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Program Summary: Research on Benzene and 1,3-Butadiene

March 1999

The purpose of this Program Summary is to describe the content and goals of HEI's research program on benzene and 1,3-butadiene, and to provide information about proposed future human studies.

Introduction

Extrapolation Issues

The assessment of human risk resulting from ambient exposures to a pollutant relies heavily on information from studies of different animal species exposed to much higher concentrations of the pollutant than those usually present under real-world conditions. Moreover, different species often display marked differences in sensitivity to the same pollutant. The information needed for accurate extrapolation from animal results to potential human effects is often lacking, leading to uncertainties in the calculation of risk. For some pollutants, human risk is estimated from epidemiologic studies of occupationally exposed workers. Even when this information is available, however, there are often uncertainties about the dose-response relation, especially in the absence of mechanistic information.

Some of the critical information needed for accurate extrapolation is an understanding of the relations, within and across species, among 1) the amount of pollutant a person or animal is exposed to (exposure concentration), 2) the amount of pollutant or its metabolites present in the body after exposure (internal dose), 3) the amount of pollutant or key metabolites that reach important target sites in the body (biologically effective dose), and 4) biological effects potentially associated with the development of cancer (such as DNA adducts, mutations, chromosomal aberrations). The examination of this information would improve our ability to determine how and why species differ in terms of sensitivity and how humans may be similar or different to other species, and the relation between exposure concentration and effects over a wide range of pollutant concentrations.

Background on HEI's Air Toxics Program

In 1993 HEI issued RFA 93-1, "Novel Approaches to Extrapolation of Health Effects for Mobile Source Toxic Air Pollutants," seeking studies to obtain data, or develop new methods, to improve extrapolation of health effects from high exposures to mobile source toxic air pollutants in animals to low ambient exposures in humans. Specifically, the RFA requested studies that would identify sensitive biomarkers of dose and early effects linked to the disease outcome for acetaldehyde, benzene, 1,3-butadiene, formaldehyde, and polycyclic organic matter. These components of mobile source emissions were targeted because they were listed in the Clean Air Act Amendments of 1990 as toxic air pollutants whose concentration in motor vehicle emissions needs to be reduced and may need to be regulated.

Although HEI was interested in funding research on all of the targeted air toxics, the research funded from this RFA includes studies on benzene and butadiene only. Common goals of these studies include understanding species differences; developing more sensitive methods for measuring dose; and understanding the relations among levels of the parent compounds, levels of several of their metabolites in blood and tissues, and the levels of genetic markers such as DNA adducts, mutations, and chromosomal alterations in target tissues.

Benzene

Research Needs

Epidemiologic investigations of populations occupationally exposed to benzene have found an increased risk of both leukemia, particularly acute myelogenous leukemia, and aplastic anemia. In humans, this leukemia may be associated with benzene-induced cell toxicity in bone marrow cells. However, it is not clear what levels of benzene are associated with these effects. Long-term studies in mice and rats have demonstrated an increased incidence of tumors in several organs. Toxic responses to benzene in the bone marrow (hyperplasia, micronuclei, leukocytopenia) have been reported in both rats and mice, with mice showing greater sensitivity than rats; this response is thought to require that benzene first be metabolized in the liver. Interspecies differences in sensitivity may be due to differences in benzene metabolism, either in the liver or in the bone marrow, or to differences in response of the bone marrow cells. Despite these indications, the evidence that benzene causes leukemia in either mice or rats is inconsistent.

Benzene in the body is either excreted or metabolized by two major pathways. One pathway leads to ring-opened metabolites, such as trans,trans-muconaldehyde and trans,trans-muconic acid; the other leads to a series of ring-hydroxylated compounds, such as phenol, catechol, and hydroquinone, and to their quinone oxidation products. Because several benzene metabolites can be detected in urine and blood of exposed animals and humans, their use as markers of benzene exposure has been proposed. However, sensitive methods for detecting very low levels of these metabolites are needed before they can be properly considered as markers of benzene exposure in humans. We also need to know which benzene metabolites, either directly or indirectly, are implicated in cancer in humans.

Some benzene metabolites can covalently bind to DNA and protein, and are thus presumed to be potentially involved in the cancer process. However, neither the relation between the benzene exposure concentration and the amounts of different metabolites in different species, nor the types of genetic changes resulting, is well understood. Moreover, the role of the various types of DNA damage associated with leukemia and toxicity to bone marrow remains to be clarified.

The current risk assessment for benzene is based primarily on the epidemiologic data from occupationally exposed populations. Effects at low, ambient exposure concentrations (averaging from < 1 to 3 ppb benzene) have been extrapolated from effects at the higher exposures (2-100 ppm) reported in the epidemiologic studies, introducing uncertainties in the risk estimates. In addition, the lack of an adequate animal model for human leukemia restricts our ability to obtain information on the development and progression of benzene-induced leukemia. Nevertheless, even if exposure to benzene leads to types of cancer other than leukemia in rodents, animal studies can still elucidate important features of benzene metabolism, levels of metabolites in various tissues (including the bone marrow), and the types of damage caused by individual benzene metabolites. Animal models can also help us develop biomarkers for use in estimating benzene exposure in humans.

Description of the HEI Research Program

The general goals of the HEI benzene research program are:

S-Phenylcysteine in Albumin as a Benzene Biomarker
William E. Bechtold, Inhalation Toxicology Research Institute
The objective of this study is to determine the feasibility of using adducts of a benzene metabolite with the cysteine group of albumin (S-phenylcysteine) in blood as a marker of benzene exposure. The major thrust of the work is to improve the sensitivity of current analytical methods and to determine the kinetics of adduct formation and removal with time. These studies will be conducted in mice exposed to benzene by inhalation. The analytical method will be further tested using human albumin to which S-phenylcysteine has been added. The ultimate goal of the study is to determine the amount of this adduct in humans exposed to different concentrations of benzene.

Quantification of Urinary Metabolites of Benzene by HPLC-MS Method: Assessment of the Relationship of Degree of Benzene Exposure to Differences in the Metabolic Activation Pathways in Humans
Assieh A. Melikian, American Health Foundation
This study aims at developing sensitive HPLC-GC methods for quantification of benzene metabolites in human urine. The metabolites selected are representative of various pathways of benzene metabolism and include ring-hydroxylated products (phenol, hydroquinone, and catechol), ring-opened products (trans,trans-muconic acid), and ring dimerized products (biphenyl), as well as a metabolite derived from the interaction of the benzene oxide intermediate with glutathione (S-phenylmercapturic acid). Initially the metabolites will be synthesized in the laboratory or obtained from the urine of animals exposed to [13C]- or [14C]-labelled benzene. The ultimate goal is to characterize variation in benzene metabolism in humans as a function of dose.

Benzene Metabolism at Doses Relevant for Human Urban Air Exposure
Kenneth W. Turteltaub, Lawrence Livermore National Laboratory and University of California at San Francisco
This study has two main objectives. One objective is to investigate how benzene, over a wide range of exposure levels, influences the metabolism in vivo, and how metabolism varies as a function of sex, strain, and species (rats and mice). The other objective is to characterize the DNA and protein adducts in bone marrow over the same range of exposure concentrations. In order to achieve the level of sensitivity necessary to detect benzene metabolites in blood and urine after the animals have inhaled benzene concentrations relevant to human ambient exposure, a new technique, accelerator mass spectrometry (AMS), will be used. The profile of metabolites in urine and blood will then be compared with the pattern of adducts found in the bone marrow in different sexes, strains, and species. Although this methodology is not directly applicable to human studies, the results of this work will improve our understanding of the relation of metabolism and macromolecular damage in the bone marrow at high and low exposure concentrations, both within an individual species and across species.

Characterization and Mechanisms of Chromosomal Alterations Induced by Benzene in Mice and Humans
David A. Eastmond, University of California at Riverside
The objective of this study is to characterize and compare the nature and persistence of chromosomal alterations induced by benzene in mice and humans. The types of chromosomal alterations formed in peripheral blood and bone marrow of mice will be examined following short-duration (8 days) exposure to benzene at levels of 50, 100, or 400 mg/kg per day. This information will be compared with the types of chromosomal alterations formed in the peripheral blood of mice after prolonged exposure to benzene, using samples collected during a recent bioassay conducted by the National Toxicology Program. This study will also investigate the potential role of the inhibition of topoisomerase II, an enzyme involved in DNA repair and replication, in the formation of chromosomal damage. Inhibition of topoisomerase II has been implicated in the mechanism of carcinogenicity for two other leukemia-producing agents, etoposide and tenoposide. This study will investigate whether the inhibition of this enzyme is involved in benzene-induced leukemia as well. Finally, the nature and persistence of chromosomal alterations in white blood cells of human workers exposed to benzene will be examined. Understanding how benzene causes genetic damage may lead to the development of a biomarker of both exposure to benzene and biologic effects.

1,3-Butadiene

Research Needs

Although 1,3-butadiene had long been considered relatively non-toxic, a set of long-term inhalation bioassays conducted by the National Toxicology Program and the International Institute of Synthetic Rubber Producers found 1,3-butadiene caused cancer in rats and mice. In these studies, 1,3-butadiene produced tumors at multiple organ sites in Sprague Dawley rats (pancreas, uterus, Zymbal gland, mammary gland, thyroid, and testis) and B6C3F1 mice (lymphoma, heart, forestomach, lung, liver, mammary gland, and ovary). In rats, neoplasms were noted only after exposure to 1,3-butadiene concentrations at or above 1000 ppm, whereas in mice, certain neoplasms were noted after exposure to much lower concentrations. For example, lung tumors were observed after exposure to 6.25 ppm 1,3-butadiene, and hemangiosarcomas of the heart after exposure to 20 ppm. Tumors were induced at multiple organ sites even after only 13 weeks of exposure to 1,3-butadiene at concentrations of 625 ppm in mice.

Quantitative differences in the metabolism of 1,3-butadiene by rats and mice may be responsible for the observed differences in tumor susceptibility in rats and mice. Mice metabolize 1,3-butadiene to the monoepoxide intermediate, 1,2-epoxy-3-butene, faster than rats, and have much greater capacity to further oxidize the monoepoxide to the diepoxide, 1,2,3,4-diepoxybutane, than do rats (see scheme of 1,3-butadiene metabolism). The diepoxide is a highly reactive compound, and is up to 100-fold more mutagenic than the monoepoxide in in vitro studies.

Epidemiologic studies for 1,3-butadiene have yielded conflicting information, with some retrospective cohort studies suggesting an increased incidence of lymphatic and hematopoietic cancers, and others finding no cases of cancer attributable to exposure to 1,3-butadiene. These studies have generally not addressed confounding exposures to other chemicals, such as styrene, often present in the occupational settings, further complicating interpretation of the results.

Because of the difficulty in interpreting the epidemiologic findings, risk assessments for 1,3-butadiene have relied upon animal studies. It is therefore important to know whether humans more closely resemble rats or mice with respect to metabolism and subsequent reactivity of 1,3-butadiene and its metabolites.

Description of the HEI Research Program

The major goals of the HEI butadiene research program are the following:

Molecular Dosimetry of Conjugated Dienes and Aliphatic Aldehydes
Ian A. Blair, Vanderbilt University
The objective of this study is to develop and apply sensitive analytical techniques (liquid chromatography coupled with mass spectrometry) for identifying the major DNA adducts derived from 1,3-butadiene and its metabolites in target tissue (lung) and urine of both mice and rats. Mice and rats differ in their sensitivity to the carcinogenic effect of butadiene. The goal of the study is to determine the role of DNA adducts derived from different butadiene metabolites in the carcinogenicity of 1,3-butadiene by comparing the types of adducts and their persistence in these two species. Some of these adducts may prove useful for assessing 1,3-butadiene uptake and metabolism in humans. Overall, this study will contribute to our understanding of species differences in tumor response and may lead to the identification of markers of butadiene internal dose.

Study of the Potential Carcinogenicity of Butadiene Diepoxide in Mice and Rats
Rogene F. Henderson, Inhalation Toxicology Research Institute
This study aims to test the hypothesis that the diepoxide metabolite of 1,3-butadiene (1,2,3,4-diepoxybutane) causes a similar incidence of lung neoplasm in both mice and rats. The parent compound, 1,3-butadiene, is a much more potent carcinogen in mice than in rats. A potential reason for the different species susceptibility is that mice metabolize much more of the 1,3-butadiene to the diepoxide than do rats. In addition to determining the carcinogenicity of the diepoxide, the investigators propose to determine whether the diepoxide induces specific mutations in the K-ras gene from both rat and mouse lung tumors. This oncogene has been selected because previous studies have shown that lung tumors induced in mice by 1,3-butadiene contained activated K-ras. If positive, this study will demonstrate that 1,2,3,4-diexpoybutane is the toxic butadiene metabolite and that the same molecular mechanism is operant in mice and rats exposed to the diepoxide. This would help explain the difference in species response to 1,3-butadiene and aid extrapolation across species.

Determination of Mutagenicity and Mutational Spectrum in Human and Rodent Cells to Assess the Role(s) of Butadiene Monoepoxide and Butadiene Diepoxide in Mediating the In Vivo Genotoxicity of 1,3-Butadiene
Leslie Recio, Chemical Industry Institute of Toxicology
The goal of this study is to define the relative mutagenicity of the two major butadiene metabolites, the monoepoxide (1,2-epoxy-3-butene) and diepoxide (1,2,3,4-diepoxybutane) metabolites, by determining the frequency and types of mutations at several "reporter" genes in transgenic mice, transgenic rats, and in human lymphoblast cells. In order to address in vivo to in vitro extrapolation issues, investigators will also examine the mutagenicity and mutational spectra in transgenic mouse cells (embryonic fibroblasts) exposed in vitro. In the human cell system, mutations at the hprt locus will be examined; in the transgenic mouse cell line, mutations at the lacI locus; in the transgenic mice and rats, lacI mutations in bone marrow cells.

1,3-Butadiene Mutagenicity and Tumorigenicity in T-Lymphocytes of Exposed Rodents
Vernon E. Walker, Wadsworth Center for Laboratories and Research, New York State Department of Health
The goal of this study is to examine the relative mutagenicity of 1,3-butadiene and its two major metabolites, butadiene monoepoxide (1,2-epoxy-3-butene) and diepoxide (1,2,3,4-diepoxybutane), in rodents. Both the frequency and types of mutations (mutational spectra) in a portion of the endogenous hprt gene will be examined in lymphocytes of rats and mice exposed to 1,3-butadiene and its epoxide metabolites. By comparing the molecular events induced by butadiene and its major metabolites in species that differ in their sensitivity to 1,3-butadiene, this study will help in across-species extrapolation.

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