Cytotoxicity In Primary Rat Hepatocytes Research Articles

Electrochemical oxidation and sensing of para benzoquinone using a novel SPE based disposable sensor

Para-benzoquinone (PBQ) is an emerging micro-contaminant owing to its chronic toxicity to plants and animals as well as its potential to induce cytotoxicity in primary rat hepatocytes and kidney cell injury. Hence, it is of utmost importance to monitor this contaminant in industrial wastewater and groundwater. In this article, we devised a unique disposable sensor that is based on a screen-printed electrode using MnO2@Co–Ni MOFs/fMWCNTs nanocomposite and is able to detect PBQ. The as-produced nanocomposite was prepared via ultrasonic assisted reflux condition and thoroughly examined by several physicochemical characterisation methods such as SEM, EDX, TEM, Raman, AFM, UV–visible, and FT-IR. Moreover, electrochemical methods like CV, DPV, EIS, and chronoamperometry were used for detecting PBQ on MnO2@Co–Ni MOFs/fMWCNTs/SPCE. Sensor performance has been investigated thoroughly and optimized to enhance the analytical potential of the fabricated sensor. DPV analysis was done on MnO2@Co–Ni MOFs/fMWCNTs that exhibit high selectivity, low peak potential, a broader linear detection range (0.005 mM–30 mM), and a LOD of 0.0027 ± 0.0005 mM. The designed electrode has shown remarkable reproducibility and excellent repeatability, with relative standard deviations of 0.12%, and 0.17%, respectively. Additionally, MnO2@Co–Ni MOFs/fMWCNTs/SPCE have been used to analyse PBQ in industrial wastewater samples, and the results have shown a significant level of recovery between 96.91 and 105.67%. Moreover, the PBQ sensor displays high applicability and was verified via the use of HPLC techniques. This disposable sensor is quick, easy, and cost-effective, so it can be useful in the future for analysing other phenolic contaminants present in environmental samples.

The metabolism and hepatotoxicity of ginkgolic acid (17 : 1) in vitro

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.

Cytotoxicity of luteolin in primary rat hepatocytes: the role of CYP3A‐mediated ortho‐benzoquinone metabolite formation and glutathione depletion

Luteolin (LUT), an active ingredient in traditional Chinese medicines and an integral part of the human diet, has shown promising pharmacological activities with a great potential for clinical use. The purpose of this study was to evaluate the role of cytochrome P450 (CYP450)-mediated reactive ortho-benzoquinone metabolites formation and glutathione (GSH) depletion in LUT-induced cytotoxicity in primary rat hepatocytes. A reactive ortho-benzoquinone metabolite was identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in rat liver microsomes (RLMs) and rat hepatocytes. Using a specific chemical inhibitor method, the CYP3A subfamily was found to be responsible for the reactive metabolite formation in RLMs. Induction of CYP3A by dexamethasone enhanced LUT-induced cytotoxicity, whereas inhibition of CYP3A by ketoconazole (Keto) decreased the cytotoxicity. The cytotoxicity and cell apoptosis induced by LUT were related to the amount of reactive metabolite formation. Furthermore, Keto inhibited the LUT-induced GSH exhaustion. The cytotoxicity was significantly enhanced by pretreatment with L-buthionine sulfoximine to deplete the intracellular GSH. A time course experiment showed that GSH depletion by LUT was not via oxidation of GSH and occurred prior to the increase in 2', 7'-dichlorofluorescein in hepatocytes. Collectively, these data suggest that CYP3A-mediated reactive metabolite formation plays a critical role in LUT-induced hepatotoxicity, and the direct GSH depletion is an initiating event in LUT-mediated cytotoxicity in primary rat hepatocytes.

Protective Effect of Ebselen on Aflatoxin B1-Induced Cytotoxicity in Primary Rat Hepatocytes

Protective Effect of Ebselen on Aflatoxin B1-Induced Cytotoxicity in Primary Rat Hepatocytes

A decrease in S-adenosyl-L-methionine potentiates arachidonic acid cytotoxicity in primary rat hepatocytes enriched in CYP2E1

Previous studies show that treatment with a polyunsaturated fatty acid, arachidonic acid (AA), or high concentrations of cycloleucine, an inhibitor of methionine adenosyltransferase (MAT), which lowers levels of S-adenosyl-L-methionine (SAM), increased toxicity in hepatocytes from pyrazole-treated rats which expressed high levels of cytochrome P450 2E1 (CYP2E1). In this study, I used concentrations of cycloleucine or AA, which by themselves do not produce any toxicity, to evaluate whether a decrease in SAM sensitizes hepatocytes to AA toxicity, especially in hepatocytes enriched in CYP2E1. Levels of SAM were lower by 50% in hepatocytes from pyrazole- compared to saline-treated rats. Cycloleucine treatment caused a 50% decline in SAM levels with both hepatocyte preparations and SAM levels were lowest in the pyrazole-treated hepatocytes. The combination of cycloleucine plus AA produced some toxicity and apoptosis in hepatocytes from saline-treated rats but increased toxicity and apoptosis was found in the hepatocytes from pyrazole-treated rats. Cytotoxicity could be prevented by incubation with SAM, the antioxidant trolox, and the mitochondrial permeability transition inhibitor trifluoperazine. The enhanced cytotoxicity could also be protected by treating rats with chlormethiazole, a specific inhibitor of CYP2E1, thus validating the role of CYP2E1. Cycloleucine plus AA treatment elevated production of reactive oxygen species (ROS) and lipid peroxidation to greater extents with the hepatocytes from pyrazole-treated rats than that from the saline-treated rats. I hypothesize that increased production of ROS by hepatocytes enriched in CYP2E1 potentiates AA-induced lipid peroxidation and toxicity when hepatoprotective levels of SAM are lowered. Such interactions, e.g. induction of CYP2E1, decline in SAM and polyunsaturated fatty acid-induced lipid peroxidation, may contribute to alcohol-induced liver injury.

In vitro evaluation of the cytotoxicity of several eremophilane constituents of Petasites hybridus in rat hepatocytes

Several Petasites hybridus eremophilanes have been tested for cytotoxicity in primary rat hepatocytes by means of the MTT assay [1, 2, 3]. (8S)-8-Hydroxyeremophil-7(11)-en-12,8-olide, (8R,9β)-2-[(Angeloyl)oxy]-9-hydroxyeremophil-7(11)-en-12,8-olide and (8S)-2-[Methacroyl)oxy]eremophil-7(11)-en-12,8-olide were not cytotoxic up to a concentration of 0.5mg/mL. No EC50 values were determinable for these less soluble compounds. (8S)-2-{[(Z)-3-(Methylsulfanyl)prop-2-enoyl]oxy}eremophil-7(11)-en-12,8-olide was not cytotoxic up to 1mg/mL. (8R)-2-[Methacroyl)oxy]eremophil-7(11)-en-12,8-olide was the most cytotoxic constituent (EC50 approx. 0.3mg/mL). The non-steroid-like 8α-conformers of both 8-H-isomeric couples of the 2-angeloyloxy and 2-methacroyloxy esters of eremophilanolide seemed to be more cytotoxic than the steroid-like 8β-H-conformers. The additionally measured cytotoxicity parameters mitochondrial dehydrogenase activity, ATP-content and LDH-leakage were considerably lower for the 8α-conformer of 2-[Methacroyl)oxy]eremophil-7(11)-en-12,8-olide than for the corresponding 8β-conformer [4]. This stereoselectivity points to a specific new cytotoxicity target.

Protective effect of ebselen on aflatoxin B1-induced cytotoxicity in primary rat hepatocytes.

Recent studies have shown that aflatoxin B1 enhances reactive oxygen species formation and causes oxidative damage, which may ultimately contribute to the cytotoxicity and carcinogenic effect of aflatoxin B1. Ebselen, 2-phenyl-1,2-benzoisoseleazol-3(H)-one, a synthetic seleno-organic compound has been shown to possess glutathione peroxidase-like activity and free radical scavenging ability. Thus present study was designed to investigate the protective effect of ebselen on aflatoxin B1-induced cytotoxicity in primary rat hepatocytes. Aflatoxin B1-induced cytotoxicity and lipid peroxidation were determined by lactate dehydrogenase leakage and malondialdehyde generation, respectively. Intracellular reactive oxygen species level was measured using the fluorescent probe 2',7'-dichlorofluorescin diacetate, and the intracellular reduced glutathione concentration was determined with a fluorometric method. Ebselen was found to display a dose-dependent protective effect on lactate dehydrogenase leakage and malondialdehyde generation caused by aflatoxin B1 exposure. The results also demonstrate that ebselen efficiently inhibits the intracellular reactive oxygen species formation in aflatoxin B1-treated hepatocytes in a dose and time-dependent manner. It was also noted that ebselen was able to increase the intracellular reduced glutathione concentration, both in the control and in aflatoxin B1-treated hepatocytes. The protection of ebselen against aflatoxin B1 cytotoxicity, however, was not affected by lowering the concentration of intracellular reduced glutathione. The overall data indicate that ebselen possesses a potent protective effect against aflatoxin B1-induced cytotoxicity, and the main mechanism involved in the protection may be its strong capability in inhibiting intracellular reactive oxygen species formation and preventing oxidative damage.