M-cadherin, a candidate gene for type 2 diabetes and related phenotypes in a KK/Ta mouse model.

The KK/Ta strain serves as a suitable polygenic mouse model for type 2 diabetes associated with fasting hyperglycaemia, glucose intolerance, hyperinsulinaemia, mild obesity and dyslipidaemia. Recently, we reported the susceptibility loci contributing to type 2 diabetes and related phenotypes in KK/Ta mice. In the present study, to identify susceptibility genes for type 2 diabetes and related disorders, GeneChip Expression Analysis was employed to survey the gene expression profile in the liver of KK/Ta and BALB/c mice. M-cadherin, a calciumdependent intercellular adhesion molecule, showed increased expression in the liver of KK/Ta mice, and sequence analysis revealed three missense mutations. The relationship between these polymorphisms and various phenotypes in 208 KK/Ta x (BALB/c x KK/Ta) F1 backcross mice was analysed. Statistical analysis revealed that M-cadherin exhibits linkage to levels of triglyceride and insulin in sera, glucose tolerance and body weight. Although it has been postulated that M-cadherin may be important for the regulation of morphogenesis of skeletal muscle cells, these results suggest that M-cadherin may influence hypertriglyceridaemia, glucose intolerance, hyperinsulinaemia and obesity in KK/Ta mice.

Inhibitory effects of alpha-carotene on proliferation of the human neuroblastoma cell line GOTO.

alpha-Carotene inhibited the proliferation of the human neuroblastoma cell line GOTO in a dose- and time-dependent manner. In addition, it was about 10 times more inhibitory than beta-carotene. Northern blot analysis indicated that alpha-carotene caused maximum suppression of the level of the N-myc messenger RNA of GOTO cells. This suppression occurred within 18 hours of alpha-carotene treatment, after which the level of the N-myc messenger RNA gradually recovered to the basal level. Analysis by flow cytometry indicated that when GOTO cells were exposed to alpha-carotene, they were arrested in the G0-G1 phase of their cell cycle. However, as the level of the N-myc messenger RNA was recovering, these cells resumed normal cycling. These results indicate that the reduction in the level of the N-myc messenger RNA caused by alpha-carotene is closely linked with G0-G1 arrest.

Potent preventive action of alpha-carotene against carcinogenesis: spontaneous liver carcinogenesis and promoting stage of lung and skin carcinogenesis in mice are suppressed more effectively by alpha-carotene than by beta-carotene.

Although beta-carotene has been considered to be a key cancer preventive agent in green and yellow vegetables, other types of carotenoids, such as alpha-carotene, may also contribute to anticarcinogenic action, since these carotenoids usually coexist with beta-carotene and are detectable in human blood and tissues. In this study, we compared the inhibitory effect of natural alpha-carotene, obtained from palm oil, with that of beta-carotene on spontaneous liver carcinogenesis in C3H/He male mice. The mean number of hepatomas per mouse was significantly decreased by alpha-carotene supplementation (per os administration in drinking water at a concentration of 0.05%, ad libitum) as compared with that in the control group (P < 0.001, Student's t test). On the other hand, beta-carotene, at the same dose as alpha-carotene, did not show any such significant difference from the control group. Furthermore, we also compared the antitumor-promoting activity of alpha-carotene with that of beta-carotene against two-stage mouse lung carcinogenesis (initiator, 4-nitroquinoline 1-oxide; promoter, glycerol). alpha-Carotene, but not beta-carotene, reduced the number of lung tumors per mouse to about 30% of that in the control group (P < 0.001, Student's t test). The higher potency of the antitumor-promoting action of alpha-carotene compared to beta-carotene was confirmed in other experimental systems; e.g., alpha-carotene was also found to have a stronger effect than beta-carotene in suppressing the promoting activity of 12-O-tetradecanoylphorbol-13-acetate on skin carcinogenesis in 7,12-dimethylbenz[a]anthracene-initiated mice. These results suggest that not only beta-carotene, but also other types of carotenoids, such as alpha-carotene, may play an important role in cancer prevention.

Immunohistological evidence for renin in human endocrine tissues.

The peroxidase-labeled antibody method and the avidin-biotin-complex method with antiserum to purified human kidney renin were used to identify renin in human endocrine tissues. Renin immunoreactivity was found in some large cells of the anterior pituitary, the zona glomerulosa and the zona reticularis of the adrenal, the Leydig cells of the testis, and the follicular epithelial cells of the thyroid and prostate glands. The specificity of the immunohistochemical reaction was confirmed by immunoabsorption tests. The specific localization of immunoreactive renin in each tissue suggests a possible role of renin in the function of these tissues.

Inhibitory effects of fucoxanthin, a natural carotenoid, on N-myc expression and cell cycle progression in human malignant tumor cells.

Fucoxanthin, a natural carotenoid prepared from brown algae, inhibited the growth of GOTO cells, a human neuroblastoma cell line. Fucoxanthin at 10 micrograms/ml reduced the growth rate of GOTO cells to 38% of the control at day 3 after drug treatment. Flowcytometric analysis revealed that fucoxanthin caused the arrest in the G0-G1 phase of cell cycle. Expression of N-myc gene was proved to be decreased by fucoxanthin as early as 4 h after treatment at 10 micrograms/ml and that may be important for the mechanism of anti-proliferative action of the carotenoid.

Inhibitory effects of natural carotenoids, alpha-carotene, beta-carotene, lycopene and lutein, on colonic aberrant crypt foci formation in rats.

Inhibitory effect of four carotenoids prevalent in human blood and tissues against the formation of colonic aberrant crypt foci was examined in Sprague-Dawley rats. They received three intrarectal doses of N-methylnitrosourea in weak 1, and a daily gavage of de-escalated doses of carotenoids during weeks 2 and 5. Lycopene, lutein, alpha-carotene and palm carotenes (a mixture of alpha-carotene, beta-carotene and lycopene) inhibited the development of aberrant crypt foci quantitated at week 6, but beta-carotene did not. The results suggested that lycopene and lutein in small doses may potentially prevent colon carcinogenesis.

Inhibition of initiation and early stage development of aberrant crypt foci and enhanced natural killer activity in male rats administered bovine lactoferrin concomitantly with azoxymethane.

The influence of concomitant administration of bovine lactoferrin (bLF) on induction of aberrant crypt foci (ACF) by azoxymethane was investigated in male F344 rats. Two percent bLF and 3% Bifidobacterium longum (B. longum), as a positive control, significantly decreased the numbers of ACF as well as the total numbers of aberrant crypts reproducibly in three independent studies (2% bLF, P < 0.01; 3% B. longum, P < 0.05). Most importantly large size foci composed of four or more crypts were always significantly decreased by 2% bLF (P < 0.05). Additional investigation of the natural killer activity of spleen cells demonstrated enhancement by bLF (P < 0.01) and B. longum (P < 0.01) in line with the levels of influence on foci induction, indicating a possible role for elevated immune cytotoxicity in the observed inhibition.

Chemoprevention by lycopene of mouse lung neoplasia after combined initiation treatment with DEN, MNU and DMH.

An investigation was conducted to assess the chemopreventive potential of lycopene (LP), a naturally occurring hydrocarbon carotenoid found in tomatoes and their products, administered during the post-initiation stage in a multiorgan carcinogenesis model. One hundred eighteen B6C3F1 mice of both sexes were subjected to combined treatment with diethylnitrosamine (DEN), N-methyl-N-nitrosourea (MNU) and 1,2-dimethylhydrazine (DMH) from day 11 after birth to week 9 (DMD treatment) (groups 1 and 2) or their vehicles (group 3). Then group 1 received LP (25 or 50 ppm in drinking water) for 21 weeks from weeks 11 to 32. Group 2 served as a carcinogen alone control and group 3 was given only LP (25 or 50 ppm). All surviving animals were sacrificed at week 32 and the major organs, including the liver, lung, kidney and colon, were histologically examined. The incidences and multiplicities of lung adenomas plus carcinomas combined in male mice in group 1 receiving 50 ppm LP were significantly decreased as compared to the DMD alone or DMD and 25 ppm LP groups (75.0 versus 18.8%, P < 0.02; 0.94 +/- 0.17 versus 0.25 +/- 0.14, P < 0.001). No such effect was observed for females. Although hepatocellular carcinomas were lacking in the DMD and LP groups while two cases were found in the DMD alone group, this difference was not statistically significant. The values for aberrant crypt foci (ACF) and tumors in the colon and kidney did not show any significant variation among the carcinogen-treated subgroups. The results suggest that LP exerts a chemopreventive effect limited to male lung carcinogenesis when given in the post-initiation stage to B6C3F1 mice.

Relationship between zonal distribution of microsomal cytochrome P-450s (P-450(17)alpha,lyase and P-450C21) and steroidogenic activities in guinea-pig adrenal cortex.

In an attempt to elucidate the functional role of the three zones of the adrenal cortex, the localization of microsomal cytochrome P-450s (P450(17)alpha,lyase and P-450C21) and their relation to steroidogenesis in the guinea-pig adrenal cortex were studied. The intracellular localization and zonal distribution of P-450(17)alpha,lyase and P-450C21 were examined by the direct peroxidase-labelled antibody technique. The cytochromes were localized immunocytochemically on the smooth-surfaced endoplasmic reticulum. P-450(17)alpha,lyase was found to be distributed in the zona fasciculata, especially in the externa, and less in the zona reticularis. The zona glomerulosa was negative for immunohistochemical staining for P-450(17)alpha,lyase. In contrast, P-450C21 was distributed in all three zones in the adrenal cortex. The cytochrome P-450 contents and their steroidogenic activities were determined with microsomes prepared from the outer zone (zonae glomerulosa and fasciculata) and from the inner zone (zona reticularis) of guinea-pig adrenals. Using an enzyme-linked immunosorbent assay, the ratio of P-450C21 content in the inner zone to that in the outer zone microsomes was estimated to be 1.24. 21-Hydroxylase activity was higher in the inner zone microsomes, which was in good agreement with the relative P-450C21 contents. The ratio of P-450(17)alpha,lyase content in the inner zone to that in the outer zone microsomes was estimated to be 0.75. 17 alpha-Hydroxylase and C17-20-lyase activities were five- to sixfold greater in the outer than in the inner zone microsomes, which could not be explained by the P-450(17)alpha,lyase contents in the two zones.(ABSTRACT TRUNCATED AT 250 WORDS)

The usefulness of rabbits as an animal model for the neuropathological assessment of neurotoxicity following the administration of vincristine.

Vincristine is an effective chemotherapeutic agent for a variety of human neoplasms, but has dose-limiting neurotoxicity. Since laboratory rodents have proven to be refractive in such neurotoxicological studies, we conducted a neuropathological and behavioral assessment in rabbits treated with vincristine at doses known to be both chemotherapeutically effective and neurotoxic in humans. Rabbits (Kbl: NZW) were given vincristine intravenously at doses of 0 (saline), 200, 250 or 300 microg/kg once a week for 6 weeks, 500 microg/kg once a week for 3 weeks, or a single 500 microg/kg administration. Detailed periodic neurologic examination revealed ataxia in a few animals. Pathologically, axonal injury progressing to fiber degeneration was observed in sensory tracts such as the posterior spinocerebellar tract and posterior funiculus, and in peripheral nerves after treatment with vincristine. These alterations were observed even after a single dose of 500 microg/kg. In the group given weekly doses of 500 microg/kg, neuronal chromatolysis was also found in the spinal cord. These results suggest the rabbit is responsive to vincristine neurotoxicity producing a predominantly sensory neuropathy and confirming earlier studies.

Suppression of lung and liver carcinogenesis in mice by oral administration of myo-inositol.

It has been reported that myo-inositol can inhibit carcinogenesis in various organs, such as the mammary gland, colon and lung. In the present study, at first, inhibitory effects of myo-inositol on lung carcinogenesis were confirmed. Then, the influence of myo-inositol on liver carcinogenesis in mice was investigated. In C3H/He male mice, the rate of spontaneous liver carcinogenesis is known to be high. Using this experimental model, the effects of oral administration of myo-inositol (added into the drinking water at the concentration of 1%) were assessed. Significant suppression of liver carcinogenesis was observed in mice treated with myo-inositol for 40 weeks. In the control group without myo-inositol administration, 88% of the animals developed liver tumors, whereas in the myo-inositol-supplemented group, the incidence of liver tumors was 38% (p < 0.05). The average number of liver tumors per mouse was also decreased significantly by myo-inositol treatment; from 7.8 in the control group to 0.8 in the myo-inositol-supplemented group (p < 0.01). Thus, myo-inositol may be useful for cancer chemoprevention in the liver, as well as the lung.

An antimicrotubule agent, TZT-1027, does not induce neuropathologic alterations which are detected after administration of vincristine or paclitaxel in animal models.

One of the major dose-limiting toxicities induced by antimicrotubule antitumor agents such as vinca alkaloids and taxanes is peripheral neuropathy. The neurotoxicity of TZT-1027 (a dolastatin 10 derivative antimicrotubule agent) was thus assessed using the animal models for antimicrotubule agent-induced neurotoxicity. Rabbits were intravenously given TZT-1027 or vincristine weekly for 5 weeks. In the mouse study, TZT-1027, vincristine or paclitaxel was intravenously given every 2 days and/or weekly. Despite the neuropathologic evidence such as myelinated axonal and fiber degeneration in the peripheral nerves and in the sensory tracts of the spinal cord following the treatment with vincristine or paclitaxel, no drug-induced alteration was observed in the TZT-1027 groups. Although there are reports that some other dolastatin derivatives with antimicrotubule activity showed no neurotoxic potential in humans, the present study represents the first demonstration in experimental animals that a dolastatin derivative has no, or at least a lower, neurotoxic potential compared to other antimicrotubule agents.

Cancer prevention by natural carotenoids.

Various natural carotenoids were proven to have anticarcinogenic activity. Epidemiological investigations have shown that cancer risk is inversely related to the consumption of green and yellow vegetables and fruits. Since beta-carotene is present in abundance in these vegetables and fruits, it has been investigated extensively as possible cancer preventive agent. However, various carotenoids which co-exist with beta-carotene in vegetables and fruits also have anti-carcinogenic activity. And some of them, such as alpha-carotene, showed higher potency than beta-carotene to suppress experimental carcinogenesis. Thus, we have carried out more extensive studies on cancer preventive activities of natural carotenoids in foods; i.e., lutein, lycopene, zeaxanthin and beta-cryptoxanthin. Analysis of the action mechanism of these natural carotenoids is now in progress, and some interesting results have already obtained; for example, beta-cryptoxanthin was suggested to stimulate the expression of RB gene, an anti-oncogene, and p73 gene, which is known as one of the p53-related genes. Based on these results, multi-carotenoids (mixture of natural carotenoids) seems to be of interest to evaluate its usefulness for practice in human cancer prevention.

Atrophic effects of antiandrogen, chlormadinone acetate (CMA) on dog prostate with spontaneous benign prostatic hyperplasia.

The atrophic effect of a synthetic steroidal antiandrogen, chlormadinone acetate (CMA), on spontaneous benign prostatic hyperplasia (BPH) in dogs was investigated. Male beagle dogs (5-8 years old) were divided into four experimental groups. Group 1 consisted of untreated controls. Groups 2 to 4 received CMA 0.03, 0.1, and 0.3 mg/kg/day, p.o., respectively, for 6 months. In group 1, glandular hyperplasia of the prostate was clearly detected. The glandular epithelial cells showed uniformly intense nuclear staining for androgen receptor (AR). AR was also localized in the nuclei of the fibro-muscular stromal cells. Immunoreactivity of 5 alpha-reductase type I was positive in most glandular epithelial cells. No fibro-muscular stromal cells were stained. Immunolocalization of 5 alpha-reductase type II was clearly detected in the interacinar fibro-muscular stromal cells, but not in the glandular epithelial cells. In groups 2 to 4, CMA produced marked atrophy of the glandular epithelium. The interacinar fibro-muscular stroma was prominent. The nuclear staining for AR in both epithelial and stromal cells was remarkably decreased. Furthermore, the immunoreaction for 5 alpha-reductase type I in most glandular epithelial cells was negative or very weak. The immunoreaction of 5 alpha-reductase type II in the interacinar fibro-muscular stromal cells was negative or very weak. These results indicate that the uptake of testosterone and/or its androgenic effect on the prostate may be suppressed by CMA. The decreased AR-immunostaining may be explained by the decrease in the number of AR and/or antibody binding sites for AR. Therefore, the atrophy after treatment with CMA may be due to shrinkage of both glandular and stromal compartments in the prostate tissue.

A significant relationship between glutathione-peroxidase (GSH-PO) localization and biological action of testosterone in rat ventral prostate.

In order to confirm the relationship between glutathione-peroxidase (GSH-PO) localization and biological testosterone action in the rat ventral prostate, immunocytochemical localization of GSH-PO in glandular epithelial cells of the rat ventral prostate was investigated. In the untreated group, GSH-PO was predominantly demonstrated in glandular epithelial cells of the ventral prostate. Intracellular localization of GSH-PO in the glandular epithelial cells was mainly observed in cytoplasmic matrix near the rough endoplasmic reticulum and was occasionally noted as a small granular structure (GSH-PO-positive granule) at the supranuclear region. In a castrated animal, the intensity of GSH-PO staining in the glandular epithelial cells was remarkably decreased. By testosterone administration to the castrated animal, GSH-PO was clearly detected in the glandular epithelial cells. Intracellular localization of GSH-PO was mainly observed in cytoplasmic matrix and the number of GSH-PO-positive granules increased remarkably. These findings suggest that immunostainable GSH-PO in the glandular epithelial cells of the rat ventral prostate is testosterone-dependent, and that its staining pattern is a useful marker for biological testosterone action.

Regulation of prostatic glutathione-peroxidase (GSH-PO) in rats treated with a combination of testosterone and 17 beta-estradiol.

In order to confirm the relationship between sex hormone administration and glutathione-peroxidase (GSH-PO) in the rat ventral prostate, the levels of GSH-PO mRNA, GSH-PO activity, and lipid peroxide (TBA) value in the ventral prostate were investigated. Male Crj:CD(SD)IGS rats were divided into six experimental groups. Group 1 consisted of intact controls. In group 2, rats were sacrificed two days after castration. In groups 3 and 4, rats were subcutaneously administered 1 mg/animal of testosterone daily for three or seven days after two days of castration, respectively. In groups 5 and 6, rats were subcutaneously administered 1 mg/animal of testosterone plus 0.01 mg/animal of 17 beta-estradiol (E2) daily for three or seven days after two days of castration, respectively. GSH-PO activity of the ventral prostate homogenate for testosterone or testosterone plus E2 administration to the castrated rat was increased and the TBA value was remarkably decreased. The prostatic GSH-PO mRNA level was diminished in the castrated rat ventral prostate, but was increased by testosterone or testosterone plus E2 administration. In particular, the GSH-PO mRNA level of testosterone plus E2-treated animals was higher than that of testosterone-treated animals. These findings strongly suggest that expression of GSH-PO in the rat ventral prostate is considered to be testosterone- or E2-dependent. Furthermore, it is suggested that the transcription of prostatic GSH-PO mRNA was regulated by testosterone or E2 and de novo synthesis of GSH-PO would thus be regulated at transcription level by testosterone or E2.

The effects of chlormadinone acetate (CMA), antiandrogen, on the pituitary, testis, prostate and adrenal gland of the dog with spontaneous benign prostatic hyperplasia.

The effect of chlormadinone acetate (CMA), a synthetic steroidal antiandrogen, on spontaneous benign prostatic hyperplasia (BPH) in dogs was investigated. Male beagle dogs (5-8 years old) were divided into four experimental group. Group 1 consisted of untreated controls. Groups 2 and 3 received CMA 0.03 and 0.1 mg/kg/day, p.o., respectively, for 6 months. In group 1, glandular hyperplasia of the prostate was clearly detected. The glandular epithelial cells showed uniformly intense nuclear staining for androgen receptor (AR). AR was also localized in the nuclei of the fibro-muscular stromal cells. In groups 2 and 3, CMA produced marked atrophy of the glandular epithelium. The interacinar fibro-muscular stroma was prominent. The nuclear staining for AR in both epithelial and stromal cells was remarkably decreased. In addition, a histopathological study showed that CMA medication for 6 months exerted no effect on the testes and adrenal glands or on immunoreactive positive cells to LH- and ACTH-antibody (pituitary LH- and ACTH-cells). Therefore, it is concluded that CMA (0.03 and 0.1 mg/kg) causes regression of spontaneous canine BPH without any histopathological effects on the testes, adrenal glands or pituitary LH- and ACTH-cells.

Application of immuno-electron microscopy to the cytologic study of benign and malignant mammary lesions. With special reference to the carcinoembryonic antigen localization patterns.

We investigated the application of immuno-electron microscopic (EM) observation of carcinoembryonic antigen (CEA), one of the surface markers of neoplastic epithelium, to the cytologic diagnosis of mammary cells in smears and imprints. Smears were stained by the indirect peroxidase-labeled antibody method after a modified Karnovsky fixation (4% paraformaldehyde containing 0.05% glutaraldehyde). In smears, CEA was immunocytochemically demonstrated on the entire cell membrane of malignant mammary cells whereas CEA was observed only on microvillous surfaces of benign mammary cells. Another characteristic immunocytochemical feature of malignant cells was the appearance of so-called intracellular lumina, of which the microvillous surfaces were CEA positive. These findings demonstrated that electron microscopic observation of the immunocytochemical localization of CEA is a quite useful tool for the cytologic diagnosis and characterization of neoplastic mammary lesions.

The effect of natural carotenoid (palm fruit carotene) intake on skin lipid peroxidation in hairless mice.

To study the effect of palm fruit carotene intake on skin lipid peroxidation, hairless mice were given ad libitum palm fruit carotene, beta-carotene, or vehicle emulsions for 15 weeks in which the carotene (0.005%, w/w) was suspended in drinking water, and then their dorsal skin was exposed to ultraviolet ray (UV). The carotene content of the skin was increased by the oral intake of palm fruit carotene or beta-carotene. In carotene-drinking mice, before the UV irradiation, the amount of thiobarbituric acid-reacting substances (TBARS) in the skin was lower than that of control (carotene untreated) mice. The skin TBARS immediately after the UV irradiation was lower in carotene-treated mice than in control mice. At 24 h after irradiation, the skin TBARS of mice that orally received palm fruit carotene was lower than that of beta-carotene mice. Immediately after the UV irradiation, the skin carotene content transiently decreased but gradual recovery was observed at 48 h. In palm fruit carotene-treated mice, the rate of carotene recovery after UV irradiation was higher than in beta-carotene-treated mice. Retinol found in the skin had also decreased after UV irradiation, and recovered gradually in both carotene-drinking groups within 48 h. These results suggested that the carotene intake, especially palm fruit carotene, prevented skin lipid peroxidation in hairless mice.

The antioxidant effect of palm fruit carotene on skin lipid peroxidation in guinea pigs as estimated by chemiluminescence-HPLC method.

To study the antioxidant effect of palm fruit carotene on skin lipid peroxidation, the guinea pigs were orally fed ad libitum palm fruit carotene, beta-carotene, or vehicle emulsions, in which carotene (0.05%, w/w) was suspended in drinking water. After treatment of carotene for 12 weeks, animals were exposed to ultraviolet ray (UV), and squalene monohydroperoxide (SqOOH)/squalene (Sq) ratios in the skin lipid were analyzed using the chemiluminescence-HPLC method. Carotene accumulation was found in the skin of guinea pigs that were orally administered palm fruit carotene or beta-carotene. After UV irradiation, especially immediately after, the rise in the SqOOH/Sq ratio was effectively suppressed in both carotene-drinking groups in contrast with the control (carotene-untreated) group. An inverse correlation between the carotene content and the SqOOH/Sq ratio in the skin was also observed. The results suggested that palm fruit carotene intake prevents skin lipid peroxidation caused by UV irradiation.