Max J. Turner

Determination of mutagenicity in tissues of transgenic mice following exposure to 1,3-butadiene and N-ethyl-N-nitrosourea.

1,3-Butadiene (BD) is carcinogenic in the B6C3F1 mouse in multiple organs, including lung and liver. We conducted a study to measure the frequency of BD mutations in mouse tissues using a transgenic mouse (Muta mouse; MM). MM is a BALB/c x DBA/2 (CD2F1) mouse that has a bacteriophage lambda shuttle vector with the target gene lacZ integrated into the mouse genome. Mice were exposed by inhalation to 625 ppm BD (6 hr/day) for 5 days and the lacZ- mutant frequency (mf) was determined in lung, bone marrow, and liver. The lacZ- mf in lung increased twofold above air-exposed control animals, but the bone marrow and liver samples did not exhibit an increase above background. N-ethyl-N-nitrosourea (250 mg/kg ip) was mutagenic in all three tissues examined. Studies on the biotransformation of BD using MM liver microsomes showed that the ratio between the rates of BD bioactivation to BD monoepoxide (BMO) and hydrolysis of BMO by epoxide hydrolases was approximately 40% less than this ratio using B6C3F1 mouse liver microsomes. Quantitation of adducts of BMO to N-terminal valine in hemoglobin (Hb) in the MM revealed an adduct level of 3.7 pmol/mg globin. Using this value, the predicted Hb adduct level in MM would be approximately one-half of that measured in the B6C3F1 mouse following similar exposures. These results indicate that BD induces mutations in vivo in a known murine target tissue, but strain differences in the biotransformation of BD should be considered in comparing the susceptibility of transgenic mouse strains to mutation.

Metabolism of 1,3-butadiene: inhalation pharmacokinetics and tissue dosimetry of butadiene epoxides in rats and mice

Significant species differences exist in the susceptibility to butadiene (BD)-induced cancer in rats and mice, and metabolism is likely a critical determinant for species sensitivity. This study measured the in vivo concentrations of, (1) BD in blood; (2) epoxybutene (EB) and diepoxybutane (DEB) in blood, lung and liver; and (3) glutathione (GSH) in lung and liver of male B6C3F1 mice and Sprague-Dawley rats during and after 6-h exposure to 62.5, 625, 1250, and 8000 (rat only) ppm BD. Mice had higher concentrations of EB and DEB in blood and tissues than did rats, DEB could not be detected in blood or tissues of rats, and the greatest depletion of GSH occurred in the lungs of mice. During exposure, the peak concentrations of EB in mice compared with rats were 4- to 8-fold higher in blood, 13- to 15-fold higher in lung, and 5- to 8-fold higher in liver. These data suggest that higher levels of BD epoxides in blood and tissues of mice compared with rats may explain, in part, the greater sensitivity of mice than rats to BD-induced carcinogenicity.

DISPOSITION OF BUTADIENE EPOXIDES IN SPRAGUE-DAWLEY RATS

1,2-Epoxybutene (BMO) and diepoxybutane (BDE) are metabolic products of 1,3-butadiene in rodents. Both BMO and BDE are suspect in the development of tumors in rats and mice. To understand the distribution and elimination of these compounds in the absence of the rate-limiting production from butadiene, the pharmacokinetics of BMO and BDE in blood were determined in adult male Sprague-Dawley rats following intravenous administration. All animals were dually cannulated in these studies. For the BMO studies, rats were dosed with 71, 143, or 286 mumol/kg BMO (n = 3 for each dose group). For the BDE studies, rats were dosed with 523 mumol/kg BDE (n = 3). All animals tolerated the BMO and BDE doses without grossly observable adverse effects. Blood was drawn at predetermined time points and extracted in methylene chloride. BDE and BMO concentrations were quantitated by gas chromatography or gas chromatography/mass spectrometry. The BMO distribution half-lives were short and ranged from 1.4 min at the lowest dose to 1.8 min at the highest dose. Volume of distribution at steady state ranged from 0.53 +/- 0.17 to 0.59 +/- 0.31 l/kg. Systemic clearances ranged from 67 +/- 17 to 114 +/- 20 ml/min per kg. The terminal elimination half-lives were also short and ranged from 5.7 to 8.5 min among the doses. The pharmacokinetic parameters after an i.v. dose of 523 mumol/kg BDE were a distribution half-life of 2.7 min, terminal elimination T1/2 of 14 min, volume of distribution at steady state of 0.73 +/- 0.06 l/kg, and systemic clearance of 76 +/- 8 ml/min per kg. These pharmacokinetic parameters demonstrate the similarity between disposition of the two epoxides in rats, that include a rapid distribution after i.v. administration into a small extravascular body compartment as well as a rapid elimination from blood. These pharmacokinetic data provide useful blood clearance information for assessing the critical physiological and biochemical determinants underlying the disposition of butadiene epoxides.

Hepatic and pulmonary glutathione conjugation of 1,2:3,4-diepoxybutane in human, rat, and mouse in vitro

1,3-Butadiene (BD) is a carcinogen in rats and mice. Previous in vitro studies showed that mouse liver microsomes formed 1,2-epoxy-3-butene (BMO) from BD and 1,2:3,4-diepoxybutane (BDE) from BMO at much higher rates than rat or human microsomes. Blood and tissue levels of BDE were significantly lower in rats than in mice following exposure to BD. Since mice are much more susceptible to cancer induced by BD than rats, these findings suggest a key role for BDE in BD-induced carcinogenicity. The aim of this study was to characterize the glutathione (GSH) conjugation of BDE by cytosol from human liver and mouse and rat liver and lung in vitro. BDE and radiolabeled GSH were incubated with cytosol. Conjugates were identified by 13C-NMR and FAB mass spectroscopy and quantitated by HPLC. The enzyme kinetics for the conjugation of BDE with GSH suggest that the higher BDE blood concentrations in mice compared with rats following inhalation exposure to BD are not due to differences in GSH conjugation of BDE.

1,4-Dichlorobenzene-Induced Nephrotoxicity: Similarity with Unleaded Gasoline (UG)-Induced Renal Effects

1,4-Dichlorobenzene (1,4-DCB) administered (150 or 300 mg/kg/day, po) 5 days per week for 104 weeks caused a dose-related increase in the incidence of renal adenomas and/or carcinomas in male rats, but not female F-344 rats or either sex of B6c3f1 mice (NTP, 1987). 1,2-Dichlorobenzene (1,2-DCB), an isomer of 1,4-DCB, caused no increase in the incidence of male rat renal tumours (NTP, 1985); the doses used were, however, 2.5 times lower than those employed for the 1,4-DCB study. Exposure to vapours of unleaded gasoline (UG) for 114 weeks also produced a doserelated increase in the incidence of renal adenomas and carcinomas in male, but not female F-344 rats or either sex of mice (Kitchen, 1984). The incidence of male rat renal tumours was dose-dependent but relatively low for both compounds. Assessment of the genotoxic properties of 1,4-DCB and UG by a battery of tests has shown that the chemicals are nongenotoxic. Our hypothesis to explain renal tumour formation is based on a multi-stage carcinogenesis model: Cells, in which spontaneous DNA alterations naturally occur, replicate following chemically-induced cell proliferation causing a mutated cell. Mutated cells undergo clonal expansion due to increased cell proliferation. This greatly increases the likelihood of additional spontaneous mutations occurring, leading to an increased incidence of renal cancer.