ASSESSING HUMAN EXPOSURES WITH BIOLOGICAL MARKERS

1. ASSESSING HUMAN EXPOSURES WITH BIOLOGICAL MARKERS Conrad D. Volz, DrPH, MPH Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh Co-Director Exposure Assessment, Center for Environmental Oncology, University of Pittsburgh Cancer Institute Scientific Director, Center for Healthy Environments and Communities Lecture 20

2. General • Biological markers represent events or changes in human biological systems as a result of exposure or disease (US NRC, 1991b). • They are classified as markers of exposure, effect, and susceptibility and are considered to represent events along a theoretical continuum from causal exposure to resulting health outcome (US NRC, 1987; Schulte, 1989). See Hatch papers for theoretical description. Major Text: WHO Exposure Assessment

3. General • Biological markers represent a means to monitor environmental exposure by characterizing an individual's total dose of a contaminant from all sources of exposure. Remember that dose is mass of contaminant over some time. • The main advantage of this strategy is in evaluation of an individual's total exposure using a measure which integrates over all exposure sources and is influenced by human behavior. • Biological markers are believed to be more predictive of health effects than external measures of exposure. • Biological markers address important exposure assessment needs: * characterizing an individual's or a population's exposure * generating population distributions of dose * identifying the environmental and demographic determinants of exposure.

4. Disadvantages of Biological Monitoring • The main disadvantage of biological markers is the difficulty in characterizing the individual sources which contribute to the subject's total exposure. • When developing and utilizing biological markers, understanding the toxicokinetics of the contaminant in the system is crucial to characterize the biological variability and to determine whether the biological marker is valid for exposure assessment purposes at the concentration of interest. • There is an ethical concern in relying on biomonitoring –especially in known occupational exposure assessment.

5. What is a Biological Marker of Exposure? A biological marker of exposure is defined as a xenobiotic substance or its metabolite(s) or the product of an interaction between a xenobiotic agent and some target molecule(s) or cell(s) that is measured within a compartment of an organism (US NRC, 1989; IPCS, 1993)-after exposure to the xenobiotic contaminant or a physical exposure, such as ionizing radiation.

6. Biological Markers of Effect and Susceptibility • Biological markers of effect are measurable biochemical, physiological, behavioral or other alterations within an organism that, depending upon the magnitude, can be recognized as associated with an established or possible health impairment or disease (IPCS, 1993). • Biological markers of susceptibility are indicators of inherent or acquired abilities of an organism to respond to the challenge of exposure to a specific xenobiotic substance (IPCS, 1993). • Example-Different genotypes of BCHE-K, PON-192, and PON-55 may be related to the severity of adverse health effects of organophosphorus pesticide exposure. (Carboxylic esterase and its associations with long-term effects of organophosphorus pesticides. Biomed Environ Sci. 2007 Aug;20(4):284-90. )

7. Situations which are bestsuited for biological monitoring. Ideally, a biological marker of exposure should be: 1. Chemical-specific. 2. Detectable in trace quantities. 3. Available by non-invasive techniques. • Inexpensive to assay. • Relate consistently and quantitatively to the extent of exposure and ideally also integrate the exposure over time (Bond et al., 1992). Currently there are very few biological markers that possess all these characteristics.

8. Sampling of Blood • Blood is frequently used for biological monitoring, especially in clinical settings such as occupational medicine. • Blood can integrate all sources of exposure, including internal sources, and provide an indication of current internal dose. • Since blood transports all agents throughout the organism, it represents an opportunity to sample all types of contaminants, such as gases, solvents, metals and fat-soluble compounds.

9. Components of Blood Available for Sampling * Whole blood consists of all the blood components and is preferable when the distribution of the analyte between plasma and cellular elements is unknown (Que Hee, 1993). * Red blood cells make up a large portion of blood and their primary role is to transport oxygen via hemoglobin throughout the body. Mature red blood cells contain no nucleus and therefore no DNA, and have a 120-day lifetime. Chemicals that interact with hemoglobin, such as carbon monoxide, are found in red blood cells.

10. Components of Blood Available for Sampling Continued • Numerous types of white blood cells are present in blood. These cells have a half-life ranging from 18-20 days to decades (Carrano & Natarajan, 1988). • White blood cells are circulating cells that have • DNA can itself be altered or its expression can be changed as a result of exposure to a genotoxic agent, so white blood cell DNA may be used for biomarkers of exposure to genotoxic agents (Carrano & Natarajan, 1988; Kelsey, 1990). Interpretation of genotoxic response is complicated because DNA damage can result in either cell death or removal of the marker by DNA repair, or may alter cell functions (Perera, 1987). Regardless of this, correlations have been seen between environmental exposures and DNA adducts. • A DNA adduct is an abnormal section of DNA which is bonded to a contaminant. DNA adducts are used as biological markers of exposure. Acetaldehyde DNA adducts are common in cigarette smokers.

11. Components of Blood Available for Sampling Continued Plasma and serum represent the non-cellular component of blood. • Plasma is a straw-coloured aqueous solution of electrolytes, non-electrolytes and macromolecules (including clotting factors). • Serum is plasma without the clotting factors (Que Hee, 1993). • Plasma represents a component of whole blood (approximately 60%), and it may contain the most biologically active fraction of blood borne contaminants, since plasma is in more immediate contact with tissues (Silbergeld, 1993). Plasma can be used for analysis of lipophilic chemicals, thereby avoiding the need for fat sampling.

12. Components of Blood Available for Sampling • Blood proteins can be sensitive monitoring tools for chemicals that bind to macromolecules including DNA (Osterman-Golkar et al., 1976; Bond et al., 1992). • Protein adducts, unlike DNA adducts, are not repaired and may prove to be a useful dosimeter of mutagen exposure (Grassman & Haas, 1993; Que Hee, 1993). • Hemoglobin and albumin are two proteins available for use in exposure assessment. Hemoglobin is located in red blood cells in high concentration and has the half-life of red blood cells (120days); albumin is present in serum and has a half-life of 21 days. • Because of their differing biological half-lives, these proteins can be used to investigate the timing of exposure.

13. Sampling of Urine • The concentrations of compounds found in urine usually reflect time-weighted averages in plasma during collection and storage in the bladder (Que Hee, 1993). • The presence of a contaminant or its metabolite in urine generally represents recent exposure, though in some cases it may represent release from storage within the body (Lauwerys, 1983). (Release of lipopylic chemicals from adipose tissue during weight reduction is an example of release from body storage.) • Urine can be analysed for metabolites of organic chemicals (e.g., benzene and styrene), metals (e.g., arsenic and mercury) and pesticides as well as for mutagenic potential (Lauwerys, 1983; Baselt, 1988; Que Hee, 1993). • Since collection of urine samples is non-invasive, some investigators feel that, when validated, urine may be a better sampling medium than blood for monitoring (Smith & Suk, 1994).

14. Types of Urine Sampling, Advantages and Disadvantages * Spot urine samples are relatively easy to collect but there may be significant variability with respect to exposure prediction as a result of metabolism, liquid consumption and kidney function. * First morning void samples have less variability since they are more concentrated than spot samples, but require motivated subjects to collect the samples. * Twenty-four hour urine samples control much of the intraindividual variability but require highly motivated subjects in order to collect useful samples (Baselt, 1988). • To make the results of urine monitoring comparable between individuals, analytical results are frequently standardized to creatinine concentration or specific weight. Standardization reduces some of the variability of body size and urinary output (Lauwerys).

15. Example Atrazine Metabolite from UrineChem Res Toxicol. 1993 Jan-Feb;6(1):107-16. • Enzyme-linked immunosorbent assays (ELISAs) are reported useful for the detection of atrazine and its principle metabolite in human urine. The ELISAs can be used with crude urine or following extraction and partial purification. • GC, MS, and HPLC techniques can be used to confirm and complement the ELISA methods for qualitative and quantitative detection of urinary metabolites. • A series of samples from workers applying this herbicide confirmed a mercapturic acid conjugate of atrazine as a major urinary metabolite. The mercapturate was found in concentrations at least 10 times that of any of the N-dealkylated products or the parent compound. • Atrazine mercapturic acid was isolated from urine using affinity extraction based upon a polyclonal antibody for hydroxy-s-triazines and yielded products sufficiently pure for structure confirmation by MS/MS. • In a pilot study monitoring applicators, a relationship between cumulative dermal and inhalation exposure and total amount of atrazine equivalents excreted over a 10-day period was observed. On the basis of these data, we propose that an ELISA for the mercapturate of atrazine could be developed as a useful marker of exposure. • Lucas AD, Jones AD, Goodrow MH, Saiz SG, Blewett C, Seiber JN, Hammock BD., Department of Entomology, University of California, Davis 95616.

16. Exhaled Breath • Breath analysis is useful for assessing recent exposure to gases (e.g., carbon monoxide) and organic vapors and solvents (e.g., acetone and toluene).

17. Types of Exhaled Breath Samples • * Exhaled breath can be a mixture of inhaled and exhaled air. If the exhaled biological marker is not present in inhaled air, then exhaled breath analysis is an effective means to measure internal exposure. For example, when alcohol has an internal source only (i.e., ingestion) a mixed breath sample is appropriate. • * Alveolar air provides a measure of the air that is in equilibrium with the blood in the deep lung (Bond et al., 1992). For analytes present in inhaled air, it is necessary to collect an alveolar air sample.

18. Saliva Sampling Glands at four locations in the mouth produce saliva; the secretion rate varies at each location. Chemicals enter saliva via passive diffusion from plasma. Therefore, saliva may become a useful tool to non-invasively characterize plasma levels of contaminants (Silbergeld, 1993). Social science research has used saliva sampling because of its ease of collection and storage (Dabbs, 1991, 1993). Contaminants found in saliva include cotinine, drugs, metals, organic solvents, pesticides and steroid hormones (Tomita & Nishimura, 1982; Nigg & Wade, 1992; Silbergeld, 1993).

19. Sampling Keratinized Tissues (hair and nails) • Keratinized tissues, primarily hair and toenails, are practical sampling media for evaluation of past exposure to metals (Bencko et al., 1986; Bencko, 1991; Subramanian, 1991; Kemper, 1993, Bencko, 1995). • Toenails are usually the medium of choice because these media: - integrate exposures over a period of months. -contain relatively larger concentrations of trace elements than blood or urine and -are easy to collect, store and transport (Garland et al., 1993; Kemper, 1993).

20. Sampling of Hair • Hair can be used to study exposure to environmental tobacco smoke (ETS). • The German Environmental Survey (Krause et al., 1992) it was concluded that in large population studies nicotine and continine in urine as well as nicotine in hair are useful indicators of exposure for different levels of active and passive smoking. • Continine and nicotine concentrations in hair have also been used to study fetal exposure by maternal smoking (Klein et al., 1993). • Hair has also successfully been used in studies evaluating exposure to organic mercury (Suzuki et al., 1989) or PCB (Que Hee, 1993). • Hair grows approximately 1 cm/30 days (Que Hee, 1993) and can be evaluated along the shaft to provide a profile of exposure over time. Since growth rates of hair differ based on body location, standardization of sampling location is crucial.

21. Ossified TissueSampling-Teeth and Bones • Teeth constitute a unique medium for assessment of past exposure. Depending on the tooth type and part of the tooth, one can reconstruct early childhood exposures to bone-seeking elements, such as lead (Rabinowitz MB, Leviton A, & Bellinger DC (1989) Blood lead – tooth lead relationship among Boston children. Bull Environ Contam Toxicol,43: 485-492.). • Electron Paramagnetic Resonance (EPR) tooth dosimetry has been used to validate dose models of acute and chronic radiation exposure. (Retrospective assessment of radiation exposure using biological dosimetry: chromosome painting, electron paramagnetic resonance and the glycophorin a mutation assay. Kleinerman RA, Romanyukha AA, Schauer DA, Tucker JD. Radiat Res. 2006 Jul;166(1 Pt 2):287-302. )

22. Bone Sampling • Bone represents both past exposure to bone-seeking elements and is a source for future internal exposure to these elements. The concentrations of elements in bone represent long-term exposure and storage of contaminants. For example, the half-life of lead in bone is approximately 10-40 years (Rabinowitz, 1991). • Although numerous elements can be detected in bone tissue using destructive analyses such as atomic absorption spectroscopy (AAS), in vivo measurement of environmental contaminants in bone has been limited to lead (e.g., Somervaille et al., 1988; Hoppin et al., 1995). • Lead concentration in bone can be analyzed non-invasively using a technique known as X-ray fluorescence (XRF) (Hu et al., 1995). • Epidemiological studies have established that bone-seeking a-particle-emitting radionuclides are effective sarcomagenic agents, increasing tumor incidence by up to 1000-fold in exposed individuals (H. S. Martland and R. E. Humphries, Osteogenic sarcoma in dial painters using luminous paint. Arch. Pathol. 7, 406 (1929) andC. Mays and H. Spiess, Bone sarcomas in patients given 224Ra. In Radiation Carcinogenesis: Epidemiology and Biological Significance (J. B. Fraumeni, Ed.), pp 241–252. Raven Press, New York, 1982.)

23. Breast Milk Sampling • Breast milk sampling represents a non-invasive method to estimate body burden of contaminants in adipose tissue. The correlation between contaminant concentrations in the lipid phase of milk and adipose tissue is good (Sim & McNeil, 1992). • Environmental studies have used breast milk to evaluate past exposure to lipophilic chemicals (e.g., pesticides and PCBs) and metals (WHO, 1996b) and to examine potential exposures for breast-feeding infants (Niessen et al., 1984; Davies & Mes, 1987; Sikorski et al., 1990; Sim & McNeil, 1992). • Organic chemicals found in breast milk have high lipid solubility, resistance to physical degradation or biological metabolism and slow or absent excretion rates (Rogan et al., 1980). Breast milk represents a major route of excretion of lipophilic chemicals for lactating women (Rogan et al., 1980; Sim & McNeil, 1992). • Concentrations of chemicals in breast milk are a function of parity, age, body mass, time of sampling, nutritional status, lactation period and fat content of milk (Rogan et al., 1986; Sim & McNeil, 1992). Breast milk results are generally standardized to milk fat levels.

24. Sampling Adipose Tissue • Exposure assessment studies using adipose tissue have been limited primarily to ecological studies comparing fat from cadavers or surgical specimens to general pollution levels. • Adipose tissue represents a long-term reservoir of lipophilic compounds that the body slowly metabolizes and may release into the bloodstream. • Unfortunately there is no non-invasive manner to sample fat stores directly, and many subjects see fat sampling as exceedingly invasive. • WHO Human Exposure Assessment

25. Sampling Feces • Most often used for bacteriological exposure sampling. • Feces are a highly fat-soluble medium that provides information on compounds of high-molecular weight that exit the body via biliary excretion (metabolism by liver and excretion via bile) and on unabsorbed chemicals that enter the body via ingestion. Main Text for Biomonitoring Material Extraction-WHO Exposure Assessment