Biomarkers of exposure and effect as indicators of potential carcinogenic risk arising from in vivo metabolism of ethylene to ethylene oxide
Vernon E. Walker1,4,9,
Kuen-Yuh Wu2,5,
Patricia B. Upton2,
Asoka Ranasinghe2,
Nova Scheller2,
Myung-Haeng Cho2,6,
Jane S. Vergnes3,7,
Thomas R. Skopek2,8 and
James A. Swenberg2
1 Department of Pathology and
2 Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill,NC 27599-7525,
3 Bushy Run Research Center, Union Carbide Corp., Export, PA 15632 and
4 Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA
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Abstract
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The purposes of the present study were: (i) to investigate the potential use of several biomarkers as quantitative indicators of the in vivo conversion of ethylene (ET) to ethylene oxide (EO); (ii) to produce molecular dosimetry data that might improve assessment of human risk from exogenous ET exposures. Groups (n = 7/group) of male F344 rats and B6C3F1 mice were exposed by inhalation to 0 and 3000 p.p.m. ET for 1, 2 or 4 weeks (6 h/day, 5 days/week) or to 0, 40, 1000 and 3000 p.p.m. ET for 4 weeks. N-(2-hydroxyethyl)valine (HEV), N7-(2-hydroxyethyl) guanine (N7-HEG) and Hprt mutant frequencies were assessed as potential biomarkers for determining the molecular dose of EO resulting from exogenous ET exposures of rats and mice, compared with background biomarker values. N7-HEG was quantified by gas chromatography coupled with high resolution mass spectrometry (GCHRMS), HEV was determined by Edman degradation and GCHRMS and Hprt mutant frequencies were measured by the T cell cloning assay. N7-HEG accumulated in DNA with repeated exposure of rodents to 3000 p.p.m. ET, reaching steady-state concentrations around 1 week of exposure in most tissues evaluated (brain, liver, lung and spleen). The doseresponse curves for N7-HEG and HEV were supralinear in exposed rats and mice, indicating that metabolic activation of ET was saturated at exposures
1000 p.p.m. ET. Exposures of mice and rats to 200 p.p.m. EO for 4 weeks (as positive treatment controls) led to significant increases in Hprt mutant frequencies over background in splenic T cells from exposed rats and mice, however, no significant mutagenic response was observed in the Hprt gene of ET-exposed animals. Comparisons between the biomarker data for both unexposed and ET-exposed animals, the doseresponse curves for the same biomarkers in EO-exposed rats and mice and the results of the rodent carcinogenicity studies of ET and EO suggest that too little EO arises from exogenous ET exposure to produce a significant mutagenic response or a carcinogenic response under standard bioassay conditions.
Abbreviations: EO, ethylene oxide; ET, ethylene; GCHRMS, gas chromatography coupled with high resolution mass spectrometry; HEV, N-(2-hydroxyethyl)valine; Hprt, hypoxanthine-guanine phosphoribosyltransferase; N7-HEG, N7-(2-hydroxyethyl)guanine; TWA, time-weighted average.
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Introduction
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Ethylene (ET) (CAS no. 74-85-1), the most used petrochemical in the world, is metabolized by mammalian cells to ethylene oxide (EO) (CAS no. 75-21-8), a direct acting alkylating agent with the potential to induce cytogenetic alterations, mutations and cancer (1). Over 95% of ET for industrial use is produced by steam cracking from naphthas, with the major ET-derived products being low density and high density polyethylene, ethylene dichloride, EO and vinyl chloride (2). ET is also used for controlling the ripening of fruit. Exogenous sources of ET, which are not occupational in nature, include burning of vegetation, degradation of agricultural wastes and refuse, active and passive smoking and incomplete combustion of fossil fuels (2). Furthermore, ET is formed endogenously from several possible sources, including lipid peroxidation of unsaturated fats, oxidation of free methionine, oxidation of hemin in hemoglobin and metabolism by intestinal bacteria (35). Numerous studies have shown that ET from both endogenous and exogenous sources is metabolized to EO in vivo in rats, mice and humans (36). EO is currently classified by the IARC as a known human carcinogen based on sufficient evidence in animals together with strong evidence in humans of a relevant mechanism for carcinogenicity, rather than on sufficient evidence in epidemiological studies of EO-exposed workers (1). Nonetheless, the relative human health risk from in vivo conversion of exogenously and endogenously produced ET to EO remains unclear.
Although there were no concentration-dependent increases in toxicity or tumor induction in a 2 year carcinogenicity study of F344 rats exposed to 0, 300, 1000 or 3000 p.p.m. ET (7), this chemical has been speculated to have some carcinogenic potential in exposed animals based upon the pharmacokinetics of ET and EO in rats and mice (810). Bolt and Filser (10) have investigated the elimination of ET in SpragueDawley rats using a closed exposure system and a two-compartment pharmacokinetic mode1. Above concentrations of ~1000 p.p.m. ET the Vmax for ET was reached, therefore, higher exposures would not yield greater conversion to EO. Exposure of rats to concentrations of ET
1000 p.p.m. correspond to a theoretical exposure to ~6 p.p.m. EO. Also, the theoretical and the experimental data coincided at exposures below 80 p.p.m. ET; exposures of ~40 p.p.m. ET correspond to 1 p.p.m. EO, which is the current Occupational Safety and Health Administration standard for EO [i.e. 8 h time-weighted average (TWA) per 40 h week].
In contrast to ET, EO has been demonstrated to be mutagenic and carcinogenic in rats and mice chronically exposed to high concentrations of this compound (1,1113). Furthermore, EO has been shown to alkylate (2-hydroxyethylate) cellular macromolecules, including proteins, RNA and DNA, and the resulting genetic damage is thought to play a critical role in induction of mutations and cancers in rodents (1). Identical reaction products, which have been observed after exposure of rodents to ET, have been attributed to its conversion to EO (8,9,14). One of the hemoglobin adducts of EO, N-(2-hydroxyethyl)valine (HEV), has been used extensively as a molecular dosimeter for exposures to EO in experimental animals and humans (1). Likewise, the major DNA adduct of EO, N7-(2-hydroxyethyl)guanine (N7-HEG), has been evaluated as a molecular dosimeter following single and multiple exposures of rats and mice to EO (see ref. 6; 1519). Also, repeated inhalation exposure to high concentrations of EO has been found to produce a linear doseresponse effect for increased mutant frequencies in the hypoxanthine guanine phosphoribosyltransferase (Hprt) gene of lymphocytes from exposed mice (18). Hence, there is a theoretical risk of a carcinogenic potential of ET based on its in vivo conversion to EO and the assumption of a linear doseresponse effect (from high to low exposures) for the events involved in EO-induced carcinogenesis.
The goals of the present study were: (i) to investigate the potential of HEV, N7-HEG, and Hprt mutant frequencies to act as quantitative indicators of the in vivo conversion of ET to EO; (ii) to generate molecular dosimetry data that might improve risk assessment for humans exposed to ET in the environment or workplace. Because alkylation of proteins and DNA during ET exposure has been ascribed to its metabolism to EO, it is important to define accurately the levels of EO produced by endogenous processes in rats, mice and humans and by repeated exogenous exposure of rats and mice to ET. Previous studies in rodents indicate that measurement of hemoglobin adducts, and perhaps DNA adducts, provides a means of determining the levels of EO resulting from exogenous exposure to ET (35,20,21). Accurate predictions should be possible because doseresponse data for HEV, N7-HEG and Hprt mutant T cells induced in EO-exposed rats and mice (1519) are available for comparisons with results from ET-exposed animals.
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Materials and methods
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Chemicals, enzymes and medium components
ET at a purity of 99.99% (vendor specified) was purchased from Matheson Gas Products (Twinsburg, OH) and remained stable for the duration of animal exposures. Pentafluorobenzyl bromide, potassium hydroxide, t-butyl nitrite, toluene and tetrabutyl ammonium sulfate were obtained from Aldrich Chemical (Milwaukee, WI). GC2 hexane, dichloromethane and ethyl acetate were obtained from Baxter Diagnostic (McGaw, IL). The sources of DNA purification grade lysis buffer (100 mM Tris, pH 8.0, 0.2 M NaCl, 0.5% N-laurosarcosine, 4 M urea and 10 mM 1,2-diaminocyclohexane-N-tetraacetic acid), 70% phenol:water:chloroform reagent, RNase T1 and RNase A and other DNA isolation and HPLC reagents have been listed elsewhere (16). Pentafluorophenyl isothiocyanate was purchased from Fluka Chemical (Ronkonkoma, NY). Formamide (spectrograde), 2-propanol (HPLC grade) and sodium carbonate (HPLC grade) were purchased from Fisher Scientific (Raleigh, NC). Diethyl ether and pentane were obtained from American Scientific (Charlotte, NC) and VWR (Marietta, GA), respectively. Other reagents used for analysis of HEV were described in a previous study (15). Medium and cell culture components for Hprt mutation assays were obtained from the indicated sources: fetal bovine serum, RPMI 1640 medium, HEPES buffer, glutamine, MEM non-essential amino acids, penicillin/streptomycin and sodium pyruvate (Life Technologies, Grand Island, NY); HL-1 medium (Ventrex Laboratories, Portland, ME); Lympholyte M (Accurate Chemical and Scientific, Westbury, NY); 2-mercaptoethanol, concanavalin A and 6-thioguanine (Sigma, St Louis, MO).
Animals, husbandry and exposures
Male F344 rats (~8 weeks old) were obtained from Harlan SpragueDawley (Indianapolis, IN); male B6C3F1 mice (~8 weeks old) were purchased from Charles River Laboratories (Portage, MI). All animals were free of virus titers, as determined by standard rodent virus antibody assays (Microbiologicals Associates, Bethesda, MD). The housing of animals conformed with NIH guidelines (NIH Publication no. 86-23, 1985). Animals were housed in stainless steel wire mesh cages in temperature and humidity controlled rooms (72 ± 4°F and 50 ± 10%) with a 12 h light/dark cycle. They had free access to food (certified Agway Prolab Animal Diet Rat, Mouse, Hamster 3000; Agway) and tap water except during periods of inhalation exposure. During an acclimation period of 2 weeks, animals were randomly divided into groups of seven rats and seven mice per treatment group, as described below. All procedures involving the use of animals were approved by the Animal Care and Use Committees of the institutions where the chemical exposures and animal experiments were performed.
Rodents were exposed to 0 or target concentrations of 40, 1000 or 3000 p.p.m. ET for 4 weeks (6 h/day, 5 days/week) using four stainless steel whole body exposure chambers with glass doors and windows for animal observation. All supply air was HEPA-filtered before being introduced into the chambers and the flow rate through each chamber was maintained at 1314 changes/h. Rodents in one chamber received filtered air only as a control group. ET from compressed gas cylinders was metered by a pressure regulator and a flow meter through stainless steel tubing for delivery into each ET exposure chamber. Each exposure chamber was monitored for concentrations of ET approximately seven times during each 6 h exposure period, using flame ionization gas chromatography. Chamber concentrations of EO were also determined in the air-only and ET chambers once weekly. Finally, additional groups of rats and mice were concurrently exposed to 200 p.p.m. EO for 4 weeks (6 h/day, 5 days/week) in a fifth inhalation chamber. Throughout the study, animals were weighed weekly and observed for overt signs of chemically induced toxicity.
Several experiments were conducted in control and ET-exposed animals. To determine the effect of repeated ET exposure on the accumulation of DNA adducts in tissues of rats and mice, groups of animals (n = 7/exposure level/species) were exposed to chamber air or nominal 3000 p.p.m. ET for 1, 2 or 4 weeks (6 h/day, 5 days/week). To determine the doseresponse characteristics for formation of both hemoglobin and DNA adducts following repeated exposure, groups of seven rats and seven mice were exposed to chamber air or nominal 40, 1000 or 3000 p.p.m. ET for 4 weeks (6 h/day, 5 days/week). Scheduled necropsies were conducted within 2 h after cessation of the last exposure. Animals were killed by exsanguination under CO2 anesthesia. At necropsy, brain, liver, lung and spleen were removed, frozen and stored at -80°C until DNA isolation. Red blood cells from animals in the doseresponse study were washed with saline and frozen at 80°C until globin isolation.
The third experiment was designed to investigate the mutagenic potential of ET at the Hprt locus of T lymphocytes following repeated exposure of rats and mice. To this end, the doseresponse effect for Hprt mutant frequencies in splenic T cells was evaluated in animals exposed to 0, 40, 1000 or 3000 p.p.m. ET for 4 weeks (6 h/day, 5 days/week). For comparison, Hprt mutant frequencies were measured in splenic T cells from positive control animals exposed to 200 p.p.m. EO for 4 weeks (6 h/day, 5 days/week). Groups of air-, ET- and EO-exposed rats (n = 7/exposure level or chemical) were necropsied at 5 weeks after the cessation of exposure while groups of concurrently exposed mice (n = 7/group) were necropsied at 8 weeks post-exposure. At necropsy, animals were killed as described and their spleens were removed asceptically for isolation of lymphocytes. At the same time, several untreated rats and mice were necropsied to provide sources of splenic lymphocytes to be used as syngeneic `feeder' cells.
Analysis of HEV in hemoglobin of rats and mice
The methods for isolation of globin, derivatization of globin samples and quantitation of HEV, using gas chromatography coupled with high resolution mass spectrometry (GCHRMS), have been described previously (3,15,2224). In brief, globin was isolated from lysed red blood cells of individual animals, an internal standard of [2H4]EO-treated rat globin was added to aliquots of globin samples and a modified Edman degradation method using pentafluorophenyl isothiocyanate was employed for cleavage and derivatization of the N-terminal valine from globin. Quantitation of HEV using GCHRMS was achieved by monitoring fragment ions of the analyte (m/z 348.0556) and an internal standard (m/z 352.0803) and the polyfluorokerosine lock mass (m/z 330.9792) (24).
Analysis of N7-HEG in tissue DNA of rats and mice
The methods for isolation of DNA, derivatization of DNA samples and quantitation of N7-HEG have been described elsewhere (19,21). In brief, N7-HEG was depurinated from individual DNA samples (spiked with [13C4]N7-HEG internal standard) by neutral thermal hydrolysis, converted to its corresponding xanthine analog, derivatized with pentafluorobenzyl bromide twice and quantified by GCHRMS. Quantitation of N7-HEG in each sample was based on the ratio of the peak area of the analyte (m/z 555.0515) to that of an internal standard (m/z 559.0649) and comparison to a calibration curve for N7-HEG divided by the amount of guanine in each liver sample and DNA content for lymphocyte samples.
Isolation and culture of Hprt mutant lymphocytes from rats and mice
The procedures used for isolation and culture of lymphocytes from rodent spleen have been described in detail previously (25,26). Briefly, lymphocytes were isolated by macerating spleens individually in medium and layering the cells on Ficoll gradients (Lympholyte M) for collection, washing and resuspension in supplemented medium. Isolated lymphocytes were stimulated overnight with mitogen and then counted and diluted to 4x105 cells/ml for seeding into 96-well microtiter plates with supplemented medium to determine cloning efficiencies and to select for Hprt mutant colonies. Spleens from individual animals were coded before processing so that Hprt mutant colonies could be scored, 10 days after plating, without reference to the treatment group. Mutant clones from control and EO-exposed rats and mice were collected and frozen for molecular analyses (V.E.Walker, T.R.Fennell, J.P.MacNeelay, P.B.Upton, T.R.Craft, D.M.Walker, Q.Meng, T.R.Skopek, and J.A.Swenberg, in preparation.)
Statistical analyses
The one-way Student's t-test was used to evaluate the differences between HEV concentrations in globin or, alternatively, N7-HEG concentrations in tissues of control versus ET-exposed animals of the same species. Student's t-test was also used to assess the differences between N7-HEG concentrations of given tissues from ET-exposed rats versus mice. The P values used to describe the differences in Hprt mutant frequencies between groups of sham-exposed and groups of ET- or EO-exposed animals were determined using the MannWhitney U-statistic. A P value <0.05 was considered significant.
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Results
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Animal exposures
The average daily inhalation chamber concentrations (± SD) for the 4 week exposure period were 39.1 ± 0.9, 966 ± 29 and 2995 ± 116 p.p.m. for the target concentrations of 40, 1000 and 3000 p.p.m. ET, respectively. The average daily inhalation chamber concentration was 202 ± 7 p.p.m. for the EO target concentration (V.E.Walker et al., in preparation). In the air-only control chamber atmosphere neither ET nor EO could be detected at or above the estimated minimum detection limits of 4 and 1 p.p.m., respectively. Chemical exposures caused no clinical signs of toxicity and the average body weight gains of the treatment groups were not significantly different from that of the control group (data not shown).
Endogenous formation of HEV and N7-HEG in rats and mice
Background levels of HEV were measured in hemoglobin of rats and mice, with the mean values (± SD) being 0.10 ± 0.01 and 0.05 ± 0.01 pmol/mg globin, respectively (Table I
). Comparable concentrations of HEV have been reported in groups of rats and mice without known exposure to hydroxyethylating agents or any known precursor (3,15).
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Table I. HEV (pmol/mg globin ± SD) in blood of F344 rats and B6C3F1 mice (8-week-old) exposed by inhalation to 03000 p.p.m. ethylene for 6 h/day, 5 days/week for 4 weeks
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Analyses for N7-HEG in DNA of control rats and mice demonstrated substantially lower levels of this adduct than were reported in earlier evaluations of rodent tissues (16,27,28). The data in Tables II and III
show that similar background levels of N7-HEG were found in all tissue samples (n = 816/tissue type) from both species, ranging narrowly around 0.20.3 pmol adduct/µmol guanine in brain, liver, lung and spleen from F344 rats and B6C3F1 mice (see ref. 19 for a sample chromatogram).
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Table II. N7-HEG (pmol/µmol of guanine ± SD) in tissues of F344 rats (8-week-old) exposed by inhalation to 03000 p.p.m. ethylene for 6 h/day, 5 days/week for 4 weeks
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Table III. N7-HEG (pmol/µmol of guanine ± SD) in tissues of B6C3F1 mice (8-week-old) exposed by inhalation to 03000 p.p.m. ethylene for 6 h/day, 5 days/week for 4 weeks
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Effects of ET exposure duration on the formation of N7-HEG in rats and mice
The effects of exposure duration on the accumulation of N7-HEG in DNA of brain, liver, lung and spleen were assessed in rats and mice exposed to 3000 p.p.m. ET for 1, 2 or 4 weeks (6 h/day, 5 days/week). N7-HEG levels increased significantly in all tissues of both species during the first week of ET exposure and then accumulated more slowly and appeared to approach steady-state concentrations between 1 and 4 weeks of exposure (Figure 1
). Concentrations of N7-HEG were greatest in liver DNA from ET-exposed rats, but the extent of adduct accumulation during repeated exposures varied <2-fold among tissues from each species. Concurrent exposures of rats and mice to 3000 p.p.m. for 4 weeks led to 2- to 3-fold higher concentrations of N7-HEG in rat DNA of all tissues compared with mouse DNA (Tables II and III
).
Effects of ET exposure concentration on the formation of HEV, N7-HEG and Hprt mutant T cells in rats and mice
The effects of exposure concentration on formation of HEV and N7-HEG were evaluated in tissues of rats and mice necropsied immediately after the cessation of 4 weeks exposure (6 h/day, 5 days/week) to 0, 40, 1000 or 3000 p.p.m. ET. Repeated ET exposure led to non-linear doseresponse curves for both HEV and N7-HEG in rats and mice. Graphs (not shown) of the data in Tables IIII

demonstrated that hemoglobin and DNA adduct concentrations were significantly increased following exposure to 40 p.p.m. ET, continued to increase with exposure to 1000 p.p.m. and approached a plateau in all tissues (with the possible exception of N7-HEG in rat liver) after exposure to 3000 p.p.m. N7-HEG concentrations in rats exposed to 1000 and 3000 p.p.m. ET were significantly higher than those in corresponding tissues of similarly exposed mice (P < 0.05), while HEV values were remarkably similar in both species at all three exposure levels. In addition, N7-HEG levels in brain and lungs of rats exposed to 40 p.p.m. ET were significantly higher than those in the same tissues of concurrently exposed mice (P < 0.05).
The effects of ET exposure concentration on Hprt mutant frequencies in splenic T cells were assessed in rats and mice necropsied 5 and 8 weeks, respectively, after cessation of 4 week exposures to 0, 40, 1000 and 3000 p.p.m. ET. Based upon results of an earlier mutagenicity study of inhaled EO in mice (18), additional animals were exposed to 200 p.p.m. EO for 4 weeks to provide positive controls for EO-induced Hprt mutant frequencies. The cloning efficiencies of T cells from ET- and EO-exposed rats and mice were indistinguishable from control values, ranging from 4.1 to 14.3% in rats and 1.2 to 4.4% in mice. The mean Hprt mutant frequencies in T cells from control rats and mice were 1.2 ± 0.3x106 and 2.0 ± 0.8x106, respectively. These cloning efficiency and mutant frequency values resemble those reported previously for splenic T cells of untreated rats and mice (18,25,26,29). Repeated exposures to ET did not increase Hprt mutant frequencies in splenic T cells of exposed animals compared with control rats and mice (i.e. 1.3 ± 0.7x106 in rats and 1.7 ± 0.5x106 in mice) necropsied 5 and 8 weeks post-exposure, respectively. Average mutant frequencies in rats exposed to 40, 1000 and 3000 p.p.m. ET were 2.1 ± 0.9x 106, 1.4 ± 0.5x106 and 1.4 ± 0.7x106, respectively, while concurrently exposed mice had mutant frequencies of 2.2 ± 0.4x106, 1.1 ± 0.4x106 and 1.7 ± 0.4x106, respectively. In contrast, exposure to 200 p.p.m. EO for 4 weeks led to Hprt mutant frequencies (means ± SD) in splenic T cells that were 5- to 6-fold over background in exposed rats (7.9 ± 4.9x106; P = 0.032) and mice (9.9 ± 2.7x106; P <0.001). Furthermore, the average induced mutant frequency (i.e. the average observed treatment mutant frequency minus the average background mutant frequency) in EO-exposed mice in this study was in line with that observed previously in mice exposed in a similar fashion to 200 p.p.m. EO (average induced mutant frequency 11.9 ± 1.1x106) as part of a doseresponse study for Hprt mutations (18).
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Discussion
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For the purposes of this discussion the data presented here will be considered in the light of: (i) the two-compartment pharmacokinetic model for elimination of ET in SpragueDawley rats (10); (ii) current knowledge of the formation of hemoglobin and DNA adducts in rodents and humans via exogenous and endogenous exposure to ET and EO. In the present study, experiments were conducted to evaluate the effects of exposure duration and exposure concentration on changes in HEV levels, N7-HEG levels and Hprt mutant frequencies after inhalation exposure to ET in male F344 rats and B6C3F1 mice. The F344 rat and the B6C3F1 mouse were selected as animal models for this research because they have been used in carcinogenicity bioassays of EO in rodents (1113), in several molecular dosimetry studies of HEV and N7-HEG in EO-exposed rodents (6,1517,19) and in development of the rodent T cell cloning/sequencing assays for Hprt mutations (25,26,29,30). The tissues evaluated for N7-HEG content in ET-exposed animals included non-target (liver) and target tissues for EO-induced cancers in rats (brain) and mice (lung and spleen as a site of T cell lymphomas). The 4 week exposure duration for the doseresponse experiments in ET-exposed animals was based upon earlier studies demonstrating accumulation of HEV, N7-HEG and Hprt mutant T cells following repeated inhalation exposure of rodents to EO (1518). The exposure concentrations of 40, 1000 and 3000 p.p.m. ET were based on data from the Bolt and Filser (10) model for elimination of ET in SpragueDawley rats and they overlapped well with the rat cancer bioassay of ET (i.e. 300, 1000 and 3000 p.p.m. ET) (7).
In time course experiments, N7-HEG appeared to reach steady-state concentrations after the first week of exposure of F344 rats and B6C3F1 mice to 3000 p.p.m. ET (current study); in comparison, earlier studies demonstrated that N7-HEG continued to accumulate in the same tissues of rodents through 4 weeks of exposure to high concentrations of EO (i.e. 300 and 100 p.p.m. in rats and mice, respectively) (6,16). The half-life values for N7-HEG in various tissues from EO-exposed rodents suggested that this DNA adduct would reach steady-state concentrations in most mouse tissues (t
= 1.02.3 days) between 5 and 10 days exposure and in most rat tissues (t
= 2.94.8 days) between 2 and 3 weeks exposure (since 98% of steady-state is achieved in 4.3 half-lives) (16). We previously hypothesized that after repeated exposure of rodents to high concentrations of EO (16), the discrepancies between the time to steady-state (i.e.
28 days) and the DNA adduct half-life values were related to the saturation of DNA repair and a greater dependence upon spontaneous depurination for loss of N7-HEG [i.e. t
7 days in vivo (16) compared with t
~ 5375 h in vitro (31,32)]. If, as suggested by Zhao et al. (33), depurination were the primary mode of N7-HEG loss at the low levels of EO produced by exposure of rodents to ET, then the time required to reach steady-state concentrations of N7-HEG should be the same after low or high concentration EO exposure because the in vivo rate of the chemical shift and loss of N7-adducted guanine should be independent of the DNA adduct load. However, if DNA repair processes have a substantial impact upon the shape of the N7-HEG formation curves, then the time required to reach a steady-state should be reduced at lower EO concentrations (16). The latter hypothesis is supported by the shapes of the formation curves for N7-HEG in ET-exposed animals, where conversion of ET to low levels of EO resulted in a plateau in N7-HEG levels in most tissues of mice and rats after ~1 week of ET exposure (Figure 1
). An alternative hypothesis is that there is greater saturation of metabolic activation of ET with length of exposure.
The doseresponse curves for both HEV and N7-HEG in ET-exposed rats and mice were supralinear (based on plots of data in Tables IIII

) (34), demonstrating that metabolism to EO was saturated in all tissues except liver at exposures
1000 p.p.m. ET. Thus, the shapes of the doseresponse curves resemble those predicted by the two-compartment pharmacokinetic model for ET in rats (10).
The N7-HEG concentrations in brain, liver, lung and spleen of rats and mice exposed to 0, 40, 1000 or 3000 p.p.m. ET for 4 weeks were compared with the doseresponse curves for N7-HEG formation in the above tissues from rats and mice exposed to 0, 3, 10, 33 and 100 p.p.m. EO for 4 weeks (19), and the levels of EO that were formed and subsequently reacted with DNA in each tissue of ET-exposed animals were derived from these curves. Data in Table IV
show that the predicted amounts of EO resulting from exogenous ET exposure were both species and tissue dependent, with the highest levels of N7-HEG generally formed in the liver. Exposure to 40 p.p.m. ET produced tissue concentrations of N7-HEG similar to those formed by 0.72.3 p.p.m. exogenous EO in rats and 3.08.8 p.p.m. EO in mice. Likewise, the amounts of N7-HEG formed in animals exposed to 3000 p.p.m. ET were equivalent to those induced by exogenous EO exposures of 6.423.3 p.p.m. in rats and 6.721.5 p.p.m. EO in mice. Depending upon the tissue, these results deviated in some cases from the estimates of 1 and 6 p.p.m. EO equivalents predicted for rats exposed to 40 and 1000 p.p.m. ET, respectively, using HEV values and the two-compartment model of Bolt and Filser (10). These deviations are probably due to a combination of species and tissue differences in metabolism and DNA repair.
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Table IV. ET exposures versus equivalent EO exposures that induce the same amounts of N7-HEG in tissues of exposed F344 rats and B6C3F1 micea
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The comparative formation of N7-HEG in tissues of rats and mice exposed to ET and EO for the same regimens of 4 weeks can be normalized on the basis of HEV, which has been considered to be a good indicator of blood levels of EO (35,36) and has been used as a dosimeter to estimate ET exposure levels from tobacco smoke and occupational exposure in humans (37,38). An approximately constant ratio between the formation of hemoglobin adducts in blood and N7-HEG in spleen and testis was assumed previously for mice treated with a range of single doses of ET or EO (9). However, in studies using repeated exposure of mice and rats to EO, the relationships between HEV and N7-HEG concentrations were found to vary with length of exposure, time interval since exposure, species type and tissue type due to differences in adduct formation and persistence (17). These discrepancies between HEV and N7-HEG concentrations in ET-exposed versus EO-exposed animals (with standardized exposures of 6 h/day, 5 days/week for 4 weeks) are illustrated by the curves in Figure 2
. When N7-HEG is normalized to HEV by plotting the doseresponse curve for the DNA adduct concentration against the hemoglobin adduct concentration, it appears that ET exposure (to 40, 1000 or 3000 p.p.m.) induces somewhat higher amounts of N7-HEG in liver and lung of rats than equivalent exposure of rats to exogenous EO (Figure 2
). Conversely, one could state that when exogenous ET and EO exposures are normalized to levels that result in the formation of equivalent numbers of N7-HEG adducts in rat liver and lung, then these study results demonstrate that ET exposure yields less HEV formation than comparable EO exposure. The relatively high N7-HEG values in liver may be partially explained by the fact that this tissue has the greatest amount of cytochrome P450 2E1 and is a major tissue for bioactivation of ET to EO (2). Intracellularly formed EO can readily react with DNA, glutathione or proteins before EO is distributed systemically, such that formation of N7-HEG is disproportionately high compared with the formation of HEV under conditions of intracellular metabolic activation of ET. This phenomenon was also observed in the lungs of rats. In contrast, the curves in Figure 2C and D
are consistent with low metabolic activities in brain and spleen of rats, and DNA damage in these tissues of ET-exposed rats is likely to be via circulation of EO from sites of ET metabolism.
There have been several earlier efforts to characterize the formation of N7-HEG in mice and rats exposed to ET (9,14), in rodents exposed to low doses of EO (16) and in animals and people with no known exposure to ET or EO (16,27,28), however, these studies were constrained by the sensitivity and/or specificity of the analytical methods for this DNA adduct (see discussions in refs 19,39). Segerbäck (9) demonstrated that N7-HEG was formed in liver, spleen and testis of male CBA mice exposed for 1 h to 50 p.p.m. 14C-labeled ET, but the dependence upon radioactivity precluded evaluation of the background levels of N7-HEG in unexposed mice and the effects of repeated ET exposure on DNA adduct accumulation in exposed mice. Elevated concentrations of N7-HEG were also reported in liver and lymphocytes of male SpragueDawley rats exposed for 12 h/day for 3 consecutive days to 300 p.p.m. ET, but the 32P-post-labeling method used for adduct measurement could not distinguish between N7-HEG and N7-methylguanine (14). Due to limitations of previous assays (19,39,40), the endogenous levels of N7-HEG reported in earlier studies of control mice and rats were 1030 times greater (16,27,28) than those found in the present work using a highly sensitive and specific GCHRMS method for measuring N7-HEG (Tables II and III
). The lower background of N7-HEG in F344 rats (0.20.3 pmol/µmol guanine) has been confirmed using a blind comparison of the GCHRMS method with an improved 32P-post-labeling method using TLC and HPLC for separation of N7-HEG and N7-methylguanine (40). Background levels of N7-HEG in F344 rats also agree reasonably well with those reported in recent studies in SpragueDawley rats (39,40).
Data in Table V
show that background levels of N7-HEG have been consistently higher in tissues of humans compared with those in tissues of unexposed rats and mice (Tables II and III
). The lowest levels of endogenous N7-HEG have been measured in human tissues using an immunochemical assay (42) and 32P-post-labeling coupled with TLC and HPLC (39; Table V
). Nonetheless, the differences in endogenous N7-HEG levels in humans versus rodents do not appear to be explained solely by differences in exogenous exposure to ET and EO from urban air pollution, tobacco smoke and other sources (43,44). Possible mechanisms explaining, in part, the higher N7-HEG values in humans may include less efficient detoxification or DNA repair or greater endogenous exposure to intracellularly metabolized sources of hydroxyethylating agents compared with rodents (20). Thus, there is a clear need for large human population studies of the relationships between background levels of DNA adducts and various host factors to provide a better understanding of background cancer risk (45,46) and to discover the significance of exogenous chemical exposure relative to endogenous exposure (44,47).
The results of the various studies of Hprt mutant frequency in splenic T cells of EO-exposed mice and rats (18,26,48; this study) are difficult to compare quantitatively because of differences in: (i) the ages and strains of the animals used; (ii) the routes of exposure; (iii) the exposure regimens (including the dose rate and dose delivered); (iv) the time allowed for mutant expression (i.e. both phenotypic expression of Hprt protein and T cell migration); (v) the number of sampling times post-exposure (48,49). EO exposure of rats and mice in this report were performed before it became apparent that, because of age-dependent trafficking of mutant T cells from or through the thymus, determining the change in mutant frequencies over time in spleen of a given rodent species (i.e. determining the area under the mutant T cell `manifestation' curve of exposed versus control animals) provides a superior estimate of the mutagenic potency of a specific exposure regime compared with measuring the mutant frequency at a single time point post-exposure (49). The rationale for the mutant expression (or sampling) time of 8 weeks post-exposure in EO-exposed mice in the current study has been discussed elsewhere (18), but was based primarily on experimental evidence suggesting that the maximum frequency of Hprt mutant T cells in spleen would occur ~8 weeks after cessation of EO exposure in this age group of mice. The sampling time of 5 weeks post-exposure to measure mutant frequencies in the same age group of rats was based upon earlier Hprt mutagenicity studies of cyclophosphamide-treated rats (29). Thus, these experiments were not optimal for estimating the species difference in the mutagenic response of rats and mice (of identical age) to the selected EO exposure regime (200 p.p.m. for 4 weeks).
The results of more recent experiments in young adult male Lewis rats exposed to EO (via single i.p. injections, 4 weeks of drinking water exposure or 4 weeks of inhalation exposure) were based upon several sampling times post-exposure (48). Although the number of animals per group and number of sampling times may not have been optimal in these studies, the results were considered in the light of the current knowledge of age-dependent differences in T cell kinetics, the `manifestation' of mutant T cells and several variables that might impact on mutant frequency measurements at a given time after exposure (48). The mutagenicity data as a whole showed that EO gives rise to a dose-dependent increase in Hprt mutant frequency under the different experimental conditions studied. Furthermore, the resulting Hprt mutagenicity data indicated that EO is a relatively weak mutagen in T cells of rats (48). Likewise, earlier doseresponse studies of Hprt mutant frequencies measured at a single time point post-exposure suggested that EO is a relatively weak mutagen in T cells of mice (18). In view of these observations, a more sensitive analysis of the species difference in mutagenic response to EO is needed and could be achieved by constructing detailed mutant T cell manifestation curves for the spleen of EO-exposed and control rats and mice by using the youngest possible adult animals, increasing the number of sampling times post-exposure and increasing the number of animals per control and treatment groups at each sampling time (49).
Additional evaluation of the results presented here along with comparisons to previous studies support the conclusion that a significant threshold of endogenous hydroxyethylation (or damage) to DNA must be exceeded in order: (i) to induce a carcinogenic response from exogenous EO in rodents; (ii) to achieve an increased cancer risk from low levels of EO in people by direct exogenous exposure or by in vivo bioactivation after exogenous ET exposure. First, the 2 year carcinogenicity study of ET in F344 rats (7) and the N7-HEG data from ET- and EO-exposed rats (Table IV
) to all intents and purposes reveal a no observed effect limit for all cancer types associated with EO in the rat. The exposureresponse characteristics of EO were best characterized in the cancer bioassay of Snellings et al. (12) (i.e. exposure to 0, 10, 33 or 100 p.p.m. EO), where incidences of `splenic leukemias' (i.e. F344 rat mononuclear cell leukemias) and brain tumors were significantly increased at
10 and
33 p.p.m. EO exposure levels, respectively. Yet, exposure of rats to 3000 p.p.m. ET for 2 years failed to induce F344 mononuclear cell leukemias (7). The DNA adduct data for ET and EO exposures (Table IV
) indicate that this exposure to 3000 p.p.m. ET was equivalent to chronic exposures to 69 p.p.m. EO in tissues of low metabolic activity. Likewise, brain tumors were not induced by chronic exposure of rats to 3000 p.p.m. ET (i.e. the equivalent of exposure to ~6 p.p.m. EO in brain) (7), just as there was no induction of brain tumors after chronic exposure of rats to 10 p.p.m. EO (12). Moreover, risk estimates based upon the exposureresponse curves for F344 rat mononuclear cell leukemia are problematical because this neoplasm is unique to this species and strain, it occurs at a high spontaneous rate, the cell of origin is unknown and there is no identifiable human counterpart. Nonetheless, these findings do not rule out the possibility that ET or low level EO exposure might induce some tumors in rodents if the sensitivity of the `standard' carcinogenicity bioassay were increased by augmenting the number of animals in the treatment and control groups.
Second, well-designed studies of persons with occupational exposure to ET [e.g. fruit store workers (37) and plastics industry workers (50)] showed that the low doses of EO arising in vivo induced small amounts of HEV (
200 pmol/g globin) compared with that produced by EO exposure at the TWA of 1 p.p.m. (2400 pmol/g globin) (36), while a well-executed biomarker study of workers exposed to 25 p.p.m. EO (TWA) demonstrated no significant increase in N7-HEG levels in the exposed group (n = 42) compared with background DNA adduct levels in the control group (n = 29) (47; Table V
). These findings are consistent with the results of comparisons between background levels of N7-HEG in human tissues (Table V
) and the doseresponse curves for N7-HEG in tissues of rodents exposed for 4 weeks (6 h/day, 5 days/week) to 0, 3, 10 or 33 p.p.m. EO (20); these comparisons indicate that the higher endogenous amounts of N7-HEG in humans (as measured in refs 39,42, as opposed to measurements in refs 21,27,41) correspond roughly to steady-state levels found in tissues of rats and mice exposed to 3 and 10 p.p.m. EO, respectively (see tables I and II
of ref. 19). These results illustrate the biological importance of physiological background levels of N7-HEG, and other alkylated and oxidized DNA bases, in human and rodent models (44,47) and call into question current regulatory procedures that neglect mechanistic information about significant endogenous DNA damage in deriving human cancer risk estimates for low exposures to chemicals such as ET and EO (43). Additional research will be needed to identify the variations in individual susceptibility to EO-induced DNA damage in humans (e.g. polymorphisms in relevant detoxification and DNA repair enzymes), to determine if the relatively high endogenous levels of EO-induced adducts in humans are due to controllable/uncontrollable sources and to clarify the relationships between risk and endogenous versus exogenous exposure.
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Notes
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5 Present address: Department of Occupational Safety and Health, China Medical College, 91 Husuesh-Shih Road, Taichung, Taiwan, Republic of China 
6 Present address: College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea 
7 Present address: Texaco Inc., EHS Division, Beacon, NY 12508, USA 
8 Present address: Merck Research Laboratories, WP45-301, West Point, PA 19486, USA 
9 To whom correspondence should be addressed at: The Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA Email: walker{at}wadsworth.org 
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Acknowledgments
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The authors are grateful to Drs Timothy Fennell and Roger Giese for their respective gifts of derivatized N-(2-hydroxyethyl)valine and N7-(2-hydroxyethyl)guanine standards. Thanks are due to Dr Dale M.Walker for critical reading of the manuscript. In addition, we appreciate the considerate comments and contributions of the peer reviewers for Carcinogenesis. This study was funded in part by a grant from the Chemical Manufacturers Association and a grant from the National Institute of Environmental Health Sciences (no. P42-ES05948). The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS.
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Received February 4, 2000;
revised May 2, 2000;
accepted May 17, 2000.