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.     

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.     

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.     

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.     

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.     

Selective immunosuppression resulting from exposure to the carcinogenic congener of benzopyrene in B6C3F1 mice.

B6C3F1 mice were exposed to two congeners of benzopyrene, either the carcinogen benzo(a)pyrene (B(a)P) or the non-carcinogen benzo(e)pyrene (B(e)P. Exposure of mice to B(a)P resulted in a reduced number of IgM and IgG antibody plaque forming cells (PFC) to the T-dependent (TD) antigen SRBC and IgM PFC's to the T-independent (TI) antigen LPS. The IgM response to hapten conjugated TI antigens was examined using TNP-LPS for reactivity of less mature B cells (B1) and TNP-Ficoll for more mature B cells (B2). Exposure to B(a)P severely depressed the TNP-Ficoll PFC response by up to 77% without altering the TNP-LPS response. These data indicated that exposure to B(a)P alters differentiation and antibody production in mature B cells to both TD and B2 TI antigens. No change in PFC was observed following exposure to B(e)P. Mishell-Dutton co-cultures confirmed that B cells were affected and that T helper cells or suppressor Mphi were not involved. Parameters of cell-mediated immunocompetence including delayed cutaneous hypersensitivity to KLH, allograft or tumour cell rejection and susceptibility to Listeria monocytogens were unaltered in B(a)P treated mice.     

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.     

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).     

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.     

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.     

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.     

Relative sensitivity of the endogenous hprt gene and lacl transgene in ENU-treated big blue™ B6C3F1 mice

Three-week-old Big Blue? (BB) B6C3F1 mice were given a single i.p. injection of ENU. Three weeks later, splenic T cells were isolated from each animal by ficoll gradient centrifugation and divided into two samples. One sample was cultured to measure hprt^? mutation and the other was used to extract DMA for lacl^? analysis. T cells from BB mice exposed to 0, 4.5, 13.5, and 40 mg ENU/kg (9 or 10 animals per group) displayed dose-related increases in the frequency of both hprt^? and lacl^? mutations. Within each treatment group, the ENU-induced mutation frequency (average observed mutation frequency minus average control frequency) was remarkably similar at the two loci. This suggests that treatments that increase mutation frequency at the endogenous hprt gene also produce similar incremental increases at the BB lacl transgene. However, because of the ten-fold higher spontaneous mutation rate at lacl, the fold-increase over background produced by ENU at this locus was significantly less than the fold-increase produced at hprt. For example, the 4.5 mg ENU/kg treatment produced a 5.2-fold increase above background at hprt (P = 0.001), whereas only a 1.5-fold increase was produced at lacl (P = 0.140). Consequently, mutagenic insults that produce up to a fivefold increase in mutation frequency at an endogenous locus may be difficult to detect at the lacl transgene. Finally, the ENU-induced response at hprt in BB mice was identical to that in generic B6C3F1 mice, suggesting that there are no inherent differences between transgenic and normal mice in their response to this mutagenic agent. © 1995 Wiley-Liss, Inc.     

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.     

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.     

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.     

Quantitation of the cancer process in C57BL/6J, B6C3F1 and C3H/HeJ mice

This study examines growth alterations in liver foci and tumor development as a basis for the different susceptibility in hepatocarcinogenesis found among different strains of mice. Male C57, B6C3F1, and C3H mice treated with a single dose (1 mg/kg) of N,N-diethylnitrosamine (DEN) at 15 days of age and followed up to 12 months displayed a strain-dependent (C3H > B6C3F1 > C57) increase in incidence, number, volume fraction, and size of foci and macroscopic lesions (masses). DEN-treated mice exhibited a time-dependent increase in foci size but not in foci number. Phenobarbital (PB) treatment (500 ppm) in the drinking water starting 2 weeks after DEN-initiation did not affect the incidence or number of masses and foci. In all 3 strains, the bromodeoxyuridine labeling index in foci correlated with foci growth, supporting the major role of cell proliferation in foci growth. Measurements of apoptosis by morphological criteria with H&E staining suggest that intrafocal apoptosis may be a late event preventing foci growth and possibly also promoting focal cell selection, whereas extrafocal apoptosis may facilitate clonal growth by removing adjacent normal cells. The onset of conversion of foci to masses also correlated with strain susceptibility to hepatocarcinogenesis.