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M Yin,
M D Wheeler,
H D Connor,
Z Zhong,
H Bunzendahl,
A Dikalova,
R J Samulski, R Schoonhoven,
R P Mason,
J A Swenberg,
R G Thurman
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ABSTRACT: Evidence has accumulated for a role of toxic oxygen radicals in the pathogenesis of ischemia-reperfusion injury in the kidney. The aim of this study was to evaluate the hypothesis that reducing postischemic renal injury is possible by delivery of the gene for the antioxidant enzyme superoxide dismutase (SOD). Female Sprague-Dawley rats received intravenous injections of recombinant adenovirus (1 x 10(9) pfu) containing the transgenes for Escherichia coli beta-galactosidase (Ad-LacZ, as control) or human Cu/Zn-SOD (Ad-SOD). Three days later, renal ischemia was produced by cross-clamping the left renal vessels for 60 min. The right kidney was removed before reperfusion and processed for the transgene. Renal SOD protein and activity in rats given Ad-SOD was 2.5-fold higher than from the animals receiving Ad-LACZ: Urinary lactate dehydrogenase concentrations were elevated by ischemia-reperfusion in the Ad-LacZ group (1403 +/- 112 U/L), yet values were 50% lower in Ad-SOD-treated rats. Free radical production was elevated by ischemia-reperfusion but was significantly lower in SOD-treated animals. Importantly, on postischemic day 1, glomerular filtration rates were reduced to 0.21 ml/min per 100 g in the Ad-LacZ group, whereas values remained significantly higher (0.39) in the Ad-SOD group. Two weeks after ischemia-reperfusion, inflammation, interstitial fibrosis, tubular atrophy and tissue levels of tumor necrosis factor alpha and interleukin-1 were significantly higher in the Ad-LacZ-treated than in Ad-SOD-treated rats. In conclusion, these results indicate that SOD expression can be increased by delivery of the sod gene to the kidney by intravenous injection and that sod gene transduction minimized ischemia-reperfusion-induced acute renal failure.
Journal of the American Society of Nephrology 01/2002; 12(12):2691-700. · 9.66 Impact Factor
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ABSTRACT: Activation of hepatic stellate cells is the earliest step in fibrogenesis. Alpha-smooth muscle actin (alpha-SMA), expressed by activated hepatic stellate cells, and C-terminal procollagen alpha1(III) propeptide (PIIICP) are early markers of fibrogenesis and should precede fibrosis.
Determine if suppression of hepatitis B virus replication with lamivudine would decrease fibrogenesis as measured by immunohistochemical markers.
Paired liver biopsies from patients with hepatitis B before and after therapy with lamivudine (n=47) or placebo (n=33) were studied. alpha-SMA and PIIICP were detected in paraffin-embedded tissue by immunohistochemistry and quantified in a blinded manner by video imaging analysis.
Liver biopsies from patients treated with lamivudine showed a significant decrease in alpha-SMA expression (1.06+/-0.23 vs. 0.58+/-0.11, pre vs. post, P<0.05). Placebo recipients had increased levels of alpha-SMA (0.82+/-0.14 vs. 1.32+/-0.21, P<0.05). PIIICP was similarly decreased after lamivudine. Among subjects whose Histologic Activity Index fibrosis score was unchanged or worsened, the mean change in alpha-SMA expression was significantly decreased in the lamivudine group compared with placebo.
Lamivudine decreased markers of hepatic stellate cell activation and collagen synthesis. Immunohistochemical techniques are sensitive for assessing fibrogenesis and will be useful in trials of antiviral and antifibrotic agents.
Journal of Hepatology 12/2001; 35(6):749-55. · 9.26 Impact Factor
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ABSTRACT: Oxygen-derived free radicals play a central role in reperfusion injury after organ transplantation, and fatty livers are particularly susceptible. Endogenous radical scavengers such as superoxide dismutase (SOD) degrade these radicals; however, SOD is destroyed rapidly when given exogenously. Therefore, an adenoviral vector encoding the Cu/Zn-SOD gene (Ad.SOD1) was used here to test the hypothesis that organ injury would be reduced and survival increased in a rat model of transplantation of fatty livers. Donors received chow diet (untreated), high-fat diet, or ethanol-containing high-fat diet. Some of the ethanol-fed donors were infected either with the gene lacZ encoding bacterial beta-galactosidase (Ad.lacZ), or Ad.SOD1. After liver transplantation, SOD activity and protein expression in liver, survival, histopathology, release of transaminases, free radical adducts in bile, and activation of NF-kappaB, IkappaB kinase (IKK), Jun-N-terminal kinase (JNK), and TNFalpha were evaluated. Ad.SOD1 treatment increased survival dramatically, blunted transaminase release, and reduced necrosis and apoptosis significantly. Free radical adducts were increased two-fold in the ethanol group compared with untreated controls. Ad. SOD1 blunted this increase and reduced the activation of NF-kappaB. However, release of TNFalpha was not affected. Ad.SOD1 also blunted JNK activity after transplantation. This study shows that gene therapy with Ad.SOD1 protects marginal livers from failure after transplantation because of decreased oxygen radical production. Genetic modification of fatty livers using viral vectors represents a new approach to protect marginal grafts against primary nonfunction.
Hepatology 01/2001; 32(6):1255-64. · 11.66 Impact Factor
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ABSTRACT: Using the differential PCR display method to select cDNA fragments that are differentially expressed after hepatic stellate cell (HSC) activation, we have isolated from activated HSCs a cDNA that corresponds to rat alphaB-crystallin. Northern blots confirmed expression of alphaB-crystallin in culture-activated HSCs but not in quiescent HSCs. Western blot analysis and immunocytochemical staining confirmed expression of alphaB-crystallin protein in activated but not quiescent HSCs. alphaB-crystallin is induced as early as 6 h after plating HSCs on plastic and continues to be expressed for 14 days in culture. Expression of alphaB-crystallin was also induced in vivo in activated HSCs from experimental cholestatic liver fibrosis. Confocal microscopy demonstrated a cytoplasmic distribution of alphaB-crystallin in a cytoskeletal pattern. Heat shock treatment resulted in an immediate perinuclear redistribution that in time returned to a normal cytoskeletal distribution. The expression pattern of alphaB-crystallin was similar to that of HSP25, another small heat shock protein, but differed from the classic heat shock protein HSP70. Therefore, alphaB-crystallin represents an early marker for HSC activation.
AJP Gastrointestinal and Liver Physiology 01/2001; 279(6):G1333-42. · 3.43 Impact Factor
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ABSTRACT: It was shown that 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (Wy-14,643), a potent peroxisome proliferator, caused rapid oxidant-dependent activation of nuclear factor kappaB (NF-kappaB) in Kupffer cells in vivo and activated superoxide production by isolated Kupffer cells. Here, we tested the hypothesis that NADPH oxidase (NADPH OX) is the source of oxidants increased by Wy-14,643. Indeed, both activation of NF-kappaB and increases in cell proliferation due to a single dose of Wy-14,643 (100 mg/kg) were prevented completely when rats were pretreated with diphenyleneiodonium (1 mg/kg), an inhibitor of NADPH OX. p47phox is a critical subunit of NADPH OX; therefore, p47phox knockout mice were used to specifically address the hypothesis of NADPH OX involvement. In livers of wild-type mice, Wy-14,643 activated NF-kappaB, followed by an increase in mRNA for tumor necrosis factor a. Importantly, these changes did not occur in p47phox knockouts. Moreover, when Kupffer cells were treated with Wy-14,643 in vitro, superoxide production was increased in cells from wild-type but not p47phox-null mice. Finally, when mice were fed a Wy-14,643-containing (0.1%) diet for 7 days, the increase in liver weight and cell proliferation caused by Wy-14,643 in wild-type mice was blocked in p47phox-null mice. Combined, these results are consistent with the hypothesis that Wy-14,643 activates NADPH OX, which leads to NF-kappaB-mediated production of mitogens that causes hepatocellular proliferation characteristic of this class of nongenotoxic carcinogens.
Cancer Research 10/2000; 60(17):4798-803. · 7.86 Impact Factor
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ABSTRACT: Activation of the transcription factor NFkappaB has been demonstrated in activated hepatic stellate cells (HSCs). We investigated the role of NFkappaB in proliferation, in activation, and in TNFalpha-induced apoptosis of HSCs.
NFkappaB activation was inhibited using an adenovirus expressing an IkappaB dominant negative protein (Ad5IkappaB) in both quiescent and activated HSCs. Quiescent HSCs were infected with Ad5IkappaB or an adenovirus expressing beta-galactosidase (Ad5LacZ). The cells were cultured for 7 days. HSCs activation was determined by cell morphology, smooth muscle alpha-actin (alpha-sma) expression, and steady-state mRNA levels of alpha1(I) collagen as assessed by Western blot and RNase protection assay, respectively. Proliferation was determined in culture-activated HSCs by 3H-thymidine incorporation and direct cell counting. Apoptosis was analyzed by infecting quiescent or activated HSCs with Ad5IkappaB or Ad5LacZ, and then treating with TNFalpha. Apoptosis was demonstrated by determining cell number, assessing nuclear morphology, TUNEL assay and caspase 3 activity.
After 7 days in culture no differences were noted between the Ad5IkappaB- and the Ad5LacZ-infected cells in the morphology, alpha-sma expression or in alpha1(I) collagen mRNA levels. Ad5IkappaB infection did not modify proliferation in activated HSCs. TNFalpha induced apoptosis only in Ad5IkappaB-infected activated, but not quiescent HSCs. Apoptosis was initially demonstrated 12 h after exposure to TNFalpha. Twenty-four h after the TNFalpha treatment, 60% of the activated HSCs were apoptotic.
NFkappaB activity is not required for proliferation or activation of HSCs; however, NFkappaB protects activated HSCs against TNFalpha-induced apoptosis.
Journal of Hepatology 08/2000; 33(1):49-58. · 9.26 Impact Factor
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ABSTRACT: Oxygen-derived free radicals play a central role in pathomechanisms of reperfusion injury after organ transplantation. Endogenous radical scavenger systems such as superoxide dismutase (SOD) degrade toxic radicals; however, SOD is degraded rapidly when given exogenously. Therefore, the hypothesis that treatment of the donor liver with an adenoviral vector encoding the Cu/Zn-SOD gene (Ad-SOD1) would lead to permanent gene expression and therefore protect the organ against injury and increase survival in a rat model of liver transplantation was tested.
Some donors were infected with Ad-SOD1, whereas untreated grafts and livers infected with the indicator gene lacZ encoding bacterial beta-galactosidase (Ad-lacZ) served as controls. After orthotopic liver transplantation, survival, serum transaminases, and histopathology were evaluated.
Approximately 80% of hepatocytes expressed beta-galactosidase 72 hr after injection of Ad-lacZ. Moreover, SOD1 gene expression and activity were increased 3- and 10-fold in the Ad-SOD1 group, respectively. After transplantation, 20-25% of rats treated with Ad-lacZ survived. In contrast, all SOD1-treated animals survived. Transaminases measured 8 hr after transplantation in Ad-SOD1 rats were only 40% of those in controls, which increased 40-fold above normal values. Approximately 20% of hepatocytes in untreated and Ad-lacZ-infected organs were necrotic 8 hr after reperfusion, whereas necrosis was nearly undetectable in grafts from rats treated with Ad-SOD1.
This study provides clear evidence for the first time that gene therapy with Ad-SOD1 increases survival and decreases hepatic injury after liver transplantation. Genetic modification of the liver represents a future approach to protect organs against injury where oxygen-derived free radicals are involved.
Transplantation 04/2000; 69(6):1051-7. · 4.00 Impact Factor
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M Yin,
I Rusyn, R Schoonhoven,
L M Graves,
E V Rusyn,
X Li,
F Li,
A D Cox,
T W Harding,
H Bunzendahl,
J A Swenberg,
R G Thurman
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ABSTRACT: Chronic rejection is influenced by a variety of risk factors, including histoincompatibility and ischemia. Glycine, a cytoprotective agent, has been shown to protect against ischemia-reperfusion injury in the liver, inactivate hepatic resident macrophages, minimize cyclosporin A-induced nephrotoxicity, and exhibit immunosuppressive properties in vitro. The aim of this study was to investigate whether dietary glycine could reduce development of chronic rejection.
Lewis recipients of Fisher-344 abdominal aortic allografts received diets that contained either 5% glycine plus 15% casein or 20% casein as control for 10 weeks. Vascular lesions of aortic isografts and allografts were evaluated quantitatively with image analysis and cell counting.
No significant vascular changes were observed in isografts (mean medial areas of 3.3 +/- 0.3x0(5) microm2). However, dramatic intimal thickening (neointimal area 2.1+/-0.3) and medial thinning (1.5+/-0.3) were observed in allografts from rats fed control diet. In contrast, glycine significantly reduced the neointima by 45% (1.2+/-0.3) and protected the media (3.5+/-0.2). This led to intima to media area ratios almost twice as large in the control group as in glycine-fed rats (2.2+/-0.4 vs. 1.1+/-0.3, P<0.05). Moreover, infiltrating leukocytes, especially macrophages, were reduced significantly in the adventitia by glycine. In addition, glycine inhibited proliferation and migration of rat aortic smooth muscle cells in culture by 45 and 60%, respectively.
These results indicate that dietary glycine minimizes histopathological changes of chronic rejection by reducing the immune response and, in part, by minimizing proliferation and migration of smooth muscle cells.
Transplantation 03/2000; 69(5):773-80. · 4.00 Impact Factor
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ABSTRACT: N-6 polyunsaturated fatty acids (N-6 PUFAs), major constituents of corn oil and natural ligands for peroxisome proliferator-activated receptors, increase the rate of growth of established tumors. It has been proposed that chemical peroxisome proliferators increase hepatocyte proliferation by mechanisms involving activation of nuclear factor-kappaB (NF-kappaB) and production of low levels of tumor necrosis factor alpha (TNFalpha) by Kupffer cells; however, how N-6 PUFAs are involved in increased cell proliferation in liver is not well understood. Here, the hypothesis that N-6 PUFAs increase production of mitogens by activation of Kupffer cell NF-kappaB was tested. A single dose of corn oil (2 ml/kg, i.g.), but not olive oil or medium-chain triglycerides (saturated fat), caused an approximately 3-fold increase in hepatocyte proliferation. Similarly, when activity of NF-kappaB in whole rat liver or isolated hepatocytes and Kupffer cells was measured at various time intervals for up to 36 h, only corn oil activated NF-kappaB. Corn oil increased NF-kappaB activity approximately 3-fold 1-2 h after treatment exclusively in the Kupffer cell fraction. In contrast, increases were small and only occurred after approximately 8 h in hepatocytes. The activation of NF-kappaB at 2 h and increases in cell proliferation at 24 h due to corn oil were prevented almost completely when rats were pretreated for 4 days with either dietary glycine (5% w/w), an agent that inactivates Kupffer cells, or the NADPH oxidase inhibitor, diphenyleneiodonium (s.c., 1 mg/kg/day). Furthermore, arachidonic acid (100 microM) activated superoxide production approximately 4-fold when added to isolated Kupffer cells in vitro. This phenomenon was not observed with oleic or linoleic acids. Interestingly, a single dose of corn oil increased TNFalpha mRNA nearly 2-fold 8 h after treatment. It is concluded that corn oil rapidly activates NF-kappaB in Kupffer cells via oxidant-dependent mechanisms. This triggers production of low levels of TNFalpha which is mitogenic in liver and promotes growth of hepatocytes.
Carcinogenesis 12/1999; 20(11):2095-100. · 5.70 Impact Factor
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ABSTRACT: Increased cell proliferation most likely plays a key role in peroxisome proliferator-induced liver cancer. Recently, Kupffer cells were shown to be responsible for Wy-14,643-induced cell proliferation. However, the mechanism by which peroxisome proliferators activate Kupffer cells is unknown. Since gut-derived endotoxin is a known activator of Kupffer cells, the hypothesis that it is involved was evaluated. Increased cell proliferation and peroxisome induction were unaffected by gut sterilization. Moreover, endotoxin was not detectable in portal blood following treatment with Wy-14,643. Therefore, it is concluded that gut-derived endotoxin is not responsible for Kupffer cell activation. To test the hypothesis that Kupffer cells are activated by Wy-14,643 directly, Kupffer cell superoxide production was measured following treatment in vitro. Wy-14,643 increased superoxide production in a dose-dependent manner (0.1 and 50 microM) with half-maximal stimulation at 2.5 microM. Diethylhexylphthalate (DEHP) and ethylhexanol did not increase superoxide production even at doses 50 times higher than Wy-14,643; however, monoethylhexylphthalate (MEHP) activated superoxide production as effectively as Wy-14,643 with half-maximal stimulation at 5 microM. Treatment with Wy-14,643 for 21 days caused a 2-fold increase in Kupffer cell superoxide production while DEHP did not. Pretreatment of Kupffer cells with staurosporine (0.01-10 pM) completely blocked generation of superoxide demonstrating that protein kinase C is required. Moreover, Wy-14,643 increased Kupffer cell protein kinase C activity 3-fold. Pretreatment of Kupffer cells with the amino acid glycine (0.01-3 mM), which blunts calcium signaling, inhibited Wy-14,643-stimulated superoxide production and increased protein kinase C activity completely. These data are consistent with the hypothesis that potent peroxisome proliferators (Wy-14,643 and MEHP) directly activate Kupffer cell production of oxidants via mechanisms involving protein kinase C. Further, peroxisome proliferator treatments that sustain elevated rates of cell proliferation (e.g. Wy-14,643) activate Kupffer cell superoxide production following long-term dietary treatment supporting the hypothesis that Kupffer cell-derived oxidants are involved in peroxisome proliferator-induced neoplasia.
Carcinogenesis 02/1999; 20(1):27-33. · 5.70 Impact Factor
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ABSTRACT: Both in situ organ manipulation during harvest and steatosis reduce survival after liver transplantation via mechanisms involving Kupffer cells; thus, their effect on survival was compared here. Moderate steatosis was induced by a single dose of ethanol to Lewis rats, while long-term administration of ethanol yielded severe steatosis in donor animals. After minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 min subsequently. Orthotopic liver transplantation was performed after 1 h of cold storage in UW solution. Ethanol increased hepatic lipid content to a level of moderate or severe steatosis that reduced survival after transplantation from 100% to approximately 70% (P < 0.05). However, gentle manipulation decreased survival to approximately 30% (P < 0.05) in livers from normal, saline-treated rats and in livers from rats fed a high-fat control diet. Moreover, after short- or long-term ethanol administration, manipulation of fatty livers decreased survival from 70% to approximately 13% (P < 0.05). Further, manipulation elevated serum transaminases, total bilirubin, and necrosis significantly about 2- to 20-fold in fatty grafts after transplantation. At the end of harvest, trypan blue distribution time and hypoxia assessed from 2-nitroimidazole binding were elevated significantly about two- to fourfold by manipulation of fatty grafts. Gadolinium chloride, a Kupffer cell toxicant, blocked the detrimental effects of manipulation. These data demonstrate for the first time that, while steatosis is detrimental for survival, organ manipulation plays a much greater role than fat in mechanisms of primary nonfunction.
Transplant International 01/1999; 12(5):351-9. · 2.92 Impact Factor
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ABSTRACT: The etiology of primary graft nonfunction and dysfunction is unknown but most likely involves Kupffer cell-dependent reperfusion injury. However, the donor operation and surgical technique may also have an effect on the outcome after transplantation. Because liver manipulation during harvest cannot be prevented completely with standard procedures, its effect on survival was assessed here.
Donor livers were harvested from female Sprague-Dawley rats (200-230 g). Briefly, after minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 subsequent minutes. At 25 min, all livers were perfused with cold University of Wisconsin solution via the portal vein, and transplantation was performed after cold storage (1 hr). In some rats, Kupffer cells were destroyed with gadolinium chloride or inactivated with dietary glycine before harvest. Survival, proteolytic activity in the rinse effluent, serum transaminases, trypan blue distribution to index microcirculation, and histology were compared.
In the nonmanipulated group, survival was 100% after transplantation; however, gentle manipulation decreased survival by 70%. Further, manipulation elevated transaminases fivefold and caused about 200% necrosis. At harvest, proteolytic activity and the time for trypan blue to distribute homogeneously were elevated three- to eightfold by manipulation. Gadolinium chloride and glycine prevented the effects of manipulation on all parameters studied.
These data indicate for the first time that brief, gentle manipulation of the donor liver has a marked detrimental effect on survival by priming or activating Kupffer cells. This may represent an important early event in pathogenesis, because Kupffer cells play an important role in primary graft nonfunction.
Transplantation 05/1998; 65(8):1015-20. · 4.00 Impact Factor
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ABSTRACT: Although NFkappaB binding activity is induced during liver regeneration after partial hepatectomy, the physiological consequence of this induction is unknown. We have assessed the role of NFkappaB during liver regeneration by delivering to the liver a superrepressor of NFkappaB activity using an adenoviral vector expressing a mutated form of IkappaBalpha. This adenovirus (Ad5IkappaB) was almost exclusively expressed in the liver and inhibited NFkappaB DNA binding activity and transcriptional activity in cultured cells as well as in the liver in vivo. After partial hepatectomy, infection with Ad5IkappaB, but not a control adenovirus (Ad5LacZ), resulted in the induction of massive apoptosis and hepatocytes as demonstrated by histological staining and TUNEL analysis. In addition, infection with Ad5IkappaB but not Ad5LacZ decreased the mitotic index after partial hepatectomy. These two phenomena, increased apoptosis and failure to progress through the cell cycle, were associated with liver dysfunction in animals infected with the Ad5IkappaB but not Ad5LacZ, as demonstrated by elevated serum bilirubin and ammonia levels. Thus, the induction of NFkappaB during liver regeneration after partial hepatectomy appears to be a required event to prevent apoptosis and to allow for normal cell cycle progression.
Journal of Clinical Investigation 03/1998; 101(4):802-11. · 15.39 Impact Factor
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ABSTRACT: Peroxisome proliferators are a group of nongenotoxic carcinogens which include a number of hypolipidemic drugs, solvents, and industrial plasticizers. Although the mechanism by which they cause cancer remains unknown, one likely possibility is that they act as tumor promoters by increasing cell proliferation. Hepatic Kupffer cells represent a rich source of mitogenic cytokines (e.g., tumor necrosis factor alpha, TNF alpha) and are stimulated by peroxisome proliferators. Since glycine prevents activation of Kupffer cells, these experiments were designed to test the hypothesis that a diet containing glycine could block the mitogenic effect of the peroxisome proliferator [[4-chloro-6-(2,3-xylidino)pyrimidinyl]thio]acetic acid (WY-14,643). The effects of a glycine-enriched diet on WY-14,643-induced increases in cell proliferation after a single dose or after feeding WY-14,643 in the diet for 3 weeks were assessed. As expected, 24 h after a single dose of WY-14,643, rates of cell proliferation increased from basal values of 0.7 +/- 0.3% to 5.1 +/- 0.5%. Glycine largely prevented the increase caused by WY-14,643 with proliferation only reaching 1.9 +/- 0.4% (p < 0.05). Acyl CoA oxidase increased from 1.4 +/- 0.1 to 3.5 +/- 0.6 nmol of H2O2 min-1 (mg of protein)-1 (p < 0.05) indicating that peroxisome-specific enzyme activity was induced about 2-fold in livers of WY-14,643-treated rats after 24 h. Unlike cell proliferation, however, acyl CoA oxidase was not affected by dietary glycine, consistent with the hypothesis that cell proliferation and peroxisome proliferation occur via different mechanisms. After 3 weeks, dietary glycine reduced basal rates of cell proliferation by about 50% and completely prevented the sustained 5-fold increase in cell proliferation caused by feeding WY-14,643. Moreover, the 3-fold increase in TNF alpha mRNA caused by WY-14,643 was blocked completely by the glycine-enriched diet. Similarly, immunohistochemical staining for TNF alpha was increased 6-fold by WY-14,643, an increase which was prevented by dietary glycine. However, the 6-fold increase in acyl CoA oxidase activity was unaffected by glycine under similar conditions demonstrating that a diet containing 5% glycine prevents increased hepatocyte proliferation caused by a potent peroxisome proliferator without affecting induction of peroxisomes. These data demonstrate that a glycine-enriched diet prevents stimulated cell proliferation most likely by inhibiting TNF alpha production and raise the possibility that dietary glycine will be effective in preventing cancer caused by nongenotoxic carcinogens such as WY-14,643.
Chemical Research in Toxicology 10/1997; 10(10):1198-204. · 3.78 Impact Factor
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ABSTRACT: WY-14,643 [4-chloro-6-(2,3-xylidino)pyrimidinylthio-acetic acid] is a well-known non-genotoxic carcinogen and peroxisome proliferator that causes liver cancer in rodents by unknown mechanisms. Its ability to sustain elevated rates of hepatocyte DNA synthesis is most likely pivotal in the ultimate development of tumors. The source of this mitogenic stimulus following treatment of rats with WY-14,643 has been hypothesized to be Kupffer cells, the resident hepatic macrophages, since they are activated by peroxisome proliferators in vivo. Therefore, these studies were designed to determine if Kupffer cells are causally responsible for WY-14 643-induced increases in hepatocyte DNA synthesis in vivo. WY-14,643 (100 mg/kg) increased DNA synthesis 8-fold 24 h after treatment; however, inactivation of Kupffer cells with methyl palmitate, a nonhydrolyzable fatty acid ester and known Kupffer cell inhibitor, completely prevented the mitogenic effect of WY-14,643. On the other hand, the ability of WY-14,643 to induce peroxisomes was not affected by methyl palmitate. These data demonstrate that induction of peroxisomes is not dependent on factors from Kupffer cells and support the idea that stimulation of DNA synthesis and induction of peroxisomes occur via distinct mechanisms. Additionally, WY-14,643 increased liver mRNA transcripts of the hepatocyte mitogen tumor necrosis factor alpha (TNF alpha) more than twofold. This increase was also prevented by inactivating Kupffer cells with methyl palmitate. Therefore, it is concluded that Kupffer cells are causally responsible for WY-14,643-induced increases in hepatocyte DNA synthesis most likely by increasing production of TNF alpha, a hepatic mitogen.
Carcinogenesis 09/1997; 18(8):1453-6. · 5.70 Impact Factor
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ABSTRACT: 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.
Carcinogenesis 05/1997; 18(4):669-74. · 5.70 Impact Factor
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ABSTRACT: 1,2,3-Trichloropropane (TCP) induces high incidences of tumors at multiple sites in mice and rats when administered chronically by gavage. The animal tumor data are being used to predict human risk from potential exposure to TCP in drinking water. Risk assessment may be affected by differences in the route of exposure. Gavage administration, which results in high bolus concentrations compared to drinking water exposure, may quantitatively affect toxicokinetics, cytotoxicity, and genotoxicity. We have examined the effects of TCP exposure by the two routes on the formation of DNA adducts and the induction of cellular proliferation. Male B6C3F1 mice were administered [14C]TCP for 1 week by gavage or in drinking water at the low dose (6 mg/kg) used in the NTP carcinogenesis bioassay. Two target organs (forestomach and liver) and two nontarget organs (glandular stomach and kidney) were examined for DNA adduct formation. Adducts were hydrolyzed from DNA, isolated by HPLC, and quantitated by measuring HPLC fractions for radioactivity. In the forestomach, liver, and kidney, gavage administration of TCP resulted in 1.4-to 2.4-fold greater yields of the major DNA adduct, previously identified as S-[1-(hydroxymethyl)-2-(N7-guanyl)ethyl]glutathione. Significant differences in cell proliferation, as determined by incorporation of bromodeoxyuridine into DNA, were also observed for the two routes. Gavage administration of TCP for 2 weeks resulted in up to a threefold greater cell proliferation rate relative to administration in drinking water. Our findings of exposure-related differences in TCP-induced DNA adduct formation and cell proliferation suggest that a risk assessment based on the existing gavage study may overestimate human risk.
Toxicology and Applied Pharmacology 10/1996; 140(1):108-14. · 4.45 Impact Factor
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