the use of biological markers in toxicologytoxicology.usu.edu/endnote/2711737d.pdftoxicology the use...

Toxicology

The Use of Biological Markers in Toxicology

By Rogene F. Henderson, William E. Bechtold, James A. Bond, and James D. Sun

1. INTRODUCTION

There is a growing interest in biological markers of exposure to chemicals and in markers of adverse health effects from such exposures. Reflecting this interest, a new journal, Biological Monitoring, is being published by The Telford Press. The National Academy of ScienceslNational Research Coun- cil’s Board of Environmental Studies and Toxicology, at the request of the Environmental Protection Agency and the Na- tional Institute of Environmental Health Sciences, has established a Committee on Biological Markers to evaluate the scientific basis, current state of development, validation, and use of biological markers in environmental health research. *

What are biological markers, or biomarkers, as they are sometimes called? The term “biomarker” refers to the use made of a piece of information, rather than to a specific type of information. A biomarker is a change in a biological system that can be related to an exposure to, or effects from, a specific xenobiotic or type of toxic material. An ideal biomarker of an exposure is one that is chemical specific, detectable in trace quantities, available by noninvasive techniques, inexpensive to assay, and quantitatively relatable to a prior exposure regimen. An ideal marker of an effect or of a disease state is unique to the disease state in question and quantitatively relatable to the degree or stage of the disease. Few if any biomarkers will have all the above characteristics, but many nonideal biomarkers can be useful for specific purposes.

Part of the current interest in this field has been sparked by the development of new techniques, such as those that allow quantitation of adducts formed by the interaction of reactive metabolites of xenobiotics and macromolecules, with the potential for such adducts acting as markers for exposure to the xenobiotics. Interest in biomarkers has also resulted in reex- amination of many types of measurements that have been done in the past for purposes other than use as biomarkers, but have potential value as biomarkers of exposures to, or effects from, xenobiotics .

Biomarkers are needed in several fields of science. The usefulness of biomarkers in epidemiology is easily apparent, because of the need for objective means of quantitating exposures to specific materials. Industrial hygienists have used biomarkers of exposures for some time, in their analyses of urine, expired air, hair, or other body tissue samples for evidence of exposures to industrial materials. Clinicians are perhaps the original founders of the field of biomarker research, because for centuries they have relied on clinical signs to guide them in their diagnoses and therapeutic regimens. The new

interest in biomarkers for clinicians is in determining early indicators of late-developing disease processes so that appropriate therapeutic and preventative measures can be initiated to halt the progress of the disease. Finally, toxicologists have an interest in biomarkers as markers of both exposure and effects in animal studies. Toxicologists are also involved in developing new biomarkers in animal systems that can be transferred for use in human epidemiology studies.

What are the most useful biomarkers that toxicologists can develop? Biomarkers might be identified for any of a continuum of events leading from exposure to a toxic chemical to the development of disease (Figure 1). The more that is known about the mechanism of a chemically induced disease, the more specific and useful are the biomarkers that can be developed to indicate where an exposed individual is on this continuum. For example, if the exact mechanism of development of a toxin- induced disease is known, the dose to the critical site can be measured and related to the early adverse alterations known to precede development of a frank disease state. In many in- stances, the exact mechanism of disease development is not known, and one must rely on markers of exposure that indicate the total internal body dose received by an individual and relate this to early adverse changes or to the later development of a frank disease state. Thus, studies to determine the mechanisms of chemically induced disease are essential for the development of useful biomarkers .

In the past, many toxicology studies were designed to monitor only the external exposure regimen and a late-developing disease. Information from such studies may be deceiv- ing, because the external dose may not be linearly related to

Rogene F. Henderson received B.A. and B.S. degrees from the Texas Christian University in Fort Worth and a Ph.D. degree from The University of Texas in Austin. Dr. Henderson is currently Supervisor of the Chemistry and Biochemical Toxicology Group at Lovelace ITRI in Albuquerque, New Mexico. She is an Adjunct Professor in the Department of Veterinary Microbiology, Pathology, and Public Health in the School of Veterinary Medicine at Purdue University in West Lafayette, Indiana. Dr. Henderson is also a Clinical Professor in the College of Pharmacy at the University of New Mexico in Albuquerque. William E. Bechtold received B.S. and Ph.D. degrees from Emory University in Atlanta, Georgia. Dr. Bechtold is currently a chemist in the Chemistry and Biochemical Toxicology Group at Lovelace ITIU in Albuquerque, New Mexico. James A. Bond received a B.A. degree from Pomona College in Claremont, California, and a Ph.D. degree from the University of Washington in Seattle. Dr. Bond is currently Supervisor of the Molecular and Cellular Toxicology Group at Lovelace ITRI and is a Clinical Assistant Professor in the College of Pharmacy at the University of New Mexico in Albuquerque. James D. Sun received a B.S. degree from the University of California at Davis and a Ph.D. degree from the University of California at Riv- erside. Dr. Sun is currently an Inhalation Toxicologist in the Chemistry and Biochemical Toxicology Group at Lovelace ITRI in Albuquerque,

, ’ ’ New Mexico.

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[ Y L INTERNAL

fully reversible)

(irreversible)

M A R K L Z O F EXPOSURE MARKERS OF EFFECT - - FIGURE 1. Biomarkers of exposure and effect.

the internal body dose or the dose to the critical site of toxicity. High doses administered by any route may exceed the ability of the body to absorb or metabolize the chemical to toxic intermediates. Thus, much of the “dose” may be wasted or not available to induce a toxic effect. In such cases, dose- response curves will flatten out at high doses, indicating similar responses at all doses above a certain point. Linear extrapolation from effects seen at doses beyond this “saturation” point to expected effects at lower doses will underestimate the toxicity of a compound.

The ideal state of knowledge is to know the relationship between each of the steps (and substeps) in the continuum illustrated in Figure 1. Markers of any stage on this continuum can then be used to predict or provide information on events at other points in the continuum. In such a situation, contents of a blood sample that can be used as a marker of internal dose may also be used to obtain information on prior exposure and to predict potential health effects. This desirable stare of knowledge is difficult to achieve, but is most nearly approached in animal studies. Herein lies the role of the toxicologist. Once such information has been obtained in animals, extrapolation to the human situation can be attempted based on limited clinical and epidemiological data and studies with human tissues m vitro.

The major focus of this review is on biological markers of exposure. Markers of effects will be mentioned only in the cases where the observed biological changes can be interpreted as both a biomarker of exposure and a biomarker of an effect. Techniques for monitoring external exposure atmospheres are a separate topic and are not to be included in this review. The ethical aspects of the use of biomarkers in humans has been reviewed elsewhere.

II. DIRECT MEASURES OF EXPOSURE MATERIAL AND METABOLITES

Markers of internal dose include traditional analyses of body samples such as urine, blood, tissues, hair, fat, and exhaled air for chemicals or their products. New techniques have allowed more sensitive assays of these samples; detailed toxicokinetic studies in animals have provided more information on dose-response relationships; physiology-based mathematical modeling of toxicokinetic parameters measured in animal studies has allowed easier interpretation and extrapolation of these animal data to humans. These are discussed below.

A. Toxicokinetic Data in Animal Studies A large body of evidence indicates that measurement of

parent compound and metabolites in tissues, blood, and excreta of animals can serve as useful markers of exposure to inhaled chemicals. Toxicokinetic studies that determine the disposition and metabolic fate of xenobiotic compounds can define the exposure/dose relationships shown in Figure 1. Numerous studies have been published in which laboratory animals were exposed to a chemical and the concentration of the chemical and its metabolites in blood, tissue, and excreta were determined over a period of time subsequent to the exp~sure .~ - ’~ Such studies can determine the range of exposure concentrations over which metabolism and excretion of a chemical are linear. For example, studies of inhaled I4C-methyl bromide indicate that the internal dose received by rats is linear for 6-h exposures up to atmospheric concentrations of 5.7 pmol/l.‘ However, if the exposure concentration is increased to 10.4 pmol/l, the internal dose does not increase over that received at 5.7 pmoY 1, due to a decrease in the ability of the rats to absorb the inhaled gas as well as a decrease in minute volume. Thus, any risk assessment based on animal toxicity studies conducted at exposure concentrations greater than 5.7 pmoV1 would underestimate the toxic potential of methyl bromide, because the internal dose received would be overestimated.

Toxicokinetic studies with benzene indicate the importance of determining not only the disposition of the parent compound, but of the metabolites as In the cited studies, it was observed that benzene metabolism leading to putative toxic metabolites (muconaldehyde and benzoquinone) was by high- affinity, low-capacity pathways, while detoxification products (phenyl conjugates) were formed by low-affinity , high-capacity pathways. At the high doses (greater than 10 mgkg administered orally, or greater than 50 ppm for 6 h by inhalation) normally used in animal toxicity studies, a shift in metabolism toward detoxification pathways occurs. Here again, risk estimates based on results of animal toxicity studies conducted with high administered doses would underestimate the relative toxicity of benzene at the low exposure concentrations normally encountered by humans.

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Toxicology

B. Toxicokinetic Sampling in Humans Many of the above studies made use of radiolabeled sub-

strates. While the use of radiolabeled chemicals affords the opportunity for increased sensitivity , I 8 these are only useful for experimental purposes in animal studies. Such techniques cannot be transferred for use in human studies. Newer methods for detecting the presence of metabolites in blood and urine of humans exposed either to single chemicals or to chemical mixtures have been reported. For example, a report by Garner et aLi9 reviewed methodologies for detecting aflatoxin B, and its metabolites in biological material in order to better assess human exposure to this carcinogen. Several methods appear promising for the detection of parent compound and metabolites in the urine, and include both physicochemical (e.g., thin- layer chromatography, high-performance liquid chromatography [HPLC]) and antibody techniques. Table 1 summarizes the level of aflatoxin in human biological materials. For the case of analysis using immunoassays, both monoclonal and polyclonal antibodies have proven suitable for measurements of exposure to aflatoxin.19 Because of the cross-reactivity of polyclonal antibodies with several aflatoxin metabolites, the use of polyclonal antibodies provides a reasonably good estimate of exposure. Monoclonal antibodies, on the other hand, provide insight into the pattern of metabolites excreted.

A single metabolite may be used as a marker for exposure to a chemical rather than looking at the total level of metabolites excreted. For example, the American Conference of Govern- mental Industrial HygienistsZo has established biological exposure indices (BEI) for a number of chemicals. Measurement of these indices allows the industrial hygienist to assess exposure to a given chemical by measuring either the parent

Table 1 Level of Aflatoxin (AF) in Human Biological Materials

AMY technique Country of study Material

ELISA Gambia Urine

RIA HPLC Trout bioassay HPLC RIA HPLC

HPLC HPLC

HPLC HPLC-RIA

Quidong, People’s Republic of China u s . Philippines Philippines Philippines Sudan Sudan U.S. Kobe, Japan Kobe, Japan U.S.

Urine Urine Urine Urine

Urine Serum Serum Serum Serum Serum

compound, or a metabolite, in exhaled breath, blood, or urine. As shown in Table 2, single metabolites in urine can often be used as markers. The BE1 value represents the level of urinary metabolite likely to be observed in specimens collected from a healthy worker who has been exposed to the threshold limit value-time weighted average (TLV-TWA) of a chemical.

Researchers in China have found a good correlation between urinary aflatoxin M, and dietary intake of aflatoxin B, in residents of an area where there is a high liver cancer in- cidence.*’ The urinary aflatoxin M, was detected by an enzyme- linked immunosorbent assay.

BecheP has reported a new method for the determination of PAH and PAH metabolites in urine. His method was applied to urine samples from an occupationally nonexposed control group and from aluminum workers with high exposure to PAH. The method involved the reduction of oxygenated PAH metabolites to the parent hydrocarbons. The results from these studies indicated that high concentrations of PAH (52 to 268 mg/m3) in the working atmosphere of aluminum plants are not reflected to a corresponding extent in the excretion of PAH in urine. The author concluded that “nevertheless, determination of PAH and PAH metabolites in urine may serve as an indicator for the human exposures to PAH.” The author questioned the relevance of PAH air-monitoring data as a measure of the hazard associated with PAH exposure in the ambient air.

Besides urine and blood, exhaled air and aspirated fat samples have been analyzed for parent compounds and metabolites as markers of exposure in humans. In a recent document published by the Environmental Protection Agency (EPA) ,23 the use of the total exposure assessment methodology (TEAM) to study the relation between volatile organic compounds in the

No. of samples analyzed

23

48 5 23 96

61 41 4/6 Positive 34/100 Positive

1

Levels of AF’

High, 266 mg AFB, equivalent per year; low, -1 rng AFB, equivalent per year

-1 mg AFB, per year High, -3 mg/year; low, -1 mg/year Maximum, -0.05 mglyear High, -5 mg/year; low, -0.4 mglyear High, -0.02 mglyear High, -0.3 rnglyear; low, -0.07 mg/year -0.08--0.7 ng/ml -3.4-12 n g / d -0.02-1.17 nglml -2.52-4.68 n g h l -3.4 nglml

Note: ELISA = enzyme-linked imrnunosohnt assay; RIA = radioimmunoassay; HPLC = high-performance liquid chromatography, (From Gamer, C., Ryder, R., and Montesano, R., Cancer Res., 45, 922, 1985. With permission.)

a Values quoted as milligrams per year assume 1 1 of urine per person per day.

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Table 2 Urinary Metabolites Used as Biological Exposure Indices for Organic Compounds

Compound Urinary index Time of measurement BE1

Benzene Ethylbenzene

Hexane Toluene

Aniline Dimethy lfonnamide Carbon disulfide

Methylchlomform

Parathion Perchiomethylene

Phenol Mandelic acid

2,5 Hexanedione Hippuric acid

P-Amino phenol N-Methyl formamide 2-Thiothiazolidine4-car~ boxylic acid

Trichloroacetic acid Trichloroethanol P-Nitrophenol Trichloroacetic acid

End of shift End of shift and end of

work week End of shift End of shift Last 4 h of shift End of shift End of shift End of shift

End of work week End of shift, work week End of shift End of work week

50 mg/l 2 g/l 1.5 g/g Creatinine 5 g/l 2.5 g/g Creatinine 3 mg/min 50 mg/l 40 mg/g Creatinine 5 mg/g Creatinine

10 mgll 30 mg/l 0.5 mg/l I mg/l

From American Conference of Governmental Industrial Hygienists, Inc., Documentation of the threshold limit values and biological exposure indices, 5th ed., 1986, BEI-I. With permission.

breath and prior exposures was reported. The amount of 11 prevalent volatile organic compounds found in the breath of 355 New Jersey residents was found to correlate with the previous 12-h average air exposures, indicating breath measurements may be capable of providing rough estimates of preceding exposures. This same group used analysis of exhaled air to determine exposure to benzene during the filling of a gasoline tank, exposure to perchloroethylene in dry-cleaning shops, exposure to chloroform from hot water in the home, and exposure to aromatic compounds in tobacco smoke.

Aspirated fat samples have been analyzed as an index of exposure to halogenated hydrocarbons. The 2,3,7,8-tetrach- lorobenzo-p-dioxin levels in the adipose tissue of Missouri residents distinguished between persons with a history of exposure to this chemical and control (presumably unexposed) persons. 74 Andersonz5 stresses the importance of analyzing both blood sampies and adipose tissue samples at the same time to provide more information on the partitioning between these two compartments. When sufficient information of this type is available, blood samples can be used to predict fat concentrations of this compound. The National Adipose Tissue Survey of the EPA’s National Human Monitoring Programz6 is an example of the excellent use that can be made of tissue banks for retrospective studies of the influence of occupation, geo- graphical location, age, and sex on the levels of halogenated hydrocarbons in human fat.

Sputum, nasal lavage fluid, and bronchoalveolar lavage fluid (BALF) may provide an indication of prior exposures to insoluble materials. Roggli et al.27 found a higher asbestos body content per million cells in BALF from patients with asbestosis than in patients with sarcoidosis or idiopathic pul- monary fibrosis. Wood and asbestos fibers have been observed in sputum of exposed persons.” However, no one has defined

the relationship between the extent of exposure and the level of these materials in respiratory tract fluids. At present, the data only provide a “yes or no” answer as to whether or not an exposure has occurred.

C. Mathematical Modeling of Toxicokinetic Data The above discussion emphasized the types of samples that

can be measured to determine the toxicokinetics of xenobiotics in animals or humans. However, whether the data are from a well-designed animal toxicokinetic study in which numerous types of samples are analyzed, or from a human study in which only limited samples are analyzed, the information can be utilized in a mathematical model to define the relationship among exposure regimens (single, repeated, varying concentrations, or doses) and the various types of biomarkers of exposure (blood, tissue, and excreta samples).

Mathematical models are useful in many ways. First, the attempt to set up the model indicates what additional information is required from experimental studies to complete the model. Thus, the model assists experimental design. Second, the model allows one to predict what will happen under varying exposure conditions without actually performing each study. Third, if the model makes use of physiological parameters such as cardiac output, alveolar ventilation, blood flow to organs, organ volumes and body weight, and of chemical parameters such as blood/air and tissuehlood partition coefficients, the model can be used to extrapolate between species, particularly between animals and humans.

The use of animal toxicokinetic data for human risk assessment is illustrated in Figure 2. Some precautions in this approach should be mentioned. In the animal studies it is important to determine how and at what rate the chemical and its metabolites are cleared from the body. This must be determined

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USE OF ANIMAL TOXICOKINETIC DATA IN HUMAN RISK ASSESSMENT

Animals

0 Percent 0 Animal absorbed toxicokinetic

data

Mathematical Validate Model for Human

Humans Model

0 Human 0 Limited physiological human data data

Animal

data TI12 to steady state physiological

0 Major routes and rates of excretion

0 T , , , for clearance

0 Effect of - exposure concentration - exposure rate - repeated exposures - route of exposures

FIGURE 2. Use of animal toxicokinetic data in human risk assessment.

for different doses or exposure regimens to find the range of doses over which the disposition and metabolism of the compound are linearly related to dose. Such information is required for extrapolation from animal studies (normally at high doses) to the low doses normally encountered by humans. The importance of determining the effect of dose on the disposition of a chemical and its metabolites is illustrated in Figure 3, taken from data reported by Sabourin et al.” in B6C3F1 mice given benzene orally. At low doses, a much higher percent of the total benzene metabolites appears to be produced by pathways leading to putative studies done in animals given high doses to expected toxicity in humans exposed to low doses would underestimate the toxicity of benzene.

It is also important to determine the effect of repeated doses on the fate of a chemical. Repeated exposures to low concentrations is the most commonly encountered human exposure regimen, and such dosing could induce enzymatic changes affecting the toxicity of a compound. Such studies can be conducted in animals.

The biggest problem faced in using animal toxicokinetic data for human risk assessment is the potential for species differences in metabolism of the compound. Figure 4 illustrates the difference in formation of putative toxic vs. nontoxic metabolites of benzene in F344 rats and B6C3F1 mice.” The mouse is the more sensitive species and forms more of the toxic metabolites. Is man, then, like the mouse or the rat, or neither? Information obtained from cultured human cells or tissue slices may help resolve such questions.

Physiologically based modeling of the disposition of volatile chemicals has been Recent work by Med- insky et al.’“*’’ extended this approach to modeling the disposition of both the parent compound and its metabolites in rats and mice administered benzene orally or by inhalation. To

extend such models to humans, however, requires some information on human metabolism of benzene (or the compound of interest). For example, rats and mice metabolize benzene differently.’*.I5 Extension of the model of Medinsky et al. 1 6 3 ’

to humans by changing the physiological parameters from those of the rat and mouse to those known for humans necessitates also choosing the appropriate metabolic parameters for humans. If one uses either rat or mouse metabolic parameters, the extension of the model will only predict how a very large rat or a very large mouse would handle benzene. Therefore, it is essential to have enough information on the metabolism of a compound by human tissue to make a valid extension of a physiological model from animals to humans.

D. Use of Biomarkers of Exposure to a Single Chemical, Styrene

Perhaps one of the best examples of research conducted to better understand the relationship between exposure concentration and “dose” is found in studies of styrene toxicity. A series of excellent studies have been published, using laboratory animals and humans, in which styrene metabolites in tissues and excreta are quantitatively measured. A few of these studies are reviewed here.

Wieczorek and PiotrowskiZ9 exposed human volunteers by inhalation to 5 , 10, 25, and 50 ppm styrene to determine if blood levels of styrene (measure of retained dose) were linearly related to exposure concentrations. Over this range of exposure levels, retention of inhaled styrene was not affected by exposure concentration and averaged about 71%. Loef et al.30 exposed human volunteers (controls and factory workers) by inhalation to 300 mg/m3 (-70 ppm) styrene for 2 h and found the total uptake of styrene to be 63%. The styrene blood concentration was lower in subjects having previous occupational exposure to styrene than in control subjects who had not worked in a factory. Of interest in these studies was the observation that levels of styrene glycol conjugated with glucuronide were about two times higher in subjects having prior exposure to styrene than in control subjects, suggesting that preexposure to styrene enhanced the rate of conjugation.

Several papers have suggested that levels of styrene metabolites in the urine of workers exposed to styrene can be used as a dose monitor. Ohtsuji and Ikeda3’ noted that both mandelic acid and phenylglyoxylic acid levels in urine are a sensitive index of exposure. Consistent with this observation, Engstrom et al.32 measured urinary mandelic acid levels in workers exposed to 4 to 100 ppm styrene and found that a linear relationship existed between excretion of mandelic acid in urine and styrene exposure up to about 0.15 Ym3 (150 ppm).

Wolff et al.33 analyzed fat (subcutaneous needle aspiration from buttock) from humans working in a styrene polymeri- zation plant (actual exposure levels not well documented), and Engstrom et al.j4 analyzed fat from human subjects exposed

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DOSE EFFECT ON METABOLISM OF BENZENE IN THE MOUSE

loo[ LOW DOSE (lmgfkg)

8o t High Dose (200mglkg) "'c 80

METABOLITES

Pathways lor Toxic Metabolites: high atfinity. low capaclty

Pathways for Detoxilication: low allinity, high capacity

FIGURE 3. Effect of dose on metabolism of benzene in the B6C3F, mouse. Toxic and nontoxic metabolites defined in Figure 4. (From Sabourin, P. J . , Bechtold, W. E. , Griffith, W. C., Bimbaum, L. S . , Lucier. G., and Henderson, R . F., Toxicol. Appl. Pharmacol., submitted. With permission.)

to 50 ppm styrene. Both found that the decline of styrene in fat was slow.

Andersen et al.3' used gas uptake measurements in a static exposure chamber to determine styrene uptake, total in vivo metabolism, and time course of metabolic enzyme induction in rats exposed to 400, 600, or 1200 ppm styrene for 24 h. Arterial blood/inhaled air concentration ratios and physiological constants were used to calculate total styrene metabolism. Using these data from animal studies, the investigators developed a physiologically based mathematical model to describe the toxicokinetics of styrene observed in the rats and to predict the same for humans.'" The calculated styrene concentrations in blood and exhaled air of humans agreed with earlier studies by Ramsey et al.37 in which four human volunteers were exposed to 80 ppm styrene for 6 h. These elegant studies illustrate the utility of animal studies for producing mathematical models for the disposition of xenobiotics in humans - models that can be validated with limited data from human studies (Figure 2). Ideally, a similar approach could be used to develop models that predict the disposition of not only the parent compound, but of the toxicologically significant metabolites as well. Such an approach has been used in animals exposed to benzene in dynamic exposure systems with benzene and its metabolites measured in blood, tissues, and excreta.I5 The data from this study were used t o develop a mathematical model predicting the disposition of benzene and its metabolites under differing

exposure conditions. l6,l7 With additional data from accidental human exposure or from studies on human tissues in vitro, this type of model could be extended to humans.

E. Use of Biomarkers of Exposure to a Complex Chemical, Cigarette Smoke

An example of the use of markers to determine the dosimetry of a complex chemical mixture is monitoring for exposure to cigarette smoke. Indicators commonly used as markers of the degree of cigarette smoke exposure are (1) carboxyhemoglobin, (2) expired air carbon monoxide (CO), (3) thiocyanate, (4) nicotine, and (5) cotinine. Each of these is discussed in turn and their validity as indicators of cigarette smoke exposure indicated.

Carboxyhemoglobin is formed when CO diffuses from the alveolar space into the capillaries and combines with hemoglobin. CO originates from incomplete combustion of the car- bonaceous material in cigarettes and binds approximately 240 times as tightly to hemoglobin as does oxygen. Carboxyhem- oglobin formation is dependent upon the amount of smoke the individual inhales.38 In rats undergoing inhalation exposure, carboxyhemoglobin levels in whole blood are roughly propor- tional to cigarette smoke exposure. Carboxyhemoglobin has been used in humans to indicate the degree of cigarette smoke e x p o ~ u r e . ~ ~ . ~ The test is rapid and inexpensive; however, a blood sample is required, and it must be measured promptly.

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SPECIES DIFFERENCES IN METABOLISM OF BENZENE (lmg/kg)

F344/N Rat

loor

100

80

60

40

20

0 TOXIC NONTOXIC

B6C3F Mouse

TOXIC NONTOXIC

ME TA B 0 LIT E S Putatlve Toxic Metabolites: hydroquinone, muconaldehyde

Nontoxic Metabolism: Phenyl conjugates

FIGURE 4. Differences in metabolism of benzene in F344IN rat and B6C3F, mouse. (From Sabourin, P. J. , Bechtold, W. E., Griffith, W. C., Birnbaum, L. S., Lucier, G., and Henderson, R. F., Toxicol. Appl. Pharmucol., submitted. With permission.)

Moreover, exposure to other sources of CO, such as vehicle emissions or wood stoves, will also raise blood carboxyhem- ~globin.~ ' The half-time for clearance from the blood in humans is about 2 to 4 h.42

CO is inhaled with the cigarette smoke, and a portion reaches the alveolar space. The alveolar gas can be sampled as end-tidal gas. Thus, the amount of CO in end-tidal air serves as an indicator of the exposure to cigarette smoke.43 Expired air CO has the advantage that it can be determined by a noninvasive and inexpensive procedure that gives immediate feed- back as to the level of CO in end-tidal expired air. However, like carboxyhemoglobin, end-tidal CO is elevated from any source of C0.41 Moreover, CO is not present in expired air for long periods after exposure. In humans, it can barely be detected 8 h after smoking a cigarette. Finally, measurement of end-tidal CO requires some cooperation from human subjects, so that it cannot easily be obtained from small children to monitor indirect exposures to sidestream smoke.

Thiocyanate is formed from the hydrogen cyanide (HCN) in mainstream cigarette smoke. It is inhaled, and a significant portion goes to the alveoli. From the alveoli it enters the blood by diffusion. Thus, some investigators have used blood thio-

cyanate as a marker of cigarette smoke exposure, or in some cases, thiocyanate in urine or saliva.".45 Thiocyanate has a long half-life in humans (approximately 2 weeks), and it can be measured noninvasively (in urine and saliva as well as in serum).& However, one can obtain thiocyanates from a variety of sources, including Few data are available from animals concerning thiocyanate levels following exposure to cigarette smoke.

Nicotine is a component of cigarettes. It is inhaled in cigarette smoke, goes to the alveoli, enters the blood, and serves as an indicator of cigarette smoke exposure.48 It can be measured noninvasively. In humans, rats, and dogs, a significant portion of the circulating nicotine has a very short half-life (less than 30 m).49 The remaining circulating nicotine has a half-life of approximately 4 h. In humans, more than 85% of nicotine is rapidly metabolized to cotinine, which accounts for the rapid clearance of nicotine.

Cotinine has many advantages as an indicator of smoke expo~ure.~@~* It is the primary metabolite of nicotine, and appears rapidly after nicotine uptake. It has a relatively long half- life in the animal (-20 h in humans; -2 to 4 h in rodents and dogs).53 It can be measured in saliva and urine, and is little

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affected by urine pH or volume. Since it can only be produced endogenously from nicotine, it is specific for cigarette smoke exposure. It is stable and can be analyzed after years of storage. Its half-life, however, does not allow its use in measuring cumulative exposures over long periods of time.

In summary, exposure to complex mixtures can be mon- itored by biomarkers, if the mixture has a relatively stable, known composition and a component of the mixture can be chosen as representative of the total mixture. A more complex problem is monitoring the dose to the critical site. In the case of cigarette smoke, neither the critical compound(s) nor the critical molecular site of action for tumor production is known. The quantitation of DNA adducts at various points in the respiratory tract is a logical approach to determining if there is a correlation between the concentration of such adducts and the most common sites of tumor formation in the respiratory tract (see below).

111. MEASURES OF PRODUCTS OF EXPOSURE

The quantitation of exposure material or its metabolites has been well documented as a means of assessing animal and human exposures to various compounds. The utility of this technique, however, is generally limited to times shortly after exposure, primarily because clearance of many chemicals from the body is rapid (<48 h). The need to assess exposure levels at times well after the actual exposure (weeks to months) has led to studies of the use of other markers of exposure, in particular DNA adducts and protein adducts. Such markers may or may not be markers of toxic effects (see discussion below).

A. DNA Adducts as Biomarkers of Exposure Many organic compounds that enter the body are highly

reactive or can undergo biological transformation to form highly reactive, metabolic intermediates. These electrophilic compounds or their metabolites can then covalently bind to various cellular macromolecules .54 Initial research in this area focused on the hypothesis that these covalent interactions were caus- ative factors in many toxic and carcinogenic responses. In many studies measurement of these macromolecular adducts after administration of the test compound was done as an indication of metabolic activation, and this was surmized to be an indication of the toxic or carcinogenic potential of the compound. Since then, it has been proposed that the covalent binding of a chemical to cellular macromolecules does not necessarily mean a toxic or carcinogenic response will result.55 Adduct formation may occur with noncritical macromolecules and not produce a deleterious effect on the cell.

A conference on “DNA Adducts: Dosimeters to Monitor Human Exposure to Environmental Mutagens and Carcino- gens” explored the application of modern methods of molec-

ular biology to human bi~monitoring.~~ At that conference the measurement of covalent adducts (i.e., protein and nucleic acids) as dosimeters was discussed. The use of DNA adducts as dosimeters was noted to stem from the underlying mechanisms by which chemicals are metabolically activated to reactive chemicals that can form covalent boi. :; with potential nucleophiles such as protein and DNA (i.e., nucleic acids). Measurement of DNA adducts is particularly relevant as a dosimeter, since DNA adduct formation, if not repaired, can be considered to be one of the first steps in the process of the conversion of a normal cell to a neoplastic cell. Wogan and G o r e l i ~ k ~ ~ pointed out that measurements of DNA adducts in a target tissue provide information on actual genotoxic exposure. Because many DNA adducts have varying degrees of stability (e.g., spontaneous removal, enzymatic repair), excretion of DNA adducts in the urine may also represent genotoxic exposure to a potential carcinogen.

Most of the research reported is related to the quantitative assessment of DNA adduct levels in cells and tissues. These consist primarily of measurements in blood lymphocytes as well as biopsy and autopsy samples. Measurements of DNA adducts in tissues and cells at various times after exposure to a potential carcinogen provide information not only on an individual’s ability to metabolically activate the chemical in a given tissue, but also on the individual’s ability to “repair” the damaged DNA. Thus, indirectly, it is possible to determine the integrated “genotoxic” dose in a given individual. Un- fortunately, the rates of removal of damaged DNA vary from cell to cell and from tissue to tissue, making it difficult to determine the genotoxic dose of a chemical.

Most of the available information on DNA adduct formation was obtained under controlled laboratory situations in which animals were exposed by varying routes of administration and DNA adducts were measured in specific tissues. De- tection of DNA adducts has been primarily by three methods: (1) physicochemical, (2) radiochemical, and (3) immunochemical. The former two methods have typically found application following administration of high doses of a chemical. This is due to the lack of sensitivity of the methods. Furthermore, the use of radiochemical methods is basically restricted to the laboratory setting and is not applicable to the monitoring of DNA adducts in humans. One major drawback to these methods is that the high doses used are typically not relevant, and in many cases can saturate metabolic systems such that the data on DNA adducts obtained at high doses may not linearly extrapolate to lower, more environmentally realistic doses. Immunochemical methods are both sensitive and specific, and have found application in many cases. Their high sensitivity coupled with their use of nonradioactive substrates makes them a good choice for detecting adducts in humans.

There have been a few reports of studies using laboratory animals that indicate that DNA adduct levels are adequate reflections of the administered dose. In some cases, DNA ad-

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duct levels were linearly related to dose over several orders of magnitude. Phillips et al." treated mice topically with dime- thylbenzanthracene (DMBA) over a range of doses of 0.025 to 1 pmol per mouse. They found that the formation of DMBA metabolite-DNA adducts in mouse skin was not linear with adminsitered dose. However, Pereira et aL5* demonstrated that the binding of benzo( a)pyrene (BaP) metabolites to epidermal DNA was linear from 0.01 to 300 pg BaP per mouse. Adri- asenssens et al.59 investigated the binding of BaP metabolites to DNA in lung, liver, and forestomach of mice treated orally with BaP doses ranging from 2 to 1350 pmol BaP per kilogram body weight. In lung and liver, the dose-response curves for the formation of BaP metabolite-DNA adducts were sigmoidal, whereas the forestomach dose-response curve was more linear. Belinsky et aL60 recently reviewed studies conducted in their laboratory in which DNA adducts in respiratory tract issue were measured following administration of 4-(methy1nitrosamino)- 1 -(3-pyridyl)- 1 -butanone (NNK), the tobacco-specific nitro- samine (Figure 5). For these studies, rats were administered NNK for 12 d at doses of 0.3 to 100 mg/kg/d. In the lung, the dose response for the formation of 06-methylguanine (06MG) was nonlinear above 3 mg NNK per kilogram. "he dose response for 06MG formation was also nonlinear above 3 mg/ kg in nasal respiratory mucosa, but appeared to be linear over a broader range of doses in nasal olfactory mucosa. The ef- ficiency of formation of this adduct (amount formed per unit dose) was highest in the respiratory mucosa after low doses, suggesting a high-affinity pathway for formation of the adduct in this tissue. More studies such as these are needed to assess the appropriate use of DNA adducts as markers of exposure.

The detection of DNA adducts in human tissues and cells by a variety of assays, including enzyme immunoassays, flu- orescent assays, and the 32P-postlabeling assay has been reviewed (see Tables 1 to 3).61 In addition, Pereira has recently reviewed some examples of studies in which DNA adducts in human peripheral blood cells have been detected in chemically exposed humans. The data from these studies clearly point to the usefulness of using DNA adducts as measures of exposures to chemicals. In these examples, adducts were detected after exposure to specific chemicals (e.g., BaP), as well as to mixtures of chemicals (e.g., PAH, cigarette smoke). The relationship between exposure history and level of DNA adducts is still unclear for most situations and points to the need for future research in this area of exposure dose-response relationships.

With respect to the detection of DNA adducts in respiratory tract tissue of humans, Perera et al.62 applied the technique of enzyme-linked immunosorbent assay (ELISA) and found detectable levels of benzo(a)pyrene-DNA adducts in human lung, lung tumors, and peripheral blood mononuclear cells of five patients diagnosed with primary lung cancer. Despite the ef- forts of these investigators to obtain detailed exposure histories to environmental sources of BaP, the authors concluded that

- '0510 20 30 40 60 80 100

300,- NASAL RESPIRATORY - MUCOSA

200 -

300,- NASAL _ _ _ RESPIRATORY - MUCOSA

200 -

300- NASAL OLFACTORY

- MUCOSA

200 -

0 10 20 30 40 60 00 100

300- NASAL OLFACTORY

- MUCOSA

200 -

0 10 20 30 40 60 00 100

N N K ( m g l k g l d a y )

FIGURE 5. Dose-response curves for formation of 06-methylguanine in tissues of the rat respiratory tract following 12 d of treatment with "K. (From Belinsky, S. A,, White, C. M . , Devereux, T. R . , and Anderson, M. W., Environ. Health Per- spect., 76, 3, 1987. With permission.)

the number of patients was too small to enable any definitive conclusions regarding exposure/dose (i.e., DNA adducts) relationships.

The detection of DNA adducts in urine has also been used as an index of exposure to chemicals. Samples are obtained in a noninvasive manner. In contrast to measuring DNA adducts in tissues and blood, measurement of DNA adducts in urine

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provides an index of the potential total genotoxic dose of a chemical. In addition, information is obtained on the ability of an individual to repair the damaged DNA.

One of the chemicals studied in this regard is aflatoxin. Autrup et reported the presence of 8,9-dihydro-8-(7- guanyI)-9-hydroxyaflatoxin B, (Am-Gua) in the urine of people exposed to aflatoxin. However, the authors concluded that the present technology was not sensitive enough to detect low levels of AFB exposure, and the method they employed (HPLC verified by synchronous scanning fluorescence spectrophotometry) appears too laborious to be used for a large epidemiological study.

6. Characterization of DNA Adducts Methods of detecting DNA adducts have been reviewed

by Wogan and GorelickJ6 (Table 3). A frequently used technique is the 32P-postlabeling assay.M The method makes use of changes in chromatographic mobility of adducted nucleo- tides to separate normal from adducted DNA bases. This method has the advantages of extreme sensitivity and the ability to

detect total adducts in a DNA sample resulting from interaction with a broad range of chemicals. The method is limited to those adducts large enough to alter the chromatography of the adducted nucleotide. Recent modifications in the method have allowed detections of smaller ad duct^.^^ A major limitation of the technique, however, is its lack of specificity. In most in- stances, the chemical identity of the detected adducted material is not known. Even if a standard for a proposed adduct is synthesized and run in the same chromatography system, co- chromatography of a number of adducts is possible.

To overcome the lack of specificity of the 32P-postlabeling technique, many investigators have used immunoassays. In such assays the degree of specificity depends on the method of development of the antibody. Chemically altered DNA or a specifically altered component base can be used as the an- tigen, against which polyclonal antisera or monoclonal antibodies can be developed. The advantage to using polyclonal sera is the relative ease with which it is obtained. However, polyclonal serum will contain antibodies derived from many cell types against a variety of antigenic determinants. Also, if

Table 3 Assays for the Detection of Carcinogen-DNA Adducts

Method

UV in line with HPLC (major benzo(a)pyrene-DNA

Fluorescence in line with HPLC (BPDE-I-tetrol) Photon counting synchronous scanning fluorimetryd Immunoassays

adduct)”

Polyclonal rabbit antibodies against BPDE-I-DNA‘ Competitive assays

RIA = Radioimmunoassays ELISA = Enzyme-linked inmunosorbent assays USERIA = Ultrasensitive radioirnmunoassays USERIA

USERIA Noncompetitive assays

Monoclonal antibodies against BPDE-I-DNA8 Competitive ELISA Noncompetitive ELISA

J’P-Postlabelingh

Limit of detection (fmole)

100,000h

31‘

5300‘ 55‘ 12‘ 10

3

19‘ 3

0.03-0.3‘

Amount of DNA used

per analysis (PP)

2600

100

1 1 1

25

0.01

0.005 0.0002

1

From Wogan and Gorelick.S6

* l0-~Deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,I0-tetrahydrobenzo(a)pyrene

‘ Data of Weinstein% Data of Rahn et al.qs Data of Vahakangas% Data of Hsu et al.” .4mount of adduct showing 50% inhibition Data of Santella et al.87 Data of Gupta et a].-

‘ ’ ’ Theoretical limit of detection

Modification of analyzed DNA,

adducts/base

2 x 10-5

1 x 10-7 1 x 10-6

1.7 x 10-3 1 . 8 x 10-5 3.9 x 10-6 1.4 x lo -?

9.7 x 10-5

1.2 x 10-3 4.9 x 10-3

1 x 1 0 - 7 x 10-8


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the immunized animal dies, the serum is no longer available. In contrast, monoclonal antibodies, while considerably more difficult and labor-intensive to produce, are derived from a single cell, are specific for a single antigenic determinant, and are available as long as the cell culture is maintained. Im- munoassays are both sensitive (Table 3) and specific.

It is becoming apparent, however, that not all DNA adducts are equally effective in producing health effects. In the future it will be important to devote more research effort to the identification of specific DNA adducts and to link such adducts to specific health effects, if any. Such research will require the tools of the analytical chemist to chemically synthesize and identify the adducts.

C. Protein Adducts as Biomarkers of Exposure Osterman-Golkar et al.% pioneered the interest in using

protein adduct formation as a possible biological monitor for chemical exposures in man. Studies by Sun and Dent67 have shown that the majority of chemical adduct formation with cellular macromolecules occurs with various proteins rather than with DNA. To date, a number of studies have been published that indicate quantitation of protein adducts can be related to the degree of exposure to a specific compound. Extensive reviews concerning this area of research have been pub- l i ~ h e d . % . ~ ~ - ~ ~ The class of proteins receiving the most attention are the proteins found in circulating blood. A primary reason for this is that blood protein samples can be obtained from subjects in a relatively noninvasive manner. Other advantages include the fact that the specific proteins found in blood are relatively abundant and easily isolated for analysis.

D. Blood Protein Adducts Although blood has some capability of metabolizing xe-

nobiotics to electrophilic intermediates ,71 blood protein adduct formation depends largely on remote formation of reactive metabolites that are sufficiently stable to be transported from the cells of the metabolizing organ into circulating blood. Thus, highly reactive metabolites may have little or no opportunity to covalently react with blood proteins.

Hemoglobin (Hb) has been the most studied of the proteins in blood for adduct formation. In a series of experiments in which rats were given radiolabeled test compounds by gavage, Pereira and Chang7* reported the levels of Hb adduct formation for 16 different carcinogenic compounds and 2 noncarcinogenic agents. They concluded that, since the level of binding to Hb for all the chemicals tested was dose dependent, Hb adducts might be a good biomarker for chemical exposures. This study and the majority of the other studies that investigated the possible use of Hb adducts as a monitor of exposure used radiolabeled test compound given at relatively high doses in a manner not normally associated with human exposure scenarios. Thus, quantitation of Hb adducts was not chemical specific (detection

was by radiolabel) and was optimized by the route and high levels of test material given.

If Hb adducts are to be used as a biomarker for human exposures to various compounds, highly sensitive chemical assays for the adducts need to be developed. Few studies have been reported that address this issue. The quantitation of Hb adducts from people exposed to the direct alkylating agent, ethylene oxide, has been r e p ~ r t e d . ~ ~ . ~ ~ This was done by hy- drolyzing isolated globin into individual amino acids and quantitating the levels of adducted histidine amino acid residues in globin obtained from blood samples taken from the exposed individuals. Using these methods, it was possible to relate Hb adduct formation with ethylene oxide exposures at concentrations in the work environment as low as 5 to 10 ppm. Similar studies have used levels of 4-aminobiphenyl-Hb adducts in people as a biomarker of tobacco smoke exposures.7s Nordqvist et al.76 measured Hb adducts in NMRI male mice administered styrene (1.1 to 4.9 nmol/kg body weight) or styiene oxide (0.037 to 1.1 nmoYkg body weight) intraperitoneally. The data from these experiments are summarized in Figure 6. There was a dose-dependent increase in the formation of Hb adducts 2 h after injection of styrene or styrene oxide. Note that the reactive metabolite, styrene oxide, formed detectable Hb adducts at a lower dose than the parent compound, styrene. These authors did not measure Hb adducts at any other time points, so it is not clear whether or not the data represent peak levels of Hb adducts. Thus, it would appear that the potential exists for using Hb adducts of styrene metabolites to monitor human exposure to this chemical. However, these methods to measure Hb adducts are labor intensive. If Hb or protein adducts are to be used routinely as biomarkers for human exposures to organic compounds, further studies are needed to simplify such analyses. The current developments in chemical methods to quantitate protein adducts will be discussed later in this section.

In relation to other blood proteins, Hb also has an advantage in that it is relatively long-lived. The turnover rate of blood Hb is related to the lifetime of erythrocytes in blood, which in man is approximately 120 d.77 Thus, it is conceivable that exposure monitoring by assessing quantities of Hb adducts can be done at times well past the last exposure period. How- ever, blood proteins other than Hb may be better suited as biomarkers for special exposure situations. Such proteins might include serum proteins such as albumin. Albumin, like Hb, is relatively abundant in blood and is easily isolated. Limited studies have been done with respect to measuring albumin a d d ~ c t s . ~ ~ , ~ ~ These studies showed that because albumin has a substantially shorter lifetime in blood (20 to 25 d) than does Hb," albumin adducts can only be used to assess relatively recent human exposures to organic compounds.

Despite the shorter half-life of albumin, this protein may have several special advantages over Hb as a biomarker of exposure. Because albumin exists in blood serum, circulating reactive chemical metabolites released into the blood by various

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13 I 0) . 1 - (I)

0 E C

-

0.1

-

-

-

0.01 I I I I 0.0 1 0.1 1 10

mmole/kg Body Weight

FIGURE 6. Formation of Hb adducts after exposure to styrene and styrene oxide in NMRI mice. (From Nordqvist, M. B . , Lof. A., Osterman-Golkar, S . , and Walles, S . A. S., Chem. B i d . Inferact., 55 , 63, 1985. With permission.)

metabolizing organs do not have to penetrate a cell membrane before being able to alkylate the “target” protein. In contrast, for Hb adduct formation to occur, reactive intermediates in blood must be stable enough to penetrate the cell membrane of a red blood cell without reacting with it and yet be reactive enough to form an adduct with Hb. Thus, albumin adduct formation may be more abundant and a more sensitive marker for the presence of reactive chemical metabolites in blood and thus exposure to the parent compound. In industrial situations, where low-level exposures may occur consistently throughout a work shift, albumin adducts present at the end of a shift may provide a more sensitive measure of exposure than Hb adducts.

Another advantage may be that serum proteins such as albumin are synthesized in the liver, which has the largest capacity for metabolizing xenobiotics. Formation of albumin adducts in the liver and their subsequent release into circulating blood may occur readily for compounds requiring metabolic conversion to reactive species. If this occurs, measured levels of serum albumin adducts may greatly exceed those of Hb adducts. This may be particularly true for those compounds that are metabolized to highly reactive intermediates for which blood levels of the metabolites are low because of their chemical instability. Studies that compare the levels of Hb vs. serum protein (albumin) adduct formation for different classes of organic compounds are needed to determine the most suitable biomarker for a given exposure scenario.

One study comparing serum albumin adducts to dietary

intake of aflatoxin B, and urinary excretion ox aflatoxin , has been reported.81 The albumin adducts in the blood of residents of an area with relatively high aflatoxin dietary intake were assayed by enzymatic proteolysis of isolated serum albumin, followed by purification of immunoreactive products by immunoaffinity chromatography. The adducts were quan- tified by radioimmunoassays. The amount of albumin adducts in the blood correlated well, both with the dietary intake of aflatoxin B, and the urinary excretion of aflatoxin M,. Thus, when there is a continuous intake of a toxin, albumin adducts can serve as biomarkers of that intake.

E. Urinary Protein Adducts While it is becoming evident that adduct formation with

intact proteins found in blood may be a useful biomarker for human exposure to organic compounds, it may also be feasible to investigate protein adducts from urine samples. As previously discussed in this review, analysis of urine for indicators of chemical exposure has commonly been for various metabolites of the parent compound in question andlor the parent compound itself. Since these chemical species are usually cleared from the body relatively quickly, such markers of chemical exposure cannot be used for an exposure that occurred in the distant past. Proteins and nucleic acids are broken down in the body by catabolic processes, and that portion of the individual breakdown products not reutilized is eliminated from the body. The majority of this elimination is by excretion into the urine


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as polypeptide fragments, amino acids, purines, pyrimidines, and, for specific biological molecules that enter into the urea cycle, urea and uric acid. Presumably, if a chemical adduct were present on these constituents, it may also be eliminated as an intact adduct by this route and be detectable as an adduct rather than simply an intermediate of metabolic transformation. In vitro studies by Sun and Dent8* have shown that when intracellular proteolysis occurs, chemical adducts on the proteins are maintained on the peptide fragment breakdown products. The rate at which breakdown product adducts might appear in urine would depend on the overall turnover rate of the class of macromolecules of interest. Development of methods to detect such breakdown product adducts in urine, however, have yet to be attempted. It is likely that the levels of the breakdown product adducts are extremely low. However, since relatively large volumes of urine can be easily obtained from exposed individuals, the sensitivity requirements for such an assay may not be a problem. Further research needs to be done to determine if urine samples can serve as a source for exposure biomarkers and if methods can be developed to quantitate these adducts.

F. Other Protein Adducts Recent work has shown that adducts formed with protam-

ine, a basic protein associated with sperm DNA, may also be potential markers of exposure close to the critical site for adverse biological effects. Sega et aLS3 have identified one of the protamine adducts formed after acrylamide exposure to be S-carboxyethylcysteine. The formation of this adduct acts to break S-S bonds in the protamine and may be the basis for the chromosome breakage induced by this compound. This represents one of the first reports linking adduct formation with a specific deleterious effect.

At present, there is good scientific evidence to suggest that quantitation of protein or protein-derived adducts may be a useful biomarker for human exposures, and may have wide applicability to a number of organic compounds. Selection of the specific biomarker to measure and the methods to use require a great deal of prior information concerning the organic compound of interest. Figure 7 suggests hypothetical relationships of the various biomarkers discussed with respect to these relative concentrations as a function of time after exposure. This figure illustrates the considerations of detectability and time after exposure one must make in selecting a biomarker of exposure. For example, measures of urinary metabolites may be useful markers of a single exposure to an organic chemical for only a day or two after the exposure. Albumin adducts formed from reactive metabolites of the compound may be detected over a 2-week period after exposure, while Hb adducts may be detected over several months. The window of time for detection of adducts formed on DNA, as well as blood protein adducts, will depend on the kinetics of formation and breakdown or repair of such adducts, information that is

limited at this time. Urinary protein and DNA which should represent the breakdown or turnover products of the adducts formed earlier, should be detectable at later times after exposure. Figure 7 illustrates hypothetical times at which markers are detectable after a single exposure, but real-life exposures are often multiple. For multiple exposures, such as might occur in an industrial situation, many of the markers of exposure may reach steady-state levels, and the relationship between the steady-state levels of the different markers could be used to monitor the exposure. Knowledge of the kinetics of formation and breakdown of the adducts should allow development of a mathematical model relating adduct levels to prior exposure conditions.

Other considerations include knowing the chemical nature of the reactive species that will form the adduct and how the compound will be metabolically handled in the body. For example, Lewalter and Korallus" demonstrated that levels of Hb adducts following aniline exposures were not only dependent on the exposure concentration, but were also dependent on the acetylator status of the exposed individual. Slow acetylators were able to break down such adducts to yield free aniline as acetanilide that was primarily excreted in the urine. Similar reports concerning the metabolic removal of Hb adducts have been published for 4-aminobiphenyl in ratss5 and humans.86 Such occurrences, if not taken into account, can lead to mis- interpretations of the data.

G. Chemical Characterization of Blood Protein Adducts

Both chemical and immunochemical methods have been devised to measure blood protein adducts. While immunolog- ical techniques show much promise, only a small amount of work has been done using monoclonal antibodies to measure adduct levels. 87

Chemical assays to identify and quantitate protein adducts (e.g., Hb or serum albumin) from several different toxicants have been developed.69 In reviewing the available literature regarding the analysis of protein adducts, it is apparent that three general strategies can be identified (Table 4).

One strategy involves isolation of the protein of interest (e.g., Hb) from animals or humans that have been exposed to a particular toxicant. A portion of the protein is treated with mild acid or base to hydrolyze the adducting species from the protein. No attempt is made to fragment the protein itself. The free molecule is isolated by extraction or by column chromatography and, following derivatization, is quantitated by HPLC or gas chromatography (GC). Toxicants amenable to this approach include aromatic amines and polycyclic aromatic hydrocarbons .".88 Bryant and co-workersS9 have measured Hb adducts of 4-aminobiphenyl (4-ABP) in smokers using this approach. The 4-ABP covalently binds to sulfhydryl groups on cysteine as a sulfonic acid amide. Base hydrolysis of the Hb releases the parent amine, which, following derivatization

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.- Exposure Conc.

} Dose

Albumin Adducts

Hemoglobin Adducts

1 10 100 1000 TIME AFTER EXPOSURE (days)

FIGURE 7. levels and time of appearance after a single exposure.

Hypothetical relationships among different biornarkers of exposure with respect to their relative

Table 4 Techniques for Analysis of Adducted Proteins

Chemical Technique adducted Strengths Limitations Ref.

Weak acid/ba$e 4-Aminobiphenyl Simple

Complete protein Acrylamide Several possible ad- hydrolysis Benzo( a)pyrene

hydrolysis Ethylene oxide duct types to quantitate; universal

Modified Edrnan Ethylene oxide Simple degradation

using pentafluoropropionamide, is measured using capillary GC with negative-ion chemical ionization mass spectrometry. Using this method for analysis. blood samples from cigarette smokers showed consistently higher levels of 4-ABP-Hb adducts than nonsmokers. ShugartX8 has described a method for measuring the binding of benzo(a)pyrene to Hb as the diol- epoxide metabolite. The benzo(a)pyrene moiety is released as a tetrol by acid hydrolysis. After extensive clean-up, the tetrol is measured by HPLC with fluorescence detection. The utility of this approach may be limited, however, due to the small

Only acid/base labile 85,88

Tedious 91 74

molecules

Only applicable to 90 compounds bound to terminal valine; can be insensitive because of low levels of adduction

percentage of tetrol released by acid hydrolysis. Overall, analytical approaches using the whole protein are particularly appealing because of the lack of extensive sample workup required.

A second strategy also involves isolation of the protein; however, instead of mild acid treatment, more harsh acidic conditions are applied that quantitatively hydrolyze the protein to individual amino acids. The mixture is partially purified by chromatography, and the adducted amino acids are quantitated by ion exchange chromatography or GC. Alkylating agents,

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such as ethylene and propylene oxide, are often analyzed in this mam~er , ’~ .~ as was a~rylamide.~’ For example, a method has been developed to determine the alkylated amino acid N- 3’-(2-hydroxypropyl)histidine in Hb, one of the prime products from exposure to propylene oxide. After isolation of the Hb, the protein is hydrolyzed with 6 N HCI and the protein hydro- lysate chromatographed on a Dowex@ 50 W column. The amino acids are derivatized and measured using GC/mass spectrometry (MS).

A third strategy, only recently described, pertains to alkylating agents that bind to terminal valine groups of Hb. After isolation of the Hb, the alkylated terminal valines are cleaved by a modified Edman degradation method using pentafluoro- phenyl i so th io~yana te .~ .~~ The amino acids are cleaved from the protein by mild base treatment. The free species then rear- ranges to a thiohydantoin, which is analyzed by GUMS. This approach shows much promise because of its sensitivity and the relatively mild degradation conditions. Its general applicability to proteins other than Hb is as yet unexplored.

One of the prime needs in the development of chemical assays for indicators of exposure is for sensitive, specific, and simple approaches to analysis. Probably the easiest approach will be to use monoclonal antibodies to identify and quantitate adducts. In principle, levels of adduction could be determined on the intact protein, without isolating bound amino acids or any analyte of interest.

Chemical approaches using the whole protein, such as the mild base hydrolysis and the modified Edman degradation, will also be important. However, only a few exposure compounds will be amenable to this approach. As of yet, approaches using mild base or acid hydrolysis have been limited to aromatic amines and polycyclic aromatic hydrocarbons. One fallacy of this approach may be the long-term stability of the adduct in vivo. If unstable on the protein, use of the adduct to quantitate prior exposure would obviously be hindered.

While the modified Edman degradation has proved useful for aliphatic epoxides, its general utility is unknown. As spec- ified above, this approach necessitates binding of the analyte on terminal valines. However, reactive species have often been observed to bind in high proportion to 98B-cysteine of the human Hb. Thus, only a minor fraction may be available for the valine group. Chemical strategies designed to quantitate binding to the cysteine group would thus be useful; one method has been described recently to measure the general binding of reactive metabolites to this amino acid.93

H. Relating Markers to a Prior Exposure Scenario Relating quantitative measures of markers of exposure to

the prior exposure history of an individual will require more information than is currently available. Required for such analyses is knowledge of the kinetics of marker formation and

breakdown after single and repeated exposures to a range of exposure concentrations of the compound of interest. Once such information is available, a mathematical model could be developed to predict marker values for various exposure scenarios. This does not mean that a unique exposure regimen can be defined for each level of a marker detected, but such a model should allow determination of the types of exposure regimens that would produce a given level of the marker of interest. Also, the model could define the level of a marker that should not be exceeded in an exposure situation if regulated external exposure conditions are being met. Eventually, if the link is made between the exposure marker and a predicted adverse health effect, the model could define the level of the marker that should not be exceeded to avoid the adverse health effect.

IV. SUMMARY AND FUTURE DIRECTIONS

The field of biomarkers of exposure and health effects is a dynamic one, with new markers being developed at a rapid pace. The prospect of using macromolecular adducts formed from reactive chemicals or their metabolites as monitors of exposure is exciting. New methodologies are making it possible to detect such adducts at even lower concentrations. However, the field has some important limitations at the present time, and these must be overcome to make full use of the approach.

First, there must be more chemical identifications of the structure of the adducts. Many methods, such as the 32P-postlabeling technique, allow one to determine the presence of adducts in a semiquantitative fashion, but, thus far, the chemical structures of only a few of the adducts detected have been determined.

Second, the toxicological significance of the adducts must be determined. Not all adducts will have the same impact on the health of the exposed individual. Some adducts, because of their abundance and stability, may be excellent markers of exposure, but may have little or no adverse health effects. Some DNA adducts may be at sites that are not essential for the functioning of the cell. Until we have more information on the potential for adverse health effects, all DNA adducts are considered equally as indicators of genotoxicity .

Third, the kinetics of the formation and breakdown of adducts must be determined. It is only when such information is available that we can begin to relate the tissue or excreta concentrations of adducts to prior exposure scenarios. If the kinetics of formation and breakdown of the adducts were known, mathematical models of the relation between the exposures and the concentration of the adducts at specific sites could be generated.

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