Evaluation of anti-platelet and anti-thrombotic effects of cilostazol with PFA-100® and Multiplate® whole blood aggregometer in vitro, ex vivo and FeCl3-induced thrombosis models in vivo.

We evaluate the anti-platelet and anti-thrombotic effects of cilostazol using Multiplate® and PFA-100® in vitro and ex vivo with freshly isolated rat whole blood and in vivo venous and arterial thrombosis models in the same species, in an effort to assess the sensitivity of the whole blood aggregometer assays without potential issues of species differences. In vitro assay of anti-platelet effects of cilostazol against collagen-induced aggregation using Multiplate® produced a graded dose-dependent inhibition curve with IC50 value of 75.4 ± 2.4 µM while it showed a highly sensitive and all-or-none type inhibition response from 25 µM in PFA-100®. Interestingly, cilostazol manifested anti-thrombotic effects in vivo at much lower plasma concentrations than the effective concentrations measured in ex vivo or in vitro aggregation tests using PFA-100® or Multiplate®. In addition, the tail bleeding time measurement demonstrated that rats have lower sensitivity to the anti-platelet effects of cilostazol than mice. These results suggest that the detailed comparative evaluation of whole blood aggregometer assays with anti-thrombotic effects in vivo should be preceded before the application of these methods for the pharmacodynamic studies of anti-thrombotic agents.

Black soybean extract can attenuate thrombosis through inhibition of collagen-induced platelet activation.

Many clinical trials have demonstrated the beneficial effects of soybean (Glycine max) on general cardiovascular health. Among a variety of soybeans, black soybean is known to display diverse biological activities superior to those of yellow and green soybeans, such as in antioxidant, anti-inflammatory and anticancer activities. However, few studies have been directed on the effect of black soybean on cardiovascular function. In this study, we aimed to investigate the effect of black soybean extract (BB) on platelet activation, a key contributor to thrombotic diseases. In freshly isolated human platelets, BB has shown potent inhibitory activity on collagen-induced platelet aggregation, while yellow soybean extract had marginal activity only. BB also attenuated serotonin secretion and P-selectin expression, which are important factors for the platelet-tissue interaction along with thromboxane A(2) formation. These in vitro results were further confirmed in an ex vivo platelet aggregation measurement and in vivo venous thrombosis model where oral administration of BB reduced collagen-induced platelet aggregation and FeCl(3)-induced thrombus formation significantly. A potential active ingredient for antiplatelet effects of BB was isolated and identified to be adenosine through bioassay-directed fractionation and NMR and ESI-MS analyses. These results indicate that black soybean can be a novel dietary supplement for the prevention of cardiovascular risks and the improvement of blood circulation.

Predose blood gene expression profiles might identify the individuals susceptible to carbon tetrachloride-induced hepatotoxicity.

Although the extent of chemical-induced liver injury differs substantially from individual to individual, it is very hard to identify susceptible population priori to chemical exposure. We report here that the gene expression of the blood samples collected predose might identify the susceptible population without actual exposure to hepatotoxicant. The innate gene expressions in the blood samples collected at predose were compared using whole-genome microarray analysis and semiquantitative PCR with the extent of hepatotoxicity following the treatment of a model hepatotoxicant, carbon tetrachloride (CCl(4)) posteriori. The expression of 18 genes was found to innately differ in the blood of the susceptible animals from the resistant to CCl(4)-induced hepatotoxicity. Of these 18 genes, three genes, NADH dehydrogenase subunit 6 (ND6), transient receptor potential cation channel, subfamily C, member 6 (Trpc6), and tetraspanin 12 (Tspan12), were found to be different reproducibly in real-time PCR analysis with independent sets of animals. Of particular note, animals with the low expression level of ND6 and Tspan12 showed significantly higher susceptibility to CCl(4)-induced hepatotoxicity indeed. This study demonstrated that blood gene expression profiling might identify the susceptible individuals to chemical-induced hepatotoxicity without actual chemical exposure, providing a novel and important methodology for the prevention of drug-induced hepatotoxicity.

Determination of spatial distribution of melamine-cyanuric acid crystals in rat kidney tissue by histology and imaging matrix-assisted laser desorption/ionization quadrupole time-of-flight mass spectrometry.

After the outbreak of acute renal failure associated with melamine-contaminated pet food, many attempts have been made to uncover the mechanism underlying the renal toxicity caused by melamine and melamine-related compounds. Using rat models, we investigated the renal crystal formation following the ingestion of a melamine-cyanuric acid mixture (M+CA, 1:1) to gain insight into the M+CA-induced renal toxicity. M+CA did not induce toxicity in precision-cut kidney slices, suggesting that M+CA does not have a direct nephrotoxicity. On the contrary, oral administration of M+CA for 3 days induced nephrotoxicity as determined by increased serum blood urea nitrogen and creatinine, reduced creatinine clearance, and enlarged kidneys in the animals treated with 50 mg/kg M+CA (melamine, 25 mg/kg, and cyanuric acid, 25 mg/kg; 2 of 10 animals) and 100 mg/kg M+CA (9 of 9 animals). While urine crystals were found in all animals treated with M+CA (25-100 mg/kg), histological examination revealed that renal crystals could be observed only in the kidneys of animals showing signs of nephrotoxicity. Remarkably, at 50 mg/kg M+CA, crystals were observed mainly in the medulla region of the kidney, while at 100 mg/kg, crystals were disseminated throughout the cortex and medulla regions. To further investigate the crystal formation by M+CA, matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-Q-TOF) imaging mass spectrometry detecting melamine distribution through monitoring the product ion (m/z 85, M + H) from melamine (m/z 127, M + H) was developed to directly obtain the image of melamine distribution in the kidney. The distribution image of melamine in kidney tissue confirmed that dense points of melamine were located only in the medulla region at 50 mg/kg M+CA, while at 100 mg/kg, they were disseminated widely from the cortex to medulla. These results demonstrated that M+CA ingestion could lead to crystal formation in kidney tubules along the osmotic gradient and that renal crystal formation is closely linked with M+CA-induced nephrotoxicity.

Expression levels of pituitary tumor transforming 1 and glutathione-S-transferase theta 3 are associated with the individual susceptibility to D-galactosamine-induced hepatotoxicity.

Although drug-induced liver injury (DILI) is frequently observed, individual variation in the susceptibility to DILI is hard to predict. Intrinsic genetic variation is considered a key element for this variation but little is known about the identity of the genes associated with DILI. In this study, pre-biopsy method was applied to uncover the key genes for D-galactosamine (GalN)-induced liver injury and a cause and effect study was conducted to elucidate the correlation between the expression of uncovered genes and GalN-induced hepatotoxicity. To identify the genes determining the susceptibility to GalN-induced hepatotoxicity, we compared the innate gene expression profiles in the liver tissue pre-biopsied before GalN treatment of the SD rats susceptible and resistant to GalN-induced hepatotoxicity, using microarray. Eight genes including Pttg1, Ifit1 and Gstt3 were lower or higher in the susceptible animals than the resistant and RT-PCR analysis confirmed it. To determine if these genes are associated with the susceptibility to GalN-induced hepatotoxicity indeed, expression levels were measured using real-time PCR in a new set of animals and the correlation with GalN-induced hepatotoxicity were analyzed. Notably, the expression of Pttg1 was significantly correlated with the severity of GalN-induced hepatotoxicity (p<0.01) and the animals with lowest and highest level of Gstt3 turned out to be the most susceptible and resistant, respectively, demonstrating that the expression of Pttg1 and Gstt3 could predict inter-individual susceptibility to GalN-induced hepatotoxicity. More importantly, this study showed the utility of pre-biopsy method in the identification of the gene for the chemical-induced hepatotoxicity.

Determination of the key innate genes related to individual variation in carbon tetrachloride-induced hepatotoxicity using a pre-biopsy procedure.

High inter-individual variation in chemical-induced liver injury is a frequent observation with many hepatotoxic chemicals, yet the mechanism underlying it remains poorly understood. Even with carbon tetrachloride (CCl(4)), a well-known model hepatotoxicant, substantial individual variations are observed in the severity of liver injury. Using microarray, many attempts have been made to identify the key genes in CCl(4)-induced liver injury but mostly, they examined the gene expression of liver after CCl(4) exposure, unable to dissect out the complicating factors from pathological changes secondary to liver injury. To more accurately identify the genes for the individual variation in CCl(4)-induced hepatotoxicity, we compared the innate gene expression of the individual liver samples pre-biopsied prior to CCl(4)-treatment with the severity of liver injury after CCl(4)-treatment. Effect of biopsy procedure and 3 week recovery period on liver function and gene expression were confirmed to be insignificant. Using this design, we found that the expression of genes associated with immunity and defense, lipid metabolism, transport and complement-mediated immunity, which are previously known to be suppressed by CCl(4)-treatment, were innately lower in the susceptible animals than resistant animals. Moreover, we demonstrated that the genes such as Gsta2, Sult2a1, Fgl1 and C6 were newly found to be innately lower in the susceptible animals to CCl(4)-hepatotoxicity. These naturally lower gene expression patterns were further confirmed by RT-PCR. We believe that this pre-biopsy design may provide a useful tool for understanding the cause of variability of hepatotoxicity and for the prediction and pre-screening of the susceptible individual to drug-induced hepatotoxicity.

The effects of BADGE and caffeine on the time-course response of adiponectin and lipid oxidative enzymes in high fat diet-fed C57BL/6J mice: correlation with reduced adiposity and steatosis.

Adiponectin, which is expressed exclusively in adipose tissue, has been shown to increase fatty acid oxidation via activation of AMP-activated kinase (AMPK) and phosphorylation of acetyl CoA carboxylase (ACC). ACC phosphorylation and carnitine palmitoyl-transferase-1 (CPT1) activity have been shown to be rate controlling factors in fatty acid oxidation. In high fat diet (HFD)-induced obese mice, we analyzed the time-course of changes in the expression of adiponectin and lipid oxidative enzymes induced by treatment with bisphenol A diglycidyl ether (BADGE) or caffeine for 8 weeks, and investigated whether the changes of adiponectin and lipid oxidative enzymes expression correlated with reduced adiposity or steatosis after 8 weeks of treatment. After 8 weeks of treatment, BADGE and caffeine had reduced body weight and epididymal adipose tissue weight in mice fed HFD, and markedly reduced the number of fatty droplets in the liver. Interestingly, the expression of adiponectin and lipid oxidative enzymes significantly increased after 2 weeks of treatment. These results indicate that the expression of adiponectin and lipid oxidative enzymes in the early stages of BADGE or caffeine treatment correlated well with the long-term anti-obesity effects.

GCG-rich tea catechins are effective in lowering cholesterol and triglyceride concentrations in hyperlipidemic rats.

The (-)-gallocatechin gallate (GCG) concentration in some tea beverages can account for as much as 50% of the total catechins, as a result of sterilization. The present study aims to examine the effects of GCG-rich tea catechins on hyperlipidemic rats and the mechanisms associated with regulating cholesterol metabolism in the liver. By performing heat epimerization of (-)-epigallocatechin gallate (EGCG), we manufactured a mixture of catechins that had a GCG content of approximately 50% (w/w). In sucrose-rich diet-induced hyperlipidemic rats, the GCG-rich tea catechins exhibited strong activity in reducing plasma cholesterol and triglyceride concentrations. Furthermore, the hepatic cholesterol and triglyceride concentrations that had increased as a result of the sucrose-rich diet were reduced due to GCG-rich tea catechins consumption. In order to investigate the hyperlipidemic mechanism of GCG-rich tea catechins, we examined the hepatic expressions of LDL receptor and HMG-CoA reductase in hyperlipidemic rats. We further evaluated the action of purified GCG on LDL receptor activity, which is a key contributor to the regulation of cholesterol concentrations. We found that purified GCG increased LDL receptor protein level and activity to a greater extent than EGCG. In conclusion, our study indicates that GCG-rich tea catechins in tea beverages may be effective in preventing hyperlipidemia by lowering plasma and hepatic cholesterol concentrations.

Effect of dietary epigallocatechin-3-gallate on cytochrome P450 2E1-dependent alcoholic liver damage: enhancement of fatty acid oxidation.

This study was designed to determine whether dietary epigallocatechin-3-gallate (EGCG), the most abundant catechin polyphenol in green tea, can protect the liver from cytochrome P450 2E1 (CYP2E1)-dependent alcoholic liver damage. Compared with an ethanol group, when EGCG was present in the ethanol diet, the formation of a fatty liver was significantly reduced and the serum aspartate transaminase (AST) and alanine transaminase (ALT) levels were much lower. Ethanol treatment significantly elevated hepatic CYP2E1 expression while simultaneously reducing hepatic phospho-acetyl CoA carboxylase (p-ACC) and carnitine palmitoyl-transferase 1 (CPT-1) levels. While EGCG markedly reversed the effect of ethanol on hepatic p-ACC and CPT-1 levels, it had no effect on the ethanol-induced elevation in CYP2E1 expression. EGCG prevents ethanol-induced hepatotoxicity and inhibits the development of a fatty liver. These effects were associated with improvements in p-ACC and CPT-1 levels. The use of EGCG might be useful in treating patients with an alcoholic fatty liver.

Hypercalcemia and high parathyroid hormone-related peptide concentration in a dog with a complex mammary carcinoma.

A 10-year-old female Dachshund was presented with a history of mammary masses, slight lethargy, polyuria, and polydipsia. Physical examination findings included masses involving the first, second, and fourth mammary glands of the left side. The mandibular, axillary, and right popliteal lymph nodes were mildly enlarged. Serum chemistry results included hypercalcemia (13.9 mg/dL, reference interval 8.0-11.5 mg/dL). Although intact parathyroid hormone (PTH) concentration (1.05 pmol/L) was below the reference interval (2-13 pmol/L), PTH-related protein (PTHrP) concentration was markedly increased (9.40 pmol/L, reference value < 2 pmol/L). The masses were surgically removed, and the histopathologic diagnosis was complex mammary carcinoma. Three weeks after surgery, serum total calcium concentration had decreased to 10.5 mg/dL. Resolution of the hypercalcemia and clinical signs supported a diagnosis of humoral hypercalcemia of malignancy associated with mammary gland carcinoma.