Mice Of C3H Strain Research Articles

Pre-clinical assessment of anti-vascular drugs using quantitative dynamic contrast-enhanced MRI

The effect of novel anti-cancer agents on the vascular properties of radiation-induced fibrosarcomas in C3H-strain mice was evaluated in vivo using dynamic contrast-enhanced (DCE) MRI. A T1-weighted volume acquisition was used to obtain DCE-MRI data. Pre-contrast T1 measurements and the contrast agent arterial input function were used to estimate the volume transfer constant (Ktrans) and extravascular extracellular volume fraction (ve). Five novel combretastatin analogues, combretastatin A-4 (CA-4) and hazelnut oil (vehicle) were tested for their effect 24 hours after administration on the tumours. Three of the five agents and the positive control (CA-4), significantly decreased tumour Ktrans and ve: Ktrans for CA-4 by 72%; fluorocombretastatin by 75%; methylcombretastatin by 68%; and ve by 40% for methylcombretastatin and 75% for the combretastatin quinone. These agents were recommended for further consideration. These results suggest that DCE-MRI may be a useful tool for pre-clinical evaluation of anti-cancer drugs.

Pathobiology of Aging Mice and GEM

The use of induced and spontaneous mutant mice and genetically engineered mice (and combinations thereof) to study cancers and other aging phenotypes to advance improved functional human life spans will involve studies of aging mice. Genetic background contributes to pathology phenotypes and to causes of death as well as to longevity. Increased recognition of expected phenotypes, experimental variables that influence phenotypes and research outcomes, and experimental design options and rationales can maximize the utility of genetically engineered mice (GEM) models to translational research on aging. This review aims to provide resources to enhance the design and practice of chronic and longevity studies involving GEM. C57BL6, 129, and FVB/N strains are emphasized because of their widespread use in the generation of knockout, transgenic, and conditional mutant GEM. Resources are included also for pathology of other inbred strain families, including A, AKR, BALB/c, C3H, C57L, C58, CBA, DBA, GR, NOD.scid, SAMP, and SJL/J, and non-inbred mice, including 4WC, AB6F1, Ames dwarf, B6, 129, B6C3F1, BALB/c,129, Het3, nude, SENCAR, and several Swiss stocks. Experimental strategies for long-term cross-sectional and longitudinal studies to assess causes of or contributors to death, disease burden, spectrum of pathology phenotypes, longevity, and functional healthy life spans (health spans) are compared and discussed.

Effect of Low Magnitude and High Frequency Mechanical Stimuli on Defects Healing in Cranial Bones

The aim of this investigation was to assess the effect of low magnitude high frequency (LMHF) mechanical vibrated stimulation on healing the defects surgically imposed on craniofacial bones. Forty 12-week-old C3H strain mice were separated into surgical and non-surgical groups. The surgical groups had a reproducible surgical bony lesion prepared in their right parietal bone. Both groups were further subdivided into vibration (experimental) and non-vibration (control) groups at 3 time points (zero hours, 2 weeks, and 4 weeks). The vibration groups were subjected to LMHF mechanical stimuli (30 Hz with peak strain 5 microepsilon) via a vibration machine for 20 minutes a day for 5 days a week for a total of 28 days. The specimens were analyzed using micro-computer tomography (micro-CT). Micro-CT volumetric measurement showed that in the surgical defects groups there was a significant decrease in the volume of the healing lesion with time (P < .001) and the linear decrease was significantly more pronounced in the vibration-treated group than the nontreated group (P < .001). Micro-CT histomorphometric measurement showed that in the nonsurgical groups there was no significant difference in microstructures of bony trabeculae between vibration-treated and nontreated groups. It is suggested that the introduction of LMHF mechanical stimuli in a healing bony lesion in the non-weight bearing bone significantly increases its healing capacity.

00/02848 Effect of volume fraction of mixture on separation characteristics in vibrated fluidized beds

The aim of this investigation was to assess the effect of low magnitude high frequency (LMHF) mechanical vibrated stimulation on healing the defects surgically imposed on craniofacial bones.Forty 12-week-old C3H strain mice were separated into surgical and non-surgical groups. The surgical groups had a reproducible surgical bony lesion prepared in their right parietal bone. Both groups were further subdivided into vibration (experimental) and non-vibration (control) groups at 3 time points (zero hours, 2 weeks, and 4 weeks). The vibration groups were subjected to LMHF mechanical stimuli (30 Hz with peak strain 5μϵ) via a vibration machine for 20 minutes a day for 5 days a week for a total of 28 days. The specimens were analyzed using micro-computer tomography (micro-CT).Micro-CT volumetric measurement showed that in the surgical defects groups there was a significant decrease in the volume of the healing lesion with time (P < .001) and the linear decrease was significantly more pronounced in the vibration-treated group than the nontreated group (P < .001). Micro-CT histomorphometric measurement showed that in the nonsurgical groups there was no significant difference in microstructures of bony trabeculae between vibration-treated and nontreated groups.It is suggested that the introduction of LMHF mechanical stimuli in a healing bony lesion in the non-weight bearing bone significantly increases its healing capacity.

Different cellular basis for the resistance of C3H and STS strain mice to the development of thymic lymphomas following fractionated whole-body irradiation: Analysis using radiation bone marrow chimeras

Purpose : B10 strain mice are susceptible to the induction of thymic lymphomas by fractionated whole-body X-irradiation (FI), whereas C3H and STS mice are resistant. The nature of the factors responsible for the strain difference in the susceptibility to thymic lymphomagenesis was investigated by using radiation bone marrow chimeras. Methods : Radiation bone marrow chimeras were constructed in the reciprocal donor-host combinations of susceptible and resistant mice with use of Thy 1 markers that allow the genetic origins of thymocytes and thymic lymphomas to be determined. Results : B10.Thy 1.1 →C3H, B10.Thy 1.1 →STS as well as B10.Thy 1.1 →B10 bone marrow chimeras manifested a high incidence of thymic lymphomas after FI-treatment, whereas C3H.Thy 1.1 →B10 and STS →B10.Thy →1.1 as well as C3H.Thy 1.1 →C3H and STS →STS chimeras manifested a low incidence of thymic lymphoma. Furthermore, FI-treatment of (B10.Thy 1.1 + C3H) →B10.Thy 1.1 mixed chimeras resulted in the generation of similar numbers of thymic lymphomas of B10 and C3H origins, whereas FI-treatment of (B10.Thy 1.1+ STS) →B10.Thy 1.1 mixed chimeras preferentially induced thymic lymphomas of B10 origin. Conclusions : (1) genetic factors responsible for the strain-dependent susceptibility and/or resistance to FI-induced lymphomagenesis exert their effects entirely on bone-marrow derived cells, (2) host environments of C3H and STS resistant mice are not inhibitory for the development of thymic lymphomas and (3) the resistance of STS mice to FI-induced thymic lymphomagenesis is an intrinsic property of thymocytes, whereas C3H and B10 thymocytes themselves are similarly susceptible for FI-induced thymic lymphomagenesis.

Limiting dilution analysis of T-cell progenitors in the bone marrow of thymic lymphoma-susceptible B10 and-resistant C3H mice after fractionated whole-body X-irradiation

. Earlier studies from this laboratory using Thy 1 congenic B10 strain mice suggested that a depletion of T cell progenitors (pre T cells) in the bone marrow in addition to the destruction of the thymus after fractionated whole body X-irradiation (IR) are the two main critical factors that cause differentiation arrest of initially repopulating intrathymic radioresistant T cell progenitors, which then lead to the appearance of preneoplastic, prelymphoma cells, and eventually to highly neoplastic thymic lymphomas under the influence of the thymic environment. In order to explore the significance of the depletion (or reduction) of T cell progenitors in the bone marrow during pathogenesis of radiation-induced thymic lymphomas, we compared the pool size of pre T cells in the bone marrow and the spleens as well as the profiles of the regenerating thymocyte populations between thymic lymphoma induction-susceptible B10 and-resistant C3H strain mice following irradiation. The results indicated that irradiation severely depleted the pre T cells in the bone marrow and the spleens of both lymphoma inductionsusceptible and-resistant mice. They also showed that in C3H mice the differentiation and maturation of intrathymic T cell progenitors which initially repopulated the depleted thymus seemed to proceed normally in spite of the poor cellularity, while this process was greatly suppressed in B10 mice. These data indicate that a depletion of pre T cells in the bone marrow combined with atrophy of the thymus in the irradiated mice is necessary, but not sufficient for development of thymic lymphoma. Implication of these findings on the possible mechanism of radiation-induced thymic lymphomagenesis is discussed.

High frequency of leukemic lymphomas with osteosarcomas but no myeloid leukemias in C3H mice after 239Pu citrate injection.

Female C3H strain mice, which do not spontaneously exhibit frequent bone tumors and myeloid leukemias, were injected intraperitoneally with various doses of 10 to 12,000 Bq/animal of monomeric 239Pu citrate to clarify lifetime carcinogenesis caused by alpha-particles distributed mainly in the skeleton. Survival time was reduced significantly at mean skeletal doses of more than 2.93 Gy and was accompanied by marked life-shortening as compared to the controls due to an earlier increase in neoplastic or non-neoplastic death. Bone tumors, almost all of which were osteosarcomas, were not found in the controls, whereas their incidence increased sharply to a maximum of 96% at 6.92 Gy, then dropped at higher doses (20% at 42.4 Gy). Although lymphoid tumors were present in 20% of the control animals, their incidence dropped to zero at 6.92 Gy, then increased at higher doses (36% at 25.5 Gy). Non-thymic, mostly pre-B cell type, leukemic lymphomas mainly occurred at early time after 239Pu-injection; whereas, in the controls thymic, lymphocytic or histiocytic lymphomas occurred only at a later time. Of the other soft tissue tumors, neither myeloid leukemias nor myelogenous neoplasms were found in the controls or the 239Pu-injected animals. Tumors affecting the lungs, liver, ovaries, and skin were much fewer or not found at mean doses of more than 2.93 Gy. These results indicate dose-dependent, differential tumor induction resulting from bone and lymphoid tumor competition after an injection of plutonium.

Animal models of beryllium-induced lung disease.

The inhalation Toxicology Research Institute (ITRI) is conducting research to improve the understanding of chronic beryllium disease (CBD) and beryllium-induced lung cancer. Initial animal studies examined beagle dogs that inhaled BeO calcined at either 500 or 1000 degrees C. At similar lung burdens, the 500 degrees C BeO induced more severe and extensive granulomatous pneumonia, lymphocytic infiltration into the lung, and positive Be-specific lymphocyte proliferative responses in vitro than the 1000 degrees C BeO. However, the progressive nature of human CBD was not duplicated. More recently, Strains A/J and C3H/Hej mice were exposed to Be metal by inhalation. This produced a marked granulomatous pneumonia, diffuse infiltrates, and multifocal aggregates of interstitial lymphocytes with a pronounced T helper component and pulmonary in situ lymphocyte proliferation. With respect to lung cancer, at a mean lung burden as low as 17 micrograms Be/g lung, inhaled Be metal induced benign and/or malignant lung tumors in over 50% of male and female F344 rats surviving > or = 1 year on study. Substantial tumor multiplicity was found, but K-ras and p53 gene mutations were virtually absent. In mice, however, a lung burden of approximately 60 micrograms (-300 micrograms Be/g lung) caused only a slight increase in crude lung tumor incidence and multiplicity over controls in strain A/J mice and no elevated incidence in strain C3H mice. Taken together, this research program constitutes a coordinated effort to understand beryllium-induced lung disease in experimental animal models.

Prevention of mammary tumorigenesis in acatalasemic mice by vitamin E supplementation.

Adult male and female acatalasemic (C3H/AnLCsbCsb), hypocatalasemic (C3H/AnLCscCsc) and normal mice of C3H strain fed on regular laboratory chow for 15 months showed an increased incidence of spontaneous mammary tumor in the decreasing order of female acatalasemic, male acatalasemic, female hypocatalasemic and male hypocatalasemic mice. Normal mice did not develop mammary tumor. We conducted a prospective study with female acatalasemic mice, which showed the highest incidence of mammary tumor, to examine the preventive effect of vitamin E on mammary tumor. Female acatalasemic mice were fed on vitamin E‐deficient (28 animals) and vitamin E‐supplemented diet (25 animals) for 29 months. The incidence of mammary tumor in mice given the vitamin E‐supplemented diet was 47%, while that in mice given vitamin E‐deficient diet was 82% (P<0.002). Mammary tumors were apparent after 9 months of vitamin E deprivation and after 14 months of vitamin E supplementation. Female normal mice did not develop mammary tumor during a comparable period of time. The mean catalase activity of mammary gland in acatalasemic mice was 18.8% of that in normal mice. The results indicate that vitamin E protects acatalasemic mice against the development of mammary tumor.

Induction of lung tumors in C3H strain mice after single or fractionated irradiation with X-rays.

Murine model for lung tumor induction was studied in C3H/He male mice, a strain with low spontaneous incidence of lung tumors. Dose-response relationships in lung tumor induction were compared following irradiation with single doses and split doses of X-rays to the thorax either at night or in the daytime. The tumor incidence after a single 1.25 Gy dose at night during the period of nocturnal activity almost reached the maximum level after a 5 Gy dose in the daytime. Proliferative activity determined by observing the labeling index with tritiated thymidine in the normal lung was low as a whole, but tended to decrease in the daytime. When the proliferative response was induced by X-irradiation, significantly higher activity was observed at night. These circadian fluctuations were thought to affect radiosensitivity and lung tumor induction in mice. When split doses or fractionated doses of X-rays were applied to the thorax, lung tumor incidence definitely increased. The incidence after two 7.5 Gy doses with a 12 hr-interval was 41%, 3-fold higher than that after a single 15 Gy dose. Moreover, fractionated whole body irradiations (three times at 3 Gy with 3-month-interval) after a single 7.5 Gy thoracic irradiation was most effective in increasing not only the incidence (47%) but also the multiplicity of the lung tumor. More than 30% of tumor-bearing mice had two or more tumors following thoracic and whole body irradiations, while only 10% of tumor-bearers had multiple tumors after single or fractionated thoracic irradiation alone.

Differential induction of bone and hematopoietic tumors in C3H mice after the injection of 239Pu citrate.

Although alpha-emitting plutonium is easily distributed in the skeleton via circulation and subsequently induces bone tumors, there is little evidence that hematopoietic neoplasias are highly induced even though bone marrow stem cells are irradiated internally by alpha particles. We injected groups of female C3H strain mice with doses of 239Pu citrate from 500 to 10000 Bq to investigate the dose-related spectrum of tumor types induced during a lifetime. Survival time was reduced strikingly in all the injected mice due to much earlier induction of bone and lymphoid tumors as compared to the control animals that showed a variety of soft tissue tumors after a longer period of survival. Induction of osterosarcomas was dose-dependent, being maximal in 70% of the animals that received a mean skeletal dose of 10 Gy or less, but was 48% or less at 20 Gy or more. Non-thymic lymphomas accompanied by lymphocytic leukemia were observed in only 4-6% of the animals that received a dose of 10 Gy or less whereas it was maximal in 17-19% at 20 Gy or more. In contrast, there were no bone tumors in the control animals, rather thymic lymphomas or histiocytic lymphomas were found very late in 20% and other soft tissue tumors, including lung, liver and ovary tumors, were noted in 60%. Neither myeloid leukemia nor other myelogenous neoplasias were found in the control and 239Pu-injected animals that received a mean skeletal dose of 3 Gy or more. These results indicate that the differential induction of bone tumors and hematopoietic tumors in mice depends on the dose range and the time after the injection of plutonium.

Genetic and phenotypic aspects of the interrelationship between cellular and humoral immune response to sheep erythrocytes in mice

The presence of the two types of immune reactivity has been shown earlier: 1st--with prevalence of delayed type hypersensitivity reaction over antibody response (in mice of C57Bl/6 strain), 2nd--with prevalence of antibody responses (in mice of CBA strain). These differences first of all have been displayed in case of using by suboptimal doses of antigen. In the present study the character of local and common immune response in mice of BALB/c, DBA/2, CC57BR, C3H strains and (CBA x C57Bl/6)F1 hybrid on different sheep erythrocyte doses has been investigated. It has been revealed, that DBA/2, BALB/c and CC57BR mice react according to the first type and mice of C3H strain--according to the second. It has been concluded, that the relationship between cellular and humoral immune response depends on the structure of H-2 complex and in F1 hybrid mice any of parents' haplotypes can predominate, depending on phenotypical factors.

Effect of leaves of Ginkgo biloba on hair regrowth in C3H strain mice

Effects of 70% ethanolic extract from leaves of Ginkgo biloba (GBE) on the hair regrowth in normal and high butter diet-pretreated C3H strain mice which posterior hair we shaved were investigated. GBE showed a promoting effect on the hair regrowth. GBE had the inhibitory effects on blood platelet aggregation, thrombin activity and fibrinolysis. GBE inhibited the increase of serum the triglyceride level in high cholesterol diet-treated rats. These results suggested that GBE promotes the hair regrowth and could be used as a hair tonic.

Basic study on the effect of nasally administered salmon calcitonin preparation on the nasal ciliary epithelium.

Nasal administration of drugs is an administration method which is worthy of attention. In such therapy, local administration should not damage the nasal mucous membranes. In the present study, we conducted an investigation by the tissue culture test method of the functional and morphological effects of new nasal calcitonin preparation, which is being developed as a nasal spray agent for osteoporosis, on the nasal ciliary epithelium. The nasal septal mucous membranes of C3H-strain mice were used, and 5 types of nasal calcitonin preparations with a microcrystalline cellulose base and additives added were used. The various drugs were added to nasal mucous membrane slices in RPMI-1640 culture medium; ciliary activity before addition and 0.5, 2, and 4 hr after was electrophotometrically measured; and morphological observation was then carried out by SEM. In the various additive groups, virtually no effect on ciliary activity was observed, and the same results were obtained when 3 and 9 times the estimated clinical spray dosage was added. Moreover, in morphological observation by SEM, in the various additive groups, no morphological changes in samples of nasal ciliary specimens were observed after 4 hr of culturing at the maximum administered dosage (9 times the estimated clinical spray dosage). Based on these findings, it was concluded that the series of calcitonin preparations with various additives had no hazardous effect on the nasal ciliary epithelium.

Mouse strain differences in susceptibility to sporidesmin‐induced biliary tract injury

Biliary tract injury was examined in four inbred strains of mice orally dosed with 500 micrograms of the fungal toxin sporidesmin. Semiquantitative histological analysis was used to assess the grade of necroinflammatory changes in the gall bladder, intra- and extrahepatic biliary tree and lobular parenchyma. Injury was greatest in the C57BL/6 and C3H strain mice and was least in SJL/J mice. In these strains injury was greatest at 4 days and had regressed by 10 days. In BALB/c mice the damage, although similar to that in SJL/J mice at 4 days, persisted at the same severity at day 10 and was accompanied by periductal fibrosis and occasionally by obliteration of ducts typical of sclerosing cholangitis. Analysis of the time-course of development of the lesions in C57BL/6 mice showed that the primary target for the toxin is the biliary epithelium. The severity of the lesions within the liver increased centripetally and the worst affected ducts were found at the confluence of the lobar ducts with the common bile duct. The variation in the degree of damage and rate of healing between strains may be due to differences in sporidesmin excretion in bile or interactions with biliary epithelial cells and/or efficacy of protective cellular repair mechanisms.

Strain differences of I-compounds in relation to organ sites of spontaneous tumorigenesis and non-neoplasticrenal disease in mice

Possible associations between the patterns and levels of I-compounds (putative endogenous, age-dependent, adduct-like DNA derivatives) and the incidence of spontaneous tumors and renal diseases in mice were investigated by 32P-postlabeling assay. Liver, lung and kidney DNAs of 8-10 month old inbred male mice of strains C57BL, C3H, CBA, A and RFM were examined. It was found that liver DNA of the C3H mouse, known to develop a high incidence of spontaneous hepatomas, and lung DNA of the A mouse, known to have a high incidence of spontaneous lung tumors, contained smaller numbers and significantly lower total levels of I-compounds as compared with the resistant C57BL mouse. Kidney DNA of C57BL, C3H and A mice presented significantly higher levels of I-compounds compared with those of CBA and RFM mice, both of which are known for exceptionally high incidences of spontaneous renal diseases. Total kidney I-compound levels in CBA and RFM mice were only 30-40% of those found in C3H, A and C57BL. Reduced I-compound levels observed in the three organs thus tended to be associated with spontaneous tumorigenesis and degenerative disease in susceptible strains, suggesting that I-compounds may be important for normal gene transcription and DNA replication. On the other hand, some of these DNA derivatives may, as adduct-like premutagenic lesions, contribute to spontaneous tumorigenesis. This was suggested by the finding that CBA mice which exhibit moderate spontaneous hepatoma incidence, had higher levels of certain I-compounds in their liver DNA although the total levels were not significantly different compared with those in C57BL mice. The observations of the present study suggest that (i) the patterns and levels of I-compounds are genetically determined and (ii) lack or excess of these DNA modifications may result in adverse health effects; however, this may not always be the case, for example, if modulating factors exist which compensate for I-compound deficiency.

Modulation of low-dose streptozotocin-induced diabetes in mice by administration of antibodies to I-A, I-E and I-J determinants

In male mice of strains C3H and C57BL/6 an experimental immune-mediated diabetes can be induced by multiple low doses of streptozotocin. The delay and partial suppression of hyperglycaemia after anti-I-A monoclonal antibody administration was dose dependent. Even saturation levels of anti-I-A did not cause complete protection from diabetes development. Administration of anti-I-E monoclonal antibody also significantly delayed the onset of hyperglycaemia. Surprisingly, the combined treatment with anti-I-A and anti-I-E did not result in better protection from diabetes. Thus, there is an I-A and I-E independent component of the disease. Furthermore, there is no restriction to either I-A or I-E. Anti-I-A was only effective when given at the beginning of the experiment, which implies that I-A molecules have a primary function during the induction of diabetes. The contribution of I-J to the disease process is different. Administration of a polyspecific alloantiserum to I-J almost completely prevented hyperglycaemia. Injections of monospecific antibodies to I-J determinants enhanced hyperglycaemia, especially when given after the induction of diabetes. This indicates that I-J is involved in initial as well as in later stages of the disease process.

A novel methodology for analysis of cell distribution in chimeric mouse organs using a strain specific antibody.

Chimeric animals are very useful for analysis of cell lineage, homeostasis in tissue architecture, and cell-cell interactions during both organogenesis and carcinogenesis. However, there is not a generally effective means for marking cells of chimeric mice. We have therefore developed a polyclonal antibody that is useful for this purpose. This antibody specifically recognizes those cells derived from C3H strain mice. The specificity of this antibody was checked by both immunoblotting and immunoadsorption methods. The antigens were immunohistochemically detected in cytoplasm of both epithelial and mesenchymal cells of C3H/HeN strain mouse in many different organs, but not the corresponding cell types from BALB/c or C57BL/10 or several other mouse strains. The validity of these antibodies as markers for C3H cells was further checked by tissue recombination experiments and in mixed cultures of mouse and rat cells. In each case the antibody recognized only the C3H mouse cells. Next, chimeric mice were prepared between strains C3H/HeN and BALB/c, and C3H/HeN and C57BL/10 mice. Chimeras 2-mo old were examined for antigen distribution using the indirect immunofluorescence method. Many tissues in chimeric mice were composed of cells that were both stained and unstained by the anti-C3H specific antigen. The chimeric patterns were classified into four types, A-D. In well-defined structural units such as intestinal crypts, small intestinal villi, kidney convoluted tubules, exocrine gland acini, ovarian follicles, thyroid gland follicles, stomach glands, adrenal cortex, lingual papillae, etc., (A) each unit was composed entirely of either positive or negative cells, or else (B) in some organs each unit was composed of both types of cells. In the uniform tissues without such distinguishable units, such as stratified squamous epithelium, mesenchymal tissue, corpora lutea, pituitary gland, Islets of Langerhans, adrenal medulla etc., (C) the tissue was composed of definite small cell groups made entirely of either positive or negative cells, or else (D) the tissue was composed of both types of cells which were intermingled with one another. These findings strongly suggest that the chimeric patterns demonstrated here reflect the cell proliferative unit in each tissue. This cell marker system has proven useful for analysis of cell lineage and cell renewal systems in many organs of chimeric mice.

Interstrain differences in susceptibility to liver carcinogenesis initiated by N-nitrosodiethylamine and its promotion by phenobarbital in C57BL/6NCr, C3H/HeNCrMTV- and DBA/2NCr mice.

Selected inbred strains of mice were compared with respect to their susceptibility to two-stage liver carcinogenesis. Five-week-old male mice of strains C57BL/6NCr (C57), C3H/HeNCrMTV- (C3H) and DBA/2NCr (DBA) were given a single i.p. injection of N-nitrosodiethylamine (DEN, 90 mg/kg body weight) or the solvent tricaprylin (10 ml/kg). Beginning 2 weeks later, half of the DEN-treated and half of the control mice were given drinking water containing 0.05% phenobarbital (PB). Ten mice from each treatment group were killed at 12, 24, 36 and 52 weeks of age (5, 17, 29 and 45 weeks exposure to PB). PB significantly increased both the number of hepatocellular foci/cm2 and the incidence of hepatocellular tumors after 17 weeks of treatment in 24-week-old DEN-initiated mice of strains C3H (0.11 +/- 0.07 versus 2.9 +/- 0.3 foci/cm2 and 20 versus 70% incidence of hepatocellular tumors) and DBA (0.09 +/- 0.09 versus 3.72 +/- 0.6 foci/cm2 and 0 versus 90% incidence of hepatocellular tumors) but was ineffective in C57 mice (0.04 +/- 0.04 versus 0.07 +/- 0.07 foci/cm2). At 36 weeks of age the incidence of liver cell tumors in mice given DEN but not PB was 10 (DBA), 10 (C57) and 50% (C3H); the incidence was increased by PB to 90% in DBA and 100% in C3H mice, but there was no increase in C57 mice. Even at 52 weeks, the low incidence of hepatocellular tumors in C57 mice given DEN only (20%) was not significantly increased by subsequent exposure to PB. Serum PB levels observed at 12, 24 and 36 weeks of age were significantly higher in DBA mice than in C57 or C3H mice. Similar results were observed in a separate study in which PB was administered in drinking water to 7-week-old male mice of these three strains for 20 days, during which period serum PB levels were measured at shorter intervals. DBA mice thus appear to be unable to metabolize PB, which itself rather than its metabolites is probably responsible for tumor-promoting effects. DBA mice were especially sensitive, while C57 mice were refractory to promotion of hepatocarcinogenesis by PB. These two strains, which differ with respect to other significant parameters for chemical carcinogenesis including inducibility for aryl hydrocarbon hydroxylase and susceptibility to promotion of hydrocarbon-initiated skin tumors by 12-O-tetradecanoylphorbol-13-acetate, thus also provide a means for analysis of the pharmacogenetics of susceptibility to hepatocellular tumor promotion.

Attenuation of Virulence of M, lepraemurium after Serial Mouse Passages During Long Years

Virulence comparison test between Hawaiian M (HM) and Hawaiian B (HB) strains was made on spread subcutaneous tissue preparations in male and female mice of C3H strain and male mice of BALB/c strain.Tested mice were inoculated subcutaneously in their back with 0.25ml of a1: 1, 000 leproma suspension (with HM or HB) and techniqe of the tissue spreading was described in our earlier report (5).In cases infected with HM bacilli, subcutaneous lesions developed gradully without reactional cell infiltration during the first 3 weeks after infection. Bacillus-containing mononuclears increased in number and became larger in size. At 3 weeks, in female C3H and male BALB/c mice, a small number of lymphocytes and a few polymorphonuc-lears were found on the specimens. After 5 weeks, lymphocytic infiltration with polymo-rphonuclears appeared around the bacillus-containing mononuclears (murine leprosy cells). Especially in male BALB/c mice, at 7 weeks, their subcutaneous lesions showed typically benign feature in which severe infiltration with lymphocytes and polymorphonuclears were observed, surrounding a small number of murine leprosy cells. At week 7, these cell reactions were occationally observed in male C3H mice.On the other hand, in almost all the cases HB bacill-induced lesions developed with time, without reactional cell infiltration, throughout the obsergation period, . After 5 weeks, the lesions showed typically malignant feature. Enlarged mononuclears, being crowded with long baiclli, were scattered or accumulated in the whole specimens. And in male BALB/c mice with HB bacilli a small number of lymphocytes appeared around these mononuclears at about 5 to 7 weeks. However, this feature was considered to be malignant so as to be non-typical.These results clearly showed that HM bacilli were much less virulent for these mice than HB bacilli.It is assumed from these findings that difference in virulence between HM and HB bacilli results from their ability to induce cell-mediated immunity in the host.Plate I Subcutaneous lesions, at week 3. (×200, Z-N stained)There are no pronounced differences in subcutaneous lesions between tested mice infected with HM and HB bacilli 3 weeks after infection.However, in female C3H and male BALB/c mice infected with HM bacilli, a small number of lymphocytes and a few polymorphonuclears are observed around murine leprosy cells (Photos 3 and 5). On the other hand, large and round-shaped murine leprosy cells are found without cell reactions in the remaining cases (Photos 1, 2, 4 and 6).Plate II Subcutaneous lesions, at week 7. (×200, Z-N stained)Remarkable differences are found in subcutaneous lesions between male BALB/c and female C3H mice infected with HM and HB bacilli, 7 weeks after infection. Severe lymphocytic infiltration with polymorphonuclears, surrounding murine leprosy cells, are found in male BALB/c mice with HM bacilli (Photo 11). This feature is typically benign. And there are numerous large murine leprosy cells in the specimens of male BALB/c with HB bacilli. However, very slight cell reaction appears around murine leprosy cells (Photo 12). Then, that feature is considered to be malignant but to be non-typical.The lesions of female C3H mice with HM bacilli (Photo 9) show similar tendencies to those of male BALB/c mice with HM bacilli. However, cell reactions are much more severe in the latter than in the former. And many large murine leprosy cells are observed in the remaining 3 groups without cell reactions (Photos 7, 8 and 10). These features are typically malignant.