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.

Molecular cytotoxic mechanisms of chlorpromazine in isolated rat hepatocytes.

Chlorpromazine (CPZ), a member of the largest class of first-generation antipsychotic agents, is known to cause hepatotoxicity in the form of cholestasis and hepatocellular necrosis in some patients. The mechanism of CPZ hepatotoxicity is unclear, but is thought to result from reactive metabolite formation. The goal of this research was to assess potential cytotoxic mechanisms of CPZ using the accelerated cytotoxicity mechanism screening (ACMS) technique with freshly isolated rat hepatocytes. This study identified CPZ cytotoxicity and inhibition of mitochondrial membrane potential (MMP) to be concentration-dependent. Furthermore, inhibition of cytochrome P450s (CYPs), including CYP2D1 and 1A2, delayed CPZ cytotoxicity, suggesting a role for CYP activation of CPZ to a toxic metabolite(s) in this model. Metabolism studies also demonstrated glucuronide and glutathione (GSH) requirement for CPZ detoxification in hepatocytes. Inactivating the 2-electron reduction pathway, NAD(P)H quinone oxidoreductase (NQO1), caused a significant increase in hepatocyte susceptibility to CPZ, indicating quinoneimine contribution to CPZ cytotoxicity. Nontoxic concentrations of peroxidase/H(2)O(2) (inflammatory model) increased cytotoxicity in CPZ-treated hepatocytes and caused additional mitochondrial toxicity. Inflammation further depleted GSH and increased oxidized glutathione (GSSG) levels. Results suggest activation of CPZ to reactive metabolites by 2 pathways in hepatocytes: (i) a CYP-catalyzed quinoneimine pathway, and (ii) a peroxidase-catalyzed oxidation of CPZ to CPZ radicals.

Glyoxal and methylglyoxal: autoxidation from dihydroxyacetone and polyphenol cytoprotective antioxidant mechanisms.

Previously, this laboratory had shown that fructose and its downstream metabolites can be enzymatically metabolized to form glyoxal and methylglyoxal. Fructose metabolites, glycoaldehyde, glyceraldehyde and hydroxypyruvate have also been shown to be autoxidizable. In this study, however, fructose did not cause protein carbonylation itself and instead protected against apparent carbonylation by Fenton's reagent; fructose did not form significant levels of dicarbonyl compounds over a period of 6 days under standard conditions (37°C, pH 7.4). In contrast, dihydroxyacetone, a fructose metabolite, caused protein carbonylation and autoxidized to form dicarbonyls, which effects were further potentiated under oxidative stress conditions (Fenton's reaction). Natural polyphenols were tested for their ability to protect against glyoxal- and methylglyoxal-induced cytotoxicity, reactive oxygen species formation and improved mitochondrial membrane potential maintenance. The polyphenols investigated were gallic acid, methyl gallate, ethyl gallate, propyl gallate, rutin and curcumin. The polyphenols were assayed using primary and GSH-depleted hepatocytes. The polyphenols were also investigated for their rescuing ability and were found to provide greater hepatoprotection when toxins were pre-incubated for 30 min before adding the polyphenols. However, rutin was less protective when rescuing hepatocytes, perhaps, because rutin metabolites may scavenge reactive oxygen species more effectively than rutin itself. The longer the alkyl group attached to the gallate compound, the more cytoprotective the polyphenol was. However, the gallates with longer alkyl groups were less able to scavenge reactive oxygen species, and to maintain the mitochondrial membrane potential.

Acrolein and chloroacetaldehyde: an examination of the cell and cell-free biomarkers of toxicity.

Cyclophosphamide and ifosfamide are two commonly used DNA-alkylating agents in cancer chemotherapy that undergo biotransformation to several toxic and non-toxic metabolites, including acrolein and chloroacetaldehyde (CAA). Acrolein and CAA toxicities occur by several different mechanisms, including ROS formation and protein damage (oxidation), however, these pathways of toxicity and protecting agents used to prevent them have yet to be compared and ranked in a single study. This research focused on the molecular targets of acrolein and CAA toxicities and strategies to decrease toxicities. Hepatocyte viability (cytotoxicity) was assessed using Trypan blue uptake; formation of reactive oxygen species (ROS) and endogenous H2O2 were also assessed in the hepatocyte model. In cell-free models (bovine serum albumin and hepatic microsomes), protein carbonylation was the measurement of toxicity. The present study demonstrated that acrolein was a more potent toxin than CAA for freshly isolated rat hepatocytes, bovine serum albumin and rat hepatic microsomes. Acrolein protein carbonylation was dependent on its concentration; as acrolein concentration increased, protein carbonylation increased in a linear trend, whereas, CAA deviated from the trend and did not cause protein carbonylation at lower concentrations (<400 µM). Aldehyde dehydrogenase (ALDH) is a major pathway for detoxifying pathway for CAA in hepatocytes, as a 3-fold increase in cytotoxicity occurred when cells were incubated with cyanamide, an ALDH inhibitor. Inhibiting ALDH or depleting GSH in hepatocytes increased cytotoxicity by about 3-fold in acrolein-treated hepatocytes. The overall effectiveness of protecting agents to prevent or suppress acrolein or CAA toxicities in cell and cell-free models were ranked in order of most effective to least effective: reducing agents (sodium borohydride, sodium bisulfite)>thiol-containing compounds (N-acetylcysteine, cysteine, glutathione, 2-mercaptoethane sulfonate [MESNA], penicillamine)>carbonyl scavengers/amines (aminoguanidine, hydralazine, hydroxylamine)>antioxidants/ROS scavengers (ascorbic acid, Trolox; only utilized in hepatocyte system). An understanding of acrolein and CAA toxicities and the ability of protecting agents to protect against toxicities may help to establish or improve existing therapeutic interventions against the side effects associated with acrolein or CAA in cyclophosphamide or ifosfamide treatment.

Metabolic mechanisms of methanol/formaldehyde in isolated rat hepatocytes: carbonyl-metabolizing enzymes versus oxidative stress.

Methanol (CH(3)OH), a common industrial solvent, is metabolized to toxic compounds by several enzymatic as well as free radical pathways. Identifying which process best enhances or prevents CH(3)OH-induced cytotoxicity could provide insight into the molecular basis for acute CH(3)OH-induced hepatoxicity. Metabolic pathways studied include those found in 1) an isolated hepatocyte system and 2) cell-free systems. Accelerated Cytotoxicity Mechanism Screening (ACMS) techniques demonstrated that CH(3)OH had little toxicity towards rat hepatocytes in 95% O(2), even at 2M concentration, whereas 50 mM was the estimated LC(50) (2h) in 1% O(2), estimated to be the physiological concentration in the centrilobular region of the liver and also the target region for ethanol toxicity. Cytotoxicity was attributed to increased NADH levels caused by CH(3)OH metabolism, catalyzed by ADH1, resulting in reductive stress, which reduced and released ferrous iron from Ferritin causing oxygen activation. A similar cytotoxic mechanism at 1% O(2) was previous found for ethanol. With 95% O(2), the addition of Fe(II)/H(2)O(2), at non-toxic concentrations were the most effective agents for increasing hepatocyte toxicity induced by 1M CH(3)OH, with a 3-fold increase in cytotoxicity and ROS formation. Iron chelators, desferoxamine, and NADH oxidizers and ATP generators, e.g. fructose, also protected hepatocytes and decreased ROS formation and cytotoxicity. Hepatocyte protein carbonylation induced by formaldehyde (HCHO) formation was also increased about 4-fold, when CH(3)OH was oxidized by the Fenton-like system, Fe(II)/H(2)O(2), and correlated with increased cytotoxicity. In a cell-free bovine serum albumin system, Fe(II)/H(2)O(2) also increased CH(3)OH oxidation as well as HCHO protein carbonylation. Nontoxic ferrous iron and a H(2)O(2) generating system increased HCHO-induced cytotoxicity and hepatocyte protein carbonylation. In addition, HCHO cytotoxicity was markedly increased by ADH1 and ALDH2 inhibitors or GSH-depleted hepatocytes. Increased HCHO concentration levels correlated with increased HCHO-induced protein carbonylation in hepatocytes. These results suggest that CH(3)OH at 1% O(2) involves activation of the Fenton system to form HCHO. However, at higher O(2) levels, radicals generated through Fe(II)/H(2)O(2) can oxidize CH(3)OH/HCHO to form pro-oxidant radicals and lead to increased oxidative stress through protein carbonylation and ROS formation which ultimately causes cell death.