Hepatotoxicity In Fish Research Articles

Cytotoxicity of pharmaceuticals and their mixtures toward scaffold-free 3D spheroid cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes

Pharmaceutical residues are widely detected in surface waters all around the world, causing a range of adverse effects on environmental species, such as fish. Besides population level effects (mortality, reproduction), pharmaceutical residues can bioaccumulate in fish tissues resulting in organ-specific toxicities. In this study, we developed in vitro 3D culture models for rainbow trout (Oncorhynchus mykiss) liver cell line (RTH-149) and cryopreserved, primary rainbow trout hepatocytes (RTHEP), and compared their spheroid formation and susceptibility to toxic impacts of pharmaceuticals. The rapidly proliferating, immortalized RTH-149 cells were shown to form compact spheroids with uniform morphology in just three days, thus enabling higher throughput toxicity screening compared with the primary cells that required acclimation times of about one week. In addition, we screened the cytotoxicity of a total of fourteen clinically used human pharmaceuticals toward the 3D cultures of both RTH-149 cells (metabolically inactive) and primary RTHEP cells (metabolically active), to evaluate the impacts of the pharmaceuticals’ own metabolism on their hepatotoxicity in rainbow trout in vitro. Among the test substances, the azole antifungals (clotrimazole and ketoconazole) were identified as the most cytotoxic, with hepatic metabolism indicatively amplifying their toxicity, followed by fluoxetine, levomepromazine, and sertraline, which were slightly less toxic toward the metabolically active primary cells than RTH-149 spheroids. Besides individual pharmaceuticals, the 3D cultures were challenged with mixtures of the eight most toxic substances, to evaluate if their combined mixture toxicities can be predicted based on individual substances’ half-maximal effect (EC50) concentrations. As a result, the classical concentration addition approach was concluded sufficiently accurate for preliminarily informing on the approximate effect concentrations of pharmaceutical mixtures on a cellular level. However, direct read-across from human data was proven challenging and inexplicit for prediction of hepatotoxicity in fish in vitro.

Dietary exposure to 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) induces oxidative damage promoting cell apoptosis primarily via mitochondrial pathway in the hepatopancreas of carp, Cyprinus carpio

To investigate the mechanisms of BDE-47 on hepatotoxicity in fish, this study examined the effects of dietary exposure to BDE-47 (40 and 4000 ng/g) on carp for 42 days. The results showed that BDE-47 significantly increased carp’s condition factor and hepatosomatic index. Pathological results revealed unclear hepatic cord structure, hepatocytes swelling, cellular vacuolization, and inflammatory cell infiltration in the hepatopancreas of carp. Further investigation showed that ROS levels significantly increased on days 7, 14, and 42. Moreover, the activities of antioxidant enzymes SOD, GSH, CAT, and GST increased significantly from 1 to 7 days, and the transcription levels of antioxidant enzymes CAT, Cu-Zn SOD, Mn-SOD, GST, and GPX, and antioxidant pathway genes Keap1, Nrf2, and HO-1 changed significantly at multiple time-points during the 42 days. The results of apoptosis pathway genes showed that the mitochondrial pathway genes Bax, Casp3, and Casp9 were significantly upregulated and Bcl2 was significantly downregulated, while the transcription levels of FADD and PERK were significantly enhanced. These results indicate that BDE-47 induced oxidative damage in hepatopancreas, then it promoted cell apoptosis mainly through the mitochondrial pathway. This study provides a foundation for analyzing the mechanism of hepatotoxicity induced by BDE-47 on fish.

Glyphosate-induced lipid metabolism disorder contributes to hepatotoxicity in juvenile common carp.

Residues of glyphosate (GLY) are widely detected in aquatic systems, raising potential environmental threats and public health concerns, but the mechanism underlying GLY-induced hepatotoxicity in fish has not been fully elucidated yet. This study was designed to explore the hepatotoxic mechanism using juvenile common carp exposed to GLY for 45d, and plasma and liver samples were collected at 15d, 30d, and 45d to analyze the assays. First, GLY-induced hepatic damage was confirmed by serum liver damage biomarker and hepatic histopathological analysis. Next, changes in oxidative stress biomarkers, gene expression levels of pro- and anti-inflammatory cytokines, and lipid metabolism-related parameters in collected samples were analyzed to clarify their roles in GLY-induced hepatic damage. Data showed that oxidative stress was an early event during GLY exposure, followed by hepatic inflammatory response. Lipid metabolism disorder was a late event during GLY exposure, as evidenced by overproduced hepatic free fatty acids, enhanced lipogenesis-related gene expression levels, reduced lipolysis-related gene expression levels, and resultant hepatic lipid accumulation. Collectively, these findings demonstrate that GLY induces hepatotoxicity in fish through involvement of oxidative stress, inflammatory response, and lipid metabolism disorder, which are intimately interrelated with each other during GLY exposure.

A protective study of curcumin associated with Cr6+ induced oxidative stress, genetic damage, transcription of genes related to apoptosis and histopathology of fish, Channa punctatus (Bloch, 1793)

Ameliorative potential of curcumin against Cr6+-induced eco-toxicological manifestations was assessed in liver of exposed Channa punctatus (Actinopterygii) in six groups for 45 d; Group I as control. Group II with 3 mg/L of curcumin; group III with 7.89 mg/L of Cr6+. Groups IV, V and VI were simultaneously co-exposed with 7.89 mg/L of Cr6+ and three different curcumin concentrations, 1, 2, and 3 mg/L, respectively. In group III, SOD-CAT, GR significantly (p < 0.05) increased; decreased GSH level; elevated MN and AC frequencies; and a significant (p < 0.05) up-regulation of cat (2.72-fold), p53 (1.73-fold), bax (1.33-fold) and apaf-1 (2.13-fold) together with a significant (p < 0.05) down-regulation of bcl-2 (0.51-fold). Co-exposure significantly (p < 0.05) brought down activities of SOD-CAT, GR, raised GSH, decreased micronuclei and apoptotic frequencies along with recovery of histopathological anomalies in liver. This study establishes the protective role of curcumin against Cr6+-induced hepatotoxicity in fish.

Sex-dependent effects of subacute mercuric chloride exposure on histology, antioxidant status and immune-related gene expression in the liver of adult zebrafish (Danio rerio)

Mercury (Hg) is a global pollutant that causes negative health effects. In order to assess Hg-induced hepatotoxicity in fish and examine whether gender differences existed in response to Hg exposure, adult zebrafish were exposed to 0, 15 and 30 μg L−1 Hg2+ for 30 days, and histology, antioxidant status and the transcription levels of several immune-related genes were examined in the liver. Hg2+ exposure caused a dose-dependent increase in histopathological lesions of the liver, including vacuolization, parenchyma disorganization and pyknotic nucleus, and these lesions were more severe in males than in females. In females, Hg2+ exposure decreased CAT activity and its mRNA levels, while increased GSH content and the expressions of sod1, gpx1a, gstr and keap1. In males, the decrease in cat1 expression and the increase in GST activity, GSH and MDA contents as well as gpx1a, gstr, nrf2 and keap1 mRNA levels were observed in Hg2+-exposed groups, but the activities of CAT, SOD and GPX were only stimulated in the 15 μg L−1 Hg2+ group. Moreover, both in females and males, Hg2+ exposure down-regulated il-8 expression while up-regulated il-10 and lyz mRNAs. However, the down-regulation of il-1β and tnfα was detected only in males under Hg2+ treatments. Thus, our results indicated that HgCl2 exposure induced histopathological damage, oxidative stress and immunotoxicity in the liver of zebrafish. Different response patterns of histology, antioxidant status and immune defenses to Hg2+ between females and males suggested sex-dependent effects of Hg, and males showed more vulnerable to Hg2+ exposure than females.

Histopathological and proteomic responses in male Chinese rare minnow (Gobiocypris rarus) indicate hepatotoxicity following benzotriazole exposure

Benzotriazole (BT) and its associated derivatives are used ubiquitously in industrial processes, and can be detected in indoor temperature coolants and in chemicals designed to inhibit corrosion. This chemical has been widely detected in aquatic environments and shows some degree of environmental persistence. Evidence has shown that BT exposure can negatively affect endocrine systems and can result in neurotoxicity in fish. However, no study has examined whether this chemical exhibits hepatotoxicity in fish, and if so, what are the underlying mechanism associated with the damage. To address this knowledge gap, we measured the liver proteome of adult male Chinese rare minnow (Gobiocypris rarus) exposed to either 0.05, 0.5, or 5 mg/L BT for 28 days. Overall, 17 proteins were induced and 9 were reduced in abundance following BT treatment (ratio > 1.5, p < 0.05). Pathway analysis revealed that cellular processes affected by BT included xenobiotic clearance, oxidative stress response, apoptosis, and translation. Moreover, transcripts related to these toxic pathways were also significantly affected by BT. In addition, rare minnows exposed to BT showed signs of hypertrophy of hepatocytes, nuclei pyknosis, and higher levels of cellular vacuolization compared to the controls, thus these early proteomic responses in the liver may be related to pathology (i.e. adverse outcome pathway). Our data demonstrate that BT dysregulates molecular responses in the liver and tissue pathology indicative of damage. This study provides new insight into BT hepatotoxicity in Chinese rare minnow.

A glyphosate-based herbicide induces histomorphological and protein expression changes in the liver of the female guppy Poecilia reticulata

Glyphosate-based herbicides (GBH) are among the most common herbicides found in aquatic systems, but limited data are available about their mode of action and hepatotoxicity in fish. This study investigated the hepatotoxicity induced by GBH in the guppy Poecilia reticulata using a histopathological assessment associated with a proteomic approach. Guppies were exposed to GBH for 24 h at 1.8 mg of glyphosate L−1, corresponding to 50% of the LC50, 96 h. The results indicate that the GBH at 1.8 mg of glyphosate L−1 induce the development of hepatic damage in P. reticulata, which is exposure-time dependent. The histopathological indexes demonstrate that GBH cause inflammatory, regressive, vascular and progressive disorders in the liver of guppies. Using 2D gel electrophoresis associated with mass spectrometry, 18 proteins that changed by GBH were identified and were related to the cellular structure, motility and transport, energy metabolism and apoptosis. The results show that the acute exposure to GBH causes hepatic histopathological damage related to protein expression profile changes in P. reticulata, indicating that a histopathological assessment associated with a proteomic analysis provides a valuable approach to assess the toxic effects of GBH in sentinel fish species.

Molecular Cloning and Expression Determination of p38 MAPK from the Liver and Kidney of Silver Carp.

The sequence of p38MAPK in silver carp (Hm-p38a) was cloned and sequenced. Additionally, the acute toxicity of crude microcystins (MCs) on silver carp and induction expression of Hm-p38a by MCs exposure were also determined in this study. The results reveal that the length of Hm-p38a is 2418 bp and it contains a 1086 bp open reading frame. Hm-p38a could encode 361 amino acids. Sequence analysis indicates that Hm-p38a contains the conserved structures of Thr-Gly-Tyr motif and substrate binding site Ala-Thr-Arg-Trp, and it is highly conserved in fish. Phylogenetic analysis reveals that Hm-p38a is more closely related to fish than mammals and it belongs to p38α subfamily. Moreover, Hm-p38a in silver carp is constitutively expressed in all examined tissues. In addition, our results indicate that MCs exposure significantly promotes the transcription of Hm-p38a in fish liver or kidney, suggesting that mitogen-activated protein kinase should also be the signal pathway of MCs hepatotoxicity in fish.

Vitamin C and Chitosan Alleviate Toxic Effects of Paraquat on Some Biochemical Parameters in Hepatocytes of Common Carp

Background: Paraquat is nonselective bipyridyl herbicide that induces hepatotoxicity through oxidative stress. Vitamin C and chitosan have antioxidant as well as radical scavenger properties and show protective effects against reactive oxygen species (ROS). In the present study, hepatoprotective effects of chitosan and vitamin C were evaluated in common carp exposed to paraquat. Methods: While exposed to 0.02 mg. L-1paraquat for 21 days, common carp were fed chitosan (1000 mg. kg‑1 feed), vitamin C (1000 mg. kg-1 feed), and vitamin C combined with chitosan. At the end of the experiment, activities of hepatic enzymes and oxidative stress biomarkers were evaluated. Results: Paraquat induces changes in the activity of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase and lactate dehydrogenase in liver tissue of fish. However, these enzymes were restored to normal levels in fish fed with vitamin C and vitamin C combined with chitosan following exposure to paraquat. Increased levels of malondialdehyde were observed in liver after exposure to paraquat, while glucose-6-phosphate dehydrogenase and catalase activities and the total antioxidant levels decreased. Administration of vitamin C combined with chitosan significantly reduced malondialdehyde levels and increased the total antioxidant capacity, glucose-6-phosphate dehydrogenase and catalase activities. Conclusion: Administration of vitamin C is effective in reducing liver toxicity of paraquat. However, administrating both vitamin C and chitosan is more effective. In other words, chitosan and vitamin C have a synergic effect. They could be used as hepatoprotective agents against paraquat-induced hepatotoxicity in fish.

Unravelling the mechanisms of mercury hepatotoxicity in wild fish (Liza aurata) through a triad approach: bioaccumulation, metabolomic profiles and oxidative stress.

Clarification of the mechanisms of mercury (Hg) toxicity in fish remains challenging. An innovative approach to this clarification is the combined assessment of metabolomic shifts, alterations in the antioxidant system and the risk of oxidative damage, and Hg bioaccumulation. This strategy was applied to the livers of golden grey mullet (Liza aurata) inhabiting an Hg-contaminated system in Aveiro Lagoon, Portugal. Marked changes in both the metabolic profile and the response to antioxidants were observed in fish exposed to Hg in the environment, indicating their compromised state of health. The metabolomics analysis showed a clear differentiation between the contaminated and uncontaminated areas, revealing that Hg has multiple levels of impact, including membrane stabilization/degradation/repair processes, osmoregulation, energy metabolism, gene expression and antioxidant protection. Research on oxidative stress biomarkers showed that Hg triggered adaptive responses in the antioxidant system as seen by an increase in the activities of glutathione-S-transferase and catalase, as well as the total glutathione content, which compensated for a decrease in glutathione peroxidase activity. The integration of metabolomics and oxidative stress endpoints allowed the identification of reduced glutathione as a first line of defence against Hg and evidenced oxidative insults in cell membranes, recognized through the increase in phosphatidylcholine degradation products (phosphocholine and glycerophosphocholine). Despite these effects, the induction of lipid peroxidation was efficiently prevented. The triad approach used here was demonstrated to be sensitive and effective towards a mechanistically based assessment of Hg hepatotoxicity in fish.

Effects of carbon tetrachloride on oxidative stress, inflammatory response and hepatocyte apoptosis in common carp (Cyprinus carpio)

In the present study, the cellular and molecular mechanism of carbon tetrachloride (CCl4)-induced hepatotoxicity in fish was investigated by studying the effects of CCl4 on the oxidative stress, inflammatory response and hepatocyte apoptosis. Common carp were given an intraperitoneal injection of 30% CCl4 in arachis oil (0.5ml/kg body weight). At 72h post-injection, blood were collected to measure glutamate pyruvate transaminase (GPT), glutamate oxalate transaminase (GOT), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione (GSH), total antioxidant capacity (T-AOC) and malondialdehyde (MDA), liver samples were taken to analyze toll-like receptor 4 (TLR4), cytochrome P450 2E1 (CYP2E1) and gene expressions of inflammatory cytokines and nuclear factor-κB (NF-κB/cREL). Cell viability and apoptosis were analyzed after treatment of the primary hepatocytes with CCl4 at 8mM. The results showed that CCl4 significantly increased the levels of GPT, GOT, MDA, TLR4 and CYP2E1, reduced the levels of SOD, GPx, CAT, GSH and T-AOC, and up-regulated the gene expressions of NF-κB/cREL and inflammatory cytokines including tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), IL-1β, IL-6 and IL-12. In vitro, CCl4 caused a dramatic loss in cell viability and induced hepatocyte apoptosis. Overall results suggest that oxidative stress lipid peroxidation, and TNF-α/NF-κB and TRL4/NF-κB signaling pathways play important roles in CCl4-induced hepatotoxicity in fish.

The protective effect of silymarin on the carbon tetrachloride (CCl4)-induced liver injury in common carp (Cyprinus carpio)

Silymarin, a mixture of bioactive flavonolignans from the milk thistle (Silybum marianum), is traditionally used in herbal medicine to defend against various hepatotoxic agents. The aim of the present study was to evaluate the protective effect of silymarin against carbon tetrachloride (CCl4)-induced liver injury in fish. Common carp, with an average initial weight of 17.0 ± 1.1g, were fed diet containing four doses of silymarin (0, 0.1, 0.5, and 1g/kg diet) for 60d. Fish were then given an intraperitoneal injection of CCl4 (30% in arachis oil) at a dose of 0.5ml/kg body weight. At 72h after CCl4 injection, blood and liver samples were collected for the analyses of serum biochemical parameters, liver index, peroxidation product, glutathione, and antioxidant enzyme activities. The results showed that administration of silymarin at 0.5 and 1g/kg diet for 60d prior to CCl4 intoxication significantly reduced the elevated activities of glutamate pyruvate transaminase, glutamate oxalate transaminase, lactate dehydrogenase (LDH), and increased the reduced levels of total protein and albumin in the serum. The reduced levels of liver index, superoxide dismutase, glutathione peroxidase, catalase, glutathione, and total antioxidant capacity were markedly increased, and malondialdehyde formation was significantly restrained in the liver. However, these parameters, except LDH, were not significantly changed in fish fed with silymarin at 0.1g/kg diet. Based on the results, it can be concluded that silymarin has protective effect against CCl4-induced hepatotoxicity in fish. It is suggested that silymarin may be used as a hepatoprotective agent to prevent liver diseases in fish.

The Liquorice Plant Extract proved CCL4-induced damages of the fish hepatoxicity

The Liquorice Plant Extract (LPE) has been used traditionally in the world for various therapeutic purposes. LPE are extensively used in gastric ulcer, anticancer, inflammation and allergy. The hepatotoxic effect of carbon tetrachloride is due to the oxidative damage by free radical production, and antioxidant property is thought to be one of the approaches of hepatoprotective therapeutic agents. Moreover, CCL4-induced hepatoxicity could be used as a model for plenty of water contamination including chlorinated hydrocarbons. Therefore, we aimed to evaluate the antioxidant effect of LPE CCl4-induced hepatotoxicity in fish. Fresh roots of LP were air-dried; powdered and ethanolic extract was prepared using suxhlet. The hepatoprotective study was carried out in Common Carp (Cyprinus Carpio) weighing (30-50g). Thirty nine fishes were equally divided into 5 groups (n = 3 in each group). Group I (control) and group II were given DMSO and CCl4 respectively, for 45 minutes. CCL4 was administered in 3-subgroup at 3 various concentrations for 10 days. Group III, group IV and V were pretreated with LPE at different concentrations for 10 days and daily 3 h, and followed the same protocol of CCL4-treated group for 45 min. At the end of 10th day all groups anesthetized with Eugenol powder and blood samples were collected in heparanized tubes. The activity of two key liver-functional-enzymes namely Glutamic-Oxalacetic Transaminase (SGOT) and Alkaline Phosphatase (ALP) were measured to evaluate the potential effect of proposed herbal agent as hepatoprotective. Additionally, total in vivo antioxidant effect o f used compound was assessed, as well. The biochemical analyses revealed that: Firstly; CCL4 lowered total antioxidant power concentration-dependently and proposed herbal chemical could prove the decreased power in dose-dependent fashion; secondly ALP activity in serum following CCL4 treatment declined dramatically and at medium concentration of used herbal extract we obtained the remarkable increase in ALP activity. Moreover SGOT activity in sham groups decreased in comparison to control and only the lowest concentration of used drug could increase the activity. In conclusion, our data suggest that the CCL4-induced toxicity could be used as a model for water

A Brief Overview of Nonneoplastic Hepatic Toxicity in Fish

Biochemical assays are not routinely used to assess liver damage in fish, therefore, a histopathological evaluation is usually required to determine the existence or extent of nonneoplastic liver toxicity. Many mammalian pathologists may be uncomfortable when requested to identify and interpret subtle liver changes in these unfamiliar animals. It may be reassuring to note that there are more similarities than differences between fish and mammals in terms of their macro- and microanatomy, physiological and biochemical characteristics, and pathologic responses to hepatotoxic substances. This brief overview addresses several topics pertaining to hepatotoxicity in fish, including: anatomic considerations, that is, how the anatomy of the fish liver may be predictive of its metabolic capacity, and also its microscopic appearance, following exposure to toxins; physiologic considerations, including comparisons between mammalian and fish livers regarding the uptake, elimination, toxification, or detoxification of xenobiotic compounds; morphologic responses to toxicity, in which some of the general types of findings that are most commonly observed in cases or studies of fish hepatotoxicity are highlighted; and last, responses of the fish liver to specific hepatotoxins.

Human influence on the global cycling of trace metals

Mercury (Hg) is a global pollutant that causes negative health effects. In order to assess Hg-induced hepatotoxicity in fish and examine whether gender differences existed in response to Hg exposure, adult zebrafish were exposed to 0, 15 and 30 μg L−1 Hg2+ for 30 days, and histology, antioxidant status and the transcription levels of several immune-related genes were examined in the liver. Hg2+ exposure caused a dose-dependent increase in histopathological lesions of the liver, including vacuolization, parenchyma disorganization and pyknotic nucleus, and these lesions were more severe in males than in females. In females, Hg2+ exposure decreased CAT activity and its mRNA levels, while increased GSH content and the expressions of sod1, gpx1a, gstr and keap1. In males, the decrease in cat1 expression and the increase in GST activity, GSH and MDA contents as well as gpx1a, gstr, nrf2 and keap1 mRNA levels were observed in Hg2+-exposed groups, but the activities of CAT, SOD and GPX were only stimulated in the 15 μg L−1 Hg2+ group. Moreover, both in females and males, Hg2+ exposure down-regulated il-8 expression while up-regulated il-10 and lyz mRNAs. However, the down-regulation of il-1β and tnfα was detected only in males under Hg2+ treatments. Thus, our results indicated that HgCl2 exposure induced histopathological damage, oxidative stress and immunotoxicity in the liver of zebrafish. Different response patterns of histology, antioxidant status and immune defenses to Hg2+ between females and males suggested sex-dependent effects of Hg, and males showed more vulnerable to Hg2+ exposure than females.