Co-administration of ethanol transiently inhibits urethane genotoxicity as detected by a kinetic study of micronuclei induction in mice.

Urethane (ethyl carbamate) is a genotoxic carcinogen that requires metabolic activation. Ethanol is known to inhibit urethane metabolism and genotoxicity. Since ethanol is eliminated rapidly in animals, the persistence of ethanol inhibition was studied in a mouse bone marrow and a peripheral blood micronucleus assays. In the bone marrow assay, male CD-1 mice were injected intraperitoneally (i.p.) with water (vehicle), urethane (1000 mg/kg), ethanol (2500 mg/kg) or urethane and ethanol (1000 and 2500 mg/kg, respectively) in single injections. Polychromatic erythrocytes (PCE) from bone marrow were obtained at 24 and 48 h after injection and scored for micronuclei. Urethane induced an increase of micronucleated PCE (MN PCE) frequency from 0.19% in the control to 8.63% at 24 h, followed by a decrease to 6.98% at 48 h. When urethane was co-administered with ethanol, the MN PCE frequency was suppressed to 0.49% at 24 h, but markedly increased to 7.35% at 48 h. This delay of MN PCE occurrence indicated that ethanol inhibition was transient. To pinpoint the duration of this delay, a peripheral blood micronucleus assay was conducted to monitor the kinetics of MN PCE induction. In this assay, male CD-1 mice were injected i.p. with water, ethanol, urethane, or urethane and ethanol as described above. Peripheral blood was scored for MN PCE at 8-h intervals for 4 days. Two additional dose groups injected with urethane or urethane and ethanol were also scored for MN PCE at 8 h intervals, but each blood sampling time was staggered 4 h later from the first four dose groups. The combined data provided MN PCE frequencies at 4-h intervals from 24 to 100 h after injection. Urethane alone induced a peak MN PCE frequency of 11.6% at 52 h. Urethane and ethanol induced a peak MN PCE frequency of 11.2% at 64 h, a delay of 12 h. Thus, ethanol delays but does not diminish urethane genotoxicity.

A pharmacokinetic study of ethanol inhibition of micronuclei induction by urethane in mouse bone marrow erythrocytes.

Urethane (ethyl carbamate) is a genotoxic carcinogen in fermented products and alcoholic beverages. The genotoxicity of urethane requires metabolic activation. Metabolism of urethane is mediated by multiple pathways, and ethanol is known to inhibit the esterase hydrolysis pathway of urethane, which accounts for over 95% of urethane metabolism. This report shows that ethanol also inhibits the induction of micronuclei by urethane in mouse bone marrow erythrocytes, presumably by inhibiting the minor pathway that generates genotoxic metabolite(s). In this study, male CD-1 mice were administered urethane, ethanol, or urethane co-administered with increasing amounts of ethanol in single intraperitoneal injections. Bone marrow polychromatic erythrocytes (PCE) obtained 24 h after injection were scored for micronuclei. The dose of urethane was 1000 mg/kg, and the doses of ethanol were 0, 625, 1250, 2000, 2250, 2500, 3000 and 3500 mg/kg. The blood ethanol level at each dose was determined. Two pharmacokinetic parameters, Cmax and AUC, were estimated for each dose. The observed Cmax of ethanol at doses of 1250, 2000, 2250, 2500, 3000 and 3500 mg/kg were 1.39, 2.84, 3.15, 3.69, 4.13 and 4.76 mg/ml, with AUCs of 1.37, 4.84, 5.88, 7.28, 10.76 and 13.51 mg.h/ml, respectively. Urethane treatment alone markedly increased the micronucleus frequency from 0.1% in the vehicle control to 2.47%. This magnitude of increase was suppressed when urethane was co-administered with ethanol at ethanol doses of 2500 mg/kg and above. At 2500, 3000 and 3500 mg/kg, the micronucleus frequencies reduced from 2.47% to 0.9, 0.44 and 0.28%, respectively. This study shows that ethanol inhibits the induction of micronuclei by urethane.

Selenium kinetics, placental transfer, and neonatal exposure in cynomolgus macaques (Macaca fascicularis).

Forty pregnant cynomolgus macaques were treated daily from gestational day 20 to 50 by nasogastric intubation of 0, 25, 150, or 300 micrograms selenium as L-selenomethionine/kg body weight. In each group, 7-8 pregnancies were terminated by hysterotomy at gestational day 100 +/- 2 and the fetuses were examined, while 2-3 pregnancies in each group were allowed to proceed to term. Selenium and soluble glutathione peroxidase were measured in: maternal, neonatal, and fetal plasma and erythrocytes; fetal kidney, liver, muscle, and placenta; and maternal breast milk. The area under the multidose maternal plasma selenium concentration:time curve, the maximum maternal plasma selenium concentration, and the maternal urinary selenium excretion rates were proportional to the L-selenomethionine dose. Selenium concentrations in all fetal and neonatal, tissues were also proportional to maternal L-selenomethionine dose. Glutathione peroxidase was affected only in maternal erythrocytes, fetal kidney, and neonatal plasma. The selenium concentration in fetal plasma was an average 33% of that in maternal plasma. Although selenium concentrations in macaque milk were doubled by the highest dose, intrauterine selenium accumulation accounted for the majority of the neonatal selenium body burden. Despite the elevated selenium concentrations in fetal tissues, neonatal blood, and milk, no deleterious effects on neonates were observed. These results suggest that primate fetuses are well protected against selenium toxicity arising from high maternal L-selenomethionine intakes.

A review of the dose-response induction of DNA adducts by aflatoxin B1 and its implications to quantitative cancer-risk assessment.

The induction of DNA adducts by aflatoxin B1 in the liver has been extensively reviewed in a quantitative cancer-risk assessment of aflatoxins (CDHS, 1990). Rat is the most sensitive species for aflatoxin tumorigenesis and liver is the most sensitive site. In vitro DNA-adduct studies were mostly on adduct identification and specificity of binding. In vivo studies provided dose-response relationship of aflatoxin B1, binding to DNA and DNA-adduct formation. Most in vivo studies were conducted in rats. The dose-response curves of DNA-adduct induction after ingestion or injection treatments in this species were reviewed. A linear dose-response relationship was observed in both injection and ingestion studies at low doses. For cancer-risk assessment, this observation is consistent with the assumption of the linear dose-response risk-assessment model for genotoxic agents, and justifies the use of this model for quantitative cancer-risk assessment for aflatoxins.

Incorporation of a micronucleus study into a developmental toxicology and pharmacokinetic study of L-selenomethionine in nonhuman primates.

Concomitant to a developmental toxicology study of selenium in long-tailed macaques (Macaca fascicularis), a transplacental bone marrow micronucleus assay was conducted in the fetuses of treated animals. Selenium was administered as L-selenomethionine by nasogastric intubation at 0, 150 or 300 micrograms/kg-day to pregnant macaques daily throughout organogenesis (gestation days 20-50). Pregnancy was terminated on gestation day 100 +/- 2 and fetuses were obtained by hysterotomy. Selenium concentrations in maternal blood were monitored throughout pregnancy and selenium concentrations in fetal blood were measured at hysterotomy. Maternal circulating selenium did not exceed 4 ppm in plasma or 3.7 ppm in erythrocytes. Selenium in cord blood was < or = 0.1 ppm in plasma and < or = 1.1 ppm in erythrocytes at 300 micrograms/kg-day. Fetal bone marrow smears were prepared from the humerus and micronucleated polychromatic erythrocytes were scored. No increase of micronucleus frequency was detected in any dose group, although signs of maternal selenosis were obvious. This finding is compared to the previous observation that micronuclei were induced in the bone marrow of adult nonpregnant macaques treated at 600 micrograms/kg-day, a lethal dose yielding blood selenium levels to 7.3 ppm in plasma and 5.7 ppm in erythrocytes after 15 days of daily treatment, when death occurred. These data demonstrate that measurement of circulating xenobiotics can be useful for the interpretation of genetic toxicology results.

Effects of excess selenomethionine on selenium status indicators in pregnant long-tailed macaques (Macaca fascicularis).

Forty pregnant long-tailed macaques were treated daily for 30 d with 0, 25, 150, or 300 micrograms selenium as L-selenomethionine/kg body weight. Erythrocyte and plasma selenium and glutathione peroxidase specific activities, hair and fecal selenium, and urinary selenium excretion were increased by and were linearly related to L-selenomethionine dose. Hair selenium was most sensitive to L-selenomethionine dose, with an 84-fold increase in the 300 micrograms selenium/(kg-d) group relative to controls (r = 0.917). Daily urinary selenium excretion (80-fold, r = 0.958), plasma selenium (22-fold, r = 0.885), erythrocyte selenium (24-fold, r = 0.920), and fecal selenium (18-fold, r = 0.911) also responded strongly to L-selenomethionine. Erythrocyte and plasma glutathione peroxidase specific activities increased 154% and 69% over controls, respectively. Toxicity was associated with erythrocyte selenium > 2.3 micrograms/mL, plasma selenium > 2.8 micrograms/mL, and hair selenium > 27 micrograms/g. Plasma, erythrocyte, and hair selenium concentrations may be useful for monitoring and preventing the toxicity of L-selenomethionine administered to humans in cancer chemoprevention trials.

Absorption, distribution and elimination of selenium as L-selenomethionine in non-human primates.

20 adult female macaques (Macaca fascicularis) were given oral doses of L-selenomethionine (L-SeMet) equivalent to 0, 25, 150, 300 and 600 micrograms selenium (Se)/kg body weight, and plasma, erythrocyte, hair, faecal and urine Se concentrations were determined. The macaques were scheduled for 30 daily oral doses of L-SeMet, but systemic toxicity necessitated dose reduction in several animals; two macaques given 600 micrograms Se/kg body weight/day for 10-15 days died, and the concentration of Se in their tissues was determined and compared with Se concentrations in tissues collected from one untreated animal. Circulating and urinary Se concentrations in control macaques were within the normal human ranges. Plasma, erythrocyte, hair and urinary Se concentrations were generally dependent on the dose of L-SeMet administered. Plasma Se reflected more immediately exposure to L-SeMet, whereas erythrocyte Se concentrations increased and decreased more slowly. In some cases, erythrocyte Se was still increasing or showed a plateau after L-SeMet treatment was discontinued. Plasma Se concentrations of 6.7-7.3 ppm were observed in the two animals that died due to acute toxicity to L-SeMet. Neither plasma nor erythrocyte GPx activity was influenced by a single L-SeMet dose, but an increase in erythrocyte GPx activity occurred with continuous exposure. Total tissue Se increased 13-28-fold in macaques given 600 micrograms Se/kg body weight/day for 10-15 days, with the liver and kidneys containing the the highest Se concentrations.

In vitro and in vivo genotoxicity of 1,3-butadiene and metabolites.

1,3-Butadiene and two major genotoxic metabolites 3,4-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB) were used as model compounds to determine if genetic toxicity findings in animal and human cells can aid in extrapolating animal toxicity data to man. Sister chromatid exchange (SCE) and micronucleus induction results indicated 1,3-butadiene was genotoxic in the bone marrow of the mouse but not the rat. This paralleled the chronic bioassays which showed mice to be more susceptible than rats to 1,3-butadiene carcinogenicity. However, 1,3-butadiene did not induce unscheduled DNA synthesis (UDS) in the rat or mouse hepatocytes following in vivo exposure. Likewise, UDS in rat and mouse hepatocytes in vitro was not induced by EB or DEB. Salmonella typhimurium gene mutation (Ames) tests of 1,3-butadiene using strains TA1535, TA97, TA98, and TA100 and employing rat, mouse, and human liver S9 metabolic systems were barely 2-fold above background only in strain TA1535 at 30% 1,3-butadiene in air with induced and uninduced rat S9 and mouse S9 (uninduced). 1,3-Butadiene was negative in in vitro SCE studies in human whole blood lymphocytes cultures treated in the presence of rat, mouse, or human liver S9 metabolic activation. In general, 1,3-butadiene is genotoxic in vivo but is a weak in vitro genotoxin.

Embryotoxicity and dose-response relationships of selenium in hamsters.

Pregnant hamsters were treated with selenite, selenate, and selenomethionine during the critical stages of embryogenesis. The dosing regimens were oral, intravenous, and osmotic minipump infusion. Malformations, mainly encephaloceles, were noted with oral and intravenous selenite and selenate but were associated with maternal toxicity manifested by inanition and weight loss. Fetal body weights and lengths were reduced in a dose-dependent manner with the inorganic forms. Single oral doses of selenomethionine above 77 mumol/kg induced similar malformations but not when the dose was delivered orally over four days nor by minipump over several days. Fetal body weights and lengths were decreased by selenomethionine in a dose-dependent manner. Maternal toxicity was pronounced with the higher doses of selenomethionine. Assigning a specific teratogenic effect to selenium is confounded by maternal toxicity.

In vivo sister chromatid exchange and micronucleus induction studies with 1,3-butadiene in B6C3F1 mice and Sprague-Dawley rats.

Male B6C3F1 mice and Sprague-Dawley rats were exposed for 2 days, 6 h/day to 1,3-butadiene (BD) by inhalation (nose only) and their bone marrow cells were evaluated for the induction of micronuclei (MN) and sister chromatid exchanges (SCEs). A significant dose-dependent increase in MN induction was observed in mice. At 100 p.p.m., the frequency of micronucleated polychromatic erythrocytes was 6-fold above control with a maximal induction of 38-fold at 10,000 p.p.m. A significant increase in SCEs was also observed in mouse bone marrow cells starting at 100 p.p.m. with a 4-fold increase over the control evident at 10,000 p.p.m. The highest tested no observed effect level for both endpoints was 50 p.p.m. In contrast, rat bone marrow cells did not exhibit significant increases in micronucleated polychromatic erythrocytes or SCEs. These results indicate that BD is genotoxic in the bone marrow of the mouse but not the rat. This paralleled the chronic bioassays which showed mice to be more susceptible than rats to BD carcinogenicity.

Density-gradient enrichment of newly-formed mouse erythrocytes. Application to the micronucleus test.

To facilitate scoring micronuclei in peripheral blood erythrocytes, we have developed a centrifugation method to concentrate polychromatic and newly-formed normochromatic erythrocytes from microliter quantities of blood in a Percoll density gradient. Erythrocytes were separated into two discrete bands in a continuous gradient generated in situ in a microhematocrit capillary tube. The upper band contained white blood cells and a mixture of polychromatic and young normochromatic erythrocytes with a density of 1.080-1.082 g/ml. More than 75% of the polychromatic erythrocytes in samples of normal blood were recovered in the upper band. Older normochromatic erythrocytes migrated to the lower band. The frequency of polychromatic erythrocytes was increased from approximately 2% in whole blood to 60-80% in the upper band. After clastogen treatments, the elevated frequencies of micronuclei in the upper band polychromatic erythrocytes were similar to those in unfractionated blood. The frequencies of micronucleated normochromatic erythrocytes in the upper band were higher than those in whole blood at 48, 72 and 96 h after clastogen treatment, consistent with the expectation that the low-density normochromatic cells are newly derived from polychromatic erythrocytes. This density-gradient centrifugation technique enhances the efficiency of scoring micronuclei in the acute peripheral blood micronucleus test.

Micronuclei in circulating erythrocytes: a rapid screen for chromosomal damage during routine toxicity testing in mice.

Micronuclei in circulating erythrocytes provide a convenient measure of genetic damage resulting from chromosomal breakage or anaphase lag in bone marrow erythroblasts. The assay is easily integrated with acute, subchronic, or chronic toxicity tests. Scoring micronucleated erythrocytes in blood rather than bone marrow permits repeated sampling, simplifies sample preparation, and provides a more favorable cell population for scoring. Micronucleated erythrocytes accumulate during repeated exposures to clastogens and reach a maximum steady-state frequency after about five weeks of continuous treatment. Measurement of micronuclei in peripheral blood is therefore ideally suited for inclusion with routine subchronic toxicity tests. The same smear made for differential white blood cell counts at or near the end of the study can be scored for micronucleated erythrocytes, minimizing the effort required for this additional information and also permitting retrospective evaluation of completed studies.

Isolation of diploid human lymphoblast mutants presumably homozygous for ouabain resistance.

From a mass culture of diploid human lymphoblasts we have isolated a subline resistant to 1 muM ouabain, and from this, a subline resistant to 10 muM ouabain. These sublines occurred spontaneously, but similar mutants were induced with 10-fold increased frequency by treatment with mutagens, in which case they could be selected as clones in soft agarose. Scatchard plot analyses of ouabain binding indicated that the subline resistant to 1 muM ouabain retained an average of 39% of the high-affinity ouabain receptors of the parental lymphoblast line, and the subline resistant to 10 muM ouabain retained an average of 8.4%. The ouabain binding site is known to be located in the Na(+),K(+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) molecule, an essential cell membrane enzyme that mediates ion transport. Studies on Na(+),K(+)-ATPase activity, using (86)Rb in the absence and presence of ouabain, indicated that our parental lymphoblasts contained one population of Na(+),K(+)-ATPase molecules highly sensitive to ouabain inhibition, the subline resistant to 10 muM ouabain contained one population relatively insensitive to ouabain, and the subline resistant to 1 muM ouabain contained both populations. Thus, the moderately resistant subline appears to be heterozygous for ouabain resistance, probably containing a structural mutation in the ouabain receptor region of the Na(+),K(+)-ATPase molecule in one of the two homologous loci for this enzyme, whereas the highly resistant subline derived from it appears to be homozygous, containing an additional mutation in the other Na(+),K(+)-ATPase locus.

Role and location of "protease I" from Escherichia coli.

Pacaud and Uriel described an enzyme from Escherichia coli ("protease I") that hydrolyzes acetyl phenylalanine naphthyl ester (APNE). We examined the possible involvement of this enzyme in intracellular protein degradation, its subcellular distribution, and its proteolytic activity. Although the APNE-hydrolyzing activity is localized primarily in the periplasm, proteolytic activity against casein was found in the periplasm, membrane, and cytoplasm with similar specific activities. The APNE-hydrolyzing enzyme did not appear to contribute to the proteolytic activity of the periplasm. A mutant deficient in APNE-hydrolyzing activity lacked all activity in the periplasm but showed a slight percentage of residual activity in the cytoplasm. Extracts of such cells were normal in their ability to hydrolyze casein. The mutant was indistinguishable from wild-type cells in its rate of protein degradation during growth or glucose starvation and in the ability to rapidly degrade puromycin-containing polypeptides. Nitrogen starvation, which increased protein breakdown severalfold, affected neither the total amount nor the distribution of APNE-hydrolyzing activity. The mutant showed no defect in its ability to cleave small phenylalanine-containing peptides released during protein degradation. The mutant and wild-type cells are equally able to hydrolyze exogenously supplied phenylalanyl peptides. These experiments suggest that the APNE-hydrolyzing enzyme is not required for protein degradation and that "protease I" is probably not a protease.