Cytokine and chemokine expression associated with steatohepatitis and hepatocyte proliferation in rats fed ethanol via total enteral nutrition

To determine the temporal relationship between alcohol-induced changes in cytokines and chemokines, development of liver pathology and stimulation of hepatocyte proliferation, male Sprague-Dawley rats were intragastrically fed low carbohydrate-containing ethanol (EtOH) diets via total enteral nutrition (TEN) for up to 49 d. Induction of EtOH metabolism and appearance of steatosis preceded development of oxidative stress, inflammation, and cell death. A transitory peak of tumor necrosis factor (TNFalpha) and interferon gamma (IFN gamma) was observed at 14 d followed by reduced expression of TNFalpha, IFN gamma and another Th1 cytokine IL-12 accompanied by reduced expression of the Th1 regulators T-bet and STAT4. After 35-49 d of EtOH, at a time when hepatocyte proliferation was stimulated, IL-12 returned to control values and a second peak of TNFalpha occurred. The Th2 cytokine IL-4 remained suppressed throughout the study and was accompanied by reductions in the Th2 regulator GATA3. There was no temporal effect of EtOH on expression of IL-6 or TGFbeta. IL-5 and IL-13 mRNA were undetectable. Chemokine CXCL-2 expression increased progressively up to 35 d and preceded the appearance of inflammatory infiltrates. These data suggest that steatosis, increased ethanol metabolism, a transient induction of the innate immune response and suppression of Th2 responses were acute consequences of ethanol treatment and were followed by suppression of Th1 responses. However, the majority of necrosis, apoptosis and a late peak of TNFalpha only occurred after 6-7 weeks of ethanol, coincided with the appearance of inflammatory infiltrates and were associated with stimulation of hepatocyte proliferation.     

Oxidative mechanisms in the pathogenesis of alcoholic liver disease

Although the capacity of ethanol to induce oxidative stress in the liver is well established, the mechanisms by which oxidative damage contributes to the pathogenesis of alcoholic liver disease (ALD) is still incompletely understood. Recent reports have implicated oxidative mechanisms in the onset of alcoholic steatosis and in the formation of Mallory's bodies. Moreover, by inducing mitochondrial alterations, oxidative stress promotes hepatocyte necrosis and contributes to alcohol-induced sensitization of hepatocyte to the pro-apoptotic action of TNF-alpha. Oxidative mechanisms play also a role in the progression of liver fibrosis by triggering the release of pro-fibrotic cytokines and activating collagen gene expression in hepatic stellate cells. Finally, immune responses towards antigens originating from the reactions of lipid peroxidation products with hepatic proteins might represent one of the mechanisms that contribute to perpetuate chronic hepatic inflammation in ALD. Altogether these observations give a rationale to the possible clinical application of antioxidants in the therapy of ALD.     

On the effect of a therapy able to modify both the growth rates in a Gompertz stochastic model

A Gompertz-type diffusion process characterized by the presence of exogenous factors in the drift term is considered. Such a process is able to describe the dynamics of populations in which both the intrinsic rates are modified by means of time-dependent terms. In order to quantify the effect of such terms the evaluation of the relative entropy is made. The first passage time problem through suitable boundaries is studied. Moreover, some simulation results are shown in order to capture the dependence of the involved functions on the parameters. Finally, an application to tumor growth is presented and simulation results are shown.     

Free-radical metabolism of carbon tetrachloride in rat liver mitochondria. A study of the mechanism of activation.

Alterations in liver mitochondria as consequence of rat poisoning with carbon tetrachloride (CCl4) have been reported over many years, but the mechanisms responsible for causing such damage are still largely unknown. Isolated rat liver mitochondria incubated under hypoxic conditions with succinate and ADP were found able to activate CCl4 to a free-radical species identified as trichloromethyl free radical (CCl3) by e.s.r. spectroscopy coupled with the spin-trapping technique. The incubation of mitochondria in air decreased free-radical production, indicating that a reductive reaction was involved in the activation of CCl4. However, in contrast with liver microsomes (microsomal fractions), mitochondria did not require the presence of NADPH, and the process was not significantly influenced by inhibitors of cytochrome P-450. The addition of inhibitors of the respiratory chain such as antimycin A and KCN decreased free-radical formation by only 30%, whereas rotenone displayed a greater effect (approx. 84% inhibition), but only when preincubated for 15 min with mitochondria not supplemented with succinate. These findings suggest that the mitochondrial electron-transport chain is responsible for the activation of CCl4. A conjugated-diene band was observed in the lipids extracted from mitochondria incubated with CCl4 under anaerobic conditions, indicating that stimulation of lipid peroxidation was occurring as a result of the formation of free-radical species.     

Strain-gauge plethysmographic measurement of the systemic bioavailability of oral nitroglycerin in liver cirrhosis

7 cirrhotic (M = 3, F = 4, mean age 55, range 35-74) and 7 healthy subjects (M = 6, F = 1, mean age 24, range 23-40) were studied. 2.5mg% nitroglycerin were administered per os. This drug is quite completely metabolized in its first pass through the liver (first pass effect). Peripheric vascular effect of nitroglycerin was evaluated by venous occlusion strain-gauge plethysmography, ECG-coupled (Rest Flow measurement RF, in ml/min/100 ml). No statistically significant differences were found between pre-drug RF in the two groups and between pre and post-drug measurements in healthy subjects. Post-drug RF decreased in cirrhotic subjects when compared either to pre-drug values or to post-drug values in normal subjects (statistically significant after the third minute, p ranging less than 0.05 and less than 0.001). The different peripheric vascular effect found in the two groups was considered as a consequence of the increased drug bioavailability in cirrhotics, caused by portosystemic shunts.     

Metabolic activation of 1,2-dibromoethane to a free radical intermediate by rat liver microsomes and isolated hepatocytes

A one-electron reductive metabolism of 1,2-dibromoethane (DBE) is described that gives rise to a free radical intermediate, which can be stabilized by a spin trapping agent and detected by electron spin resonance spectroscopy. Using rat liver microsomes or isolated hepatocytes from phenobarbitone pretreated animals, under hypoxic conditions, it has been possible to trap a free radical intermediate and identify it by using ¹³C-DBE. Inhibition experiments have demonstrated that the site of activation is the microsomal drug metabolizing system.     

In vitro toxicity and metabolism of 2',3'-dideoxycytidine, an inhibitor of human immunodeficiency virus infectivity.

U937 human monoblastoid cell growth was inhibited in a concentration-dependent manner by 2',3'-dideoxycytidine (ddCyd) (an antiretroviral drug) up to 500 microM. Cell growth inhibition was associated with a pronounced increase in cell volume, however this was not due to cell ATP or NAD+ depletion that could effect osmotic balance or DNA repair. This ddCyd toxicity paralleled the accumulation of ddCyd into acid soluble material where 2',3'-dideoxycytidine-5'-triphosphate (ddCTP) was the predominant labelled nucleotide up to an extracellular ddCyd concentration of 150 microM. At higher ddCyd concentrations, the amount of 2',3'-dideoxycytidine-5'-diphosphate (ddCDP) became predominant over ddCTP. This increase of phosphorylated dideoxycytidine in U937 cells was also associated with an increased incorporation of the drug into cell DNA suggesting a possible toxicity mechanism. That ddCyd does indeed become cytotoxic to human cell by incorporation into DNA was shown by incubating human resting and stimulated lymphocytes with ddCyd. While the drug does not affect cell viability in resting cells it strongly affects cell proliferation upon phytohemagglutinin (PHA) addition.     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

Differential effect of the amino acid cystine in cultured mammalian and bacterial cells exposed to oxidative stress.

The effect of cystine in the cytotoxic response of cultured Chinese hamster ovary and Escherichia coli cells to challenge with hydrogen peroxide has been investigated. It was found that this amino acid could either protect or sensitize cells, depending on the cellular system. In fact, although a reduction in the growth-inhibitory effect of hydrogen peroxide was observed in mammalian cells, a marked increase in the susceptibility to oxidative stress was induced by cystine in bacteria. None of the amino acid precursors of glutathione, e.g., glutamate, glycine or cysteine, afforded protection in the mammalian cell system, whereas cysteine, but not glycine or glutamate, markedly sensitized bacteria to hydrogen peroxide-induced cell killing. In mammalian cells, methionine, an amino acid which is converted to cysteine, was also unable to modify the oxidative response. The results presented indicate that cystine displays differential effects in oxidatively injured mammalian or bacterial cells and suggest that the mechanism whereby the amino acid modulates the lethal action of hydrogen peroxide differs in the two cellular systems.