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Molecular Dosimetry of Ethylene Oxide: Formation and Persistence of 7-(2-Hydroxyethyl)guanine in DNA following Repeated Exposures of Rats and Mice¹

Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709 [V. E. W., T. R. F.]; Department of Environmental Sciences and Engineering [P. B. U., J. A. S.] and Department of Pathology [V. E. W., T. R. S.], University of North Carolina, Chapel Hill, North Carolina 27599; International Agency for Research on Cancer [V. P., D. E. G. S.], 69372 Lyon Cedex 08, France; and Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 [V. E. W.]

The formation of 7-(2-hydroxyethyl)guanine (7-HEG) in DNA of target and nontarget tissues was investigated in male B6C3F1 mice (20/group) and F344 rats (10/group) exposed to 0, 3, 10, 33, 100, or 300 (rats only) ppm ethylene oxide (ETO) by inhalation for 6 h/day for 4 weeks (5 days/week) and mice exposed to 100 ppm ETO for 1 or 3 days or 1, 2, or 4 weeks (5 days/week). The persistence of 7-HEG was studied in mice killed up to 7 days after cessation of the 4-week time-course study. In addition, the formation of O⁶-(2-hydroxyethyl)guanine and 3-(2-hydroxyethyl)adenine was evaluated in rats exposed to 300 ppm ETO. DNA samples from control and treated animals were analyzed for 7-HEG using neutral thermal hydrolysis, microconcentration, and high-performance liquid chromatography separation with fluorescence detection. Fluorescence-linked high-performance liquid chromatography was used for O⁶-(2-hydroxyethyl)guanine quantitation, and immunochromatography and gas chromatography-mass spectrometry were used for 3-(2-hydroxyethyl)adenine detection. Analysis of DNA from tissues of control mice and rats revealed the presence of peaks equivalent to 2–6 pmol 7-HEG/mg DNA. In mice exposed to 100 ppm ETO, 7-HEG accumulated to a similar extent in target and nontarget tissues, with adduct concentrations ranging from 17.5 ± 3.0 (SE) (testis) to 32.9 ± 1.9 (lung) pmol adduct/mg DNA after 4 weeks of exposure. Concurrent exposures of mice and rats to 100 ppm ETO for 4 weeks led to 2- to 3-fold lower concentrations of 7-HEG in mouse DNA in all tissues compared to rat DNA. 7-HEG disappeared slowly in a nearly linear fashion from the DNA of mouse kidney (t_1/2 = 6.9 days) and rat brain and lung (t_1/2 = 5.4–5.8 days), which was consistent with the loss of adduct mainly by chemical depurination. In contrast, a more rapid removal of 7-HEG from other mouse (t_1/2 = 1.0-2.3 days) and rat (t_1/2 = 2.9-4.8 days) tissues was consistent with adduct loss by depurination and DNA repair. Dose-response relationships for 7-HEG were nonlinear in both mice and rats, with the alkylating efficiency of ETO increasing at high exposures. In rats exposed to 300 ppm ETO, O⁶-(2-hydroxyethyl)guanine and 3-(2-hydroxyethyl)adenine accumulated to steady-state concentrations of approximately 1 pmol adduct/mg DNA; these levels were 250- to 300-fold less than the concentrations of 7-HEG found in the same tissues after 4 weeks of exposure (V. E. Walker et al., Mutat. Res., 233: 151–164, 1990). Comparisons of the formation, persistence, and dose-response curves for 7-HEG in tissues of both mice and rats suggested that saturation of DNA repair occurred at the concentrations of ETO used in the time-course studies and that repeated exposures to lower concentrations of ETO should lead to species and tissue differences in the molecular dose of 7-HEG. However, the overall assessment of DNA adduct formation in exposed rats and mice suggests that the species and tissue specificity for ETO-induced carcinogenesis is dependent upon factors in addition to DNA adduct formation.

¹ This is the second of two papers on this topic. Funded in part by the Ethylene Oxide Industry Council of the Chemical Manufacturers Association. V. E. W. was a recipient of a graduate fellowship from the Department of Pathology, Duke University.

² To whom requests for reprints should be addressed, at the University of North Carolina, Department of Pathology, CB# 7525, Chapel Hill, NC 27599-7525.

Received 2/20/92. Accepted 6/ 9/92.

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