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.     

Altered hematopoietic stem cell development in male B6C3F1 mice following exposure to 1,3-butadiene

The effects of the murine lymphomagen, 1,3-butadiene (BD), on the proliferation and differentiation of hematopoietic stem cells were examined in male B6C3F1 mice. Exposure to 1250 ppm BD for 6 weeks resulted in no demonstrable alteration in the frequency of spleen colony-forming units (CFU-S); however, colonies derived from treated animals were smaller than those from controls. The absence of any difference in the frequency of CFU-GM after 6 weeks exposure suggests that BD produces an alteration in the relative proportion of immature to mature pluripotent stem cells in BD-exposed animals. This was confirmed by the examination of the effects of BD on stem cell development in long-term bone marrow culture. After 14 days, the number of CFU-GM derived from cultures of animals exposed for 6 weeks was reduced compared to controls. However, at 28 days an increase relative to controls was observed. This shift in the course of differentiation of the granulocyte/macrophage precursor cell, as assessed by the CFU-GM, provides further evidence that there is an increase in the relative frequency of primitive or immature stem cells in BD-treated mice. After a 30-31 week exposure to BD, a decrease in the numbers of both CFU-S and CFU-GM was observed. These findings indicate that BD causes alterations in stem cell development and suggest that alterations in bone marrow stem cells may play an essential role in the pathogenesis of BD-induced thymic lymphoma.     

Genotoxicity and cytotoxicity in male B6C3F1 mice following exposure to mixtures of 1,3-butadiene and styrene

1,3-Butadiene and styrene are oxidized, in part, by cytochrome P450 2E1 and have been shown to metabolically interact in rodents exposed by inhalation to mixtures of both compounds. Because the reactive metabolites of butadiene and styrene are thought to be responsible for the toxicity of each compound, metabolic interactions may alter the response in animals exposed to mixtures of butadiene and styrene compared with the response in animals exposed to butadiene alone or styrene alone. The purpose of this study was to quantitate alterations in genotoxicity and cytotoxicity in male B6C3F1 mice exposed to mixtures of butadiene and styrene. Male B6C3F1 mice were exposed to 6.25, 62.5, 200, or 625 ppm butadiene alone, 50 ppm styrene alone, or mixtures of 6.25, 62.5, 200, or 625 ppm butadiene and 50 ppm styrene. Genotoxicity was assessed by quantitating the frequency of micronucleated polychromatic erythrocytes in bone marrow. Cytotoxicity was assessed by counting total spleen and thymus cells and by quantitating the frequency of polychromatic erythrocytes in the peripheral blood. Butadiene and mixtures of butadiene and styrene were genotoxic in mice, as shown by a significant increase in the frequency of micronucleated polychromatic erythrocytes. The increased frequency following exposure to mixtures of butadiene and styrene was not significantly different compared with the frequency following exposure to butadiene alone. Styrene and mixtures of butadiene and styrene were cytotoxic in mice, as shown by significantly decreased number of spleen cells. Exposure to mixtures of butadiene and styrene with butadiene concentrations of 62.5 or 625 ppm significantly reduced the number of thymus cells. Exposure to 200 ppm or 625 ppm butadiene alone, or to mixtures of 200 ppm or 625 ppm butadiene and 50 ppm styrene, significantly reduced the frequency of polychromatic erythrocytes in the peripheral blood. The results of the study demonstrate that exposure to mixtures of butadiene and styrene does not reduce the respective genotoxicity of butadiene or cytotoxicity of styrene. Environ. Mol. Mutagen. 29: 335–345, 1997. © 1997 Wiley-Liss, Inc.     

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.     

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.     

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

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

Selective activation of endogenous ecotropic retrovirus in hematopoietic tissues of B6C3F1 mice during the preleukemic phase of 1,3-butadiene exposure

1,3-Butadiene (BD), a comonomer used in the production of synthetic rubber, is a rodent carcinogen. We have observed a marked increase in the incidence of thymic lymphoma in male B6C3F1 relative to NIH Swiss mice chronically exposed to BD in the absence of demonstrable differences in bone marrow (target organ) toxicity. Increased expression of murine leukemia virus (MuLV) antigens was also observed on lymphomas from BD-exposed B6C3F1 mice. Because NIH Swiss mice do not usually express endogenous retroviruses and their ecotropic proviral sequences are not intact, these findings provide presumptive evidence of a role for endogenous retrovirus sequences in BD-induced lymphoma in the B6C3F1 mouse. The present study was conducted to examine the expression and behavior of endogenous retroviruses in these strains during the preleukemic phase of BD exposure. Chronic exposure to BD (1250 ppm) 6 hr/day, 5 days/wk for 3 to 21 weeks increased markedly the quantity of ecotropic retrovirus recoverable from bone marrow, thymus, and spleen of B6C3F1 mice. However, expression of other endogenous retroviruses (xenotropic, MCF-ERV) was not enhanced. No viruses of any type were found in similarly treated NIH Swiss mice. The mechanism of this increase in ecotropic retrovirus in B6C3F1 mice is believed to be de novo activation in greater numbers of cells because changes in the Fv-1 tropism of the replicating viruses or changes in Fv-1 host restriction were not found. Endogenous retroviruses are thus implicated in BD-induced leukemogenesis in B6C3F1 mice. Further studies will examine the role of retrovirus in BD-induced leukemogenesis and the mechanisms of activation of ecotropic proviral sequences in murine cells.     

Mutations of ras protooncogenes and p53 tumor suppressor gene in cardiac hemangiosarcomas from B6C3F1 mice exposed to 1,3-butadiene for 2 years

1,3-Butadiene is a multisite carcinogen in rodents. Incidences of cardiac hemangiosarcomas were significantly increased in male and female B6C3F1 mice that inhaled 1,3-butadiene (BD) for 2 years. Eleven BD-induced cardiac hemangiosarcomas were examined for genetic alterations in ras protooncogenes and in the p53 tumor suppressor gene. Nine of 11 (82%) BD-induced hemangiosarcomas had K-ras mutations and 5 of 11 (46%) had H-ras mutations. All of the K-ras mutations were G-->C transversions (GGC-->CGC) at codon 13; this pattern is consistent with reported results in BD-induced lung neoplasms and lymphomas. Both K-ras codon 13 CGC mutations and H-ras codon 61 CGA mutations were detected in 5 of 9 (56%) hemangiosarcomas. The 11 hemangiosarcomas stained positive for p53 protein by immunohistochemistry and were analyzed for p53 mutations using cycle sequencing of polymerase chain reaction (PCR) amplified DNA isolated from paraffin-embedded sections. Mutations in exons 5 to 8 of the p53 gene were identified in 5 of 11 (46%) hemangiosarcomas, and all of these were from the 200- or 625-ppm exposure groups that also had K-ras codon 13 CGC mutations. Our data indicate that K-ras, H-ras, and p53 mutations in these hemangiosarcomas most likely occurred as a result of the genotoxic effects of BD and that these mutations may play a role in the pathogenesis of BD-induced cardiac hemangiosarcomas in the B6C3F1 mouse.     

Mutational specificity: Assessment of 1,3-butadiene mutagenicity in the bone marrow of B6C3F1 lacl transgenic mice (Big Blue®): A review of mutational spectrum and LACL mutant frequency after a 5-day 625 PPM 1,3-butadiene exposure

1,3-Butadiene (BD) is a carcinogen that is bioactivated to at least two genotoxic metabolites. In the present article, we review briefly our previous studies on the in vivo, mutagenicity and mutational spectra of BD in bone morrow and extend these studies to examine the effect of exposure time (5-day vs. 4-week exposure to 625 ppm BD used in previous studies) on the lacl mutant frequency in the bone marrow. Inhalation exposure to BD at 625 ppm and 1,250 ppm was mutagenic in vivo inducing an increase in the transgene mutant and mutation frequency in the bone marrow. Analysis of the mutational spectrum in BD-exposed and air control mice demonstrated that BD exposure induced an increased frequency of mutations at A:T base pairs. There was no difference in the lacl mutant frequency determined in the bone marrow between a short-term exposure to BD (5 days) and a longer-term exposure (4 weeks). These data taken together demonstrate that inhalation exposure to BD induces in vivo somatic cell mutation. © 1996 Wiley-Liss, Inc.     

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.     

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.     

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.     

Pi-class glutathione-S-transferase-positive hepatocytes in aging B6C3F1 mice undergo apoptosis induced by dietary restriction.

Liver sections from aging ad libitum-fed and diet-restricted B6C3F1 male mice were evaluated immunohistochemically for pi-class glutathione S-transferase (GST-II). GST-II immunostaining of hepatocytes was diffuse and occurred in periportal regions of hepatic acinus, whereas perivenous areas were weakly stained or were stain-free. Expression of GST-II was significantly diminished in diet-restricted mice in all age groups and was associated with a marked decrease in liver tumor development. As most spontaneous liver tumors were GST-II positive, it can be speculated that they developed from GST-II positive initiated hepatocytes. To determine whether dietary restriction induces apoptosis in GST-II-positive hepatocytes, 24-month-old ad libitum-fed mice were introduced to 40% diet restriction. After 1 week of diet restriction, a decrease in GST-II expression was associated with a threefold increase in the frequency of apoptotic bodies as detected by terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling of DNA fragments. A two-step immunohistochemical procedure revealed that approximately 70% of apoptotic bodies were GST-II positive. These results suggest that spontaneous, potentially preneoplastic hepatocytes in tumor-prone B6C3F1 mice are eliminated by apoptosis with dietary restriction.     

Spontaneous lesions in control B6C3F1 mice and recommended sectioning of male accessory sex organs

Because sampling of the paired lobes (ventral, dorsal, lateral, and anterior) of the mouse prostate has often been inconsistent, comparisons among different investigations have lacked validity. The absence of site identification for prostatic lesions has made reported incidences relatively nonspecific. We present here the lobe-specific incidences and degree of severity of spontaneous lesions in prostate, coagulating gland (anterior prostatic lobe), seminal vesicles, and ampullary glands in 612 control B6C3F1 mice from 12 recent National Toxicology Program 2-year carcinogenicity and toxicity studies conducted in 1 of 4 different laboratories. Lymphocytic infiltration, inflammation, epithelial hyperplasia, mucinous cyst, and mucinous metaplasia were observed in the dorsolateral lobes. Lymphocytic infiltration, inflammation, epithelial hyperplasia, and edema were present in the ventral lobes. Lymphocytic infiltration, acinar dilatation, inflammation, epithelial hyperplasia, and atrophy occurred in the coagulating glands. No neoplastic lesions were observed in the prostate or coagulating gland. Lymphocytic infiltration, acinar dilatation, inflammation, atrophy, adenoma, adenocarcinoma, and a granular cell tumor were observed in the seminal vesicles. Lymphocytic infiltration was also present in the ampullary glands. The results of our survey indicate that the amounts of glandular tissues were not present consistently in slides from the different laboratories. Landmarks for uniform tissue trimming are needed. We therefore suggest an optimal trimming and embedding method for mouse prostate and seminal vesicles to ensure adequate, consistent sampling.     

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.     

Characterization of Hprt mutations in cDNA and genomic DNA of T-cell mutants from control and 1,3-butadiene-exposed male B6C3F1 mice and F344 rats

A multiplex PCR procedure for analysis of genomic DNA mutations in the mouse hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene was developed and then used with other established methods for the coincident identification of large- and small-scale genetic alterations in the Hprt gene of mutant T-cell isolates propagated from sham- and 1,3-butadiene (BD)-exposed mice and rats. The spectra data for RT-PCR/cDNA analysis and multiplex PCR of genomic DNA from Hprt mutants were combined, and statistical analyses of the mutant fractions for the classes of mutations identified in control versus exposed animals were conducted. Under the assumption that the mutant fractions are distributed as Poisson variates, BD exposure of mice significantly increased the frequencies of (1) nearly all types of base substitutions; (2) single-base deletions and insertions; and (3) all subcategories of deletions. Significantly elevated fractions of G:C-->C:G and A:T-->T:A transversions in the Hprt gene of BD-exposed mice were consistent with the occurrence of these substitutions as the predominant ras gene mutations in multiple tumor types increased in incidence in carcinogenicity studies of BD in mice. BD exposure of rats produced significant increases in (1) base substitutions only at A:T base pairs; (2) single-base insertions; (3) complex mutations; and (4) deletions (mainly 5' partial and complete gene deletions). Future coincident analyses of large- and small-scale mutations in rodents exposed to specific BD metabolites should help identify species differences in the sources of deletion mutations and other types of mutations induced by BD exposures in mice versus rats.     

In vivo mutagenicity and mutation spectrum in the bone marrow and testes of B6C3F1 lacI transgenic mice following inhalation exposure to ethylene oxide

The lacI mutant frequency and mutation spectrum were determined in the bone marrow and testes of B6C3F1 lacI transgenic mice exposed by inhalation to ethylene oxide (EO). Groups of male transgenic lacI B6C3F1 mice were exposed to 0, 25, 50, 100 or 200 p.p.m. EO for up to 48 weeks (6 h/day, 5 days/week) and were killed at 12, 24 or 48 weeks of EO exposure for determination of lacI mutant frequency. In the bone marrow, the lacI mutant frequency was significantly increased at the two highest exposure levels (100 and 200 p.p.m.) and at the 48 week exposure time point. The shape of the exposure-response curve for lacI mutant frequency in the bone marrow was non-linear. DNA sequence analysis of the bone marrow mutation spectrum revealed that only AT-->TA transversions occurred at an increased frequency in EO-exposed mice: 25.4% in EO-exposed mice for 48 weeks (200 p.p.m.) compared with 1.4% in air controls. In testes, the lacI mutant frequency was increased at a single exposure level of 200 p.p.m. for 24 weeks. At 48 weeks, the lacI mutant frequency in testes was significantly increased to an equal degree at 25, 50 and 100 p.p.m. EO but not at 200 p.p.m. Analysis of the testes mutation spectrum in air control mice and in mice exposed to 200 p.p.m. EO for 48 weeks revealed that no single mutational type occurred at an increased frequency. In the testes, there was a small increase across all mutational types that was sufficient to increase the overall lacI mutation frequency although not significant individually. The mutation spectrum in testes of EO-exposed mice also revealed that the increased lacI mutant frequency observed at 25 or 50 p.p.m. EO was not due to an increase in mutant siblings (clonality). These data demonstrate that inhalation exposure to EO for up to 48 weeks produces distinct mutagenic responses in bone marrow and testes.     

Systemic vascular disease in male B6C3F1 mice exposed to particulate matter by inhalation: studies conducted by the National Toxicology Program

Epidemiological studies suggest an association between ambient particulate matter and cardiopulmonary diseases in humans. The mechanisms underlying these health effects are poorly understood. To better understand the potential relationship between particulate-matter-induced inflammation and vascular disease, a 2-phase retrospective study was conducted. Phase one included the review of heart, lung, and kidney tissues from high-dose and control male B6C3F1 mice exposed by inhalation to 9 particulate compounds for a 2-year period. The results showed that high-dose males developed significantly increased incidences of coronary and renal arteritis over controls in 2 of the 9 studies (indium phosphide and cobalt sulfate heptahydrate), while marginal increases in arteritis incidence was detected in 2 additional studies (vanadium pentoxide and gallium arsenide). In contrast, arteritis of the muscular arteries of the lung was not observed. Morphological features of arteritis in these studies included an influx of mixed inflammatory cells including neutrophils, lymphocytes, and macrophages. Partial and complete effacement of the normal vascular wall architecture, often with extension of the inflammatory process into the periarterial connective tissue, was observed. Phase 2 evaluated the heart, lung, kidney, and mesentery of male and female B6C3F1 mice from the 90-day studies of the 4 compounds demonstrating arteritis after a 2-year period. The results showed arteritis did not develop in the 90-day studies, suggesting that long-term chronic exposure to lower-dose metallic particulate matter may be necessary to induce or exacerbate arteritis.     

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.