Evaluation of azathioprine-induced cytotoxicity in an in vitro rat hepatocyte system.

Azathioprine (AZA) is widely used in clinical practice for preventing graft rejection in organ transplantations and various autoimmune and dermatological diseases with documented unpredictable hepatotoxicity. The potential molecular cytotoxic mechanisms of AZA towards isolated rat hepatocytes were investigated in this study using "Accelerated Cytotoxicity Mechanism Screening" techniques. The concentration of AZA required to cause 50% cytotoxicity in 2 hrs at 37°C was found to be 400 µM. A significant increase in AZA-induced cytotoxicity and reactive oxygen species (ROS) formation was observed when glutathione- (GSH-) depleted hepatocytes were used. The addition of N-acetylcysteine decreased cytotoxicity and ROS formation. Xanthine oxidase inhibition by allopurinol decreased AZA-induced cytotoxicity, ROS, and hydrogen peroxide (H2O2) formation and increased % mitochondrial membrane potential (MMP). Addition of N-acetylcysteine and allopurinol together caused nearly complete cytoprotection against AZA-induced hepatocyte death. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl), a known ROS scavenger and a superoxide dismutase mimic, and antioxidants, like DPPD (N,N'-diphenyl-p-phenylenediamine), Trolox (a water soluble vitamin E analogue), and mesna (2-mercaptoethanesulfonate), also decreased hepatocyte death and ROS formation. Results from this study suggest that AZA-induced cytotoxicity in isolated rat hepatocytes may be partly due to ROS formation and GSH depletion that resulted in oxidative stress and mitochondrial injury.

Molecular mechanism of 17α-ethinylestradiol cytotoxicity in isolated rat hepatocytes.

17α-Ethinylestradiol (17-EE) is used in formulations of contraceptives and hormone replacement therapy because it is an estradiol derivative. However, it has been associated with an increase in the risk of liver cancers and injury. The carcinogenic properties of 17-EE are similar to that of other estrogens, but the molecular mechanism of liver injury is still unclear. It is important to identify any secondary toxic mechanisms that can be used to prevent or treat the toxicity. The LC50 of 17-EE toward isolated rat hepatocytes was determined to be 150 ± 8 µmol/L. Accelerated cytotoxicity mechanism screening (ACMS) techniques using isolated rat hepatocytes showed that CYP1A inhibitors decreased cytotoxicity, whereas tyrosinase increased toxicity; this suggests that the toxic mechanism involved is the oxidation of 17-EE. A hepatocyte inflammation model also increased 17-EE-induced mitochondrial toxicity, as well as the formation of ROS and H2O2. Cytotoxicity was increased when inhibitors of quinone reduction, catechol-O-methylation, glucuronidation, glutathione conjugation, and sulfation were co-incubated with 17-EE. The hepatocytes could be rescued with antioxidants and quinone trapping agents, thereby suggesting a role for quinoid moiety induced oxidative stress in 17-EE induced cytotoxicity. These mechanisms for 17-EE hepatotoxicity could provide a new perspective for the treating 17-EE-induced liver injury.

Modeling xenobiotic susceptibility to hepatotoxicity using an in vitro oxidative stress inflammation model.

Evidence suggests xenobiotic exposure during periods of inflammation can increase an individual's susceptibility to toxicity. The present study aimed to validate an in-vitro inflammatory model to identify compounds that increase hepatotoxicity during inflammation. Using freshly isolated hepatocytes exposed to a low nontoxic flow of H2O2 using glucose (G) and glucose oxidase (GO) and supplementing it with either peroxidase or Fe(II), the effects of inflammation on 2 classes of drugs known to cause hepatotoxicity were examined: nitroaromatics (nimesulide, nilutamide, flutamide) and aromatic amines (clozapine, thioridazine). Co-incubation with G/GO and the nitroaromatics increased toxicity that was further increased when peroxidase was present. While the aromatic amines did not increase cytotoxicity with G/GO alone, they demonstrated significant increases in cytotoxicity when incubated with peroxidase+G/GO. Liver injury is commonly observed with alcohol abusers; therefore, the effects of inflammation on ethanol, and its metabolite acetaldehyde, were observed. Both ethanol and acetaldehyde increased cytotoxicity, which was further increased when Fe(II) was present. These results implicate H2O2, a cellular mediator of inflammation, as a potential risk factor for hepatotoxicity. A H2O2-enhanced hepatocyte-system in the presence of peroxidase or Fe(II) may prove useful for a more robust screening of xenobiotics for assessing potential toxicity associated with inflammation.

A-CD estrogens. I. Substituent effects, hormone potency, and receptor subtype selectivity in a new family of flexible estrogenic compounds.

Long-term use of estrogen supplements by women leads to an increased risk of breast and uterine cancers. Possible mechanisms include metabolism of estradiol and compounds related to tumor-initiating quinones, and ligand-induced activation of the estrogen receptors ERα and ERß which can cause cancer cell proliferation, depending on the ratio of receptors present. One therapeutic goal would be to create a spectrum of compounds of variable potency for ERα and ERß, which are resistant to quinone formation, and to determine an optimum point in this spectrum. We describe the synthesis, modeling, binding affinities, hormone potency, and a measure of quinone formation for a new family of A-CD estrogens, where the A-C bond is formed by ring coupling. Some substituents on the A-ring increase hormone potency, and one compound is much less quinone-forming than estradiol. These compounds span a wide range of receptor subtype selectivities and may be useful in hormone replacement therapy.