from occupational exposure to many volatile chemicals. To investigate whether occupational exposure to styrene was associated with olfactory impairment, we examined olfactory function in 2 groups: workers in a German reinforced-plastics boat-manufacturing facility having a minimum of 2 years of styrene exposure (15-25 ppm as calculated from urinary metabolite concentrations, with historical exposures up to 85 ppm) and a group of age-matched workers from the same facility with lower styrene exposures. The results were also compared with normative data previously collected from healthy, unexposed individuals. Multiple measures of olfactory function were evaluated using a standardized battery of clinical assessments from the Monell-Jefferson Chemosensory Clinical Research Center that included tests of threshold sensitivity for phenylethyl alcohol (PEA) and odor identification ability. Thresholds for styrene were also obtained as a measure of occupational olfactory adaptation. Styrene exposure history was calculated through the use of past biological monitoring results for urinary metabolites of styrene (mandelic acid [MA], phenylglyoxylic acid [PGA]); current exposure was determined for each individual using passive air sampling for styrene and biological monitoring for styrene urinary metabolites. Current mean effective styrene exposure during the day of olfactory testing for the group of workers who worked directly with styrene resins was 18 ppm styrene (standard deviation [SD] = 14), 371 g/g creatinine MA + PGA (SD = 289) and that of the group of workers with lower exposures was 4.8 ppm (SD = 5.2), 93 g/g creatinine MA+PGA (SD = 100). Historic annual average exposures for all workers were greater by a factor of up to 6x. No differences unequivocally attributable to exposure status were observed between the Exposed and Comparison groups or between performance of either group and normative population values on thresholds for PEA or odor identification. Although odor identification performance was lower among workers with higher ongoing exposures, performance on this test is not a pure measure of olfactory ability and is influenced by familiarity with the stimuli and their sources. Consistent with exposure-induced sensory adaptation, however, elevated styrene thresholds were significantly associated with higher occupational exposures to styrene. In summary, the present study found no evidence among a cross-section of reinforced-plastics workers that current or historical exposure to styrene was associated with a general impairment of olfactory function. When taken together with prior studies of styrene-exposed workers, these results suggest that styrene is not a significant olfactory toxicant in humans at current exposure levels.