Study of hepatotoxins influence in vitro on basic biochemical indicators of liver functional state

An antimicrobial drug of the fluoroquinolone group, ciprofloxacin, is widely used in Ukraine. However, some researchers have noted the probable hepatotoxicity of this drug with prolonged use or use of high doses of ciprofloxacin. The aim of this study was to compare the effects of carbon tetrachloride, as a classical model of hepatocyte injury, with the effects of ciprofloxacin. The aim of the study was to investigate the biochemical parameters of the liver when simulating toxic damage to hepatocytes with carbon tetrachloride or ciprofloxacin. Materials and methods. The study was carried out on isolated rat hepatocytes, in whose culture medium carbon tetrachloride or ciprofloxacin was added. After incubation in the supernatant and cell homogenate, the activities of marker enzymes of cytolysis were determined: ALT, AST, γ-GTP, LF, LDH, DC and MDA. Results. The introduction of ciprofloxacin into the culture of hepatocytes at a concentration of LC50 caused changes in biochemical parameters similar to those caused by carbon tetrachloride. Thus, an increase in ALT, AST, γ-GTP, LF, LDH, DC and MDA was observed when CCl4 or ciprofloxacin was added to the culture. Conclusion. Incubation of rat hepatocytes with carbon tetrachloride or ciprofloxacin caused an increase in the level of enzymes and lipid peroxidation products. These parameters are indicators of gross changes in cells, which are the result of impaired keto acid formation, impaired redox reactions, impaired glycogen production

In- vitro Hepatoprotective Action of the Crude Extracts of Luffa amara (Whole Fruit) and Rheum emodi (Rhizomes) in CCl4 Intoxicated Rat Hepatocytes

Aim: The objective of this in-vitro study involves evaluating the protective action of the extracts of L. amara (LA) (whole fruits including seeds) and R. emodi (RE) (rhizomes) at various concentrations on isolated primary rat hepatocytes. Methods: The pulverised dried whole fruits of L. amara (LA) and rhizomes of R.emodi (RE) were extracted successively with petroleum ether (PE), ethanol (EE) and distilled water (AE) and vacuum dried. These extracts of LA petroleum ether (PE), ethanolic (EE) and aqueous (AE) extracts and RE obtained were subjected to in vitro studies at doses of 25, 75, 100, and 150 µg/ml and silymarin (250 µg/ml) in CCl4 (1%) intoxicated primary hepatocytes monolayer cultures the hepatoprotective action of all the extracts of both plants at different doses was carried out using isolated rat hepatocytes which were subjected to CCl4 intoxication followed by estimating/ measuring the changes in serum biochemical markers – SGPT, SGOT, ALP, Total proteins (TP), total bilirubin (TB), albumin (ALB) and triglycerides (TGL). Results: Hepatoprotective activity against CCl4 was demonstrated in the rat primary monolayer hepatocyte culture using MMT assay with the ethanolic extracts of both plants showing more hepatocyte protective action compared to the aqueous and petroleum ether extracts by reducing the elevated serum marker levels. Alcoholic and aqueous extracts were found to express more protective action towards CCl4 intoxicated isolated primary rat hepatocytes in a dose dependant manner. Conclusion: Based on the result, it is suggested that the extract with the most hepatocyte protective action at a dose of 150µg is LA ethanolic extract (viability=88.24%), followed by LA aqueous extract (viability=84.31%), RE ethanolic extract (viability=88.24%) and RE aqueous extract (viability=88.24%) - which are comparable to the reference silymarin with viability at 92.15%. the petroleum ether extracts of both plants showed least hepatic cell viability with LA pet ether extract at 49.02% and RE pet ether extract at 47.85%

Optimization of primary hepatocyte isolation for the pharmacological characterization of metabotropic glutamate receptor (mGluR) s ubtype 5: A study on Reduction

To minimize the number of animals used during experiments, it is important to choose the suitable enzyme according to the final goal. In our work, we demonstrated the superiority of collagenase IV in the maintenance of functional transmembrane receptor, thus the pharmacological activity, in isolated rat hepatocytes.

Role of Elevated Intracellular S-Adenosylhomocysteine in the Pathogenesis of Alcohol-Related Liver Disease

Background: The earliest manifestation of alcohol-related liver disease (ALD) is steatosis, characterized by the accumulation of lipid droplets (LDs) in hepatocytes. Findings from our laboratory have indicated that many pathological changes, including steatosis, correlate with the alcohol-induced hepatocellular increases in S-adenosylhomocysteine (SAH). Based on these considerations, we hypothesized that an experimental increase in intracellular SAH alone will result in similar steatotic changes to those seen after alcohol exposure. Methods: Freshly isolated rat hepatocytes grown on collagen-coated plates were exposed to serum-free medium containing 50 µmol/L oleic acid and varying concentrations of 3-deazaadenosine (DZA) to experimentally elevate intracellular SAH levels. Results: Overnight exposure to DZA treatment dose-dependently increased hepatocellular triglyceride accumulation, which was also evident by morphological visualization of larger-sized LDs. The rise in triglycerides and LDs accompanied increases in mRNA and protein levels of several LD-associated proteins known to regulate LD number and size. Furthermore, DZA treatment caused a decline in the levels of lipases that prevent fat accumulation as well as increased the expression of factors involved in lipogenesis and fatty acid mobilization. Collectively, our results indicate that the elevation of intracellular SAH is sufficient to promote fat accumulation in hepatocytes, which is similar to that seen after alcohol exposure.

Isolation of rat hepatocytes for pharmacological studies on metabotropic glutamate receptor (mGluR) subtype 5: a comparison between collagenase I versus collagenase IV

Isolated hepatocytes can be obtained from the liver by collagenase infusion, a procedure that could affect cell isolation as well as the integrity of membrane receptors. In this respect we compared metabotropic glutamate subtype 5 receptor (mGluR5) protein expression and activity in rat hepatocytes isolated by two collagenases, type I and type IV. Hepatocytes were isolated from male Wistar rats (200-250 g) using collagenase I or collagenase IV and after isolation, viability and morphology of rat hepatocytes were assessed measuring mGluR5 protein expression by Western blot analyses. mGluR5 activation was evaluated by inositol-1-phosphate (IP-1) accumulation after treatment with the mGluR5 orthosteric agonist ACPD or the selective antagonist MPEP. No difference in cellular viability and morphology was observed using collagenase I when compared with collagenase IV. An increase in mGluR5 protein expression was observed in hepatocytes isolated using collagenase IV with respect to collagenase I. Moreover, hepatocytes treated with ACPD and with MPEP presented higher levels of IP-1 when isolated using collagenase IV compared to collagenase I. These results indicate that collagenase IV better preserves the activity of surface proteins such as mGluR5 in isolated rat hepatocytes, an in vitro model useful to reduce the use of experimental animals, in line with the 3R ethical framework.

Contrasting Role of Dose Increase in Modulating Sofosbuvir-Induced Hepatocyte Toxicity

Sofosbuvir, the oral direct-acting antiviral, is a medication, which used as the effective treatment for hepatitis C infection. Although sofosbuvir thought to have few adverse-effects, there have been some experiences of serious drug-induced hepatotoxicity. In this research, the molecular/cellular pathways that lead to sofosbuvir-induced hepatotoxicity were evaluated on isolated rat hepatocytes. Rat hepatocytes were isolated using collagenase reperfusion technique. In evaluating the different pathways of sofosbuvir-induced hepatotoxicity, ROS formation, mitochondrial membrane potential collapse, lysosomal membrane damage, glutathione depletion, and the percentage of apoptosis versus necrosis were determined. Our data demonstrated that the cytotoxic effect of sofosbuvir in lower concentrations (25, 50 and 100 µM) is mediated by above stream pathways. On the other hand, sofosbuvir acts in opposite directions at higher concentrations (400 µM) and acts as an antioxidant and hepatoprotective. We concluded that sofosbuvir while looking toxic and pro-oxidant in lower concentrations, acts as protective and antioxidant in higher concentrations.

Subcellular Organelle Toxicity Caused by Arsenic Nanoparticles in Isolated Rat Hepatocytes

Background: Arsenic, an environmental pollutant, is a carcinogenic metalloid and also an anticancer agent. Objective: To evaluate the toxicity of arsenic nanoparticles in rat hepatocytes. Methods: Freshly isolated rat hepatocytes were exposed to 0, 20, 40, and 100 µM of arsenic nanoparticles and its bulk counterpart. Their viability, reactive oxygen species level, glutathione depletion, mitochondrial and lysosomal damage, and apoptosis were evaluated. Results: By all concentrations, lysosomal damage and apoptosis were clearly evident in hepatocytes exposed to arsenic nanoparticles. Evaluation of mitochondria and lysosomes revealed that lysosomes were highly damaged. Conclusion: Exposure to arsenic nanoparticles causes apoptosis and organelle impairment. The nanoparticles have potentially higher toxicity than the bulk arsenic. Lysosomes are highly affected. It seems that, instead of mitochondria, lysosomes are the first target organelles involved in the toxicity induced by arsenic nanoparticles.