n(7)-hydroxyethylguanine and 2-hydroxyethylvaline

Although ethylene oxide is a proven genotoxic carcinogen in experimental animals, its human carcinogenicity is still being debated. Alkylations (hydroxyethylation) of DNA and proteins by ethylene oxide are well established. Ethylene oxide is metabolically formed from ethylene, which is a natural body constituent. Thus, endogenous sources of ethylene/ethylene oxide contribute to background alkylations of physiological macromolecules. There are now experimentally well established data sets on the background hydroxyethylations of the N-terminal valine of hemoglobin and of the 7-N position of guanine in DNA, in laboratory animals as well as in humans: A review of these data leads to the conclusion that these background levels display remarkable consistency between the different species studied and, as far as DNA adducts are concerned, also between different tissues. From the existing database it can be deduced that in rats a hemoglobin alkylation, equivalent to the level of normal background, would be caused by repetitive external atmospheric exposures to ethylene oxide (6 hr/day, 5 days/week for several weeks) of about 30 ppb. On the contrary, in the same species, a DNA alkylation, equivalent to the level of normal background, would be caused by similar repetitive exposures to ethylene oxide at about 1-2 ppm. This paradox is unresolved. It points, however, to the biological importance of endogenous DNA alkylations and questions current regulatory procedures of assessing the risk of minute doses of exogenous carcinogens.

Topics: Animals; Carcinogens; DNA; Ethylene Oxide; Guanine; Hemoglobins; Humans; Valine

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), N:7-(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 (GC-HRMS), HEV was determined by Edman degradation and GC-HRMS 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 dose-response 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 dose-response 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.

Topics: Animals; Biomarkers; Biotransformation; Carcinogens; DNA; DNA Damage; Dose-Response Relationship, Drug; Ethylene Oxide; Ethylenes; Guanine; Hemoglobins; Hypoxanthine Phosphoribosyltransferase; Inhalation Exposure; Male; Mice; Mice, Inbred Strains; Mutation; Rats; Rats, Inbred F344; T-Lymphocytes; Valine

The analytical potential of gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry (HRMS) for characterization and quantitation of DNA and hemoglobin adducts was demonstrated using three model compounds: N2, 3-ethenoguanine (EG), 7-(2-hydroxyethyl)guanine (7-HEG), and N-(2-hydroxyethyl)valine (HEV). At a resolving power of 10 000, the signal-to-noise (S/N) ratios obtained from quantitative selected ion monitoring (SIM) experiments using biological samples were comparable to or better than existing unit mass resolution experiments due to the reduction of chemical noise from the use of narrower mass windows. The specificity gained by HRMS was essential for quantitation of ultratrace amounts near the limit of detection since coeluting interferences of the analyte or internal standard can lead to inaccurate measurement of response factors. The limit of detection (LOD) was 100 amol (S/N = 5) using a pure standard of TTB2-EG. The LOD for complete assays using spiked samples was 500 amol (S/N = 5) for EG and 600 amol (S/N = 5) injected for 7-HEG. The standard deviation (SD) for the HRMS quantitative measurements was typically less than 10%. The SD for the complete biological assays as determined by spiking replicate samples was less than 15%. This method has adequate sensitivity and specificity to accurately measure DNA and protein adducts as low as endogenous concentrations in rodent and human tissues.

Topics: Animals; Calibration; DNA Adducts; Gas Chromatography-Mass Spectrometry; Guanine; Humans; Indicators and Reagents; Proteins; Rats; Valine

Experiments involving ethylene oxide (ETO) have been used to support the concept of using adducts in hemoglobin as a surrogate for DNA adducts in target tissues. The relationship between repeated exposures to ETO and the formation of N-(2-hydroxyethyl)valine (HEtVal) in hemoglobin and 7-(2-hydroxyethyl)guanine (7-HEG) in DNA was investigated in male rats and mice exposed by inhalation to 0, 3, 10, 33, or 100 ppm ETO for 6 hr/day for 4 weeks, or exposed to 100 ppm (mice) or 300 ppm (rats) for 1, 3, 5, 10, or 20 days (5 days/week). HEtVal was determined by Edman degradation, and 7-HEG was quantitated by HPLC separation and fluorescence detection. HEtVal formation was linear between 3 and 33 ppm ETO and increased in slope above 33 ppm. The dose-response curves for 7-HEG in rat tissues were linear between 10 and 100 ppm ETO and increased in slope above 100 ppm. In contrast, only exposures to 100 ppm ETO resulted in significant accumulation of 7-HEG in mice. Hemoglobin adducts were lost at a greater rate than predicted by normal erythrocyte life span. The loss of 7-HEG from DNA was both species and tissue dependent, with the adduct half-lives ranging from 2.9 to 5.8 days in rat tissues (brain, kidney, liver, lung, spleen, testis) and 1.0 to 2.3 days in all mouse tissues except kidney (t1/2 = 6.9 days). The concentrations of HEtVal were similar in concurrently exposed rats and mice, whereas DNA from rats had at least 2-fold greater concentrations of 7-HEG than DNA from mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; DNA; DNA Damage; Dose-Response Relationship, Drug; Ethylene Oxide; Guanine; Hemoglobins; Male; Mice; Rats; Rats, Inbred F344; Time Factors; Valine