Hyaline glomerulopathy in B6C3F1 mice.

Hyaline glomerulopathy is a spontaneous disease of undetermined etiology that occurs sporadically in various strains of aging mice. In our laboratory, this disease was observed with unusual ultrastructural features as an incidental finding in 2 female B6C3F1 mice from 2 carcinogenicity bioassays. Microscopically, renal lesions were characterized by marked diffuse enlargement and prominent hyalinization of the glomeruli, equally affecting both kidneys. Affected glomeruli were PAS positive, but were negative for amyloid by the Congo red method. Immunocytochemical staining revealed weakly positive glomerular deposits with polyclonal anti-mouse IgG-IgM-IgA cocktail. Ultrastructurally, there were characteristic subendothelial osmiophilic deposits composed of loosely-packed linear structures in the glomeruli. Lamellae, which appeared as fibrils in perpendicular sections, were relatively uniform, measured 6.1-17.01 nm in diameter, and formed single or double-layered structures. The ultrastructural and immunocytochemical characteristics are suggestive of a spontaneous immune-mediated mechanism in a strain of mouse commonly used in toxicology studies.     

Primary cardiac hemangiosarcomas induced by 1,3-butadiene in B6C3F1 hybrid mice

Proliferative vascular lesions of the heart were found in mice exposed chronically to 1,3-butadiene by inhalation with an overall incidence of 30% in males and 43% in females. Based on histological criteria, the lesions were subclassified as endothelial hyperplasia with an incidence of 7% in males and 13% in females and hemangiosarcoma with an incidence of 23% and 30%, respectively. A dose-relationship for both lesions was observed in females, but not in males. The absence of a dose response in males was most likely due to the lower survival rate for high-dose animals (14%) when compared to the lower-dose animals (22%). Endothelial hyperplasia was characterized by widened vascular spaces lined by a single layer of plump endothelial cells. When cellular pleomorphism and piling up of endothelial nuclei were observed, the lesion was diagnosed as hemangiosarcoma. Ultrastructural examination of hemangiosarcomas revealed lumen formation, intercellular junctions and cytoplasmic filaments. Pinocytotic vesicles which are 1 of the characteristics of endothelial cells could not be identified with certainty. Weibel-Palade bodies were not detected in the neoplastic endothelium. Metastatic lesions were observed in liver, lung and kidney. To date, 1,3-butadiene is the only carcinogen reported that induces proliferative vascular lesions in the heart of mice.     

Epithelial-induced intrapulpal denticles in B6C3F1 mice

Multiple intrapulpal denticles were observed in maxillary incisors of control and treated B6C3F1 mice used in a chronic inhalation study. Histologically, the denticles originated from multiple small budlike projections emanating from the epithelial sheath, immediately adjacent to the pulp chamber. The denticles were round to ovoid in shape with a central cavity surrounded by tubular dentin. Immature denticles contained epithelial cells within the central cavity, whereas mature denticles were either devoid of cells or contained cell fragments. A layer of columnar odontoblasts surrounded the outer surface of each denticle. Denticles advanced in a coronal direction as the incisors grew. With continued incisor growth, some denticles impacted the tooth wall and were associated with defects in dentinogenesis, altered tooth shape, and microfractures. Some denticles became partly or entirely incorporated into the dentin of the tooth. Intrapulpal denticle formation may represent a previously unidentified alteration that could contribute to the development of dental dysplasia in mice by interfering with normal tooth development and predisposing affected teeth to malformation and biomechanical failure with fracture.     

Spontaneous tumors in aging (C57BL/6N x C3H/HeN)F1 (B6C3F1) mice

Spontaneous tumors in untreated (C57BL/6N x C3H/HeN)F1 (B6C3F1) mice used as controls in carcinogenicity tests were recorded. In both sexes, the development of spontaneous tumors was age-related. In 244 male mice, the most common tumors were hyperplastic nodules of the liver, hepatocellular carcinomas, malignant lymphomas/leukemias, lung adenomas, and adenocarcinomas. In 246 female mice, the most common tumors were malignant lymphomas/leukemias, pituitary adenomas, neoplastic nodules of the liver, hepatocellular carcinomas, and lung adenomas. Hepatocellular carcinomas metastasized in 20.3% of the animals with these tumors. Few other tumors except malignant lymphomas and leukemias metastasized. Various tumors of other organs and/or tissues were found at low incidences.     

Epididymal sperm granuloma induced by chronic administration of 2-methylimidazole in B6C3F1 mice

Two-year mouse and rat bioassay studies of 2-methylimidazole (2-MI) conducted by the National Toxicology Program revealed that epididymal sperm granuloma(SG)s occurred only in male B6C3F1 mice in a dose-related manner. The present study characterized 2-MI-induced SGs in these epididymides. Groups of 50 male B6C3F1 mice were fed diets containing 0, 625, 1250, or 2500 ppm 2-MI for 105 weeks; the doses were equivalent to average daily doses of approximately 13, 40, or 130 mg/kg. Testes and epididymides were histopathologically reexamined. 2-Methylimidazole increased the incidence of epididymal SGs (0%, 0%, 6%, 12%, respectively). Histologically, most of the SGs exhibited rupture of epididymal ducts with focal aggregations of macrophages in interstitia. Lesions occurred in the proximal caput of the epididymis and/or efferent ducts, not in the corpus and cauda. In the testis, incidences of germinal epithelial atrophy (GEA) increased dose-relatedly (2%, 8%, 16%, 28%, respectively). All mice with epididymal SG developed testicular GEA. The grading scores of testicular GEA tended to be more severe in mice with SGs than those without. No epididymal SG or testicular GEA was observed in 6-month-interim-sacrificed mice. The results imply that 2-year treatment of B6C3F1 mice with 2-MI can induce epididymal SGs, primarily followed by more severe testicular GEA. The potential mechanism of SG induction by 2-MI is discussed.     

Subchronic toxicology studies of hexachloro-1,3-butadiene (HCBD) in B6C3F1 mice by dietary incorporation

Two-week repeated-dose and 13-week subchronic studies of HCBD were conducted in B6C3F1 mice. Groups of five mice/sex received 0, 30, 100, 300, 1,000, or 3,000 ppm HCBD in feed for 15 days. Toxic responses, primarily in the higher dose groups, included abnormal clinical signs (lethargy, hunched posture, rough coat, sensitivity to light, and/or incoordination), mortality (all mice in the top two dose groups died by day 7), body and organ weight depression, and gross and histopathological changes. The most prevalent microscopic lesion, seen in all HCBD-treated mice of both sexes, was renal tubular cell necrosis and/or regeneration. Regeneration was seen only in the lower dose groups. Thirteen-week studies were conducted in which groups of 10 mice/sex received 0, 1, 3, 10, 30, or 100 ppm HCBD in feed. No treatment-related clinical signs or mortality were observed. Body weight gain was reduced in the 30- and 100-ppm males (-49 and -56, respectively), and the 100-ppm females (-47). Significant reduction in kidney weights was seen in the 30- and 100-ppm males and 100-ppm females. A treatment-related increase in tubular cell regeneration in the renal cortex occurred in both male and female mice. This lesion was characterized by an increase both in number and basophilic staining intensity of the tubular epithelial cells. Regeneration was seen in the outer stripe of the outer medulla and extended into the medullary rays (pars recta); severity increased with dose. Female mice were more susceptible to the toxicity of HCBD than male mice. Although no adverse effects were observed at the 10-ppm level for male mice in the subchronic study, the regenerative lesion was present in female mice at 1 ppm, the lowest dose administered.     

A comparative immunohistochemical study of spontaneous and chemically induced pheochromocytomas in B6C3F1 mice

Spontaneously occurring and chemically induced pheochromocytomas are rare in mice. That the mouse pheochromocytoma is a more appropriate animal model than that of the rat for study of human medullary adrenal tumors has been suggested. The expression of phenylethanolamine-N-methyltransferase (PNMT), the enzyme responsible for production of epinephrine from norepinephrine, is common to both mouse and human pheochromocytomas. This investigation assessed the expression of the immunohistochemical markers PNMT, tyrosine hydroxylase (TH), and chromogranin A (CGA) in spontaneously occurring and chemically induced pheochromocytomas in the B6C3F1 mouse. Spontaneous tumors were derived from control animals from 10 different studies and the pheochromocytomas from treated groups from 4 different studies. All tumors were positive for maximal TH expression. A highly significant difference in PNMT expression (p<0.01) occurred between spontaneously occurring pheochromocytomas classified as benign or “malignant” by the criteria of toxicologic pathology. Chemically induced tumors showed intermediate PNMT staining. A marked reduction in CGA expression occurred in pheochromocytomas induced by technical grade pentachlorophenol, compared to the other three chemicals and the spontaneously occurring tumors. These findings suggest that immunohistochemistry is a reliable tool in investigating the functional capabilities of pheochromocytomas in mice. PNMT expression is a tightly regulated component of the chromaffin cell phenotype and appears to be readily lost in mouse pheochromocytomas, particularly those with aggressive characteristics.     

Lack of chloroform-induced DNA repair in vitro and in vivo in hepatocytes of female B6C3F1 mice

Chloroform has been shown to induce hepatocellular carcinomas in female B6C3F₁ mice when administered by gavage, but not when given in drinking water. When administered in corn oil at the carcinogenic doses of 238 and 477 mg/kg, chloroform induced necrosis and sustained regenerative cell proliferation in the liver. To investigate the mode of action of tumor induction in the target cells, the ability of chloroform to induce unscheduled DNA synthesis (UDS) was examined in the in vitro and in vivo hepatocyte DNA repair assays. In the in vitro assay, primary hepatocyte cultures from female B6C3F₁ mice were incubated with concentrations from 0.01 to 10 mM chloroform in the presence of ³H-thymidine. UDS was assessed by quantitative autoradiography. No induction of DNA repair was observed at any concentration. In the in vivo assay, animals were treated by gavage with 238 and 477 mg/kg chloroform in corn oil. Primary hepatocyte cultures were prepared 2 and 12 hr later, incubated with ³H-thymidine, and assessed for induction of UDS as above. No DNA repair activity was seen at either dose or at either timepoint. These negative results in the target organ are consistent with the concept that neither chloroform nor its metabolites are directly DNA reactive and that the carcinogenicity of chloroform is secondary to induced cytolethality and regenerative cell proliferation. © 1994 Wiley-Liss, Inc.     

Age-related changes in the intrinsic rate of apoptosis in livers of diet-restricted and ad libitum-fed B6C3F1 mice.

Cancer incidence increases progressively with age. This observation suggests that a mechanistic relationship may exist at the cellular level between these two apparently diverse processes. Indirect evidence for this fundamental relationship is derived from the fact that interventions that retard the rate of aging simultaneously retard the incidence of many forms of cancer. Dietary restriction of rodents is a noninvasive manipulation that reproducibly retards most physiological indices of aging as well as the incidence of spontaneous and chemically induced tumors. As such, it provides a powerful model in which to study common mechanistic processes associated with both aging and cancer. In a recent study, we established that chronic dietary restriction induces an increase in the spontaneous rate of apoptotic cell death in hepatocytes of 12-month-old B6C3F1 mice and is associated with a significant reduction in the subsequent development of spontaneous hepatoma in this genetically susceptible strain. The purpose of the present investigation was to extend and confirm these original observations by determining whether the increased rate of spontaneous apoptosis with chronic dietary restriction is maintained throughout the life span in this strain. We quantified the spontaneous apoptotic rate by histological examination of liver sections from diet-restricted and ad libitum-fed B6C3F1 mice at age intervals of 12, 18, 24, and 30 months. The incidence of apoptotic bodies was enumerated in non-tumor-bearing mice by scoring 50,000 hepatocytes per liver by in situ end-labeling immunohistochemistry and was expressed as the mean incidence per 100 cells. The rate of apoptotic cell death was found to be elevated with age in both diet groups; however, the rate of apoptosis was significantly and consistently higher in the diet-restricted mice, relative to the ad libitum-fed mice, regardless of age. It has been proposed that apoptosis, or physiological cell death, provides a protective mechanism whereby DNA-damaged or potentially neoplastic cells are selectively eliminated. Thus, interventions that increase cellular sensitivity to apoptotic cell death would tend to protect genotypic and phenotypic stability with age; on the other hand, the failure to initiate or respond to appropriate signals for apoptosis would tend to accelerate the accumulation of age-associated genetic lesions and age-related neoplasia. An increase in the intrinsic rate of apoptotic cell death may contribute, in part, to decreased tumor incidence and increased life span potential with dietary restriction.     

Characterisation of busulphan-induced myelotoxicity in B6C3F1 mice using flow cytometry

Three experiments were carried out to investigate the myelotoxicity of busulphan in female B6C3F₁ mice using the Technicon H^*1 and the Sysmex R-1000 flow cytometers, instruments which produce a full blood count and a differential leucocyte count, and an automated reticulocyte count, respectively. In Experiment 1, a single dose of busulphan was administered at levels from 0 to 60 mg/kg and blood parameters measured at day 14. In Experiment 2, four doses of busulphan, from 0 to 40 mg/kg, were given at fortnightly intervals, and blood samples taken at days 14 and 42. In the third experiment, a single dose of busulphan was given at 0, 35 or 45 mg/kg and sequential blood, marrow and spleen samples examined up to day 10. In the first experiment there was a dose-related depression in the numbers of all leucocyte types. Values for Hb, RBC and HCT were not affected, whereas MCV and percentage macrocytic erythrocytes were increased, and MCHC was decreased, at high dose levels. Platelet numbers showed marked dose-related decreases. There were dose-related decreases in the numbers of all leucocyte types in Experiment 2 at days 14 and 42. Large unstained cell (LUC) numbers were reduced, and the mean neutrophil peroxidase index (MPXI) was increased, at high busulphan levels. Hb, RBC and HCT were reduced, whereas MCV, MCH and percentage macrocytic and percentage hypochromic erythrocytes were increased, in a dose-related fashion. Reticulocyte numbers showed a dose-related upward trend, but platelet counts illustrated a dose-related decrease, at days 14 and 42. In Experiment 3, busulphan caused a depression with a ‘U’-shaped curve, in the numbers of monocytes, eosinophils, lymphocytes and neutrophils. Decreased values and ‘U’-shaped curves were also seen for Hb, RBC, HCT and reticulocyte counts. Reticulocyte fluorescence ratio analysis showed that the high fluorescence ratio (HFR) was affected first and most profoundly. Calculation of the reticulocyte maturation index also demonstrated a dose-related effect on the earliest reticulocytes, and a ‘rebound’ effect. Total nucleated cell counts of the spleen and femur showed decreasing cell numbers and ‘U’-shaped responses with 45 mg/kg busulphan. This series of three experiments has established the use of a 6 week dosing regimen, with busulphan administered at fortnightly intervals, to induce myelotoxicity in a range of haematological parameters in female B6C3F₁ mice. We consider the use of the newly-developed flow cytometers and associated software, and the measurement of ‘non-standard parameters’ such as LUC, HFR and MPXI, to be particularly effective in the charcterisation of these busulphan-induced haematological changes.     

Long-term haematological alterations in female B6C3F1 mice treated with busulphan

Female B6C3F₁ mice (12–14 weeks old) were dosed with busulphan (BU) by the intraperitoneal route at dose levels between 10 and 40 mg/kg on four occasions at 14 day intervals. Six weeks after the final dose, mice were given normal drinking water or drinking water containing chloramphenicol succinate (CAPS) at 4 mg/ml. Animals were killed at eight timepoints after the final BU dose, the last samples being taken at day 485 or 497 and a range of haematological parameters measured. During the experiment, no differences could be detected between mice receiving BU and CAPS and those receiving BU alone. Animals surviving to the end of the study displayed moderate leucopenia but no reduction in marrow cellularity nor was any effect on erythropoiesis apparent. Lymphocyte numbers were reduced in peripheral blood and bone marrow, and splenic cellularity was also reduced. Increased mortality was seen in animals which had received 40 mg BU/kg. In the first four months after BU dosing, animals killed due to ill health were pancytopenic with hypocellular marrows; deaths that occurred thereafter were due to lymphoma.     

Long-term effects of busulphan on lymphocyte subpopulations in female B6C3F1 mice

Female B6C3F₁ mice (12–14 weeks old) were dosed with busulphan (BU) by the intraperitoneal route at a range of dose levels between 10 and 40 mg/kg on four occasions at 14 day intervals. Six weeks after the final dose, mice were given normal drinking water or drinking water containing chloramphenicol succinate (CAP.S) at 4 mg/ml. Animals were killed at eight timepoints after the final BU dose, the final samples being taken at day 485 or 497. A range of haematological parameters were determined and lymphocyte subsets analysed. The inclusion of CAP.S in the drinking water did not affect the changes to haematological parameters or lymphocyte subsets induced by BU. Lymphocyte count was reduced to 51%–66% of control values in animals receiving 40 mg BU/kg on all occasions except day 85. However, the lymphocyte count was increased to 107%–143% of control values in animals receiving 10 mg BU/kg on all occasions except day 85. The large unstained cell (LUC) count was reduced to between 25%–47% of control values in animals receiving 40 mg BU/kg on all occasions, however, the LUC count was not increased at low BU dosage. At day 485/497, B lymphocyte counts were reduced to 48% (p<0.01), 64% (NS) and 55% (p<0.05) of control values in mice treated with 40, 33 and 25 mg BU/kg, respectively. T lymphocyte counts were not affected by BU treatment. We have, therefore, demonstrated in the present study that administration of BU at high levels to the B6C3F₁ mouse induces a long-lasting and specific depletion of peripheral B lymphocytes; also, BU at low dose levels causes a mild but persistent lymphocytosis.     

Pyridostigmine bromide (PYR) alters immune function in B6C3F1 mice

Pyridostigmine bromide (PYR) is an anticholinesterase drug indicated for the treatment of myasthenia gravis and neuromuscular blockade reversal. It acts as a reversible cholinesterase inhibitor and was used as a pretreatment for soldiers during Operation Desert Storm to protect against possible nerve gas attacks. Since that time, PYR has been implicated as a possible causative agent contributing to Gulf War Illness. PYR's mechanism of action has been well-delineated with regards to its effects on the nervous system, yet little is known regarding potential effects on immunological function. To evaluate the effects of PYR on immunological function, adult female B6C3F1 mice were gavaged daily for 14 days with PYR (0, 1, 5, 10, or 20 mg/kg/day). Immune parameters assessed were lymphoproliferation, natural killer cell activity, the SRBC-specific antibody plaque-forming cell (PFC) response, thymus and spleen weight and cellularity, and thymic and splenic CD4/CD8 lymphocyte subpopulations. Exposure to PYR did not alter splenic and thymus weight or splenic cellularity. However, 20 mg PYR/kg/day decreased thymic cellularity with decreases in both CD4+/CD8+ (20 mg/kg/day) and CD4-/CD8- (10 and 20 mg/kg/day) cell types. Functional immune assays indicated that lymphocyte proliferative responses and natural killer cell activity were normal; whereas exposure to PYR significantly decreased primary IgM antibody responses to a T-cell dependent antigen at the 1, 5, 10 and 20 mg/kg treatment levels for 14 days. This is the first study to examine the immunotoxicological effects of PYR and demonstrate that this compound selectively suppresses humoral antibody responses.     

Alterations in hematopoietic responses in B6C3F1 mice caused by drinking a mixture of 25 groundwater contaminants.

Myelotoxicity parameters were monitored in female B6C3F1 mice exposed to 0, 1, 5, and 10% of a chemical mixture stock in drinking water for 2.5 to 31.5 weeks. The mixture consisted of 25 groundwater contaminants frequently found near toxic waste dumps, as determined by U.S. Environmental Protection Agency (EPA) surveys. Water consumption, body and organ weights, and hematological and histopathological examinations were conducted. No animals developed overt signs of toxicity after 2.5 weeks of treatment. No significant effect on bone marrow cellularity was observed after 2.5, 15.5, or 31.5 weeks of exposure; however, mice exposed to 5% or higher concentrations of the chemical mixture stock solution for 15.5 weeks showed significant suppression of granulocyte-macrophage progenitor cells (CFU-GM) and erythroid precursors (CFU-E) with no changes in body weight, histopathological or hematological parameters. Decreases occurred in erythrocyte mean corpuscular volume of mice exposed to a 10% solution for 15.5 weeks and to 5 and 10% solutions following 31.5 weeks of treatment. In addition, dose-related decreases were found in body, liver, and thymus weights in the 5 and 10% solutions exposure groups after 31.5 weeks. These results suggest that bone marrow may be a sensitive indicator for long-term, low-level exposure of multiple chemicals in mice. Furthermore, long-term exposure to highly contaminated groundwater may present a subtle risk to the hematopoietic system.     

Predominant K-ras codon 12 G --> A transition in chemically induced lung neoplasms in B6C3F1 mice

Based on long-term toxicity and carcinogenicity studies in B6C3F1 mice conducted by the National Toxicology Program, 2,2-Bis(bromomethyl)-1,3-propanediol (BMP) and tetranitromethane (TNM) have been identified as carcinogens. Following 2 yr of exposure to 312, 625, or 1,250 ppm BMP in feed, or exposure to 0.5 or 2 ppm TNM by inhalation, increased incidences of lung neoplasms were observed in B6C3F1 mice at all exposure concentrations compared to unexposed mice. The present study characterizes genetic alterations in the K-ras protooncogene in BMP- and TNM-induced lung neoplasms, respectively, and compares the findings to spontaneous lung neoplasms from corresponding control mice. The frequencies of the K-ras mutations were 57% (29/51) in BMP-induced lung neoplasms compared to 15% (3/20) in lung neoplasms from dosed feed control mice, and 54% (14/26) in TNM-induced lung neoplasms compared to 60% (3/5) in lung neoplasms from inhalation control mice. G --> A transitions at the second base of the K-ras codon 12 (GGT --> GAT) were the most frequent pattern of K-ras mutations identified in BMP-induced (20/29) and TNM-induced lung neoplasms (13/14), which differed from the mutational patterns identified in the lung neoplasms from unexposed control mice. These results indicate that mutations in the K-ras gene are involved in B6C3F1 lung carcinogenesis following BMP- and TNM-exposure, and the high frequency and specificity of the ras mutation profile in lung neoplasms (G --> A transition) may be due to in vivo genotoxicity by the parent compounds or their metabolites.     

Inhibition of intracellular peroxides and apoptosis of lymphocytes in lupus-prone B/W mice by dietary n-6 and n-3 lipids with calorie restriction

Our earlier studies have shown that calorie restriction and n-3 fatty acids inhibit autoimmune disease and prolong life span. Experiments were designed to study the alteration of apoptosis and its mediators in B/W mice fed either n-6 fatty acids [5% corn oil (CO)] or n-3 fatty acids [5% fish oil (FO)] and either allowed access to the diet ad libitum (AL) or restricted in caloric intake by 40% (CR), from 4 weeks of age. At 4 months (young) and 9 months (old) mice were killed, splenic lymphocytes were isolated, and apoptosis was measured with Annexin V and PI staining. Apoptosis was decreased in splenic lymphocytes from both young and old CR mice compared to their respective AL-fed control groups regardless of fat source. Increasing apoptosis with age was observed in CO/AL, CO/CR, and FO/AL mice which correlated closely with significantly higher cellular peroxides measured by flow cytometry using dichlorofluourescein diacetate (DCFH-DA), whereas in both CO/CR and FO/CR peroxide levels remained low in old mice. Furthermore, CR increased the proliferative response of splenic lymphocytes and decreased the Fas (CD95) and Fas-L protein expression in CD4+ lymphocytes from old mice. Higher levels of Fas and Fas-L expression were observed in old mice compared to young mice. Bcl-2 levels were elevated in both young and old CR groups compared to the respective AL groups. Calorie restriction prevented the loss of CD8 cells in old mice fed both the CO and the FO diet. In summary, CR resulted in decreased apoptosis accompanied by alterations in Fas, Fas-L, and Bcl-2 expression in old mice, increased life span, and delayed onset of kidney disease.     

Comparative cytogenetic analysis of bone marrow damage induced in male B6C3F1 mice by multiple exposures to gaseous 1,3-butadiene

Groups of male B6C3F1 mice (N = 12) were exposed to ambient air or to gaseous 1,3-butadiene (BD) at 6.25, 62.5, and 625 ppm for 10 exposure days (6 hr + T90/day). Exposure to BD induced in bone marrow: 1) a significant increase in the frequency of chromosomal aberrations (CA); 2) a significant elevation in the frequency of sister chromatid exchanges (SCE); 3) a significant lengthening of the average generation time (AGT); 4) a significant depression in the mitotic index (MI); and, as measured in the peripheral blood, 5) a significant increase in the proportion of circulating polychromatic erythrocytes (%PCE), and 6) a significant increase in the level of micronucleated PCE (MN-PCE) and micronucleated normochromatic erythrocytes (MN-NCE). The most sensitive indicator of genotoxic damage was the frequency of SCE (significant at 6.25 ppm), followed by MN-PCE levels (significant at 62.5 ppm), and then by CA and MN-NCE frequencies (significant at 625 ppm). The most sensitive measure of cytotoxic damage was AGT (significant at 62.5 ppm), followed by %PCE (significant at 625 ppm), and then by MI (significant by trend test only). Because each cytogenetic endpoint was evaluated in every animal, a correlation analysis was conducted to evaluate the degree of concordance among the various indicators of genotoxic and cytotoxic damage. The extent of concordance ranged from a very good correlation between the induction of MN-PCE and the induction of SCE (correlation coefficient r = 0.9562) to the lack of a significant correlation between the depression in the MI and any other endpoint (r less than 0.37).     

Genetic alterations in brain tumors following 1,3-butadiene exposure in B6C3F1 mice

The nervous system of the B6C3F1 mouse has rarely been a target for chemical carcinogenesis in the National Toxicology Program (NTP) bioassays. However, 6 malignant gliomas and 2 neuroblastomas were observed in B6C3F1 mice exposed to 625 ppm 1,3-butadiene (NTP technical reports 288 and 434). These mouse brain tumors were evaluated with regard to the profile of genetic alterations that are observed in human brain tumors. Alterations in the p53 tumor suppressor gene were common. Missense mutations were observed in 3/6 malignant gliomas and 2/2 neuroblastomas and were associated with loss of heterozygosity. Most of the mutations occurred in exons 5-8 of the p53 gene and were G-->A transitions, and did not involve CpG sites. Loss of heterozygosity at the Ink4a/Arf gene locus was observed in 5/5 malignant gliomas and 1/1 neuroblastoma, while the PTEN(phosphatase and tensin homologue) gene locus was unaffected by deletions. One of 2 neuroblastomas had a mutation in codon 61 of H-ras, while H-ras mutations were not observed in the malignant gliomas examined. Only 1 brain tumor has been reported from control mice of over 500 NTP studies. This malignant glioma showed no evidence of alterations in the p53 gene or K- and H-ras mutations. It is likely that the specific genetic alterations observed were induced or selected for by 1,3-butadiene treatment that contributed to the development of mouse brain tumors. The observed findings are similar in part to the genetic alterations reported in human brain tumors.     

Benzene-induced micronuclei in erythrocytes: an inhalation concentration-response study in B6C3F1 mice

High concentrations (300–1000 p.p.m) of benzene have beenshown to induce an increase in the frequency of micronucleatederythrocytes in mice. This study investigated the mutagenicityof benzene at lower concentrations, including the current limitfor occupational exposure, 1 p.p.m. The frequencies of micronucleartedpolychromatic erythrocytes (MPCE) in the bone marrow and bloodand micronucleated normochromatic erythrocytes (MNCE) in theblood of male B6C3F1 mice were Measured following inhalationof benzene at 0, 1, 10, 100 or 200 p.p.m. during an 8 week exposureperiod. Only 100 and 200 p.p.m. benzene induced a statisticallysignificant increased frequency of micronucleated erythrocytesin the bone marrow and blood. The frequency of MPCE plateauedat week 2 with 43/1000 (100 p.p.m.) and 86/1000 (200 p.p.m.)in the bone marrow as compared with 10/1000 for controls. Thefrequency of MNCE in the blood progressively increased to 13.4/1000(100 p.p.m.) and 32.5/1000 (200 p.p.m.) at week 8 as comparedwith 1.8/1000 for controls. Cytotoxicity of replicating andmaturing erythrocytes by 100 and 200 p.p.m. benzene delayedthe accumulation of MNCE in the blood. There was not a stastisticallysignificant increase in the frequency of micronucleated erythrocytes,as an indicator of mutagenicity, with inhalation of 1 or 10p.p.m. benzene over an 8 week period. A quadratic curve fitthe bone marrow MPCE data of mice exposed to up to 200 p.p.m.benzene with a high correlation (R² = 0.94) and could not berejected based on lack of fit. ¹To whom correspondence should be addressed: