Dibromoacetic acid (DBA) is a by-product of drinking water disinfection that alters spermatogenesis in adult male rats. To identify a mechanism by which DBA alters spermatogenesis, seminiferous tubules representing specific groups of spermatogenic stages were exposed either in vivo or in vitro, and structural and functional consequences were evaluated. Seminiferous tubules representing stages I-V, VI-VIII, and IX-XIV were isolated from testes of adult rats and cultured overnight in conditions of reduced oxygen and temperature. For in vivo exposures, seminiferous tubules were recovered from animals that had received 250 mg/kg DBA via gavage for 5 days. For in vitro exposures, 180 and 600 microM concentrations were tested; these concentrations bracket the concentration of DBA observed within the testis following in vivo exposure. Protein synthesis was evaluated by 35S-methionine labeling overnight and quantitative analysis of radiolabeled proteins in mini, 2-dimensional (2D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Radio-inert cultures were processed for light and electron microscopy. Morphologicaf evaluation indicated that all spermatogenic stages of the seminiferous tubules from control animals were well maintained during the isolation and culture period. Although no treatment-related lesions were observed following in vivo exposure, histological alterations were observed at the lowest in vitro exposure. There was a significant diminution (P < .05) in the synthesis of 4 cytosolic proteins following both in vivo and in vitro exposures. Diminution in these proteins was restricted to stages I-V and IX-XIV of spermatogenesis, suggesting that proteins involved in the early stages of spermiogenesis are uniquely sensitive to DBA exposure. Because histology and protein synthesis were affected by relevant in vitro exposures, this indicates that DBA is capable of altering spermatogenesis directly.

The drinking water disinfection by-product, dibromoacetic acid (DBA) has been reported to affect gonadal functions in the male rat. However, there is little information regarding the influence of DBA on female reproductive activity. Consequently, the present study investigated the effects of DBA on estrous cyclicity and the impact in vitro of DBA on ovarian follicular steroid secretion. Regularly cycling animals were dosed with DBA (0 to 270 mg/kg/day) for 14 days and estrous cyclicity was monitored during treatment and for an additional 2-week posttreatment interval. A dose-related alteration in cyclicity was observed at 90 and 270 mg/kg/day, which persisted through the posttreatment monitoring in the high dose group. An in vitro exposure of preovulatory follicles to DBA was then used to assess the influence of DBA on steroid release. To select a concentration for use, a single oral exposure to 270 mg/kg was administered, and the mean blood levels were determined over a 5-h interval. For this in vitro work, pairs of preovulatory follicles from PMSG-primed immature rats were exposed to 0 or 50 microg/mL DBA over a 24-h period and evaluated for estradiol and progesterone release under baseline and hCG-stimulated conditions. The influence of tumor necrosis factor (TNFalpha) exposures under these conditions was also determined. In the nonstimulated condition, DBA was found to increase the release of estradiol, but had no detectable effect in response to hCG. Progesterone, however, showed marked suppression under hCG stimulation following exposure to DBA, while nonstimulated secretion was unaffected. TNFalpha by itself also suppressed stimulated progesterone release, but had no additional effect in combination with DBA. The data suggest that one factor in the disruption in estrous cyclicity could be an alteration in steroid production, which was characterized by separate effects on both estradiol and progesterone secretion.

Exposure of pregnant CD-1 mice to methanol (MeOH) by inhalation on gestation days (gd) 6-15 results in dose-related increases in fetal cleft palate, exencephaly, and skeletal defects. Here, critical periods for the developmental toxicity of MeOH were assessed in pregnant CD-1 mice exposed to 10,000 ppm MeOH or filtered air for 7 hr/day on 2 consecutive days during gd 6-13, or to single day (7 hr) exposures to 10,000 ppm MeOH during gd 5-9. Mice received water but not food during exposure. Maternal blood MeOH was determined at times during, at the end of, and subsequent to a single 7 hr exposure on gd 7. On gd 17, remaining mice were weighed, killed, and gravid uteri removed. Live, dead, and resorbed fetuses were counted, and live fetuses were examined, weighed, and preserved in 70% ethanol. All fetuses were examined externally and for cleft palate, eviscerated, and stained with Alizarin red for skeletal examination. Pregnant mice lost an average of 0.3-2.9 g during 7 hr exposure to either filtered air or MeOH, but a MeOH treatment effect was evident only with 2-day exposure on gd 7-8. Peak maternal blood MeOH concentration (at the end of exposure) was approximately 4 mg/ml, and MeOH was cleared from maternal blood within 24 hr. Some fully resorbed litters were observed with 2-day MeOH exposures on gd 6-7 or 7-8, or 1-day exposure on gd 7. With 1-day MeOH exposure on gd 7, the number live was lower than with exposure on any other day. As previously reported, cleft palate, exencephaly, and skeletal defects were the fetal anomalies observed in this mouse strain. Cleft palate occurred with 2-day exposures on gd 6-7 through gd 11-12 (peak on gd 7-8), and with 1-day exposure on gd 5 through gd 9 (peak on gd 7). Exencephaly occurred with 2-day exposures on gd 6-7 through gd 8-9 (peak gd 6-7) or 1-day exposure on gd 5 through gd 8 (peak on gd 7). Skeletal elements malformed included the exoccipital (peak gd 6-7, gd 5), atlas (peak gd 6-7, gd 5,6), axis (peak gd 6-7, gd 7), cervical vertebra 7 with a rib (peak gd 6-7, gd 7), and lumbar vertebra 1 with a rib (peak gd 7-8, gd 7). An increased incidence of fetuses with 25 presacral vertebrae (normal = 26) was observed with methanol exposure on gd 5, whereas an increased incidence of fetuses with 27 presacral vertebrae was observed with MeOH exposure on gd 7. These results indicate that gastrulation and early organogenesis represent a period of increased embryonal sensitivity to methanol.

5-Fluorouracil (5-FU) is a chemotherapeutic agent known to retard embryonic growth and induce cleft palate and limb deformities. The predominant mechanism underlying its toxic action is thought to be inhibition of thymidylate synthetase (TS), and hence thymidine triphosphate (dTTP) synthesis, resulting in alteration of the balance of deoxynucleotide (dNTP) pools and disruption of DNA synthesis. Indeed, previously we demonstrated retarded cell-cycle progression concurrent with a 60% decrease in TS activity in rat whole embryos following maternal exposure to 40 mg/kg 5-FU on Gestational Day 14 and in the murine erythroleukemic cell (MELC) suspension culture following exposure to 5-25 microM 5-FU for 2 hr. In the study described herein, we used high-performance liquid chromatography (HPLC) to demonstrate in both of these model systems that 5-FU exposure results in similar patterns of dNTP perturbations: a prolonged decrease in dTTP and dGTP levels and an increase in dCTP and dATP. In addition, we used centrifugal elutriation to synchronize MELC in the phases of the cell cycle (G0/G1 and early S) most sensitive to 5-FU to investigate the ability of nucleoside supplementation to mitigate 5-FU-induced toxicity. Our data indicate that following a 2-hr exposure to 5-25 microM 5-FU, supplementation with 1-10 microM thymidine (TdR) for 24 hr partially reverses 5-FU-induced toxicity as evidenced by increased cellular proliferation and cell-cycle progression and amelioration of 5-FU-induced perturbations of protein synthesis and cellular membrane permeability compared to unsupplemented 5-FU-exposed cells. However, TdR concentrations >/=100 microM inhibited growth or were cytotoxic. In comparison, supplementation with 10 microM-10 mM of deoxycytidine (CdR) was not toxic, but effected a dose-dependent recovery from 5-FU-induced toxicity. At 1-100 microM, neither deoxyadenosine nor deoxyguanosine supplementation reduced 5-FU-induced toxicity; at higher concentrations, both purine nucleotides inhibited cell growth. Although these results support the hypothesis that 5-FU disrupts the MELC cell cycle by depleting dTTP (a perturbation that is reversible by TdR supplementation), they also indicate that CdR supplementation offers an additional recovery pathway.

The prospect of widespread human exposure associated with its use as an alternative fuel has sparked concern about the toxic potential of inhaled methanol (MeOH). Previous studies have revealed congenital malformations in rats following inhaled MeOH (Nelson et al. (1985). Fundam. Appl. Toxicol. 5, 727-736) but these studies did not include postnatal behavioral assessment. In the present study, pregnant Long-Evans rats were placed in exposure chambers containing 15,000 ppm MeOH or air for 7 hr/day on Gestational Days (GD) 7-19. The total alveolar dose of methanol was estimated at about 6.1 g/kg/day, for a total dose of about 42.7 g/kg for the entire study. Maternal body weights were recorded daily and blood methanol concentrations were determined at the end of exposure on GD 7, 10, 14, and 18. Following birth (Postnatal Day 0 [PND 0]), a number of tests were performed at various points in development, including: offspring mortality and body wt (PND 1, 3), motor activity (PND 13-21, 30, 60), olfactory learning (PND 18), behavioral thermoregulation (PND 20-21), T-maze learning (PND 23-24), acoustic startle response (PND 24, 60), reflex modification audiometry (PND 60), pubertal landmarks (PND 31-56), passive avoidance (PND 72), and visual-evoked potentials (PND 160). Maternal blood MeOH levels, measured from samples taken within 15 min after removal from the exposure chamber, declined from about 3.8 mg/ml on the first day of exposure to 3.1 mg/ml on the 12th day of exposure. MeOH transiently reduced maternal body wt (4-7%) on GD 8-10, and offspring BW (5%) on PND 1. No other test revealed significant effects of MeOH. Prenatal exposure to high levels of inhaled MeOH appears to have little effect on this broad battery of tests beyond PND 1 in the rat. (C) 1995 society of Toxicology

The developmental toxicity of the alternative motor vehicle fuel methanol was assessed in mice by the inhalation route. Pregnant CD-1 mice were exposed to 1,000, 2,000, 5,000, 7,500, 10,000, or 15,000 ppm methanol for 7 hr/day on days 6-15 of gestation. Sham-exposed controls were exposed to filtered air under similar conditions. Additional control groups were left in their home cages either unhandled or food-deprived for 7 hr/day to match the food deprivation experienced by the exposed mice. Dams were observed twice daily and weighed on alternate days during the exposure period. Blood methanol concentrations were determined in some mice on gestation days 6, 10, and 15. On day 17, the remaining mice were weighed and killed and the gravid uteri removed. Implantation sites, live and dead fetuses and resorptions were counted, fetuses were examined externally and weighed as a litter. Half of each litter was examined for skeletal morphology and the other half of each litter was examined for internal soft tissue anomalies. One dam died in each of the 7,500, 10,000, and 15,000 ppm methanol exposure groups, but no dose-response relationship was evident for maternal death. The sham-exposed and food-deprived controls as well as all methanol exposed dams gained less weight than did unexposed dams fed ad libitum, but methanol did not exacerbate this effect. Significant increases in the incidence of exencephaly and cleft palate were observed at 5,000 ppm and above, increased embryo/fetal death at 7,500 ppm and above (including an increasing incidence of full-litter resorptions), and reduced fetal weight at 10,000 ppm and above. A dose-related increase in cervical ribs or ossification sites lateral to the seventh cervical vertebra was significant at 2,000 ppm and above. Thus, the NOAEL for the developmental toxicity in this study was 1,000 ppm. A log-logistic dose response model was applied to the incidence data for exencephaly, cleft palate, resorption and cervical rib, and maximum likelihood estimates (MLEs) and benchmark dosages (BDs, the lower 95% confidence interval of the MLEs) corresponding to 1% and 5% added risk above background were calculated. The MLE for 5% added combined risk of having either exencephaly or cleft palate or being resorbed was 3667 ppm, and the corresponding BD was 3,078 ppm. For cervical rib, the 5% added risk values for the MLE and BD were 824 and 305 ppm, respectively. The BDs for 1% added risk were 1915 ppm for exencephaly, cleft palate or resorption, and 58 ppm for cervical rib.(ABSTRACT TRUNCATED AT 400 WORDS)

Two experiments were conducted in which the acute effects of inhaled methanol on serum hormones associated with reproductive function in the male rat were evaluated. In the first experiment, rats exposed to methanol (0, 200, 5000 and 10 000 ppm) for 6 h were killed at the end of the exposure period (6 h) or the following morning (24 h). Also, because the process of exposure itself could modify neuroendocrine function, the effect of the handling associated with placing the rat in the exposure chamber was evaluated further by dividing the exposed animals into acclimated (2 weeks of prior handling) and non-acclimated groups. At 6 h, an effect of prior handling was noted in the sham-exposed rats, with serum luteinizing hormone (LH) of the non-acclimated group being greater than that of the acclimated group. Serum LH concentrations were altered by methanol exposure, but the direction of change and the exposure level at which an effect was noted differed between the acclimated and non-acclimated rats. Methanol (5000 ppm) reduced serum LH in the non-acclimated animals, while 10 000 ppm increased LH in the acclimated rats. Follicle stimulating hormone (FSH) and testosterone were unchangd by methanol in rats killed at 6 h. Thus, this experiment did not confirm earlier reports that exposure to 200 ppm for 6 h reduced serum testosterone. At 24 h, an effect of prior handling was still present in the hormonal measures, with serum and interstitial fluid testosterone concentrations being greater in the non-acclimated rats. Also, there was a dose × handling interaction with methanol exposure inducing an increase in serum testosterone in the non-acclimated rats (up to 5000 ppm) and a decrease in the acclimated rats (up to 10 000 ppm). In the second experiment, groups of acclimated and non-acclimated rats were exposed to 0 or 5000 ppm methanol for 1, 2 and 6 h killed immediately after removal from the chamber. Serum LH, testosterone and FSH values were not different in sham- vs methanol-exposed rats at any time point. As in experiment 1, an effect of prior handling was noted. In general, the concentrations of these hormones and serum prolactin in the non-acclimated rats were greater than those observed for acclimated rats. Methanol exposure resulted in increased prolactin concentrations under both conditions. These results demonstrate that while acute exposure to methanol (5000 ppm) can cause significant alterations in serum hormones, the direction and magnitude of the changes are dependent on whether or not the animal has been acclimated to the test situation.

The organochlorine insecticide lindane (gamma-hexachlorocyclohexane) induces hepatomas in select strains of mice including two of three phenotypic classes of (YS X VY) F1 hybrid mice. In contrast, lindane does not induce hepatomas in rats and other strains of mice. It has been suggested that variations in the biotransformation of lindane may play a role in the different susceptibility of rodents to lindane-induced hepatomas. This study reports the effect of chronic treatment with 160 ppm dietary lindane on the comparative metabolism and disposition of this insecticide in obese yellow Avy/a, lean pseudoagouti Avy/a, and lean black a/a phenotypes of (YS X VY) F1 hybrid female mice at 17, 30, 56, and 86 wk of age. At 24 h prior to necropsy, all mice were dosed po with 18 mg lindane (containing 55 muCi [U-14C]lindane)/kg. Urine, feces, and expired air were sampled for analysis. Data indicated that metabolism of lindane and excretion of its metabolites by these mice differ significantly from those of rats that are resistant to lindane-induced hepatomas. Treatment of the mice with 160 ppm lindane in the diet appeared to saturate the elimination pathways and resulted in an increased tissue burden of the insecticide and its metabolites in the older animals. Results indicate that differences in lindane metabolism and disposition observed in the (YS X VY) F1 hybrid mice were associated with chronic lindane treatment, aging, and obesity but not with genotype.

Previous studies have indicated that acute exposure to ambient concentrations of ozone (O3) as low as 196 μg/m3 (0.1 ppm) increases pentobarbital (PEN)-induced sleeping time in female mice. To elucidate potential mechanisms involved, additional studies were performed. A 3 h exposure to 9800 μg O3m3 (5 ppm) did not affect brain concentrations of PEN at time of awakening, even though sleeping time was increased. Exposure for 3 h to 9800 μg O3m3 (5 ppm) did not alter the pattern of brain or plasma metabolites of PEN. Pentobarbital clearance followed first-order kinetics with a one-compartment model. Mice exposed to 9800 μg O3m3 (5 ppm) for 3 h had a 106% increase in the plasma half-life of pentobarbital; at 1960μg O3m3 (1 ppm) for 3 h, a 71% increase was observed. It therefore appears possible that PEN-induced sleeping time might be increased due to an decrease in hepatic metabolism of PEN.

Exposure of male rats to 0 (air), 1, 1.75, and 3 ppm ozone (O3) 5 hr/day for a total of 10 days resulted in a positive linear relationship between ozone concentration and the concentrations of serum total lipoprotein free cholesterol (FCh) and high-density lipoprotein total cholesterol (HDL-Ch). The latter response was reflected in both its free (HDL-FCh) and esterified (HDL-ChE) components. On the other hand, serum triglycerides (TG) showed a marked decreasing linear trend with increasing ozone concentration. As judged by decreased body weights with no accompanying differences in feed consumption, apparent metabolic rate increased as ozone concentration increased. In another experiment, male rats were exposed 5 hr/day to either air or 1 ppm O3 for a total of 15 days. Groups of animals from each exposure were sampled at times ranging from immediately after to 44 hr postexposure. In agreement with the concentration response study, effects of O3 included increases in serum total cholesterol (Ch), HDL-Ch and HDL-FCh, and a decrease in TG. In addition, the degree of effects appeared to be maintained over the 44-hr period and to be greater than that observed at 1 ppm O3 in the concentration-response study.

Lindane (gamma-hexachlorocyclohexane) has been shown to produce hepatomas in some strains of mice but not in others. Genetic factors and/or altered metabolism may play a role in the susceptibility to lindane-induced hepatomas. This study reports the effect of age and obesity on the comparative metabolism and disposition of lindane in obese yellow Avy/a and in lean pseudoagouti Avy/a and black a/a phenotypes of (YS x VY) F1 hybrid female mice at 8, 17, 30, 56, and 86 wk of age. At 24 h prior to sacrifice the mice were dosed p.o. with 18 mg lindane (containing 55 microCi [U-14C]lindane/kg). Aging altered the biotransformation of lindane such that while the excretion of lindane and its metabolites declined, the proportion of conjugated and polar metabolites increased. Tissue storage was elevated in older animals. In the yellow Avy/a mice, which are known to have a predisposition to the formation of hepatomas, there was accelerated and prolonged growth, reduced metabolite excretion, a greater proportion of conjugated metabolites, and higher dechlorinase activity compared to that of their pseudoagouti Avy/a and black a/a siblings.

In two complete replicates of a 2 × 2-fractorial-designed experiment involving chlorobenzene and γ-hexachlorocyclohexane (lindane), the hepatotoxicity induced by a challenge dose of chlorobenzene was altered by the pretreatments due to selective changes in various metabolis pathways. Pretreatment with either toxicant, alone or in combination, elevated the relative metabolism of 1.12 g chlorobenzene/kg to conjugated and polar metabolites. The relative importance of these pathways was increased most by pretreatment with chlorobenzene + lindane and least with chlorobenzene. The incidence and severity of chlorobenzene-induced hepatocellular necrosis was dependent on how much the pretreatments increased excretion of these metabolites relative to that of p-chlorophenol, since the conjugates and polar metabolites represent an inactivation of the toxic chlorobenzene-3,4-epoxide whereas p-chlorophenol reflects its formation. Thus these changes in the metabolic pathways resulted in either (i) a marginally significant decrease in hepatotoxicity (chlorobenzene pretreatment); (ii) significant reduction in both the incidence and severity of the lesions (lindane pretreatment); or (iii) absence of centrilobular hepatocellular necrosis in all but 1 of 12 rats where a minimal degree of necrosis was present (chlorobenzene + lindane pretreatment). In this study, the effect of pretreatment with xenobiotics on chlorobenzene-induced hepatotoxicity was dependent on how much the pretreatments altered the inactivation of chlorobenzene-3,4-epoxide relative to its formation.

Vitamin C-deficient and normal guinea pigs were exposed to various concentrations of NO2 or air, and lavage fluid was obtained and analyzed for protein and lipid content. Exposure of normal animals to 752, 1880, 5640, or 9400 μg NO2/m3 (0.4, 1.0, 3.0, or 5.0 ppm) for 72 hr did not alter the protein or lipid content of lung lavage fluid. However, exposure of vitamin C-deficient animals to the same concentrations of NO2 caused marked increases in lavage proteins and lipids at all but the 752 μg/m3 (0.4 ppm) level. At 9400 μg NO2/m3 (5.0 ppm), 50% of the exposed vitamin C-deficient animals died, and pathologic study of the lungs showed proteinaceous edema fluid in the alveoli. Lungs from air-exposed animals and normal animals exposed to NO2 appeared healthy. No effects were seen at 752 μg NO2 (0.4 ppm) in either normal or deficient animals even when the time of exposure was extended to 1 week. At 9400 μg NO2/m3 (5 ppm) effects could be seen in vitamin C-deficient animals even when the exposure period was shortened to 3 hr. Assessment of protein and lipid content of lavage fluid provided a sensitive method for determining subtle changes in the lung following NO2 exposure.

An attempt was made to distinguish different patterns of microsomal enzyme induction by phenobarbital, β-naphthoflavone, and Aroclor 1254 on the biotransformation and excretion of the organochlorine insecticide lindane. Treated groups of six weanling female Sprague-Dawley rats, individually housed in metabolism cages, received diets containing either 500 ppm Aroclor 1254, 356 ppm phenobarbital, or 418 ppm β-naphthoflavone. After 1 week all animals, except one group of controls, were dosed p.o. with 1.89 mg lindane (containing 1.63 μCi [U-14C]lindane). Twenty-four hours later the rats were sacrificed and urine, feces, expired air, and tissue samples were taken for analysis of radioactivity. Hepatic cytochrome P-450 content, microsomal phospholipid content, and the enzyme activity involved in the dehydrogenation of lindane, the dechlorination of lindane, and the hydroxylation of the intermediate hexachlorocyclohexene were determined in vitro. Moreover, the effect of pretreatment on the excretion of radioactivity and the distribution of eight lindane metabolites was examined. Even though the rate of lindane metabolism was unchanged by the β-naphthoflavone pretreatment, results of the study indicated that all three pretreatments significantly altered lindane metabolism. The pretreatments differed from one another in that they selectively altered specific metabolic pathways.