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To assess modification of thioacetamide-induced hepatotoxicity in mice fed a high-fat diet, male C57BL/6J mice were fed a normal rodent diet or a high-fat diet for 8 weeks and then treated once intraperitoneally with thioacetamide at 50 mg/kg body weight. At 24 and 48 hours after administration, massive centrilobular hepatocellular necrosis was observed in mice fed the normal rodent diet, while the necrosis was less severe in mice fed the high-fat diet. In contrast, severe swelling of hepatocytes was observed in mice fed the high-fat diet. In addition, mice fed the high-fat diet displayed more than a 4-fold higher number of BrdU-positive hepatocytes compared with mice fed the normal rodent diet at 48 hours after thioacetamide treatment. To clarify the mechanisms by which the hepatic necrosis was attenuated, we investigated exposure to thioacetamide and one of its metabolites, the expression of CYP2E1, which converts thioacetamide to reactive metabolites, and the content of glutathione S-transferases in the liver. However, the reduced hepatocellular necrosis noted in mice fed the high-fat diet could not be explained by the differences in exposure to thioacetamide or thioacetamide sulfoxide or by differences in the expression of drug-metabolizing enzymes. On the other hand, at 8 hours after thioacetamide administration, hepatic total glutathione in mice fed the high-fat diet was significantly lower than that in mice fed the normal diet. Hence, decreased hepatic glutathione amount is a candidate for the mechanism of the attenuated necrosis. In conclusion, this study revealed that thioacetamide-induced hepatic necrosis was attenuated in mice fed the high-fat diet. [ABSTRACT FROM AUTHOR]

We investigated the role of glutathione S-transferases Mu 1 (GSTM1) in acetaminophen (APAP)-induced hepatotoxicity using Gstm1-null mice. A single oral administration of APAP resulted in a marked increase in plasma alanine aminotransferase accompanied by hepatocyte necrosis 24 hr after administration in wild-type mice, but its magnitude was unexpectedly attenuated in Gstm1-null mice. Therefore, it is suggested that Gstm1-null mice are resistant to APAP-induced hepatotoxicity. To examine the mechanism of this resistance in Gstm1-null mice, we measured phosphorylation of c-jun N-terminal kinase (JNK), which mediates the signal of APAP-induced hepatocyte necrosis, by Western blot analysis 2 and 6 hr after APAP administration. A marked increase in phosphorylated JNK was observed in wild-type mice, but the increase was markedly suppressed in Gstm1-null mice. Therefore, it is suggested that sup-pressed phosphorylation of JNK may be a main mechanism of the resistance to APAP-induced hepatotoxicity in Gstm1-null mice, although other possibilities of the mechanism cannot be eliminated. Additionally, phosphorylation of glycogen synthase kinase-3β and mitogen-activated protein kinase kinase 4, which are upstream kinases of JNK in APAP-induced hepatotoxicity, were also suppressed in Gstm1-null mice. A decrease in liver total glutathione 2 hr after APAP administration, which is an indicator for exposure to N-acetyl-p-benzoquinoneimine, the reactive metabolite of APAP, were similar in wild-type and Gstm1-null mice. In conclusion, Gstm1-null mice are considered to be resistant to APAP-induced hepatotoxicity perhaps by the suppression of JNK phosphorylation. This study indicates the novel role of GSTM1 as a factor mediating the cellular signal for APAP-induced hepatotoxicity. [ABSTRACT FROM AUTHOR]

Toxicokinetics (TK) is usually performed by measurement of the total drug concentrations in plasma. However, free drug concentrations in plasma are considered to correlate directly with toxicodynamics (TD). In the present study, to evaluate the applicability of TK/TD analysis based on free drug concentrations, we investigated the TK/TD of clofibrate, which binds to albumin with a higher ratio, using an albumin-deficient mutant strain, Nagase analbuminemia rats (NAR). TK, blood chemistry, histopathology, drug and fatty acid metabolizing enzymes and microarray analysis in the liver were examined after a 4-day oral administration of clofibrate. Compared to Sprague-Dawley (SD) rats, the parent strain of NAR, 4.1-fold higher AUC0-24hr, based on free drug concentrations (3445 versus 844 μ·g hr/ml) was observed in NAR when both rats showed the same level of AUC0-24hr, based on the total drug concentrations (4436 versus 4237μg·hr/ml). Additionally, more severe hepatocellular hypertrophy, increase in aspartate transaminase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), decrease in total cholesterol (T.CHO), phospholipid (PL), triglyceride (TG), and non-esterified fatty acid (NEFA), and increase in the mRNA levels of fatty acid metabolizing enzymes (FAOS, CAT, and CPT) were observed in NAR at the same dose. These results demonstrated that NAR developed more severe toxicities and pharmacological effects than SD rats correlating with the higher AUC of the free drug concentrations. The results also suggested that TK/TD analysis based on the free drug concentration is appropriate to interpret the relationship between exposure and toxicity in cases of protein binding saturation including protein decrease or species differences on protein binding, especially when drugs showing a higher protein binding ratio are dosed. [ABSTRACT FROM AUTHOR]

We have reported the existence of low glutathione S-transferase (GST) dogs whose GST activity to 1,2-dichloro-4-nitrobenzene (DCNB) as a substrate (GST-D activity) is quite low, and have also reported significant individual differences in dog liver GST-D activity. The dogs were classified as “low”, “middle”, or “high” GST dogs based on their GST-D activity. In the present study, in order to investigate the causes of quite low GST-D activity in low GST dogs and the individual differences in dog GST-D activity, glutathione (GSH) conjugation of DCNB was kinetically analyzed. Moreover, liver cytosolic proteins whose expression levels were significantly lower in low GST dogs than in high GST dogs were identified by two-dimensional difference gel electrophoresis (2D-DIGE) and LC tandem mass spectrometry (LC/MS/MS). Interestingly, Vmax values for this reaction well reflected their GST-D activities in all groups, i.e. they were 3.8, 80.6, and 169.2 nmol/min/mg protein in the low, middle, and high GST dogs, respectively. However, Km values were almost the same (260.0–283.7 μM) among these groups. These suggest that GSH conjugation of DCNB should be catalyzed by the same enzyme in all the dogs, and individual differences in the GST-D activity should be the result of individual differences in the expression level of the GST isozyme, which catalyzes conjugation of DCNB. In 2D-DIGE, the expression levels of the two protein spots were significantly lower in the low GST dogs than in the high GST dogs. Positive good correlation ( r>0.800) was observed between GST-D activity and expression levels of these two protein spots. Moreover, expression levels were quite low in low GST dogs. These two proteins were both identified as the theta class GST isozyme, YdfYdf, which specifically catalyzes GSH conjugation of DCNB in dog livers. In the present study, we present two novel findings based on an enzyme kinetic study and protein-expression analysis: (1) GSTYdfYdf is expressed at quite a low level in the liver of low GST dogs, and (2) individual differences in dog liver GST-D activity would be due to individual differences in the expression level of GSTYdfYdf. Considering these findings, low GST dogs might have high susceptibility, including an unexpected toxicity at abnormal exposure to chemicals metabolized by GSTYdfYdf. [ABSTRACT FROM AUTHOR]

Hepatic P450 monooxygenase activities, assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities, show obvious daily fluctuations in male rats with high values during the dark period and low values during the light period. We have already confirmed that the ACD activities are controlled by the suprachiasmatic nucleus (SCN), which is well known as the oscillator of circadian rhythm. Recently, it is reported that circadian oscillators exist not only in the SCN but also in peripheral organs. To date, it is unclear which circadian oscillators predominantly drive the daily fluctuations of hepatic ACD activities. To address this question, we examined the effects of restricted feeding, which uncouples the circadian oscillators in the liver from the central pacemaker in the SCN, on the daily fluctuations in hepatic ACD activities in male rats. Here we show that restricted feeding inverts the oscillation phase of the daily fluctuations in hepatic ACD activities. Regarding the hepatic P450 content, there were no fluctuations between the light and dark periods under ad libitum and restricted feeding conditions. Therefore, it is considered that the daily fluctuations in hepatic ACD activities are predominantly driven by the circadian factors in peripheral organs rather than by the oscillator in the SCN directly. [ABSTRACT FROM AUTHOR]

Indium nitride (InN) luminescence is substantially enhanced by the introduction of a multilayer graphene interlayer, mitigating the lattice mismatch between the InN epilayer and the Gallium nitride (GaN) template on a sapphire substrate via weak van der Waals interaction between graphene and nitride layers. The InN epilayers are deposited by radio-frequency plasma-assisted molecular beam epitaxy (MBE), and are characterized by spatially-resolved photoluminescence spectroscopy using confocal microscopy. A small blue shift of the emission band from the band gap evidences a low density of equilibrium carriers, and a high quality of InN on multilayer graphene. A deposition temperature of ~375 °C is determined as optimal. The granularity, which is observed for the InN epilayers deposited on multilayer graphene, is shown to be eliminated, and the emission intensity is further enhanced by the introduction of an aluminum nitride (AlN) buffer layer between graphene and InN. [ABSTRACT FROM AUTHOR]

In order to characterize the hepatic effects of phenobarbital (PB) and clofibrate (CPIB) in dogs, PB and CPIB were administered to male beagle dogs for 14 days, and biochemical and histopathological examinations and comprehensive genomic and proteomic analyses, including GeneChip® analysis and proteomics analysis using the 2-dimension difference gel electrophoresis (2D-DIGE) technique, were performed. Both compounds caused centrilobular hepatocellular hypertrophy, which were related to smooth endoplasmic reticulum (SER) proliferation in PB-treated dogs and to mitochondrial proliferation in CPIB-treated dogs. In the PB-treated dogs, drug-metabolizing enzyme induction was observed by Western blot and genomic analyses. CYP proteins could not be detected by the 2D-DIGE analysis, but increases in several endoplasmic reticulum (ER)-related proteins were observed. In the CPIB-treated dogs, drug-metabolizing enzyme induction was not clearly observed by any of Western blot, genomic and proteomic analyses. Genomic and proteomic analyses revealed that mitochondrial genes and proteins, including carnitine palmytoiltransferase II, acyl-CoA deheydrogenase and hydroxyacyl-CoA dehydrogenase, pyruvate carboxylase and ATP synthase beta chain were induced. There is a relatively good correlation among the morphology and the genomic and proteomic data, but some differences exist between the genomic and proteomic data. Comprehensive evaluation using these techniques in addition to morphological evaluation may provide a useful tool for safety assessment of the liver. [ABSTRACT FROM AUTHOR]