Involvement of cyclooxygenase-2 in the potentiation of allyl alcohol-induced liver injury by bacterial lipopolysaccharide.

Bacterial endotoxin (lipopolysaccharide; LPS) augments the hepatotoxicity of a number of xenobiotics including allyl alcohol. The mechanism for this effect is known to involve the inflammatory response elicited by LPS. Upregulation of cyclooxygenase-2 (COX-2) and production of eicosanoids are important aspects of inflammation, therefore studies were undertaken to investigate the role of COX-2 in LPS-induced enhancement of liver injury from allyl alcohol. Rats were pretreated (iv) with a noninjurious dose of LPS or sterile saline vehicle and 2 h later were treated (ip) with a noninjurious dose of allyl alcohol or saline vehicle. COX-2 mRNA was determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and liver injury was assessed from activities in serum of alanine and aspartate aminotransferases (ALT and AST, respectively) and from histology. Liver injury was observed only in rats cotreated with LPS and allyl alcohol. Serum ALT activity was increased by 4 h after administration of LPS and continued to increase through 8 h. COX-2 mRNA was detectable at low levels in livers from rats receiving only the vehicles at any time up to 8 h. Expression of COX-2 mRNA was increased by 30 min after administration of LPS and remained elevated through 6 h. Allyl alcohol treatment alone caused an increase in COX-2 mRNA at 4 h (2 h after allyl alcohol) that lasted less than 2 h. In livers from rats cotreated with LPS and allyl alcohol, levels of COX-2 mRNA were greater than levels seen with either LPS or allyl alcohol alone. The increased expression of COX-2 mRNA was accompanied by an increase in the concentration of prostaglandin (PG) D(2) in plasma. Plasma PGD(2) concentration was increased to a greater extent in rats treated with LPS plus allyl alcohol compared to allyl alcohol or LPS alone. Pretreatment with the COX-2 selective inhibitor, NS-398, abolished the increase in plasma PGD(2) and reduced the increase in ALT and AST activities observed in rats cotreated with LPS and allyl alcohol. NS-398 did not affect liver injury from allyl alcohol alone administered at a larger, hepatotoxic dose. In addition, ibuprofen, a nonselective inhibitor of cyclooxygenases, did not protect against liver injury from LPS plus allyl alcohol. In isolated hepatocytes PGD(2), but not PGE(2), reduced the concentration of allyl alcohol required to cause half-maximal cytotoxicity. These results suggest that products of COX-2 play a role in the augmentation of allyl alcohol-induced liver injury by LPS.

Pentoxifylline attenuates bacterial lipopolysaccharide-induced enhancement of allyl alcohol hepatotoxicity.

Small amounts of exogenous lipopolysaccharide (LPS) (10 ng/kg-100 microg/kg) enhance the hepatotoxicity of allyl alcohol in male Sprague-Dawley rats. This augmentation of allyl alcohol hepatotoxicity appears to be linked to Kupffer cell function, but the mechanism of Kupffer cell involvement is unknown. Since Kupffer cells produce tumor necrosis factor-alpha (TNF alpha) upon exposure to LPS, and this cytokine has been implicated in liver injury from large doses of LPS, we tested the hypothesis that TNF alpha contributes to LPS enhancement of allyl alcohol hepatotoxicity. Rats were treated with LPS (10-100 microg/kg iv) 2 h before allyl alcohol (30 mg/kg ip). Co-treatment with LPS and allyl alcohol caused liver injury as assessed by an increase in activity of alanine aminotransferase in plasma. Treatment with LPS caused an increase in plasma TNF alpha concentration, which was prevented by administration of either pentoxifylline (PTX) (100 mg/kg iv) or anti-TNF alpha serum (1 ml/rat iv) one h prior to LPS. Only PTX protected rats from LPS-induced enhancement of allyl alcohol hepatotoxicity; anti-TNF alpha serum had no effect. Exposure of cultured hepatocytes to LPS (1-10 microg/ml) or to TNF alpha (15-150 ng/ml) for 2 h did not increase the cytotoxicity of allyl alcohol (0.01-200 microM). These data suggest that neither LPS nor TNF alpha alone was sufficient to increase the sensitivity of isolated hepatocytes to allyl alcohol. Furthermore, hepatocytes isolated from rats treated 2 h earlier with LPS (i.e., hepatocytes which were exposed in vivo to TNF alpha and other inflammatory mediators) were no more sensitive to allyl alcohol-induced cytotoxicity than hepatocytes from naïve rats. These data suggest that circulating TNF alpha is not involved in the mechanism by which LPS enhances hepatotoxicity of allyl alcohol and that the protective effect of PTX may be due to another of its biological effects.

Bacterial endotoxin enhances the hepatotoxicity of allyl alcohol.

Lipopolysaccharide (LPS), or bacterial endotoxin, causes liver damage at relatively large doses in rats. Smaller doses, however, may influence the response to other hepatotoxicants. The purpose these studies was to examine the effect of exposure to relatively all doses of LPS on the hepatotoxic response to allyl alcohol, which causes periportal necrosis in laboratory rodents through an known mechanism. Rats were pretreated with LPS (100 micrograms/kg) 2 hr before treatment with a minimally toxic dose of allyl alcohol mg/kg), and liver toxicity was assessed 18 hr later from activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in plasma and from histologic changes in liver sections. Plasma ALT and AST activities were not elevated significantly in rats treated with vehicle, LPS, or allyl alcohol alone, but pronounced increases were observed in rats treated with LPS and allyl alcohol. Significant liver injury occurred as early as 2 hr after allyl alcohol treatment in LPS-pretreated rats and peaked at 6 hr. LPS treatment did not affect the activity of alcohol dehydrogenase and did not affect the rate of production of NADH in isolated livers perfused with allyl alcohol; thus, LPS does not appear to increase the metabolic bioactivation of allyl alcohol into acrolein. On the other hand, pretreatment with 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, abolished the hepatotoxicity of allyl alcohol in LPS-treated rats, indicating that production of acrolein was needed for LPS enhancement of the toxicity of allyl alcohol. Pretreatment of rats with gadolinium chloride (10 mg/kg), a known inactivator of Kupffer cell phagocytic function, decreased LPS augmentation of the response to allyl alcohol. These data indicate that LPS markedly enhances the hepatotoxic response to allyl alcohol. Furthermore, the results suggest that the LPS-induced enhancement of allyl alcohol hepatotoxicity occurs through a Kupffer cell-dependent mechanism.

Quantitation of the host cell infiltration kinetics of the nonimmunogenic colon 26 tumor by multiparameter flow cytometry.

In this study, we examined the kinetics of host cell infiltration into the nonimmunogenic Colon 26 tumor. We found that 1 x 10(4) cells were required to produce tumors in 100% of mice. The vivo doubling time was 42.5 h, and a barely palpable tumor contained 8 x 10(6) cells. No evidence of concomitant immunity was found. The number of host cells infiltrating the in vivo tumors increased at the same rate as the number of tumor cells, but averaged only 22% of total cells. Cycling T lymphocytes were present in the host cell infiltrate of this tumor. In addition, approximately 50% of in vivo Colon 26 cells were Thy-1.2 positive. The observed characteristic of low immunogenicity makes it a useful murine model for studying human malignant tumors.