- Email: [email protected]
Insights into the toxicokinetics and toxicodynamics of 1,3-butadiene
Chemico-Biological Interactions 135– 136 (2001) 599– 614
www.elsevier.com/locate/chembiont
Insights into the toxicokinetics and toxicodynamics of 1,3-butadiene James A. Bond *, Michele A. Medinsky ToxCon, 5505 Frenchman’s Creek Dr., Durham, NC 27713, USA
Abstract 1,3 Butadiene (BD) is a colorless gas used in the production of synthetic rubber and plastics. BD is carcinogenic in rats and mice, however, there are striking species differences in cancer potency and spectrum of tumors, with mice being more susceptible to tumor induction than rats. Epidemiology studies suggest an excess incidence of leukemia in workers in the styrene–butadiene rubber industry. Consideration of mechanisms of BD carcinogenicity can provide insights into differences in cancer potency between rodents and serve to elucidate the extent to which BD exposure may cause cancer in humans. Mechanistic research in the areas of biochemical toxicology, molecular biology, molecular dosimetry, and susceptibility factors can impact BD cancer risk assessment for humans. This research has focused on quantitating species differences in the metabolism of BD and BD epoxides, defining molecular lesions produced by BD epoxides, identifying biomarkers for BD exposure to explore metabolic pathways in humans, and determining potential risk factors for sensitive subpopulations. BD is activated by P450 isozymes, including CYP2E1, to at least two genotoxic metabolites, epoxybutene (EB) and diepoxybutane (DEB). Dosimetry data from several laboratories on EB and DEB following inhalation exposure to BD indicate that blood concentrations of EB were four– eight-fold higher in mice compared with rats and that blood concentrations of DEB were 25 – 100-fold higher in mice than in rats. The higher levels of these two DNA-reactive metabolites in mice compared with rats probably contribute to the species differences in carcinogenic effects of BD between mice and rats. In vitro metabolism studies of BD in rats, mice, and human tissues indicate that there are significant quantitative species differences in the metabolic activation of BD to EB and DEB and the detoxication of EB and DEB. Activation/detoxication ratios calculated using in vitro kinetic constants reveal that ratios in mice were greater than in both rats and humans. In vitro data are consistent with in vivo dosimetry data and cancer potency for rodents, and suggest that
* Corresponding author: Tel.: + 1-919-5446384; fax: + 1-919-5446384. E-mail address: [email protected] (J.A. Bond). 0009-2797/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 1 ) 0 0 1 9 9 - 5
600
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
humans may be at a decreased risk. Data on mutagenicity and mutational spectra of BD epoxides show mechanistic differences between EB- and DEB-induced mutational events suggesting involvement of DEB in the development of cancer. Concentrations of DEB that are genotoxic in vitro are within the range of concentrations measured in mice in vivo, whereas concentrations of EB that are genotoxic in vitro are ten –100-fold greater than concentrations observed in vivo. Characterization of molecular events indicate that EB-induced genotoxicity is due to point mutations and small deletions, while DEB induces point mutations, small deletions, and large-scale deletions involving many base pairs. The extent to which epoxybutanediol is involved in BD carcinogenesis is not known. Molecular dosimetry studies in rodents and humans have focused on urinary metabolites and DNA and hemoglobin adducts. Data from these studies are consistent with in vivo and in vitro metabolism data providing further support for the differences in metabolic activation and deactivation of BD and BD epoxides across species and the role of DEB in tumor development. Research on potential susceptibility factors points to other P450 isozymes, in addition to CYP2E1, that are involved in both the metabolic activation and mutagenicity of BD. Taken together, mechanistic data on BD toxicokinetics and toxicodynamics provide an integrated insight into critical steps in initiation of cancer, metabolites responsible for cancer, sensitive biomarkers for exposure, and potential risk factors for individual susceptibility. Available evidence suggests that BD is unlikely to be a human carcinogen at the low exposure concentrations currently encountered in the environment or workplace. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Butadiene; Carcinogensis; Toxicodynamics; Toxicokinetics
1. Introduction 1,3-Butadiene (BD) is an industrial monomer used in the production of rubber and plastics. BD has been detected in cigarette smoke and automobile emissions [1,2] and is currently listed as one of the 189 hazardous air pollutants in the 1990 Clean Air Act Amendments [3]. BD is carcinogenic in two laboratory animal species, the B6C3F1 mouse and Sprague–Dawley rat [4–6]. Mice developed tumors from exposure to BD concentrations that were as much as three orders of magnitude lower than those that caused cancer in rats [5]. Epidemiological findings indicate an excess incidence of leukemia in workers in the styrene– butadiene rubber industry. However, these workers were exposed to other chemicals such as styrene, benzene, and diethyldithiocarbamate, thus, it is not possible to attribute the excess cancers solely to BD exposure [7,8]. The extensive and diverse data sets available for BD can be best integrated and interpreted within the context of an exposure–dose–response paradigm as illustrated in Fig. 1. Mechanistic data linking exposure and dose deal primarily with toxicokinetic processes such as absorption, distribution, metabolism, and elimination. Significant research has been conducted on understanding species differences in the toxicokinetics of inhaled BD. For BD, issues of species differences in internal dose are particularly important. Mechanistic data linking internal dose and response (i.e., cancer) deal primarily with the interaction of the target dose with critical cellular components (i.e., DNA) and the subsequent cascade of events
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
601
leading to a tumorigenic response. The extent to which toxicokinetic and toxicodynamic mechanisms are similar in laboratory animals and humans dictates the appropriateness of the use of animal toxicity data for human health risk assessment of BD. Consideration of mechanisms of BD carcinogenicity can provide insights into differences in cancer potency between rodents and serve to elucidate the extent to which BD exposure may cause cancer in humans. Mechanistic research in the areas of biochemical toxicology, molecular biology, molecular dosimetry, and susceptibility factors can impact BD cancer risk assessment for humans. Research in these four areas has focused on quantitating species differences in the metabolism of BD and BD epoxides, defining molecular lesions produced by BD epoxides, identifying biomarkers for BD exposure, to explore metabolic pathways in humans and determining potential risk factors for sensitive subpopulations. The impact of research in each of these areas in elucidating mechanisms of BD carcinogenicity is discussed below.
2. Biochemical toxicology BD requires metabolic activation to DNA-reactive epoxides that can bind to DNA and initiate tumor formation. BD is metabolized to the reactive epoxides 1,2-epoxy-3-butene (EB), 1,2-epoxy-3,4-butanediol (EBD), and 1,2:3,4-diepoxybutane (DEB) [8–12]. These epoxides have the potential of reacting with biomolecules such as DNA and proteins or can be detoxified by enzymatic hydrolysis and conjugation with glutathione (GSH) [8,13–17]. In vitro studies using tissues from rodents and humans have shown that patterns of BD metabolism vary among species [8–10,16,17] and among individuals within a species such as humans [9,10,16,17]. Variation is noted in all enzymes studied including cytochrome P450 (CYP450)-mediated oxidation of BD and EB [9,10],
Fig. 1. Exposure –dose –response paradigm for integrating human and laboratory data for butadiene risk assessment.
602
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
Fig. 2. Initial rates of butadiene metabolism by specific enzymes vary among mice, rats and humans. P450, cytochrome P450; EH, epoxide hydrolase; GST, glutathione-S-transferase; Initial rates are calculated as the ratio of the maximum velocity for the respective enzyme (Vmax) to the concentration at one-half maximum velocity (Km). Data were taken from Csana´ dy et al. [9].
hydrolysis of EB and DEB by epoxide hydrolase [9,16], and conjugation of EB and DEB with GSH via glutathione-S-transferase [9,17]. In general, these studies have shown that initial rates of CYP450 oxidation of BD to EB are approximately six– eight times faster in mice compared with rats and humans (Fig. 2). Initial rates, which are tangential to the linear portion of the Michaelis–Menten rate plot [18], can be calculated by dividing the maximum rate of metabolism (Vmax) by the concentration at one-half the maximum rate (Km ). The resulting ratio, Vmax/Km, is a pseudo-first order rate constant for metabolism at low substrate concentrations [18]. Detoxication of epoxides also varies across species [9,16,17]. For example, human tissues detoxify EB preferentially through epoxide hydrolase, whereas mice detoxify EB preferentially through glutathione-S-transferase (Fig. 2). Oxidation of EB and detoxication of DEB show similar species differences [10,16,17]. Additionally, differences in the intracellular localization of epoxide hydrolase and glutathione-Stransferase enzymes may also contribute to differences in relative efficiencies for detoxication across species. Epoxide hydrolase is situated on the endoplasmic reticulum in close proximity to CYP450 increasing the probability that the epoxides formed may be preferentially hydrolyzed prior to release into the cytoplasm. In contrast, the glutathione-S-transferase isozyme most active in detoxifying the BD epoxides is located in the cytoplasm and thus will only react with the epoxides after the epoxides are released from the endoplasmic reticulum. These differences in intracellular localization of enzymes may offer an explanation as to why in vivo detoxification of BD epoxides is more efficient in humans compared with rodents. Integration of differences in overall rates of epoxide formation and removal can be achieved by calculating activation/detoxication ratios for each of the various species. For these ratios, the initial rate for oxidation is divided by the sum of the initial rates for hydrolysis and GSH-conjugation [19]. The resulting activation/
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
603
Fig. 3. Activation/detoxication ratios integrate overall rates of epoxide formation and removal of epoxybutene (EB) and diepoxybutane (DEB) for mice, rats and humans. Ratios were calculated by dividing the Vmax/Km for oxidation by the sum of the Vmax/Km for hydrolysis and GSH-conjugation. Data were taken from Csana´ dy et al. [9] and Seaton et al. [10].
detoxication ratio is ten-fold higher for mice compared with rats and humans for both EB and DEB formation/removal (Fig. 3). Humans have the lowest ratio for DEB formation/removal compared with mice and rats suggesting a lower risk for DEB-related tumors for humans compared with that for rats and mice [19]. The studies described above were conducted using in vitro methods and tissue samples. The relevance of in vitro observations to predicting relative epoxide levels after exposure of rodents and humans to BD gas can be assessed by examining results of studies in which rats and mice were exposed to a range of BD concentrations for various times up to 6 h [11,12,20,21]. The results of these studies conducted in two laboratories are summarized in Table 1. In vivo concentrations of EB in blood of mice are four–eight times higher that those measured in rats. Blood levels of DEB are approximately 30-fold higher in mice than rats. Thus, in vivo blood concentrations of epoxides measured after BD exposure reflect the differences in activation/detoxication ratios determined from in vitro studies. The correlation Table 1 Steady-state blood concentrations of butadiene epoxides in rats and mice exposed to butadiene gas by inhalationa Butadiene (ppm)
62.5 625 1250 8000
Epoxybutene (mM)
Diepoxybutane (mM)
Mice
Rats
Mice
Rats
0.45 3.7 8.6 –c
0.06 0.9 1.0 4.0
0.4 1.9 2.5 –
0.014 NDb ND 0.017
a Data were taken from Himmelstein et al. [11] and Thornton-Manning et al. [12]. Exposure concentrations ranged from 4 to 6 h. b ND, not detected [11]. c Not determined.
604
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
Fig. 4. Mechanistic differences between epoxybutene (EB)- and diepoxybutane (DEB)-induced mutations in human TK6 cells. Cells were exposed to EB or DEB in vitro; background mutations were determined in untreated cells. White and light gray portions of the bars indicate total number of base substitutions. Specific A·TT·A transversions are noted. Dark gray and black portions of the bars indicate total number of deletions. Specific partial 5% deletions are noted. Data for EB and DEB exposed cells were not corrected for background mutations. Data were taken from Recio et al. [23].
of in vitro– in vivo data for rodents adds confidence to predictions of human metabolism of BD in vivo based on in vitro data derived from human tissues. Both patterns and ratios of in vitro metabolism in human samples are more similar to those of rats than mice suggesting that humans would have significantly lower epoxide levels than mice following exposure to BD, and thus be at lower risk for BD-induced tumors.
3. Molecular biology Molecular biology techniques have been employed to determine mutational mechanisms responsible for BD-induced carcinogenicity. These studies can also be used to provide insights into the BD epoxide, or combination of BD epoxides, responsible for the mutations specific to BD exposure and thus potentially responsible for the initiation of the carcinogenic effect. For example, mutational spectra studies in which human TK6 lymphoblastoid cells (TK6 cells) were exposed to solutions of EB and DEB in vitro have shown that EB-induced genotoxicity is due primarily to point mutations and small deletions [22,23]. In contrast, exposure of TK6 cells to DEB induces point mutations, small deletions, and large-scale deletions involving many base pairs [23,24]. As summarized in Fig. 4, background mutations in unexposed TK6 cells consist of a variety of base substitutions and deletions [23]. In vitro exposure of TK6 cells to EB results in a significant increase above background in the overall numbers of base substitutions and in particular a significant increase in A·T T·A transversions [22,23]. In vitro exposure of TK6
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
605
cells to DEB results in a significant increase above background in overall numbers of base deletions and in particular a significant increase in partial 5% deletions [23,24]. Assessment of the types of mutational spectra formed in rodents exposed to carcinogenic concentrations of BD can indicate which epoxide, or combination of epoxides, are predominately responsible for the development of tumors. In that way, human health risk assessments can be related to the effective epoxide dose at the target site rather than to external concentration of BD. In vitro mutagenesis studies can be used to directly compare the relative potencies of the BD epoxides for inducing specific types of mutations without the complicating factors such as metabolism and excretion that can occur in vivo. For example, the TK6 cells described above do not contain an active CYP2E1 and thus, the base substitutions and deletions observed are due to exposure to the specific epoxide and are not the result of metabolic activation to a more mutagenic species. In vitro studies have also been used to compare the relative abilities of DEB and EB to cause DNA damage through a clastogenic mechanism [25]. Clastogenicity was assessed by quantitating the number of micronuclei (MN) formed. Micronuclei are chromosome fragments that remain isolated from the chromosomes during cell division and thus are not incorporated into the nuclei of the daughter cells. Murg and colleagues [25] measured MN formed per 1000 cultured human lymphocytes following in vitro exposure to concentrations of epoxides of up to 10 mM for DEB and 300 mM for EB. DEB was far more potent in producing MN than EB (Fig. 5). In addition, DEB concentrations that are genotoxic to cultured human lymphocytes (Fig. 5) are within the range of DEB concentrations measured in mice exposed to BD by inhalation (Table 1). In contrast, concentrations of EB that produce elevated numbers of MN in vitro are ten–30-fold greater than EB concentrations measured in BD-exposed mice. The greater clastogenic potency of DEB compared with EB is
Fig. 5. Micronuclei per 1000 cultured human lymphocytes treated in vitro with epoxybutene or diepoxybutane solutions. The level of micronuclei in control (0 mM) samples is noted. Black bars indicate diepoxybutane treated cells. White bars indicate epoxybutene treated cells. Asterisk indicated significant increase in the formation of micronuclei as compared with untreated controls. Data were taken from Murg et al. [25].
606
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
likely related to the greater ability of DEB to cause deletions in DNA sequences [23]. Research has also been conducted on a third BD epoxide, epoxybutanediol (EBD), although not to the extent as that with EB and DEB. Both in vitro studies with human TK6 cells [26] and in vivo studies in mice that received injections of EB, DEB, or EBD [27,28] have shown the relative genotoxic potency of the three epoxides to be DEB\ \EB \ EBD. In vitro DEB is up to 100 times more potent than EB and up to 300 times more potent than EBD [26]. In vivo studies assessing the clastogenicity of each epoxide in bone marrow erythrocytes of mice after intraperitoneal injection showed the relative potencies of the three epoxides to be DEB \ EB \EBD [27,28]. On a molar basis DEB was about three times as effective as EB in inducing clastogenic damage and EB was about four times as effective as EBD. Other studies determined that DEB was positive in the dominant lethal assay at a dose as low as 36 mg/kg. However, neither EB nor EBD induced effects up to doses of 120 and 240 mg/kg, respectively [27]. The difference in relative potencies between in vivo and in vitro studies can be explained by consideration of toxicokinetic mechanisms that may be operational in vivo that are not involved in vivo. DEB and EB are readily eliminated by enzymatic hydrolysis and GSH-conjugation in vivo. Metabolic elimination reduces the exposure of target DNA to the mutagenic epoxides and thus the total mutations formed. EB can also be oxidized by CYP450 in vivo to DEB; thus, any mutations formed in vivo after EB administration may be a combined result of both EB and DEB exposure. In vitro these enzymes are less likely to be present in the exposed cell lines. Thus, enzymatic processes for the formation and removal of epoxides would not be operational. Disappearance of epoxides in vitro would be expected to occur only by slower, chemical hydrolytic mechanisms, potentially increasing the total exposure of the cells to the epoxides compared with the exposure of target cells that would occur after injection of epoxides.
4. Molecular dosimetry Biomolecules, such as water-soluble metabolites eliminated in urine and electrophilic metabolites bound to DNA or hemoglobin, can be used to estimate human exposure to BD. When coupled with similar measurements in laboratory animals, insights into potential differences in metabolic pathways between humans and laboratory animals can be obtained. For example, the most abundant urinary metabolites eliminated by rats, mice, and humans after BD exposure are 1,2-dihydroxybutyl mercapturic acid (DHBMA) and a mixture of monohydroxy-3-butenyl mercapturic acids (MHBMA) [29,30]. However, the ratios of these metabolites in urine vary widely among the three species (Table 2) and illustrate that the relative importance of hydrolytic metabolism differs among the species with humans \ \rats \ mice [29,30]. Humans eliminate predominately DHBMA; rats eliminate approximately equal amounts of DHBMA and MHBMA; and mice eliminate proportionately more MHBMA than DHBMA. DHBMA is formed by hydrolysis
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
607
Table 2 Comparison of biomarker data for urinary metabolites and hemoglobin adducts for rats, mice and humans after exposure to butadiene by inhalationa Biomarker
Rat
Mouse
DHBMA/DHBMA + MHBMAb MHBValc (pmol/g Hb/ppmh) THBVald (pmol/g Hb/ppmh)
0.48 0.086 2.3
Human
Insight
0.38
0.983
Hydrolysis predominates in humans
0.22
0.0047
Activation/detoxication ratio lowest in humans
2.4
Epoxybutanediol levels lower in humans than mice; epoxybutanediol likely source for adducts
14
a Data taken from Van Sittert et al. [29]. Exposure concentrations vary across species, thus the data are normalized for differences in exposure. b DHBMA, 1,2-dihydroxybutyl mercapturic acid; MHBMA, Monohydroxy-3-butenyl mercapturic acid. c MHBVal, Monohydroxybutenyl valine adduct. d THBVal, Trihydroxybutyl valine adduct.
of EB to 1,2-dihydroxy-3-butene followed by conjugation with GSH and MHBMA is formed via conjugation of EB with GSH. These findings indicate that in humans a much greater proportion of EB is metabolized by hydrolysis and a much smaller proportion detoxified by direct conjugation with GSH compared with rats and mice. Differences in urinary metabolite ratios across species are consistent with differences in the relative rates of enzymatic hydrolysis and GSH-conjugation measured in vitro (Fig. 2). The high rate of hydrolysis of EB in humans, as well as the lower activation/deactivation ratio for EB seen in vitro (Fig. 3), is also reflected in the very low levels of monohydroxy-3-butenyl valine adducts (MHBVal) detected in humans compared with rats and mice (Table 2). These adducts are formed by reaction of EB with the N-terminal valine of hemoglobin (Hb). When adjusted for exposure duration and concentration, relative levels of MHBVal were mice\ rats\ \ humans [29]. Formation of MHBVal adducts depends upon the concentration of free EB that is available to react with Hb. The concentration of EB is determined by its relative rates of formation and removal or the activation/detoxication ratio for EB. Interspecies differences in levels of MHBVal seen in vivo are consistent with both interspecies differences in the activation/detoxication ratio determined in vitro and with differences in blood concentrations of EB measured in vivo in rats and mice (Table 1). Taken together these data on in vitro metabolism, Hb adducts, and urinary metabolites suggest that blood concentrations of circulating epoxides such as EB should be lowest in humans compared with rats and mice placing the human at the lowest risk from BD exposure. Formation of DNA adducts by carcinogens or their DNA-reactive metabolites is thought to be an initial step in carcinogenesis. If information is known regarding the DNA adduct or adducts responsible for initiation of cancer, then one can estimate the molecular dose of a carcinogenic compound by measuring the number
608
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
of DNA adducts induced by a given exposure. Numerous DNA adducts of BD epoxide metabolites have been reported in the literature. Adducts that appear to be formed in the highest amounts are those which react with the guanine base at the N-7 position [13,31]. N-7 guanine adducts detected in tissues of rats and mice following exposure to BD concentrations ranging from 20 to 625 ppm reflect interspecies differences in BD metabolism (Fig. 6). Greater amounts of N-7-(2,3,4trihydroxybut-1-yl) – guanine (THB-Gua) and N-7-(hydroxybutenyl)– guanine (EBGua) were detected in tissues of mice compared with rats [13]. THB-Gua adducts were far more abundant than EB-Gua adducts. It has been proposed that the THB-Gua adducts are formed from the reaction of EBD with DNA [13]. Other indirect evidence for the existence in vivo of significant levels of EBD is that from data on trihydroxybutyl valine adducts (THBVal) in Hb (Table 2). THBVal adducts are more prevalent in rodents and humans than are MHBVal adducts and have been proposed to result from the reaction of EBD with the N-terminal valine of Hb. To date no studies have assayed for EBD in blood or tissues of rodents exposed to BD. Molecular dosimetry studies highlight the need for measurements of EBD blood or tissues concentrations in vivo. Finally, an additional DNA-adduct, N7-(1-(hydroxymethyl)-2,3-dihydroxypropyl) guanine, has been detected in rodents exposed to BD in vivo [31]. This adduct is of interest because it is the major DNA adduct found in both rats and mice after exposure to BD, but it is not detected in rodents exposed to EB. The BD-epoxide involved in the formation of these adducts remains to be determined.
Fig. 6. Number of DNA adducts per million bases in lung tissue of rats and mice exposed to various concentrations of butadiene by inhalation. Squares are data for rat lung; circles are data for mouse lung. Filled symbols are data for trihydroxybutyl guanine adducts and open symbols are data for hydroxybutenyl guanine adducts. Data were taken from Koc et al. [13].
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
609
Fig. 7. In vitro rate of diepoxybutane (DEB) formation by microsomes from various cDNA-expressed human CYP isozymes incubated with epoxybutene. Data were taken from Seaton et al. [10].
5. Susceptibility factors Studies using microsomal suspensions prepared from liver samples obtained from humans of various backgrounds and ages have shown a 60-fold variability in oxidation of EB to DEB [10] and a fourfold variability in the oxidation of BD to EB [9]. The origin of this interhuman variability may derive from multiple sources. For example, correlation analysis comparing rates of BD oxidation and activity of a specific CYP450 isozyme, cytochrome P4502E1 (CYP2E1) shows a significantly positive correlation between CYP2E1 activity and rate of BD oxidation [9]. This correlation suggests that expression of this particular isozyme is a significant indicator of the potential for EB formation and thus, potential risk. However, correlation between CYP2E1 activity and BD oxidation rate was not complete, suggesting that other isozymes might be responsible for BD oxidation. The role of other P450 isozymes in both BD oxidation to EB and EB oxidation to DEB has been investigated. Microsomes from human B-lymphoblastoid cells expressing cDNAs encoding several human cytochrome P450 enzymes were exposed to EB or BD in vitro and rates of oxidation were determined [10,32]. Data for EB oxidation to DEB, shown in Fig. 7, indicates that both CYP2E1 and CYP3A4 have significant affinity for EB resulting in high rates of oxidation [10]. Similar studies using BD as a substrate implicated both CYP2E1 and CYP2A6 in BD oxidation to EB in vitro [32]. Taken together, both in vitro studies suggest that CYP2E1 expression is an important indicator of individual susceptibility, but is not the sole isozyme responsible for the conversion of BD to electrophilic and mutagenic metabolites. The studies described above were conducted in vitro and their implications for understanding the mechanisms of BD carcinogenicity can be assessed by determining the extent to which specific CYP450 isozymes metabolize BD in vivo. In vivo studies, using either specific metabolic inhibitors or transgenic animals in which expression of specific isozymes is eliminated can provide information critical to
610
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
assessing the extent to which in vitro correlations between isozyme activity and rates of epoxide formation are maintained under in vivo exposure situations [33,34]. Such studies have been conducted in transgenic mice (cyp2e1 − / − ) that lack the gene for expression of the CYP2E1 enzyme [34]. Both cyp2e1 − / − mice and the parental normal strain of the mice with an active gene for CYP2E1 (cyp2e1 + / + ) were exposed to BD in a closed chamber (Fig. 8). Mice pretreated with specific enzyme inhibitors were also exposed to BD in a similar manner [33]. The concentration of BD in the chamber declined over time due to uptake of BD from the chamber into the mice and metabolism of BD. Thus, disappearance of BD from the chamber is directly related to the extent of BD uptake by the animal and the rate of BD metabolism. As seen in Fig. 8, the most rapid rates of BD disappearance from the chamber were observed when cyp2e1 + / + mice were exposed to BD. The slowest rates of BD disappearance from the chamber were seen in mice pretreated with 1-aminobenzotriazole (ABT), a non-specific inhibitor of CYP450 isozymes. Since BD metabolism was completely eliminated in ABT-treated mice, the disappearance of BD from the chamber is due solely to the uptake of BD into blood and partitioning into tissues, especially fat, which has the highest solubility for BD [35]. For cyp2e1 − / − mice the rate of BD disappearance was intermediate between the ABT pretreated mice and the cyp2e1 + / + mice indicating that in vivo another P450 isozyme or isozymes, in addition to CYP2E1, is responsible for BD oxidation. These results are consistent with in vitro observations and suggest that expression of other P450 isozymes in addition to CYP2E1 may be important susceptibility factors for potential risk after exposure to BD. In vitro work with microsomal
Fig. 8. Disappearance of butadiene from a closed chamber containing mice pretreated with the CYP450 inhibitor 1-aminobenzotriazole (ABT), transgenic mice which lack the gene for expression of the CYP2E1 enzyme (cyp2e1 − / − ), or the parental strain of mice with an active gene for CYP2E1 (cyp2e1 + / + ). The rate of disappearance of butadiene is the result of uptake of butadiene into the mouse tissues only (ABT), uptake into tissues and non-CYP2E1 mediated oxidation of butadiene (cyp2e1 − / − ), or uptake into tissues, non-CYP2E1 and CYP2E1 mediated oxidation of butadiene (cyp2e1 + / + ). Data were taken from Jackson et al. [33,34].
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
611
Fig. 9. Micronucleated polychromatic erythrocytes (PCE) per 1000 PCE in bone marrow of mice exposed to butadiene gas in a closed chamber and euthanized 24 h after the start of the exposure. Control, No pretreatment, no butadiene exposure; BD, No pretreatment, butadiene exposure; DCE + BD, Intraperitoneal injection of 1,1-trans-dichloroethylene (DCE) prior to butadiene exposure; ABT + BD, Intraperitoneal injection of 1-aminobenzotriazole (ABT) prior to butadiene exposure. DCE is a specific inhibitor of CYP2E1. ABT is a non-specific inhibitor of all CYP450. * Indicates significantly different from other treatment groups at a PB 0.05. Data were taken from Jackson et al. [33].
suspensions prepared from mouse lung, liver, and kidney samples suggests that these additional isozymes may be CYP2A5 and CYP4B1 [36]. CYP2A5 is the mouse analog to the human isozyme CYP2A6; both isozymes are involved in the hydroxylation of coumarin. CYP4B1 is active in mouse kidney. The gene for human CYP4B1 has been cloned; the cDNA have been expressed and determined to be inactive [36]. The relationship between level of enzyme activity and genotoxic potency was also evaluated in mice exposed to BD under closed chamber conditions [33]. In these studies mice were pretreated with ABT or 1,2-trans-dichloroethylene (DCE), a specific CYP2E1 inhibitor, prior to exposure to BD in a closed chamber. Mice that were not pretreated with enzyme inhibitors, but were exposed to BD, in the closed chamber, were used as positive controls and mice, pretreated with inhibitors, but not exposed to BD were used as negative controls. At the end of the exposure mice were removed from the chamber. Genotoxicity was assessed at 24 h after the start of exposure by quantitating MN in bone marrow polychromatic erythrocytes [33]. ABT pretreatment reduced the level of MN in BD-exposed mice to that of the unexposed controls (Fig. 9). DCE pretreatment reduced the number of MN in BD-exposed mice compared to that seen in the BD-exposed controls, but not to the level seen in the ABT treated mice. Thus, although inhibition of CYP2E1 decreased BD-mediated genotoxicity, it did not prevent clastogenic damage due to BD exposure, consistent with the results from metabolism studies suggesting that other P450 isoforms are involved in BD activation.
612
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
Since formation of MN is due primarily to DEB [23,25], and not EB, these studies suggest that elevated MN levels in the absence of CYP2E1-mediated oxidation must be the result of the oxidation of EB to DEB by CPY450 isozymes other than CYP2E1. In vitro studies with cDNA expressed human CPY450 suggest that CYP3A4 may be the other isozyme responsible for oxidation of EB to DEB in humans. Taken together in vitro and in vivo studies suggest that differences in expression of CYP2E1, CYP2A6, and CYP3A4 in humans may be important susceptibility factors for individual exposure to BD. As noted previously, these isozymes appear to be correlated with both formation of reactive epoxides and expression of clastogenic effects. However, other enzymes also play a role in the overall metabolism of BD, and in vitro studies have also demonstrated differences in the activities of these enzymes among the individual human tissue samples assessed. For example, activity of epoxide hydrolase varied among 12 human samples studied, by a factor of sixfold for hydrolysis of EB [9] and threefold for hydrolysis of DEB [16]. Similarly, activity of glutathione-S-transferase varied across human samples analyzed being on the order of seven or fivefold for conjugation of EB or DEB, respectively [9,17]. The enzymes discussed above demonstrate the steps in the toxicokinetic processes for which there might exist potential risk factors for sensitive subpopulations. Increased rates of oxidation and decreased rates of detoxication might be expected to increase levels of reactive epoxides in target tissues and thus affect individual risk. Less attention has been paid to potential steps in the toxicodynamic processes that might also alter risk. For example, enzymes responsible for repair of mismatched DNA bases resulting from epoxide adducts or repair of single strand breaks in DNA might also show differences among individuals and thus be additional risk factors for susceptibility.
6. Conclusions In summary, mechanistic studies provide insights into critical genotoxic events in the initiation of cancer, metabolites responsible for cancer, sensitive biomarkers for exposure, and potential risk factors for individual susceptibility. Consideration of mechanisms of BD carcinogenicity can provide insights into differences in cancer potency between rodents and serve to elucidate the extent to which BD exposure may cause cancer in humans. Mechanistic research in the area of biochemical toxicology has pointed to the role of DEB in BD carcinogenesis and to the likely pathways for BD metabolism in humans. Molecular biology studies suggest a major role for at least DEB in BD genotoxicity/carcinogenicity. Molecular dosimetry studies involving hemoglobin adducts and urinary metabolites show differences in human/rodent metabolism that parallel differences highlighted in biochemical toxicology studies and suggest, in general, humans to be at decreased risk from BD exposure. However, studies of specific susceptibility factors indicate that expression of specific isozymes involved in BD formation can alter risk.
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
613
References [1] K.D. Brunnemann, M.R. Kagan, J.E. Cox, D. Hoffman, Analysis of 1,3-butadiene and other gas-phase components in cigarette mainstream and sidestream smoke by gas chromatography – mass selective detection, Carcinogenesis 11 (1990) 1863 – 1868. [2] N. Pelz, A.M. Dempster, P.R. Shore, Analysis of low molecular weight hydrocarbons including 1,3-butadiene as engine exhaust gases using an aluminum oxide porous-layer open-tubular fused-silica column, J. Chrom. Sci. 28 (1990) 230 – 235. [3] Environmental Protection Agency, Preliminary draft list of categories and subcategories under Section 112 of the Clean Air Act, Fed. Reg. 56 (1991) 28548 – 28557. [4] J.E. Huff, R.L. Melnick, H.A. Solleveld, J.K. Haseman, M. Powers, R.A. Miller, Multiple organ carcinogenicity of 1,3-butadiene in B6C3F1 mice after 60 weeks of inhalation exposure, Science 227 (1985) 548 –549. [5] R.L. Melnick, J. Huff, B.J. Chow, R.A. Miller, Carcinogenicity of 1,3-butadiene in B6C3F1 mice at low exposure concentrations, Cancer Res. 50 (1990) 6592 – 6599. [6] P.E. Owen, J.R. Glaister, I.F. Gaunt, D.H. Pullinger, Inhalation toxicity studies with 1,3-butadiene. III. Two-year toxicity/carcinogenicity studies in rats, Am. Ind. Hygiene Assoc. J. 48 (1987) 407–413. [7] E. Delzell, N. Sathiakumar, M. Hovinga, M. Macaluso, J. Julian, R. Larson, P. Cole, D.C. Muir, A follow-up study of butadiene production workers, Toxicology 113 (1996) 182 – 189. [8] M.W. Himmelstein, J.F. Acquavella, L. Recio, M.A. Medinsky, J.A. Bond, Toxicology and epidemiology of 1,3-butadiene, Crit. Rev. Toxicol. 27 (1997) 1 – 108. [9] G.A. Csana´ dy, F.P. Guengerich, J.A. Bond, Comparison of the biotransformation of 1,3-butadiene and its metabolite, butadiene monoepoxide, by hepatic and pulmonary tissues from humans, rats and mice, Carcinogenesis 13 (1992) 1143 – 1153. [10] M.J. Seaton, M.H. Follansbee, J.A. Bond, Oxidation of 1,2-epoxy-3-butene to 1,2:3,4-diepoxybutane by cDNA-expressed human cytochromes P450 2E1 and 3A4 and human, mouse, and rat liver microsomes, Carcinogenesis 16 (1995) 2287 – 2293. [11] M.W. Himmelstein, M.J. Turner, B. Asgharian, J.A. Bond, Comparison of blood concentrations of 1,3-butadiene and butadiene epoxides in mice and rats exposed to 1,3-butadiene by inhalation, Carcinogenesis 15 (1994) 1479 –1486. [12] J.R. Thornton-Manning, A.R. Dahl, W.E. Bechtold, W.C. Griffith Jr., R.F. Henderson, Disposition of butadiene monoepoxide and butadiene diepoxide in various tissues of rats and mice following a low-level inhalation exposure to 1,3-butadiene, Carcinogenesis 16 (1995) 1723 – 1731. [13] H. Koc, N.Y. Tretyakova, V.E. Walker, R.F. Henderson, J.A. Swenberg, Molecular dosimetry of N-7 guanine adduct formation in mice and rats exposed to 1,3-butadiene, Chem. Res. Toxicol. 12 (1999) 566 –574. [14] K.A. Richardson, H.J.J.J. Megens, J.D. Webb, J.J. Van Sittert, Biological monitoring of butadiene exposure by measurement of haemoglobin adducts, Toxicology 113 (1996) 112 – 118. [15] K.A. Richardson, M.M.C.G. Peters, B.A. Wong, H.J.J.J. Megens, P.A. Van Elburg, E.D. Booth, et al., Quantitative and qualitative differences in the metabolism of 14C-1,3-butadiene in rats and mice: relevance to cancer susceptibility, Toxicol. Sci. 49 (1999) 86 – 201. [16] P.J. Boogaard, J.A. Bond, The role of hydrolysis in the detoxification of 1,2:3,4-diepoxybutane by human, rat, and mouse liver and lung in vitro, Toxicol. Appl. Pharmacol. 141 (1996) 617 – 627. [17] P.J. Boogaard, S.C. Sumner, J.A. Bond, Glutathione conjugation of 1,2:3,4-diepoxybutane in human liver and mouse liver and lung in vitro, Toxicol. Appl. Pharmacol. 136 (1996) 307 – 316. [18] G.L. Kedderis, Toxicokinetics: biotransformation of toxicants, in: J. Bond (Ed.), General Principles. In: Comprehensive Toxicology, vol. 1, Elsevier Science, UK, 1997, pp. 115 – 148. [19] J.A. Bond, M.W. Himmelstein, M. Seaton, P. Boogaard, M.A. Medinsky, Metabolism of butadiene by mice, rats, and humans: a comparison of physiologically based toxicokinetic model predictions and experimental data, Toxicology 113 (1996) 48 – 54. [20] M.W. Himmelstein, B. Asgharian, J.A. Bond, High concentrations of butadiene epoxides in livers and lungs of mice compared to rats exposed to 1,3-butadiene, Toxicol. Appl. Pharmacol. 132 (1995) 281–288.
614
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
[21] W.E. Bechtold, M.R. Strunk, J.R. Thornton-Manning, R.F. Henderson, Analysis of butadiene, butadiene monoxide, and butadiene dioxide in blood by gas chromatography/gas chromatography/ mass spectroscopy, Chem. Res. Toxicol. 8 (1995) 182 – 187. [22] A.-M. Steen, K.G. Meyer, L. Recio, Characterization of hprt mutations following 1,2-epoxy-3butene exposure of human TK6 cells, Mutagenesis 12 (1997) 359 – 364. [23] L. Recio, C.J. Saranko, A.-M. SteenPart II: Roles of two metabolites of 1,3-butadiene in mediating its in vivo genotoxicity, HEI Research Report 1,3-Butadiene: Cancer, mutations and adducts, 92 (2000) 49 –87 [24] A.-M. Steen, K.G. Meyer, L. Recio, Analysis of hprt mutations occurring in human TK6 lymphoblastoid cells following exposure to 1,2:3,4-diepoxybutane, Mutagenesis 12 (1997) 61 – 67. [25] M.N. Murg, M. Schuler, D.A. Eastmond, Evaluation of micronuclei and chromosomal breakage in the 1cen –q12 region by the butadiene metabolites epoxybutene and diepoxybutane in cultured human lymphocytes, Mutagenesis 14 (1999) 541 – 546. [26] J.E. Cochrane, T.R. Skopek, Mutagenicity of butadiene and its epoxide metabolites. I. Mutagenic potential of 1,2-epoxybutene, 1,2:3,4-diepoxybutane and 3,4-epoxy-1,2-butanediol in cultured human lymphoblasts, Carcinogenesis 15 (1994) 713 – 771. [27] I.-D. Adler, U. Kliesch, L. Nylund, K. Peltonen, In vitro and in vivo mutagenicity of the butadiene metabolites butadiene diolepoxide, butadiene monoepoxide and diepoxybutane, Mutagenesis 12 (1997) 339 –345. [28] I.-D. Adler, U. Kliesch, C. Tiveron, F. Pacchierotti, Clastogenicity of diepoxybutane in bone marrow cells and germ cells of mice, Mutagenesis 10 (1995) 535 – 541. [29] N.J. van Sittert, H.J.J.J. Megens, W.P. Watson, P.J. Boogaard, Biomarkers of exposure to 1,3-butadiene as a basis for cancer risk assessment, Toxicol. Sci. 56 (2000) 189 – 202. [30] W.E. Bechtold, M.R. Strunk, I.Y. Chang, J.B. Ward Jr., R.F. Henderson, Species differences in urinary butadiene metabolites: comparisons of metabolite ratios between mice, rats, and humans, Toxicol. Appl. Pharmacol. 127 (1994) 44 – 49. [31] P.J. Boogaard, K.P. DeKloe, N.J. Van Sittert, N7-(1-(Hydroxymethyl)-2,3-dihydroxypropyl) guanine is the major DNA adduct after exposure to 1,3-butadiene but not 1,2-epoxy-3-butene, Toxicol. Sci. 54 (2000) S229. [32] R.J. Duescher, A.A. Elfarra, Human liver microsomes are efficient catalysts of 1,3-butadiene oxidation: Evidence for major roles by cytochromes P450 2A6 and 2E1, Arch. Biochem. Biophys. 311 (1994) 342 –349. [33] T.E. Jackson, P.D. Lilly, L. Recio, P.M. Schlosser, M.A. Medinsky, Inhibition of cytochrome P450 2E1, decreases but does not eliminate, genotoxicity mediated by 1,3-butadiene, Toxicol. Sci. 55 (2000) 266 –273. [34] T.E. Jackson, M.A. Medinsky, B.E. Butterworth, F.J. Gonzalez, L. Recio, The use of cytochrome P450 2E1 knockout mice to develop a mechanistic understanding of carcinogen biotransformation and toxicity, CIIT Activities 19 (1999) 1 – 9. [35] M.A. Medinsky, T.L. Leavens, G.A. Csana´ dy, M.L. Gargas, J.A. Bond, In vivo metabolism of butadiene by mice and rats: a comparison of physiological model predictions and experimental data, Carcinogenesis 15 (1994) 1329 – 1340. [36] A.A. Elfarra, R.J. Krause, R.A. Kemper, Cellular and molecular basis for species, sex and tissue differences in 1,3-butadiene metabolism, Chem.-Biol. Interact. 135 – 136 (2001) 239 – 248 (this volume).
.
www.elsevier.com/locate/chembiont
Insights into the toxicokinetics and toxicodynamics of 1,3-butadiene James A. Bond *, Michele A. Medinsky ToxCon, 5505 Frenchman’s Creek Dr., Durham, NC 27713, USA
Abstract 1,3 Butadiene (BD) is a colorless gas used in the production of synthetic rubber and plastics. BD is carcinogenic in rats and mice, however, there are striking species differences in cancer potency and spectrum of tumors, with mice being more susceptible to tumor induction than rats. Epidemiology studies suggest an excess incidence of leukemia in workers in the styrene–butadiene rubber industry. Consideration of mechanisms of BD carcinogenicity can provide insights into differences in cancer potency between rodents and serve to elucidate the extent to which BD exposure may cause cancer in humans. Mechanistic research in the areas of biochemical toxicology, molecular biology, molecular dosimetry, and susceptibility factors can impact BD cancer risk assessment for humans. This research has focused on quantitating species differences in the metabolism of BD and BD epoxides, defining molecular lesions produced by BD epoxides, identifying biomarkers for BD exposure to explore metabolic pathways in humans, and determining potential risk factors for sensitive subpopulations. BD is activated by P450 isozymes, including CYP2E1, to at least two genotoxic metabolites, epoxybutene (EB) and diepoxybutane (DEB). Dosimetry data from several laboratories on EB and DEB following inhalation exposure to BD indicate that blood concentrations of EB were four– eight-fold higher in mice compared with rats and that blood concentrations of DEB were 25 – 100-fold higher in mice than in rats. The higher levels of these two DNA-reactive metabolites in mice compared with rats probably contribute to the species differences in carcinogenic effects of BD between mice and rats. In vitro metabolism studies of BD in rats, mice, and human tissues indicate that there are significant quantitative species differences in the metabolic activation of BD to EB and DEB and the detoxication of EB and DEB. Activation/detoxication ratios calculated using in vitro kinetic constants reveal that ratios in mice were greater than in both rats and humans. In vitro data are consistent with in vivo dosimetry data and cancer potency for rodents, and suggest that
* Corresponding author: Tel.: + 1-919-5446384; fax: + 1-919-5446384. E-mail address: [email protected] (J.A. Bond). 0009-2797/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 1 ) 0 0 1 9 9 - 5
600
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
humans may be at a decreased risk. Data on mutagenicity and mutational spectra of BD epoxides show mechanistic differences between EB- and DEB-induced mutational events suggesting involvement of DEB in the development of cancer. Concentrations of DEB that are genotoxic in vitro are within the range of concentrations measured in mice in vivo, whereas concentrations of EB that are genotoxic in vitro are ten –100-fold greater than concentrations observed in vivo. Characterization of molecular events indicate that EB-induced genotoxicity is due to point mutations and small deletions, while DEB induces point mutations, small deletions, and large-scale deletions involving many base pairs. The extent to which epoxybutanediol is involved in BD carcinogenesis is not known. Molecular dosimetry studies in rodents and humans have focused on urinary metabolites and DNA and hemoglobin adducts. Data from these studies are consistent with in vivo and in vitro metabolism data providing further support for the differences in metabolic activation and deactivation of BD and BD epoxides across species and the role of DEB in tumor development. Research on potential susceptibility factors points to other P450 isozymes, in addition to CYP2E1, that are involved in both the metabolic activation and mutagenicity of BD. Taken together, mechanistic data on BD toxicokinetics and toxicodynamics provide an integrated insight into critical steps in initiation of cancer, metabolites responsible for cancer, sensitive biomarkers for exposure, and potential risk factors for individual susceptibility. Available evidence suggests that BD is unlikely to be a human carcinogen at the low exposure concentrations currently encountered in the environment or workplace. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Butadiene; Carcinogensis; Toxicodynamics; Toxicokinetics
1. Introduction 1,3-Butadiene (BD) is an industrial monomer used in the production of rubber and plastics. BD has been detected in cigarette smoke and automobile emissions [1,2] and is currently listed as one of the 189 hazardous air pollutants in the 1990 Clean Air Act Amendments [3]. BD is carcinogenic in two laboratory animal species, the B6C3F1 mouse and Sprague–Dawley rat [4–6]. Mice developed tumors from exposure to BD concentrations that were as much as three orders of magnitude lower than those that caused cancer in rats [5]. Epidemiological findings indicate an excess incidence of leukemia in workers in the styrene– butadiene rubber industry. However, these workers were exposed to other chemicals such as styrene, benzene, and diethyldithiocarbamate, thus, it is not possible to attribute the excess cancers solely to BD exposure [7,8]. The extensive and diverse data sets available for BD can be best integrated and interpreted within the context of an exposure–dose–response paradigm as illustrated in Fig. 1. Mechanistic data linking exposure and dose deal primarily with toxicokinetic processes such as absorption, distribution, metabolism, and elimination. Significant research has been conducted on understanding species differences in the toxicokinetics of inhaled BD. For BD, issues of species differences in internal dose are particularly important. Mechanistic data linking internal dose and response (i.e., cancer) deal primarily with the interaction of the target dose with critical cellular components (i.e., DNA) and the subsequent cascade of events
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
601
leading to a tumorigenic response. The extent to which toxicokinetic and toxicodynamic mechanisms are similar in laboratory animals and humans dictates the appropriateness of the use of animal toxicity data for human health risk assessment of BD. Consideration of mechanisms of BD carcinogenicity can provide insights into differences in cancer potency between rodents and serve to elucidate the extent to which BD exposure may cause cancer in humans. Mechanistic research in the areas of biochemical toxicology, molecular biology, molecular dosimetry, and susceptibility factors can impact BD cancer risk assessment for humans. Research in these four areas has focused on quantitating species differences in the metabolism of BD and BD epoxides, defining molecular lesions produced by BD epoxides, identifying biomarkers for BD exposure, to explore metabolic pathways in humans and determining potential risk factors for sensitive subpopulations. The impact of research in each of these areas in elucidating mechanisms of BD carcinogenicity is discussed below.
2. Biochemical toxicology BD requires metabolic activation to DNA-reactive epoxides that can bind to DNA and initiate tumor formation. BD is metabolized to the reactive epoxides 1,2-epoxy-3-butene (EB), 1,2-epoxy-3,4-butanediol (EBD), and 1,2:3,4-diepoxybutane (DEB) [8–12]. These epoxides have the potential of reacting with biomolecules such as DNA and proteins or can be detoxified by enzymatic hydrolysis and conjugation with glutathione (GSH) [8,13–17]. In vitro studies using tissues from rodents and humans have shown that patterns of BD metabolism vary among species [8–10,16,17] and among individuals within a species such as humans [9,10,16,17]. Variation is noted in all enzymes studied including cytochrome P450 (CYP450)-mediated oxidation of BD and EB [9,10],
Fig. 1. Exposure –dose –response paradigm for integrating human and laboratory data for butadiene risk assessment.
602
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
Fig. 2. Initial rates of butadiene metabolism by specific enzymes vary among mice, rats and humans. P450, cytochrome P450; EH, epoxide hydrolase; GST, glutathione-S-transferase; Initial rates are calculated as the ratio of the maximum velocity for the respective enzyme (Vmax) to the concentration at one-half maximum velocity (Km). Data were taken from Csana´ dy et al. [9].
hydrolysis of EB and DEB by epoxide hydrolase [9,16], and conjugation of EB and DEB with GSH via glutathione-S-transferase [9,17]. In general, these studies have shown that initial rates of CYP450 oxidation of BD to EB are approximately six– eight times faster in mice compared with rats and humans (Fig. 2). Initial rates, which are tangential to the linear portion of the Michaelis–Menten rate plot [18], can be calculated by dividing the maximum rate of metabolism (Vmax) by the concentration at one-half the maximum rate (Km ). The resulting ratio, Vmax/Km, is a pseudo-first order rate constant for metabolism at low substrate concentrations [18]. Detoxication of epoxides also varies across species [9,16,17]. For example, human tissues detoxify EB preferentially through epoxide hydrolase, whereas mice detoxify EB preferentially through glutathione-S-transferase (Fig. 2). Oxidation of EB and detoxication of DEB show similar species differences [10,16,17]. Additionally, differences in the intracellular localization of epoxide hydrolase and glutathione-Stransferase enzymes may also contribute to differences in relative efficiencies for detoxication across species. Epoxide hydrolase is situated on the endoplasmic reticulum in close proximity to CYP450 increasing the probability that the epoxides formed may be preferentially hydrolyzed prior to release into the cytoplasm. In contrast, the glutathione-S-transferase isozyme most active in detoxifying the BD epoxides is located in the cytoplasm and thus will only react with the epoxides after the epoxides are released from the endoplasmic reticulum. These differences in intracellular localization of enzymes may offer an explanation as to why in vivo detoxification of BD epoxides is more efficient in humans compared with rodents. Integration of differences in overall rates of epoxide formation and removal can be achieved by calculating activation/detoxication ratios for each of the various species. For these ratios, the initial rate for oxidation is divided by the sum of the initial rates for hydrolysis and GSH-conjugation [19]. The resulting activation/
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
603
Fig. 3. Activation/detoxication ratios integrate overall rates of epoxide formation and removal of epoxybutene (EB) and diepoxybutane (DEB) for mice, rats and humans. Ratios were calculated by dividing the Vmax/Km for oxidation by the sum of the Vmax/Km for hydrolysis and GSH-conjugation. Data were taken from Csana´ dy et al. [9] and Seaton et al. [10].
detoxication ratio is ten-fold higher for mice compared with rats and humans for both EB and DEB formation/removal (Fig. 3). Humans have the lowest ratio for DEB formation/removal compared with mice and rats suggesting a lower risk for DEB-related tumors for humans compared with that for rats and mice [19]. The studies described above were conducted using in vitro methods and tissue samples. The relevance of in vitro observations to predicting relative epoxide levels after exposure of rodents and humans to BD gas can be assessed by examining results of studies in which rats and mice were exposed to a range of BD concentrations for various times up to 6 h [11,12,20,21]. The results of these studies conducted in two laboratories are summarized in Table 1. In vivo concentrations of EB in blood of mice are four–eight times higher that those measured in rats. Blood levels of DEB are approximately 30-fold higher in mice than rats. Thus, in vivo blood concentrations of epoxides measured after BD exposure reflect the differences in activation/detoxication ratios determined from in vitro studies. The correlation Table 1 Steady-state blood concentrations of butadiene epoxides in rats and mice exposed to butadiene gas by inhalationa Butadiene (ppm)
62.5 625 1250 8000
Epoxybutene (mM)
Diepoxybutane (mM)
Mice
Rats
Mice
Rats
0.45 3.7 8.6 –c
0.06 0.9 1.0 4.0
0.4 1.9 2.5 –
0.014 NDb ND 0.017
a Data were taken from Himmelstein et al. [11] and Thornton-Manning et al. [12]. Exposure concentrations ranged from 4 to 6 h. b ND, not detected [11]. c Not determined.
604
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
Fig. 4. Mechanistic differences between epoxybutene (EB)- and diepoxybutane (DEB)-induced mutations in human TK6 cells. Cells were exposed to EB or DEB in vitro; background mutations were determined in untreated cells. White and light gray portions of the bars indicate total number of base substitutions. Specific A·TT·A transversions are noted. Dark gray and black portions of the bars indicate total number of deletions. Specific partial 5% deletions are noted. Data for EB and DEB exposed cells were not corrected for background mutations. Data were taken from Recio et al. [23].
of in vitro– in vivo data for rodents adds confidence to predictions of human metabolism of BD in vivo based on in vitro data derived from human tissues. Both patterns and ratios of in vitro metabolism in human samples are more similar to those of rats than mice suggesting that humans would have significantly lower epoxide levels than mice following exposure to BD, and thus be at lower risk for BD-induced tumors.
3. Molecular biology Molecular biology techniques have been employed to determine mutational mechanisms responsible for BD-induced carcinogenicity. These studies can also be used to provide insights into the BD epoxide, or combination of BD epoxides, responsible for the mutations specific to BD exposure and thus potentially responsible for the initiation of the carcinogenic effect. For example, mutational spectra studies in which human TK6 lymphoblastoid cells (TK6 cells) were exposed to solutions of EB and DEB in vitro have shown that EB-induced genotoxicity is due primarily to point mutations and small deletions [22,23]. In contrast, exposure of TK6 cells to DEB induces point mutations, small deletions, and large-scale deletions involving many base pairs [23,24]. As summarized in Fig. 4, background mutations in unexposed TK6 cells consist of a variety of base substitutions and deletions [23]. In vitro exposure of TK6 cells to EB results in a significant increase above background in the overall numbers of base substitutions and in particular a significant increase in A·T T·A transversions [22,23]. In vitro exposure of TK6
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
605
cells to DEB results in a significant increase above background in overall numbers of base deletions and in particular a significant increase in partial 5% deletions [23,24]. Assessment of the types of mutational spectra formed in rodents exposed to carcinogenic concentrations of BD can indicate which epoxide, or combination of epoxides, are predominately responsible for the development of tumors. In that way, human health risk assessments can be related to the effective epoxide dose at the target site rather than to external concentration of BD. In vitro mutagenesis studies can be used to directly compare the relative potencies of the BD epoxides for inducing specific types of mutations without the complicating factors such as metabolism and excretion that can occur in vivo. For example, the TK6 cells described above do not contain an active CYP2E1 and thus, the base substitutions and deletions observed are due to exposure to the specific epoxide and are not the result of metabolic activation to a more mutagenic species. In vitro studies have also been used to compare the relative abilities of DEB and EB to cause DNA damage through a clastogenic mechanism [25]. Clastogenicity was assessed by quantitating the number of micronuclei (MN) formed. Micronuclei are chromosome fragments that remain isolated from the chromosomes during cell division and thus are not incorporated into the nuclei of the daughter cells. Murg and colleagues [25] measured MN formed per 1000 cultured human lymphocytes following in vitro exposure to concentrations of epoxides of up to 10 mM for DEB and 300 mM for EB. DEB was far more potent in producing MN than EB (Fig. 5). In addition, DEB concentrations that are genotoxic to cultured human lymphocytes (Fig. 5) are within the range of DEB concentrations measured in mice exposed to BD by inhalation (Table 1). In contrast, concentrations of EB that produce elevated numbers of MN in vitro are ten–30-fold greater than EB concentrations measured in BD-exposed mice. The greater clastogenic potency of DEB compared with EB is
Fig. 5. Micronuclei per 1000 cultured human lymphocytes treated in vitro with epoxybutene or diepoxybutane solutions. The level of micronuclei in control (0 mM) samples is noted. Black bars indicate diepoxybutane treated cells. White bars indicate epoxybutene treated cells. Asterisk indicated significant increase in the formation of micronuclei as compared with untreated controls. Data were taken from Murg et al. [25].
606
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
likely related to the greater ability of DEB to cause deletions in DNA sequences [23]. Research has also been conducted on a third BD epoxide, epoxybutanediol (EBD), although not to the extent as that with EB and DEB. Both in vitro studies with human TK6 cells [26] and in vivo studies in mice that received injections of EB, DEB, or EBD [27,28] have shown the relative genotoxic potency of the three epoxides to be DEB\ \EB \ EBD. In vitro DEB is up to 100 times more potent than EB and up to 300 times more potent than EBD [26]. In vivo studies assessing the clastogenicity of each epoxide in bone marrow erythrocytes of mice after intraperitoneal injection showed the relative potencies of the three epoxides to be DEB \ EB \EBD [27,28]. On a molar basis DEB was about three times as effective as EB in inducing clastogenic damage and EB was about four times as effective as EBD. Other studies determined that DEB was positive in the dominant lethal assay at a dose as low as 36 mg/kg. However, neither EB nor EBD induced effects up to doses of 120 and 240 mg/kg, respectively [27]. The difference in relative potencies between in vivo and in vitro studies can be explained by consideration of toxicokinetic mechanisms that may be operational in vivo that are not involved in vivo. DEB and EB are readily eliminated by enzymatic hydrolysis and GSH-conjugation in vivo. Metabolic elimination reduces the exposure of target DNA to the mutagenic epoxides and thus the total mutations formed. EB can also be oxidized by CYP450 in vivo to DEB; thus, any mutations formed in vivo after EB administration may be a combined result of both EB and DEB exposure. In vitro these enzymes are less likely to be present in the exposed cell lines. Thus, enzymatic processes for the formation and removal of epoxides would not be operational. Disappearance of epoxides in vitro would be expected to occur only by slower, chemical hydrolytic mechanisms, potentially increasing the total exposure of the cells to the epoxides compared with the exposure of target cells that would occur after injection of epoxides.
4. Molecular dosimetry Biomolecules, such as water-soluble metabolites eliminated in urine and electrophilic metabolites bound to DNA or hemoglobin, can be used to estimate human exposure to BD. When coupled with similar measurements in laboratory animals, insights into potential differences in metabolic pathways between humans and laboratory animals can be obtained. For example, the most abundant urinary metabolites eliminated by rats, mice, and humans after BD exposure are 1,2-dihydroxybutyl mercapturic acid (DHBMA) and a mixture of monohydroxy-3-butenyl mercapturic acids (MHBMA) [29,30]. However, the ratios of these metabolites in urine vary widely among the three species (Table 2) and illustrate that the relative importance of hydrolytic metabolism differs among the species with humans \ \rats \ mice [29,30]. Humans eliminate predominately DHBMA; rats eliminate approximately equal amounts of DHBMA and MHBMA; and mice eliminate proportionately more MHBMA than DHBMA. DHBMA is formed by hydrolysis
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
607
Table 2 Comparison of biomarker data for urinary metabolites and hemoglobin adducts for rats, mice and humans after exposure to butadiene by inhalationa Biomarker
Rat
Mouse
DHBMA/DHBMA + MHBMAb MHBValc (pmol/g Hb/ppmh) THBVald (pmol/g Hb/ppmh)
0.48 0.086 2.3
Human
Insight
0.38
0.983
Hydrolysis predominates in humans
0.22
0.0047
Activation/detoxication ratio lowest in humans
2.4
Epoxybutanediol levels lower in humans than mice; epoxybutanediol likely source for adducts
14
a Data taken from Van Sittert et al. [29]. Exposure concentrations vary across species, thus the data are normalized for differences in exposure. b DHBMA, 1,2-dihydroxybutyl mercapturic acid; MHBMA, Monohydroxy-3-butenyl mercapturic acid. c MHBVal, Monohydroxybutenyl valine adduct. d THBVal, Trihydroxybutyl valine adduct.
of EB to 1,2-dihydroxy-3-butene followed by conjugation with GSH and MHBMA is formed via conjugation of EB with GSH. These findings indicate that in humans a much greater proportion of EB is metabolized by hydrolysis and a much smaller proportion detoxified by direct conjugation with GSH compared with rats and mice. Differences in urinary metabolite ratios across species are consistent with differences in the relative rates of enzymatic hydrolysis and GSH-conjugation measured in vitro (Fig. 2). The high rate of hydrolysis of EB in humans, as well as the lower activation/deactivation ratio for EB seen in vitro (Fig. 3), is also reflected in the very low levels of monohydroxy-3-butenyl valine adducts (MHBVal) detected in humans compared with rats and mice (Table 2). These adducts are formed by reaction of EB with the N-terminal valine of hemoglobin (Hb). When adjusted for exposure duration and concentration, relative levels of MHBVal were mice\ rats\ \ humans [29]. Formation of MHBVal adducts depends upon the concentration of free EB that is available to react with Hb. The concentration of EB is determined by its relative rates of formation and removal or the activation/detoxication ratio for EB. Interspecies differences in levels of MHBVal seen in vivo are consistent with both interspecies differences in the activation/detoxication ratio determined in vitro and with differences in blood concentrations of EB measured in vivo in rats and mice (Table 1). Taken together these data on in vitro metabolism, Hb adducts, and urinary metabolites suggest that blood concentrations of circulating epoxides such as EB should be lowest in humans compared with rats and mice placing the human at the lowest risk from BD exposure. Formation of DNA adducts by carcinogens or their DNA-reactive metabolites is thought to be an initial step in carcinogenesis. If information is known regarding the DNA adduct or adducts responsible for initiation of cancer, then one can estimate the molecular dose of a carcinogenic compound by measuring the number
608
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
of DNA adducts induced by a given exposure. Numerous DNA adducts of BD epoxide metabolites have been reported in the literature. Adducts that appear to be formed in the highest amounts are those which react with the guanine base at the N-7 position [13,31]. N-7 guanine adducts detected in tissues of rats and mice following exposure to BD concentrations ranging from 20 to 625 ppm reflect interspecies differences in BD metabolism (Fig. 6). Greater amounts of N-7-(2,3,4trihydroxybut-1-yl) – guanine (THB-Gua) and N-7-(hydroxybutenyl)– guanine (EBGua) were detected in tissues of mice compared with rats [13]. THB-Gua adducts were far more abundant than EB-Gua adducts. It has been proposed that the THB-Gua adducts are formed from the reaction of EBD with DNA [13]. Other indirect evidence for the existence in vivo of significant levels of EBD is that from data on trihydroxybutyl valine adducts (THBVal) in Hb (Table 2). THBVal adducts are more prevalent in rodents and humans than are MHBVal adducts and have been proposed to result from the reaction of EBD with the N-terminal valine of Hb. To date no studies have assayed for EBD in blood or tissues of rodents exposed to BD. Molecular dosimetry studies highlight the need for measurements of EBD blood or tissues concentrations in vivo. Finally, an additional DNA-adduct, N7-(1-(hydroxymethyl)-2,3-dihydroxypropyl) guanine, has been detected in rodents exposed to BD in vivo [31]. This adduct is of interest because it is the major DNA adduct found in both rats and mice after exposure to BD, but it is not detected in rodents exposed to EB. The BD-epoxide involved in the formation of these adducts remains to be determined.
Fig. 6. Number of DNA adducts per million bases in lung tissue of rats and mice exposed to various concentrations of butadiene by inhalation. Squares are data for rat lung; circles are data for mouse lung. Filled symbols are data for trihydroxybutyl guanine adducts and open symbols are data for hydroxybutenyl guanine adducts. Data were taken from Koc et al. [13].
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
609
Fig. 7. In vitro rate of diepoxybutane (DEB) formation by microsomes from various cDNA-expressed human CYP isozymes incubated with epoxybutene. Data were taken from Seaton et al. [10].
5. Susceptibility factors Studies using microsomal suspensions prepared from liver samples obtained from humans of various backgrounds and ages have shown a 60-fold variability in oxidation of EB to DEB [10] and a fourfold variability in the oxidation of BD to EB [9]. The origin of this interhuman variability may derive from multiple sources. For example, correlation analysis comparing rates of BD oxidation and activity of a specific CYP450 isozyme, cytochrome P4502E1 (CYP2E1) shows a significantly positive correlation between CYP2E1 activity and rate of BD oxidation [9]. This correlation suggests that expression of this particular isozyme is a significant indicator of the potential for EB formation and thus, potential risk. However, correlation between CYP2E1 activity and BD oxidation rate was not complete, suggesting that other isozymes might be responsible for BD oxidation. The role of other P450 isozymes in both BD oxidation to EB and EB oxidation to DEB has been investigated. Microsomes from human B-lymphoblastoid cells expressing cDNAs encoding several human cytochrome P450 enzymes were exposed to EB or BD in vitro and rates of oxidation were determined [10,32]. Data for EB oxidation to DEB, shown in Fig. 7, indicates that both CYP2E1 and CYP3A4 have significant affinity for EB resulting in high rates of oxidation [10]. Similar studies using BD as a substrate implicated both CYP2E1 and CYP2A6 in BD oxidation to EB in vitro [32]. Taken together, both in vitro studies suggest that CYP2E1 expression is an important indicator of individual susceptibility, but is not the sole isozyme responsible for the conversion of BD to electrophilic and mutagenic metabolites. The studies described above were conducted in vitro and their implications for understanding the mechanisms of BD carcinogenicity can be assessed by determining the extent to which specific CYP450 isozymes metabolize BD in vivo. In vivo studies, using either specific metabolic inhibitors or transgenic animals in which expression of specific isozymes is eliminated can provide information critical to
610
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
assessing the extent to which in vitro correlations between isozyme activity and rates of epoxide formation are maintained under in vivo exposure situations [33,34]. Such studies have been conducted in transgenic mice (cyp2e1 − / − ) that lack the gene for expression of the CYP2E1 enzyme [34]. Both cyp2e1 − / − mice and the parental normal strain of the mice with an active gene for CYP2E1 (cyp2e1 + / + ) were exposed to BD in a closed chamber (Fig. 8). Mice pretreated with specific enzyme inhibitors were also exposed to BD in a similar manner [33]. The concentration of BD in the chamber declined over time due to uptake of BD from the chamber into the mice and metabolism of BD. Thus, disappearance of BD from the chamber is directly related to the extent of BD uptake by the animal and the rate of BD metabolism. As seen in Fig. 8, the most rapid rates of BD disappearance from the chamber were observed when cyp2e1 + / + mice were exposed to BD. The slowest rates of BD disappearance from the chamber were seen in mice pretreated with 1-aminobenzotriazole (ABT), a non-specific inhibitor of CYP450 isozymes. Since BD metabolism was completely eliminated in ABT-treated mice, the disappearance of BD from the chamber is due solely to the uptake of BD into blood and partitioning into tissues, especially fat, which has the highest solubility for BD [35]. For cyp2e1 − / − mice the rate of BD disappearance was intermediate between the ABT pretreated mice and the cyp2e1 + / + mice indicating that in vivo another P450 isozyme or isozymes, in addition to CYP2E1, is responsible for BD oxidation. These results are consistent with in vitro observations and suggest that expression of other P450 isozymes in addition to CYP2E1 may be important susceptibility factors for potential risk after exposure to BD. In vitro work with microsomal
Fig. 8. Disappearance of butadiene from a closed chamber containing mice pretreated with the CYP450 inhibitor 1-aminobenzotriazole (ABT), transgenic mice which lack the gene for expression of the CYP2E1 enzyme (cyp2e1 − / − ), or the parental strain of mice with an active gene for CYP2E1 (cyp2e1 + / + ). The rate of disappearance of butadiene is the result of uptake of butadiene into the mouse tissues only (ABT), uptake into tissues and non-CYP2E1 mediated oxidation of butadiene (cyp2e1 − / − ), or uptake into tissues, non-CYP2E1 and CYP2E1 mediated oxidation of butadiene (cyp2e1 + / + ). Data were taken from Jackson et al. [33,34].
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
611
Fig. 9. Micronucleated polychromatic erythrocytes (PCE) per 1000 PCE in bone marrow of mice exposed to butadiene gas in a closed chamber and euthanized 24 h after the start of the exposure. Control, No pretreatment, no butadiene exposure; BD, No pretreatment, butadiene exposure; DCE + BD, Intraperitoneal injection of 1,1-trans-dichloroethylene (DCE) prior to butadiene exposure; ABT + BD, Intraperitoneal injection of 1-aminobenzotriazole (ABT) prior to butadiene exposure. DCE is a specific inhibitor of CYP2E1. ABT is a non-specific inhibitor of all CYP450. * Indicates significantly different from other treatment groups at a PB 0.05. Data were taken from Jackson et al. [33].
suspensions prepared from mouse lung, liver, and kidney samples suggests that these additional isozymes may be CYP2A5 and CYP4B1 [36]. CYP2A5 is the mouse analog to the human isozyme CYP2A6; both isozymes are involved in the hydroxylation of coumarin. CYP4B1 is active in mouse kidney. The gene for human CYP4B1 has been cloned; the cDNA have been expressed and determined to be inactive [36]. The relationship between level of enzyme activity and genotoxic potency was also evaluated in mice exposed to BD under closed chamber conditions [33]. In these studies mice were pretreated with ABT or 1,2-trans-dichloroethylene (DCE), a specific CYP2E1 inhibitor, prior to exposure to BD in a closed chamber. Mice that were not pretreated with enzyme inhibitors, but were exposed to BD, in the closed chamber, were used as positive controls and mice, pretreated with inhibitors, but not exposed to BD were used as negative controls. At the end of the exposure mice were removed from the chamber. Genotoxicity was assessed at 24 h after the start of exposure by quantitating MN in bone marrow polychromatic erythrocytes [33]. ABT pretreatment reduced the level of MN in BD-exposed mice to that of the unexposed controls (Fig. 9). DCE pretreatment reduced the number of MN in BD-exposed mice compared to that seen in the BD-exposed controls, but not to the level seen in the ABT treated mice. Thus, although inhibition of CYP2E1 decreased BD-mediated genotoxicity, it did not prevent clastogenic damage due to BD exposure, consistent with the results from metabolism studies suggesting that other P450 isoforms are involved in BD activation.
612
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
Since formation of MN is due primarily to DEB [23,25], and not EB, these studies suggest that elevated MN levels in the absence of CYP2E1-mediated oxidation must be the result of the oxidation of EB to DEB by CPY450 isozymes other than CYP2E1. In vitro studies with cDNA expressed human CPY450 suggest that CYP3A4 may be the other isozyme responsible for oxidation of EB to DEB in humans. Taken together in vitro and in vivo studies suggest that differences in expression of CYP2E1, CYP2A6, and CYP3A4 in humans may be important susceptibility factors for individual exposure to BD. As noted previously, these isozymes appear to be correlated with both formation of reactive epoxides and expression of clastogenic effects. However, other enzymes also play a role in the overall metabolism of BD, and in vitro studies have also demonstrated differences in the activities of these enzymes among the individual human tissue samples assessed. For example, activity of epoxide hydrolase varied among 12 human samples studied, by a factor of sixfold for hydrolysis of EB [9] and threefold for hydrolysis of DEB [16]. Similarly, activity of glutathione-S-transferase varied across human samples analyzed being on the order of seven or fivefold for conjugation of EB or DEB, respectively [9,17]. The enzymes discussed above demonstrate the steps in the toxicokinetic processes for which there might exist potential risk factors for sensitive subpopulations. Increased rates of oxidation and decreased rates of detoxication might be expected to increase levels of reactive epoxides in target tissues and thus affect individual risk. Less attention has been paid to potential steps in the toxicodynamic processes that might also alter risk. For example, enzymes responsible for repair of mismatched DNA bases resulting from epoxide adducts or repair of single strand breaks in DNA might also show differences among individuals and thus be additional risk factors for susceptibility.
6. Conclusions In summary, mechanistic studies provide insights into critical genotoxic events in the initiation of cancer, metabolites responsible for cancer, sensitive biomarkers for exposure, and potential risk factors for individual susceptibility. Consideration of mechanisms of BD carcinogenicity can provide insights into differences in cancer potency between rodents and serve to elucidate the extent to which BD exposure may cause cancer in humans. Mechanistic research in the area of biochemical toxicology has pointed to the role of DEB in BD carcinogenesis and to the likely pathways for BD metabolism in humans. Molecular biology studies suggest a major role for at least DEB in BD genotoxicity/carcinogenicity. Molecular dosimetry studies involving hemoglobin adducts and urinary metabolites show differences in human/rodent metabolism that parallel differences highlighted in biochemical toxicology studies and suggest, in general, humans to be at decreased risk from BD exposure. However, studies of specific susceptibility factors indicate that expression of specific isozymes involved in BD formation can alter risk.
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
613
References [1] K.D. Brunnemann, M.R. Kagan, J.E. Cox, D. Hoffman, Analysis of 1,3-butadiene and other gas-phase components in cigarette mainstream and sidestream smoke by gas chromatography – mass selective detection, Carcinogenesis 11 (1990) 1863 – 1868. [2] N. Pelz, A.M. Dempster, P.R. Shore, Analysis of low molecular weight hydrocarbons including 1,3-butadiene as engine exhaust gases using an aluminum oxide porous-layer open-tubular fused-silica column, J. Chrom. Sci. 28 (1990) 230 – 235. [3] Environmental Protection Agency, Preliminary draft list of categories and subcategories under Section 112 of the Clean Air Act, Fed. Reg. 56 (1991) 28548 – 28557. [4] J.E. Huff, R.L. Melnick, H.A. Solleveld, J.K. Haseman, M. Powers, R.A. Miller, Multiple organ carcinogenicity of 1,3-butadiene in B6C3F1 mice after 60 weeks of inhalation exposure, Science 227 (1985) 548 –549. [5] R.L. Melnick, J. Huff, B.J. Chow, R.A. Miller, Carcinogenicity of 1,3-butadiene in B6C3F1 mice at low exposure concentrations, Cancer Res. 50 (1990) 6592 – 6599. [6] P.E. Owen, J.R. Glaister, I.F. Gaunt, D.H. Pullinger, Inhalation toxicity studies with 1,3-butadiene. III. Two-year toxicity/carcinogenicity studies in rats, Am. Ind. Hygiene Assoc. J. 48 (1987) 407–413. [7] E. Delzell, N. Sathiakumar, M. Hovinga, M. Macaluso, J. Julian, R. Larson, P. Cole, D.C. Muir, A follow-up study of butadiene production workers, Toxicology 113 (1996) 182 – 189. [8] M.W. Himmelstein, J.F. Acquavella, L. Recio, M.A. Medinsky, J.A. Bond, Toxicology and epidemiology of 1,3-butadiene, Crit. Rev. Toxicol. 27 (1997) 1 – 108. [9] G.A. Csana´ dy, F.P. Guengerich, J.A. Bond, Comparison of the biotransformation of 1,3-butadiene and its metabolite, butadiene monoepoxide, by hepatic and pulmonary tissues from humans, rats and mice, Carcinogenesis 13 (1992) 1143 – 1153. [10] M.J. Seaton, M.H. Follansbee, J.A. Bond, Oxidation of 1,2-epoxy-3-butene to 1,2:3,4-diepoxybutane by cDNA-expressed human cytochromes P450 2E1 and 3A4 and human, mouse, and rat liver microsomes, Carcinogenesis 16 (1995) 2287 – 2293. [11] M.W. Himmelstein, M.J. Turner, B. Asgharian, J.A. Bond, Comparison of blood concentrations of 1,3-butadiene and butadiene epoxides in mice and rats exposed to 1,3-butadiene by inhalation, Carcinogenesis 15 (1994) 1479 –1486. [12] J.R. Thornton-Manning, A.R. Dahl, W.E. Bechtold, W.C. Griffith Jr., R.F. Henderson, Disposition of butadiene monoepoxide and butadiene diepoxide in various tissues of rats and mice following a low-level inhalation exposure to 1,3-butadiene, Carcinogenesis 16 (1995) 1723 – 1731. [13] H. Koc, N.Y. Tretyakova, V.E. Walker, R.F. Henderson, J.A. Swenberg, Molecular dosimetry of N-7 guanine adduct formation in mice and rats exposed to 1,3-butadiene, Chem. Res. Toxicol. 12 (1999) 566 –574. [14] K.A. Richardson, H.J.J.J. Megens, J.D. Webb, J.J. Van Sittert, Biological monitoring of butadiene exposure by measurement of haemoglobin adducts, Toxicology 113 (1996) 112 – 118. [15] K.A. Richardson, M.M.C.G. Peters, B.A. Wong, H.J.J.J. Megens, P.A. Van Elburg, E.D. Booth, et al., Quantitative and qualitative differences in the metabolism of 14C-1,3-butadiene in rats and mice: relevance to cancer susceptibility, Toxicol. Sci. 49 (1999) 86 – 201. [16] P.J. Boogaard, J.A. Bond, The role of hydrolysis in the detoxification of 1,2:3,4-diepoxybutane by human, rat, and mouse liver and lung in vitro, Toxicol. Appl. Pharmacol. 141 (1996) 617 – 627. [17] P.J. Boogaard, S.C. Sumner, J.A. Bond, Glutathione conjugation of 1,2:3,4-diepoxybutane in human liver and mouse liver and lung in vitro, Toxicol. Appl. Pharmacol. 136 (1996) 307 – 316. [18] G.L. Kedderis, Toxicokinetics: biotransformation of toxicants, in: J. Bond (Ed.), General Principles. In: Comprehensive Toxicology, vol. 1, Elsevier Science, UK, 1997, pp. 115 – 148. [19] J.A. Bond, M.W. Himmelstein, M. Seaton, P. Boogaard, M.A. Medinsky, Metabolism of butadiene by mice, rats, and humans: a comparison of physiologically based toxicokinetic model predictions and experimental data, Toxicology 113 (1996) 48 – 54. [20] M.W. Himmelstein, B. Asgharian, J.A. Bond, High concentrations of butadiene epoxides in livers and lungs of mice compared to rats exposed to 1,3-butadiene, Toxicol. Appl. Pharmacol. 132 (1995) 281–288.
614
J.A. Bond, M.A. Medinsky / Chemico-Biological Interactions 135–136 (2001) 599–614
[21] W.E. Bechtold, M.R. Strunk, J.R. Thornton-Manning, R.F. Henderson, Analysis of butadiene, butadiene monoxide, and butadiene dioxide in blood by gas chromatography/gas chromatography/ mass spectroscopy, Chem. Res. Toxicol. 8 (1995) 182 – 187. [22] A.-M. Steen, K.G. Meyer, L. Recio, Characterization of hprt mutations following 1,2-epoxy-3butene exposure of human TK6 cells, Mutagenesis 12 (1997) 359 – 364. [23] L. Recio, C.J. Saranko, A.-M. SteenPart II: Roles of two metabolites of 1,3-butadiene in mediating its in vivo genotoxicity, HEI Research Report 1,3-Butadiene: Cancer, mutations and adducts, 92 (2000) 49 –87 [24] A.-M. Steen, K.G. Meyer, L. Recio, Analysis of hprt mutations occurring in human TK6 lymphoblastoid cells following exposure to 1,2:3,4-diepoxybutane, Mutagenesis 12 (1997) 61 – 67. [25] M.N. Murg, M. Schuler, D.A. Eastmond, Evaluation of micronuclei and chromosomal breakage in the 1cen –q12 region by the butadiene metabolites epoxybutene and diepoxybutane in cultured human lymphocytes, Mutagenesis 14 (1999) 541 – 546. [26] J.E. Cochrane, T.R. Skopek, Mutagenicity of butadiene and its epoxide metabolites. I. Mutagenic potential of 1,2-epoxybutene, 1,2:3,4-diepoxybutane and 3,4-epoxy-1,2-butanediol in cultured human lymphoblasts, Carcinogenesis 15 (1994) 713 – 771. [27] I.-D. Adler, U. Kliesch, L. Nylund, K. Peltonen, In vitro and in vivo mutagenicity of the butadiene metabolites butadiene diolepoxide, butadiene monoepoxide and diepoxybutane, Mutagenesis 12 (1997) 339 –345. [28] I.-D. Adler, U. Kliesch, C. Tiveron, F. Pacchierotti, Clastogenicity of diepoxybutane in bone marrow cells and germ cells of mice, Mutagenesis 10 (1995) 535 – 541. [29] N.J. van Sittert, H.J.J.J. Megens, W.P. Watson, P.J. Boogaard, Biomarkers of exposure to 1,3-butadiene as a basis for cancer risk assessment, Toxicol. Sci. 56 (2000) 189 – 202. [30] W.E. Bechtold, M.R. Strunk, I.Y. Chang, J.B. Ward Jr., R.F. Henderson, Species differences in urinary butadiene metabolites: comparisons of metabolite ratios between mice, rats, and humans, Toxicol. Appl. Pharmacol. 127 (1994) 44 – 49. [31] P.J. Boogaard, K.P. DeKloe, N.J. Van Sittert, N7-(1-(Hydroxymethyl)-2,3-dihydroxypropyl) guanine is the major DNA adduct after exposure to 1,3-butadiene but not 1,2-epoxy-3-butene, Toxicol. Sci. 54 (2000) S229. [32] R.J. Duescher, A.A. Elfarra, Human liver microsomes are efficient catalysts of 1,3-butadiene oxidation: Evidence for major roles by cytochromes P450 2A6 and 2E1, Arch. Biochem. Biophys. 311 (1994) 342 –349. [33] T.E. Jackson, P.D. Lilly, L. Recio, P.M. Schlosser, M.A. Medinsky, Inhibition of cytochrome P450 2E1, decreases but does not eliminate, genotoxicity mediated by 1,3-butadiene, Toxicol. Sci. 55 (2000) 266 –273. [34] T.E. Jackson, M.A. Medinsky, B.E. Butterworth, F.J. Gonzalez, L. Recio, The use of cytochrome P450 2E1 knockout mice to develop a mechanistic understanding of carcinogen biotransformation and toxicity, CIIT Activities 19 (1999) 1 – 9. [35] M.A. Medinsky, T.L. Leavens, G.A. Csana´ dy, M.L. Gargas, J.A. Bond, In vivo metabolism of butadiene by mice and rats: a comparison of physiological model predictions and experimental data, Carcinogenesis 15 (1994) 1329 – 1340. [36] A.A. Elfarra, R.J. Krause, R.A. Kemper, Cellular and molecular basis for species, sex and tissue differences in 1,3-butadiene metabolism, Chem.-Biol. Interact. 135 – 136 (2001) 239 – 248 (this volume).
.