Anal. Chem. 2001, 73, 2745-2760
Biological Monitoring: Exquisite Research Probes, Risk Assessment, and Routine Exposure Measurement William M. Draper
Sanitation and Radiation Laboratory, California Department of Health Services, 2151 Berkeley Way, Berkeley, California 94704 Review Contents Reviews and General Articles Organic Compounds Volatile Organic Compounds Industrial Chemicals Agrochemicals Incomplete Combustion Products Naturally Occurring Toxins and Agents Metals and Inorganic Compounds Genetic Polymorphisms Effect and Disease Biomarkers and Epidemiologic Studies Organic Compounds Metals and Inorganic Compounds Conclusions Literature Cited
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In the previous Analytical Chemistry industrial hygiene application review (A1), the entire field was surveyed with coverage including instruments, sensors, radiation dosimetry, sorbents/ filter media, and quality assurance. Consistent with a change of editorial philosophy and other considerations, this year’s 2-year industrial hygiene review is much more limited in scope, focusing only on advancements in biological monitoring. At this time, biological monitoring plays at most a supporting or complementary role in industrial hygiene practice in the United Statessin total there are only ∼50 compounds with biological exposure indexes (BEI) (A2) compared to ∼700 with TLVs. Most of the industrial hygiene standard methods utilize mature techniques. Biological monitoring, in contrast, is developing rapidly, so much so that it is difficult to predict the state of the technology even 5 years in the future. Two years from now, however, we can predict with confidence that there will have been many significant developments in the identification and application of new biological markers, as well as a better appreciation for complexities in their use and interpretation. There have been significant innovations in instrumental analysis over the past decade, especially at the fringes of biology. Many of these advancements are directly applicable in the determination of biological markers. Decades of research on toxic substances, much of it focused on the development of new pharmaceuticals, agrochemicals, and food additives, but also basic scientific research, have broadened our understanding of chemical toxicology, metabolism, pharmacokinetics, and mechanisms at the molecular level. 10.1021/ac010394s CCC: $20.00 Published on Web 05/02/2001
© 2001 American Chemical Society
Aside from a growing importance in industrial hygiene and occupational medicine, biological monitoring warrants review because of its importance in epidemiologic research. Biological markers applied in the fields of occupational and environmental epidemiology greatly illuminate our understanding of the effects and health risks of chemical exposure. Without studying biological markers in humans, our knowledge of toxicology is limited to studies in experimental animals, and interspecies differences are sometimes substantial. This review is an outline of recent trends in published biological monitoring research. Much of this research suggests that the more conventional approaches used in industrial hygiene (e.g., air monitoring, passive monitoring, or personal breathing zone monitoring) in many cases provide adequate worker protection, are simpler to implement, and avoid some privacy and ethics concerns. Biological markers are recommended for routine use in industrial hygiene investigations because they are able to detect and quantify dermal exposures. Moreover, they detect exposures incurred simultaneously from multiple exposure routes and quantify aggregate exposure. Biological monitoring also is powerful in its ability to detect subtle effects and disease processes, an important feature in biomedical research. An excellent primer on the use of biological monitoring methods in industrial hygiene practice is provided by Teass and co-workers (A3). As biological monitoring techniques or probes are developed and applied, we appreciate more and more the complications in their interpretationsthis is an expected outcome of advancements in our understanding of chemical toxicology. Individual human differences (e.g., genotypic/phenotypic variation or polymorphism) are tremendousschemicals undergo differential absorption, distribution, metabolism, and elimination. Consider the variation in response to a single chemical administered directly to the bloodstream in an inbred strain of laboratory animal, each of approximately identical weight, age, dietary status, “lifestyle”, etc. Any biological response or end point, such as acute lethal dose (LD50), varies over a substantial range from individual to individual organism. A section of this review discusses research on genetic polymorphisms because of the importance of this phenomenon to both interpretation and application of biomarkers. Biological markers must be specific to the chemical of interest. Aitio (A4) outlines three types of biomarker specificity: analytical specificity (well known to analytical chemists and pertaining to the ability of the method to respond specifically to the analyte in Analytical Chemistry, Vol. 73, No. 12, June 15, 2001 2745
a complex matrix); metabolic specificity (the extent to which the marker is the product of the chemical of interest); and source specificity (the extent to which the sources of exposure can be attributed to the workplace, lifestyle factors, diet, ecological exposures, etc). Aggressive research and development efforts have produced analytical instruments with remarkable specificity, notably mass spectrometers, high-resolution mass spectrometers, tandem mass spectrometers, and even commercially available MS10 mass spectrometers! Through the use of powerful techniques including isotope dilution mass spectrometry, parts-per-trillion (ppt) detection and quantitation of foreign substances is routinely achievable in biological tissues and fluids. The limits are defined only by the creativity and skill of the chemistsand sometimes the size of the laboratory budget. Absolute accuracy and precision are sought after, but they are not sufficient. If the biomarker is common to the metabolism of other chemicals (especially a common environmental chemical or dietary ingredient), then the biological marker may not be useful. Information on this type of “confounding” is critical for interpretation of biomonitoring data. Examples noted in the following pages include the occurrence of 2-thiothiazolidine-4carboxylic acid (TTCA), a carbon disulfide metabolite, in vegetables and the finding that trans,trans-muconic acid, a specific benzene marker for “ecological” exposures, is a metabolite of ascorbic acid. Abstinence from certain foods can limit interference in certain cases. The appeal of biological markers in epidemiologic research is that they provide an authoritative and unbiased means of sorting subjects into exposure groups. The “old-fashioned” approach of using questionnaires for classification is often the only one available, but self-reported information is recognized to be unreliable. Misclassification of exposures is a fundamental problem and frequent occurrence in environmental epidemiology. Aitio predicts that biological monitoring can only survive as a routine part of occupational medicine if techniques are available “that cost next to nothing per analysis” (A4). This means that techniques that are not laboratory-based are going to be more and more importantstest kits, dipsticks, indicators, badges, portable devices, probes, monitors, indicating devices, and realtime monitors are needed so that cost is reduced, and information is available to the worker in time to avoid hazardous chemical exposures. This review article summarizes research appearing in the literature during 1999 and 2000. The majority of this information has been gleaned from Chemical Abstracts. The review is arranged in five sections: (1) general articles and reviews, (2) exposure biomarkers for organic compounds, (3) biomarkers of metals and inorganic substances, (4) genetic polymorphisms, and (5) effect and disease biomonitoring. In total ∼700 abstracts from the time period were examined and ∼150 are discussed because of their novel focus, technical innovation, and significance or general interest. Owing to the large number of important research papers appearing, however, no claim is made that the coverage is comprehensive. Hopefully, the selection will provide readers with a feeling for current trends and future prospects in this active research field. 2746
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REVIEWS AND GENERAL ARTICLES Biological monitoring in occupational medicine was the subject of two reviews, one by Rudiger (B1) and a second by Aitio (A4). Rudiger cites 47 references and touches on sources of biological material, quality assessment in the laboratory, reference values, and other matters. Aitio looked to the future in his end-of-themillennium perspective and considered the entire array of biological markers, especially exposure, effect, and susceptibility markers in future occupational health programs. Aitio and Kallio evaluated practical applications of exposure and effect monitoring (B2). Some effect markers are in routine use in occupational medicine (e.g., renal effects and Cd, hemoglobin synthesis and Pb, and acetylcholinesterase inhibition and organophosphorus (OP) pesticide exposure). Yet, in the case of Cd and Pb, effect markers offer little or no advantage over metal analysis in biological samples. Correlational studies are often a cornerstone in developing biological markers and establish the relationship between the exposure concentration and the biological marker in the biological sample over time. The U.S. Environmental Protection Agency (USEPA) evaluated a commercially available, 24-h personal breathing zone sampler effective for collecting small particles and semivolatiles (B3). With this apparatus, 16 PAHs have recoveries ranging from 94 to 108%, and particles of 100 000 people to chronic, high arsenic levels. Aside from the need to reduce exposure in these populations, epidemiologists are interested in studying them to better understand arsenic’s human health effects. Gebel notes deficiencies in our understanding of arsenic’s tumorigenicity, ethnic and racial differences in susceptibility, and interactions with other water contaminants (e.g., antimony) and nutritional factors such as selenium and zinc. Further epidemiologic studies bolstered with biological monitoring data and chemical speciation are needed to improve our understanding of arsenic’s health effects. Kortenkamp (B18) summarized the current status and future prospects for Cr(VI) biomonitoring. His review encompassed a conceptual framework for carcinogen biomonitoring, biological markers of Cr(VI) internal dose and effect, and cytogenetic surveillance. Because of chrome’s toxicological profile inhalation toxicokinetics, information on deposition in lung tissue, and upper respiratory tract (URT) effects monitoring was emphasized. Blood lead, principally red blood cell (RBC) lead, is the most widely used measure of lead absorbed dose and body burden. Sakai provides a useful overview of the array of exposure, effect, and susceptibility markers that are available for use in studying human lead exposure (B19). Administration of chelating agents reveals the mobilizable pool that is present in blood and soft tissues, not bone. Multiple effect markers are discussed, including those that probe heme biosynthesis and activities of nucleotide metabolizing enzymes. The influence of genetic polymorphisms is also discussed. A useful review on health risks of platinum emissions from automobile exhaust catalysts was published (B20). The report describes characteristics of emissions, allergic responses, and biological monitoring pertaining to Pt. Epidemiologists have devised means to reconstruct past exposure based on job classification, years of employment, and historical work practices. Brooks presents a conceptual framework for use of biological markers to characterize past exposures to radiation (B21). The title of the article indicates that exposure, sensitivity, and disease markers may be relevant. Even when there is an established causal relationship between exposure to an agent and disease, however, disease markers are of limited use in estimating exposure because multiple factors are involved in etiology. Endocrine disrupting chemicals are of increasing interest, in part, due to observations of effects in wildlife. Moline and co-workers discuss research methods for studying chemical effects on male reproductive health (B22). The authors consider evidence of effects (e.g., testicular cancer, hypospadis and cryptorchidism, Analytical Chemistry, Vol. 73, No. 12, June 15, 2001
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semen quality) and current research methods. They note a need for both relevant exposure and effect markers related to male reproductive health. Reproductive health in women was the subject of a review by Kesner and co-workers (B23). In particular, they report development of specific and sensitive, time-resolved fluorescence immunoassays for measuring hormonal profiles in urine (e.g., lutenizing hormone (LH), follicle stimulating hormone (FSH), estrone 3-glucuronide, and pregnanediol 3-glucuronide). Practical application of these markers in field studies is discussed. Non-Hodgkin’s lymphoma (NHL) incidence has been on the rise for several decades for unknown reasons. Occupational epidemiology studies have established an association between benzene exposure and both acute myeloid leukemia and NHL. O’Conner et al. hypothesize that environmental benzene exposure originating primarily with vehicle exhaust may explain NHL incidence in the general public (B24). They note challenges in quantifying chronic, low-level human exposures. 32P Postlabeling is the sort of technique needed, but at this time, no benzeneDNA adduct has been identified. Primary liver cancer (PLC) is of multifactorial etiology with various known risk factors including hepatitis B and HIV infection. Various chemicals have been classified as human liver carcinogens by the International Agency for Research on Cancer (IARC) based on epidemiologic data and experimental animal studies. Wogan (B25) reviewed impacts of chemicals on liver cancer risk with a focus on molecular epidemiology. Lesage and Perrault (B26) review the environmental monitoring of substances causing occupational asthma, which include isocyanates, acid anhydrides, aldehydes, amines, fluxes, certain metals, and other substances. Their comprehensive overview treats relevant biological monitoring techniques as well. Stejskal and Stejskal consider the role of metal exposures in development of autoimmune disorders, particularly multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and rheumatoid arthritis (B27). Since metal-induced sensitization may result from chronic low-level metal exposure, longitudinal studies of metal-sensitive individuals may be more useful that the traditional case control studies in the future. ORGANIC COMPOUNDS Volatile Organic Compounds. Biological monitoring of volatile organic compounds (VOCs) and solvents in the workplace is of continued interest. Exposure to these compounds is pervasive and difficult to control due to volatility, the potential for both inhalation and dermal exposure, a multitude of applications in various industries (e.g., dry cleaning, degreasing applications, paints and coatings, chemical synthesis, and petroleum refining), and the volumes used. This discussion pertains to modern “clean” industries as well as the conventional onesstechnicians in semiconductor “clean rooms”, for example, are exposed to methylchloroform, toluene, benzene, acetone, trichloroethylene, and arsine. Most emissions occur in unpredictable, upset conditions or events, and therefore, biological monitoring may be more effective than air sampling for evaluating typical exposures. In this section, solvents are grouped with other types of VOCs including petroleum products (e.g., benzene and fuel oxygenates), halogenated and aromatic solvents, and related substances. 2748
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Some of the most useful solvents are also toxic and represent acute and chronic health hazards in the workplace. Cancer hazard (e.g., benzene, toluene, chloroform, trichloroethylene, and methylene chloride), toxicity to various organs and tissues (e.g., benzene/bone marrow, TCE/kidney disease, etc), particularly the liver and kidney, and reproductive toxicity (e.g., glycol ethers and acetone) are some examples. The approaches that have become most useful in VOC biomonitoring include analysis of exhaled breath, blood, and urine, and in many cases, the parent compound is determined, especially from blood. Favorable Henry’s law constants make headspace and solid-phase microextraction (SPME) useful in the analysis of biological fluids. Purge-and-trap sample introduction suffers from sample foaming and cross-contamination. VOCs are very widespread. Drinking water disinfection introduces trihalomethanes (THMs), haloacetonitriles, haloketones and haloacetic acids, chloropicrin, and other disinfection byproducts (DBPs) in low parts-per-billion quantities to drinking water, for example. Inhalation exposure in hot showers and swimming pools represent other sources of exposure. Consumer use of petroleum products leads to frequent low-level exposure to hydrocarbons such as benzene/toluene/ethylbenzene/xylene (BTEX) as well as fuel oxygenates such as methyl tert-butyl ether (MTBE). Paint thinners, dry cleaning, and use of other consumer products may also be important exposure sources. Because of their occurrence, establishing the population reference range is critical for understanding the significance of biological marker data and successful use of biological monitoring in exposure measurement (A3). The reference range is the distribution of biological marker values in the “normal” population where exposure is incurred ecologically, e.g., via food, drinking water, consumer products, polluted air, etc. Passarelli and coworkers, for example, estimated methanol reference values in the general population of Sao Paulo, Brazil (C1). Urinary methanol concentrations (determined by headspace GC-FID) were distributed log-normally and had a geometric mean of 2.1 mg/L and a reference range of 0.5-4.8 mg/L ((2 SD). Males and females were similar, and 95% of the values were below 4.8 mg/L. Consultation with a biostatistician would be of obvious importance in conducting a reference range study. Benzene exposure has been quantitated using a two-dimensional LC method for urinary trans,trans-muconic acid (t,t-MA). Limits of detection of only 4 ppt are reported using a nonspecific UV absorbance detector (C2). Anion-exchange SPE, ion chromatography, and an eight-port switching valve are utilized. Qu and co-workers used HPLC tandem MS to determine a variety of urinary benzene exposure markers including S-phenylmercapturic acid (s-PMA), t,t-MA, hydroquinone, catechol, and benzenetriol (C3). In their investigation of exposures in Chinese glue factories and shoe manufacturing, all but benzenetriol were elevated in workers. Consistent with other studies, s-PMA and t,t-MA are preferred for biological monitoring for low-level benzene exposures because the background level is much lower in controls. In an extensive review article, Dor and others recommend urine benzene and s-PMA as the optimal biomarkers for ecological benzene exposures (C4). Brugnone and co-workers examined benzene exposures in Italian gas station and refinery workers by determination of blood
benzene and personal breathing zone monitoring (C5). Group mean blood levels were 110, 132, and 99 ng/L in the general population, workers at the end of shift (EOS), and workers at the beginning of shift (BOS), respectively. Blood benzene levels were not elevated when workplace exposure concentrations were 3 times higher (exposed workers). Another technique that has gained increasing attention is headspace SPME. The technique understandably has many proponents because of its high sensitivity, minimal sample preparation, and automation potential. Schimming and co-workers determined benzene and toluene in blood with limits of quantitation of 5 and 25 ng/L (ppt), respectively. These detection limits are sufficient for determination of both occupational and ecological exposures (C6). The linear dynamic range for blood benzene determination is 5-5000 ppt. Plebani and colleagues (C7) have refined the GC/MS determination of benzene in exhaled breath using a two-step, Tedlar sampling technique that improves control over breath volume and optimizes moisture removal. Cryogenic trapping is claimed to reduce analysis time and cost and lower detection limits. The 1.5 ng/L detection limit is 1/200th of the American Conference of Governmental and Industrial Hygienists (ACGIH) BEI. Johnson and co-workers report that employees using hot wire and cool rod devices to wrap meat and other foods in plastic are exposed to benzene and PAHs (C8). Urinary t,t-MA was elevated in workers using the devices. An important observation was that some of the subjects had unexplained levels of the biomarker corresponding to 2 ppm benzene exposures. The authors suggested that a non-benzene-related source of the metabolite could account for the findingsfor a possible explanation see ref C18. Carbon disulfide exposure is quantified by analysis of urinary 2-thiothiazolidine-4-carboxylic acid (TTCA). Kivisto reported that TTCA is found in some cruciferous vegetables (e.g., 0.6-5 mg/ kg) and acts as a dietary confounder in estimating low-level CS2 exposures (C9). The upper reference limit (URL) of TTCA in the unexposed Finnish population, 0.3 mmol/mol creatinine, was established by study of 116 individuals. After a normal meal of the crucifers, the TTCA levels can exceed the URL, but do not reach a biomonitoring action level set as low as 2 mmol/mol creatinine. An improved analytic method based on columnswitching HPLC rather than the liquid-liquid extraction method currently used is more reliable for analysis at low TTCA concentrations. South Korean investigators examined the effect of exposure route and coexposure to toluene on EOS urinary N-methyformamide (NMF), a biological marker for N,N-dimethylformamide (DMF), for workers in the synthetic fiber, fiber coating, synthetic leather, and print manufacturing industries (C10). Coexposure to toluene lowers urinary NMF. The authors recommend two exposure indexes, one for those exposed by inhalation (24 mg/g creatinine) and another for workers with concurrent dermal exposure (39 ng/g creatinine). This research demonstrates the ability of biological monitoring to integrate multiple exposures, but it seems impractical to try to apply different exposure indexes. Research in Taiwan suggests that the ACGIH BEI may be too high for DMF (C11). Kuo and colleagues evaluated synthetic leather worker exposure to DMF, epichlorhydrin, and toluene.
Personal breathing zone and area air sampling revealed many instances of exceeding the DMF permissible exposure limit (37%), while urinary NMF was generally below the BEI. There are various urinary metabolites that could be used as biological markers of DMF. N-Hydroxymethyl-N-methylformamide (HMMF) is the major metabolite. Demethylation yields NMF, formamide, and hydroxymethylformamide. A conjugate, N-acetylS-(N-methylcarbamoyl)cysteine (AMCC), is excreted along with small amounts of the parent compound. Kafferlein and co-workers describe a single GC-NPD method capable of detecting three of them, HMMF, NMF, and AMCC (C12), with limits of detection ranging from 0.5 to 1 mg/L. DMF toxicokinetics are such that NMF (t1/2 ) 5.1 h) is a good predictor of exposure incurred in the just-completed shift; AMCC (t1/2 g 16 h) averages exposure over a period of days. The authors recommend AMCC for biomonitoring because of its half-life and relevance to DMF toxicity. Detailed knowledge of toxicokinetics allows the optimal development and utilization of biological markers. Nihlen and Johanson (C13) used a PBPK model to probe the behavior of biological markers for the fuel oxygenate, ethyl tert-butyl ether (ETBE). ETBE and its metabolite, tertiary-butyl alcohol (TBA), in exhaled breath, urine, and blood were evaluated. The authors simulated extremes in exercise and fluctuating exposure scenarios and considered the time course of elimination. Heavy exercise increases biomarker levels by 2- (EOS) and 3-fold (next morning). Exhaled breath or blood ETBE are highly responsive to exposure fluctuations, in contrast to urine or blood TBA, which reflect exposures incurred over a greater time interval. In considering the model predictions and practical matters, they conclude that urinary TBA is the optimal exposure marker. Jarnberg and Johanson (C14) investigated inhalation exposure to 1,2,4-trimethylbenzene (TMB), also using a PBPK model with the goal of understanding the kinetics of elimination of blood TMB (B-TMB), exhaled TMB (E-TMB), and urinary dimethyhippuric acid (U-DMHA). Moderate exercise (100 W) doubled the excretion at the EOS while the next morning U-DMHA was elevated 5-fold. EOS E-TMB was highly responsive to fluctuating exposures unlike venous blood TMB or U-DMHA. The blood half-life of ethylbenzene is very short (t1/2 ) 0.5 ( 0.1 h), too short to be of much value in biomonitoring (C15). Ethylbenzene urinary metabolites, mandelic acid (MA) and phenylglyoxylic acid (PGA), have half-lives of ∼5 h and are better suited as a biological markers. Knecht and co-workers recommend a biological tolerance value (BAT) of 2 g of ∑MA + PGA/g of creatinine at the German maximum allowable exposure concentration (MAK). Nihlen and Droz developed and validated a toxicokinetic model for inhaled methyl formate (MF) for use in developing urinary methanol and formate as exposure markers (C16). Formic acid (FA) is reabsorbed, except at high doses where the process becomes saturated. MF has a reduced background and at low MF doses urinary methanol is linear with dose. Both biological markers have merit, however, as urinary FA is a “critical indicator” of health effects. Berod and co-workers studied controlled human MF exposures in an exposure chamber (C17). A combination of monitoring devices including personal breathing zone air monitoring, FT-IR, and FID were used to determine exposure concentraAnalytical Chemistry, Vol. 73, No. 12, June 15, 2001
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Table 1. Biological Monitoring of Volatile Organic Compounds/Solvents compound: analysis, matrix, and comments benzene: t,t-MA is a urinary metabolite of sorbic acid and consumption of sorbic acid-preserved foods represents a substantial interference benzene: HPLC/UV method for prephenylmercapturic and phenylmercapturic acid (s-PMA) described benzene: heat shock proteins in plasma determined by Western dot blotting suggested as benzene exposure markers benzene: thorough review of exposure biomarkers at “ecological” levels published in Portuguese BTEX: benzene in blood and urine determined by SPME compared with urinary t,t-MA chlorobenzene: SPME GC/MS used to determine urinary p-chlorophenolsmultiple extraction factors were optimized to arrive at an 8 µg/L detection limit cyclohexane/cyclohexanol/cyclohexanone: exposure measurement for these industrial chemicals is based on urinary 1,2- and 1,4-cyclohexanediol analysis. A GC method is used following enzymatic cleavage of conjugatessmaximum excretion occurs at 0-6-h postexposure with a biological half-life of 14-18 h cyclohexane: researchers examine a suite of urinary metabolites including cyclohexane, cyclohexanol, cisand trans-1,2- and 1,4-cyclohexanediolsstime-weighted cyclohexane in air (determined using carbon cloth passive sampler) is correlated to trans-1,2-cyclohexanediol, which is responsive to exposures of
