Ultrastructural alterations in the liver and intestine of carp Cyprinus carpio induced orally by ultra-low doses of endosulfan.

In order to elucidate the importance of food-borne chemical contamination in fish, cytological and ultrastructural alterations in hepatocytes and enterocytes of common carp Cyprinus carpio L. exposed for 5 wk to 0.5 microgram endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9a-hexahydro-6,9-methano-2,4,3-benzo- dioxyanthiepin-3-oxide) kg-1 food dry weight, equivalent to an ultra-low dosis of 15 ng kg-1 fish d-1, were investigated by means of light and electron microscopy. Observations on liver alterations were quantified by morphometric analysis. Livers show enlargement of the nucleolus, increase in number and size of both Golgi fields and rough endoplasmic reticulum (ER) lamellae, as well as proliferation of peroxisomes and lysosomes. Taken together, these alterations represent the morphological equivalent of a general stimulation of hepatic metabolism. Proliferation of the smooth ER is indicative of the onset of biotransformation processes under the influence of food-borne endosulfan. Further pathological processes in the liver were evident by glycogen and lipid depletion, invasion of phagocytic macrophages, and accumulation of myelinated bodies in endothelial cells of hepatic sinusoids. In the intestinal tract, exposure to endosulfan is associated with a complete lack of chylomicrons in the epithelial lining, which indicates disturbance of intestinal absorption. The reaction of the gut epithelium also included considerable distension of the intercellular space and an elevated number of lysosomal inclusions in enterocytes. An increased rate of mucous cell precursors was detectable, and macrophages were numerous. Results are consistent with endosulfan resorption by the intestinal epithelium and the coexistence of gut and liver ultrastructural changes at extremely low doses. Thus, the substantiation of pathological alterations in organs sequentially in contact with toxicants appears useful as a biomarker of pollutant exposure and effect. With regard to a chemical spill into the Rhine river at Basel, Switzerland, in November 1986, endosulfan, as a component of the mixture of toxic substances, may well have contributed to the overall toxicity of the chemicals released during the accident and the subsequent fish kill, less as a toxicant in itself than as a stimulant for the toxicity of other xenobiotics.     

Use of hepatocytes cultured in microcarriers for correction of acute hepatic insufficiency

The ability of hepatocytes cultured on Biosilon microcarriers to secrete albumin and to conjugate bilirubin were examined for the purpose of developing an artificial liver support system. Cultured hepatocytes were able to synthesize 100-120 micrograms albumin per 10(5) cells in 24 hours and to conjugate about 20 micrograms/hr of bilirubin for at least 5 days. Rats with liver failure caused by i.p. injection of CCL4 or D-galactosamine were subjected to hemosorption via minicolumns containing 2 ml of Biosilon microcarriers with 40 X 10(6) cultured hepatocytes. The procedure was performed 20-24 h after hepatotoxins injection and lasted for 3 h at a flow rate of 60 ml/h. This reduced mortality from 100% to 20% after 48% and to 40% after 7 days in case of CCl4 and from 100% to 40% after 48 h and 7 days in case of D-galactosamine. Our results suggest that hepatocytes cultured on microcarriers may be efficiently applied to correction of fulminant hepatic failure caused by different hepatotoxins.     

肝脏移植与肠道微生态变化的研究进展

摘要：肝移植已成为公认的治疗终末期肝疾病的有效手段。由于“肝－肠轴”和“肝脏－微生态轴”的存在,肝移植术后感染和移植肝损伤与肠道微生态变化密切相关。本研究综述了肝脏移植与肠道微生态变化的研究进展,主要包括大鼠肝移植中缺血再灌注损伤与肠道微生态变化、大鼠肝移植后移植排斥损伤与肠道微生态变化、大鼠肝损伤肝移植与肠道微生态变化的互作关系,以及临床肝移植围手术期、术前术后等与肠道微生态变化的研究。同时,指出了临床肝脏移植与肠道微生态变化可能的研究方向,提出了肝移植后微生物标志物的鉴定及潜在的靶向微生物治疗的策略。     

Experimental hyperprolinemia induces mild oxidative stress, metabolic changes, and tissue adaptation in rat liver

The present study investigated the effects of chronic hyperprolinemia on oxidative and metabolic status in liver and serum of rats. Wistar rats received daily subcutaneous injections of proline from their 6th to 28th day of life. Twelve hours after the last injection the rats were sacrificed and liver and serum were collected. Results showed that hyperprolinemia induced a significant reduction in total antioxidant potential and thiobarbituric acid-reactive substances. The activities of the antioxidant enzymes catalase and superoxide dismutase were significantly increased after chronic proline administration, while glutathione (GSH) peroxidase activity, dichlorofluorescin oxidation, GSH, sulfhydryl, and carbonyl content remained unaltered. Histological analyses of the liver revealed that proline treatment induced changes of the hepatic microarchitecture and increased the number of inflammatory cells and the glycogen content. Biochemical determination also demonstrated an increase in glycogen concentration, as well as a higher synthesis of glycogen in liver of hyperprolinemic rats. Regarding to hepatic metabolism, it was observed an increase on glucose oxidation and a decrease on lipid synthesis from glucose. However, hepatic lipid content and serum glucose levels were not changed. Proline administration did not alter the aminotransferases activities and serum markers of hepatic injury. Our findings suggest that hyperprolinemia alters the liver homeostasis possibly by induction of a mild degree of oxidative stress and metabolic changes. The hepatic alterations caused by proline probably do not implicate in substantial hepatic tissue damage, but rather demonstrate a process of adaptation of this tissue to oxidative stress. However, the biological significance of these findings requires additional investigation.     

A combined healthy strategy for successful weight loss,weight maintenance and improvement of hepatic lipid metabolism

Obesity is critically related with the development of metabolic and pathophysiological alterations among which non-alcoholic fatty liver disease (NAFLD) is of especial relevance. Although there are numerous strategies to successfully treat obesity, the prevention of weight regain still remains challenging for individuals who have undergone weight loss programs. In such context, diet and physical activity are considered essential for the regulation of body weight and lipid metabolism. In this study, rats were fed a high-fat diet (HFD) to induce obesity and alterations in hepatic lipid metabolism. Obese rats were then treated with single or combined strategies of caloric restriction, physical exercise, and/or pharmacological treatment with an appetite suppressant, to lose weight, reverse the obesity-related alterations in hepatic morphology and lipid metabolism and maintain the beneficial effects of the interventions used. HFD induced excess body weight, hepatic steatosis, altered fatty acid profile, dysregulated gene expression of lipogenic and lipolytic enzymes, as well as plasma markers of liver damage, and modifications in liver antioxidant enzyme activity. Such alterations were ameliorated by caloric restriction in combination with a mixed training protocol and/or food-intake inhibitor administration during a weight loss intervention period of 3 weeks, and the beneficial effects remained after 6 weeks of weight maintenance, with some interesting interactions observed. In conclusion, weight loss strategies assayed were efficient at correcting the obesogenic action of a HFD and related alterations in hepatic functionality through different molecular mechanisms. The beneficial effects were also evident along the post-intervention maintenance period to avoid body weight regain.     

Prolonged induction of hepatic haem oxygenase and decreases in cytochrome P-450 content by organotin compounds.

The administration of organotin compounds to rats in single doses causes a significant and prolonged induction of haem oxygenase and a sustained decrease in haemoprotein content in the liver. The extent of induction of hepatic haem oxygenase varied between 3 and 5-fold at 72h after a single injection of water-insoluble organotins of differing structure. The alterations in haem metabolism produced by tricyclohexyltin hydroxide were studied in detail. The effects were dose-dependent, with doses as low as 3.75 mg/kg body wt. resulting in significant induction of haem oxygenase and a decrease in cytochrome P-450 and cytochrome b5 contents at 72h in the liver. The effects with time of a single dose of tricyclohexyltin on various parameters of liver haem metabolism were also examined. The organotin produced a substantial and very prolonged induction of haem oxygenase accompanied by a steady decline in cytochrome P-450 content for periods up to 8 days. The long duration of action of these organotins with respect to induction of haem oxygenase and depletion of cellular haemoprotein content provides a highly sensitive metabolic system with which to define further the toxic potential of organometals as well as to study the adaptive responses in liver to long-term perturbations of haem metabolism by foreign chemicals.     

Early Histological and Functional Effects of Chronic Copper Exposure in Rat Liver

Cu is an essential trace element capable of producing toxic effects in animals and man when ingested acutely or chronically in excess. Although chronic Cu exposure is increasingly recognized as a public health issue, its early effects remain largely unknown. We approached the significance of a moderate chronic Cu load in young rats to correlate early hepatic histopathological changes with functional alterations of liver cells. For this purpose, supplementation with 1200 ppm of Cu in rat food for 16 weeks was chosen. In these conditions, Cu load elicited a significant decrease in growth curves. There were mild light microscopy alterations in Cu-treated rats, although increasing intracellular Cu storage was correlated with longer Cu exposure both by histological and biochemical measurements. Ultrastructural alterations included lysosomal inclusions as well as mitochondrial and nuclear changes. Liver perfusion studies revealed higher rates of basal O₂ consumption and colloidal carbon-induced O₂ uptake in Cu-treated rats, with enhanced carbon-induced O₂/carbon uptake ratios and NF-κB DNA binding activity. These changes were time-dependent and returned to control values after 12 or16 weeks. It is concluded that subchronic Cu loading in young rats induces early hepatic morphological changes, with enhancement in Küpffer cell-dependent respiratory burst activity and NF-κB DNA binding, cellular responses that may prevent or alleviate the hepatotoxicity of the metal.     

Modification of hepatic vitamin E stores in vivo. I. Alterations in plasma and liver vitamin E content by methyl ethyl ketone peroxide

Since experiments with freshly isolated rat hepatocytes have shown that cellular vitamin E is consumed in response to insult by compounds that induce an oxidative stress only after cellular glutathione (GSH) concentrations have been substantially depleted, experiments were performed to determine whether this sequence of events occurred in response to oxidative insult in vivo. The role that plasma vitamin E plays in the response to chemically induced oxidative injury in vivo was also assessed. Treatments with 40 mg/kg of methyl ethyl ketone peroxide (MEKP) quickly induced lipid peroxidation in vivo and from one to 4 h after treatment caused a depression in the plasma content of vitamin E and the liver content of GSH, as well as signs of toxicity (elevations in serum activities of alanine and aspartate aminotransferases). At these time points however, the liver content of vitamin E was either indistinguishable from or slightly elevated from controls. By 12 to 24 h after treatment the liver content of vitamin E was reduced by 20-25% whereas values for all other indicators had returned toward control levels. Pretreatment of rats with L-buthionine-S,R-sulfoximine, an inhibitor of GSH by 4 or 24 h after treatment, did not alter the time course or extent of hepatic vitamin E depletion that was observed after treatment with MEKP. Other compounds that induce oxidative stress and lipid peroxidation to the liver, carbon tetrachloride and menadione, did not provoke an alteration in hepatic vitamin E levels as compared to controls 1 day after treatment. These findings indicate that depletion of hepatic vitamin E may not occur as an immediate consequence of oxidative insult to the liver and that the depletion of hepatic vitamin E levels may not be related to the extent of prior GSH depletion. Moreover, these findings suggest that alterations in the plasma concentration of vitamin E may not reflect concurrent alterations in hepatic vitamin E levels. A mechanism whereby liver vitamin E stores are mobilized for the maintenance of plasma vitamin E levels is proposed.     

Long-Term Selenium-Deficient Diet Induces Liver Damage by Altering Hepatocyte Ultrastructure and MMP1/3 and TIMP1/3 Expression in Growing Rats

The effects of selenium (Se)-deficient diet on the liver were evaluated by using growing rats which were fed with normal and Se-deficient diets, respectively, for 109 days. The results showed that rats fed with Se-deficient diet led to a decrease in Se concentration in the liver, particularly among male rats from the low-Se group. This causes alterations to the ultrastructure of hepatocytes with condensed chromatin and swelling mitochondria observed after low Se intake. Meanwhile, pathological changes and increased fibrosis in hepatic periportal were detected by hematoxylin and eosin and Masson’s trichrome staining in low-Se group. Furthermore, through immunohistochemistry (IHC) staining, higher expressions of metalloproteinases (MMP1/3) and their tissue inhibitors of metalloproteinases (TIMP1/3) were observed in the hepatic periportal of rats from the low-Se group. However, higher expressions of MMP1/3 and lower expressions of TIMP1/3 were detected in hepatic central vein and hepatic sinusoid. In addition, upregulated expressions of MMP1/3 and downregulated expressions of TIMP1/3 at the messenger RNA (mRNA) and protein levels also appeared to be relevant to low Se intake. In conclusion, Se-deficient diet could cause low Se concentration in the liver, alterations of hepatocyte ultrastructure, differential expressions of MMP1/3 and TIMP1/3 as well as fibrosis in the liver hepatic periportal.     

Effects of liver failure on the enzymes in the branched-chain amino acid catabolic pathway

Branched-chain alpha-keto acid dehydrogenase (BCKDH) complex catalyzes the committed step of the catabolism of branched-chain amino acids (BCAA). The liver cirrhosis chemically induced in rats raised the activity of hepatic BCKDH complex and decreased plasma BCAA and branched-chain alpha-keto acid concentrations, suggesting that the BCAA requirement is increased in liver cirrhosis. Since the effects of liver cirrhosis on the BCKDH complex in human liver are different from those in rat liver, further studies are needed to clarify the differences between rats and humans. In the valine catabolic pathway, crotonase and beta-hydroxyisobutyryl-CoA hydrolase are very important to regulate the toxic concentration of mitochondrial methacrylyl-CoA, which occurs in the middle part of valine pathway and highly reacts with free thiol compounds. Both enzyme activities in human and rat livers are very high compared to that of BCKDH complex. It has been found that both enzyme activities in human livers were significantly reduced by liver cirrhosis and hepatocellular carcinoma, suggesting a decrease in the capability to dispose methacrylyl-CoA. The findings described here suggest that alterations in hepatic enzyme activities in the BCAA catabolism are associated with liver failure.     

Role of melatonin in the oxidative damage prevention at different times of hepatic regeneration

The process of regenerating liver is the result of a balance between stimulating factors and inhibitors of hepatocyte proliferation. Melatonin and its metabolites have been found to protect tissues against oxidative damage generated by a variety of toxic agents and metabolic processes. Furthermore, studies in liver of rats showed a decrease in the liver mitochondrial hydroxylation of drugs returning to the normal state after the administration of antioxidants. This study was designed to determine, in experimental animals, whether the administration of an antioxidant agent such as melatonin could prevent cells events leading to tissue injury and hepatic dysfunction after partial hepatectomy (PH). Biliary flow (BF), oxidative stress in hepatic tissue and Na⁺/K⁺ATPase activities in whole plasma membrane were determined. PH decreased the Na⁺/K⁺ATPase activity. PH significantly reduced the BF (36%) and promoted oxidative stress with an increase of lipoperoxidation and decrease of glutathione peroxidase and catalase activities. Treatment with melatonin prevented the decrease of BF in rats with hepatectomy and normalized the Na⁺/K⁺ATPase activity. Moreover, melatonin markedly attenuated oxidative stress produced by PH. This may be the results of the higher efficacy of melatonin in scavenging various free radicals and also because of its ability in stimulating the antioxidant enzymes. We suggest that oxidative stress before and during liver regeneration has a crucial role in cholestasis, apoptotic/necrotic hepatocellular damage and the impairment in liver transport function induced by PH and that melatonin could modulate the degree of oxidative stress and through it prevent the alterations in liver function carrier. Copyright © 2012 John Wiley & Sons, Ltd.     

Three-Dimensional Imaging of Hepatic Sinusoids in Mice Using Synchrotron Radiation Micro-Computed Tomography

Hepatic sinusoid, the smallest vessel in the liver, plays important roles in hepatic microcirculation. Although the structure of the hepatic sinusoids affects diverse functions of the liver, little is known about morphological alterations in the sinusoids under pathological conditions. In this study, we show that the structure of hepatic sinusoids can be identified three-dimensionally in normal and carbon tetrachloride-injured mouse liver, using the absorption mode of synchrotron radiation micro-computed tomography. We observed that the hepatic sinusoidal structure on tomographic slice images was similar to that on histological images of normal and acutely injured mice. Moreover, centrilobular necrosis and structural alterations of the sinusoids in the necrotic region were detectable on tomographic slice and volume-rendered images of the acutely injured mice. Furthermore, quantitative analyses on 3D volume-rendered images of the injured sinusoid revealed decrease in the volume of the sinusoid and connectivity of the sinusoidal network. Our results suggest that the use of synchrotron radiation micro-computed tomography may improve our understanding of the pathogenesis of hepatic diseases by detecting the hepatic sinusoids and their alterations in three-dimensional structures of the damaged liver.     

Acute fructose intake suppresses fasting-induced hepatic gluconeogenesis through the AKT-FoxO1 pathway

Excessive intake of fructose increases lipogenesis in the liver, leading to hepatic lipid accumulation and development of fatty liver disease. Metabolic alterations in the liver due to fructose intake have been reported in many studies, but the effect of fructose administration on hepatic gluconeogenesis is not fully understood. The aim of this study was to evaluate the acute effects of fructose administration on fasting-induced hepatic gluconeogenesis. C57BL/6J mice were administered fructose solution after 14 h of fasting and plasma insulin, glucose, free fatty acids, and ketone bodies were analysed. We also measured phosphorylated AKT and forkhead box O (FoxO) 1 protein levels and gene expression related to gluconeogenesis in the liver. Furthermore, we measured glucose production from pyruvate after fructose administration. Glucose-administered mice were used as controls. Fructose administration enhanced phosphorylation of AKT in the liver, without increase of blood insulin levels. Blood free fatty acids and ketone bodies concentrations were as high as those in the fasting group after fructose administration, suggesting that insulin-induced inhibition of lipolysis did not occur in mice administered with fructose. Fructose also enhanced phosphorylation of FoxO1 and suppressed gluconeogenic gene expression, glucose-6-phosphatase activity, and glucose production from pyruvate. The present study suggests that acute fructose administration suppresses fasting-induced hepatic gluconeogenesis in an insulin-independent manner.     

Functions of autophagy in normal and diseased liver

Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. Investigations specifically employing the liver or hepatocytes as experimental models have contributed significantly to our current knowledge of autophagic regulation and function. The diverse cellular functions of autophagy, along with unique features of the liver and its principal cell type the hepatocyte, suggest that the liver is highly dependent on autophagy for both normal function and to prevent the development of disease states. However, instances have also been identified in which autophagy promotes pathological changes such as the development of hepatic fibrosis. Considerable evidence has accumulated that alterations in autophagy are an underlying mechanism of a number of common hepatic diseases including toxin-, drug- and ischemia/reperfusion-induced liver injury, fatty liver, viral hepatitis and hepatocellular carcinoma. This review summarizes recent advances in understanding the roles that autophagy plays in normal hepatic physiology and pathophysiology with the intent of furthering the development of autophagy-based therapies for human liver diseases.     

Increased UDP-glucuronyltransferase and gamma-glutamyltranspeptidase in enzyme-altered rat liver lesions produced by low doses of aflatoxin B1

Preneoplastic liver lesions were produced in female Wistar rats by low doses of aflatoxin B1 (Model 1: administration of 37.5 micrograms/kg 12 and 24 h after partial hepatectomy; Model 2: continuous application of 3.5 micrograms/kg in tap water daily for 28 days with partial hepatectomy after 14 days. The animals then received sodium phenobarbital, 0.1% in tap water, for 180 to 400 days). In both models numerous altered hepatic foci (AHF) and hyperplastic nodules (HN) were detected enzyme histochemically by their negative ATPase and positive gamma-glutamyltranspeptidase reactions. Immunohistochemically these lesions were also UDP-glucuronyltransferase positive. Increased UDP-glucuronyltransferase adds to permanent alterations of a number of drug metabolizing enzymes observed in a variety of different tumor models. These alterations are responsible for the toxin-resistant phenotype (Faber 1984b). Increased gamma-glutamyltranspeptidase was detected both enzyme histochemically and immunohistochemically; whereas gamma-glutamyltranspeptidase activity was present in both AHF/HN and in periportal areas by enzyme histochemistry, the immunohistochemical method selectively stained gamma-glutamyltranspeptidase in AHF and HN. Immunohistochemically detectable UDP-glucuronyltransferase and gamma-glutamyltranspeptidase are markers of putative precancerous liver lesions which may be useful in the analysis of the prestages of liver carcinogenesis.     

Action of streptokinase on the hemostatic indices of rabbits with a toxic lesion of the liver due to carbon tetrachloride

The rabbits with CCl4-induced hepatic failure have revealed changes in hemostasis responses to streptokinase administration. The main distinction of hepatic dystrophy was the depression of plasma fibrinolytic activity accompanying the decrease in fibrinogen and antiplasmin concentrations. Streptokinase administration to rabbits with productive inflammatory liver disorders produced changes in hemostasis identical to those observed in intact rabbits, fibrinogen levels, however, remained unchanged. The common feature of all the toxic liver disorders is the increase of antithrombin III levels after streptokinase administration, whereas the antithrombin levels in the control animals were decreased.     

Changes in the passive electrical properties of the erythrocytes during hemosorption

Passive electrical properties of erythrocytes were studied during hemosorption in vivo. It was shown that specific conduction and capacity of the erythrocyte plasma membrane were reduced after hemosorption. Incubation of erythrocyte suspension with free fatty acids resulted in an increase in specific conduction and capacity of the plasma membrane. That effect was eliminated after the passing of erythrocytes through a column with activated charcoal.     

Non-invasive oxidative stress markers for liver fibrosis development in the evolution of toxic hepatitis

Oxidative stress is related to the liver fibrosis, anticipating the hepatic stellate cells' (HSC) activation. Our aim was to correlate oxidative stress markers with the histological liver alterations in order to identify predictive, noninvasive parameters of fibrosis progression in the evolution of toxic hepatitis.CCl4 in sunflower oil was administered to rats intragastrically, twice a week. After 2, 3, 4 and 8 weeks of treatment, plasma levels of malondialdehyde (MDA), protein carbonyls (PC), hydrogen donor capacity (HD), sulfhydryl groups (SH), and glutathione (GSH) were measured and histological examination of the liver slides was performed. Dynamics of histological disorders was assessed by The Knodell score. Significant elevation of inflammation grade was obtained after the second week of the experiment only (p=0.001), while fibrosis started to become significant (p=0.001) after 1 month of CCl4 administration. Between plasma MDA and liver fibrosis development a good correlation was obtained (r=0.877, p=0.05). Correlation between PC dynamics and liver alterations was marginally significant for inflammation grade (r=0.756, p=0.138). HD evolution revealed a marginally inverse correlation with inflammation grade (r=-0.794, p=0.108). No correlations could be established for other parameters with either inflammation grade or fibrosis stage.Our study shows that MDA elevation offers the best prediction potential for fibrosis, while marginal prediction fiability could be attributed to high levels of plasma PC and low levels of HD.     

Changes in the expression of genes related to apoptosis and fibrosis pathways in CCl4-treated rats

Chronic liver diseases are accompanied by changes in the biochemical pathways related to the regulation of apoptosis and extra-cellular matrix deposition. The present study was designed to investigate, using low density arrays, changes in the hepatic gene expression together with hepatic biochemical and histological alterations in rats that had liver impairment induced by chronic exposure to CCl₄. Further, we examined the possible recovery of genetic and pathological changes following the cessation of the hepatotoxic injury. Experimental fibrosis was induced in male Wistar rats by CCl₄ administration. Animals were subdivided into two groups. One group was given CCl₄ and animals were killed at 8 and 12 weeks of treatment. The other group was treated with CCl₄ for 6 weeks, the CCl₄ was then stopped and, subsequently, subgroups of animals were killed after 1 and 2 weeks of recovery. CCl₄ administration over 12 weeks was associated with significant changes in B-cell leukemia/lymphoma 2, procollagen type I α 2, matrix metalloproteinases 3 and 8, tissue inhibitors of metalloproteinases 1, 2, and 3 and the inhibitor of apoptosis 4 gene expressions. Recovery after CCl₄ cessation was associated with changes in procollagen type I α 2, matrix metalloproteinase 7, tissue inhibitors of metalloproteinases 1 and 2, inhibitor of apoptosis 4, and survivin gene expressions. This study shows an association between changes in the expression of several genes regulating hepatic cell apoptosis, the fibrosis process, and the recovery of the hepatic function after removal of the toxic injury.     

Characterization of the protective effects of melatonin and related indoles against alpha-naphthylisothiocyanate-induced liver injury in rats

The protective effect of melatonin, 6-hydroxymelatonin and N-acetylserotonin against alpha-naphthylisothiocyanate (ANIT)-induced liver injury was investigated and compared in rats injected once with the hepatotoxicant (75 mg/kg body weight). In rats injected with ANIT alone, liver injury with cholestasis developed within 24 h, as indicated by both serum levels of alanine aminotransferase (SGPT) and aspartic acid aminotransferase (SGOT) activities and serum total bilirubin concentration. The administration of melatonin or 6-hydroxymelatonin (10 mg/kg body weight) to ANIT-injected rats reduced significantly the serum levels of both SGPT and SGOT and the serum total bilirubin concentration. For all hepatic biochemical markers, melatonin was more effective that 6-hydroxymelatonin. By comparison, the administration of N-acetylserotonin (10 mg/kg body weight) to ANIT-injected rats did not reduce the serum levels of either hepatic enzymes or the serum total bilirubin concentration. In ANIT-injected rats, hepatic lipid peroxidation (LPO) was significantly higher than in control animals and this increase was significantly reduced by either melatonin, 6-hydroxymelatonin or N-acetylserotonin. Furthermore, ANIT treatment caused a significant reduction in liver microsomal membrane fluidity and this reduction was completely reversed by the three indoles. The liver from ANIT-injected rats showed several histopathological alterations; above all there was an acute infiltration of polymorphonuclear neutrophils and an increase in the number of apparent apoptotic hepatocytes. The concurrent administration of melatonin reduced the severity of all morphological alterations, specially the neutrophil infiltration and the number of presumed apoptotic cells. On the contrary, the administration of 6-hydroxymelatonin or N-acetylserotonin did not provide any protective effect in terms of the histopathological alterations. These results indicate that melatonin protects against ANIT-induced liver injury with cholestasis in rats, and suggests that this protective effect is likely due to its antioxidant properties and above all to its capacity to inhibit liver neutrophil infiltration, a critical factor in the pathogenesis of ANIT-induced liver injury. 6-hydroxymelatonin, although able to provide partial protection against the ANIT-induced hepatic injury, probably through its antioxidant properties by mechanisms that are unclear, was unable to reduce neutrophil infiltration. Finally, N-acetylserotonin in the experimental conditions of this study, only exhibited some antioxidant protection but had no protective effect against ANIT-induced hepatic damage.