Characterizing the mechanism of thiazolidinedione-induced hepatotoxicity: An in vitro model in mitochondria.

OBJECTIVE: To characterize the mechanism of action of thiazolidinedione (TZD)-induced liver mitochondrial toxicity caused by troglitazone, rosiglitazone, and pioglitazone in HepaRG cells. METHODS: Human hepatoma cells (HepaRG) were treated with troglitazone, rosiglitazone, or pioglitazone (12.5, 25, and 50µM) for 48h. The Seahorse Biosciences XF24 Flux Analyzer was used to measure mitochondrial oxygen consumption. The effect of TZDs on reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected by flow cytometry. The mitochondrial ultrastructure of HepaRG cells was observed under a transmission electrical microscope (TEM). mtDNA content was evaluated by real-time PCR, and ATP content and mitochondrial respiratory chain (MRC) complex I, II, III, IV activity were measured via chemiluminescence. Results were considered statistically significant at p<0.05. RESULTS: Among the three drugs, troglitazone exhibited the highest potency, followed by rosiglitazone, and then pioglitazone. The TZDs caused varying degrees of mitochondrial respiratory function disorders including decreases in oxygen consumption, MRC activity, and ATP level, and an elevation in ROS level. TZD treatment resulted in mtDNA content decline, reduction in MMP, and alterations of mitochondrial structure. CONCLUSION: All investigated TZDs show a certain degree of mitochondrial toxicity, with troglitazone exhibiting the highest potency. The underlying mechanism of TZD-induced hepatotoxicity may be associated with alterations in mitochondrial respiratory function disorders, oxidative stress, and changes in membrane permeability. These parameters may be used early in drug development to further optimize risk:benefit profiles.

Studies on the characteristics and mechanisms of testicular toxicity induced by Hydroxyurea.

OBJECTIVE: Apoptosis plays a dominant role in both spontaneous spermatogenesis and germ cell death. This study was aimed to investigate the functions of related genes in testicular germ cell death induced by Hydroxyurea (HU). METHOD: Wild-type (WT) and FasL transgenic (TG) DBA/C57BL mice were intraperitoneal injected with 400 mg/kg HU. Twelve hours later, testes were collected. Histomorphology of testis was observed by staining with Periodic Acid Schiff (PAS). Apoptosis was assessed by TUNEL assay. mRNA and protein levels of related genes were evaluated by quantitative RT-PCR and Western blot, respectively. RESULTS: The 2 × 2 factorial design comparative experiments between the WT and TG mice showed that the TG mice exhibited a higher basal apoptotic index. The basal mRNA levels of Fas and FasL and protein levels of Fas, FasL, Caspase-3, Caspase-8 and Caspase-9 in the TG mice were also higher than that in the WT mice. Twelve hours after injection of HU, the testicular tubules exhibited no significantly morphological changes but apoptosis index remarkably increased in both the WT and TG mice, with the latter having the higher amplitude. Although, HU up-regulated the mRNA of apoptosis-related genes, such as Fas and FasL, in both the TG and WT mice, the increased amplitude was more obvious in the TG mice. By Western blot analysis, apoptosis-related proteins Fas, FasL Caspase-3, Caspase-8 and Caspase-9 were significantly increased in both the WT and TG mice, with the TG mice exhibiting a greater up-regulation. CONCLUSION: Germ cell apoptosis induced by the HU treatment may be related to the FasL-mediated signal transduction pathway.

Plasma and liver proteomic analysis of 3Z-3-[(1H-pyrrol-2-yl)-methylidene]-1-(1-piperidinylmethyl)-1,3-2H-indol-2-one-induced hepatotoxicity in Wistar rats.

3Z-3-[(1H-pyrrol-2-yl)-methylidene]-1-(1-piperidinylmethyl)-1,3-2H-indol-2-one (Z24), a synthetic anti-angiogenic compound, inhibits the growth and metastasis of certain tumors. Previous works have shown that Z24 induces hepatotoxicity in rodents. We examined the hepatotoxic mechanism of Z24 at the protein level and looked for potential biomarkers. We used 2-DE and MALDI-TOF/TOF MS to analyze alternatively expressed proteins in rat liver and plasma after Z24 administration. We also examined apoptosis in rat liver and measured levels of intramitochondrial ROS and NAD(P)H redox in liver cells. We found that 22 nonredundant proteins in the liver and 11 in the plasma were differentially expressed. These proteins were involved in several important metabolic pathways, including carbohydrate, lipid, amino acid, and energy metabolism, biotransformation, apoptosis, etc. Apoptosis in rat liver was confirmed with the terminal deoxynucleotidyl transferase dUTP-nick end labeling assay. In mitochondria, Z24 increased the ROS and decreased the NAD(P)H levels. Thus, inhibition of carbohydrate aerobic oxidation, fatty acid beta-oxidation, and oxidative phosphorylation is a potential mechanism of Z24-induced hepatotoxicity, resulting in mitochondrial dysfunction and apoptosis-mediated cell death. In addition, fetub protein and argininosuccinate synthase in plasma may be potential biomarkers of Z24-induced hepatotoxicity.

Systems toxicology used in nanotoxicology: mechanistic insights into the hepatotoxicity of nano-copper particles from toxicogenomics.

In some studies, nano-copper particles have been found to be acutely toxic to exposed mice, with the liver and kidney being the target tissues. However, the characteristics of subacute toxicity from repeated nano-copper exposure in rats and the molecular mechanism of its hepatotoxicity at the genomic level remain unclear. We investigated the mechanisms of nano-copper-induced hepatotoxicity, which were identified from hepatic gene expression profiles that were phenotypically anchored to conventional toxicological outcomes, and identified biomarkers of nanotoxicity caused by nano-copper. Male Wistar rats were administered nano-copper or micro-copper at different doses for five days. Subsequently, we examined conventional toxicological parameters including body weight, clinical chemistry, and histopathology, and also used microarrays to identify gene expression changes in rat liver. High dose nano-copper induced increases in alanine aminotransferase, aspartate aminotransferase, triglyceride, total bilirubin, total bile acid levels, and a decrease in body weight. Histopathological studies of the liver indicated scattered, dotted hepatocytic necrosis in all rats in the high dose nano-copper group. Identified genes from the group receiving the high dose were functionally categorized, and results showed that genes related to oxidoreductase activity, metabolism, and signal transduction were involved in the development of the observed phenotypes. The results also suggest that altered gene expression patterns induced by exposure to a low, subtoxic dose of nano-copper may reveal signs of cell stress or subtle cell injury indicative of overt toxicity at higher doses. Results in this study provide new insights into the toxicology of nano-copper particles and illustrate how toxicogenomic approaches are providing an unprecedented amount of mechanistic information on molecular responses to nano-copper, as well as how they are likely to impact hazard and risk assessment. Gene expression changes are likely to be more sensitive indicators of potential adverse effects than traditional measurements of toxicity.

Overview of developmental and reproductive toxicity research in China: history, funding mechanisms, and frontiers of the research.

Reproductive and developmental toxicology (DART) is the discipline that deals with adverse effects on male and female resulting from exposures to harmful chemical and physical agents. DART research in China boasted a long history, but presently has fallen behind the western world in education and research. The funding mechanisms for DART research in China were similar to that for other toxicological disciplines, and the funding has come from research grants and fellowships provided by national, ministerial, and provincial institutions. Finally, the frontiers of DART research in China could be summarized as follows: (1) use of model animals such as the zebrafish and roundworm, and use of cutting-edge techniques such as stem cell culture, as well as transgenic, metabonomic, and virtual screening to study the mechanisms of developmental toxicity for some important toxicants in China; (2) use of model animals and other lower-level sentinel organisms to evaluate and monitor the developmental toxicogical risk of environmental chemicals or pollutants; (3) epidemiological studies of some important reproductive hazards; (4) in-depth studying of the reproductive and developmental toxicity of some important environmental chemicals; and (5) evaluation and study of the reproductive and developmental toxicity of traditional Chinese medicines.

Toxicological evaluation of z24, a novel indolin-2-ketone compound, in cultured human liver cells using toxicogenomic techniques.

The current study was designed to investigate the toxicity of 3Z-3-[((1)H-pyrrol-2-yl)-methylidene]-1-(1-piperidinylmethyl)-1, 3-2H-indol-2-one (Z24), a novel synthetic indolin-2-ketone small molecule compound, using toxicogenomic techniques (complementary DNA [cDNA] microarray). Bioinformatic analysis suggested that the main functions of genes with altered expression were consistent with liver cell regeneration, apoptosis, metabolism of energy and fat, and the death receptor (DR)-mediated apoptosis-signaling pathway. Death receptor 4, Bcl-2, Bcl-xl, caspase 3, and cytochrome C, which are involved in the DR-mediated apoptosis-signaling pathway, were altered after Z24 treatment as determined by Western blotting analysis. When hepatocarcinoma cell line (HepG2 cells) treated with Z24 at 0.248 mmol/L for 24 hours, DNA fragmentation reached a maximum, and examination of cell morphology showed typical signs of apoptosis. These results indicate that Z24 can initiate apoptosis in hepatocytes, which in turn causes hepatotoxicity. A possible toxicological mechanism is that apoptosis was induced in hepatocytes by initiating the DR-mediated signal transduction pathway. Apoptosis of hepatocytes might lead to impairment of energy and lipid metabolism and provoke hepatocyte necrosis or inflammation, resulting in hepatotoxicity.

MiR-96-5p inhibition induces cell apoptosis in gastric adenocarcinoma.

BACKGROUND: Gastric adenocarcinoma (GAC) mortality rates have remained relatively changed over the past 30 years, and it continues to be one of the leading causes of cancer-related death. AIM: To search for novel miRNAs related to GAC prognosis and further investigate the effect of miR-96-5p on MGC-803 cells. METHODS: The miRNA expression profile data of GAC based on The Cancer Genome Atlas were obtained and used to screen differently expressed miRNAs (DEMs) and DEMs related to GAC prognosis. Then, the expression of DEMs related to GAC prognosis was identified in GAC tumor samples and adjacent normal samples by qRT-PCR. The target gene, ZDHHC5, of miR-96-5p was predicted using TargetScan, miRTarBase, and miRDB databases and confirmed by luciferase reporter assay. Furthermore, MGC-803 cells were transfected with inhibitor NC, miR-96-5p inhibitor, si-ZDHHC5, or miR-96-5p inhibitor + si-ZDHHC5, and then cell apoptosis was detected by flow cytometry. The expression of ZDHHC5, Bcl-2, and COX-2 was detected using western blotting. RESULTS: A total of 299 DEMs and 35 DEMs related to GAC prognosis were screened based on The Cancer Genome Atlas. Then compared with adjacent normal samples, the levels of miR-96-5p, miR-222-5p, and miR-652-5p were remarkably increased, while miR-125-5p, miR-145-3p, and miR-379-3p levels were reduced in GAC tumor samples (P < 0.01), which were consistent with bioinformatics analysis. Furthermore, ZDHHC5 was defined as a direct target gene of miR-96-5p. miR-96-5p inhibition increased the number of apoptotic cells as well as promoted the expression of ZDHHC5, Bcl-2, and COX-2 in MGC-803 cells (P < 0.01). After ZDHHC5 inhibition, the number of apoptotic cells and the expression of ZDHHC5, Bcl-2, and COX-2 were reduced. The addition of an miR-96-5p inhibitor partly reversed these effects (P < 0.01). CONCLUSION: Our findings identified six miRNAs related to GAC prognosis and suggested that downregulated miR-96-5p might induce cell apoptosis via upregulating ZDHHC5 expression in MGC-803 cells.

Integrated metabolomic analysis of the nano-sized copper particle-induced hepatotoxicity and nephrotoxicity in rats: a rapid in vivo screening method for nanotoxicity.

Despite an increasing application of copper nanoparticles, there is a serious lack of information concerning their impact on human health and the environment. In this study, the biochemical compositions of urine, serum, and extracts of liver and kidney tissues of rats treated with nano-copper at the different doses (50, 100, and 200 mg/kg/d for 5 d) were investigated using (1)H NMR techniques with the pattern recognition methods. Serum biochemical analysis and histopathological examinations of the liver and kidney of all the rats were simultaneously performed. All the results indicated that the effects produced by nano-copper at a dose of 100 or 50 mg/kg/d were less than those induced at a higher dose of 200 mg/kg/d. Nano-copper induced overt hepatotoxicity and nephrotoxicity at 200 mg/kg/d for 5 d, which mainly involved scattered dot hepatocytic necrosis and widespread renal proximal tubule necrosis. Increased citrate, succinate, trimethylamine-N-oxide, glucose, and amino acids, accompanied by decreased creatinine levels were observed in the urine; furthermore, elevated levels of lactate, 3-hydroxybutyrate, acetate, creatine, triglycerides, and phosphatide and reduced glucose levels were observed in the serum. The predominant changes identified in the liver tissue aqueous extracts included increased lactate and creatine levels together with reduced glutamine and taurine levels, and the metabolic profile of the kidney tissue aqueous extracts showed an increase in lactate and a drop in glucose. In the chloroform/methanol extracts of the liver and kidney tissues, elevated triglyceride species were identified. These changes suggested that mitochondrial failure, enhanced ketogenesis, fatty acid beta-oxidation, and glycolysis contributed to the hepatotoxicity and nephrotoxicity induced by nano-copper at 200 mg/kg/d for 5 d. An increase in triglycerides in the serum, liver and kidney tissues could serve as a potential sensitive biomarker reflecting the lipidosis induced by nano-copper. The data generated from the current study completely supports the fact that an integrated metabolomic approach is promising for the development of a rapid in vivo screening method for nanotoxicity.

NMR-spectroscopy-based metabonomic approach to the analysis of Bay41-4109, a novel anti-HBV compound, induced hepatotoxicity in rats.

An integrated metabonomics study using high-resolution (1)H nuclear magnetic resonance (NMR) spectroscopy has been applied to investigate the biochemical composition of urine, serum, liver tissue aqueous extracts (acetonitrile/water) and liver tissue lipidic extracts (chloroform/methanol) obtained from control and Bay41-4109 treated rats (10, 50, 400mg.kg(-1).d(-1) for 5 days, i.g.). Principal components analysis was used to visualize similarities and differences in biochemical profiles. The results showed the effects induced by Bay41-4109 at 400mg.kg(-1).d(-1) are different from those induced at 10, 50mg.kg(-1).d(-1). The biochemical profiles of 400mg.kg(-1).d(-1) group might reflect the hepatotoxicity of Bay41-4109 more exactly. The elevation in the level of 3-HB, lactate, 2-hydroxy-acetol and d-glucose was found in the urine, and the levels of VLDL/LDL(CH(2))(n), VLDL/LDL-CH(3), 2-oxo-3-methyl-n-valerate, 3-HB, lactate, acetate, taurine, 2-hydroxy-isovalerate in serum were increased significantly in 400mg.kg(-1).d(-1) group. The predominant changes identified in liver tissue aqueous extracts included an increase in the signal intensities of lactate, 3-amino-isovalerate, pyruvate, choline, trimethylamine-N-oxide (TMAO) and a reduction in the intensities of taurine, hippurate and d-glucose. In liver tissue chloroform/methanol extracts, there was a remarkably increase in many of the lipid signals including the triglyceride terminal methyl, methylene groups, and CH(2)CO, N(+)(CH(3))(3), CH(2)OPO(2), CH(2)OCOR. These observations all provide evidence that fatty acid metabolism disorder and mitochondrial inability might contribute to the hepatotoxicity of Bay41-4109. The application of (1)H NMR spectroscopy to an array of biological samples comprising urine, serum and liver tissue extracts yields new insight into the hepatotoxicity of xenobiotics.

Study of a novel indolin-2-ketone compound Z24 induced hepatotoxicity by NMR-spectroscopy-based metabonomics of rat urine, blood plasma, and liver extracts.

Antiangiogenic compound has been believed to be an ideal drug in the current cancer biological therapy, but the angiogenesis inhibitors suffer setback for unknown toxicity now. A novel synthetic indolin-s-ketone small molecular compound, 3Z-3-[((1)H-pyrrol-2-yl)-methylidene]-1-(1-piperidinylmethyl)-1,3-2H-indol-2-one (Z24) can inhibit angiogenesis in new blood vessels. The hepatotoxicity effects of Z24 oral administration (dosed at 60, 130 and 200 mg/kg) have been investigated in female Wistar rats by using metabonomic analysis of (1)H NMR spectra of urine, plasma and liver extracts, as well as by clinical chemistry analysis, liver histopathology and electron micrographs examination. The (1)H NMR spectra of the biofluids were analyzed visually and via pattern recognition by using principal component analysis. The metabonomic trajectory analysis on the time-related hepatotoxicity of Z24 was carried out based on the (1)H NMR spectra of urine samples, which were collected daily predose and postdose over an 8-day period. Urinary excretion of citrate, lactate, 2-oxo-glutarate and succinate increased following Z24 dosing. Increased plasma levels of lactate, TMAO and lipid were observed, with concomitant decrease in the level of glucose and phosphatidylcholine. Metabolic profiling on aqueous soluble extracts of liver tissues with the high dose level of Z24 showed an increase in lactate and glutamine, together with a decrease in glucose, glycogen and choline. On the other hand, studies on lipid soluble extracts of liver tissues with the high dose level of Z24 showed increased level in lipid triglycerides and decreased level in unsaturated fatty acids and phosphatidylcholine. Moreover, the most notable effect of Z24 on the metabolism was the reduction in the urinary levels of creatinine and TMAO and the increase in acetate, citrate, succinate and 2-oxo-glutamate with time dependence. The results indicate that in rats Z24 inhibits mitochondrial function through altering the energy and lipid metabolism, which results in the accumulation of free fatty acids and lactate because of the lack of aerobic respiration. These data show that the metabonomic approach represents a promising new technology for the toxicological mechanism study.