Absorption, disposition kinetics, and metabolic pathways of cyclohexene oxide in the male Fischer 344 rat and female B6C3F1 mouse

Cyclohexene oxide (CHO) is a monomer intermediate used in the synthesis of pesticides, pharmaceuticals, and perfumes. Although CHO has a variety of industrial uses where direct human exposure is possible, very little is known about its fate in the body. Therefore, the objectives of this study were to determine the absorption, distribution, metabolism, and excretion of cyclohexene oxide after oral, intravenous, and dermal exposure in male Fischer 344 rats and female B6C3F, mice. After intravenous administration of [14C]CHO (50 mg/kg), CHO was rapidly distributed, metabolized, and excreted into the urine. Plasma concentrations of CHO rapidly declined and were below the limit of detection within 60 min. Average (+/- SD) values for terminal disposition half-life, apparent volume of distribution at steady-state, and systemic body clearance were: 19.3 +/- 1.6 min; 0.44 +/- 0.08 liter/kg; and 31.3 +/- 0.5 ml/kg * min, respectively. After oral administration of [14C]CHO (10 and 100 mg/kg), it was found that 14C-equivalents were rapidly excreted in the urine of both species. At 48 hr, the majority of the dose (73-93%) was recovered in urine, whereas fecal elimination accounted for only 2-5% of the dose. At no time after oral administration was parent CHO detected in the blood. However, its primary metabolite cyclohexane-1,2-diol was present for different lengths of time depending on the dose. Four metabolites were detected and identified in mouse urine by MS: cyclohexane-1,2-diol; cyclohexane-1,2-diol-O-glucuronide; N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine; and cyclohexane-1,2-diol-O-sulfate. The sulfate conjugate was not present in rat urine. Topical application of [14C]CHO (60 mg/kg) provided poor absorption in both species. The majority of 14C-equivalents applied dermally were recovered from the charcoal skin trap (approximately 90% of the dose). Only 4% of the dose was absorbed, and the major route of elimination was via the urine. To evaluate the toxicity of CHO, animals were given daily doses of CHO orally and topically for 28 days. No statistically significant changes in final body weights or relative organ weights were noted in rats or mice treated orally with CHO up to 100 mg/kg or up to 60 mg/kg when given topically. Very few lesions were found at necropsy, and none were considered compound related. In conclusion, regardless of route, CHO is rapidly eliminated and excreted into the urine. Furthermore, after either oral or dermal administration, it is unlikely that CHO reaches the systemic circulation intact due to its rapid metabolism, and is therefore unable to cause toxicity in the whole animal under the test conditions used in this study.     

Immunological evaluation of the mycotoxin patulin in female B6C3F1 mice

Patulin is a mycotoxin produced by many fungal species of the genera Penicillium, Aspergillus and Bryssochamys. Previous literature reports have suggested that patulin is toxic to the immune system. The studies presented were conducted to provide a comprehensive assessment of the effects of patulin on the immune system. Unlike previous reports, the doses of patulin used (0.08, 0.16, 0.32, 0.64, 1.28 and 2.56 mg/kg) were based on predicted human exposure levels. Female B6C3F1 mice were exposed orally to patulin for 28 days. Effects were not observed on final body weight or body weight gain. Relative weight of the liver, spleen, thymus, kidneys with adrenals, and lungs was not affected. Peripheral blood leucocyte and lymphocyte counts were decreased by approximately 30% in the two highest dose groups. The leucocyte differential was not altered. Total spleen cell, total T-cell (CD3+), helper T-cell (CD4+CD8-), B-cell (surface immunoglobulin+) and monocyte (MAC-3+) counts were not changed. Cytotoxic T-cell (CD8+CD4-) counts were increased 50% only by the highest dose. Natural killer cell (NK1.1+CD3-) and monocyte (MAC-1+) counts were increased 30% and 24%, respectively, only in the 0.08 mg/kg group. Humoral immune function as assessed by antibody-forming cell response and serum IgM titre to sheep erythrocytes, and cell-mediated immune function evaluated utilizing natural killer cell activity and the mixed lymphocyte reaction were not altered. Oral exposure to patulin for 28 days did not alter the ability of female B6C3F1 mice to mount either a cell-mediated or humoral immune response.     

Metabolism of trichloroethylene in B6C3F1 mouse and human liver slices

Human and B6C3F1 mouse liver tissue was exposed to trichloroethylene (TCE) to determine metabolic rate constants. Using a novel volatile exposure system based on precision-cut tissue explants, TCE biometabolism was measured by appearance of a major oxidative product trichloroacetic acid (TCA). TCE metabolic rate was linear in this system to 150 minutes, allowing calculation of Michaelis-Menten kinetic parameters, Km and Vmax. Both human and mouse liver explants tolerated exposure to TCE up to 750 microM without evidence of cytotoxicity. Km values for mouse and human tissue were 215 and 30.6 microM TCE, respectively, and Vmax estimates were 6.14 and 0.47 ng TCA produced per mg protein*min-1, mouse and human, respectively. These results are consistent with other reports in describing the greater capacity of mice to metabolize TCE. Metabolic differences such as these must be considered when interpreting the implications of TCE-induced toxicity in rodent models for human health assessment.     

Metabolism of bromodichloroacetate in B6C3F1 mice

Trichloroacetate (TCA), dichloroacetate (DCA), and bromodichloroacetate (BDCA) are byproducts of the chlorination of drinking water. TCA acts primarily as a peroxisome proliferator, but DCA produces tumors at doses less than required for peroxisome proliferation. BDCA does not induce peroxisome proliferation even at high doses. This study attempts to determine whether differences in the metabolism of the trihaloacetates (THAs) may contribute to their differing toxicological properties. Studies were performed in male B6C3F1 mice given [14C1,2]TCA, [14C1]BDCA, and [14C1,2]DCA by gavage. The replacement of a Cl by a Br greatly enhances THA metabolism. Much less radiolabel from BDCA is retained in the carcass after 24 hr than from TCA. Radiolabel from BDCA is largely found in the urine, with oxalate being the major metabolite. TCA is largely eliminated unchanged in the urine. There are dose-related changes in the rate of CO2 production from BDCA. The initial rate of CO2 production is reduced from 4.1 +/- 0.3 hr-1 at 5 and 20 mg/kg to 2.7 +/- 0.6 hr-1 at 100 mg/kg, but the net conversion to CO2 in 24 hr is greater at the highest dose. As would be predicted, substitution Br for Cl on TCA greatly increased its metabolism.     

Loss of tumor-promoting activity of unleaded gasoline in N-nitrosodiethylamine-initiated ovariectomized B6C3F1 mouse liver

Unleaded gasoline (UG) vapor (2056 ppm) increased the incidence of liver tumors in a chronic bioassay and exhibited tumor-promoting activity in N-nitrosodiethylamine (DEN)-initiated female mouse liver. Estrogen inhibited mouse liver tumor development and the hepatocarcinogenic and tumor-promoting dose of UG produced uterine changes suggestive of estrogen antagonism. To directly test the hypothesis that UG-induced tumor-promoting ability is secondary to its interaction with the mouse liver tumor inhibitor, estrogen, we compared the tumor-promoting ability of UG in ovariectomized (Ovex) mice with the hepatic tumor-promoting ability of UG in intact mice. Ovaries were surgically removed at 4 weeks of age. Exposure to wholly vaporized UG (2018 ppm) under bioassay and tumor-promoting conditions began at 8 weeks of age. After 4 months of exposure, UG increased relative liver weight and hepatic microsomal cytochrome P450 pentoxyresourfin-O-dealkylase and ethoxyresorufin-O-deethylase activity to a similar extent in intact and Ovex mice. Non-focal hepatocyte proliferation, as measured by the incorporation of bromo-deoxyuridine, was not changed by UG exposure and was similar in all treatment groups. After 4 months of exposure to DEN-initiated mice, UG significantly increased the volume fraction of liver occupied by foci (three-fold) as compared to control intact mice. As expected, volume of foci was elevated in DEN/Ovex/control mice as compared to DEN/intact/control mice. In DEN/Ovex mice UG did not significantly increase the focal volume fraction. Thus, the tumor promoting activity of UG, as demonstrated by increased volume fraction of liver occupied by hepatic foci in intact mice, is greatly attenuated in Ovex mice. The volume fraction data in Ovex mice support the hypothesis that the tumor promoting activity of UG is dependent upon the interaction of UG with ovarian hormones. These data also indicate that hepatic microsomal cytochrome P450 PROD and EROD induction, hepatomegaly and non-focal hepatic LI are not specific markers of hepatic tumor promoting activity of UG.     

Effects of dichloroacetate on glycogen metabolism in B6C3F1 mice

Dichloroacetate (DCA) is a by-product of drinking water chlorination. Administration of DCA in drinking water results in accumulation of glycogen in the liver of B6C3F1 mice. To investigate the processes affecting liver glycogen accumulation, male B6C3F1 mice were administered DCA in drinking water at levels varying from 0.1 to 3 g/l for up to 8 weeks. Liver glycogen synthase (GS) and glycogen phosphorylase (GP) activities, liver glycogen content, serum glucose and insulin levels were analyzed. To determine whether effects were primary or attributable to increased glycogen synthesis, some mice were fasted and administered a glucose challenge (20 min before sacrifice). DCA treatments in drinking water caused glycogen accumulation in a dose-dependent manner. The DCA treatment in drinking water suppressed the activity ratio of GS measured in mice sacrificed at 9:00 AM, but not at 3:00 AM. However, net glycogen synthesis after glucose challenge was increased with DCA treatments for 1-2 weeks duration, but the effect was no longer observed at 8 weeks. Degradation of glycogen by fasting decreased progressively as the treatment period was increased, and no longer occurred at 8 weeks. A shift of the liver glycogen-iodine spectrum from DCA-treated mice was observed relative to that of control mice, suggesting a change in the physical form of glycogen. These data suggest that DCA-induced glycogen accumulation at high doses is related to decreases in the degradation rate. When DCA was administered by single intraperitoneal (i.p.) injection to na?ve mice at doses of 2-200 mg/kg at the time of glucose challenge, a biphasic response was observed. Doses of 10-25 mg/kg increased both plasma glucose and insulin concentrations. In contrast, very high i.p. doses of DCA (> 75 mg/kg) produced progressive decreases in serum glucose and glycogen deposition in the liver. Since the blood levels of DCA produced by these higher i.p. doses were significantly higher than observed with drinking water treatment, we conclude that apparent differences with data of previous investigations is related to substantial differences in systemic dose and/or dose-time relations.     

Immunosuppression in adult female B6C3F1 mice by chronic exposure to ethanol in a liquid diet

The overall objective of these studies was to characterize the effects of ethanol on the immunocompetence of adult female B6C3F1 mice. To obtain a significant suppression in the antibody response to SRBC, splenocytes from untreated mice had to be directly exposed to concentrations of ethanol from 0.3% to 3.0%, or to acetaldehyde at concentrations greater than 0.03%. We do not believe that these results are consistent with a role by a direct effect by either ethanol or its primary metabolite because these concentrations are higher than what could be obtained as reasonable blood levels. For in vivo exposure, we employed a pair-feeding regimen which was based on a liquid diet containing 5% ethanol (v/v) that provided 36% of the caloric intake as ethanol. Our results indicated that there was a definite temporal relationship to the consequent suppression of the antibody response to SRBC in that no effect was observed after 14 days exposure, and that the magnitude of the suppression increased from 18% after 21 days to 70% after 42 days. We also monitored the liver for histopathology and observed that the ethanol-induced liver damage was restricted to steatosis (fatty liver), which was also manifested with time and which was most pronounced after 42 days exposure. In contrast to our results with the in vivo antibody response, we saw no effect on mitogen-induced proliferation by splenocytes from ethanol-treated mice. These results prompted us to measure in vitro antibody responses by splenocytes from ethanol-treated mice. We saw no suppression of the in vitro antibody responses to SRBC, DNP-Ficoll or LPS after any length of exposure to ethanol, and speculated that the basis for the suppression of the in vivo antibody response was an indirect consequence of exposure. We subsequently determined that when normal splenocytes were cultured in 5% serum from ethanol-exposed mice (42-day group), there was a > 80% suppression relative to the serum from the pair-fed controls. As important controls for these studies, we have demonstrated that there was no difference between the responses of normal lymphocytes cultured in 5% normal mouse serum and in 5% serum taken from the pair-fed restricted controls. A determination of the ethanol content in the serum from ethanol-exposed mice (42-day group) indicated that the amount of ethanol present in these cultures was < 0.003%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacokinetics of a novel benzodiazepine partial inverse agonist in the F344 rat, SD rat and B6C3F1 mouse

1. The pharmacokinetics of a novel benzodiazepine partial inverse agonist (S-8510) were studied in the Fischer 344 (F344) rat and B6C3F1 mouse to obtain information for the planning of carcinogenicity studies. Sprague-Dawley (SD) rats were also included for comparison. 2. Clear non-linear elimination of S-8510 was observed after single oral administration of S-8510 in all animals tested (F344 rat, 1-50 mg/kg; SD rat and B6C3F1 mouse, 1-150 mg/kg). 3. Exposure of S-8510 after single oral administration was in the order F344 rat > B6C3F1 mouse > SD rat. 4. Multiple oral administration to F344 rat and B6C3F1 mouse decreased the exposure to S-8510. 5. These results indicate that it is very important to evaluate pharmacological and toxicological studies based on exposure and to be careful in selecting the species and strains of animal used in toxicology studies.     

Periodontal inflammatory and cystlike lesions in BDF1 and B6C3F1 mice

Periodontal inflammatory and cystlike lesions were detected in aged BDF1 and B6C3F1 mice. Lesions were unilateral, single, and located in the labial region of the incisor teeth. The largest cystlike lesion deformed the skull. Histologically, the cystlike lesions were classified into three types: predominantly an inflammatory reaction, predominantly a cyst formation, and a mixed lesion. These periodontal inflammatory and cystlike lesions were pathogenetically similar and were considered sequential changes; the lesions showing both cyst formation and inflammatory reaction seemed to be intermediate along a path toward formation of large cysts. The cysts probably were derived from periodontal tissue and developed as a result of inflammatory reaction to foreign bodies, including hair. The term "murine periodontal cyst" is proposed for this lesion. Detailed pathogenetic studies on murine odontogenic cystic lesions may be warranted.     

Haloacetate-induced oxidative damage to DNA in the liver of male B6C3F1 mice

Brominated and chlorinated haloacetates (HAs) are by-products of drinking water disinfection. Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic in rodents, but the brominated analogs have received little study. Prior work has indicated that acute doses of the brominated derivatives are more potent inducers of oxidative stress and increase the 8-hydroxydeoxyguanosine (8-OH-dG) content of the nuclear DNA in the liver. Since, DCA and TCA are also known as weak peroxisome proliferators, the present study was intended to determine whether this activity might be exacerbated by peroxisomal proliferation. Classical responses to peroxisome proliferators, cyanide-insensitive acyl-CoA oxidase activity and increased 12-hydroxylation of lauric acid, were elevated in a dose-related manner in mice maintained on TCA and clofibric acid (positive control), but not with DCA, dibromoacetate (DBA) or bromochloroacetate (BCA). Administration of the HAs in drinking water to male B6C3F1 mice for periods from 3 to 10 weeks resulted in dose-related increases in 8-OH-dG in nuclear DNA of the liver with DBA and BCA, but not with TCA or DCA. These findings indicate that oxidative damage induced by the haloacetates is, at least in part, independent of peroxisome proliferation. In addition, these data suggest that oxidative damage to DNA may play a more important role in the chronic toxicology of brominated compared to the chlorinated haloacetates.     

Toxicity of fumonisin B1 to B6C3F1 mice: a 14-day gavage study

Fumonisin B1 (FB1) is a fungal toxin produced by members of the genus Fusarium. Ingestion of FB1 causes species-specific neurotoxic, nephrotoxic, hepatotoxic and pulmonary effects. The clinical, haematological and pathological responses of adult male and female B6C3F1 mice to FB1 were assessed following 14 daily gavage doses ranging from 1 to 75 mg FB1/kg body weight/day. There were no consistent sex-related changes. Although all responses were modest, the most notable effects of FB1 were on the liver, bone marrow, adrenals and kidneys. In the liver, hepatocellular single cell necrosis, mitosis and anisokaryosis were observed, accompanied by elevated serum ALT. In the kidneys, minor histopathological changes were confined to female mice, while mild decreases in ion transport and increases in blood urea nitrogen were seen only in males. Small changes in glutathione levels were observed in the kidneys and livers of male mice. Adrenal cortical cell vacuolation was observed at 15 mg FB1/kg and higher in females and from 35 mg FB1/kg in males. Serum cholesterol was elevated in both male and female mice, possibly due to FB1-induced changes in lipid metabolism in the liver and adrenals. Although bone marrow cell numbers were unchanged, increases in vacuolated myeloid cells and lymphocytes were observed in female mice. In general, the degree of changes observed indicate that mice are not as sensitive a model of FB1 toxicity as rats.     

DNA adduct formation in the bone marrow of B6C3F1 mice treated with benzene

We used P1-enhanced 32P-postlabeling to investigate DNA adduct formation in the bone marrow of B6C3F1 mice treated intraperitoneally with benzene (BZ). No adducts were detected in the bone marrow of controls or mice treated with various doses of BZ once a day. After twice-daily treatment with BZ, 440 mg/kg, for 1 to 7 days, one major and two minor DNA adducts were detected. The relative adduct levels ranged from 0.06-1.46 x 10(-7). In vitro treatment of bone marrow from B6C3F1 mice with various doses of hydroquinone (HQ) for 24 h also produced three DNA adducts. These adducts were the same as those formed after in vivo treatment of bone marrow with BZ. Co-chromatography experiments indicated that the principal DNA adduct detected in the bone marrow of B6C3F1 mice was the same as that detected in HL-60 cells treated with HQ. This finding suggests that HQ may be the principal metabolite of BZ leading to DNA adduct formation in vivo. DNA adduct 2 corresponds to the DNA adduct formed in HL-60 cells treated with 1,2,4-benzenetriol. DNA adduct 3 remains unidentified. After a 7-day treatment with BZ, 440 mg/kg twice a day, the number of cells per femur decreased from 1.6 x 10(7) to 0.85 x 10(7), indicating myelotoxicity. In contrast, administration of BZ once a day produced only a small decrease in bone marrow cellularity. These studies demonstrate that metabolic activation of BZ leads to the formation of DNA adducts in the bone marrow. Further investigation is required to determine the role of DNA adducts and other forms of DNA damage in the myelotoxic effects of exposure to BZ.     

Inhalation toxicity of 1,6-hexanediamine dihydrochloride in F344/N rats and B6C3F1 mice

1,6-Hexanediamine (HDA) is a high production volume chemical which is used as an intermediate in the synthesis of paints, resins, inks, and textiles and as a corrosion inhibitor in lubricants. Two- and 13-week studies of the toxicity of the dihydrochloride salt of HDA (HDDC) were conducted in male and female Fischer 344/N rats and B6C3F1 mice using whole-body inhalation exposure. Both species were evaluated for histopathologic and reproductive effects, and rats were examined for clinical chemistry and hematologic changes. In the 2-week inhalation studies, animals were exposed to 10-800 mg HDDC/m3, 6 hr per day. All rats, all female mice, and two of five male mice in the high-exposure group died before the end of the study. Surviving mice in this group had a dose-dependent depression in body weight gain. Clinical signs were primarily related to upper respiratory tract irritation and included dyspnea and nasal discharge in both species. Treatment-related histopathologic lesions included inflammation and necrosis of the laryngeal epithelium of both species and the tracheal epithelium of mice, as well as focal inflammation and ulceration of the respiratory and olfactory nasal mucosa. In the 13-week inhalation studies, animals were exposed to HDDC at concentrations of 1.6-160 mg/m3 for 6 hr per day, 5 days per week. In addition to the base study groups, a supplemental group of rats at each exposure level was included to assess the effect of HDDC on reproduction. No treatment-related changes in organ weights or organ-to-body-weight ratios occurred in rats, and no treatment-related clinical signs or gross lesions were seen in either species. Chemical-related microscopic lesions were limited to the upper respiratory tract (larynx and nasal passages) in the two highest exposure groups and were similar in both species. These lesions included minimal to mild focal erosion, ulceration, inflammation, and hyperplasia of the laryngeal epithelium, in addition to degeneration of the olfactory and respiratory nasal epithelium. HDDC caused no significant changes in sperm morphology or vaginal cytology and no significant adverse effects on reproduction in rats or mice. Hematologic and clinical chemistry changes in rats were minor and sporadic and were not accompanied by related histologic findings. HDDC did not increase the frequency of micronucleated erythrocytes in mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

A 13-week subchronic toxicity study of bisphenol A in B6C3F1 mice

A 13-week subchronic toxicity study of bisphenol A (BPA) was performed in male and female B6C3F1 mice at dose levels of 0, 0.2, 0.5, 1.0, 2.0 and 4.0% in the diet, to facilitate dose selection for a subsequent carcinogenicity study. Mice were randomly allocated to 6 groups, each consisting of 10 males and 10 females. Two 0.2% group males and two 4.0% group females died during the experimental period. Suppression of body weight gain and increase in food consumption were observed in males and females of the 4.0% groups. Hematological examination revealed decrease in number of erythrocytes, volume of hemoglobin and value of hematocrit in males and females of the groups receiving 1.0% or above, and an increase in number of platelets in males of 4.0% group. Decrease in number of erythrocytes and hematocrit value was also noticed in females of 0.5% group. Histopathologically, cystic dilatation, degeneration or regeneration of renal tublues were found in males and females of 1.0% or higher groups, multinucleated hepatocytes were increased in mice of both sexes treated with any dose of BPA, and fibrous osteodystrophy was observed in males and females of the 4.0% groups. Based on the results of the present study, it was concluded that the maximum tolerance dose (MTD) of BPA is 0.2% in diet, because the dose level of 0.5% proved to exert significant hematological toxicity.     

Oncogenicity testing of 2-ethylhexanol in Fischer 344 rats and B6C3F1 mice

An increasing number of studies, both experimental and epidemiologic, have provided evidence that filtering glaucoma surgery may be less effective than initially described. Of a number of risk factors for failure, duration and number of antiglaucoma drugs prior to surgery seem to play a critical role and highly accumulated antiglaucoma topical treatments significantly reduce success rates. Histopathological studies have confirmed that topically applied drugs may exert toxic effects to the corneoconjunctival surface, and induce chronic infraclinical inflammation, as shown by the presence of immune and inflammatory infiltrates in multitreated eyes. The origin of topical inflammation has not yet been clearly determined, but a common component of ophthalmic drugs, the benzalkonium chloride used as preservative in almost all antiglaucoma preparations, has shown strong evidence of toxicity. A number of questions remain to be investigated, but suppression of preservatives from chronically applied drugs should be a critical issue in the near future.     

Alteration in cell kinetics in control B6C3F1 mice infected with Helicobacter hepaticus

The discovery of Helicobacter hepaticus infection, H. hepaticus hepatitis, and increased incidence of liver tumors in control males from several recent National Toxicology Program B6C3F1 mouse carcinogenicity bioassays raised questions regarding the suitability of these bioassays for hazard identification. The purpose of this study was to determine if changes in cell proliferation and death at terminal sacrifice might be linked to the increased liver tumor incidences among control males. In control males, enhanced rates of hepatocyte proliferation, as assessed by immunostaining for proliferating cell nuclear antigen (PCNA), and apoptosis, as assessed from hematoxylin and eosin- and TUNEL-stained preparations, were seen in 3 bioassays with H. hepaticus hepatitis. One bioassay with H. hepaticus infection without attendant hepatitis and one bioassay without H. hepaticus or hepatitis did not have elevated rates of hepatocyte proliferation or apoptosis. There was no significant effect on PCNA cell proliferation indices or apoptosis in females. The present findings are indicative of a clear association between the presence of H. hepaticus infection with attendant hepatitis, increased cell proliferation and apoptosis, and increased incidences of hepatocellular neoplasia in males but not in females. Thus, the interpretation of liver tumor responses in H. hepaticus-infected studies is considered to be confounded in male mice. The lack of enhanced cell proliferation or hepatocellular neoplasia in control females suggests that bioassay results from females are valid for hazard identification. Furthermore, the absence of enhanced cell proliferation in lungs and kidneys of male and females suggests that neoplastic effects at these sites are not exacerbated by H. hepaticus infection.     

Influence of dietary restriction on ionotropic glutamate receptors during aging in C57B1 mice

The present study was designed to determine whether the memory sparing effects of dietary restriction during aging could be through an effect on ionotropic glutamate receptors. Quantitative autoradiography was performed on 3, 10, and 26 month old mice to examine the density changes of NMDA, AMPA and kainate binding sites in aging animals. Spatial memory performance was also tested in these mice with the use of the Morris water maze. The 10 and 26 month olds were either ad libitum-fed or diet-restricted (60% of ad libitum-fed calories). Ad libitum-fed, 26 month old mice had significant decreases in NMDA-displaceable [3H]glutamate in all ten cortical, two out of seven hippocampal, and two out of four subcortical regions, as compared to 3 month olds. Diet-restricted, 26 month old mice only differed significantly from young in three cortical and two subcortical regions. The aged ad libitum-fed mice exhibited significantly poorer performance in the spatial memory task than all other groups. The diet-restricted 26 month olds only performed significantly worse than 3 month olds and diet-restricted 10 month olds. These results suggest that some of the memory sparing effects of dietary restriction on aged animals may be due to an influence on NMDA receptors.     

Comparison of methods of identifying Helicobacter hepaticus in B6C3F1 mice used in a carcinogenesis bioassay

In a long-term rodent bioassay evaluating the carcinogenicity of triethanolamine, there was equivocal evidence of carcinogenic activity in male B6C3F1 mice, based on a marginal increase in the number of hepatocellular adenomas and hepatoblastomas. Interpretation was complicated by the presence of Helicobacter hepaticus in selected silver-stained liver sections which also had histological evidence of karyomegaly and oval cell hyperplasia. An increase in numbers of liver tumors, as evidence of carcinogenic activity, was also noted in female mice. However, H. hepaticus was not considered a complicating factor, because the livers of the female mice did not have histological features compatible with H. hepaticus infection. A retrospective analysis of 51 liver tissue samples from the original carcinogenicity study was conducted to determine the incidence of H. hepaticus infection and to evaluate different diagnostic approaches for assessing the presence of H. hepaticus in livers lacking characteristic lesions. In an initial evaluation of seven mice with liver tumors, argyrophilic bacteria resembling H. hepaticus were observed in liver sections, associated with characteristic liver lesions of hepatocytic karyomegaly and oval cell hyperplasia. Frozen liver tissue was available from four of these mice; all were confirmed to be infected with H. hepaticus by culture and PCR. In a larger subsequent analysis using frozen liver tissues from 44 mice without characteristic hepatic lesions, H. hepaticus-specific DNA was amplified from the livers of 21 of 44 of the mice (47%), compared to 14 of 44 of the mice (32%) having H. hepaticus cultured from their frozen liver tumors. The results of H. hepaticus culture and H. hepaticus-specific PCR concurred (i.e., both positive and negative results) in 84% of the cases. Microscopic detection of immunofluorescence-labeled or silver-stained bacteria in liver sections was relatively insensitive compared to either culture or PCR detection. This study confirms the widespread prevalence of H. hepaticus in mice, its potential to confound experimental results, and the need to include diagnostic testing for H. hepaticus in a murine health monitoring program.     

Mechanism of the paradoxical, inhibitory effect of phenobarbital on hepatocarcinogenesis initiated in infant B6C3F1 mice with diethylnitrosamine

Phenobarbital (PB), a classical rodent hepatopromoter, remarkably enhances hepatocarcinogenesis initiated by diethylnitrosamine (DEN) in adult B6C3F1 mice. However, it is also known to strongly inhibit liver tumor development in the B6C3F1 mice initiated with DEN in their infancy. The present study aimed to elucidate the unknown biological mechanisms for this paradoxical, inhibitory effect of PB on B6C3F1 mouse hepatocarcinogenesis. Male 12-day-old infant B6C3F1 mice were injected i.p. with DEN and, at 6 weeks of age, divided into PB-treated (PB+ group) and untreated (PB- group) animals. At 24 weeks, PB treatment was ceased for half of the PB+ animals (PB+/- group) and started for half of the PB- animals (PB-/+ group). Finally, all mice were sacrificed at 36 weeks and examined for the development of liver tumors. The mean multiplicity of gross tumors in the PB+ group was only one-fifteenth of that for the PB- group. PB-/+ animals developed fewer than half of the tumors found in PB- mice, indicating that the PB effect depends solely on the treatment duration, rather than the animal age. The effect was proven to be reversible, because the mean tumor multiplicity for the PB+/- group was seven times larger than that for the PB+ group. Stereological analysis revealed the mean volume of hepatocellular proliferative lesions in the PB- animals to be 7.7- and 4.1-fold the values for the PB+ and PB-/+ groups, respectively. The mean proliferating cell nuclear antigen-labeling indices for hepatocellular adenomas in PB+ and PB-/+ animals were also one-third of that for tumors in PB- animals, whereas no significant differences were observed with regard to the mean apoptotic index. In conclusion, the inhibitory effect of PB seemed to be primarily caused by the suppression of tumor cell proliferation. Irrespective of the group, most lesions observed were basophilic hepatocellular adenomas or foci, positive for Bcl-2 oncoprotein. They were thus distinct from the eosinophilic Bcl-2- lesions that predominate with PB promotion after the initiation of adult B6C3F1 mice. This age-dependent nature of initiation, together with the differential responses of Bcl-2+ and Bcl-2- lesions, may be responsible for the apparently contradictory outcomes of PB treatment in infant and adult B6C3F1 mice.