Molecular dosimetry of endogenous and ethylene oxide-induced N7-(2-hydroxyethyl) guanine formation in tissues of rodents
Kuen-Yuh Wu1,3,
Asoka Ranasinghe1,
Patricia B. Upton1,
Vernon E. Walker1,2 and
James A. Swenberg1,4
1 Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599 and
2 Wadsworth Center for Laboratory and Research, New York State Department of Health, Empire State Plaza, PO Box 509, Albany, NY 12201, USA
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Abstract
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The formation of N7-(2-hydroxyethyl)guanine (7-HEG) in DNA was investigated previously in target and non-target tissues of F-344 rats and B6C3F1 mice exposed to
ISOdia
10 p.p.m. concentrations of ethylene oxide (EO) using fluorescence-linked high-performance liquid chromatography [V.E.Walker et al. (1992) Cancer Res., 52, 42384334]. In order to study the doseresponses for 7-HEG at lower exposures, a highly sensitive and specific gas chromatography coupled with high-resolution mass spectrometry (GCHRMS) assay was developed. DNA was extracted from liver, brain, lung and spleen of B6C3F1 mice and F-344 rats exposed to 0, 3, 10, 33 or 100 p.p.m. EO for 4 weeks (6 h/day, 5 days/week). Analysis of DNA from control rodents showed that endogenous 7-HEG varied from 0.2 ± 0.1 to 0.3 ± 0.2 pmol/µmol guanine in tissues of rats and mice. 7-HEG exhibited tissue- and species-specific doseresponse relationships in EO-exposed animals. Linear doseresponse relationships were evident in mouse liver, brain and spleen at exposures between 3 and 100 p.p.m.. Mouse lung exhibited a slightly sublinear response between 33 and 100 p.p.m. EO. The relationships were linear in liver and spleen of rats between 3 and 100 p.p.m. EO, but were slightly sublinear in brain and lung between 33 and 100 p.p.m. EO. The number of 7-HEG adducts present in rats exposed to 3 p.p.m. EO was 5.312.5 times higher than endogenous 7-HEG in unexposed controls. In contrast, mice exposed to 3 p.p.m. EO only had 1.3- to 2.5-fold greater numbers of 7-HEG adducts. The factors driving the exposureresponse relationships are also likely to affect carcinogenic and mutagenic responses of rodents to EO. Likewise, a better understanding of the relationships between 7-HEG derived from low exposures to EO and endogenously formed 7-HEG may be important for the accurate extrapolation of risk to humans.
Abbreviations: 7-HEG, N7-(hydroxyethyl)guanine; EO, ethylene oxide; FD, fluorescence detection; GCHRMS, gas chromatography coupled with high-resolution mass spectrometry; HPLC, high-performance liquid chromatography; PFBBr, pentafluorobenzyl bromide; SCE, sister chromatid exchange; TLC, thin-layer chromatography.
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Introduction
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Ethylene oxide (EO) is an important industrial chemical and gaseous sterilant (1). EO is positive in routine genotoxicity studies and causes cancer in laboratory animals (1). EO is currently classified by the IARC as a known human carcinogen, and is regulated for occupational exposure at 1 p.p.m. in many countries (1). The IARC classification was based on limited epidemiology and positive genotoxicity data in humans and rodents, rather than sufficient evidence in epidemiological studies. The supporting evidence from the human biomarker studies used sister chromatid exchanges (SCE), chromosomal aberrations and micronuclei, which are not chemical specific (25). Epidemiological studies showed that cancer mortality was weakly, inconclusively or not associated with EO exposure in studies of 28 900 US workers, even though 86% of this cohort had their first exposure to EO during or before 1977 when EO was regulated at 50 p.p.m. (69). Due to limited quantitative exposure data in epidemiological studies and the lack of chemical specificity of SCE, chromosomal aberrations and micronuclei, the risk for human carcinogenicity at low EO exposures is associated with considerable uncertainty.
Carcinogenesis is considered a multistage process. Among the earliest cellular changes caused by chemical carcinogens is the formation of DNA adducts, which may play an important role in the initiation and progression of carcinogenesis (10). Molecular dosimetry of DNA adducts in experimental animals can provide essential information on the relationships between exposure and effect and may permit extension of the observable range for relevant scientific information to concentrations lower than can be achieved in carcinogenesis bioassays (11,12). N7-(2-hydroxyethyl)guanine (7-HEG) is the major EO-induced adduct formed in vitro and in vivo (13,14). Alkylation at N7 of guanine, with subsequent production of abasic sites by spontaneous or enzymatic depurination, may promote base substitutions, frameshift mutations or large deletions (15,16). The doseresponse relationships for 7-HEG were reported to be linear in mice and rats repeatedly exposed to EO from 10 to 100 p.p.m. and sublinear from 100 to 300 p.p.m. in rats (13). At exposures below 10 p.p.m. in rats and 33 p.p.m. in mice, adduct measurements were uncertain or not reported due to the limitations of sensitivity and specificity of the previous assays (13,17,18).
In addition to exogenous sources, 7-HEG is also derived from endogenously formed EO arising from metabolism of ethylene generated by intestinal bacteria, lipid peroxidation of unsaturated fats, hemin and methionine (19). Previous studies have shown that endogenous 7-HEG is present in rodent tissues, but the amounts reported were near the limit of detection of the assays used (13,20). The objective of this study was to utilize a highly sensitive and specific assay, using gas chromatography coupled with high-resolution mass spectrometry (GCHRMS), to specifically quantitate endogenous and EO-induced 7-HEG. Specific quantitation of 7-HEG in DNA of rodents at low as well as high EO exposures will better characterize this adduct as a biomarker for experimental animal and human biological monitoring studies and clarify its doseresponse relationships at low exposures (12).
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Materials and methods
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Chemicals
EO (99% purity) was purchased from National Welders Supply Co. (Raleigh, NC). 7-HEG standard (>98% purity) was obtained from Chemsyn Science Laboratories (Lenexa, KS). Pentafluorobenzyl bromide (PFBBr), potassium hydroxide, tert-butyl nitrite and tetrabutyl ammonium sulfate (Bu4HSO4) were obtained from Aldrich (Milwaukee, WI). GC2 hexane, dichloromethane and ethyl acetate were obtained from Baxter Diagnostic (McGaw, IL). The sources of DNA extraction reagents, purification-grade sterilized Dulbecco's phosphate-buffered saline solution, 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, RNase A and other DNA isolation and high-performance liquid chromatography (HPLC) reagents have been listed elsewhere (13).
Animal exposures
Details were described by Walker et al. (13). Briefly, 9-week-old male B6C3F1 mice and F-344 rats were randomly divided into groups in 8 m3 stainless steel and glass inhalation chambers. Ten animals in each group were exposed to 0, 3, 10, 33 or 100 p.p.m. of EO for 4 weeks (6 h/day; 5 days/week). EO was introduced into the chamber through a calibrated Fisher and Porter flow meter (Warminster, PA) and diluted using filtered chamber supply air to achieve the target concentrations. The EO concentrations inside the chamber were monitored continuously using a Miran infrared spectrophotometer (9.2 µm wavelength and 25 m pathlength). Control animals were exposed to chamber supply air with 0 p.p.m. EO in sham exposures. The average inhalation chamber concentrations for the 4 week exposure periods were 2.97 ± 0.34, 10.0 ± 0.5, 31.8 ± 3.2 and 99 ± 6 p.p.m. (mean ± SD). After the cessation of EO exposures, animals were immediately killed by exsanguination. Organs were harvested, packed in foil and stored at 80°C.
DNA isolation
DNA was extracted from the whole lung, spleen and brain, or up to 2 g of liver from rats and mice using an automated phenolic extraction procedure (13). Briefly, DNA was isolated by one phenolchloroform extraction, two chloroform extractions and isopropanol precipitation using a 340A Nucleic Acid Extractor (Applied Biosystems, Foster City, CA). DNA pellets were dissolved overnight in 34 ml of sterilized distilled deionized water at 4°C. These DNA solutions were homogenized by shearing through a 20 gauge needle connected to a 5 ml syringe. DNA concentrations were analyzed using a UV 160U spectrophotometer (Shimadzu, Columbia, MD) and calculated using calf thymus DNA as a standard and from the equation: DNA concentration = 50 µg/mlxabsorbance at 260 nmxdilution factor (13).
DNA content was further characterized for each sample by guanine analysis. Twenty microliters of DNA solution were removed from each sample and hydrolysed in 500 µl of 0.1 N HCl solution at 70°C for 30 min. Guanine concentrations were determined with HPLC using an Alltech (Deerfield, IL) strong cation exchange column (250x4.6 mm; 10 µm) and 0.1 M ammonium formate, 10% methanol (pH 2.8) as the mobile phase with a flow rate of 1.8 ml/min and an Applied Biosystem UV detector (Ramsey, NJ) with the wavelength set at 254 nm (13).
Derivatization and quantitation of 7-HEG
The DNA samples were derivatized according to Saha et al. (21), with minor modifications (22). DNA samples (10300 µg) were spiked with 0.1 pmol [13C4]7-HEG. Each sample was dried in a Speed-Vac (Savant model SVC100; Farmingdale, NY) and dissolved in HPLC-grade water at 4°C overnight. 7-HEG was released from the DNA backbone using neutral thermal hydrolysis (at 100°C for 15 min). Each sample was immediately placed in an ice bath, treated with 150 µl of 1 N cold HCl solution and centrifuged at 1200 g and 0°C for 15 min using a Sorvall Instruments RC5C centrifuge (DuPont, Wilmington, DE). The DNA backbone was washed with 100 µl of 1 N cold HCl solution and centrifuged a second time. Both supernatants from each sample were combined and dried under vacuum, and 7-HEG was converted to N7-(2-hydroxyethyl)xanthine by the reaction with tert-butylnitrite in 6 N HCl solution (degassed with nitrogen). Then, each sample was dried in vacuo. Each residue was subjected to liquidliquid extraction using ethyl acetate and water. The aqueous phase was dried in vacuo. Each residue was reacted with 150 µl of 10% PFBBr in acetonitrile and 5 mg K2CO3 (dehydrated at 60°C at least 1 h) at room temperature for 20 h, followed by evaporation under nitrogen at 85°C. Each sample was further derived with 10 µl PFBBr in 150 µl dichloromethane and 50 µl of 1 mg/µl Bu4NHSO4 in 1 N KOH at room temperature for 20 h. The sample was then dried with nitrogen at 45°C and extracted three times with ethyl acetate. The combined supernatants were dried with nitrogen at 85°C. The residue was dissolved in 50% ethyl acetate/hexane solution and subjected to solid-phase extraction using silica gel (EM Science, Darmstadt, Germany; particles sizes: 0.0630.2 mm). The eluent was dried under nitrogen at 85°C and raised in dry toluene for quantitation. The derivatized samples were quantitated using a Hewlett-Packard 5890 gas chromatograph connected to the mass spectrometer with a J & W Scientific (Folsom, CA) DB-5 ms capillary column (15 m, 0.32 mm and 0.1 µm film thickness). The mass spectrometer was a VG 70,250SEQ operated in the selective-ion-monitoring (SIM) mode and tuned to achieve a resolving power of 10 000 using perfluorotributylamine (CF-43; Scientific Instrument Services, Ringoes, NJ) as a calibration reagent as described previously (22,23). Helium was used as a carrier gas and the head pressure was set at 10 p.s.i. The source temperature was set at 250°C and methane (5x105 mbar) was used as the reagent gas for electron capture negative chemical ionization. One microliter of derivatized sample was injected through a tight-press injection liner. The GC temperature was increased from 70 to 300°C over 10 min and held for 3 min to evaporate residues from the injected sample. The isotope dilution method was used to determine 7-HEG by monitoring m/z 555.0515 (analyte) and m/z 559.0649 (internal standard). Quantitation of 7-HEG was based on the ratio of the peak area of the analyte to that of the internal standard and comparison with a calibration curve for 7-HEG (22).
Statistical analysis
The one-way Student's t-test was used to evaluate differences in amounts of 7-HEG between controls and EO-exposed rodents. The null hypothesis, that means of 7-HEG were equal, was rejected at P < 0.05. Linear regression was used to fit the doseresponse relationships for 7-HEG in tissues of rodents using SAS software.
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Results
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Previous methods for measuring 7-HEG have been limited in both specificity and sensitivity. This study utilizes a newly developed method that couples electrophore labeling with GCHRMS for the measurement of 7-HEG (22). The sensitivity of this method is affected by the yield of derivatization. Conversion of 7-HEG to N7-(2-hydroxyethyl)xanthine by nitrosation increased the derivatization efficiency ~2.5-fold (21). Derivatization efficiency decreased with the presence of salt (such as ammonium formate and ammonium phosphate). The yield of derivatization went up with an increase of either derivatization time or the amount of PFBBr used. However, reactions between undesirable compounds and PFBBr also were promoted, increasing background. These chemical interferences might overlap with the analyte or internal standard peak so that sensitivity and specificity could drop dramatically. 150 microliters of 10% PFBBr in acetonitrile in the first and 10 µl of PFBBr in the second derivatization for 20 h were optimal for the analysis of endogenous 7-HEG. The derivatization efficiency varied from ~2.5 to 15% (estimated by the comparison of the peak area of the amount of [13C4]7-HEG spiked in each sample with that of 500 fg of derivatized standard solution).
Quantitation of endogenous 7-HEG
This assay was able to quantitate endogenous 7-HEG with excellent sensitivity, specificity, precision and reproducibility. Figure 1
is a representative chromatogram from spleen DNA of a control rat. Analyses of chromatograms from tissue samples of control animals revealed average background concentrations of 7-HEG varying from 0.2 to 0.3 pmol/µmol of guanine in lung, brain, spleen and liver from B6C3F1 mice and F-344 rats (Tables I and II
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Fig. 1. A representative chromatogram from spleen DNA of a control rat. (A) The analyte channel (m/z = 555.0515) and (B) the internal standard channel (m/z = 559.0649), demonstrating that the analyte and internal standard peaks coelute. The content of 7-HEG in this sample was 0.3 pmol/µmol of guanine.
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Table I. 7-HEG (pmol/µmol of guanine ± SD) in tissues of F-344 rats exposed to ethylene oxide from 3 to 100 p.p.m. for 4 weeks
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Table II. 7-HEG (pmol/µmol of guanine ± SD) in tissues of B6C3F1 mice exposed to ethylene oxide from 3 to 100 p.p.m. for 4 weeks
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Quantitation of 7-HEG in EO-exposed rodents
The effects of repeated EO exposures on the formation of 7-HEG in DNA of liver, spleen, brain and lung from rats and mice exposed to 3100 p.p.m. EO are presented in Tables I and II
, respectively. The amount of 7-HEG increased in all four tissues of rats and mice from 3 to 100 p.p.m. EO. The extent of adduct accumulation during repeated exposures did not vary >3-fold in target tissues (spleen and brain of rats and lung of mice) and non-target tissues (liver and lung of rats and liver, spleen and brain of mice) for carcinogenesis (2426). Statistically greater amounts of 7-HEG accumulated in tissues of rats than in mice exposed to the same concentrations of EO (Tables I and II
). These results agree with a previous study that demonstrated tissue and species differences in formation and removal of 7-HEG (13).
Repeated exposures to EO produced a similar pattern of doseresponse curves in tissues of rats and mice. Doseresponses were linear at exposures between 3 and 100 p.p.m. in the liver, brain, spleen and lung of mice as determined by linear regression analysis (Figure 2
). However, 7-HEG was significantly higher in lung than liver and spleen of mice exposed to 10100 p.p.m. EO (P < 0.05). For rats, the doseresponse curves for 7-HEG in all four tissues were linear between 3 and 100 p.p.m. when examined using linear regression (Figure 3
). When 7-HEG was compared across tissues, brain and lung exhibited evidence for a slightly sublinear response between 33 and 100 p.p.m. EO. There were significantly greater amounts of 7-HEG in brain and lung than in liver and spleen of rats at 100 p.p.m. EO (P < 0.005).

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Fig. 2. The doseresponse relationships for 7-HEG (pmol/µmol of guanine) in DNA of mice exposed to 0, 3, 10, 33 or 100 p.p.m. EO for 4 weeks (6 h/day, 5 day/week). Symbols, means; bars, standard deviations (n > 4).
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Fig. 3. Doseresponse for 7-HEG (pmol/µmol of guanine) in DNA following 4 weeks (6 h/day, 5 day/week) of exposures of rats to 0, 3, 10, 33 or 100 p.p.m. ethylene oxide. Symbols, means; bars, standard deviations (n > 4).
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Discussion
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Advantages of the GCHRMS assay
The main limitations of previous assays for 7-HEG were sensitivity and specificity (27,28). 32P-post-labeling has comparable sensitivity (0.11.0 fmol) with GCHRMS, but 7-HEG and N7-methylguanine were not completely resolved on the thin-layer chromatography (TLC) plates (29). Recently, 7-HEG and N7-methylguanine have been separated using TLC and HPLC (30). In addition, depurination of this adduct must be carefully monitored in the 32P-post-labeling assay. Immunoassays often lacked sensitivity and suffered from extensive cross-reactivity of their antibodies with N7-methylguanine (31). HPLC with fluorescence detection (HPLCFD) and electrochemical detection (HPLCECD) have been unable to routinely quantitate endogenous 7-HEG (13,32). Derivation of 7-HEG with N-methyl-trimethylsilyl-trifluoroacetamide was not very stable, and analysis of this derivative using electron impact ionization mass spectrometry (EI-GC/MS) demonstrated a detection limit of ~35 fmol on column (20).
Sensitivity of the present GCHRMS assay was significantly increased over the EI-GC/MS assay because the addition of three pentafluorobenzyl (PFB) groups dramatically improved adduct volatility, GC separation and efficiency to absorb thermal electrons in the source to specifically generate the most abundant fragment [PFB2-7-HEG] (2123). Specificity was significantly improved by using HRMS to eliminate specific and non-specific interferences originating from reagents, complex biological matrixes and/or derivatization steps (23). Accurate quantitation was based on the ion abundance ratio measurement of the coeluted analyte and a stable isotope-labeled internal standard. This sensitivity and specificity are very important when analyzing trace amounts of DNA adducts produced by very low exposures or arising endogenously.
HPLCFD with a detection limit of ~20 pmol was not able to quantitate 7-HEG in rats exposed to EO concentrations below 10 p.p.m. and in mice exposed to EO below 33 p.p.m. (13). Furthermore, when DNA from the same tissue samples was analyzed, quantitation of 7-HEG using HPLCFD showed ~1.3- to 2.5-fold higher values than were obtained using GCHRMS (Tables I and II
). These differences may be in part due to the ability of this GCHRMS assay to eliminate coeluted interferences, resulting in lower values than the HPLCFD method.
Endogenous 7-HEG in rodents
Endogenous DNA damage is likely to be involved in spontaneous mutation and cancer formation (33,34). As such, knowledge of endogenous 7-HEG may provide a better understanding of background cancer risk. Endogenous levels of 5.6 ± 3.0 pmol 7-HEG/mg DNA have been reported in the white blood cells of SpragueDawley rats, and 26 pmol adduct/mg DNA in tissues of F344 rats and B6C3F1 mice (13,18). Compared with the data in Tables I and II
, the earlier results are likely to be over-estimations due to the lack of specificity and sensitivity of the previous methods. The data from the present study demonstrate much lower amounts of endogenous 7-HEG in tissues of rats and mice (0.20.3 pmol/µmol guanine). This finding has been confirmed using a blinded comparison of the present method with the newly improved 32P-post-labeling method for 7-HEG (35). These rodent values are lower than GCHRMS data on human liver and lymphocytes, which have mean endogenous 7-HEG values of 13 pmol/µmol guanine (22,36). On the other hand, endogenous N-(2-hydroxyethyl)valine adducts are similar in rodents and humans (1,22,37; K.-Y.Wu, A.Ranasinghe, P.B.Upton, N.Scheller, V.E.Walker, J.S.Vergnes and J.A.Swenberg, manuscript submitted for publication). Reasons for the higher amounts of endogenous 7-HEG in humans compared with rodents are presently unknown. Possible mechanisms include less efficient detoxification or DNA repair, or greater endogenous exposure to intracellularly metabolized sources of hydroxyethylating agents, such as ethylene (K.-Y.Wu, A.Ranasinghe, P.B.Upton, N.Scheller, V.E.Walker, J.S.Vergnes and J.A. Swenberg, manuscript submitted for publication). Consideration of these issues may be important in human biological monitoring and risk assessment, since occupational exposures are regulated at 1 p.p.m. EO in many countries; yet the endogenous amounts of 7-HEG in humans are equivalent to that produced by ~10 p.p.m. EO in rodents.
7-HEG in EO-exposed rodents
Significant dose-dependent accumulations of 7-HEG at repeated exposures below 33 p.p.m. of EO were measured in this study. In contrast, significant increases in cancer incidence were not detected at these exposures in 2 year carcinogenicity bioassays (24,25). This phenomenon suggests that 7-HEG should be a sensitive biomarker for EO exposures that would be useful for extending the doseresponse for biologically based risk assessment. Differences in 7-HEG in target and non-target tissues were not greater than 3-fold and were consistent with efficient pulmonary uptake of EO (13), rapid and even distribution of EO to all tissues, except possibly testis (38), and the ability of EO to act as a direct alkylating agent (39). These results agreed with the previous dosimetry study of 7-HEG and suggest that the formation of DNA adducts was not the only determining factor in EO carcinogenicity (13).
Tissue-specific accumulation of 7-HEG represents the summation of EO distribution, detoxification and DNA repair in individual tissues. If one assumes similar rates of chemical depurination across tissues, tissues with greater half-life values of 7-HEG may have lower base excision repair rates, resulting in greater accumulation of 7-HEG. For example, higher 7-HEG in rat lung and brain corresponded to longer half-life values in brain (5.4 days) and lung (5.8 days) than liver (3.9 days) and spleen (2.9 days) of rats (13). Similar relationships between the amounts of 7-HEG and its half-life values were also true between rats and mice (13).
The linear doseresponses in liver, brain and spleen of mice and liver and spleen of rats suggest that detoxification and DNA repair were not saturated at EO exposures from 3 to 100 p.p.m.. Methylpurine DNA glycosylase (MPG) is thought to be the principal pathway for repair of N7-alkylguanines (40). Induction of MPG has been observed in cultured cells and rats exposed to DNA-damaging agents (41,42), but species differences in repair have not been reported.
Detoxication of EO involves two different pathways: glutathione conjugation and hydrolysis with epoxide hydrolase (43). The distribution of detoxication enzymes in various tissues could affect detoxication rates and subsequent 7-HEG formation. For example, glutathione content is 4-fold higher in liver than in brain and lung of rats (44). This lower glutathione content might account for higher amounts of 7-HEG in brain and lung than in liver of rats. The amounts of glutathione have been reported to be depressed by ~20% in liver and lung of F-344 rats exposed to 100 p.p.m. of EO for 4 h (45). Glutathione depletion was also reported to be significant in lung of B6C3F1 mice exposed to
50 p.p.m. EO for 4 h (46). The extent of glutathione depletion and rate of recovery may be important factors in the efficiency of DNA alkylation by EO. The low amounts of epoxide hydrolase in lung suggest that detoxication could be depleted even at the low concentrations of EO employed in this study (45). Together, the slight slope change of the doseresponse curve for 7-HEG in lungs of mice suggests that detoxication may be an important factor in the responses of 7-HEG at concentrations that were carcinogenic.
When the amount of 7-HEG induced by exposure to 3 p.p.m. EO was compared with that present in unexposed control animals, a major difference was found between rats and mice. Rats exposed to 3 p.p.m. EO had 5.3, 12.5, 9.0 and 8.0 times more 7-HEG in liver, spleen, brain and lung, respectively, than was present in the same tissues of unexposed rats. In contrast, mice exposed to 3 p.p.m. EO only had 1.3, 2.5, 2.0 and 1.7 times more 7-HEG in liver, spleen, brain and lung, respectively. The increase in 3 p.p.m. EO mouse liver 7-HEG was not significantly different from controls. This species difference may reflect more efficient detoxication and/or DNA repair of EO in mice. Obtaining similar data for humans would clearly enhance our ability to more accurately predict the effects of low exposures of EO.
Ehrenberg and Törnqvist (47) have suggested that there are two major problems associated with EO research: (i) sensitivity of the analytical methods and (ii) knowledge of the shape of doseresponse curves at very low doses. The present study directly addresses these issues. This GCHRMS method, with superior sensitivity and specificity, can be used to study the doseresponse for 7-HEG in tissues of rodents exposed to EO from 0 p.p.m. to the highest exposure used. This advance is possible since the use of GCHRMS tuned to a resolving power of 10 000 reduces chemical interferences to a minimum (22,23). This methodology can be expanded to use [13C2]EO exposure to accurately determine the relationships between low exposures and endogenous 7-HEG formation. Likewise, interindividual differences in detoxication and DNA repair can be evaluated. Together, such data should significantly improve biologically based risk assessment for EO.
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Acknowledgments
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The authors are grateful to Dr Roger Giese for his gift of derivatized N7-(2-hydroxyethyl)guanine standard. This study was funded in part by the Chemical Manufacturers Association and a grant from NIEHS (P42-ES05948).
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Notes
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3 Present address: Department of Occupational Safety and Health, China Medical College, No. 91 Hsuesh Shih Road, Taichung, Taiwan 
4 To whom correspondence should be addressed Email: james_swenberg{at}unc.edu 
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Received September 3, 1998;
revised April 2, 1999;
accepted May 13, 1999.