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.     

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.     

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.     

Bioactivation of xenobiotics by formation of toxic glutathione conjugates

Evidence has been accumulating that several classes of compounds are converted by glutathione conjugate formation to toxic metabolites. The aim of this review is to summarize the current knowledge on the biosynthesis and toxicity of glutathione S-conjugates derived from halogenated alkanes, halogenated alkenes, and hydroquinones and quinones. Different types of toxic glutathione conjugates have been identified and will be discussed in detail: (i) conjugates which are transformed to electrophilic sulfur mustards, (ii) conjugates which are converted to toxic metabolites in an enzyme-catalyzed multistep mechanism, (iii) conjugates which serve as a transport form for toxic quinones and (iv) reversible glutathione conjugate formation and release of the toxic agent in cell types with lower glutathione concentrations. The kidney is the main, with some compounds the exclusive, target organ for compounds metabolized by pathways (i) to (iii). Selective toxicity to the kidney is easily explained due to the capability of the kidney to accumulate intermediates formed by processing of S-conjugates and to bioactivate these intermediates to toxic metabolites. The influences of other factors participating in the renal susceptibility are discussed.     

Mercapturic acids: recent advances in their determination by liquid chromatography/mass spectrometry and their use in toxicant metabolism studies and in occupational and environmental exposure studies

This review describes recent selected HPLC/MS methods for the determination of urinary mercapturates that are useful as noninvasive biomarkers in characterizing human exposure to electrophilic industrial chemicals in occupational and environmental studies. High-performance liquid chromatography/mass spectrometry is a sensitive and specific method for analysis of small molecules found in biological fluids. In this review, recent selected mercapturate quantification methods are summarized and specific cases are presented. The biological formation of mercapturates is introduced and their use as indicators of metabolic processing of reactive toxicants is discussed, as well as future trends and limitations in this area of research.     

The relevance of xenobiotic metabolism in the interindividual susceptibility to chemicals

Biotransformation enzymes may catalyze either detoxication or bioactivation reactions; indeed, many xenobiotics exert their toxic effects after metabolic activation to electrophilic chemicals, interacting with nucleophilic sites on cellular macromolecules. On the other hand, by increasing xenobiotic hydrophilicity, the drug-metabolizing enzymes favors excretion of lipophilic chemicals, not allowing their bioaccumulation up to toxic levels. The expression of the enzymes of the drug-metabolizing system is modulated by genetic, pathological, developmental, environmental and dietary factors. Genetic polymorphism resulting in interindividual and interethnic variation in xenobiotic metabolism is responsible for differences in the susceptibility to chemical-induced toxicity and carcinogenicity, allowing the identification of people at increased risk. Moreover, differences in drug metabolism may correspond to variability in drug response during pharmacological therapy, which can be manifest either as adverse reactions or as a lack of benefit.     

Degradation of glutathione S-conjugates in Physcomitrella patens is initiated by cleavage of glycine

Glutathione-dependent detoxification is a key pathway that allows plants to efficiently remove toxic compounds like heavy metals or electrophilic xenobiotics. Under persistent exposure to toxins plants need to respond to continuous demand with efficient synthesis of glutathione (GSH) and ideally fast and efficient removal of potentially toxic glutathione S-conjugates. With the aim of studying the respective degradation pathway in Physcomitrella patens we initially characterized fluorescence labeling of protonema cultures with GSH-specific xenobiotic monochlorobimane (MCB). Incubation of protonema with 200 μM MCB for 24 h resulted in a steady increase of total bimane label, which was not confined to glutathione S-bimane (GS-B), but predominantly present in γ-glutamylcysteine S-bimane (γ-EC-B) and cysteine S-bimane (Cys-B). Pulse-chase experiments identified γ-EC-B and Cys-B as degradation products of GS-B, suggesting initial cleavage of the C-terminal glycine, followed by cleavage of the γ-glutamyl bond. The amount of GS-B formed, increased linearly at 90 nmol GSH g fw?1 h?1 for 24 h and after ～1.5 h already surpassed the amount of GSH present in control protonema. This demand-driven biosynthesis of GSH depends on sufficient supply of sulfate in the incubation medium.     

Hepatic glutathione and glutathione S-conjugate transport mechanisms.

Glutathione (GSH) plays a critical role in many cellular processes, including the metabolism and detoxification of oxidants, metals, and other reactive electrophilic compounds of both endogenous and exogenous origin. Because the liver is a major site of GSH and glutathione S-conjugate biosynthesis and export, significant effort has been devoted to characterizing liver cell sinusoidal and canalicular membrane transporters for these compounds. Glutathione S-conjugates synthesized in the liver are secreted preferentially into bile, and recent studies in isolated canalicular membrane vesicles indicate that there are multiple transport mechanisms for these conjugates, including those that are energized by ATP hydrolysis and those that may be driven by the electrochemical gradient. Glutathione S-conjugates that are relatively hydrophobic or have a bulky S-substituent are good substrates for the canalicular ATP-dependent transporter mrp2 (multidrug resistance-associated protein 2, also called cMOAT, the canalicular multispecific organic anion transporter, or cMrp, the canalicular isoform of mrp). In contrast with the glutathione S-conjugates, hepatic GSH is released into both blood and bile. GSH transport across both of these membrane domains is of low affinity and is energized by the electrochemical potential. Recent reports describe two candidate GSH transport proteins for the canalicular and sinusoidal membranes (RcGshT and RsGshT, respectively); however, some concerns have been raised regarding these studies. Additional work is needed to characterize GSH transporters at the functional and molecular level.     

Reductive alkylation of DNA by mitomycin A, a mitomycin with high redox potential

The mitomycins are a group of antitumor antibiotics that covalently bind to DNA upon reductive activation. Mitomycin A (1b; MA) is more toxic than its clinically useful mitomycin C (1a; MC). The greater toxicity of mitomycin A has been previously attributed to its higher reduction potential. In this report, the DNA alkylation products of reductively activated MA were isolated and characterized by conversion to the known 7-amino mitosene-deoxyguanosine adducts. The three major adducts formed were identified as a monoadduct, N2-(2"beta-amino-7"-methoxymitosen-1"alpha-yl)- 2'-deoxyguanosine (5), a decarbamoyl monoadduct, N2-(2"beta-amino-10"-decarbamoyl-7"-methoxymitosen-1"alpha-y l)-2'- deoxyguanosine (6), and a bisadduct, N2-(2"beta-amino-10"-deoxyguanosin-N2-yl-7-methoxymitosen-1" alpha- yl)-2'-deoxyguanosine (7). Under all reductive activation conditions employed, MA selectively alkylated the 2-amino group of guanine in DNA, like MC. In addition, both MA and MC alkylated DNA and cross-linked oligonucleotides to a similar extent. However, variations in the reductive activation conditions (H2/PtO2, Na2S2O4, or enzymatic) affected the distribution of the three major MA adducts in a different manner than the distribution of MC adducts was affected. A mechanism is proposed wherein the 7-methoxy substituent of MA allows initial indiscriminate activation of either of the drugs' two electrophilic sites. While oxygen inhibited cross-linking by MC, similar aerobic conditions exhibited little influence on the cross-linking ability of MA. Hence, the greater toxicity of MA may be influenced by increased and nonselective activation and cross-link formation in both aerobic and anaerobic cells. This effect is a direct consequence of the higher redox potential of MA as compared to MC.     

DNA adduct formation by 12 chemicals with populations potentially suitable for molecular epidemiological studies.

DNA adduct formation, route of absorption, metabolism and chemistry of 12 hazardous chemicals are reviewed. Methods for adduct detection are also reviewed and approaches to sensitivity and specificity are identified. The selection of these 12 chemicals from the Environmental Protection Agency list of genotoxic chemicals was based on the availability of information and on the availability of populations potentially suitable for molecular epidemiological study. The 12 chemicals include ethylene oxide, styrene, vinyl chloride, epichlorohydrin, propylene oxide, 4,4'-methylenebis-2-chloroaniline, benzidine, benzidine dyes (Direct Blue 6, Direct Black 38 and Direct Brown 95), acrylonitrile and benzyl chloride. While some of these chemicals (styrene and benzyl chloride, possibly Direct Blue 6) give rise to unique DNA adducts, others do not. Potentially confounding factors include mixed exposures in the work place, as well the formation of common DNA adducts. Additional research needs are identified.     

Developmental neurotoxicity of industrial chemicals

"A Silent Pandemic : Industrial Chemicals Are Impairing the Brain Development of Children Worldwide" Fetal and early childhood exposures to industrial chemicals in the environment can damage the developing brain and can lead to neurodevelopmental disorders (NDDs)--autism, attention deficit disorder (ADHD), and mental retardation. In a new review study, published in The Lancet, Philip Grandjean and Philip Landrigan from the Harvard School of Public Health systematically examined publicly available data on chemical toxicity in order to identify the industrial chemicals that are the most likely to damage the developing brain. The researchers found that 202 industrial chemicals have the capacity to damage the human brain, and they conclude that chemical pollution may have harmed the brains of millions of children worldwide. The authors conclude further that the toxic effects of industrial chemicals on children have generally been overlooked. In North Amercia, the commission for environmental cooperation, and in European Union the DEVNERTOX projects had reached to the same conclusions. We analyse this review and discuss these rather pessimistic conclusions.     

The distribution of glutathione in the rat liver lobule.

A quantitative cytochemical method was developed for measuring the GSH (reduced glutathione) content of hepatocytes in different regions of the rat liver lobule. Use of this method enabled us to show that GSH is not evenly distributed within the rat liver lobule. The hepatocytes located within 100 micrometer of the central vein contain much less GSH than do those in other regions of the rat liver lobule. We suggest that this partially explains the peculiar susceptibility of these cells to electrophilic attack by toxic metabolites formed via the microsomal cytochrome P-450 system.     

Curcumin-glutathione interactions and the role of human glutathione S-transferase P1-1

Curcumin (diferuloylmethane), a yellow pigment of turmeric with antioxidant properties has been shown to be a cancer preventative in animal studies. It contains two electrophilic alpha, beta-unsaturated carbonyl groups, which can react with nucleophilic compounds such as glutathione (GSH), but formation of the GSH-curcumin conjugates has not previously been demonstrated. In the present studies, we investigated the reactions of curcumin with GSH and the effect of recombinant human glutathione S-transferase(GST)P1-1 on reaction kinetics. Glutathionylated products of curcumin identified by FAB-MS and MALDI-MS included mono- and di-glutathionyl-adducts of curcumin as well as cyclic rearrangement products of GSH adducts of feruloylmethylketone (FMK) and feruloylaldehyde (FAL). The presence of GSTP1-1 significantly accelerated the initial rate of GSH-mediated consumption of curcumin in 10 mM potassium phosphate, pH 7.0, and 1 mM GSH. GSTP1-1 kinetics determined using HPLC indicated substrate inhibition (apparent K(m) for curcumin of 25+/-11 microM, and apparent K(i) for curcumin of 8+/-3 microM). GSTP1-1 was also shown to catalyze the reverse reaction leading to the formation of curcumin from GSH adducts of FMK and FAL.     

Sulfhydryl compounds inhibit the cyto- and geno-toxicity of o-phenylphenol metabolites in CHO-K1 cells

The effects of cysteine and reduced glutathione (GSH) on the genotoxicity of o-phenylphenol (OPP) and its metabolites, phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), were examined using the frequency of sister-chromatid exchanges (SCEs) and chromosome aberrations in CHO-K1 cells as parameters. Cytotoxic (cell-progression delay) and cytogenetic effects induced by a 3-h treatment with OPP, PHQ (100 micrograms/ml) or PBQ (50 micrograms/ml) with S9 mix after a 27-h expression time were inhibited by cysteine or GSH (3-10 mM). Materials corresponding to the cysteine or GSH adducts were found by HPLC in each incubation mixture. In the culture without S9 mix, PHQ and PBQ showed severe cytotoxicity since no metaphases could be obtained at doses over 25 and 5 micrograms/ml, respectively, and the sulfhydryl compounds inhibited the toxicity by the formation of adducts with PBQ and by inhibiting the formation of PBQ in the case of PHQ. With PHQ, the sulfhydryl compounds appeared to inhibit autooxidation. However, the sulfhydryl compounds did not inhibit the cytotoxic and cytogenetic effects caused by OPP in the cell mixture without S9 mix, but on the contrary intensified them. No adduct formation was detected in the incubation solution. On the basis of these results, it is considered that electrophilic quinone (PBQ) and/or semiquinone (phenylsemiquinone, PSQ) radicals, capable of binding to nucleophilic small molecules (such as cysteine and GSH) or (biological) macromolecules, are produced from metabolite PHQ in metabolic oxidation of OPP, and induce cyto- and geno-toxic effects in the cells. The cyto- and geno-toxic effects of OPP itself to the cells are clearly independent of any electrophilic radical reaction.     

Assessing safety of agricultural chemicals

The objective of occupational health and safety programs in the chemical industry is to protect human health and the environment. Through studies in laboratory animals, bacteria, insects, and cultured cells, toxicologists collect data which allow them to predict which chemicals are likely to be hazardous to humans. The effects of acute (single high level exposures), subchronic (up to 6 months duration to sublethal levels), and chronic (lifetime to low level) exposures are studied. Toxicity involving all organ systems is evaluated. Specific end points such as skin sensitization, birth defects, mutations, accumulation in tissues, carcinogenesis, and other toxic effects are evaluated. The data collected are used to provide information for forming guidelines regarding the safe manufacture, handling, use, and disposal of agricultural and other chemicals. The results of toxicology studies are also used to support the registration of products with government agencies in Canada and other countries.     

Use of proteomics to demonstrate a hierarchical oxidative stress response to diesel exhaust particle chemicals in a macrophage cell line

Epidemiological studies demonstrate an association between short term exposure to ambient particulate matter (PM) and cardiorespiratory morbidity and mortality. Although the biological mechanisms of these adverse effects are unknown, emerging data suggest a key role for oxidative stress. Ambient PM and diesel exhaust particles (DEP) contain redox cycling organic chemicals that induce pro-oxidative and pro-inflammatory effects in the lung. These responses are suppressed by N-acetylcysteine (NAC), which directly complexes to electrophilic DEP chemicals and exert additional antioxidant effects at the cellular level. A proteomics approach was used to study DEP-induced responses in the macrophage cell line, RAW 264.7. We demonstrate that in the dose range 10-100 microg/ml, organic DEP extracts induce a progressive decline in the cellular GSH/GSSG ratio, in parallel with a linear increase in newly expressed proteins on the two-dimensional gel. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electrospray ionization-liquid chromatography/mass spectrometry/mass spectrometry analysis, 32 newly induced/NAC-suppressed proteins were identified. These include antioxidant enzymes (e.g. heme oxygenase-1 and catalase), pro-inflammatory components (e.g. p38MAPK and Rel A), and products of intermediary metabolism that are regulated by oxidative stress. Heme oxygenase-1 was induced at low extract dose and with minimal decline in the GSH/GSSG ratio, whereas MAP kinase activation required a higher chemical dose and incremental levels of oxidative stress. Moreover, at extract doses >50 microg/ml, there is a steep decline in cellular viability. These data suggest that DEP induce a hierarchical oxidative stress response in which some of these proteins may serve as markers for oxidative stress during PM exposures.     

Exposure-dependent accumulation of N-(2-hydroxypropyl)valine in hemoglobin of F344 rats exposed to propylene oxide by the inhalation route

The detection of hemoglobin adducts by mass spectrometry is a very sensitive and specific measurement of the extent of covalent binding of electrophilic chemicals. The exposure-dependent accumulation of N-(2-hydroxypropyl)valine (N-HPVal) in globin of rats exposed to propylene oxide (PO) (0, 5, 25, 50, 300 or 500 ppm) by the inhalation route was measured to assess the utility of Hb adducts as biomarkers of exposure. Analysis of N-HPVal by gas-chromatography tandem mass spectrometry showed a linear exposure-dependent response for adduct accumulation in globin of rats exposed to PO for 3 days (6 h/day). After 20 days of exposure (6 h/day; 5 days/week), the exposure-response curve was slightly sub-linear. DNA adducts had been measured in several organs of the same animals in a companion study. The dose-response for accumulation of DNA adducts was similar to that obtained for Hb adducts. However, the number of DNA adducts varied by 17-fold between different tissues. The highest number of DNA adducts was found in respiratory nasal tissue, followed by lung and then liver. These data demonstrate that hemoglobin adducts provide a sensitive dosimeter for systemic exposure, but cannot be used to predict the extent of DNA binding in individual tissues. Furthermore, the exposure-response curve for both hemoglobin and DNA adduct accumulation does not reflect the tumor incidence curve for PO, providing evidence that the assessment of risk to cancer is more complex than simple biomarker measurements. When the present rat data were compared with recent N-HPVal measurements in humans, similar binding was found.     

Physical activity and lung cancer among non-smokers: a pilot molecular epidemiological study within EPIC

The association between physical activity, potential intermediate biomarkers and lung cancer risk was investigated in a study of 230 cases and 648 controls nested within the European Prospective Investigation of Cancer and Nutrition. Data on white blood cell aromatic-DNA adducts by ³²P-post-labelling and glutathione (GSH) in red blood cells were available from a subset of cases and controls. Compared with the first quartile, the fourth quartile of recreational physical activity was associated with a lower lung cancer risk (odds ratio (OR) 0.56, 95% confidence interval (CI) 0.35–0.90), higher GSH levels (+1.87 μmol GSH g^?1 haemoglobin, p = 0.04) but not with the presence of high levels of adducts (OR 1.05, 95% CI 0.38–2.86). Despite being associated with recreational physical activity, in these small-scale pilot analyses GSH levels were not associated with lung cancer risk (OR 0.95, 95% CI 0.84–1.07 per unit increase in GSH levels). Household and occupational activity was not associated with lung cancer risk or biomarker levels.     

A rapid method to screen fungi for resistance to toxic chemicals

Although many species of fungi are able to degrade highly toxic chemicals, only a few species have been evaluated for resistance to toxic effects of these chemicals. In this paper we demonstrate the successful application of a method to rapidly screen several species of fungi for toxicity to chemicals or mixtures of chemicals using pentachlorophenol (PCP) as a model toxic compound. Cellulose antibiotic assay disks were soaked in solutions containing different concentrations of PCP (5, 10, 25, 50, and 80 mg l^–1) and then placed in a triangular pattern outside the growing edge of the mycelia of eighteen species of white rot fungi. The plates were incubated and observed for development of inhibition zones (non-growth areas) around the disks. The short-term (24 h) growth of all eighteen species of fungi was inhibited by 5–10 mg-PCP l^–1, a range similar to that observed using previously reported techniques. Long-term growth studies using this screening method were not useful since PCP diffused from the disk into the agar, decreasing the applied dose.     

Biomarkers in risk assessment of asbestos exposure

Developments in the field of molecular epidemiology and toxicology have given valuable tools for early detection of impending disease or toxic condition. Morbidity due to respiratory distress, which may be due to environmental and occupational exposure, has drawn attention of researchers worldwide. Among the occupational exposure to respiratory distress factors, fibers and particles have been found to be main culprits in causing diseases like asbestosis, pleural plaques, mesotheliomas and bronchogenic carcinomas. An early detection of the magnitude of exposure or its’ effect using molecular end points is of growing importance. The early inflammatory responses like release of the inflammatory cells collected by non-invasive methods give an indication of the unwanted exposure and susceptibility to further complications. Since free radicals like O₂⁻, OH, OOH, NO, NOO, etc. are involved in the progression of asbestos-related diseases and lead to cytogenetic changes, an evaluation of antioxidant states reducing equivalents like GSH and ROS generation can be a good biomarker. The cytogenetic end points like chromosomal aberration, micronucleus formation and sister chromatid exchange give indication of genetic damage, hence they are used as effective biomarkers. New techniques like fluorimetric analysis of DNA unwinding, alkaline elution test, fluorescent in situ hybridization and comet assay are powerful tools for early detection of initiation of disease process and may help in planning strategies for minimizing morbidity related to asbestos fiber exposure. The present review article covers in detail possible biomarkers for risk assessment of morbidity due to fibers/particles in exposed population.