Chemical-induced Hepatotoxicity Research Articles

Schisandra chinensis in Liver Disease: Exploring the Mechanisms and Therapeutic Promise of an Ancient Chinese Botanical

Background: Schisandra chinensis is a traditional Chinese herbal medicine that has been used for centuries for liver health. The active lignans in Schisandra, including schisandrin and gomisins, have exhibited anti-inflammatory, antioxidant, and hepatoprotective properties in preliminary studies. With rising rates of chronic liver diseases globally, there is interest in the potential therapeutic role of Schisandra. Purpose: To comprehensively review the current evidence for Schisandra chinensis in treating liver injury and disease and synthesize implications for future human research. Main body: Schisandra extracts decreased inflammatory cytokines and oxidative stress markers and increased endogenous antioxidant activity in animal models, suggesting utility in mitigating liver inflammation and damage. Additional preclinical studies demonstrated attenuated liver enzyme levels, necrosis, and fibrosis progression in chemical-induced hepatotoxicity with Schisandra treatment. Enhanced cytochrome P450 activity, glutathione production, and glycogen synthesis were also observed, improving detoxification and regeneration capacity. Small human trials in hepatitis and nonalcoholic fatty liver disease showed improved liver enzymes and symptoms with Schisandra supplementation but were limited in quality and sample size. Conclusion: Schisandra chinensis has biologically relevant mechanisms that warrant further human research on its role as a hepatoprotective phytotherapy. Well-designed, large-scale clinical trials are needed to establish efficacy and safety for liver disease applications.

Reproducibility and Robustness of a Liver Microphysiological System PhysioMimix LC12 under Varying Culture Conditions and Cell Type Combinations.

The liver is one of the key organs for exogenous and endogenous metabolism and is often a target for drug- and chemical-driven toxicity. A wide range of experimental approaches has been established to model and characterize the mechanisms of drug- and chemical-induced hepatotoxicity. A number of microfluidics-enabled in vitro models of the liver have been developed, but the unclear translatability of these platforms has hindered their adoption by the pharmaceutical industry; to achieve wide use for drug and chemical safety evaluation, demonstration of reproducibility and robustness under various contexts of use is required. One of these commercially available platforms is the PhysioMimix LC12, a microfluidic device where cells are seeded into a 3D scaffold that is continuously perfused with recirculating cell culture media to mimic liver sinusoids. Previous studies demonstrated this model's functionality and potential applicability to preclinical drug development. However, to gain confidence in PhysioMimix LC12's robustness and reproducibility, supplementary characterization steps are needed, including the assessment of various human hepatocyte sources, contribution of non-parenchymal cells (NPCs), and comparison to other models. In this study, we performed replicate studies averaging 14 days with either primary human hepatocytes (PHHs) or induced pluripotent stem cell (iPSC)-derived hepatocytes, with and without NPCs. Albumin and urea secretion, lactate dehydrogenase, CYP3A4 activity, and metabolism were evaluated to assess basal function and metabolic capacity. Model performance was characterized by different cell combinations under intra- and inter-experimental replication and compared to multi-well plates and other liver platforms. PhysioMimix LC12 demonstrated the highest metabolic function with PHHs, with or without THP-1 or Kupffer cells, for up to 10-14 days. iPSC-derived hepatocytes and PHHs co-cultured with additional NPCs demonstrated sub-optimal performance. Power analyses based on replicate experiments and different contexts of use will inform future study designs due to the limited throughput and high cell demand. Overall, this study describes a workflow for independent testing of a complex microphysiological system for specific contexts of use, which may increase end-user adoption in drug development.

TCDD dysregulation of lncRNA expression, liver zonation and intercellular communication across the liver lobule

The persistent environmental aryl hydrocarbon receptor agonist and hepatotoxin TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces hepatic lipid accumulation (steatosis), inflammation (steatohepatitis) and fibrosis. Thousands of liver-expressed, nuclear-localized lncRNAs with regulatory potential have been identified; however, their roles in TCDD-induced hepatoxicity and liver disease are unknown. We analyzed single nucleus (sn)RNA-seq data from control and subchronic (4 wk) TCDD-exposed mouse liver to determine liver cell-type specificity, zonation and differential expression profiles for thousands of lncRNAs. TCDD dysregulated >4000 of these lncRNAs in one or more liver cell types, including 684 lncRNAs specifically dysregulated in liver non-parenchymal cells. Trajectory inference analysis revealed major disruption by TCDD of hepatocyte zonation, affecting >800 genes, including 121 lncRNAs, with strong enrichment for lipid metabolism genes. TCDD also dysregulated expression of >200 transcription factors, including 19 Nuclear Receptors, most notably in hepatocytes and Kupffer cells. TCDD-induced changes in cell–cell communication patterns included marked decreases in EGF signaling from hepatocytes to non-parenchymal cells and increases in extracellular matrix-receptor interactions central to liver fibrosis. Gene regulatory networks constructed from the snRNA-seq data identified TCDD-exposed liver network-essential lncRNA regulators linked to functions such as fatty acid metabolic process, peroxisome and xenobiotic metabolism. Networks were validated by the striking enrichments that predicted regulatory lncRNAs showed for specific biological pathways. These findings highlight the power of snRNA-seq to discover functional roles for many xenobiotic-responsive lncRNAs in both hepatocytes and liver non-parenchymal cells and to elucidate novel aspects of foreign chemical-induced hepatotoxicity and liver disease, including dysregulation of intercellular communication within the liver lobule.

A systematic review on hepatoprotective potential of grape and polyphenolic compounds: molecular mechanism and future prospective

The hepatoprotective potential of grape and polyphenols has been scientifically well demonstrated in several experimental studies. However, there is no systematic review performed on their hepatoprotective activity. Based on scientific evidence, the goal of this systematic review was to describe and make inferences on the therapeutic advantages of grape and polyphenols against various chemical hepatotoxicities. Grape and polyphenols from preclinical and clinical studies were included for the online search utilising electronic databases such as PubMed, Web of Science, Scopus, Springer Nature, and Google Scholar. The searched words and terms were: “Hepatotoxicity” OR “Hepatoprotective” OR “Liver injury” AND “Hepatotoxic drug” AND “Grape” OR “Grapefruit” OR “Grape seed” OR “Grape peels” OR “Bioactive compounds” OR “Polyphenols” AND “Preclinical studies” OR “Clinical studies”. A total of sixty-five studies were included based on the rigorous inclusion and exclusion criteria. The systematic review revealed that grape and polyphenolic compounds could significantly alleviate hepatic markers (ALP, ALT and AST), antioxidant status (CAT, GPx, GSH and SOD), hepatic anti-inflammatory status (TNF-α, IL-1, IL-6, COX-2, and NF-κB pathways) and apoptosis markers [caspase-3 (CASP) and caspase-9] in chemical induced-hepatotoxicity. Furthermore, improvements in the structural, functional, and physiological characteristics of the liver were observed after treatment with grape and polyphenols, reflecting modulation in the cellular and molecular alterations. Conclusively, this study supports the therapeutic potential of grape and polyphenols for the management of hepatotoxicity in various conditions based on available scientific evidence.

Reproducibility and robustness of high-throughput S1500+ transcriptomics on primary rat hepatocytes for chemical-induced hepatotoxicity assessment

Cell-based in vitro models coupled with high-throughput transcriptomics (HTTr) are increasingly utilized as alternative methods to animal-based toxicity testing. Here, using a panel of 14 chemicals with different risks of human drug-induced liver injury (DILI) and two dosing concentrations, we evaluated an HTTr platform comprised of collagen sandwich primary rat hepatocyte culture and the TempO-Seq surrogate S1500+ (ST) assay. First, the HTTr platform was found to exhibit high reproducibility between technical and biological replicates (r greater than 0.85). Connectivity mapping analysis further demonstrated a high level of inter-platform reproducibility between TempO-Seq data and Affymetrix GeneChip data from the Open TG-GATES project. Second, the TempO-Seq ST assay was shown to be a robust surrogate to the whole transcriptome (WT) assay in capturing chemical-induced changes in gene expression, as evident from correlation analysis, PCA and unsupervised hierarchical clustering. Gene set enrichment analysis (GSEA) using the Hallmark gene set collection also demonstrated consistency in enrichment scores between ST and WT assays. Lastly, unsupervised hierarchical clustering of hallmark enrichment scores broadly divided the samples into hepatotoxic, intermediate, and non-hepatotoxic groups. Xenobiotic metabolism, bile acid metabolism, apoptosis, p53 pathway, and coagulation were found to be the key hallmarks driving the clustering. Taken together, our results established the reproducibility and performance of collagen sandwich culture in combination with TempO-Seq S1500+ assay, and demonstrated the utility of GSEA using the hallmark gene set collection to identify potential hepatotoxicants for further validation.

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.

Structural characterization of phosphorylated Pleurotus ostreatus polysaccharide and its hepatoprotective effect on carbon tetrachloride-induced liver injury in mice

This study aimed to explore the basic structural features of phosphorylated Pleurotus ostreatus polysaccharide (PPOP) and study the protective effect of PPOP on liver injury induced by carbon tetrachloride in male Kunming mice. The phosphorylated polysaccharide was prepared from the natural polysaccharide extracted from Pleurotus ostreatus (POP). The structures of PPOP and POP were characterized by FT-IR, ESEM spectroscopy, and Congo red test. Chemical composition analysis revealed that PPOP was mainly composed of rhamnose, galacturonic acid, and xylose in a molar ratio of 0.10: 1.98: 1.00. Structural analysis indicated that PPOP had multi-strand structure and the absorption peaks of PO and P-O-C. Furthermore, animal experiments showed that the hepatoprotective effect of PPOP against liver injury was reflected by decreasing the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total cholesterol, trilaurin, and low-density lipoprotein cholesterol in the serum, increasing the content of high-density lipoprotein cholesterol and albumin in blood, reducing the content of malondialdehyde and promoting the activity of antioxidant enzymes in liver. PPOP exhibited stronger hepatoprotective effect and antioxidant activity in vivo than POP. The final results indicated that PPOP could be used in the treatment of chemical-induced hepatotoxicity based on the above biological research.

Predicting direct hepatocyte toxicity in humans by combining high-throughput imaging of HepaRG cells and machine learning-based phenotypic profiling.

Accurate prediction of drug- and chemical-induced hepatotoxicity remains to be a problem for pharmaceutical companies as well as other industries and regulators. The goal of the current study was to develop an in vitro/in silico method for the rapid and accurate prediction of drug- and chemical-induced hepatocyte injury in humans. HepaRG cells were employed for high-throughput imaging in combination with phenotypic profiling. A reference set of 69 drugs and chemicals was screened at a range of 7 concentrations, and the cellular response values were used for training a supervised classifier and for determining assay performance by using tenfold cross-validation. The results showed that the best performing phenotypic features were related to nuclear translocation of RELA(RELA proto-oncogene, NF-kB subunit; also known as NF-kappa B p65), DNA organization, and the F-actin cytoskeleton. Using a subset of 30 phenotypic features, direct hepatocyte toxicity in humans could be predicted with a test sensitivity, specificity and balanced accuracy of 73%, 92%, and 83%, respectively. The method was applied to another set of 26 drugs and chemicals with unclear annotation and their hepatocyte toxicity in humans was predicted. The results also revealed that the identified discriminative phenotypic changes were related to cell death and cellular senescence. Whereas cell death-related endpoints are widely applied in in vitro toxicology, cellular senescence-related endpoints are not, although cellular senescence can be induced by various drugs and other small molecule compounds and plays an important role in liver injury and disease. These findings show how phenotypic profiling can reveal unexpected chemical-induced mechanisms in toxicology.

Hepatocurative effect of aqueous extract of Scoparia dulcis (sweet broom) root on carbon tetrachloride-induced hepatotoxicity in albino rats

Most acute and chronic liver diseases today are due to drugs and other chemical-induced hepatotoxicity. A great number of herbal preparations have become increasingly popular for the treatment of liver disorders due to the absence of reliable hepatoprotective drugs in modern medicine. The aim of this work is to carry out acute toxicity study and investigate the hepatocurative effects of aqueous root extract of Scoparia dulcis against carbon tetrachloride (CCl4)-induced hepatoxicity in albino rats. Serum biomarkers of hepatic injury, alanine aminotransferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP), albumine (ALB), and bilirubin, were assayed according to standard methods. The results of the biochemical studies of blood samples of CCl4-treated rats showed significant (P ˂ 0.05) increase in the levels of ALT and AST and decrease in ALB level reflecting the liver injury caused by CCl4 whereas blood samples from rats treated with aqueous root extract of Scoparia dulcis showed decreased levels of ALT and AST and increase in ALB indicating the protection of hepatic cells. Histopathological features showed decreased infiltration of inflammatory cells, staetosis, and level of fibrosis in the liver tissues of the treated compared to the untreated groups. This further confirms the hepatocurative effect of Scoparia dulcis extract. The lethal dose (LD50) of the root extract was greater than 5000 mg/kg.

Investigation of hepatoprotective effect of some algae species on carbon tetrachloride-induced liver injury in rats

The aim of this study was to investigate hepatoprotective effect of some algae species such as Spirulina platensis, Chlorella vulgaris, Laminaria japonica, Sargassum sp. on experimental acute hepatotoxicity model that induced with carbon tetrachloride (CCl4) in rats. Algaes at a dose of 200 mg/kg and Silymarin at a dose of 25 mg/kg were orally administered for 7 days followed by CCl4 at a single dose (0.5 ml/kg), at the 8th day to cause experimental acute hepatotoxicity. Levels of biochemical (AST, ALT etc.), lipid peroxidation (MDA), antioxidant (GSH, CAT, GPx) parameters and histopathological examination were carried out to investigate the hepatoprotective effects of algae. In Sp group ALT and ALP levels were significantly decreased compared with CCl4 (p < .05). Histological liver structures of Sp group were similar to the control group. MDA, GPx and CAT levels of Sp and La groups were significantly different compared with CCl4 (p < .05). Based on these results, algae species able to minimise the toxic effects of CCl4 and especially S. platensis could be used in the purpose of protection against chemical-induced hepatotoxicity.

Comparison of microRNA expressions for the identification of chemical hazards in in vivo and in vitro hepatic injury models

Biofluid-based biomarkers provide an efficient tool for hazard identification of chemicals. Here, we explored the potential of microRNAs (miRNAs) as biomarkers for hepatotoxicity of chemicals by linking in vitro to in vivo animal models. A search of the literature identified candidate circulating miRNA biomarkers of chemical-induced hepatotoxicity. The expression of candidate miRNAs (miR-122, miR-151a, miR-192, miR-193a, miR-194, miR-21, miR-29c), was determined by real-time reverse transcription-polymerase chain reaction in in vivo acute liver injury induced by acetaminophen, and then were further compared with those of in vitro cell assays. Candidate miRNAs, except miR-29c, were significantly or biologically upregulated by acetaminophen, at a dose that caused acute liver injury as confirmed by hepatocellular necrosis. Except miR-122 and miR-193a, other miRNAs elevated in in vivo models were confirmed by in vitro models using HepG2 cells, whereas they failed by in vitro models using human primary hepatocytes. These findings indicate that certain miRNAs may still have the potential of toxicological biomarkers in linking in vitro to in vivo hepatotoxicity.

Zebrafish larva as a reliable model for in vivo assessment of membrane remodeling involvement in the hepatotoxicity of chemical agents.

The easy-to-use in vivo model, zebrafish larva, is being increasingly used to screen chemical-induced hepatotoxicity, with a good predictivity for various mechanisms of liver injury. However, nothing is known about its applicability in exploring the mechanism called membrane remodeling, depicted as changes in membrane fluidity or lipid raft properties. The aim of this study was, therefore, to substantiate the zebrafish larva as a suitable in vivo model in this context. Ethanol was chosen as a prototype toxicant because it is largely described, both in hepatocyte cultures and in rodents, as capable of inducing a membrane remodeling leading to hepatocyte death and liver injury. The zebrafish larva model was demonstrated to be fully relevant as membrane remodeling was maintained even after a 1-week exposure without any adaptation as usually reported in rodents and hepatocyte cultures. It was also proven to exhibit a high sensitivity as it discriminated various levels of cytotoxicity depending on the extent of changes in membrane remodeling. In this context, its sensitivity appeared higher than that of WIF-B9 hepatic cells, which is suited for analyzing this kind of hepatotoxicity. Finally, the protection afforded by a membrane stabilizer, ursodeoxycholic acid (UDCA), or by a lipid raft disrupter, pravastatin, definitely validated zebrafish larva as a reliable model to quickly assess membrane remodeling involvement in chemical-induced hepatotoxicity. In conclusion, this model, compatible with a high throughput screening, might be adapted to seek hepatotoxicants via membrane remodeling, and also drugs targeting membrane features to propose new preventive or therapeutic strategies in chemical-induced liver diseases. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of pharmaceutical formulations containing thyme on carbon tetrachloride-induced liver injury in rats.

BackgroundHerbal supplements are widely used in the treatment of various liver disases, but some of them may also induce liver injuries. Regarding the infuence of thyme and its constituents on the liver, conflicting results have been reported in the literature. The objective of this study was to examine the influence of two commonly used pharmaceutical formulations containing thyme (Thymus vulgaris L.), tincture and syrup, on carbon tetrachloride-induced acute liver injury in rats.MethodsChemical composition of investigated formulations of thyme was determined by gas chromatography and mass spectrometry. Activities of enzyme markers of hepatocellular damage in serum and antioxidant enzymes in the liver homogenates were measured using the kinetic spectrophotometric methods. Liver morphology was characterized by light microscopy using routine hematoxylin and eosin staining.ResultsThymol was found to be predominant active constituent in both tincture and syrup. Investigated thyme preparations exerted antioxidant effects in liver by preventing carbon tetrachloride-induced increase of lipid peroxidation. Furthermore, co-treatment with thyme preparations reversed the activities of oxidative stress-related enzymes xanthine oxidase, catalase, peroxidase, glutathione peroxidase and glutathione reductase, towards normal values in the liver. Hepatotoxicity induced by carbon tetrachloride was reflected by a marked elevation of AST and ALT activities, and histopathologic alterations. Co-administration of thyme tincture resulted in unexpected exacerbation of AST and ALT values in serum, while thyme syrup managed to reduce activites of aminotransferases, in comparison to carbon tetrachloride-treated animals.ConclusionsDespite demonstrated antioxidant activity, mediated through both direct free radical scavenging and activation of antioxidant defense mechanisms, thyme preparations could not ameliorate liver injury in rats. Molecular mechanisms of diverse effects of thyme preparations on chemical-induced hepatotoxicity should be more in-depth investigated.

Altered protein S-glutathionylation identifies a potential mechanism of resistance to acetaminophen-induced hepatotoxicity.

Acetaminophen (APAP) is the most commonly used over-the-counter analgesic. However, hepatotoxicity induced by APAP is a major clinical issue, and the factors that define sensitivity to APAP remain unclear. We have previously demonstrated that mice nulled for glutathione S-transferase Pi (GSTP) are resistant to APAP-induced hepatotoxicity. This study aims to exploit this difference to delineate pathways of importance in APAP toxicity. We used mice nulled for GSTP and heme oxygenase-1 oxidative stress reporter mice, together with a novel nanoflow liquid chromatography-tandem mass spectrometry methodology to investigate the role of oxidative stress, cell signaling, and protein S-glutathionylation in APAP hepatotoxicity. We provide evidence that the sensitivity difference between wild-type and Gstp1/2(-/-) mice is unrelated to the ability of APAP to induce oxidative stress, despite observing significant increases in c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation in wild-type mice. The major difference in response to APAP was in the levels of protein S-glutathionylation: Gstp1/2(-/-) mice exhibited a significant increase in the number of S-glutathionylated proteins compared with wild-type animals. Remarkably, these S-glutathionylated proteins are involved in oxidative phosphorylation, respiratory complexes, drug metabolism, and mitochondrial apoptosis. Furthermore, we found that S-glutathionylation of the rate-limiting glutathione-synthesizing enzyme, glutamate cysteine ligase, was markedly increased in Gstp1/2(-/-) mice in response to APAP. The data demonstrate that S-glutathionylation provides an adaptive response to APAP and, as a consequence, suggest that this is an important determinant in APAP hepatotoxicity. This work identifies potential novel avenues associated with cell survival for the treatment of chemical-induced hepatotoxicity.

Adhesion of platelets through thromboxane A₂ receptor signaling facilitates liver repair during acute chemical-induced hepatotoxicity.

Platelets have been suggested to play an important role in liver regeneration and repair after hepatic resection and acute liver injury. However, the underlying mechanisms of liver repair remain elusive. Signaling through thromboxane prostanoid (TP) receptor participates in inflammation and tissue injury through platelet aggregation. On the other hand, TP receptor signaling also is involved in tissue repair and tumor growth through angiogenesis. The present study was examined whether or not TP receptor signaling contributes to liver repair and sinusoidal restoration from acute liver injury through platelet adhesion to the hepatic sinusoids. Carbon tetrachrolide (CCl4) was used to induce acute liver injury in TP receptor knockout mice (TP(-/-) mice) and their wild-type littermates (WT mice). Compared with WT mice, TP(-/-) mice exhibited delayed in liver repair and sinusoidal restoration after CCl4 treatment, which were associated with attenuated hepatic expression of pro-angiogenic factors. Intravital microscopic observation revealed that adhering platelets to the sinusoids was increased in WT livers during the repair phase as compared with TP(-/-) livers, and platelet adhesion was dependent on TP receptor signaling. The levels of hepatocyte growth factor (HGF) in platelets from WT mice treated with CCl4 for 48h were greater than those form TP(-/-) mice, and HGF enhanced the expression of angiogenic factors in cultured human umbilical vein endothelial cells (HUVECs). These results suggested that TP receptor signaling facilitates liver repair and sinusoidal restoration from acute liver injury through HGF release from platelets adhering to the sinusoids.

Differential Effects of Trovafloxacin on TNF-α and IL-6 Profiles in a Rat Hepatocyte–Kupffer Cell Coculture System

Immune-mediated chemical-induced hepatotoxicity is often overlooked as a potential mode of action due to the lack of appropriate in vitro models. For this reason, we have established a coculture system of rat primary hepatocytes and Kupffer cells (KCs) that can be used to examine chemical-induced inflammatory reactions resulting in acute hepatocellular toxicity. Cocultures were maintained for 24 or 48 hr with various concentrations of lipopolysaccharide (LPS) and trovafloxacin (TVX), a compound associated with immune-mediated hepatotoxicity in vivo. Cytokine (IL-6 and TNFα) levels, CYP3A activity, and cell viability (ATP, LDH release) were measured to monitor endotoxin- and TVX-induced changes in metabolic capacity and cell integrity. LPS-treated cocultures showed marked downregulation of CYP3A activity, which correlated with upregulation of IL-6 and TNF-α production. TVX/LPS-treated cocultures exhibited concentration-dependent hepatocellular injury, represented by decreased ATP content and increased LDH leakage. The IL-6-induced downregulation of CYP3A was impaired in cocultures cotreated with TVX/LPS, which coincided with a concentration-dependent decline in IL-6 production. However, TNF-α production was initially upregulated in TVX/LPS-treated cocultures and KC monocultures before showing a gradual decline. The data from these studies suggest that shifts in cytokine profiles, especially IL-6/TNF-α ratios, may play a role in immune-mediated chemical-induced hepatotoxicity. Coculture of hepatocytes and KC represents a valuable in vitro tool to assess adverse liver effects resulting from indirect adaptive immune responses during chemical exposure.

Cyclooxygenase-1 serves a vital hepato-protective function in chemically induced acute liver injury.

Cyclooxygenase-1 (COX-1) is the constitutive form of the COX enzyme family, which produces bioactive lipids called prostanoids. Although the role of COX-2 in liver diseases has been studied, little is known about the function of COX-1 in liver injury. We aimed to investigate the role and mechanism of COX-1 in acute liver injury. Carbon tetrachloride (CCl(4)) was administered to induce acute liver injury in wild-type or COX-1-deficient mice. Both genetic (partially or completely) deletion of COX-1 expression and pharmacological inhibition of COX-1 activity in mice exacerbated acute liver injury induced by CCl(4), revealing the (1) histopathological changes and increased serum levels of aminotransferases; (2) oxidative stress in the liver partly through the action of cytochrome P450 2E1-dependent pathway; (3) enhanced inflammatory and chemoattractive responses with increased number of activated macrophages; and (4) increased apoptosis through both intrinsic and extrinsic apoptotic pathways. These pathological changes were partly through the modulation of transcription factor-dependent pathways (eg, NF-κB and C/EBP-α). Pre-treatment with prostaglandin E2 (PGE(2)) or 5-lipoxygenase (5-LO) inhibitor in homozygous COX-1 knockout mice significantly ameliorated CCl(4)-induced hepatic injury. In addition, level of hepato-protective molecules (eg, OSM and OSMR) and associated liver regeneration pathway were significantly inhibited by the deficiency of COX-1 but restored by the addition of PGE(2) or the inhibition of 5-LO. Furthermore, the alternative arachidonic acid metabolism pathway of 5-LO, which induced additional inflammation in the liver, was activated in response to the deficiency of COX-1. In conclusion, basal expression of COX-1 is essential for the protection of liver against chemical-induced hepatotoxicity and required for hepatic homeostatic maintenance.

A Micropatterned Hepatocyte Coculture Model for Assessment of Liver Toxicity Using High-Content Imaging Analysis

The current landscape of in vitro models used to identify drug- or chemical-induced hepatotoxicity relies heavily on cell culture models consisting of HepG2, induced pluripotent stem cell-derived, or primary hepatocytes. While these in vitro models offer powerful approaches for predicting toxicity, each system has challenges, including variable metabolic capacity, brief ex vivo life span in culture, and adoption with standard automated microscopy high-content screening (HCS) systems to measure reproducibility data at the single-cell level. In this report we introduce a novel primary hepatocyte coculture model, HepatoPac™, as an alternative to current model systems for evaluation of in vitro hepatotoxicity in 96-well microtiter plate format examined by HCS. The coculture model consists of primary hepatocytes that are micropatterned to form a discrete microarchitecture or "hepatocyte islands" that are surrounded by supporting fibroblasts resulting in long-term viability and metabolic function of primary hepatocytes. Using multiple HCS image capture and image analysis strategies, we established methods to interrogate various morphometric parameters, such as size, shape, and intensity, at the island or single-cell level. We applied these approaches to identify subpopulations of both fibroblasts and hepatocytes that exhibited alterations in nuclear parameters, cell permeability, mitochondria function, and apoptosis using known reference control compounds and an eight-point dose curve. Subpopulation analysis with additional bioprobe sets can provide a powerful means of addressing differential cell and tissue susceptibilities during compound profiling. Our data show that the HepatoPac is amendable for HCS imaging applications and provides a unique approach for studying hepatotoxicity over prolonged periods of time.

The protective role of pomegranate juice against carbon tetrachloride–induced oxidative stress in rats

Most pomegranate (Punica granatum Linn., Punicaceae) fruit parts are known to possess enormous antioxidant activity. The present study was carried out to determine the phenolic and flavonoid contents of Derik pomegranate juice and determine its effect against carbon tetrachloride (CCl4)-induced toxicity in rats. Animals were divided into four groups (n=6): group I: control, group II: CCl4 (1ml/kg), group III: CCl4+pomegranate juice and group IV: CCl4+ursodeoxycholic acid (UDCA). Treatment duration was 4weeks, and the dose of CCl4 was administered once a week to groups II, III and IV during the experimental period. CCl4-treated rats caused a significant increase in serum enzyme levels, such as aspartate aminotransferase, alanine aminotransferase and total bilirubin, and decrease in albumin, when compared with control. Administration of CCl4 along with pomegranate juice or UDCA significantly reduces these changes. Analysis of lipid peroxide (LPO) levels by thiobarbutiric acid reaction showed a significant increase in liver, kidney and brain tissues of CCl4-treated rats. However, both pomegranate juice and UDCA prevented the increase in LPO level. Histopathological reports also revealed that there is a regenerative activity in the liver and kidney cells. Derik pomegranate juice showed to be hepatoprotective against CCl4-induced hepatic injury. In conclusion, present study reveals a biological evidence that supports the use of pomegranate juice in the treatment of chemical-induced hepatotoxicity.

Hepatoprotective effects of Gentiana asclepiadea L. extracts against carbon tetrachloride induced liver injury in rats

This study is an attempt to evaluate the hepatoprotective activity of Gentiana asclepiadea L. against carbon tetrachloride-induced liver injury in rats. Methanol extracts of aerial parts (GAA) and roots (GAR) of G. asclepiadea at doses of 100, 200, and 400mg/kgb.w. were orally administered to Wistar rats once daily for 7days before they were treated with CCl4. The hepatoprotective activity of the extracts in this study was compared with the reference drug silymarin. In CCl4 treated animals, GAA and GAR significantly decreased levels of serum transaminases, alkaline phosphatase and total bilirubin, and increased the level of total protein. Treatment with the extracts resulted in a significant increase in the levels of catalase, superoxide dismutase and reduced glutathione, accompanied with a marked reduction in the levels of malondialdehyde, as compared to CCl4 treated group. The histopathological studies confirmed protective effects of extracts against CCl4-induced liver injuries. No genotoxicity was observed in liver cells after GAA treatment, while GAR showed only slight genotoxic effects by comet assay. Phytochemical analysis revealed the presence of sweroside, swertiamarin and gentiopicrin in high concentrations in both extracts. It could be concluded that the use of G. asclepiadea extracts in the treatment of chemical-induced hepatotoxicity.