The effect of exposure regimen and duration on benzene-induced bone-marrow damage in mice. I. Sex comparison in DBA/2 mice

In the mouse, the concurrent evaluation of micronuclei frequencies in peripheral blood polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) permits an assessment of both recently-induced and chronically-accumulated bone-marrow damage. This assay system was used to evaluate on a weekly basis the effect of exposure duration (1-13 weeks, 6 h per day) and exposure regimen (Regimen 1:5 exposure days per week; Regimen 2:3 exposure days per week) on the ability of 300 ppm benzene to induce genotoxic damage in the bone marrow of male and female DBA/2 mice. In addition, an analysis of the percentage of PCE in peripheral blood was used to evaluate benzene-induced alterations in the rate of erythropoiesis. Exposure to benzene induced a marked increase in the frequency of micronucleated PCE (MN-PCE), an effect which was considerably greater in male mice than in female mice. In both sexes, the induction of MN-PCE was independent of exposure regiment and of exposure duration. Exposure to benzene also resulted in an exposure duration-dependent increase in the frequency of MN-NCE. The frequency of MN-NCE increased more slowly in female than in male mice and, within each sex, more slowly in Regimen 2 animals. Apparent steady-state conditions for MN-NCE frequencies were attained by about the fifth week of exposure in female mice exposed by either regimen and in male mice exposed by Regimen 2. Steady-state conditions for MN-NCE frequencies in male mice exposed to benzene by Regimen 1 did not occur during the duration of the study. An analysis of %PCE data revealed an initial severe depression in the rate of erythropoiesis in both sexes, with the return in the production of PCE to control levels being dependent on both sex and exposure regimen. Suppression of PCE production occurred throughout the course of the study in Regimen 2 males, while the percentage of PCE returned to control levels sporadically after 5 weeks in Regimen 1 males and within 5 weeks in females, regardless of regimen. Thus, while the sex-dependent induction of genotoxic damage by multiple exposures to benzene over a 13-week period was independent of exposure regimen and duration, the induction of cytotoxic damage was both sex- and regimen-dependent. The most severe depression of erythropoiesis occurred in male DBA/2 mice exposed to benzene by the more intermittent regimen (i.e., 3 days/week versus 5 days/week).(ABSTRACT TRUNCATED AT 400 WORDS)     

Benzene increases protein-bound 3-nitrotyrosine in bone marrow of B6C3F1 mice

Benzene, an environmental pollutant, is myelotoxic and leukemogenic in humans. The molecular mechanisms that can account for its biological effects have not been fully elucidated. We hypothesize that one of the underlying mechanism involves nitration of proteins by peroxynitrite and/or by bone marrow myeloperoxidase-dependent pathways in nitric oxide (NO) metabolism. Using 3-nitrotyrosine [Tyr(NO(2))] as a biomarker for NO-induced damage to proteins, we examined the effects of benzene on the levels of Tyr(NO(2)) in bone marrow in vivo. Groups of 8 weeks old B6C3F(1) male mice were given a single i.p. injection of benzene (50, 100, 200 or 400mg/kg bodyweight) in corn oil. The mice in control groups received either no treatment or a single injection of the vehicle. The mice were killed 1h after treatment and proteins were isolated from bone marrow, lung, liver and plasma. The proteins were enzymatically hydrolyzed; amino acids were separated and purified by high pressure liquid chromatography, derivatized, and quantified by electron capture-negative chemical ionization-gas chromatography/mass spectrometry (EC-NCI-GC/MS). In the GC/MS assay, 3-nitro-l-[(13)C(9)]tyrosine was used as an internal standard and l-[(2)H(4)]tyrosine served to monitor artifactual formation of 3-nitrotyrosine during sample preparation and analysis. We found that treatment of mice with benzene elevates nitration of tyrosine residues in bone marrow proteins. There was a dose (50-200mg benzene/kg b.w.)-dependent increase in protein-bound Tyr(NO(2)) formation (1.5- to 4.5-fold); however, the levels of Tyr(NO(2)) at 400mg benzene/kg b.w. were significantly higher than control but lower than that formed at 200mg benzene/kg b.w. The results of this study, for the first time, indicate that benzene increases protein-bound 3-Tyr(NO(2)) in bone marrow in vivo, and support our previous finding that benzene is metabolized to nitrated products in bone marrow of mice; collectively, these results may in part account for benzene-induced myelotoxicity.     

Food restriction-like effects of dietary dehydroepiandrosterone. Hypothalamic neurotransmitters and metabolites in male C57BL/6 and (C57BL/6 x DBA/2)F1 mice

Dehydroepiandrosterone (DHEA) is a precursor of sex hormones in mammals. Dietary DHEA serves to prevent or inhibit various diseases and also lengthens life spans of animals. Moreover, dietary DHEA inhibits food intake in certain strains of mice. We administered DHEA (0.45% w/w of food) to C57BL/6 (B6) and (B6 x DBA/2)F1 (BDF1) mice for 5 weeks. Food intake was inhibited in both strains of mice during the first week. Thereafter, B6, but not BDF1, mice consumed less food. Because hypothalamic serotonin and/or dopamine regulate appetite, satiety and other behaviors, the hypothesis tested was that hypothalamic concentration of serotonin, dopamine and/or their metabolites are affected differentially in B6 and BDF1 mice fed DHEA. In another study, mice were fed the AIN-76A diet with or without DHEA for 1 and 7 days or were pair-fed to DHEA-fed mice for 7 days. On Day 1 of DHEA feeding (acute effects) hypothalamic levels of serotonin, dopamine, and metabolites were unchanged in B6 mice, but levels of dopamine were increased and levels of dopamine metabolites were decreased in BDF1 mice. On Day 7 of DHEA feeding, levels of serotonin were increased in BDF1 but not B6 mice. On Day 7 of pair-feeding there were decreased levels of hypothalamic dopamine metabolites in BDF1 but not B6 mice. Paraventricular nuclei of BDF1 mice had decreased levels of serotonin but not of dopamine in all groups. Serum levels of DHEA and its metabolite, 5-androstene-3beta,17beta-diol, correlated significantly only with serotonin concentrations in BDF1 mice. The salient findings of these experiments are that DHEA inhibits food intake to a greater extent in B6 than in BDF1 mice. However, alterations of hypothalamic neurotransmitters were greater in BDF1 than in B6 mice. Because BDF1 and B6 mice share B6 genes, relevant gene(s) derived from DBA/2 mice might mediate the different responses detected.     

Forestomach ulcers in Crj:B6C3 (C57BL/6NCrj x C3H/HeNCrj) F1 mice

An unusually high incidence of forestomach ulcers was observed in a mouse strain that is used frequently for long-term toxicology studies. Examination of 98 untreated male and 98 untreated female B6C3F1 hybrid mice, the majority of which were between 105 and 113 weeks of age, revealed forestomach ulcers in 52% of the males and 54% of the females. Glandular stomach ulcers were uncommon, being found in only four female mice. The incidence of the ulcers increased with age. The etiology of the lesion is unknown.     

Protective effect of bone marrow and spleen suspensions on radiation-induced leukemogenesis in C57BL/6 mice

Summary The present experiments are an attempt to precise the type and localization of the cells involved in the protective effect of hemopoietic suspensions against the radiation-induced thymic lymphosarcoma (TLS) of C57BL/6 mice. Inocula containing variable numbers of BM or spleen CFUs from 60-day-old and 360-day-old donors were tested. According to their origin, the suspensions differed with respect to the CFU replication rate, the CFU ability to differentiate towards the T lineage and the content of the suspensions in thymic precursors. Two levels of inhibition were observed: BM suspensions from 60-day-old donors containing 1,500 CFUs had the best protective effect: 14.5% of TLS; 1,500 CFUs from 360-day-old donors were slightly but not significantly less efficient (28.5%). The second level of inhibition (36–46% of TLS) was obtained with all the following inocula:a) 1,200 and 300 spleen CFUs or 300 and 95 BM CFUs from 60-day-old donors,b) 1,500 spleen CFUs from aged donors. Seventy-six spleen CFUs from 60-day-old donors, 120 BM or 175 spleen CFUs from aged donors had no effect. These results suggest that in addition to the high replication rate of the BM CFUs as compared with spleen CFUs, cells endowed with an optimal protective effect are present in BM suspensions and are either absent or present in very small amount in spleen suspensions. These cells which induce an early repopulation of the thymus might correspond to thymic precursors.     

Ankylosis of hock joints in group caged male B6C3F1 mice

Enlarged hock joints were observed during 1983 in B6C3F1 mice of chronic toxicity and carcinogenicity studies sponsored by the National Toxicology Program (NTP). Subsequently, approximately 9,500 B6C3F1 mice on 32 NTP chemical toxicity and carcinogenicity studies were evaluated for this condition by clinical examination. Group caged male B6C3F1 mice had thickening and reduced mobility of the hock joints at prevalences of 1.2% up to 6 months of age; 23% at 6 to 12 months of age; and 62% at 13 to 26 months of age. Group caged female B6C3F1 mice had a prevalence of 2% or less. Histologically, affected mice had periarticular exostoses on the bones of the hock joints, with formation of bony bridges around joints and deposition of new bone in joint spaces, resulting in partial or complete ankylosis. Individually caged male and female B6C3F1 mice were not affected. The cause of the ankylosis was not determined, but its occurrence in the NTP studies has been reduced by individual caging.     

The effect of murine B16F10 melanoma on the biodistribution of 99mTc-MDP in male C57BL/6J mice.

The biodistribution of radiopharmaceuticals used in diagnostic imaging can be altered by a wide variety of factors. We studied the effect of murine B16F10 melanoma on the biodistribution in mice of 99mTechnetium-methylenediphosphonic acid (99mTc-MDP). Viable B16-F10 cell lines (1 x 10(5)) were inoculated subcutaneously in the dorsal region of 8-12 week-old male isogenic C57BV/6j mice. 14-16 days after inoculation, 99mTc-MDP was injected in the ocular plexus and after 0.5 hr the animals were rapidly sacrificed. The organs and tumor were isolated, the mass determined and the percentage per gram of injected activity (%ATI/g) calculated. The results shown that the %ATI/g:i/ has not been altered in inguinal lymph nodes, prostate, pancreas, testis, seminal vesicle, bladder, kidney, stomach, small intestine, spleen, thymus, heart, lung, brain and muscle; but ii/ significantly decreased in thyroid, bone, blood and liver. In conclusion, the B16F10 melanoma can alter the 99mTc-MDP uptakes in some organs.     

Effects of maternal strain on ethanol responses in reciprocal F1 C57BL/6J and DBA/2J hybrid mice

Variations in maternal behavior, either occurring naturally or in response to experimental manipulations, have been shown to exert long-lasting consequences on offspring behavior and physiology. Despite previous research examining the effects of developmental manipulations on drug-related phenotypes, few studies have specifically investigated the influence of strain-based differences in maternal behavior on drug responses in mice. The current experiments used reciprocal F1 hybrids of two inbred mouse strains (i.e. DBA/2J and C57BL/6J) that differ in both ethanol (EtOH) responses and maternal behavior to assess the effects of maternal environment on EtOH-related phenotypes. Male and female DBA/2J and C57BL/6J mice and their reciprocal F1 hybrids reared by either DBA/2J or C57BL/6J dams were tested in adulthood for EtOH intake (choice, forced), EtOH-induced hypothermia, EtOH-induced activity and EtOH-induced conditioned place preference (CPP). C57BL/6J and DBA/2J mice showed differences on all EtOH responses. Consistent with previous reports that maternal strain can influence EtOH intake, F1 hybrids reared by C57BL/6J dams consumed more EtOH during forced exposure than did F1 hybrids reared by DBA/2J dams. Maternal strain also influenced EtOH-induced hypothermic responses in F1 hybrids, producing differences in hybrid mice that paralleled those of the inbred strains. In contrast, maternal strain did not influence EtOH-induced activity or CPP in hybrid mice. The current findings indicate that maternal environment may contribute to variance in EtOH-induced hypothermia and EtOH intake, although effects on EtOH intake appear to be dependent upon the type of EtOH exposure.     

Evaluation of chromosomal aberrations in bone marrow of 1C3F1 mice

The chemical caprolactam (CAP) was tested for induction of chromosomal aberrations in bone marrow of male and female 1C3F1 mice at a single dose of 1000 mg/kg by oral intubation of an aqueous solution at a volume of 0.1 ml/10 g of body weight. Bone marrow was sampled from groups of 10 animals 24, 30 and 48 h after treatment. CAP did not produce chromosomal aberrations under the present experimental conditions. At the same time, benzo[a]pyrene, used as a positive control, significantly increased the aberration rates at a dose of 63 mg/kg.     

Utero-ovarian infection in aged B6C3F1 mice

An unusually high number of ovarian masses and cysts with purulent material were observed in the B6C3F1 mice on 2 year chemical carcinogenicity studies sponsored by the National Cancer Institute-National Toxicology Program. To determine possible etiology, some of these lesions were cultured for bacteria and a majority yielded Klebsiella sp. Necropsy records of 14,029 female mice in 91 chronic studies necropsied from 1979 to 1983 at six toxicology testing laboratories were reviewed to determine the incidence of lesions and distribution of this disease. Animals for these studies were obtained from barrier production colonies of six suppliers. The incidence of this lesion was low in animals less than 14 months of age, increased with age and reached a peak in 24-26 month old mice. Most animals having this lesion either died or were sacrificed in moribund condition, indicating that this is a life shortening disease of aged B6C3F1 mice. The incidence of lesions ranged from less than 1% to 70% in different chronic studies. There was a marked difference in the incidence in mice from different suppliers and the incidence rate was 2.6 to 15% depending on the source of the animals. The incidence of this lesion in some testing laboratories was several-fold higher than in others and ranged from 0.9 to 20%. The proportion of mice with this lesion was low in some laboratories irrespective of the source of the animals, whereas in other laboratories the incidence was several-fold higher with animals from some, but not all suppliers, indicating testing laboratory-supplier interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative toxicity and biotransformation of lindane in C57BL/6 and DBA/2 mice

DBA/2 mice, previously identified as "unresponsive" to aromatic hydrocarbons which induce microsomal enzymes in C57BL/6 mice, are more vulnerable to the convulsant effect of repeated doses of lindane than similarly treated C57BL/6 mice. Death in convulsions and higher blood and brain lindane concentrations indicate that less efficient disposition of lindane itself accounts for the greater vulnerability of the DBA/2 mice. The same two principal chlorophenolic metabolites of lindane were identified in the blood and tissues of both strains, but the time-courses of blood concentrations in response to repeated lindane dosing were different.     

Spontaneous, asymmetrical microphthalmia in C57B1/6J mice

An investigation was undertaken to evaluate ocular size and shape by objective and subjective criteria to establish the incidence and laterality of spontaneous microphthalmia in the C57B1/6J mouse strain. The purpose was to provide useful information of normal and abnormal ocular development in a laboratory strain frequently used for teratogenic studies. Ten-week-old C57B1/6J mice were time mated and sacrificed at gestational day 14. Eye normality was evaluated subjectively using veterinary ophthalmology methods, and objectively by a computer derived method. A distinct laterality was noted in the right eye when compared to the left eye. Degree of laterality varied with assessment methodology (19.0% vs. 6.9% and 8.9% vs. 4.0% for the subjective and objective methods respectively). Mean eye area and histogram distributions for the right eye were consistently smaller than the left eye for all anatomical rating groups, thereby suggesting that asymmetry found in the C57B1/6J may be due to an inherent discrepancy in eye size in the strain. The data from this study suggest that when C57B1/6J mouse eyes are used as end points for teratological testing each eye should be treated as an independent unit of measure.     

Aging and glucose homeostasis in C57BL/6J male mice

Age-dependent changes in glucose homeostasis were assessed in specific pathogen-free C57BL/6J male mice. Increased islet size and pancreatic insulin content in old (21-25-month-old) mice were associated with lower nonfasting plasma glucose levels and improved clearance of either an oral or an i.p. administered glucose load in comparison with young, mature (4-5-month-old) males. The almost twofold increase in islet size correlated with a twofold increase of glucose-stimulated insulin secretion from perifused islets from 25-month-old males compared with 5-month-old males. These aging male mice did not become obese, and there were no fibrotic changes associated with the hyperplastic islets observed in the old males. Thus, the findings that glucose tolerance did not deteriorate with age, coupled with the lack of evidence for impaired beta cell responsiveness to glucose in old males, suggest that deterioration in glucose homeostasis is not an inevitable consequence of aging in the mouse.     

Carcinogenicity test in B6C3F1 mice after parental and prenatal exposure to 50 Hz magnetic fields

Some epidemiological studies suggest association of childhood cancer with occupational exposure of the parents to magnetic fields. To test this relationship, 50 each of C57BL/6J female and C3H/HeJ male mice were exposed for 2 and 9 weeks, respectively, to 50 Hz sham (group A), 0.5 (group B), and 5 mT (group C) sinusoidal alternating magnetic fields. They were mated under the exposure for up to 2 weeks, and the exposure was continued until parturition. All the B6C3F1 offspring, without adjusting numbers of animals, were clinically observed without exposure to magnetic field for a nominal 78 weeks from 6-8 weeks of age after weaning and then euthanized for pathological examination according to a routine carcinogenicity test. 540 pups entered the test, and the survival rate was 96.7%. No F1 mouse died of tumoral diseases before a male in A group died of stomach cancer at 43 weeks of age. The first animal death in the exposed groups due to tumor occurred at 71 weeks of age. Eighteen animals died before necropsy at 84-86 weeks of age. No significant difference was detected in the final number of survivors and incidence of tumors between groups A and B, or A and C. Concerning reproduction total implants in group B were less than in group A and the difference was on the borderline of significance (P=.05). This difference was not reproduced in a later duplicate experiment.     

Inhibitory effect of Boerhaavia diffusa on experimental metastasis by B16F10 melanoma in C57BL/6 mice

Administration of the aqueous methanol (3:7) extract of B.diffusa was found to be effective in reducing the metastases formation by B16F10 melanoma cells. Prophylactic administration of the extract (0.5 mg/dose) inhibited the metastases formation by about 95% as compared to untreated control animals. There was 87% of inhibition in the lung metastases formation in syngenic C57BL/6 mice, when the extract was administered simultaneously with tumour challenge. Biochemical parameters such as lung collagen hydroxyproline, hexosamines and uronic acid levels were also reduced significantly (P < 0.001) in the treated animals. Levels of serum sialic acids and gamma-glutamyltranspeptidase that are markers of neoplastic proliferation were also reduced in the tumour plus extract treated animals. More over treatment with the extract enhanced the survival of the animals more than double that of untreated control animals. When a non-toxic concentration of the extract was treated directly to the B16F10 cells in vitro, it inhibited the cell proliferation as estimated by the 3H - thymidine uptake assay. From the Zymogram analysis using culture supernatant from the extract treated cells it became evident that the components of the extract inhibited the expression or activity of gelatinases A and B (MMP-2 and MMP-9). Since the MMPs are intimately associated with cell invasion and angiogenesis, inhibition of these functions along with the anti-proliferative activity (cytostatic) may be contributing to the antimetastatic property shown by B. diffusa.     

Strain differences in response to acute hypoxia: CD-1 versus C57BL/6J mice.

Some mammals respond to hypoxia by lowering metabolic demand for oxygen and others by maximizing efficiency of oxygen usage: the former strategy is generally held to be the more effective. We describe within the same species one outbred strain (CD-1) that lowers demand and another inbred strain (C57BL/6J) that maximizes oxygen efficiency to markedly extend hypoxic tolerance. Unanesthetized adult male mice (Mus musculus, CD-1 and C57BL/6J) between 20 and 35 g were used. Sham-conditioned (SC) C57BL/6J mice survived severe hypoxia (4.5% O(2), balance N(2)) roughly twice as long as SC CD-1 mice (median 211 and 93.5 s, respectively; P < 0.0001). Following acute hypoxic conditioning (HC), C57BL/6J mice survived subsequent hypoxia 10 times longer than HC CD-1 mice (median 2,198 and 238 s respectively; P < 0.0001). Therefore, C57BL/6J mice are both naturally more tolerant to hypoxia and show a greater increase in hypoxic tolerance in response to hypoxic conditioning. Indirect calorimetry indicates that CD-1 mice lower mass-specific oxygen consumption (V'o(2) in ml O(2).kg(-1).min(-1)) and carbon dioxide production (V'co(2) in ml CO(2).kg(-1).min(-1)) in response to HC (P = 0.002 and P < 0.0001, respectively), but C57BL/6J mice maintain V'o(2) and V'co(2) after HC. Respiratory exchange ratio and fluorometric assay of plasma ketones suggest that C57BL/6J mice rapidly switch to ketone metabolism, a more efficient substrate, while CD-1 mice reduce overall metabolic activity. We conclude that under severe hypoxia in mice, switching fuel, possibly to ketones, while maintaining V'o(2), may confer a greater survival advantage than simply lowering demand.     

C57BL/6J and DBA/2J mice vary in lick rate and ingestive microstructure

Fluid licking in mice is an example of a rhythmic behavior thought to be under the control of a central pattern generator. Inbred strains of mice have been shown to differ in mean or modal interlick interval (ILI) duration, suggesting a genetic-based variation. We investigated water licking in the commonly used inbred strains C57BL/6J (B6) and DBA/2J (D2), using a commercially available contact lickometer. Results from 20-min test sessions indicated that D2 mice lick at a faster rate than B6 mice (10.6 licks/s vs. 8.5 licks/s), based on analysis of the distribution of short-duration ILIs (50-160 ms). This strain difference was independent of sex, extent of water deprivation or total number of licks. D2 mice also displayed a faster lick rate when the strains were tested with a series of brief (5 s) trials. However, when ingestion over the entire 20-min session was analyzed, it was evident that D2 mice had an overall slower rate of ingestion than B6 mice. This was because of the tendency for D2 mice to have more very long pauses (>30 s) between sequences of licking bursts. Overall, it appeared that D2 mice licked more efficiently, ingesting more rapidly during excursions to the spout that were fewer and farther between.     

Strain differences in some immune parameters can be obscured by circadian variations and laboratory routines: studies of male C57BL/6J, Balb/c and CB6 F1 mice

This is a report on the potential influence of circadian changes and laboratory routines on some immune parameters (thymic and splenic weights, the numbers of bone marrow, peripheral blood, and peritoneal leukocytes) in: (1) males of C57BL/6J, Balb/c, and CB6 F1 mice kept under identical laboratory conditions; (2) males of CB6 mice kept under the same laboratory conditions, except for opposite light/dark regimes, either light/dark (LD) or dark/light (DL). All the animals were purchased from the same supplier and adapted for 4-5 weeks to strictly controlled housing conditions. Some parameters were similar at certain time points but statistically significantly different at others due to strain-specific daily variations. In order to make the interstrain comparisons more reliable, the data collected around the day/night cycle were pooled for calculations of mean values. Several immune parameters of CB6 mice kept under DL conditions were significantly different than those in mice under the conventional LD conditions. In conclusion, the extrapolation of results (especially in the field of neuroimmunology) to other strains (or species) should be done with great caution; and all interstrain (interspecific) comparisons, especially those from various laboratories, should always be related to specific time points and laboratory conditions.