Serum CETP status is independently associated with reduction rates in LDL-C in pitavastatin-treated diabetic patients and possible involvement of LXR in its association.

BACKGROUND: Statins decrease cholesteryl ester transfer protein (CETP) levels, which have been positively associated with hepatic lipid content as well as serum low density lipoproteins-cholesterol (LDL-C) levels. However, the relationship between the CETP status and statin-induced reductions in LDL-C levels has not yet been elucidated in detail. We herein examined the influence of the CETP status on the lipid-reducing effects of pitavastatin in hypercholesterolemic patients with type 2 diabetes mellitus as well as the molecular mechanism underlying pitavastatin-induced modifications in CETP levels. METHODS: Fifty-three patients were treated with 2 mg of pitavastatin for 3 months. Serum levels of LDL-C, small dense (sd) LDL-C, and CETP were measured before and after the pitavastatin treatment. The effects of pitavastatin, T0901317, a specific agonist for liver X receptor (LXR) that reflects hepatic cholesterol contents, and LXR silencing on CETP mRNA expression in HepG2 cells were also examined by a real-time PCR assay. RESULTS: The pitavastatin treatment decreased LDL-C, sdLDL-C, and CETP levels by 39, 42, and 23%, respectively. Despite the absence of a significant association between CETP and LDL-C levels at baseline, baseline CETP levels and its percentage change were an independent positive determinant for the changes observed in LDL-C and sdLDL-C levels. The LXR activation with T0901317 (0.5 µM), an in vitro condition analogous to hepatic cholesterol accumulation, increased CETP mRNA levels in HepG2 cells by approximately 220%, while LXR silencing markedly diminished the increased expression of CETP. Pitavastatin (5 µM) decreased basal CETP mRNA levels by 21%, and this was completely reversed by T0901317. CONCLUSION: Baseline CETP levels may predict the lipid-reducing effects of pitavastatin. Pitavastatin-induced CETP reductions may be partially attributed to decreased LXR activity, predictable by the ensuing decline in hepatic cholesterol synthesis. TRIAL REGISTRATION: UMIN Clinical Trials Registry ID UMIN000019020.

Ibadronate may prevent colorectal carcinogenesis in mice with ulcerative colitis.

An increased incidence of colorectal carcinoma is known to occur in patients with ulcerative colitis, which displays a cycle of recurrence-remission in the colorectal mucosa. Repeated oral doses of 3% dextran sulfate sodium subsequent to a single intraperitoneal injection of azoxymethane induces chronic ulcerative colitis, resulting in an increased incidence of high-grade dysplasia and submucosal-invasive adenocarcinomas in the mouse colorectum. The features of the colitis induced in this animal model are very similar to the ulcerative colitis in patients in terms of both clinical and histopathological characteristics. Bisphosphonates are known to increase bone mass by suppressing bone turnover in postmenopausal women. A novel single-nitrogen bisphosphonate, ibandronate, is effective in preventing skeletal events in patients with bone metastases from colorectal cancer. Decreasing the bone mineral affinity of bisphosphonates is an effective therapeutic strategy to inhibit skeletal tumor growth in vivo. In the present study, the preventative effects of the bisphosphonate ibadronate on colorectal carcinogenesis in mice treated with azoxymethane and dextran sulfate sodium was investigated. Additive treatment with bisphosphonate prevented the shrinkage of colorectum which was affected by a cycle of recurrence-remission in colorectal mucosa, resulting in a reduced incidence of colorectal dysplasia and a reduced expression of thymidine kinase mRNA in the colorectum. Taken together, the present results indicate that ibadronate may inhibit colorectal carcinogenesis and its development by inhibiting colorectal epithelial cell proliferation and the neoplastic process.

Ethyl-eicosapentaenoic acid reduces liver lipids and lowers plasma levels of lipids in mice fed a high-fat diet.

Fatty liver disease is characterized by a wide spectrum of liver damage, i.e. simple steatosis may progress to advanced fibrosis and to cryptogenic cirrhosis via steatohepatitis, and ultimately to hepatocellular carcinoma. Eicosapentaenoic acid (EPA), a marine-derived n-3 fatty acid like docosahexaenoic acid (DHA), is an anti-thrombotic and hypolipidemic agent, and is an antagonist of platelet aggregation and an inhibitor of cholesterol and lipoprotein. In an attempt to confirm the hypolipidemic action of this agent, the effects of EPA on liver and plasma levels of lipids in mice fed a high-fat diet were investigated. EPA markedly reduced the fatty droplets in the liver cells and the liver weight, also lowering plasma levels of total cholesterol, free total cholesterol, phospholipids and triglyceride. It is suggested that n-3 fatty acid intake and fish consumption may be able to prevent the occurrence not only of metabolic syndrome, but also of fatty liver and non-alcohlic steatohepatitis.

Down regulation by a low-zinc diet in gene expression of rat prostatic thymidylate synthase and thymidine kinase.

BACKGROUND: Zinc has a wide spectrum of biological activities and its deficiency is related to various abnormalities of cell metabolism. METHODS: Wistar male rats, at age of 4 weeks, were fed a low-zinc diet for six weeks. The levels of bromodeoxyuridine incorporated into the prostatic DNA and the mRNA expression levels of prostate thymidylate synthase and thymidine kinase were examined. RESULT: The low-zinc diet caused a marked reduction in the body growth and organ weights, resulted in a low hematopoiesis, hypo-albuminemia and hypocholesterolemia. Although there were few differences in plasma biochemical markers, plasma levels of luteinizing hormone and testosterone were reduced by the low-zinc diet. Bromodeoxyuridine-immunoreactive (S-phase) cells and mRNA expression levels of thymidylate synthase and thymidine kinase in the prostate cells were markedly affected by the low-zinc diet. CONCLUSION: A low-zinc diet appears to reduce the body growth and organ weights including prostate, causing low plasma levels of luteinizing hormone and testosterone and reduction in prostate DNA replication in growing-rats.

Chemopreventive effect of a vitamin D(3) analog, alfacalcidol, on colorectal carcinogenesis in mice with ulcerative colitis.

An increased incidence of colorectal carcinoma is known to occur in patients with ulcerative colitis (UC), which displays a cycle of recurrence-remission in the colorectal mucosa. Repeated oral doses of 3% dextran sulfate sodium subsequent to a single intraperitoneal injection of azoxymethane induced a chronic UC resulting in an increased incidence of high-grade dysplasia and submucosal-invasive adenocarcinomas in the mouse colorectum. The active form of vitamin D(3) is a calcium-regulating hormone that increases serum calcium levels and intestinal calcium absorption. It has been reported that there is an inverse correlation between serum levels of the active metabolite of vitamin D and colorectal carcinoma stage. The features of the colitis induced in this animal model are very similar to the UC in patients in terms of both clinical and histological characteristics. Treatment with a vitamin D(3) analog, alfacalcidol, in mice prevented colitis and carcinogenesis; this is shown by inhibition of the decrease in colorectal length and inhibition of the increased incidence of colorectal dysplasia, with a reduction in the mRNA expression of the DNA-synthesizing enzyme, thymidine kinase, in colorectal tissues.

Effects of three Chinese herbal medicines on plasma and liver lipids in mice fed a high-fat diet.

Chinese herbal medicines, Inchinko-to, Bofu-tsusho-san and Dai-saiko-to, containing 3, 18 and 8 components, respectively, have since long been used as an anti-inflammatory, antipyretic, choleretic and diuretic agent for liver disorders and jaundice, as an anti-obesity agent, a hypocholesterolemic agent for liver disorders and a therapeutic and/or preventive agent for cholesterol gallstone disease with hypertriglycerid-emia in China and Japan, respectively. In the present study, we investigated the effects of these three herbal medicines in young male mice fed a high-fat diet. Plasma levels of lipids and the numbers of the fatty droplets in the liver cytoplasm were markedly lowered by the diets supplemented with three herbal medicines. The liver weights and the body growth were reduced by the diet supplemented with Dai-saiko-to, which slightly affected the concentrations of total protein, albumin, creatinine or calcium, and the activity of lactate dehydrogenase. Thus, Dai-saiko-to, besides Bofu-tsusho-san, seems effective in the activities of anti-obesity, anti-hyperlipidemia and anti-hyperlipids in liver cytoplasm, when used carefully.

Preventive effect of fluvastatin on ulcerative colitis-associated carcinogenesis in mice.

An increased incidence of colorectal carcinoma is known to occur in patients with ulcerative colitis (UC), which displays a cycle of recurrence-remission in the colorectal mucosa. Fluvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, is a hypocholesterolemic agent effective in animals and humans. Repeated administration of 3% dextran sulfate sodium subsequent to a single intraperitoneal injection of azoxymethane induces chronic UC resulting in an increased incidence of high-grade dysplasia and submucosal-invasive adenocarcinomas in the mouse colorectum. The effects of fluvastatin as an antioxidant on colorectal carcinogenesis in mice with UC were investigated. Treatment with fluvastatin in mice with UC abolished the anemia caused by colorectal carcinogenesis, and markedly lowered plasma lipid levels resulting in a reduction of colitis and carcinogenesis, shown by inhibition of the decrease in colorectal length, the increased number of foci of gland loss with inflammatory cell infiltration indicating the severity of UC and incidence of colorectal dysplasia, respectively, with a reduction in anti-8-hydroxy-2'-deoxyguanosine (8-OHdG) antibody (a biological marker of in vivo oxidative DNA damage)-positive cells of the colorectal mucosa and the activity of the DNA-synthesizing enzyme thymidine kinase in colorectal tissues.

Effects of colestimide and/or Bofu-tsusho-san on plasma and liver lipids in mice fed a high-fat diet.

Hypercholesterolemia is known to enhance the risk of coronary heart disease and fatty liver. Colestimide is an anion-exchange resin, which is not absorbed in the small intestine, decreases the intestinal reabsorption of bile acids synthesized from cholesterol in the liver and consequently increases bile acid excretion into the feces. Bofu-tsusho-san, a traditional Japanese herbal remedy, contains 18 components and has long been used as an anti-obesity agent. In the present study, we investigated the effects of colestimide and/or Bohu-tsusho-san in young male mice fed a high-fat diet. The high-fat diet supplemented with both colestimide and Bofu-tsusho-san markedly reduced the plasma levels of lipids, the liver weight and number of fatty droplets in the liver cytoplasm, and the body growth, compared with animals fed a high-fat diet alone. Neither medicine affected the blood biochemistry. Thus, the hypocholesterolemic action of colestimide, sometimes bringing light flatulence, which is improved by simultaneous administration of Bofu-tsusho-san, which activates the thermogenesis of brown adipose tissue, is suggested to reduce body mass and liver lipids, lowering the plasma levels of lipids.

Increased plasma levels of zinc in obese adult females on a weight-loss program based on a hypocaloric balanced diet.

Zinc is required for many biological functions including DNA synthesis, cell division, gene expression and the activity of various enzymes in humans and animals. Zinc concentrations in the plasma and erythrocytes are lower and urinary zinc excretion and serum insulin levels are higher in subjects with obesity. The effects of a weight-loss program based on a hypocaloric balanced diet were investigated on 23 obese females, who had a body mass index of more than 25.0 and had dieted for 6 months at the Nutrition Clinic, Institute of Nutrition Sciences, Kagawa Nutrition University, Tokyo, Japan. The subjects ranged in age from 29 to 76 (54.3 +/- 13.0) years old. The hypocaloric balanced diet significantly reduced the body weight, body mass index, body fat percentage and amount of body fat with a slight lowering of blood pressure and plasma levels of triglyceride. Interestingly, the plasma concentrations of zinc were markedly enhanced at the end of the program.

Paradoxical effect of cytosine arabinoside on mouse leukemia cell line L1210 cells.

We investigated the effects of 1-beta-D-arabinofuranosylcytosine (ara-C) on the growth of murine leukemic L1210 cells, which were cultured with high (2.0 x 10(3) ng/ml), middle (100 ng/ml) and low doses (5.0 ng/ml) of ara-C. In the analysis by flow cytometry, high dose ara-C arrested the cell cycle in the G0/G1-phase. Middle and low doses ara-C induced a block in the S-phase, that was not completely blocked by the low dose. Analysis of DNA fragmentation revealed that ara-C dose-dependently induced apoptosis, which was only slightly induced by the low dose. We measured activities of cellular thymidylate synthase (TS) and thymidine kinase (TK) after 24-h culture. Low and middle doses, but not high dose ara-C markedly enhanced TS activity to 2.9- in low and 5.3-fold in middle doses ara-C, and TK activity to 1.3- in low and 2.2-fold in middle doses, respectively, compared with those of the control. The cells accumulated in the S-phase by 48-h culture with low dose ara-C and markedly proliferated compared to that of the control in ara-C-free medium. These results indicate that non-high dose ara-C enhances DNA-synthesizing enzyme activities in L1210 cells, and withdrawal of the non-high dose ara-C results in paradoxical cell proliferation. Thus, daily intramuscular injections with an insufficient dose of ara-C may induce cells into S-phase, resulting in the proliferation of leukemic cells.

Probucol, a hypocholesterolemic agent, prevents the development of uterine adenomyosis induced by pituitary grafting in mice.

This in vivo experimental study was designed to investigate the effects of probucol, a hypocholesterolemic agent, on uterine adenomyosis which is frequently induced by pituitary grafting in mice. SHN mice, which are known to develop uterine adenomyosis spontaneously, and much sooner after pituitary grafting, were used and histopathological study on the uteri in pituitary gland-implanted mice with or without probucol treatment was performed. Four out of 10 pituitary gland-implanted mice developed uterine adenomyosis with dilated blood vessels, but none of the probucol-treated mice. There were no differences between pituitary-grafted mice with or without probucol treatment in body weight and wet weights of uterus, ovaries, kidney and liver except spleen. Probucol markedly reduced the serum levels of total cholesterol, free cholesterol, free fatty acids, phospholipids and triglycerides and, thus, this agent inhibited the incidence of uterine adenomyosis induced by pituitary grafting in mice.

Androgenic effects on adrenocortical responsiveness in neonatal rats.

We are increasingly exposed to environmental and occupational hazardous chemicals, which modulate hormonal activity and/or mutagenicity in mammals. In the present study, we investigated the effects of sex-steroid hormones on adrenocortical responsiveness to adrenocorticotropic hormone (ACTH) in neonatal rats. The levels of corticosterone increased with the dose of ACTH in adrenal cells of males and females in vitro. Although castration markedly augmented the responsiveness in male rats, testosterone-replacement in the castrated male rats inhibited the enhancement and, furthermore, the treatment with testosterone suppressed the responsiveness in 14-day-old intact female rats, too. Castration enhanced the level of ACTH receptor mRNA to 3-fold of that in intact male rats at 14 days of age, but replacement treatment with testosterone in castrated male rats lowered the elevated levels. These findings suggest that: 1) the hyporesponsiveness of adrenocorticosteroid in the stress hyporesponsive period of neonates might be dependent on the reduction of ACTH receptor mRNA, and 2) endocrine-disrupting chemicals, with characters of androgens, estrogens or gonadotropin-releasing hormones, might affect the responsiveness to ACTH and the ACTH receptor mRNA expression levels in adrenal cells of neonates.

Effects of angiogenesis inhibitor TNP-470 on the development of uterine adenomyosis in mice.

OBJECTIVE: To investigate the effects of angiogenesis inhibitor TNP-470 on uterine microvessels in mice. Pituitary grafting frequently induced uterine adenomyosis. DESIGN: In vivo experimental study. SETTING: Department of Biological Sciences, University of Tokyo and Medical Research Institute, Tokyo Medical and Dental University. ANIMAL(S): SHN mice, which are known to develop uterine adenomyosis spontaneously, and also very soon after pituitary grafting. INTERVENTION(S): Immunohistochemical study on uterine blood vessels using an antibody to von Willebrand factor in pituitary gland-implanted mice with or without TNP-470. MAIN OUTCOME MEASURE(S): Reduced incidence of uterine adenomyosis. RESULT(S): Twelve of 15 mice developed uterine adenomyosis with dilated blood vessels, but none of the TNP-470-treated mice with shrunken microvessels. The number of bromodeoxyuridine immunoreactive cells and activities of thymidylate synthase and thymidine kinase in uterine tissues were markedly reduced in TNP-470-treated mice. CONCLUSION(S): TNP-470, a potent inhibitor of the development of vascular endothelium, reduced the development of endometrial blood vessels resulting in a lowered incidence of uterine adenomyosis induced by pituitary grafting in mice, and reduced the increase in S-phase cells and enzyme activity for pyrimidine nucleotide synthesis.

Preventive effect of ferulic acid on bone loss in ovariectomized rats.

An extract from corn germ induced a positive response in the pigeon crop sack test, used for the detection of prolactin-like substances. One of the substances extracted was identified as ferulic acid, which was reported to affect serum gonadotropin levels in ovariectomized male rats. To evaluate the effects of ferulic acid on bone loss, ovariectomized female rats of the Sprague-Dawley strain at age 35 weeks were given ferulic acid and/or 17a-ethynylestradiol daily for 8 weeks, and serum hormone levels and tibial bone mineral density were measured. In metaphysis of the tibia, which was abundant in cancellous bone and more reflective of BMD than whole tibia, the BMD was markedly reduced by ovariectomy and enhanced by the treatment with estrogen or ferulic acid in the ovariectomized rats. The treatment slightly increased the serum levels of estrogen and progesterone and alkaline phosphatase activity, which was reduced by estrogentreatment, i.e. the mechanism of bone formation by ferulic acid was suggested to be different from that by estrogens. These results indicate that ferulic acid promotes bone remodeling, leading to a predominantly osteoblastic phase, besides bone resorption by osteoclasts.

Sex-differences in adrenocortical responsiveness during development in rats.

It is known that the stress hyporesponsive period (SHRP), which seems to be related to an immature hypothalamo-pituitary-adrenal (HPA) regulatory system, occurs during the first 2 weeks after birth in rats. In the present study, we investigated the effects of sex-steroid hormones on adrenocortical responsiveness to adrenocorticotropic hormone (ACTH) in neonatal rats. The levels of cyclic adenosine 3',5'-monophosphate (cAMP), corticosterone, and adenylate cyclase activity increased with the dose of ACTH in adrenal cells of males and females in vitro. The ACTH responsiveness in adrenal cells increased with age (7-35 days of age), that is, the loss in responsiveness to ACTH just after birth began to recover in 14-35-day-old rats, but the responsiveness in 14-day-old rats was attenuated in males compared with females. Although castration markedly augmented the responsiveness in male rats, testosterone-replacement in the castrated male rats inhibited the enhancement. Furthermore, the responsiveness in 14-day-intact female rats was suppressed by treatment with testosterone. Expression levels of ACTH receptor mRNA in adrenals increased with age in the female rat, but not in the male. Castration enhanced the level of ACTH receptor mRNA to three-fold of that in intact male rats at 14 days of age, but replacement treatment with testosterone in castrated male rats lowered the elevated levels. Testicular androgens are thought to evoke a gender-specific response in neonates, and the temporal decrease of adrenal ACTH-responsiveness might be due to the topically immature adrenal system as well as the central nervous system in mammals.

Human colorectal malignancy and oral UFT.

Thymidylate synthase (EC 2.1.1.45) and thymidine kinase (EC 2.71.21) are key enzymes involved in de novo and salvage pathways for pyrimidine nucleotide synthesis. Both enzyme activities are increased in rapidly proliferating normal, fetal and neoplastic tissues. In a previous study, the activities of thymidylate synthase and thymidine kinase were relatively predominant in the poorly-and well-differentiated types of a gastric cancer. In the present study of patients with colorectal cancer, the serum thymidine kinase activities are elevated in cases at a clinically late stage, and in cases with a recurrence and a distant metastasis associated with abundant blood supply, i.e. metastasis to the liver, lung and bone. Well-differentiated colorectal cancer shows higher thymidine kinase activity than moderately-well-differentiated type as was previously shown in gastric cancer patients. Furthermore, neoadjuvant chemotherapy using the 5-fluorouracil derivative UFT demonstrates a stronger suppression of increased activities of thymidylate synthase in the tumorous tissues than in the non-tumorous mucosa.

Early progression from dimethyl sulfoxide-induced G(0)/G(1) arrest in L(1210) cells.

Recently, dimethyl sulfoxide (DMSO) has been used as a convenient cryoprotectant for stem cells in stem cell transplantation using allogenic peripheral blood or umbilical cord blood. As the stem cells have a multipotency, clarification of the extent of cell proliferation after transplantation is difficult. In the present study, DMSO gradually induced G(0)/G(1) arrest in mouse leukemia L(1210) cells with good cell viability. After removal of DMSO, the cells proliferated appropriately, resulting in expression of the DNA-synthesizing enzymes thymidylate synthase and thymidine kinase within 6h, and the cells entering into S phase within 12h. The sequence was followed by the marked activation of both enzymes within 24h and the increase of bromodeoxyuridine (BrdU) immunoreactive (S phase) cells with rapid cell proliferation within 36 h. In conclusion, mouse leukemia L(1210) cells, which were treated with 1.5% DMSO for 96 h, tolerated the treatment and reversed the cell cycle arrest within 36 h.

The more an ulcerative colitis is repeated, the more the risk of colorectal carcinogenesis is increased in mice.

The incidence of colorectal carcinogenesis is increased in patients with ulcerative colitis. We investigated the effects of repetitive diarrhea on colorectal carcinogenesis in mice singly pretreated by a low-dose of chemical carcinogen. Mucosal changes were investigated in mice pretreated with a single intraperitoneal injection of 8.0 mg/kg body weight of azoxymethane prior to a repetitive oral administration of 3% dextran sulphate sodium to induce chronic diarrhea. Repetitive treatment with dextran sulphate sodium induced a cycle of chronic diarrhea-remission in the colorectum. Five submucosal-invasive adenocarcinomas and 65 high-grade dysplasias were found in 15 mice that underwent azoxymethane-pretreatment with 3 cycles of 3% dextran sulphate sodium-exposure, i.e. colorectal carcinogenesis was observed mainly in the left side of the large intestine within 11 weeks after the initial treatment with carcinogen. Consequently, thymidine kinase was expressed and activated, and an increase in bromodeoxyuridine-immuno-reactive (S-phase) cells was observed in the regenerating mucosa and the colorectum with tumorous lesions. Colorectal carcinogenesis developed with the increasing duration of diarrhea induced by 3% dextran sulphate sodium in mice pretreated by a single injection of a small dosage of azoxymethane during the comparatively short period of this experimental colitis system.