Effects of oral administration of ciclosporin A on skin carcinogenesis: a study using the two-stage carcinogenesis protocol in mice.

BACKGROUND: Development of malignant skin neoplasms in patients receiving cyclosporin A (CsA) has been reported. The relationship between the pathogenesis of skin carcinogenesis and the dose of CsA is still unclear. AIM: To clarify the effect of oral administration of CsA, especially its dose, on skin carcinogenesis. METHODS: Hr-1 hairless mice were assigned to the following four groups: (i) control group (n = 8), given vehicle intragastrically six times/week and acetone applied to the skin of the back; (ii) chemical-alone (n = 11), given vehicle intragastrically + application of 7,12-dimethylbenz[a]anthracene (DMBA) once week and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) twice week to the back; (iii) CsA-alone group (n = 8), given CsA intragastrically (10 mg/kg) six times/week and vehicle applied to the back twice week; and (iv) CsA + chemical group (n = 8), given 10 mg/kg CsA intragastrically + topical DMBA and TPA. The number of papules > 3 mm in diameter that had developed on the back after 15 weeks was counted. The mean epidermal thickness and number of dermal infiltrates were determined. The same experiments were performed using CsA at doses of 5 and 20 mg/kg. RESULTS: Oral administration of either 10 or 20 mg/kg CsA significantly enhanced the formation of papillomas by DMBA and TPA, but no enhancement was observed when 5 mg/kg CsA was administered. The mean epidermal thickness and number of dermal infiltrates were significantly greater in the CsA + chemical group than in the chemical-alone group. CONCLUSION: These data suggest that oral administration of CsA in excess of a certain dose can accelerate tumour development in mice.

Identification of Gaucher cells in the chorionic villi associated with recurrent hydrops fetalis.

Recurrent non-immune hydrops fetalis has rarely been reported. In order to detect the risk of recurrence in a subsequent pregnancy, one should carefully consider the possibility of an inborn error of metabolism. In such cases, placental examination may be useful in detecting such metabolic storage disorders in the fetus, which usually present as vacuolization of placental cells. We describe a rare case of recurrent hydrops that was detected by placental examination. Through light microscopy, electron microscope (EM) studies and beta-glucocerebrosidase activity the disease was identified as Gaucher's disease.

Adhesion molecule detection in a case of early cerebral malaria: immunohistochemical and electron microscopic findings.

A case of early cerebral malaria caused by Plasmodium falciparum was studied. P-selectin glycoprotein ligand 1 (PL1) was detected along the inner surface of the infected red blood cells (IRBCs), which ordinarily are not positive for PL1 immunohistochemically, suggesting PL1 being the product of parasite. The electron microscopic finding showed granular deposits in the corresponding lesion, consistent with PL1 deposition, in the IRBCs firmly attached to the endothelium of small cerebral vessels. Most of the IRBCs were round shaped as though they lost their capacity to change shape. The therapeutic strategy was expected against adhesion molecules such as PL1 and for maintaining or restoring the metamorphic capacity of IRBCs.

Serum uric acid and the renin-angiotensin system in hypertension.

To study whether the renin-angiotensin system is related to hyperuricemia in hypertension, the serum concentration of uric acid was determined in 96 patients with various types of hypertension and various degrees of plasma renin activity (PRA). In malignant hypertension, both PRA and the serum uric acid level were higher than in essential hypertension; but in primary aldosteronism or desoxycorticosterone-excess hypertension, they were lower than in the essential type. In renovascular hypertension, PRA was higher than in essential hypertension, but the serum uric acid levels were similar. There were no differences in PRA and serum uric acid concentration between Cushing's syndrome and essential hypertension. The serum uric acid level in high-renin essential hypertension was higher than in either the normal-renin or the low-renin type. There was a significant correlation between serum uric acid concentration and PRA in the basal state, and between the change in PRA and the change in serum uric acid induced by administration of furosemide. Apparently the close correlation between the renin-angiotensin system and the concentration of serum uric acid is related to changes in extracellular fluid volume, although an intrarenal effect of angiotensin II cannot be excluded.

Tissue polypeptide antigen expression in human prostate tumors.

Thirty-seven specimens of benign and malignant prostatic tumors were studied for the localization of tissue polypeptide antigen (TPA) by an avidin-biotin-peroxidase complex technique. In addition, 23 metastases of prostatic carcinoma in other organs and 12 nonepithelial tumors of prostate also were studied. All benign and malignant tumors of epithelial origin, including their metastasis, stained positively. Nonepithelial tumors were uniformly negative. In the metastatic lesions, small foci of tumor cells and even single tumor cells could be identified by TPA staining. Immunohistochemical localization of TPA appeared to be a useful tool for assessing the micrometastases of prostatic carcinoma in other organs, especially lymph nodes, or elucidating the epithelial origin of an otherwise undifferentiated prostatic cancer.

Immunohistochemical localization of CA 125 antigen in formalin-fixed paraffin sections of ovarian tumors with the use of Pronase.

The OC 125 monoclonal antibody was used to localize CA 125 antigen in routine paraffin sections of ovarian tumors with the use of a modified avidin-biotin-peroxidase complex (ABC) technic. Pretreatment of the paraffin sections with Pronase allowed subsequent detection of CA 125 antigen. OC 125 stained 4 (80%) of 5 benign and borderline serous ovarian tumors, 12 (86%) of 14 serous adenocarcinomas, and 3 (23%) of 13 benign and malignant mucinous ovarian tumors. The pattern of distribution of CA 125 antigen was mostly at the intraluminal and peripheral cell surfaces, while intracytoplasmic staining also was seen. Overall, CA 125 antigen detectability rate in paraffin sections was found to be compatible with those reported in frozen sections. The method allows retrospective immunohistochemical examination of a large number of cases with ovarian tumors.

Immunohistochemical and ultrastructural localization of T antigen in ovarian tumors.

Thomsen-Friedenreich (T) antigen, the immediate precursor antigen of the human blood group MN system, has been found in many carcinomas, but it is suppressed in normal tissues and nonmalignant diseases. Using a monoclonal antibody specific for the T epitope and an indirect immunoperoxidase technique at light and electron microscopic levels, the authors studied the expression of T antigen and its potential diagnostic value in ovarian tumors. Among 30 serous and mucinous ovarian cystadenocarcinomas, 20 (67%) were positive and 10 (33%) were negative for T antigen. In carcinomas, positive rates increased in parallel with the tumor grade and were 37%, 75% and 80% for grade 1, 2, and 3 tumors, respectively. Of the nine patients with metastasis, seven (78%) had positive and two had negative reactions in their primary and metastatic tumors. T antigen staining was observed at the intraluminal cell surfaces and peripheral cell membranes. The ultrastructural localization of T antigen revealed electron-dense reaction products at the cell surface and microvillous surfaces. Of the ten benign ovarian tumors, three (30%) were weakly positive and seven (70%) were negative for T antigen. These findings indicate a positive correlation between the presence of immunoreactive T antigen and conventional unfavorable prognostic indicators in ovarian carcinoma. The surface location of T antigen suggests that it may have a functional role at the cell membrane and the membrane may be involved in secretion (shedding) of T antigen. Detection of T antigen may be a useful marker of prognosis in ovarian carcinoma.

Inhibitory effect of oleanolic acid on 12-O-tetradecanoylphorbol-13-acetate-induced gene expression in mouse skin.

Oleanolic acid (OA) has been shown to inhibit mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). This study was designed to examine the effect of OA on the TPA-induced expression of the ornithine decarboxylase (ODC) gene as well as other genes. OA inhibited the induction of ODC activity and mRNA level produced by TPA in the skin of female CD-1 mice. Preapplication of OA (10 mumol) to the mouse dorsal skin produced an approximately 50% decrease in TPA (8 nmol)-induced epidermal ODC activity, as well as ODC gene expression. These results suggest that OA inhibits TPA-induced ODC mainly at the transcriptional level. In addition to ODC, TPA also stimulated metallothionein (MT) gene expression in mouse skin. A dose of 2.5 mumol of OA diminished the TPA-induced MT mRNA 50%. Treatment with OA (10 mumol) after TPA (8 nmol) application also inhibited ODC and MT gene expression which suggests that OA does not compete with TPA for its receptor. OA pretreatment also prevented c-fos gene expression. All of these findings suggest that OA diminishes some signal transduction pathways of TPA to suppress target gene expression in mouse skin. This study suggests that OA might be a general inhibitor against TPA-stimulated gene expression in mouse skin.

Comparative studies of the effects of stilbene compounds on hepatic ornithine decarboxylase and S-adenosylmethionine decarboxylase induction in rats.

Trans-Stilbene oxide (TSO, 2 mmol/kg, ip.) induced ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) to 60-fold and 5-fold of the controls, respectively, in the liver of rats. Parallel to ODC induction, there was a marked increase in putrescine content to 50-fold of the control levels. Cis-Stilbene oxide (CSO), a stereoisomer of TSO, also produced the induction of ODC and SAMDC and the increase in putrescine content. There was no difference in the ability to induce ODC and SAMDC between TSO and CSO with respect to the extents of induction and the time needed to reach maximal levels. Trans-Stilbene (TS), a mother compound of TSO, did not show such an effect on ODC, while cis-stilbene (CS) induced both ODC and SAMDC. Treatment with glutathione inhibited TSO- and CSO-mediated induction of ODC and SAMDC. These findings add new information concerning the abilities of TSO, CSO and CS on hepatic polyamine metabolism.

Induction of ornithine decarboxylase and S-adenosylmethionine decarboxylase by sulfobromophthalein in rats.

The administration of sulfobromophthalein (BSP, 0.5 mmol/kg, ip.) increased ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities to 30-fold and 5-fold, respectively, of the controls at 12 hr in the liver of rats. Parallel to the increase in ODC, there was an increase in hepatic putrescine content. However, spermine content tended to decrease. BSP increased ODC and SAMDC activities and putrescine content, but decreased spermine content, in a dose-dependent manner. Pretreatment of rats with actinomycin D and cycloheximide almost completely blocked the BSP-mediated increase of ODC and SAMDC activities. Pretreatment with glutathione (GSH) failed to inhibit BSP-mediated increase of ODC and SAMDC activities. In addition, the administration of BSP-GSH conjugate (0.5 mmol/kg, iv.) did not produce the increase of ODC and SAMDC activities. Pretreatment with phenobarbital and 3-methylcholanthrene did not inhibit BSP-mediated increase of ODC and SAMDC. The results indicate that BSP could cause changes in hepatic polyamine content due to the induction of ODC and SAMDC.

Nutritionally and chemically induced impairment of sulfate activation and sulfation of xenobiotics in vivo.

Sulfation requires 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor. In the search for methods to inhibit sulfation reactions via impairment of PAPS synthesis, two experimental conditions have been tested in rats. A low-sulfur diet, which does not deplete hepatic glutathione, reduced inorganic sulfate but not PAPS levels in the liver and moderately decreased sulfation of acetaminophen. Administration of molybdate, which is an alternative substrate for intestinal and renal sulfate transport as well as for ATP-sulfurylase, depleted both sulfate and PAPS in liver and markedly inhibited sulfation of acetaminophen. Therefore, administration of molybdate may be used as an experimental tool to study the role of sulfation in the fate and effect of xenobiotics.

Synergistic induction of rat hepatic ornithine decarboxylase by multiple doses of cobalt chloride.

The level of rat hepatic ornithine decarboxylase (ODC) induced by repetitive administration of Co2+ was determined by affinity labeling with [3H]difluoromethylornithine. Such a treatment with Co2+ ion induced ODC level to a 10-fold greater extent than single dose of the metal ion or well-known inducers of the enzyme, such as thioacetamide or carbon tetrachloride. The half life of ODC activity induced by repetitive treatment with Co2+ (95 min) was substantially increased to about 10-fold over the value obtained from the enzyme induced by single treatment with the metal ion (10 min). ODC activity induced by repetitive treatment with Co2+ was separated into two peaks by DEAE-Sepharose column chromatography. The two independently collected fractions of ODC peaks exhibited different affinity for pyridoxal 5'-phosphate in vitro and sensitivity to cycloheximide in vivo.

Induction of metallothionein synthesis by glutathione depletion after trans- and cis-stilbene oxide administration in rats.

To investigate the relationship between glutathione (GSH) depletion and metallothionein (MT) synthesis, the effects of substrates and an inhibitor of GSH S-transferases on concentrations of hepatic GSH, zinc (Zn) and MT were studied in rats. Trans-stilbene oxide (TSO) is an inducer of drug metabolizing enzymes and also a substrate of GSH S-transferase, whereby it covalently reacts with and depletes GSH. The hepatic GSH level was decreased to 25% of the control 2 h after injection of TSO, and returned to the control level by 24 h. TSO significantly increased hepatic concentrations of Zn and MT in a dose-dependent manner. Two isoforms of MT (MT-I and MT-II) were increased by TSO; MT-II was the dominant form. Pretreatment with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, enhanced MT synthesis itself as well as that induced by TSO and cis-stilbene oxide (CSO). On the contrary, infection into rats of perfluorodecanoic acid (PFDA), an inhibitor of GSH S-transferase, resulted in a decrease in basal levels of Zn, and prevented the increase in MT synthesis by TSO and CSO. These results suggest that the decrease of GSH concentration in the liver which causes oxidative stress conditions may be related to MT induction.

Effect of exogenous hydrogen peroxide on myocardial function and structure in isolated rat heart.

A time- and dose-dependent effect of exogenous hydrogen peroxide was determined on myocardial function, structure, high energy phosphate and lipid peroxidation in the isolated perfused rat heart. Hydrogen peroxide induced a dose-dependent decrease in cardiac function whereas 200 microM hydrogen peroxide reduced +dP/dt to 50% of control value after 10 mins. The effect of 300 microM hydrogen peroxide was more severe after 15 mins; changes observed with this dose were reversible within 10 mins of perfusion, becoming irreversible after 15 mins. Lipid peroxidation and severe morphological damage were observed after 10 mins of perfusion with 300 microM hydrogen peroxide. When 16 mEq potassium ions were added in the perfusion buffer during hydrogen peroxide perfusion, the degree of tissue damage and loss of ATP were attenuated. However, lipid peroxidation was not inhibited by high potassium ions. When 0.25 microM N,N'-diphenyl-1,4-phenylenediamine, a potent antioxidant, was added to the perfusate, lipid peroxidation was totally inhibited and the degree of tissue damage was decreased. However, depletion of tissue ATP and functional deterioration were not influenced. These results suggest that hydrogen peroxide-mediated ATP loss was independent of lipid peroxidation.

Effects of the reduction of cytoplasm of mouse 2-cell embryos on blastocele formation timing and developmental ability in vitro and in vivo.

Experiments were conducted to test the hypothesis that the nucleo/cytoplasmic ratio of mouse embryos determines the time of blastocele formation. Half the volume of 2-cell stage embryos was removed from each blastomere by micropipette to alter the nucleo/cytoplasmic ratio. Reduced embryos whose nucleo/cytoplasmic ratio increased and non-treated control embryos were cultured in vitro to compare the timing of division to the 4-cell stage and blastocele formation. Reduced 2-cell embryos formed blastoceles significantly earlier than the controls (49.0 +/-2.9 vs 52.2 +/-6 h) and with fewer cells, although division into the 4-cell stage was significantly delayed (11.4 +/-4.4 vs 9.0+/-2.4 h). The cell number of blastocysts 70 h after treatment and developmental ability of blastocysts after transfer to pseudopregnant recipients were the same for the reduced and control groups. The present study indicates that the nucleo/cytoplasmic ratio of embryos may possibly be an important factor that determines the time of blastocele formation.

Immunocytochemical localization of xanthine oxidase in rat myocardium.

Monoclonal antibodies (MAb's) to xanthine oxidase (XO) (from bovine milk) were produced by hybridoma technique. Culture supernatants were initially screened using enzyme-linked immunoabsorbant assay (ELISA). Forty four positive clones were subcloned and further characterized by ELISA and immunoblot analysis. Out of fifteen clones, which were positive in immunoblot analysis, one clone N2-26 was also positive in immunocytochemical studies. Indirect immunoperoxidase and enzyme histochemistry staining showed that XO activity is present in endothelial cells of capillaries, small blood vessels and also in interstitial cells. Electron microscopy revealed that diaminobenzidine reaction product was distributed in the cytoplasm of interstitial cells and endothelial cells of capillaries and small blood vessels. This is the first report of the presence of XO in interstitial cells and endothelial cells of small blood vessels. Allopurinol, which inhibits the xanthine oxidase activity, did not have any effect on the immunocytochemical staining. Our results in normal rat heart suggest that XO activity is confined to interstitial cells, endothelial cells of capillaries and small blood vessels.

[Identification and susceptibility of clinically isolated yeast-like fungi].

We studied the identification and susceptibility of clinically isolated yeast-like fungi at Showa University Hospital from April 1988 to March 1989. Clinically significant of yeast-like fungi were observed in 7.1% of specimens from outpatients, 13.0% of inpatients. In both outpatients and inpatients, yeast-like fungi were isolated mainly from sputum and urine. But, one third of them were considered as non-pathogenic and not identified. The species of isolates were, Candida albicans 57%, Candida tropicalis 14% and Candida glabrata 8% in both inpatients and outpatients, and these species shared most part. The isolation frequency of Candida parapsilosis was higher in blood and cerebrospinal fluid (CSF) specimen than the others. The susceptibility test by agar dilution method indicated most of the isolates were susceptible to Amphotericin B and Miconazole (MIC less than or equal to 25 micrograms/ml). There was no difference in MIC between predominantly isolated fungi and commonly isolated fungi. Notably, isolates from blood and CSF showed a significant high tolerance against Amphotericin B and Miconazole than from the other specimens. The MICs of Fluconazole were shown to be very high (greater than 100 micrograms/ml) in normal Sabouraud agar, were decreased dose-dependently by human sera in the medium. These findings indicated the component(s) of sera enhanced the anti-fungal activity of Fluconazole.

Review: Exploration of placentation from human beings to ocean-living species.

This review covers four topics. 1) Placental pathology in Himalayan mountain people. To determine morphological changes of the placenta at high altitude, pathological examination was made of 1000 Himalayan placentas obtained in Nepal and Tibet and the results compared with Japanese placentas delivered at sea level. Characteristic findings in the placental villi of the Himalayan group included high incidences of villous chorangiosis and chorangioma. These processes were clarified by ultrastructural observation. 2) Placentation in Sirenians. The giant Takikawa sea cow, which lived 5 million years ago, was discovered on Hokkaido, Japan. It was an ancestor of the dugong as well as the manatees. Sirenia, the sea cow group, shares a common ancestor with Proboscidea, the elephants, even though they now inhabit quite different environments. A comparison was made of their zonary endothelial type of placentation. 3) Placentation in sharks and rays. The remarkable placentation of hammerhead sharks and manta rays is described. 4) Placentation in the Antarctic minke whale. Placental tissue samples of this whale were obtained from the Japan Institute of Cetacean Research. In an ultrastructural study of the utero-placental junction, microfilamental processes of the allantochorionic zone and crypt formation were visualized.