DS Mice Research Articles

114 Anemia as a predictor of death and rehospitalization in patients with advanced decompensated heart failure: insights from the OPTIME-CHF study

tion, transmitted over several generations. This L345P missense mutation has a dominant-negative effect on filament formation, causing myopathy and cardiomyopathy. A transgenic mouse strain (DM mice), carrying the desmin L345P mutation, was developed in order to evaluate the effect of this mutation on cardiac and skeletal muscle pathology and function. Transgenic animals with normal desmin gene were used as a control (DS mice). Expression of transgenic desmin was confirmed by immunohistochemistry and Western blot analysis using HA-tag antibody. Here, we present data on morphological analyses of heart muscle in DM and DS animals. Material and Methods: DM and DS mice 28 and 69 weeks old were analysed. Myocardial cryostat 10 μm sections were stained with hematoxilin-eosin, Van-Gieson and AgNOR technique. Results: In 28 week old DM mice an increase of stromal cell number due to lymphocyte and fibroblast-like cell infiltration, was observed. Cardiomyocytes, embedded in collagen fibers, and focal amorphic protein depositions in arterial adventitia and perivascular spaces were often found. In 69 week old DM mice these depositions were more prominent, being present in vascular walls, perivascular space and between muscle fibers. The latter were accompanied by granuloma formations, and caused a disruption of tissue architecture. Histiocyte-like and macrophage-like cells were observed in perivascular and intermyocyte spaces. These changes were not found in DS mice 28 or 69 weeks old. Conclusions: The L345P desmin mutation causes focal protein deposition in vascular walls, perivascular areas and intermyocyte spaces, accompanied by an increase in stromal cell numbers and formation of granulomas. These changes may be responsible for the development of desmin-related cardiomyopathy.

The differentially expressed C21orf5 gene in the medial temporal-lobe system could play a role in mental retardation in Down syndrome and transgenic mice

Mental retardation represents the more invalidating pathological aspect of Down syndrome, DS, and has a hard impact in public health. Modifications in DS brain, concerning abnormal size, neuronal differentiation, and cell density, cause changes in the neurophysiology and behavior of DS patients, and could be determined by dosage imbalance of genes localized in the DS critical region, DCR. Among these genes, C21orf5 showed high homology with Caenorhabditis elegans Pad1 involved in cellular differentiation and patterning. To shed light on C21orf5 role in DS, we performed molecular characterization of human and mouse orthologs, their spatio-temporal expression during development and in adult, and overexpression in DS and transgenic mice. C21orf5 was widely expressed early in embryogenesis in the nervous system. Later, its expression became differential and increased in mesencephalon and rhomboencephalon. This developmental expression profile evolves selectively in adult brain with higher signals in hippocampus, cerebellum, perirhinal, and entorhinal cortex, compared to the other cortical regions. Cellular specificity was detected in hippocampus with higher C21orf5 mRNA level in CA3 cells. Our findings appoint C21orf5 as candidate gene for mental retardation: • Its overexpression in DS cells may contribute to gene imbalance in DS. • Its specific expression in normal and its mirroring pattern in transgenic mice correspond to abnormal regions in DS patients and to neurological phenotype of transgenic mice. • Altered cortical lamination in transgenic mice and the Pad1 ortholog function suggest a potential role of C21orf5 in cell differentiation. • Its patterned differential expression in the medial temporal-lobe system, including hippocampal formation and perirhinal cortex involved in memory storage, and learning and memory defects in the transgenic mice suggest a specialized role for C21orf5 in cognitive processes. These evidences suggest that C21orf5 is an attractive candidate gene contributing to neurological alterations responsible for mental retardation in DS patients.

Disorganization in mice and humans

Disorganization (Ds) is an autosomal dominant mouse mutant that produces a remarkable array of birth defects. So variable is the phenotype that no two mice appear identical. Ds also has markedly reduced penetrance, with 85-99% of Ds mice having no apparent anomalies. Paired structures are often affected, but always asymmetrically. Although the Ds gene has yet to be identified, it is thought that Ds is a gain-of-function mutation, and that Ds malformations are thought to arise through a two-hit mechanism. Unlike the two-hit model that has been used to describe the development of retinoblastoma, the "second hit" for Ds is thought not to arise in the other Ds allele. Although there is a long list of anomalies seen in Ds mice, two stand out as most characteristic: hamartomatous skin papillae, and mirror-image limb duplications. Through the observation of these unusual anomalies in human cases, the possibility of a human homologue of Ds was suggested. However, in reviewing types of anomalies seen in Ds mice, it is apparent that cases with these unusual defects represent only one end of the spectrum of the Ds phenotype. Ds may be the genetic basis for more usual and seemingly sporadic human birth defects as well.

Reduction of tumorigenicity by an interferon-gamma-gene-transduced tumor on another syngeneic tumor in a murine model.

To evaluate the effect of interferon-gamma-gene-transduced cells, DS mice were inoculated into their footpads with syngeneic mammary adenocarcinoma SC42 admixed with interferon-gamma producing mammary adenocarcinoma SC115Kgamma, which had been established by an interferon-gamma-gene transduction in another syngeneic mammary adenocarcinoma SC115 using retroviral vectors. These mice rejected both tumor cells and developed resistance to subsequent challenges with either SC115 or SC42 cells inoculated into their opposite posterior footpads. These results thus indicate that systemic immunological memory to each of the independent tumor cell lines developed in these mice. Although the SC42 cells admixed with irradiated SC115Kgamma cells were rejected by these mice, the SC42 cells admixed with irradiated SC115neoR, in which the neo-gene had been transduced, were observed to proliferate. Tumor rejection was reversed by an in vivo administration of anti-interferon-gamma antibody, thus suggesting that locally produced interferon-gamma plays an important role in tumor elimination and immunological memory induction. In conclusion, interferon-gamma-gene-transduced tumor cells are therefore considered to have a therapeutic potential for other types of malignant tumor cell lines.

Suppressive effects of antihistaminic and/or anti-PAF agents on passive anaphylactic shock in mice sensitized with allogeneic monoclonal IgE and IgG1 antibodies and hyperimmune serum

For the immunopharmacological characterization of murine passive anaphylactic shock, the effects of antihistaminics and/or anti-platelet-activating factor (anti-PAF) agents were studied on the shock mediated by allogeneic monoclonal IgE and IgG1 antibodies and hyperimmune serum. IgE antibody-mediated shock was strongly suppressed by cyproheptadine (10 mg/kg, ip) in every strain regardless of the age and sex of the mice and the presence or absence of a shock potentiator. As far as tested with CTS, DS, and B6D2F1 mice, IgE antibody-mediated shock was also suppressed by the other two antihistamines, triprolidine (10 mg/kg, ip) and oxatomide (100 mg/kg, Po). This type of shock was not suppressed by an anti-PAF agent, CV-6209 (3.3 mg/kg, iv), when tested on aged CTS mice given no shock potentiator and young DS mice given a potentiator such as Bordetella pertussis organisms or DL-propranolol. IgG1 antibody-mediated shock was also suppressed by cyproheptadine in general except for CTS mice. Suppression in the DL-propranolol-treated DS and C3H/He mice was not very marked on sensitization with undiluted or slightly diluted IgG1 ascites but quite striking on sensitization with properly diluted ascites. In contrast with the effect of cyproheptadine, suppression by CV-6209 was obvious in aged CTS mice but not in young DL-propranolol-treated DS mice. The shock in DL-propranolol-treated DS mice sensitized with undiluted or slightly diluted ascites was completely abolished by the combined use of these two agents. These results suggest that histamine and/or PAF play a major role in IgE antibody- and IgG1 antibody-mediated shock. However, so far as tested in young DS mice, the shock mediated by serum differed in drug susceptibility from that mediated by the monoclonal antibodies. In the absence of shock potentiators, prevention was produced by cyproheptadine in the males which had been sensitized with the 1:4 or 1:8 dilution of the immune serum. In the presence of DL-propranolol, prevention was not produced even by the combined treatment with cyproheptadine and CV-6209. Therefore, it is likely that some mediators other than histamine and PAF, whose release is triggered by antibody isotypes other than IgE and IgG1, play a greater role for the shock mediated by hyperimmune serum than for the shock mediated by IgE or IgG1 antibody, especially in the presence of shock potentiators.

Disorganization in mice and humans and its relation to sporadic birth defects.

Disorganization (Ds) is a mouse mutant best known for producing an exceptional variety of unusual developmental anomalies, such as mirror-limb duplications and hamartomatous skin papillae. So great is the range of malformations that no two affected mice are identical. Several patients with a similar variety of exceptional anomalies have been reported, raising the possibility of the existence of a human homologue of Ds. However, although these human cases represent the most striking findings seen in Ds mice, they do not represent the full range of defects. Most affected mice have only a single malformation, and most of these malformations are similar to both common (neural tube defects, orofacial clefting, gastroschisis, limb reductions) and rare (anophthalmia, duplicated rectum) human birth defects. It is therefore possible that the full spectrum of the human homologue of Ds includes not only patients with the unusual combination of anomalies but also common sporadic birth defects. We suggest that the low penetrance (approximately 0-30%) and highly variable expression of Ds make it a paradigm for understanding the genetic basis for many seemingly sporadic birth defects.

Heterogeneity of drug susceptibility of mouse active anaphylactic shock.

To determine the main mediators for mouse anaphylactic shock, the suppression of active anaphylactic shock by cyproheptadine, an antagonist of histamine/serotonin, and CV-6209, an antagonist of platelet-activating factor (PAF), was systematically evaluated using various mouse strains. The suppression was quite heterogeneic depending on the mouse strain and method for sensitization. When sensitization was done with bovine serum albumin (BSA) plus Bordetella pertussis organisms, anaphylactic shock in C57BL/6N mice was suppressed by cyproheptadine but not by CV-6209. In contrast, shock in C3H/HeN and WBB6F1-W/Wv mice was suppressed by CV-6209 but not by cyproheptadine. Shock in BALB/c mice was suppressed by both agents but that in WBB6F1-(+)/+ mice was not at all by either one. DS and BDF1 mice showed differences due to the length of the sensitization period; shock was not suppressed by cyproheptadine with a shorter sensitization (9 to 11 days) but clearly suppressed with prolonged sensitization of 14 to 15 days. Shock in DS, WBB6F1-(+)/+ and BDF1 mice, which was not suppressed by either agent alone, was strongly suppressed by the combination of the two agents. In animals which had been sensitized with BSA emulsified with Freund's complete adjuvant (FCA) and given DL-propranolol as a shock potentiator, a similar pattern of drug susceptibility as that in the pertussis-vaccinated animals was obtained in general, although a discrepancy was seen with regard to the suppression of CV-6209 in C3H/HeN and BDF1 mice. Synergistic suppression by CV-6209 and cyproheptadine was also very clear with the WBB6F1-(+)/+ mice sensitized using FCA. In addition, results quite similar to those with cyproheptadine were obtained with the other two antihistamines, triprolidine almost devoid of antiserotonin activity and oxatomide, in testing with BDF1 mice sensitized using Bordetella pertussis organisms. From these findings, the conclusion is that histamine and PAF play major roles solely or in combination in mouse active anaphylactic shock.

Experimental study of the effects of hormonal therapy and intralesional injections of interleukin 2, activated macrophages on mouse prostate cancer models

In order to establish a more effective and safer therapy for androgen-dependent prostate cancer, to be used in addition to hormonal therapy, the anti-tumor effects of intralesionally administered macrophages activated with recombinant interferon-gamma (INF-gamma), alone or in combination with recombinant interleukin-2 (IL-2) were studied in mouse prostate cancer models. Firstly, in terms of cellular adoptive immunotherapy, phagocytosis against Latex bead and cytotoxicity against Shionogi 115 cancer cell line (SC115) of macrophages activated with INF-gamma for 24 hour were investigated. One ml of 0.25% glycogen solution was intraperitoneally administered to male DS mice. Three days later, fluid was aspirated from the abdominal cavity and macrophages were separated for use in this experiment. Phagocytosis INF-gamma-dose-dependently increased and macrophages activated with 100 U/ml INF-gamma phagocytosed 78.3 +/- 4.5% (mean +/- SD) Latex bead. Cytotoxicity (modified MTT assay) of SC 115 by macrophages activated with 100 U/ml INF-gamma increased remarkably in comparison with non-activated macrophages and there was a significant increase in the effector-to-target-cell ratio to 40 in the activated group 77 +/- 4.3% (mean +/- SD) relative to 50 +/- 6.3% (mean +/- SD) in the non-activated group. Based on these in vitro findings, hormonal therapy and adoptive local immunotherapy, alone or together, were studied in mouse prostate cancer models. The prostate cancer model was prepared through the subcutaneous transplantation of SC115 in male DS mice and the treatments were initiated after tumors were palpable. The therapy protocols were as follows: Group I control and Group II received 20 mg/kg/day diethylstilbestrol diphosphate (DES-P) subcutaneously for 10 days, Group III received DES-P in combination with ten thousand units of IL-2 administered five times intralesionally, Group IV received DES-P in combination with 2 x 10(6) macrophages activated with 100 U/ml INF-gamma administered three times intralesionally, Group V received DES-P and IL-2 in combination with activated macrophages. The therapeutic efficiencies were evaluated by calculating the tumor volume and survival time. The results of the tumor volume on the 40th day post tumor transplantation were as follows (mean +/- SD): Group I 7,049 +/- 1,477 mm3, Group II 4,495 +/- 654 mm3, Group III 2,050 +/- 724 mm3, Group IV 2,782 +/- 970 mm3, Group V 1,555 +/- 514 mm3. The therapeutic groups showed significant tumor reduction relative to the control. Furthermore, intralesionally IL-2, the activated macrophages injected groups, alone or together, were more effective relative to the group receiving only DES-P.(ABSTRACT TRUNCATED AT 400 WORDS)

Disorganization is a completely dominant gain-of-function mouse mutation causing sporadic developmental defects

Disorganization ( Ds) is an exceptional mutation because of its diverse and profound developmental effects. Although other mouse mutations produce similar congenital defects, extreme pleiotropism, random occurrence, developmental independence of multiple defects, and type of anomaly make Ds unique. Examples of developmental defects include cranioschisis, rachischisis, thoracoschisis, exencephaly, hamartomas, and anomalies of appendages, digestive, genital and urinary tracts, sense organs, limbs and girdles, tail and pharynx. No other mutation in the mouse has such broad effects. Ds is therefore an important model for studying not only the genetic control of lineage determination and pattern formation, but also the occurrence of sporadic congenital defects. To characterize the effects of gene dosage, we examined the viability and phenotype of Ds homozygotes and the phenotype of +/+/ Ds trisomic fetuses. Occurrence of homozygotes was tested by intercrossing Ds/+ heterozygotes, typing genetic markers that flank Ds, and examining homozygotes for morphological abnormalities. Not only were Ds homozygotes found in their expected frequency, homozygotes were not more severely affected than heterozygotes. Trisomies provide a direct test for determining whether Ds is a gain-of-function mutation. Trisomic fetuses were derived by crossing Ds/ Ds homozygous mice to hybrid mice that were heterozygous for two related Robertsonian translocations. Two trisomic fetuses had developmental defects characteristic of DS mice. Together these results demonstrate that Ds is a completely dominant, gain-of-function mutation.

Attempts to transfer immunity against Clonorchis sinensis in nude and DS mice

The effects of peritoneal exudate cells (PEC) and sera of athymic nude and DS mice infected with Clonorchis sinensis metacercariae or sensitized by injection of metabolic products into footpad on transfer of immunity against the fluke to the syngeneic mice were studied. There was no significant difference in eggs per gram pattern between the sensitized and control groups, and between nude and DS mice. However, the worm burdens were slightly greater in nude mice than in DS mice. Also, a few plaque forming cells were found in only DS mice given PEC and serum from Group II DS mice. In the light of these results, it is likely that PEC and sera of nude or DS mice which are deficient, at least partially, in the cellular immune system are unable to transfer immunity against C. sinensis to syngeneic recipients.

Age-Dependent Difference in Susceptibility to IgE Antibody- and IgG1Antibody-Mediated Passive Anaphylactic Shock in the Mouse

The age dependence of the susceptibility to passive anaphylactic shock was studied in the mouse. Anti-BPO IgE monoclonal antibody produced potent systemic sensitization sufficient for provocation of lethal shock in most aged (6 to 10 months) CTS, DS and C57BL/6J mice but only in a very few young (1.5 to 2.5 months) mice. A similar trend was found in the NOD strain, though it was not as definite as in the above three strains. Age-dependent potentiation of the IgE antibody-mediated anaphylactic shock was not found in both sexes of five other strains, C3H/He, DBA/2, NON, BALB/c and B6D2F1. However, the potentiation became obvious even in these strains, when they were treated with Bordetella pertussis before the antigen challenge. Age-dependent potentiation was also clear with IgG1 antibody-mediated anaphylactic shock in DS females and NON males. In contrast, no age-dependent difference was seen for the shock induced by PAF which is estimated to be the main mediator for anaphylactic shock in the mouse. This suggests that the age-dependent potentiation of anaphylactic shock does not seem to be due to elevated susceptibility to the mediator but to its increased release. The sex-dependent differences was also studied and found to be particularly clear in the case of IgG1 antibody-mediated anaphylactic shock in young DS and aged NON mice.

Effects of antiandrogens on growth of androgen-dependent mouse mammary tumor (Shionogi carcinoma 115) in vivo and in vitro

Binding affinities of modified steroidal anthrasteroids, 3 β-hydroxy3 aβ, 6-dimethyl- 2,3,3a,4,5,8,9,10,10a β,11,11a β,11,11b α-dodecahydro-1H-cyclopental[ a]anthracene-8-one ( 1) and 3a β,6-ndimethyl-2,3,3a,4,5,8,9,10,10a β,11a β-dodecahydro-1H-cyclopenta[ a]anthracene- 3,8-dione ( 2), the steroid oxendolone and the nonsteroid AA560, for the androgen receptor (AR) of Shionogi carcinoma 115 (SC115) and their effects on the growth of SC115 were investigated in vivo and in vitro. The inhibitory effects of these compounds on testosterone 5α-reductase of SC115 tissues were also measured. The relative binding affinities of these compounds were 3.17−0.03% of that of dihydrotestosterone, and their rank order was ( 1) > AA560 > oxendolone ⪢ ( 2) . In the presence of 10 −9M testosterone, anthrasteroids and AA560 inhibited the growth of SC115 cells at 10 −7 M in a serum-free medium, but oxendolone did not. In the absence of testosterone, ( 1), ( 2) and oxendolone promoted cell growth at 10 −6, 10 −7 and 10 −7 M, respectively. However, AA560 nearly completely blocked cell growth at 10 −5 M. At a 2 mg daily dose for 13 days, ( 1) and AA560 powerfully inhibited tumor growth in castrated DS mice treated with testosterone propionate but oxendolone had almost no effect. Anthrasteroids and oxendolone showed weak but significant agonistic activity in vivo. Anthrasteroids markedly inhibited 5α-reductase activity of SC115, oxendolone weakly and AA560 not at all. The remarkable antiandrogenic activities of ( 1) and AA560 may partially result from their higher affinities for the AR of SC115 but other yet unknown mechanisms may also contribute to these activities.

Development of spindle-shaped cells and chondroid cells from androgen-dependent Shionogi carcinoma 115. A light and electron microscopic study.

Androgen-dependent Shionogi carcinoma 115 (SC115) is an undifferentiated medullary carcinoma consisting of compact round cells. However, when host male DS mice were castrated 2 weeks after tumor transplantation, tumors composed of compact round cells, spindle-shaped cells and chondroid cells grew 4 weeks after castration. Compact round cells with desmosomes were arranged in solid nests and exhibited immunoreactivity for keratin protein. Spindle-shaped cells had prominent rough endoplasmic reticulum, and appeared to secrete collagen. Chondroid cells had the characteristics of chondrocytes. The light and electron microscopic features were highly suggestive of a transition from compact round cells to spindle-shaped cells, and from spindle-shaped cells to chondroid cells. The histology of this tumor thus suggests that SC115 cells are able to change into chondroid cells via spindle-shaped cells.

Initial sensitivity and tolerance to ethanol in mice genetically selected for diazepam sensitivity.

The benzodiazepine (BZ) receptor is coupled with a GABA-receptor chloride-ionophore complex. The BZs augment the GABA-induced increase in chloride conductance, which leads to postsynaptic inhibition. This effect is believed to be responsible for antianxiety, sedative, muscle relaxant, and anticonvulsant effects, but the mechanisms underlying these behavioral effects are poorly understood. Various other sedative-hypnotics, including ethanol and barbiturates, interact with this system, probably contributing to their behavioral effects. We have recently conducted a selective breeding program to develop lines of mice which are diazepam-resistant (DR) and sensitive (DS) (Gallaher EJ, Hollister LE, Gionet SE, Crabbe JC. Psychopharmacology, 93:25-30, 1987); when tested for the duration of rotarod impairment after 20 mg/kg diazepam the DR line was impaired for 71 +/- 13 min compared with 200 +/- 18 min in the DS line. In the current study we tested mice from the DR and DS lines to determine if BZ sensitivity generalized to ethanol. DS mice became ataxic with lower brain ethanol concentrations, and recovered at later times and with lower blood ethanol concentrations, than did DR mice, indicating that sensitivity differences did extend to ethanol. Following a series of sequential doses over 5 to 6 hr DS mice developed minimal rapid tolerance, whereas DR mice developed considerable tolerance. By the end of the day DS mice were therefore much more sensitive to ethanol than were DR mice; this difference was greater in males than in females. High dose ethanol toxicity was studied by assaying brain ethanol concentrations at the cessation of respiration; no differences were found between lines or sexes.

Amitrole: Strain differences in morphological response of the liver following subchronic administration to mice

Strain differences in the induction of hepatocellular preneoplastic lesions by amitrole were examined in NOD, ICR and DS mice. Amitrole was administered to mice in drinking water at a dose of 1% for 6 months. After 3 months, hyperplastic nodules (HN) and severe fibrosis were prominent in NOD mice but not in other strains. On examination at 6 months, both number and size of HN were greatest in the NOD strain. Furthermore, a hepatocellular carcinoma was found in a NOD mouse, suggesting that this strain is more susceptible to amitrole-induced hepatocarcinogenesis than are ICR or DS mice.

Stimulatory effects of dexamethasone and indomethacin on growth of androgen-dependent Shionogi carcinoma 115 in the mouse

The effects of dexamethasone and indomethacin on the growth of androgen-dependent Shionogi carcinoma 115 were studied. When castrated male DS mice were treated with dexamethasone for 2 weeks at daily doses of 1–5 mg/kg starting from 3 days before tumor inoculation, the tumor weight as well as the transplantability was significantly increased. The tumor grown in dexamethasone-treated mice had morphological, biochemical and biological characteristics similar to those of the original Shionogi carcinoma 115. Indomethacin at daily doses of 2–10 mg/kg also stimulated the tumor growth in castrated DS mice, but the effect was less pronounced than that of dexamethasone. In allogeneic male ICR mice, dexamethasone promoted tumor growth, but indomethacin had no effect. These results suggest that tumor growth in the mouse is stimulated not only by androgens but also by glucocorticoids through the suppression of immune responses of the host and direct action on the tumor cells.

Adjuvant chemo-endocrine therapy for androgen-dependent mammary tumor in mice.

About 500 male DS mice grafted with androgen-dependent Shionogi carcinoma 115 (SC115) were used. When the tumor diameter reached about 20 mm (approximately 25 days after transplantation), excision of the tumor and/or castration were carried out. The injection of cyclophosphamide (80 mg/kg body weight X 3 at 7-day intervals) was started from the day after excision. In mice with excised tumor, adjuvant chemo-endocrine therapy was the most effective treatment examined; cumulative 120-day mortalities after transplantation of tumors in non-treated, adjuvant endocrine therapy, adjuvant chemotherapy and adjuvant chemo-endocrine therapy groups were 91%, 29%, 21% and 0%, respectively. Castration induced development of clusters of androgen-independent cancer cells in androgen-dependent SC115 tumor. In mice without tumor excision, the chemo-endocrine therapy was again the most effective treatment, though 86% of mice died by the 120th day after tumor transplantation. These findings suggest the usefulness of adjuvant chemo-endocrine therapy for achieving complete remission in hormone-dependent tumors.

Insignificance of pituitary for growth of androgen-dependent mouse mammary tumor

Proliferation of Shionogi carcinoma 115 cells under various endocrine conditions was determined 11 days after transplantation of the tumor in 100 male DS mice by measuring the incorporation of 5-[ 125I]-iodo-2'-deoxyuridine ([ 125I]-IdUrd) into the whole tumor. The [ 125I]-IdUrd uptake almost disappeared after castration. In castrated-hypophysectomized mice, the [ 125I]-IdUrd uptake in mice given testosterone was much higher than that in mice given oestradiol-17β, progesterone, cortisol or prolactin, which was almost undetectable. The [ 125I]-IdUrd uptake into the whole tumor stimulated by testosterone was not increased significantly by the addition of prolactin or other pituitary hormones. These results show that growth of Shionogi carcinoma 115 in vivo is stimulated by androgens but not by oestrogens, progestins, glucocorticoids or prolactin.

STEROID-INDUCED INTRAHEPATIC CHOLESTASIS IN MICE

It has been reported that administration of orally-active steroids such as norethandrolone (1, 2), norethisterone (3), methyltestosterone (4) and methandienone (5) can cause in some patients a type of cholestatic jaundice characterized by accumulation of bile in the liver without extrahepatic biliary obstruction (intrahepatic cholestasis), occurence of canalicular bile-plugs, absence of either parenchymal necrosis or portal inflammation, and an elevation of conjugated bilirubin in the serum. In addition, Schaffner and Popper (6) have shown that ultrastructural alterations of the liver comprizing a widespread dilatation of the biliary canaliculi and distortion of the canalicular microvilli are invariably associated with the intrahepatic cholestasis. Several authors have reported experimental studies in relation to the cholestatic effect of steroids. Schaffner and Popper (6) have demonstrated the occurence of canalicular dilatation in the liver of rats following administration of norethandrolone. Retardation of bromosulphalein excretion is known to occur in animals treated with methyltestosterone and norethandrolone (7-9). However, the experimental model with definite manifestations of intrahepatic bile-stasis such as conjugated hyperbilirubinemia or canalicular bile-plugs has not been established. In the course of studying the acute toxicity of norethisterone, 17α-ethinyl-19-nortestosterone, we found a consistent appearance of jaundice in DS mice following administration of a large amount of norethisterone as well as other C17 α-alkyl or alkynyl substituted steroids. This experiment attempted to evaluate the pathological entities of the norethisterone-induced jaundice in mice in comparison with the intrahepatic cholestasis in men caused by various steriod drugs.