* Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824; and
Institute for Environmental Toxicology and National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824
Received November 24, 1999; accepted January 10, 2000
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ABSTRACT |
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Key Words: tumor necrosis factor-alpha (TNF); liver damage; Kupffer cells; allyl alcohol.
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INTRODUCTION |
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We have recently demonstrated that the hepatotoxicity of allyl alcohol is enhanced by pretreatment with quite small doses of LPS and that this augmented response is prevented by inhibition of the function of another cellular mediator of inflammation, Kupffer cells (Sneed et al., 1997). These results indicate that properly functioning Kupffer cells are important in the mechanism of LPS-induced enhancement of allyl alcohol hepatotoxicity and evoke interest in whether inflammatory mediators released by these cells participate in augmenting toxicity.
Kupffer cells are the resident macrophages of the hepatic sinusoids (Bouwens and Wisse, 1992; Jones and Summerfield, 1988
; Wisse et al., 1996
) and have a major role in clearing the hepatic portal blood of intestinally derived LPS (Fox et al., 1989
; Toth and Thomas, 1992
). These macrophages respond to LPS with production of mediators such as cytokines (e.g., tumor necrosis factor-alpha [TNF
], interleukin-1, and interleukin-6), reactive oxygen species, and prostaglandins (Decker, 1990
). Kupffer cells play a critical role in liver injury from large doses of LPS as evidenced by the observation that inhibition of their function with gadolinium chloride (GdCl3) affords protection (Brown et al., 1997
; Iimuro et al., 1994
; Pearson et al., 1997
). Cytokines are also essential to LPS-induced responses. For example, inhibition of TNF
synthesis or activity attenuates LPS-mediated liver injury and lethality in baboons (Tracey et al., 1987
), mice (Beutler et al., 1985
) and rats (Hewett et al., 1993
). These results indicate that TNF
is important in the pathogenesis of tissue injury from large doses of LPS and raise the possibility that TNF
may be a factor in the ability of LPS to enhance the hepatotoxicity of xenobiotics. The present study was undertaken to test the hypothesis that TNF
participates in the potentiation of allyl alcohol hepatotoxicity by LPS. Two approaches were taken to inhibit the effects of TNF
in animals treated with LPS and allyl alcohol: pentoxifylline (PTX) was given to inhibit synthesis of TNF
, and an antiserum directed against TNF
was administered to neutralize TNF
activity.
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MATERIALS AND METHODS |
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Isolation of hepatocytes.
Hepatocytes were isolated by collagenase digestion (Klaunig et al., 1981; Seglen, 1973
), placed in Williams' medium E supplemented with 10% fetal calf serum and 0.1% gentamicin, and plated in 6-well primaria plates (Falcon Laboratories) at a density of 5 x 105 cells per well. In some experiments, hepatocytes were obtained from the livers of rats treated 2 h earlier with LPS (4 mg/kg iv). For all experiments, viability of the isolated cells was
85% as measured by trypan blue exclusion.
The hepatocytes were allowed to stabilize in culture for 3 h, the medium was removed, and the cells were washed once with Williams' medium E supplemented only with 0.1% gentamicin. A final volume of 2 ml per well of the latter medium was used in the remainder of the study. Allyl alcohol was added to the hepatocyte cultures at the concentrations indicated in the figures and in the Results section. Hepatocyte injury was assessed 90 or 180 min after addition of allyl alcohol. In studies in which hepatocyte cultures were incubated with LPS or TNF, these agents were added 2 h prior to treatment with allyl alcohol.
Assessment of hepatocyte cytotoxicity.
Cytotoxicity was assessed from release of alanine aminotransferase (ALT) into the medium. The medium was collected from the wells, and 2 ml of 1% TritonX-100 were added to each well and allowed to remain for at least 5 min at room temperature. The wells were scraped thoroughly with a rubber policeman to remove all cells, and the resulting solution was sonicated to further cell lysis. All samples (both medium and lysate) were centrifuged for 10 min at 600 x g. The activity of ALT in all cell-free supernatant fluids was determined by the method of Wroblewski and LaDue (1956) using Sigma Diagnostics Kit No. 59-UV. The activity in the medium (i.e., ALT released) was expressed as a percentage of the total (medium plus lysate) activity (Ganey et al., 1994; Ho et al., 1996
).
Treatment of animals with pentoxifylline.
Rats were treated intravenously with PTX (100 mg/kg; Sigma Chemical Co., St. Louis, MO) or with an equivalent volume of sterile saline (Abbott Laboratory, IL) 1 h prior to treatment with LPS (100 µg/kg; Escherichia coli, serotype 0128:B12; Sigma Chemical Company, St. Louis, MO). This treatment protocol for PTX has been shown previously to prevent the LPS-induced rise in plasma TNF activity (Hewett et al., 1993
). Two h after administration of LPS, allyl alcohol (30 mg/kg; Aldrich Chemical Co., St. Louis, MO) or its sterile saline vehicle was injected intraperitoneally. Liver injury was assessed 6 h later. This dose of allyl alcohol and regimen for co-treatment with LPS were chosen based on previous studies demonstrating little injury from allyl alcohol alone and enhanced hepatotoxicity in co-treated rats (Sneed et al., 1997
).
Assessment of alcohol dehydrogenase activity in liver homogenates.
Rats were treated with PTX (100 mg/kg iv) or saline vehicle 90 min prior to treatment with LPS (100 µg/kg iv). Two h after treatment with LPS or saline vehicle, animals were killed. The liver was removed and homogenized in a solution of 0.05 M HEPES (pH 8.4) and 0.33 mM dithiothreitol. The homogenate was centrifuged at 100,000 x g for 45 min. The supernatant fluid was collected, and activity of alcohol dehydrogenase (ADH) was measured spectrophometrically (366 nm) by monitoring the reduction of nicotinamide adenine dinucleotide (NAD) using ethanol as a substrate (Krebs et al., 1969). Protein concentration in the supernatant fluid was determined using the bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL), which uses the method of Smith and coworkers (1985).
Treatment of animals with anti-TNF serum.
Serum directed against TNF (anti-TNF
serum) was produced in New Zealand White rabbits (Hewett et al., 1993
). Rats were treated intravenously with anti-TNF
serum (1 ml diluted with 1 ml of saline) 1 h before treatment with LPS (10 µg/kg iv). This treatment protocol has been shown previously to prevent the LPS-induced increase in plasma TNF
activity (Hewett et al., 1993
). Two h after administration of LPS, allyl alcohol (30 mg/kg) or sterile saline was injected intraperitoneally. Liver injury was assessed 6 h later.
Assessment of hepatotoxicity in vivo.
Rats were anesthetized with sodium pentobarbital (50 mg/kg ip), and blood was collected from the abdominal aorta into syringes containing sodium citrate (final concentration, 0.38%). ALT activity was determined in plasma using Sigma Diagnostics Kit No. 59-UV.
Determination of activity of TNF.
Ninety min after administration of LPS or its saline vehicle, as described above for experiments with PTX and anti-TNF serum, blood was collected from rats for determination of the activity of TNF
. Plasma was prepared, serially diluted, and incubated for 22 h in the presence of the TNF
-sensitive fibrosarcoma cell line, WEHI 164 clone 13 (Eskandari et al., 1990
; Espevik and Nissen-Meyer, 1986
). The extent of cell lysis was measured with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO) using a Bio-Tek plate reader.
Statistical analysis.
Data are expressed as means ± SEM. For all in vitro studies, N represents the number of repetitions, each performed on a different day with cells from different animals. For all in vivo studies, n represents the number of individual animals used, and experiments were repeated at least once. Homogeneous data were analyzed by one-way or two-way analysis of variance (ANOVA) for in vivo studies and by repeated measures ANOVA for in vitro studies. ALT content varies among isolations of hepatocytes, thus introducing variability among experimental days. The repeated measures ANOVA was used to remove the contribution of this variability from the statistical analysis to allow detection of treatment-related differences. Individual means for all data analyzed by ANOVA were compared using Tukey's omega test. When variances were not homogeneous, data were analyzed using Kruskal-Wallis ANOVA on ranks, and Dunn's test was used to assess significance. Data expressed as percentages were transformed by the arc sine square root method prior to analysis. The criterion for statistical significance was p 0.05.
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RESULTS |
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There was no significant elevation in plasma ALT activity in animals in the saline/saline, LPS/saline or saline/allyl alcohol groups, irrespective of pretreatment with control or anti-TNF serum (Fig. 3
). Plasma ALT activity was significantly elevated in animals cotreated with LPS and allyl alcohol compared to animals treated with LPS alone or allyl alcohol alone. There was no significant difference in ALT activity between cotreated animals pretreated with control serum and anti-TNF
serum.
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DISCUSSION |
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This hypothesis is consistent with results of studies in which inhibition of Kupffer cell function prevented enhancement of allyl alcohol hepatotoxicity by LPS (Sneed et al., 1997). One of the inflammatory mediators produced by LPS-activated Kupffer cells is the proinflammatory cytokine, TNF
, which plays a critical role in liver injury from large doses of LPS (Beutler et al., 1985
; Hewett et al., 1993
; Tracey et al., 1987
). Accordingly, we examined the role of TNF
in LPS potentiation of allyl alcohol hepatotoxicity.
The methylxanthine, PTX, inhibits the synthesis of TNF (Dezube et al., 1993
; Doherty et al., 1991
; Han et al., 1990
; Noel et al., 1990
; Semmler et al., 1993
; Zabel et al., 1989
, 1993
), and results presented here (Table 1
) confirm this. Administration of PTX prior to LPS treatment protected animals from the enhanced hepatotoxicity of allyl alcohol. These data suggested that TNF
might be involved in the mechanism by which LPS augments the hepatotoxicity of allyl alcohol. PTX, however, has multiple pharmacological effects; therefore, a more specific approach, neutralization of TNF
with an anti-TNF
serum, was used to test further whether inhibition of TNF
afforded protection. The anti-TNF
serum did not diminish LPS enhancement of allyl alcohol hepatotoxicity despite complete neutralization of circulating TNF
activity. Protection by PTX and lack of protection by antiserum-induced neutralization of TNF
have also been observed in a rat model of intestinal injury induced by nonsteroidal anti-inflammatory drugs (Reuter and Wallace, 1999
) and in a model of bacteria-induced lung injury in rabbits (Miyazaki et al., 1999
).
One explanation for the disparate results observed with PTX and anti-TNF serum in these studies is that, since PTX inhibits synthesis of TNF
, it affords a more complete blockade of TNF
action in the liver, whereas TNF
is still produced by Kupffer cells after treatment with anti-TNF
serum and can act locally before neutralization by the anti-serum. Thus, autocrine or paracrine hepatic effects of TNF
may still occur. An alternative explanation is that TNF
is not involved in the mechanism by which LPS enhances the hepatotoxicity of allyl alcohol. This explanation is supported by results from two series of in vitro experiments presented here. In the first, exposure of isolated hepatocytes to TNF
did not alter the cytotoxic response to allyl alcohol (Fig. 4
), indicating that direct effects of TNF
on hepatocytes are not sufficient to increase sensitivity to allyl alcohol. Others have also shown that TNF
alone is not cytotoxic to isolated hepatocytes, and that cell damage requires the addition of other cytokines or induction of oxidative stress in the cells (Adamson and Billings, 1992
; Sieg and Billings, 1997
). In the second series of experiments, hepatocytes isolated from rats treated 2 h earlier with LPS were used. Since TNF
activity in plasma reaches a peak 90 min after administration of LPS, these hepatocytes were exposed to TNF
in vivo. Despite this exposure to TNF
and other mediators evoked by treatment with LPS, allyl alcohol was neither more potent nor more toxic in these cells. Maximal cytotoxicity was observed at the same concentration of allyl alcohol (100 µM) in both cell populations, and the concentration of allyl alcohol required to achieve half-maximal cytotoxicity was greater, not less, in hepatocytes from LPS-treated rats compared to those from naïve rats. These results suggest that exposure in vivo to LPS-induced mediators for up to 2 h is not sufficient to increase sensitivity of hepatocytes toward allyl alcohol.
If TNF is not involved in the mechanism by which LPS enhances allyl alcohol hepatotoxicity, then the protective effect produced by PTX is due to one or more of the other pharmacological properties of this drug. One possibility explored in this study was that PTX inhibited toxicity by decreasing activity of ADH and thereby decreasing the formation of the toxic metabolite of allyl alcohol. In fact, hepatic ADH activity was not different in vehicle- and PTX-treated rats (Table 2
). Another possibility is that PTX decreased Kupffer cell function through its inhibition of phosphodiesterase, which increases intracellular levels of cyclic adenosine monophosphate (cAMP). Increases in cAMP have been associated with inhibition of macrophage function (Taffet et al., 1989
), and decreased Kupffer cell function protects against hepatotoxicity from LPS plus allyl alcohol (Sneed et al., 1997
). PTX also improves blood flow in tissues (Ward and Clissold, 1987
). This effect of PTX has been demonstrated to be protective in one model of sepsis in which high mortality was associated with hemodynamic shock (Yang et al., 1999
). In addition, PTX reduces the levels of toxic free radicals, attenuates the expression of inducible nitric oxide synthase (Wu et al., 1999
) and decreases the respiratory burst of neutrophils (Kowalski et al., 1999
). A combination of the above factors may be involved in the ability of PTX to protect animals from LPS-enhanced allyl alcohol hepatotoxicity.
In summary, inflammatory mediators may participate in the ability of LPS to enhance the hepatotoxicity of certain xenobiotics. In LPS-induced enhancement of allyl alcohol hepatotoxicity, however, circulating TNF does not appear to play a major role. The observation that TNF
may not be involved in the mechanism by which LPS enhances the toxic response to allyl alcohol is interesting, because it suggests (1) that although hypotheses can be formulated based on what is known about organ injury from larger, toxic doses of LPS, the mechanisms may not be the same for smaller doses that augment the toxicity of other chemicals, and (2) that select, and not all, components of inflammation are critical to this enhanced response. The drug PTX protects animals from the LPS-mediated enhancement of allyl alcohol-induced liver injury and may do so by affecting the responses of Kupffer cells to the presence of LPS.
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ACKNOWLEDGMENTS |
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NOTES |
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