Altered expression of CYP in TSOD mice: a model of type 2 diabetes and obesity.

To investigate the pharmacokinetic characteristics in TSOD (Tsumura, Suzuki, obese, diabetes) mice, a model of type 2 diabetes and obesity, the expressions of major hepatic CYP enzymes in TSOD and TSNO (Tsumura, Suzuki, non-obesity; control) mice were compared. The 7-month-old TSOD mice, which represented severe obesity/diabetes-related pathophysiology, showed higher expressions of Cyp2c and Cyp3a compared with TSNO mice, while those of Cyp1a and Cyp2e were lower. Cyp3a metabolic activity was also higher in TSOD mice. In the 7-month-old liver, pregnane X receptor (PXR) (nuclear receptor) and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) (cofactor) mRNA expression were higher in TSOD mice, possibly playing a role in the altered expression of Cyp3a. This specifically altered CYP expression in TSOD mice suggests that the biotransformation of drugs metabolized by these CYP enzymes differs from that in normal animals. Based on these findings, further investigation on the relationship between altered CYP expression and pathophysiology may be useful in elucidating changes in pharmacokinetics in obese/diabetic patients.

Chronic stress attenuates glucocorticoid negative feedback: involvement of the prefrontal cortex and hippocampus.

Disruption of the glucocorticoid negative feedback system is observed in approximate one half of human depressives, and a similar condition is induced in animals by chronic stress. This disruption is thought to involve down-regulation of glucocorticoid receptors (GRs) in the feedback sites of the brain. However, the responsible site of the brain has not been well elucidated. Here we examined the effects of chronic stress induced by water immersion and restraint (2 h/day) for 4 weeks followed by recovery for 10 days on the GR levels in the prefrontal cortex (PFC), hippocampus, and hypothalamus of rats using a Western immunoblot technique. In the PFC, the cytosolic GR levels were decreased, but the nuclear GR levels were not changed. In the hippocampus, the levels of cytosolic and nuclear GRs were increased. However, there were no marked changes in the GR levels in the hypothalamus. The changes in the cytosolic GR levels were confirmed at the mRNA level by an in situ hybridization technique. We next examined the suppressive effects of dexamethasone (DEX) infusions into these regions on the circulating corticosterone levels. When DEX was infused into the PFC or hippocampus of the chronically stressed rats, the suppressive response to DEX was abolished, but the response was normal in the hypothalamus. In addition, when DEX was injected systemically to the chronically stressed rats, the suppressive response to DEX was significantly attenuated. These results suggest that the abnormal changes in GRs in the higher centers of the hypothalamo-pituitary-adrenal axis are involved in the chronic stress-induced attenuation of the feedback. Since dysfunction of the PFC or hippocampus is implicated in the pathogenesis of depression, the present findings would help to understand the mechanisms underlying the disrupted feedback system and its relation to brain dysfunction in depression.

Gomisin A, a lignan component of Schizandora fruits, inhibits development of preneoplastic lesions in rat liver by 3'-methyl-4-dimethylamino-azobenzene.

The effects of gomisin A, a lignan component of Schizandra fruits, on development of preneoplastic lesions in the liver after a short-term (3 weeks) feeding of 3'-methyl-4-dimethyl-aminoazobenzene (3'-MeDAB) to male Donryu rats were investigated, and compared with the effects of phenobarbital. Gomisin A inhibited both increases of the level of glutathione-S-transferase placental form (GST-P) and the number and size of GST-P positive foci in the liver increased after treatment with 3'-MeDAB. Moreover, although the population of diploid nuclei was increased and that of tetraploid nuclei was decreased by pretreatment with 3'-MeDAB, gomisin A returned this to near the normal ploidy pattern. But phenobarbital increased the level of GST-P and the number and size of GST-P positive foci with little affect on the ploidy population changed by 3'-MeDAB. Thus, the effect of gomisin A on hepatocarcinogenesis was inhibitory in contrast with that of phenobarbital. This study suggests that gomisin A is a candidate for a chemopreventive drug inhibiting the promotion process in hepatocarcinogenesis.

Effects of the Japanese herbal medicine Keishi-bukuryo-gan and 17beta-estradiol on calcitonin gene-related peptide-induced elevation of skin temperature in ovariectomized rats.

The effects of a Japanese herbal medicine, Keishi-bukuryo-gan, and 17beta-estradiol on calcitonin gene-related peptide (CGRP)-induced elevation of skin temperature were investigated in ovariectomized (OVX) rats. Ovariectomy not only potentiated CGRP-induced elevation of skin temperature and arterial vasorelaxation but also induced a lower concentration of endogenous CGRP in plasma and up-regulation of arterial CGRP receptors, suggesting that lowered CGRP in plasma due to ovarian hormone deficiency increases the number of CGRP receptors and consequently amplifies the stimulatory effects of CGRP to elevate skin temperature. Oral Keishi-bukuryo-gan (100-1000 mg/kg, once a day for 7 days) restored a series of CGRP-related responses observed in OVX rats by normalizing plasma CGRP levels in a dose-dependent manner as effectively as s.c. injection. 17Beta-estradiol (0.010 mg/kg, once a day for 7 days). However, Keishi-bukuryo-gan did not affect the lower concentration of plasma estradiol and the decreased uterine weight due to ovariectomy, although the hormone replacement of 17beta-estradiol restored them. These results suggest that Keishi-bukuryo-gan, which does not confer estrogen activity on plasma, may be useful for the treatment of hot flashes in patients for whom estrogen replacement therapy is contraindicated, as well as menopausal women.

Characteristics of primary cultured neurons from embryonic mutant El mouse cerebral cortex.

To elucidate the differences between neurons of epileptogenic animals and those of normal animals, cellular characteristics of neurons of mutant strain El mice which are highly susceptible to seizures were investigated using immunocytochemical techniques. In neurons of 3-day primary cultures, the control ddY mouse neurons showed dividing stages in about 0.2% of neurofilament (NF)-positive neurons, whereas no dividing neurons were observed among the NF-positive El mouse neurons. In 7-day cultures, localization of GD3 ganglioside in the proliferating control ddY mouse neurons was observed, but there was no GD3 ganglioside in the mutant El mouse neuron. The content of GD3 ganglioside detected by high-performance thin-layer chromatography of El mouse cultured cells was ca. 1/4 of that of ddy mice. These findings suggest that neurons of the El mouse are differentiated earlier than those of the control ddY mouse.

Effects of cycloheximide on delayed neuronal death in rat hippocampus.

The effect of cycloheximide, a protein synthesis inhibitor, on hippocampal selective neuronal death was morphologically studied in rats subjected to 10 min forebrain ischemia using a 4-vessel occlusion model. Neuronal damage in the hippocampal CA1 subfield 72 h after ischemic insult was dramatically decreased by the lasting inhibition of protein synthesis through consecutive administration of cycloheximide. Cycloheximide, which was administered once within the first 24 h of recirculation, showed protective action on ischemic cell necrosis and its most potent effect was observed when injected at 12 h of post-ischemia. After 36 h of recirculation, however, treatment with cycloheximide could no longer prevent cell death. The possibility is considered that hippocampal delayed neuronal death following transient ischemia is caused by abnormal protein(s).

Inhibitory effect of TJ-960 (SK) on pentylenetetrazol-induced EEG power spectrum changes.

Effects of the Japanese kampo medicine 'Shosaiko-to-go-keishika-shyakuyaku-to' (TJ-960), which is a mixture of 9 herbal drugs and is practically equivalent to 'Saiko-keishi-to' (SK), on the pentylenetetrazol (PTZ)-induced EEG power spectrum changes were examined. The EEG power spectrum change with 2 PTZ administrations at an 80 min interval was clearly inhibited by oral administration of 1.0 mg/kg of TJ-960. By separate single administrations of the main component herbal drugs, Bupleuri radix, Cinnamomi cortex, Paeoniae radix and Zingiberis rhizoma, only Paeoniae radix showed statistically significant inhibition of PTZ-induced EEG power spectrum changes at a proportional dose of 1.0 mg/kg of TJ-960. The other main component herbal drugs showed no statistically significant inhibitory effect although they had a tendency to inhibit. These findings suggest that the Japanese Kampo medicine, TJ-960 (SK), has an inhibitory effect on PTZ-induced power spectrum changes and one of the component herbal drugs, Paeoniae radix, is the important component drug.

Effect of anticonvulsants on pentylenetetrazol-induced power spectrum changes in electroencephalograms in rats.

The effect of anticonvulsants on pentylenetetrazol (PTZ)-induced EEG power spectrum changes was examined. When the minimum dose of PTZ (15 mg/kg) was administered intravenously twice, with an interval of 80 min, a clear EEG power spectrum change was observed after the second PTZ administration, irrespective of whether or not the first PTZ administration evoked marked EEG changes. We defined the values F/N, S/N and S/F. The value F/N, obtained by dividing the power spectrum area after the first PTZ administration by the normal power spectrum area, was 1.06 +/- 0.05 (mean +/- S.E.). The value S/N, obtained by dividing the power spectrum area after the second PTZ administration by the normal power spectrum area, was 2.00 +/- 0.21. The value S/F, obtained by dividing S/N by F/N, was 1.86 +/- 0.16. The S/F value was almost constant regardless of whether or not the first PTZ administration could evoke marked EEG changes. The effect of anticonvulsants was examined by S/F value changes, and 100 mg/kg of PHT completely inhibited the double PTZ effect. Phenobarbital, ethosuximide and sodium valproate also inhibited the double PTZ effect. Using the S/F value of the EEG power spectrum with minimum dose double PTZ administration, quantitative evaluation is possible for anticonvulsant drugs.

Deoxycholic acid as an endogenous risk factor for hepatocarcinogenesis and effects of gomisin A, a lignan component of Schizandra fruits.

Although hepatocarcinogensis has been reported to be promoted by exogenous administration of bile acids, the relation of endogenous bile acids to hepatocarcinogenesis is not completely understood. This study investigates the relationship between serum concentration of bile acids, the appearance of preneoplastic change, glutathione S-transferase placental form (GST-P)-positive foci in the liver of male Donryu rats which had been fed 0.06% 3'-methyl-4-dimethylamino-azobenzene (3'-MeDAB), and the effects of gomisin A, previously reported to inhibit the tumor promotion process. During the feeding of 3'-MeDAB for 5 weeks, the concentrations of serum bile acids were found to have increased significantly to several times the levels found at the start of the experiment. The increase of serum bile acids, especially deoxycholic acid (DCA), and the appearance of preneoplastic lesions, the number and area of GST-P-positive foci in the liver, were significantly inhibited by simultaneous oral administration of gomisin A (30 mg/kg). When DCA (100 mg/kg) was orally administered after an initiation by 3'-MeDAB, serum bile acids and preneoplastic changes were significantly increased, these increases were inhibited by combined feeding of 0.03% gomisin A in the diet. There were good correlations between the serum concentration of DCA and the number of GST-P-positive foci in the liver in both experimental protocols. These results confirm that DCA is an endogenous risk factor for hepatocarcinogenesis and suggest that anti-promoter effect of gomisin A is based on improving metabolism of bile acids, including DCA.

Occurrence of autoimmune antibodies to liver microsomal proteins associated with lethal hepatitis in LEC rats: effects of TJN-101 ((+)-(6S,7S,R-biar)- 5,6,7,8-tetrahydro-1,2,3,12-tetramethoxy-6,7-dimethyl-10,11- methylenedioxy-6-dibenzo[a,c]cyclooctenol) on the development of hepatitis and the autoantibodies.

Long Evans Cinnamon (LEC) rats, that spontaneously develop hepatitis, were found to possess autoantibodies to liver microsomal proteins (anti-LM) before the development of hepatitis. Anti-LM antibody was assumed to appear in association with the lethal hepatitis in the LEC rats. Thus, the purpose of this study was to investigate the effects of an anti-hepatitis drug on the development of hepatitis and the occurrence of the antibody in LEC rats. Mortality, blood biochemical parameters and the titer of serum anti-LM antibody were measured. In control LEC rats, 4 of 8 rats died before 20 weeks of age. In rats treated with TJN-101 ((+)-(6S,7S,R-biar)-5,6,7,8-tetrahydro-1,2,3,12-tetramethoxy -6,7-dimethyl-10,11 - methylenedioxy-6-dibenzo[a,c]cyclooctenol), 4 of 7 rats died of hepatitis, but the time of death was delayed by 7-10 weeks compared to the control rats. The titer of the anti-LM antibody increased 3-7 weeks before death in the non-survivors in control and TJN-101-treated rats, supporting the idea that anti-LM antibody occurs in association with acute lethal hepatitis.

Effects of medicinal plants on hemopoietic cells.

It has been found that Juzentaihoto (TJ-48), one of the traditional Japanese kampo (herbal) medicines, improves the general condition of cancer patients receiving chemotherapy and radiation therapy. We analyze how TJ-48 elicits such effect, and show that oral administration of TJ-48 accelerates recovery from hemopoietic injury induced by radiation and the anti-cancer drug mitomycin C. The effects are found to be due to its stimulation of spleen colony-forming units. Based on the present findings, we propose that the administration of TJ-48 should be of benefit to patients receiving chemotherapy, radiation therapy or bone marrow transplantation.

Regulation of hepatic macrophage function by oral administration of xiao-chai-hu-tang (sho-saiko-to, TJ-9) in rats.

The effect of Xiao-Chai-Hu-Tang (Sho-saiko-to, TJ-9), the extract of a mixture of 7 herbs, on hepatic macrophage function was studied using rats. Hepatic macrophages were activated by injection of Corynebacterium parvum or 70% partial hepatectomy. Oral administration of TJ-9 for 3 weeks did not affect the ability of these macrophages to produce superoxide anions evaluated in situ by liver perfusion with nitro blue tetrazolium (NBT) and phorbol myristate acetate (PMA). However, the similar administration of TJ-9 attenuated the blocking of the activation after partial hepatectomy produced by pretreatment with gum arabic, a polysaccharide of high molecular weight. When gum arabic was added to the medium of rat hepatic macrophages cultured with normal rat sera, their ability to produce superoxide anions was reduced in a dose-related manner. This reduction was attenuated by changing the sera to the sera obtained from rats given oral doses of TJ-9 for 3 weeks. These results suggest that TJ-9 may improve the blocked function of hepatic macrophages in activation.

Protective effects of TJ-960 herbal mixture on hippocampal neuron damage induced by cobalt focus in the cerebral cortex of rats.

In the cobalt focus experimental epilepsy model, severe hippocampal neuron damage occurs with marked EEG changes. The effects of TJ-960, a herbal medicine formulation, were studied on neuron damage in the CA1 area of rat hippocampus. Continuous oral administration of TJ-960 from one month prior to the cobalt application showed almost complete protection against hippocampal neuron damage induced by cobalt application to the cerebral cortex. TJ-960 also completely inhibited the EEG changes as well as the brain edema induced by cobalt application.

Automatic monitoring system for the measurement of body weight, food and water consumption and spontaneous activity of a mouse.

The simultaneous recording system for body weight, food and water consumption and behavior (spontaneous motor activity and drinking and feeding behavior) of a mouse was developed. The body weight and food consumption were measured by force transducers. Food and water consumption and drinking and feeding behavior were measured by an infrared luminous diode and a phototransistor. Spontaneous motor activity was measured by photosensors. The system control and data acquisition were performed by using a personal computer. Every parameter could be monitored with a desired time interval. All the data collected by this system revealed apparent circadian rhythm. In conclusion, this system would be a powerful tool for pharmacological and/or toxicological research.

[Determination of gomisin A (TJN-101) and its metabolite in rat serum by gas chromatography-mass spectrometry].

Gomisin A (TJN-101) is one of the lignan components isolated from Schisandra Fruits. A high sensitive and precise method for the determination of TJN-101 and its major metabolite (Met. B) in the rat serum was developed by selected ion monitoring (SIM) with gas chromatography-mass spectrometry (GC/MS) using a fused silica capillary column (SPB-1, Supelco). A 100 microliter serum sample was used for the solid phase extraction. The calibration curves of TJN-101 and Met.B both showed a good linearity between 2.0 and 2000.0 ng/ml. The analytical precision (intra-assay, C.V. less than 4.7%), recoveries (98.4 +/- 10.1%), and detection limit (2 ng/ml) of TJN-101 indicated that this system was suited for the determination of TJN-101 in biological fluid. In case of Met.B, the same results as TJN-101, were obtained. After oral administration of TJN-101 at a dose of 10 mg/kg to male rats, the average values of the maximal serum concentration of TJN-101 and Met.B were 1446.1 +/- 131.8 and 317.4 +/- 18.5 ng/ml, respectively. The serum concentrations of these substances could be monitored sufficiently for 8 h after dosing.

[Studies on the metabolic fate of gomisin A (TJN-101). I. Absorption in rats].

Gomisin A (TJN-101) is one of the lignan components isolated from Schisandra Fruits and expected to have some efficacies in clinical treatment of hepatitis. The serum concentrations of TJN-101 and Met. B, which was identified as a demethylenated substance and one of the major metabolites of TJN-101 in rats, were investigated. After intravenous administration at doses of 1.6, 4.0 and 10 mg/kg of body weight, the serum concentration of TJN-101 decreased biphasically, and the terminal elimination half-life at each dose was about 70 min. Dose-dependency was observed for the area under the concentration-time curve (AUC). On the other hand, the serum concentration of TJN-101 increased rapidly and reached maximum within 15 to 30 min when administered orally. This result was supported by the in situ roop method. The Cmax and the AUC values were not exactly dose-dependent, but the values increased with a dose-up of TJN-101. The biotransformation of TJN-101 to Met. B, was very rapid in both intravenous and oral administrations. The AUC value of Met. B after oral administration of TJN-101 at a dose of 1.6 mg/kg was relatively larger than any other dosages. It suggested that TJN-101 was extensively underwent the first pass effect in rats. More than 80% of TJN-101 was bound with rat serum protein in vitro and in vivo. Therefore, it seems to be necessary to pay attention when it was administered concurrently with high protein binding drugs.

[Studies on the metabolic fate of gomisin A (TJN-101). II. Absorption and excretion in CCl4 treated rats].

The absorption and excretion of gomisin A (TJN-101) in rats whose livers were injured by carbon tetrachloride (CCl4) were investigated. After intravenous administration of TJN-101 at a dose of 5 mg/kg, the terminal elimination half-life was 1.5 h in the CCl4-treated rats, which was two times that in normal rats. The mean area under the blood concentration-time curve (AUC) value of TJN-101 in the CCl4-treated rats was twice that in normal rats, and this difference was significant (p less than 0.05). Therefore, the total body clearance of TJN-101 in the CCl4-treated rats decreased less than half of that in normal rats. Similar results were observed when it was administered orally. In the CCl4-treated rats, the serum concentration of Met. B, which was identified as a demethylenated substance and one of major metabolites, tended to decrease more than that in normal rats. On the other hand, the cumulative biliary excretion ratio of TJN-101 in 24 h after dosing in the CCl4-treated rats was 2.5 times that in normal rats. The excretion rate of Met. B in the bile in the CCl4-treated rats tended to be delayed. However, the quantitative variance of biliary excretion of Met. B was not found in both groups. The urinary excretion of TJN-101 or Met. B in 72 h after dosing in the CCl4-treated rats was lower than that in normal rats. Similar results were also observed in excretion in the feces.(ABSTRACT TRUNCATED AT 250 WORDS)