Novel role of oxidants in the molecular mechanism of action of peroxisome proliferators.

Peroxisome proliferators are nongenotoxic rodent carcinogens that act as tumor promoters by increasing cell proliferation; however, their precise mechanism of action is not well understood. Oxidative DNA damage caused by leakage of hydrogen peroxide (H2O2) from peroxisomes was hypothesized initially as the mechanism by which these compounds cause liver tumors. It seems unlikely that oxidants of peroxisomal origin explain the mechanism of action of peroxisome proliferators because treatment with these compounds in vivo does not lead to increased H2O2 production. On the other hand, Kupffer cell-derived oxidants, such as superoxide, may play a role in initiating tumor nerosis factor-alpha (TNF-alpha) production that leads to hepatocyte proliferation. Peroxisome proliferators have been shown to activate Kupffer cells both in vitro and in vivo, and the use of Kupffer cell inhibitors such as methyl palmitate and dietary glycine have demonstrated that Kupffer cells are responsible for hepatocyte proliferation by mechanisms involve TNF-alpha. Moreover, peroxisome proliferators activate the transcription factor NF-kappaB, one of the major regulators of TNF-alpha expression, in Kupffer cells. Importantly, activation of NF-kappaB by peroxisome proliferators was shown to be oxidant-dependent, leading to the hypothesis that oxidants of Kupffer cell origin are involved in the mechanism of action. Many of the effects of peroxisome proliferators, including peroxisome induction and hepatomegaly, involve the peroxisome proliferator-activated receptor-alpha (PPARalpha). Recently, it was shown that peroxisome proliferator-induced cell proliferation and tumors require the PPARalpha. However, PPARalpha is not involved in TNF-alpha production by Kupffer cells because it is not expressed in this cell type. How it is involved in liver tumor remains unclear and one possible explanation is that both Kupffer cell TNF-alpha and parenchymal cell PPARalpha are required. Collectively, recent data are consistent with the hypothesis that oxidants play a role in signaling hepatocellular proliferation due to peroxisome proliferators via activation of NF-kappaB and incrase in mitogenic cytokines such as TNF-alpha.

Apoptosis in hematopoietic cells (FL5.12) caused by interleukin-3 withdrawal: relationship to caspase activity and the loss of glutathione.

The mechanism of cell death caused by cytokine deprivation remains largely unknown. FL5.12 cells (a murine prolymphocytic cell line), following interleukin-3 (IL-3) withdrawal, undergo a decrease in intracellular glutathione (GSH) that precedes the onset of apoptosis. In the present study, the induction of apoptosis following IL-3 withdrawal or GSH depletion with DL-buthionine-[S,R,]-sulfoximine (BSO) was examined. Both conditions caused time-dependent increases in phosphatidylserine externalization, acridine orange and ethidium bromide staining, decreases in mitochondrial membrane potential, processing and activation of caspase-3 and proteolysis of the endogenous caspase substrate poly(adenosine diphosphate ribose)polymerase (PARP). Apoptosis induced by IL-3 deprivation but not BSO also caused lamin B1 cleavage, suggesting activation of caspase-6. Despite a more profound depletion of GSH after BSO than withdrawal of IL-3, the extent of apoptosis was somewhat lower. Benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone (z-VAD.fmk) blocked this caspase activity and prevented cell death after BSO exposure but not after IL-3 deprivation. Following IL-3 withdrawal, the caspase inhibitors z-VAD.fmk and boc-asp(OMe)fluoromethylketone (boc-asp.fmk) prevented the cleavage and activation of caspase-3, the breakdown of lamin B1 and partially mitigated PARP degradation. However, the externalization of phosphatidylserine, the fall in mitochondrial membrane potential and subsequent apoptotic cell death still occurred. These results suggest that IL-3 withdrawal may mediate cell death by a mechanism independent of both caspase activation and the accompanying loss of GSH.

Peroxisomes are involved in the swift increase in alcohol metabolism.

The purpose of this study was to determine whether catalase-dependent alcohol metabolism is activated by alcohol (i.e., swift increase in alcohol metabolism). When ethanol or the selective substrate for catalase, methanol, was given (5.0 g/kg) in vivo 2 to 3 h before liver perfusion, methanol and oxygen metabolism were increased significantly. This increase was blocked when the specific Kupffer cell toxicant GdCl3 was administered 24 h before perfusion. These data support the hypothesis that catalase-dependent alcohol metabolism is activated by acute alcohol and that Kupffer cells are involved. Ethanol treatment in vivo increased ketogenesis from endogenous fatty acids nearly 3-fold and increased plasma triglycerides and hepatic acyl CoA synthetase activity; all increases were blocked by GdCl3. These findings support the hypothesis that ethanol increases H2O2 supply for catalase-dependent alcohol metabolism by increasing fatty acid supply. Infusion of oleate stimulated oxygen uptake 1.5-fold and methanol metabolism 4-fold, but these parameters were not altered by GdCl3. Moreover, the effects of ethanol treatment were blocked by the cyclooxygenase inhibitor indomethacin, and prostaglandin E2 (PGE2) was increased more than 200% in media from cultured Kupffer cells from rats treated with ethanol in vivo. Furthermore, lipoprotein lipase activity in retroperitoneal fat pads, which is known to be inhibited by PGE2, was reduced 70% by ethanol. These data are consistent with the hypothesis that Kupffer cells play a key role in activation of catalase-dependent alcohol metabolism, most likely by producing mediators (e.g., PGE2) that inhibit lipoprotein lipase, increase the supply of fatty acids to the liver, and increase generation of H2O2 via peroxisomal beta-oxidation.

Bcl-2 and Bcl-xL in peroxide-resistant A549 and U87MG cells.

Overexpression of the bcl-2 and the related bcl-xL protooncogene proteins enhance cell survival by inhibiting apoptosis induced by many agents including oxidants. Whether these proteins contribute to survival in oxidant-resistant cells is not known. The current study assessed the expression of bcl-2 and bcl-xL proteins in human glioblastoma U87MG cells and human lung adenocarcinoma A549 cells selected for resistance to 0, 50, 100, 200, and 400 microM H2O2 by exposure to this oxidant one time each passage for 9 months. When examined 7 to 32 days after cessation of peroxide exposure (times when peroxide resistance was maintained), bcl-2 protein levels were significantly increased in most peroxide-resistant U87MG cells. However, the increase was not dose dependent and was not accompanied by an increase in mRNA levels. A549 cells did not express significant levels of bcl-2 protein, although bcl-2 mRNA was detectable. A549 cells expressed large amounts of bcl-xL and immunohistochemistry demonstrated extensive localization of this protein around the nucleus. However, expression of this protein was not altered in peroxide-resistant lines nor was bcl-2 protein increased to a measurable level. U87MG cells also expressed bcl-xL but it was not altered in peroxide-resistant cells. Although the increased bcl-2 protein in peroxide-resistant U87MG cells may contribute to their oxidant tolerance, the lack of a dose-response relationship, the failure to induce bcl-xL protein, and the absence of any bcl-2 or bcl-xL protein induction in peroxide-resistant A549 cells suggest these genes are not primary factors in oxidant resistance.

Bcl-xL overexpression attenuates glutathione depletion in FL5.12 cells following interleukin-3 withdrawal.

Bcl-xL and bax are bcl-2-related genes whose protein products either inhibit or promote apoptosis. Oxidative damage, including the loss of glutathione, has been implicated in the induction of apoptosis. The ability of the Bcl proteins to affect GSH was assessed in control, bax- and bcl-xL-transfected FL5.12 cells [an interleukin (IL)-3-dependent murine prolymphocytic cell line]. Overall levels of GSH were approximately the same in control and bcl-xL transfectants during the 6 h incubation period, although levels increased in bcl-xL transfectants 24 h after replating. GSH in cells overexpressing bax was reduced by approximately 36%. There were no consistent differences between these cell lines in the activities of superoxide dismutase, catalase, glutathione peroxidase or glutathione reductase. Following IL-3 withdrawal, a condition known to cause apoptosis in these cells, a rapid loss of intracellular GSH occurred in control and bax transfectants, which preceded the onset of apoptosis. GSH depletion could not be attributed to intracellular oxidation but rather seemed to occur due to a translocation out of the cell. Cells overexpressing bcl-xL did not lose significant amounts of GSH upon withdrawal of IL-3, and no apoptosis was evident. These results suggest a possible role for GSH in the mechanism by which bcl-xL prevents cell death.

Antibodies to tumor necrosis factor alpha prevent increases in cell replication in liver due to the potent peroxisome proliferator, WY-14,643.

Several structurally dissimilar hypolipidemic drugs, plasticizers and halogenated hydrocarbons induce peroxisomes in hepatocytes, and cause hepatocellular adenoma and carcinoma in rats and mice. The mechanism by which these agents act is unknown, although recent studies have suggested a link between increased cell proliferation and hepatic cancer caused by peroxisome proliferators. Here, we demonstrate that neutralizing antibodies to tumor necrosis factor alpha (TNF alpha) block increases in protein kinase C and cell proliferation due to [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (WY-14,643), a hypolipidemic drug and potent peroxisome proliferator that causes tumors. WY-14,643 moderately elevated the level of TNF alpha mRNA in the liver. TNF alpha was detected immunohistochemically exclusively in Kupffer cells. These results demonstrate that WY-14,643 acts as an indirect mitogen on hepatocytes via TNF alpha. We propose that the Kupffer cell, a major source of TNF alpha in the liver, is involved in the mechanism of the mitogenic effect of WY-14,643.

Potent peroxisome proliferators inhibit beta-oxidation in the isolated perfused rat liver.

It is unknown whether peroxisome proliferators decrease hepatic fatty acid oxidation via uncoupling of respiration or if they inhibit extramitochondrial fatty acyl CoA synthesis. Therefore, the purpose of this study was to examine both processes simultaneously using the isolated perfused liver, a whole cell preparation where enzymes and biochemical processes can be monitored continuously under nearly physiological conditions. Accordingly, ketone body formation (beta-hydroxybutyrate + acetoacetate) from lipid metabolism and oxygen uptake, which is increased by uncoupling agents, were monitored at the same time. 2-Bromooctanoate, a known inhibitor of acyl CoA synthetase, decreased ketone body formation in a dose-dependent manner without altering cellular respiration (half-maximal inhibition, approximately 25 microM) and concomitantly increased protein kinase C nearly fourfold also in a dose-dependent fashion. Ketogenesis was also blocked maximally 50-66% with mono(ethylhexyl)phthalate, 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (WY-14,643), and nafenopin, potent peroxisome proliferators and tumor promoters. These compounds also increased protein kinase C three- to fourfold without altering oxygen uptake significantly. Thus, lipid metabolism appears to be the prime target of potent peroxisome proliferators most likely on actions via acyl CoA synthetase rather than oxidative phosphorylation. In contrast, weak peroxisome proliferators and tumor promoters, di(ethylhexyl)phthalate and 2-ethylhexanol, did not affect ketogenesis, oxygen consumption, or protein kinase C at similar concentrations. Additionally, octanoate increased ketone body formation in the presence of nafenopin. Because octanoate is metabolized by mitochondrial acyl CoA synthetase independent of carnitine acyltransferase, these results indicate that nafenopin does not inhibit mitochondrial beta-oxidation. Taken together, it is concluded that potent peroxisome proliferators preferentially block ketogenesis without altering cellular respiration in the liver. This phenomenona, which occurs due to inhibition of acyl CoA synthetase, leads to an elevation of free fatty acids that stimulates protein kinase C and promotes cell proliferation.

Paradoxical increase in peroxisomal cyanide-insensitive respiration following dietary exposure to WY-14,643 in the perfused liver.

WY-14,643, a lipid-lowering drug, increases basal rates of oxygen uptake in perfused livers. Because peroxisomes consume oxygen for H2O2 production and are induced by WY-14,643 treatment, it is possible that peroxisomal beta-oxidation can account for some of this increase in cellular respiration. Therefore, cyanide, an inhibitor of mitochondrial cytochrome oxidase, was infused into livers of WY-14,643-fed rats (0.1% WY-14,643 in laboratory rat chow for 1, 21, and 105 days) to assess peroxisomal cyanide-insensitive respiration. As expected, the addition of cyanide abolished oxygen uptake nearly completely; however, after approximately 20 min oxygen consumption unexpectedly returned to basal levels in 105-day WY-14,643-treated animals but not in untreated controls. Urea synthesis, a process dependent upon ATP, was decreased and remained low during cyanide infusion in livers from both groups, indicating that mitochondria were not responsible for this unusual increase in oxygen uptake in the presence of cyanide. Methanol metabolism, which requires oxygen to form H2O2, was decreased from 37 +/- 5 to 6 +/- 1 micromol/g/hr in all groups treated with cyanide; however, it was increased significantly about 20 min later to 25 micromol/g/hr in livers from WY-14,643-treated rats, indicating that oxygen for peroxisomal H2O2 production is involved in cellular respiration in the presence of cyanide. Fasting abolished the recovery of both oxygen uptake and methanol metabolism in WY-14,643-fed rats, suggesting that ATP for acyl CoA synthetase, an enzyme which metabolizes fatty acids to acyl CoA compounds, is provided by glycolysis. Indeed, oleate significantly increased methanol metabolism in fed control rats from 8 +/- 4 to 26 +/- 3 micromol/g/hr in the presence of cyanide, indicating that fatty acid supply is necessary for peroxisomal respiration. Taken together, these experiments demonstrate that when mitochondrial respiration is inhibited, livers from rats fed WY-14,643 chronically have the unique ability of metabolizing fatty acids through the peroxisome using glycolytic ATP.

Peroxisome proliferators activate Kupffer cells in vivo.

The mechanism by which peroxisome proliferators increase cell replication and cause liver tumors in rodents remains unknown. When activated, Kupffer cells, the resident hepatic macrophages, release a variety of mitogenic stimuli that could theoretically increase cell proliferation in nearby hepatocytes. Therefore, in the present study we evaluated the effect of two potent peroxisome proliferators, nafenopin and WY-14,643, on Kupffer cell activation in vivo. Kupffer cell phagocytosis was determined continuously by monitoring rates of colloidal carbon uptake in the isolated, perfused liver after drug treatment in vivo. In the absence of peroxisome proliferators, colloidal carbon increased rates of oxygen uptake from 88 +/- 10 to 110 +/- 11 mumol/g/h. Livers from rats treated with either nafenopin (2-24 h) or WY-14,643 (24 h) were perfused for approximately 15 min with Krebs-Henseleit buffer and then with buffer containing colloidal carbon (2 mg/ml). Five h after nafenopin treatment (100 mg/kg i.g.), basal rates of colloidal carbon uptake of 136 +/- 12 mg/g/h were increased to 188 +/- 12 and remained elevated after 24 h (203 +/- 3 mg/g/h). Nafenopin also increased rates in a dose-dependent manner (one-half-maximal response, approximately 75 mg/kg). Similarly, WY-14,643 elevated rates of colloidal carbon uptake 1.8-fold over controls. Functional parameters of Kupffer cells were also affected. For example, WY-14,643 increased plasma nitrite significantly. This study demonstrates clearly that nafenopin and WY-14,643 activate Kupffer cell phagocytosis, suggesting a role for cell-to-cell communication in the stimulation of cell replication by peroxisome proliferators.

Expression of the peroxisome proliferator-activated receptor gene is decreased in experimental alcoholic liver disease.

Peroxisome proliferator-activated receptor (PPAR) and retinoid x receptor (RXR) play important roles in fatty acid metabolism. The present study examined the regulation of retinoic acid receptor (RAR alpha, beta, and gamma), RXR (alpha, beta, and gamma), PPAR, cytochrome P450 2E1 (CYP2E1), catalase, and beta-actin gene expression in chronic alcoholic liver disease in the rat. The results demonstrated that the expression of genes for RAR and RXR isoforms and catalase were not altered by ethanol in the fatty liver. In contrast, the levels of PPAR and CYP2E1 mRNAs were down- and up-regulated by ethanol in the liver, respectively. The levels of CYP2E1 mRNAs correlated positively with blood alcohol levels (BAL). In addition, ethanol induced expression of beta-actin mRNA was also proportional to the BAL. The level of PPAR mRNA and the content of polyunsaturated fatty acid decreased in ethanol-fed rat livers. Decreased PPAR gene expression in ethanol-fed rats might result from a decrease in the content of polyunsaturated fatty acid in the liver. However, the activities of enzymes involved in hepatic lipid metabolism, including acyl CoA synthetase, acyl CoA oxidase, catalase, and protein kinase C, were not changed by ethanol treatment. The significance of down-regulation of PPAR gene in alcohol liver disease is discussed.

Inactivation of Kupffer cells prevents early alcohol-induced liver injury.

It is well recognized that consumption of alcohol leads to liver disease in a dose-dependent manner; however, the exact mechanisms remain unclear. Hypoxia subsequent to a hypermetabolic state may be involved; therefore, when it was observed recently that inactivation of Kupffer cells prevented stimulation of hepatic oxygen uptake by alcohol, the idea that Kupffer cells participate in early events that ultimately lead to alcohol-induced liver disease became a real possibility. The purpose of this study was to test that hypothesis. Male Wistar rats were exposed to ethanol continuously by means of intragastric feeding for up to 4 weeks using the model developed by Tsukamoto and French. In this model, ethanol causes fatty liver, necrosis and inflammation--changes characteristic of alcohol-induced liver disease in human beings. Kupffer cells were inactivated by twice weekly treatment with gadolinium chloride (GdCl3), a selective Kupffer cell toxicant. AST levels were elevated to 192 +/- 13 and 244 +/- 56 IU/L in rats exposed to ethanol for 2 and 4 wk, respectively (control value, 88 +/- 7). This injury was prevented almost completely by GdCl3 treatment. Fatty changes, inflammation and necrosis were also all reduced dramatically by GdCl3 treatment. The average hepatic pathological score of rats treated with ethanol for 4 wk was 4.3 +/- 0.6, which was reduced significantly in ethanol- and GdCl3-treated rats to 1.8 +/- 0.5 (p < 0.05). Rates of ethanol elimination were elevated 2- to 3-fold in rats exposed to ethanol for 2 to 4 wk. This elevation was blocked by GdCl3 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxisomal proliferators inhibit acyl CoA synthetase and stimulate protein kinase C in vivo.

The mechanism by which hypolipidemic drugs and industrial plasticizers cause hepatic tumors in rodents remains unknown. It is known, however, that protein kinase C is elevated during hepatic cell turnover, and sustained cellular replication is correlated with an increased incidence of hepatic tumors. Therefore, several peroxisomal proliferators varying in their tumorigenic potency in chronic feeding studies were examined for their ability to increase protein kinase C activity. Intragastric administration of (4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio)acetic acid (Wy-14,643; 100 mg/kg) increased protein kinase C activity threefold in 5 hr and fivefold in 10 hr. Perfluorooctanoate also increased protein kinase C activity significantly in microsomes at 5 hr. Wy-14,643 and perfluorooctanoate also diminished acyl CoA synthetase activity significantly, with Wy-14,643 exhibiting competitive type kinetics. Other peroxisomal proliferators were examined [e.g., ciprofibrate, clofibrate, 2-ethylhexanol, valproate, and di(ethylhexyl)phthalate (DEHP)] and collectively an inverse relationship between their ability to stimulate protein kinase C activity and inhibit acyl CoA synthetase was observed (r = -0.80). All chemicals examined had no direct effect on protein kinase C activity in vitro. Interestingly, those compounds which are more potent as hepatocarcinogens (e.g., Wy-14,643) in long-term feeding studies decreased acyl CoA synthetase and elevated protein kinase C activity to a greater extent than their weaker counterparts (e.g., DEHP). It is proposed that inhibition of acyl CoA synthetase by peroxisomal proliferators elevates free fatty acids which stimulate protein kinase C activity and ultimately promote tumor formation.

The nongenotoxic hepatocarcinogen Wy-14,643 is an uncoupler of oxidative phosphorylation in vivo.

Wy-14,643 is a potent nongenotoxic hepatic carcinogen and peroxisome proliferator in rodents; however, the mechanism by which it causes tumors remains unknown. In previous work it was demonstrated that Wy-14,643 caused a dose-dependent uncoupling of oxidative phosphorylation (half-maximal effect = 100 microM) in isolated mitochondria (Keller et al., 1992, Biochim. Biophys. Acta, 1162, 237-244); therefore, the purpose of this study was to determine if uncoupling occurred in vivo under conditions which lead ultimately to tumors. Rats were fed Wy-14,643 (0.1%) in ground laboratory chow for 1, 21, 75, and 105 days. As expected, activity of the peroxisomal marker enzyme, acyl-CoA oxidase, was increased about eightfold in liver homogenates during the first 3 weeks of treatment, confirming the induction of peroxisomes. Basal rates of oxygen uptake by the perfused liver were increased significantly by Wy-14,643 treatment at all time points studied, consistent with the hypothesis that oxidative phosphorylation was uncoupled. Basal rates of oxygen uptake of about 130 mumol/g/hr were increased by over 20 mumol/g/hr in rats fed Wy-14,643 in their diet for 10 weeks. Concomitantly, rates of urea synthesis from ammonia, a process highly dependent on ATP supply, were reduced significantly in the perfused liver from 104 mumol/g/hr in control livers to 13 mumol/g/hr in livers from rats treated with Wy-14,643 for 75 days. Taken together, these data indicate that energy supply is disrupted in vivo due to uncoupling of oxidative phosphorylation by Wy-14,643.

Wy-14,643 stimulates hepatic protein kinase C activity.

The mechanism by which hypolipidemic drugs and industrial plasticizers cause hepatic tumors in rodents remains unknown. Protein kinase C is elevated during hepatic cell turnover, and sustained cellular replication has been shown to correlate with an increase in hepatic tumors. Therefore, the effect of [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) on protein kinase C activity was examined. Female Sprague-Dawley rats were given 100 mg/kg Wy-14,643 in olive oil (i.g.), while control rats received equal volumes of oil vehicle. After 24 h, the activity of protein kinase C was estimated in isolated hepatic fractions by measuring the binding of 3H-phorbol-12,13-dibutyrate, a specific ligand for protein kinase C. Administration of Wy-14,643 significantly increased protein kinase C activity nearly 2-fold in microsomal fractions. Thus, it is possible that Wy-14,643 increases cell proliferation and causes tumors by mechanisms involving protein kinase C.