Anita K. Costa

Comparative toxicity of halothane, isoflurane, hypoxia, and phenobarbital induction in monolayer cultures of rat hepatocytes.

Hypoxia, phenobarbital induction, and halothane anesthesia have been implicated in the pathogenesis of hepatotoxicity in the rat model. However, a controversy exists over the role of halothane in liver injury; does it act by reducing hepatic blood flow, thereby inducing hypoxia, or do its metabolites initiale the injury? These variables are difficult to separate during in vivo halothane exposure. In the present experiments, effects of halothane on hepatic perfusion were eliminated by exposing confluent monolayers of hepatocytes isolated from Fisher 344 rats livers, both with and without phenobarbital pretreatment, to 1.5% halothane or 2.0% isoflurane in 1%, 2%, or 4% (control) oxygen. Isoflurane exposure was included for a control of anesthetic effects on hepatocytes, because it is known to be metabolized minimally and probably is not associated with hepatic dysfunction. Oxygen levels were chosen to approximate those that may occur in the liver in vivo. Cell death was assayed via aspartate aminotransferase (AST) release, both immediately following a 2-h oxygen ± anesthetic exposure and 6 h post-exposure. Per cent cell death data were analyzed using multiple regression techniques. Results obtained immediately, and 6 h after, exposure demonstrate that low oxygen levels, halothane, and phenobrbital were each highly significant factors (P <.001) in relation to cell death, in agreement with the halothane-phenobarbital-hypoxia rat model. A toxic effect of isoflurane was not observed under identical experimental conditions. The results of the study clearly indicate that the origin of cell death in hepatocyte monolayers is multi-factorial; hypoxia, phenobaribital induction, and halothane exposure each contribute to the hepatocyte damage observed in our in vitro model.

Oxygen concentration-dependent metabolism of leukotriene B4 by hepatocyte monolayers

Leukotriene B4 was found to be metabolized by rat hepatocyte monolayers at a rate that was linear with increasing substrate concentration from 74 to 740 nM leukotriene B4. The rates of metabolism were dependent on the O2 concentration and were 315, 213, 80, and 36 pmol leukotriene B4 per min per nmol cytochrome P-450 at 20% (212 microM), 4% (42.5 microM), 2% (21.2 microM), and 1% (10.6 microM) O2, respectively. The metabolic rate was not linear with respect to O2 concentration; however, half maximal rate occurred at 4% O2, and O2 concentration found in the pericentral region of normally oxygenated liver. These results suggest that in vivo conditions of hypoxia or ischemia that lead to blood O2 concentrations less than 4% may drastically decrease hepatic clearance of leukotriene B4.

Inhibition of protein kinase C phosphorylation by mono- and divalent cations.

The effect of a matrix of concentrations of Ca2+ (0.01, 0.1, 0.5, 5 mM), Mg2+ (0.2, 0.5, 1, 2, 5, 10 mM), and Na+ (50, 100, 150 mM) on the phosphorylation of histone H-1 by protein kinase C was measured in the presence of 5 mol % diacylglycerol and Mg-ATP in both phosphatidylserine micelles and liposomes formed from a 1:4 mixture of phosphatidylserine and phosphatidylcholine. Monovalent cations (150 mM) reduced activity by 60 and 84% in the micelle and liposome assay systems, respectively. Inhibition was also observed with 5 mM Ca2+ and 10 mM Mg2+. The phosphorylating activity was compared with computer calculations of the negative electrostatic potentials (psi o) of the phospholipid membranes in the presence of the cations.

Toxicity of calcium lonophore A23187 in monolayers of hypoxic hepatocytes

Increased cytoplasmic calcium has been implicated in hepatic necrosis induced by cytochrome P-450-mediated halocarbon metabolism; hepatic necrosis is exacerbated hypoxia. It is known that addition of the calcium ionophore A23187 to a cell can mimic the metabolic causes of increased cytoplasmic calcium. In the present experiments, confluent monolayers of rat hepatocytes were exposed to A23187 (0.5-50 microM) under conditions of normoxia and 18-fold during a 4-h incubation at 0.5% (5 microM) O2 compared with 20% (212 microM) O2. The ED50 values of A23187 were 0.92, 1.84, 10.98, and 16.81 microM at 0.5, 1, 2, and 20% O2, respectively. The results demonstrate that small reductions in the oxygen concentrations normally encountered by pericentral hepatocytes (i.e., below 28 microM) may severely decrease the ability of these already compromised hepatocytes to manage perturbations in calcium homeostasis.

Toxicity oft-butylhydroperoxide in hepatocyte monolayers exposed to hypoxia and reoxygenation

SummaryInasmuch as it is known that the toxicity of anesthetic agents is potentiated by hypoxia and that the reductive metabolism of these agents results in the formation of lipid hydroperoxides, we investigated the toxicity of hydroperoxides under low-oxygen concentrations. We found that hypoxia exacerbates the toxicity oft-butyl hydroperoxide, shifting the dose-response curve oft-butyl hydroperoxide vs. lysis of hepatocytes approximately an order of magnitude to the left. Furthermore, although at the end of a 4-h exposure to 0.5% O2 hepatocyte monolayers seemed normal by three indices (release of51Cr and serum glutamate transaminase or exclusion of trypan blue), they were completely lysed after an additional 20 h reoxygenation at 20%. O2. In contrast, monolayers exposed to 2% O2 for 4 h seemed normal after 20 h reoxygenation. However, cells exposed to both a subtoxic dose of hydroperoxide and 4 h of 2% O2, although seeming healthy at the end of the hypoxic period, were completely lysed within 20 h after reoxygenation.

Interaction of hypoxia and carbon tetrachloride toxicity in hepatocyte monolayers

The toxicity of carbon tetrachloride (CCl4) in monolayer cultures of primary hepatocytes was investigated at oxygen concentrations that prevail in the liver under conditions that range from normoxia to hypoxia: 0.5, 1, 2, and 20% O2. CCl4 was administered in the vapor phase at concentrations that produce aqueous concentrations at 37 degrees C of 0.4, 2.0, and 4.0 mM. Damage was assayed by leakage of aspartate transaminase and the inclusion of Trypan Blue immediately after the 2-hr incubation and after an additional 6-hr incubation in 20% O2. Only in the case of 0.5% O2 and 4 mM CCl4 were the monolayers damaged (18%) immediately after the 2-hr exposure; all other exposed cells were undamaged at that time point and the dose response of cell death as a function of CCl4 and oxygen concentration was not evident until the 6-hr time point. The monolayers exposed to 4 mM CCl4 and 1, 2, or 20% O2 exhibited little immediate damage but were all 100% dead 6 hr later. The monolayers exposed to 2 mM CCl4 and 0.5, 1, 2, or 20% O2 were 53, 48, 40, and 22 +/- 2% dead after 6 hr, respectively. These results suggest that effects of CCl4 exposure, for example alterations in the function or synthesis of essential proteins, require several hours to affect cell viability.

Toxicity of 1,2-dibromoethane in primary hepatocyte monolayer cultures: Lack of dependence on oxygen concentration☆

Hepatic 1,2-dibromoethane (DBE) metabolism proceeds via two pathways: oxidation by cytochrome P-450 and direct conjugation with the ubiquitous tripeptide glutathione (GSH) via the GSH S-transferases. The toxicity of DBE in monolayers of hepatocytes was assessed to establish whether the toxicity of this compound is increased under conditions of reductive metabolism at low oxygen concentrations. Our previous studies with t-butyl hydroperoxide and the calcium ionophore A23187 suggested that hypoxia would exacerbate toxicity that was mediated through lipid peroxidation or loss of calcium homeostasis. Monolayers of hepatocytes were exposed for 2 hr to 0, 14, 140, 1400, or 14,000 ppm of DBE in an atmosphere of either 1, 2, or 20% oxygen. Toxicity was measured by leakage of aspartate aminotransferase (AST) and trypan blue exclusion. The time course of the development of cytotoxicity was examined by assaying cell death both immediately following a 2-hr exposure and 24 hr later. The LC50 of DBE vapor was found to be approximately 14,000 ppm when assayed immediately after exposure but only 140 ppm when assayed 24 hr after exposure. The similarity of the percentages of DBE-induced cell death after incubations at 1, 2, and 20% oxygen demonstrates that the toxicity of DBE is oxygen-independent. We conclude that while DBE is highly toxic to rat hepatocytes, hypoxia does not appear to contribute to the toxicity of DBE, even under conditions of low oxygen concentrations. This result is in direct contrast to a previous report where we showed that the toxicity of halothane is potentiated under hypoxic conditions.