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 (CCl₄), 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 CCl₄-induced liver injury but mostly, they examined the gene expression of liver after CCl₄ 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 CCl₄-induced hepatotoxicity, we compared the innate gene expression of the individual liver samples pre-biopsied prior to CCl₄-treatment with the severity of liver injury after CCl₄-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 CCl₄-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 CCl₄-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.     

Deletion of circadian gene Per1 alleviates acute ethanol-induced hepatotoxicity in mice

The severity of ethanol-induced liver injury is associated with oxidative stress and lipid accumulation in the liver. Core circadian clock is known to mediate antioxidative enzyme activity and lipid metabolism. However, the link between circadian clock and ethanol-induced hepatotoxicity remains unclear. Here we showed that extents of acute ethanol-induced liver injury and steatosis in mice exhibit circadian variations consistent with hepatic expression of Period (Per) genes. Mice lacking clock gene Per1 displayed less susceptible to ethanol-induced liver injury, as evidenced by lower serum transaminase activity and less severe histopathological changes. Ethanol-induced lipid peroxidation was alleviated in Per1?/? mice. However, Per1 deletion had no effect on antioxidants depletion caused by ethanol administration. Ethanol-induced triglycerides (TG) accumulation in the serum and liver was significantly decreased in Per1?/? mice compared with that in wild-type (WT) mice. Analysis of gene expression in the liver revealed peroxisome proliferators activated receptor-gamma (PPARγ) and its target genes related to TG synthesis are remarkably down-regulated in Per1?/? mice. HepG2 cells were treated with ethanol at 150 mM for 3 days. Per1 overexpression augmented lipid accumulation after treatment with ethanol in HepG2 cells, but had no effect on ethanol-induced oxidative stress. Expression of genes related to lipogenesis, including PPARγ and its target genes, was up-regulated in cells overexpressing Per1. In conclusion, these results indicated that circadian rhythms of ethanol-induced hepatotoxicity are controlled by clock gene Per1, and deletion of Per1 protected mice from ethanol-induced liver injury by decreasing hepatic lipid accumulation.     

Curcumin attenuates isoniazid-induced hepatotoxicity by upregulating the SIRT1/PGC-1α/NRF1 pathway

As a serious infectious disease, tuberculosis threatens global public health. Isoniazid is the first-line drug not only in active tuberculosis but also in its prevention. Severe hepatotoxicity greatly limits its use. Curcumin, extracted from turmeric, has been found to relieve isoniazid-induced hepatotoxicity. However, the mechanism of isoniazid-induced hepatotoxicity and the protective effects of curcumin are not yet understood completely. We established both cell and animal models about isoniazid-induced hepatotoxicity and investigated the new mechanism of curcumin against isoniazid-induced liver injury. The experimental data in our study demonstrated that curcumin ameliorated isoniazid-mediated liver oxidative stress. The protective effects of curcumin were demonstrated and confirmed to be correlated with upregulating SIRT1/PGC-1α/NRF1 pathway. Western blot revealed that while inhibiting SIRT1 by the siRNA1 (a SIRT1 inhibitor), the expressions of SIRT1, PGC-1α/Ac-PGC-1α, and NRF1 decreased, and the protective effect that curcumin exerted on isoniazid-treated L-02 cells was significantly attenuated. Furthermore, curcumin improved liver functions and reduced necrosis of the isoniazid-treated BALB/c mice, accompanied by downregulating oxidative stress and inflammation in liver. Western blot revealed that curcumin treatment activates the SIRT1/PGC-1α/NRF1 pathway in the isoniazid-treated BALB/c mice. In conclusion, we found one mechanism of isoniazid-induced hepatotoxicity downregulating the SIRT1/PGC-1α/NRF1 pathway, and curcumin attenuated this hepatotoxicity by activating it. Our study provided a novel approach and mechanism for the treatment of isoniazid-induced hepatotoxicity.     

Identification of endogenous reference genes for RT‐qPCR analysis of plasma microRNAs levels in rats with acetaminophen‐induced hepatotoxicity

Circulating microRNA (miRNA) expression profiles have been reported to be promising biomarkers for drug‐induced liver injury in preclinical and clinical practice. Proper normalization is critical for accurate miRNAs expression analysis. Herein, using SYBR green quantitative real‐time PCR (RT‐qPCR), we evaluated the expression stability of six candidate reference genes including two commonly used small RNAs (U6, 5S) and four miRNAs (let‐7a, miR‐92a, miR‐103 and miR‐16) in plasma of rats with acetaminophen‐induced hepatotoxicity. Data were analysed using geNorm, Normfinder, BestKeeper and comparative delta‐Ct statistical models, and the results consistently show that miR‐103 is the most stably expressed reference gene. Whereas the commonly used housekeeping genes 5S or U6 are all not suitable normalizers, because 5S exhibits extensive variability in expression and U6 has a low expression level across the plasma samples. Then the effect of reference genes on normalization of plasma miR‐122 was assessed; when normalized to the most stable reference gene there were significant differences between the acetaminophen‐treated group and the vehicle group. However, when the data were normalized to a less stably expressed gene, miR‐16, a biased result was obtained. Therefore, we recommend that miR‐103 as suitable reference gene for plasma miRNAs analysis for acetaminophen‐induced liver injury. Data presented in this paper are crucial to successful biomarker discovery and validation for the diagnosis of the early stage of acetaminophen hepatotoxicity. Copyright © 2013 John Wiley & Sons, Ltd.     

Proanthocyanidin protects against cisplatin‐induced oxidative liver damage through inhibition of inflammation and NF‐κβ/TLR‐4 pathway

Although cisplatin (CIS) is a highly effective anticancer drug, hepatotoxicity is one of the most common adverse effects associated with its use. Recently, reactive oxygen species (ROS) and inflammation are suggested to be key factors in the pathophysiology of CIS‐induced acute liver damage. The aim of this study is to investigate the possible protective effect of proanthocyanidin (PRO) against CIS‐induced acute hepatotoxicity. Rats were divided into four groups: 1, Control; 2, PRO; 3, CIS; and 4, PRO + CIS. Biochemical studies and histopathology were used to assess liver damage. ROS, inflammatory cytokines, nuclear factor kappa beta (NF‐κβ), inducible cyclooxygenase enzyme (COX‐2), inducible nitric oxide synthase (iNOS), toll‐like receptor‐4 (TLR‐4) gene expression, and apoptotic markers were also assessed. PRO pretreatment protected the liver against CIS‐induced toxicity as indicated by decreased plasma levels of liver function enzymes and the normal liver histopathology observed in the PRO + CIS group. PRO pretreatment also diminished indicators of oxidative stress in the liver, including nitric oxide (NO) and malondialdehyde (MDA). It also increased the antioxidants, reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) in the liver. Plasma interleukin‐1 beta (IL‐1β), IL‐6, and tumor necrosis factor‐alpha (TNF‐α) were all reduced. Liver gene expression of NF‐κβ, COX‐2, iNOS, and TLR‐4 were all downregulated. Furthermore, PRO administration downregulated the liver expression of the apoptotic marker, Bax, while upregulated the antiapoptotic marker, Bcl2. In conclusion, our results revealed that PRO may protect against CIS‐induced acute liver damage mainly through inhibition of ROS, inflammation, and apoptosis.     

Hepatoprotective role of PXR activation and MRP3 in cholic acid-induced cholestasis

BACKGROUND AND PURPOSE: Activation of the pregnane X receptor (PXR) has been shown to protect against cholestatic hepatotoxicity. As PXR alters the expression of numerous hepatic bile acid transporters, we sought to delineate their potential role in hepatoprotection. EXPERIMENTAL APPROACH: Wild-type (PXR+/+) and PXR-null (PXR-/-) mice were fed a 1% cholic acid (CA) diet with or without the PXR activator, PCN. Liver function was assessed along with the corresponding changes in hepatic gene expression. KEY RESULTS: CA administration caused significant hepatotoxicity in PXR+/+ mice and was associated with induction of several FXR and PXR regulated genes, which encode for bile acid transport and metabolizing proteins. Compared to CA alone, co-administration of PCN to CA-fed PXR+/+ mice significantly decreased hepatotoxicity and was associated with induction of MRP3 mRNA as well as CYP3A11 mRNA and functional activity. Unexpectedly, PXR-/- mice, which expressed significantly higher basal and CA-induced levels of MRP2, MRP3, OSTalpha, OSTbeta, OATP2 and CYP3A11, were dramatically less sensitive to CA hepatotoxicity than PXR+/+ mice. CONCLUSIONS: Protection of PXR+/+ mice against CA-induced hepatotoxicity by PCN is associated with the induction of MRP3 and CYP3A11 expression. Resistance against CA-induced hepatotoxicity in PXR-/- mice may result from higher basal and induced expression of bile acid transporters, particularly MRP3. These findings emphasize the importance of transport by MRP3 and metabolism as major protective pathways against cholestatic liver injury.     

Acute hepatotoxicity: a predictive model based on focused illumina microarrays.

Drug-induced hepatotoxicity is a major issue for drug development, and toxicogenomics has the potential to predict toxicity during early toxicity screening. A bead-based Illumina oligonucleotide microarray containing 550 liver specific genes has been developed. We have established a predictive screening system for acute hepatotoxicity by analyzing differential gene expression profiles of well-known hepatotoxic and nonhepatotoxic compounds. Low and high doses of tetracycline, carbon tetrachloride (CCL4), 1-naphthylisothiocyanate (ANIT), erythromycin estolate, acetaminophen (AAP), or chloroform as hepatotoxicants, clofibrate, theophylline, naloxone, estradiol, quinidine, or dexamethasone as nonhepatotoxic compounds, were administered as a single dose to male Sprague-Dawley rats. After 6, 24, and 72 h, livers were taken for histopathological evaluation and for analysis of gene expression alterations. All hepatotoxic compounds tested generated individual gene expression profiles. Based on leave-one-out cross-validation analysis, gene expression profiling allowed the accurate discrimination of all model compounds, 24 h after high dose treatment. Even during the regeneration phase, 72 h after treatment, CCL4, ANIT, and AAP were predicted to be hepatotoxic, and only these three compounds showed histopathological changes at this time. Furthermore, we identified 64 potential marker genes responsible for class prediction, which reflected typical hepatotoxicity responses. These genes and pathways, commonly deregulated by hepatotoxicants, may be indicative of the early characterization of hepatotoxicity and possibly predictive of later hepatotoxicity onset. Two unknown test compounds were used for prevalidating the screening test system, with both being correctly predicted. We conclude that focused gene microarrays are sufficient to classify compounds with respect to toxicity prediction.     

Dopamine Receptor D2 and Associated microRNAs Are Involved in Stress Susceptibility and Resistance to Escitalopram Treatment

Background:

Early life stress has been demonstrated to increase the risk of developing depression in adulthood. However, the roles and associated molecular mechanisms of stresses in the onset and relapse of depression have yet to be fully elucidated.

Methods:

Depression-like behaviors were induced in rats by maternal deprivation and chronic unpredictable stress. Depression- and anxiety-like behaviors of rats, dopamine receptor D2 level, and microRNAs expression in rats’ brain tissues were measured.

Results:

Chronic unpredictable stress alone induced depression-like behaviors in rats, but maternal deprivation enhanced the effect of chronic unpredictable stress. Escitalopram significantly decreased depression-like behaviors in chronic unpredictable stress rats but was less effective in maternal deprivation with chronic unpredictable stress rats. Maternal deprivation increased dopamine receptor D2 messenger RNA expression and decreased microRNA-9 expression in the striatum. Chronic unpredictable stress increased dopamine receptor D2 mRNA and protein levels and decreased microRNA-9 expression in the nucleus accumbens. Furthermore, maternal deprivation enhanced the effect of chronic unpredictable stress on dopamine receptor D2 gene and microRNA-9 expression. Chronic unpredictable stress increased the expression of microRNA-326 in the nucleus accumbens but decreased it in the striatum, whereas maternal deprivation elevated microRNA-326 expression in the striatum. Escitalopram normalized microRNA-326 expression but had no effect on the expression of microRNA-9, dopamine receptor D2 mRNA, and dopamine receptor D2 protein in both the nucleus accumbens and striatum. The in vitro study showed that only microRNA-9 directly targeted the 3’ untranslated region of dopamine receptor D2 mRNA and inhibited dopamine receptor D2 protein expression.

Conclusion:

Early life stress enhanced the susceptibility to late life stress and resistance to escitalopram treatment through decreasing microRNA-9 expression and subsequently upregulating dopamine receptor D2 expression in the nucleus accumbens. microRNA-326 may be a novel target of escitalopram.     

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.     

Subchronic effects of valproic acid on gene expression profiles for lipid metabolism in mouse liver

Valproic acid (VPA) is used clinically to treat epilepsy, however it induces hepatotoxicity such as microvesicular steatosis. Acute hepatotoxicity of VPA has been well documented by biochemical studies and microarray analysis, but little is known about the chronic effects of VPA in the liver. In the present investigation, we profiled gene expression patterns in the mouse liver after subchronic treatment with VPA. VPA was administered orally at a dose of 100 mg/kg/day or 500 mg/kg/day to ICR mice, and the livers were obtained after 1, 2, or 4 weeks. The activities of serum liver enzymes did not change, whereas triglyceride concentration increased significantly. Microarray analysis revealed that 1325 genes of a set of 32,996 individual genes were VPA responsive when examined by two-way ANOVA (P<0.05) and fold change (>1.5). Consistent with our previous results obtained using an acute VPA exposure model (Lee et al., Toxicol Appl Pharmacol. 220:45-59, 2007), the most significantly over-represented biological terms for these genes included lipid, fatty acid, and steroid metabolism. Biological pathway analysis suggests that the genes responsible for increased biosynthesis of cholesterol and triglyceride, and for decreased fatty acid beta-oxidation contribute to the abnormalities in lipid metabolism induced by subchronic VPA treatment. A comparison of the VPA-responsive genes in the acute and subchronic models extracted 15 commonly altered genes, such as Cyp4a14 and Adpn, which may have predictive power to distinguish the mode of action of hepatotoxicants. Our data provide a better understanding of the molecular mechanisms of VPA-induced hepatotoxicity and useful information to predict steatogenic hepatotoxicity.     

Distinct roles of tumor necrosis factor-alpha and nitric oxide in acute liver injury induced by carbon tetrachloride in mice

Macrophages are known to release a number of different inflammatory mediators with cytotoxic potential. In the present studies we analyzed the role of two macrophage-derived mediators, tumor necrosis factor-alpha (TNF-alpha) and nitric oxide, in liver injury induced by carbon tetrachloride (CCl4). Treatment of mice with CCl4 resulted in a dose- and time-dependent induction of centrilobular hepatic necrosis. This was observed within 12 h with 0.3 ml/kg CCl4 and was correlated with increases in serum transaminase levels. CCl4 administration also caused increases in hepatic TNF-alpha mRNA expression and serum TNF-alpha levels, as well as inducible nitric oxide synthase (NOS II) protein expression in the liver. To study the role of TNF-alpha and nitric oxide in hepatotoxicity, we used knockout mice lacking the gene for the 55-kDa TNF-alpha receptor (TNFR1/p55), the TNF-alpha cytokine, or NOS II. We found that CCl4 was significantly less effective in inducing hepatotoxicity in mice lacking TNFR1/p55 or the TNF-alpha cytokine. In contrast, CCl4-induced liver injury was increased in knockout mice lacking the gene for NOS II. This was associated with an increase in hepatic TNF-alpha mRNA expression and serum TNF-alpha levels. These data suggest that the hepatoprotective effects of nitric oxide in this model may be due in part to inhibition of TNF-alpha.     

Aberrant expression of miRNA-192-5p contributes to N,N-dimethylformamide-induced hepatic apoptosis

Excessive exposure to N,N-dimethylformamide (DMF) can lead to occupational liver poisoning in workers; however, the underlying mechanism is not fully clarified. The importance of microRNAs (miRNAs) in chemical-induced hepatotoxicity has been demonstrated. To determine whether miRNAs are also involved in DMF-induced hepatotoxicity, we systematically analyzed the miRNA expression profiles in DMF-treated (75 and 150 mm ) HL-7702 liver cells and controls by high-throughput sequencing. Among the altered miRNAs, miR-192-5p was the most significantly upregulated in HL-7702 cells after DMF exposure and was involved in DMF-mediated cell apoptosis. By contrast, suppression of miR-192-5p in HL-7702 cells attenuated the apoptosis induced by DMF. Furthermore, the anti-apoptotic gene (NIN1/RPN12 binding protein 1 homolog [NOB1]) was predicted to be a potential miR-192-5p target according to bioinformatics analysis. The direct interaction between miR-192-5p and NOB1 was confirmed by the dual-luciferase activity assay in HEK293FT cells. Overexpression of miR-192-5p efficiently reduced NOB1 mRNA and protein expression in HL-7702 cells. Alteration in NOB1 expression influenced DMF-induced hepatotoxicity by affecting hepatic apoptosis. In addition, the inverse correlation between miR-192-5p expression levels and NOB1 expression was further confirmed in DMF-exposed mouse liver tissue samples. These observations demonstrated that promotion of apoptosis from the suppression of NOB1 by miR-192-5p overexpression was responsible for the DMF-induced hepatotoxicity. This work provides the molecular mechanism at the miRNA level for hepatic apoptosis induced by DMF.     

Aryl hydrocarbon receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin enhances liver damage in bile duct-ligated mice

The environmental pollutant 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) is known to cause a wide variety of toxic effects, including hepatotoxicity, by way of the aryl hydrocarbon receptor (AHR). Although inducible expression of cytochrome P450 (CYP) 1A1 and CYP1A2 is associated with liver injury caused by high-dose TCDD, the specific role of the AHR-CYP1 cascade in hepatotoxicity remains unclear. We investigated the effects of AHR activation under conditions of cholestasis. We administered oral TCDD to mice at a dose that can effectively induce Cyp1 gene expression without overt liver toxicity and then ligated their bile ducts. TCDD pretreatment enhanced bile duct ligation (BDL)-induced increases in liver and plasma bile acids, bilirubin, and aminotransferases. Histology of TCDD-pretreated BDL mice revealed massive hepatic necrosis without any increase in number of apoptotic cells. Whereas induction of AHR-target genes by TCDD was observed similarly in sham-operated as well as in BDL mice, TCDD pretreatment of BDL mice altered the expression of hepatic genes involved in bile acid synthesis and transport. Increased plasma proinflammatory cytokines, tumor necrosis factor and interleukin-1β, in BDL mice were further elevated by TCDD pretreatment. Liver injury by TCDD plus BDL, such as increased plasma bile acids, bilirubin and aminotransferases, liver necrosis, and increased tumor necrosis factor production, was exaggerated in Cyp1a1/1a2(-/-) double knockout mice. These findings indicate that TCDD aggravates cholestatic liver damage and that the presence of CYP1A1 and CYP1A2 plays a protective role in liver damage caused by TCDD and BDL.