Evaluation of liver cell proliferation during ciprofibrate-induced hepatocarcinogenesis

To determine if the carcinogenic potential of peroxisome proliferators is dependent upon their ability to induce cell proliferation, we have investigated the extent of cell proliferation in the livers of rats fed ciprofibrate, a peroxisome proliferator. Male rats were maintained on a diet containing ciprofibrate (0.025% w/w) and killed at selected intervals following 1 week of continuous [3H]thymidine labeling. Evaluation of labeling indices demonstrated a significant increase in cell proliferation during the first week but not in rats killed at the end of 5 and 20 weeks of treatment. Increases in hepatocyte nuclear labeling were found at 40 and 70 weeks of ciprofibrate administration which coincided with the appearance in livers of putative preneoplastic and neoplastic lesions. In a short-term feeding study, ciprofibrate and ethoxyquin were fed to rats at a dietary concentration of 0.025% and 0.5%, respectively, either alone or in combination for 7 days. Ciprofibrate and ethoxyquin either alone or in combination produced marked hepatomegaly and a significant increase in DNA synthesis as demonstrated by [3H]thymidine incorporation and autoradiographic studies. DNA synthesis in the group receiving ciprofibrate and ethoxyquin simultaneously, was slightly more than in animals that received either compound alone, suggesting a synergistic effect, although chronic feeding of these agents together resulted in inhibition of liver carcinogenesis (Rao, M. S. et al. (1984) Cancer Res., 44, 1072-1076). The results of this study further suggest that cell proliferation induced by peroxisome proliferators may be less important in carcinogenesis than peroxisome proliferation induced by these compounds.     

Formation of 8-hydroxydeoxyguanosine in liver DNA of rats following long-term exposure to a peroxisome proliferator

The mechanism by which nongenotoxic peroxisome proliferators induce hepatocellular carcinomas in rats and mice remains intriguing. The available experimental evidence suggests that the proliferation of peroxisomes and induction of peroxisome-associated enzymes results in oxidative stress which then leads to tumorigenesis. However, so far no direct evidence for oxidative DNA damage in livers of peroxisome proliferator-treated animals has been established. In the present study we have examined the DNA obtained from the livers of rats treated with ciprofibrate, a potent peroxisome proliferator, for variable periods of time for 8-hydroxydeoxyguanosine (8-OH-dG), an adduct that results from the damage of DNA caused by hydroxyl radical. Administration of ciprofibrate in diet at a concentration of 0.025% for 16, 28, 36, or 40 weeks resulted in progressive increases in the levels of 8-OH-dG. At 16, 28, and 40 weeks of ciprofibrate treatment, the 8-OH-dG in the liver DNA was significantly increased as compared to controls. This increase in 8-OH-dG levels is attributed to persistent peroxisome proliferation resulting from chronic ciprofibrate treatment as no increase in 8-OH-dG was found in liver DNA of rats that received a single large dose of ciprofibrate. The results of this study clearly demonstrate, for the first time, that persistent proliferation of peroxisomes leads to specific oxidative DNA damage.     

Peroxisome proliferator-induced alterations in the expression and modification of rat hepatocyte plasma membrane proteins

Rats were fed the peroxisome proliferator ciprofibrate (0.025%), and the effects on the expression, modification, and localization of seven domain-specific integral proteins of the rat hepatocyte plasma membrane were assessed using a combination of immunoblotting, -precipitation, and -fluorescence. Ciprofibrate caused the down-regulation of five of the plasma membrane proteins (the epidermal growth factor receptor, the asialoglycoprotein receptor, HA 321, HA 4, and dipeptidylpeptidase IV) and induced the expression of a more basic, lower-Mr isoform of the basolateral plasma membrane protein CE 9. Pulse labeling, chemical deglycosylation, and 125I-wheat germ lectin blotting suggested that the ciprofibrate-induced isoform of CE 9 differed in the posttranslational modification of its oligosaccharides and contained more sialic acid. These changes in hepatocyte surface differentiation were first observed between Days 1 and 5 on the ciprofibrate-containing diet, coincident with other aspects of the pleiotropic response of the hepatocyte to peroxisome proliferators, e.g., the induction of the Mr 78,000 peroxisome proliferation-associated protein. The effects were reversed within 2-3 weeks upon removal of ciprofibrate. The three other peroxisome proliferators tested, di(2-ethylhexyl)phthalate, clofibrate, and Wy-14,643, were found to exert most of these same effects on the expression and modification of the hepatocyte plasma membrane proteins, but the compounds differed in relative potency. The ciprofibrate-induced decreases in the concentrations of the epidermal growth factor receptor, the asialoglycoprotein receptor, HA 321, and HA 4 were similar to the selective down-regulation of these proteins observed transiently during the period of hepatocyte proliferation following two-thirds hepatectomy. Other compounds frequently used in studies of liver enzyme induction and carcinogenesis, the antioxidants ethoxyquin and butylated hydroxyanisole and the liver tumor promoter phenobarbital, were not as effective as ciprofibrate or two-thirds hepatectomy at causing the down-regulation of these proteins. The induction of the lower-Mr isoform of the basolateral plasma membrane protein CE 9 was not observed following two-thirds hepatectomy or upon the feeding of the antioxidants or phenobarbital but was specific to the feeding of the peroxisome proliferators.     

Peroxisome proliferators increase ethanol catabolism through utilization of the catalase pathway

We investigated the effects of ciprofibrate, a potent peroxisome proliferator, on ethanol metabolism in mice. The blood alcohol levels of mice fed a liquid diet containing both ciprofibrate and ethanol were markedly depressed compared with mice fed the ethanol-containing diet alone. Ciprofibrate markedly induced enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, hydrogen peroxide, and to a lesser extent catalase in both control and ethanol-diet fed mice. Northern blot analysis indicated no significant upregulation of cytochrome P450IIE1 mRNA by ciprofibrate. Our study suggests that peroxisome proliferators increase ethanol catabolism through hydrogen peroxide production, thus allowing utilization of the catalase pathway. These findings indicate that catalase has the potential to provide a significant pathway for ethanol metabolism under conditions of peroxisome proliferation.     

Differences between the promoting activities of the peroxisome proliferator WY-14,643 and phenobarbital in rat liver

In order to characterize the promoting activity of the peroxisome proliferator [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (WY-14,463), male F344 rats which received a single 150-mg/kg dose of diethylnitrosamine (DEN) were fed 0.1% WY-14,643 or 0.05% phenobarbital in the diet for 11, 22, or 54 wk. WY-14,643 promoted the development of ATPase-deficient foci but not GGTase-positive or G6Pase-deficient foci, in contrast to phenobarbital which promoted development of foci detected by all three markers. The mode of promotion of ATPase-deficient foci by WY-14,643 was distinctly different from that of phenobarbital. WY-14,643 primarily increased mean volume of foci at 11 and 22 wk, while phenobarbital primarily increased the numerical density of foci at these time points. At 54 wk, the yield of hepatic neoplasms per liver was higher in rats fed WY-14,643 than in rats fed phenobarbital. To evaluate the possibility that the promotional activity of WY-14,643 was more effective at a later stage in hepatocarcinogenesis, rats receiving a dose of DEN and then phenobarbital in the diet for 11 wk were changed to a diet containing WY-14,643 for an additional 11 or 43 wk. However, WY-14,643 feeding from wk 11 to 22 caused a reduction in volume density of ATPase-deficient foci relative to the volume density of foci at 11 wk. In addition, feeding WY-14,643 from wk 11 to 54 caused similar yields of hepatic neoplasms whether or not phenobarbital was fed for the initial 11 wk. WY-14,643 induced hepatic peroxisome proliferation as indicated by palmitoyl CoA oxidase activity regardless of prior treatment with DEN and/or phenobarbital. The yield of neoplasms in rats not receiving DEN was greater in rats fed WY-14,643 for wk 11 to 54 than in rats fed WY-14,643 for wk 1 to 54. In summary, the peroxisome proliferator WY-14,643 was a more efficient promoter of hepatocarcinogenesis in DEN-initiated rats than phenobarbital. The promotional activity of WY-14,643, when evaluated by stereological analysis and by changing promoters, is distinct from that of phenobarbital, perhaps suggesting different cellular and/or molecular mechanisms of promotion. Understanding the role of promotion by WY-14,643 and other peroxisome proliferators may be important in understanding the mechanism of their hepatocarcinogenicity.     

Effect of the peroxisome proliferators ciprofibrate and perfluorodecanoic acid on hepatic cell proliferation and toxicity in Sprague--Dawley rats

The objective of this study was to compare the effects of perfluorodecanoicacid (PFDA) and ciprofibrate on the induction of hepatic toxicityand on hepatocellular proliferation in rats. In the first study,rats were first subjected to partial hepatectomy and then injectedwith [³H]thymidine (20 µCi/injection) at 23, 24, 25, 47,48 and 49 h afterwards. After a 2 week recovery period, ratswere injected with one of four levels of PFDA (03, 1.0, 3.0or 10 mg/kg/injection) in four i.p. doses every 14 days, orwere fed 0.01% or 0.003% ciprofibrate. Six days after the lastPFDA injection and three days before the animals were killed,an osmotic minipump containing 20 mg/ml 5-bromo-2’-deoxyuridine(BrdU) was implanted s.c for the measurement of DNA synthesis.Peroxisomal fatty acyl-CoA oxidase activity was significantlyenhanced in both PFDA and ciprofibrate-treated groups in a dose-dependentmanner. Hepatotoxicity, measured as the loss of [³H]thymldinefrom hepatic DNA, was not significantly affected by any of thetreatments. Hepatic DNA synthesis was significantly increasedonly in rats receiving the highest dose of PFDA. In order todetermine the time course of ciprofibrate- and PFDA-inducedcell proliferation, we conducted another study with more timepoints. Rats were fed 0.01% ciprofibrate or were injected every14 days with 3 or 10 mg PFDA/kg body weight for 10 days, 24days, 6 weeks, 26 weeks or 54 weeks. Cell proliferation wasquantified as in the first study. Ciprofibrate increased cellproliferation at the early but not the later time points, whereasPFDA increased cell proliferation at most times throughout thestudy. This study demonstrates that PFDA and ciprofibrate donot selectively induce hepatic toxicity and that their effectson cell proliferation do not correlate with their carcinogenicor promoting activities.     

Formation of 5-hydroxymethyl-2'-deoxyuridine in hepatic DNA of rats treated with gamma-irradiation, diethylnitrosamine, 2-acetylaminofluorene or the peroxisome proliferator ciprofibrate

The peroxisome proliferator ciprofibrate was tested for its ability to induce DNA damage in the form of 5-hydroxymethyl-2'-deoxyuridine (HMdU), an adduct that results from the reaction of thymine in DNA with hydroxyl radicals. In order to quantify HMdU, DNA containing [3H]thymidine of high specific activity had to be obtained. Since hepatocytes normally have a very low rate of DNA synthesis, rats were subjected to partial hepatectomy to stimulate DNA synthesis and then were administered [methyl-3H]thymidine by three p.o., i.p. or i.v. injections 20, 22 and 24 h after partial hepatectomy; or by slow infusion through the portal vein, starting 20 h after partial hepatectomy for 4 h. The specific activity of DNA in rats receiving [3H]thymidine through the portal vein was considerably higher than in rats receiving p.o., i.p. or i.v. injections. Rats were then exposed to various doses of gamma-irradiation after partial hepatectomy and infusion of [6-3H]thymidine through the portal vein. DNA from the liver was extracted, enzymatically hydrolyzed and analyzed by HPLC. The percentage of HMdU in DNA increased in a dose-dependent manner. Rats were then treated with the carcinogens 2-acetylaminofluorene (AAF) or diethylnitrosamine (DEN) in conjunction with partial hepatectomy and infusion of [methyl-3H]thymidine. There was an increase in HMdU formation after a single administration of DEN or AAF. Another group of rats was fed a diet containing the peroxisome proliferator ciprofibrate for 3 weeks. After partial hepatectomy and infusion of [6-3H]thymidine, these rats were fed the same ciprofibrate-containing diet for 2-4 more weeks. HMdU was detected in DNA at 2-4 weeks after [6-3H]thymidine infusion, but the level at 4 weeks was nearly 50% less than at 2 weeks. This study shows that oxidative DNA damage in the form of HMdU is induced in the liver by gamma-irradiation, DEN, AAF and peroxisome proliferation.     

Blockade of alpha-1 adrenergic receptor inhibits hepatic DNA synthesis stimulated by tumor promoters

Studies with regenerating liver and hepatocyte cultures have shown that the alpha-1 adrenergic receptor (A1AR) is involved in the early events which transmit a mitogenic signal to hepatocytes after 2/3 partial hepatectomy. In this study, we investigated the role of A1AR in DNA synthesis associated with the augmentative hyperplasia stimulated by the xenobiotic hepatic tumor promoters phenobarbital (PB) and alpha-hexachlorocyclohexane (alpha-HCH), and the peroxisome proliferator ciprofibrate. Male F344 rats were treated with each of the three xenobiotics to stimulate hepatic DNA synthesis. When either phenobarbital or alpha-HCH administration was preceded and accompanied by the A1AR antagonist prazosin, DNA synthesis was significantly inhibited, as measured by [3H]thymidine incorporation or 5-bromo-2'-deoxyuridine (BrdU) nuclear labeling index. There was no inhibition of DNA synthesis by prazosin in the ciprofibrate treated group. The inhibition of hepatic DNA synthesis by prazosin was accompanied by non-significant changes in the number of alpha-1 binding sites in the PB and alpha-HCH treated groups, but a significantly reduced number of alpha-1 binding sites in the ciprofibrate treated group. These studies suggest that A1AR is involved in generating the mitogenic signal leading to hepatic DNA synthesis induced by xenobiotic hepatic tumor promoters phenobarbital and alpha-HCH. A1AR is not involved in the mitogenic pathway generated by the peroxisome proliferator ciprofibrate.     

Inhibitory effect of antioxidants ethoxyquin and 2(3)-tert-butyl-4-hydroxyanisole on hepatic tumorigenesis in rats fed ciprofibrate, a peroxisome proliferator

The objective of this study was to test the hypothesis that hepatocarcinogenesis by peroxisome proliferators, a novel class of chemical carcinogens, is mediated either directly by carcinogenic H2O2, generated by peroxisomal oxidase(s) or indirectly by free radicals produced from H2O2, and that antioxidants could retard or inhibit neoplasia by scavenging active oxygen (super-oxide radicals O(2), hydrogen peroxide, hydroxyl radicals HO, and singlet oxygen 1O2). Accordingly, the effect of synthetic antioxidants 2(3)-tert-butyl-14-hydroxyanisole and ethoxyquin on the peroxisome proliferator 2-[4-(2,2-dichlorocyclopropyl)phenoxy]2-methyl-propionic acid (ciprofibrate)-induced hepatic tumorigenesis has been examined in male Fischer 344 rats. Rats were fed either a 2(3)-tert-butyl-4-hydroxyanisole (0.5% w/w)- or ethoxyquin (0.5% w/w)-containing diet with or without ciprofibrate (10 mg/kg of body weight) for 60 weeks. Rats fed ciprofibrate (10 mg/kg of body weight) in the diet or fed a diet with no added chemicals served as controls. Results of this study demonstrated that ethoxyquin markedly inhibited the hepatic tumorigenic effect of ciprofibrate, as evidenced by a decreased incidence of tumors, a decreased number of tumors per liver, and a reduced tumor size. 2(3)-tert-Butyl-4-hydroxyanisole also caused a significant decrease in the incidence and number of hepatocellular carcinomas that were larger than 5 mm. The present data suggest that the inhibitory effect of antioxidants on ciprofibrate-induced hepatic tumorigenesis may be due to H2O2 and free radical-scavenging property of ethoxyquin and 2(3)-tert-butyl-4-hydroxyanisole, since these antioxidants do not prevent peroxisome proliferation and induction of H2O2-generating peroxisomal enzymes in livers of rats fed ciprofibrate. Whether the inhibitory effect of antioxidants is exercised on the presumptive H2O2 initiation process and/or on the postinitiation growth phase of foci and nodules in liver is, at present, unknown.     

Response of hepatocytes transplanted into syngeneic hosts and heterotransplanted into athymic nude mice to peroxisome proliferators

The development of a transplantation system by which rat hepatocytes can be implanted and remain viable in the dorsal fascia of two-thirds hepatectomized syngeneic hosts provides an opportunity to examine whether such transplanted hepatocytes retain the capacity to recognize and respond to the peroxisome proliferators 2-[4-(2,2- dichlorocyclopropyl )phenoxy]-2- methylpropionic acid (ciprofibrate), a hypolipidemic drug, and di-(2-ethylhexyl)phthalate (DEHP), an industrial plasticizer. Male F344 rats with transplanted rat hepatocytes were fed a control diet or a diet containing either 0.05% ciprofibrate (w/w) or 2% DEHP (v/w). After 4 weeks, the animals were sacrificed, and transplanted hepatocytes revealed a significant increase in the numerical density of peroxisomes in both ciprofibrate- and DEHP-fed rats. The volume density of peroxisomes in transplanted hepatocytes increased 9.2- and 5.3-fold, respectively, in ciprofibrate- and DEHP-fed rats, whereas the volume density of mitochondria remained essentially unchanged. The magnitude of increase in peroxisome volume density in transplanted hepatocytes was comparable to increases in the volume density of these organelles in the liver parenchymal cells of syngeneic hosts. The present study also demonstrates that hepatocytes isolated from cat liver and heterotransplanted into partially hepatectomized athymic nude mice retain their biological integrity and respond to the peroxisome proliferative effect of ciprofibrate. This observation suggests the possibility that hepatocytes obtained from small segments of liver of humans, primates, and other species and heterotransplanted into nude mice might provide a valuable model system for toxicological evaluation of chemicals. These studies suggest that hepatocytes, irrespective of their location in the body, recognize the peroxisome proliferator or its active metabolite(s), which stimulates the expression of peroxisome-specific genes in these cells.     

Peroxisome proliferation and lipid peroxidation in rat liver

Male F344 rats were fed a diet containing the peroxisome proliferators 2-[4-(2,2-dichlorocyclopropyl)phenoxy]-2-methylpropionic acid [ciprofibrate (0.025%)] or [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid [Wy-14643 (0.1%)] for up to 14 months to determine whether hepatic peroxisome proliferation caused by these agents results in the induction of membrane lipid peroxidation in the liver. Peroxidative damage of membrane lipids from whole liver, postnuclear, heavy-particle, microsomal, and nuclear membranes was evaluated by determining the extent of formation of conjugated dienes (ultraviolet absorption, 233 nm). Increased generation of diene conjugates was noted in whole-liver, postnuclear, and heavy-particle membrane lipids of rats fed peroxisome proliferators for 6 months or longer when compared to controls. An additional, more intense absorption profile in the ultraviolet absorption range of approximately 276 nm was noted in the membrane lipids derived from whole liver, postnuclear, and heavy particle pellets, but not in the nuclear and microsomal membrane lipids of livers with peroxisome proliferation. Although the exact chemical nature of this delta 276 nm peak is not clear, it is attributed to the formation of ketone dienes and/or conjugated trienes. The excess lipid peroxidation correlates with the previous observation of accumulation of abundant quantities of lipofuscin in hepatocytes of rats chronically exposed to peroxisome proliferators. The generation of conjugated dienes and ketone dienes and/or trienes together with increased levels of H2O2 generation by peroxisomal enzymes, and decreased levels of hepatic glutathione peroxidase, glutathione reductase, and glutathione-S-transferases, enzymes responsible for the defense against H2O2 damage, suggest the occurrence of membrane lipid peroxidation and oxidative stress in livers of rats treated with carcinogenic peroxisome proliferators.     

The peroxisome-proliferator ciprofibrate induces hypergastrinemia without raising gastric pH

The ECL-cell hyperplasia and ECL-cell carcinoids occurring duringlong-term treatment with ciprofibrate, have been attributedto hypergastrinemia secondary to an inhibitory effect on acidsecretion. However, nobody has given any explanation of themechanism by which ciprofibrate and related phenoxyisobutyratederivates inhibit acid secretion. Moreover, the reported inhibitionof acid secretion has only been moderate, in contrast to theprofound inhibition of acid secretion needed to induce similarECL-cell changes. To re-examine the effect of ciprofibrate ongastric acidity and serum gastrin, we randomly assigned 33 maleFisher rats into three treatment groups (100 or 20 mg/kg/dayof ciprofibrate and control) during a period of 4 weeks. Dailyassessments of gastric acidity was done by gastric intubation,using a tube with a diameter of 2.0 mm allowing the introductionof an infant pH-catheter. Measurements were done in all animals5 days a week. Ciprofibrate did not raise gastric pH. On thecontrary, the highest dose increased the acidity. Serum gastrinlevels measured in blood taken by vein puncture before the initiationof the drug treatment and on the last day of the 4 week treatmentperiod, revealed a dose-related significant hypergastrinemiceffect of ciprofibrate. The slight increase in gastric acidityin the ciprofibrate high-dose group is most likely due to thehypergastrinemia provoked by the drug. This hypergastrinemiais therefore not secondary to an inhibition of acid secretion,but may be due to a direct effect of ciprofibrate on the G-cell.The ECL-cell hyperplasia and the ECL-cell carcinoids, whichdevelop during treatment with peroxysome-proliferators are thusdue to hypergastrinemia, which is not secondary to inhibitionof acid secretion.     

Induction of peroxisome proliferation and hepatic tumours in C57BL/6N mice by ciprofibrate, a hypolipidaemic compound

The hepatic effects of ciprofibrate, a potent peroxisome proliferator, were evaluated in male C57BL/6N mice, a mouse strain with very low incidence of spontaneous liver tumour development. Dietary feeding of ciprofibrate (0.0125% or 0.025% w/w) for 2 weeks resulted in a marked proliferation of peroxisomes (9-fold increase) and several-fold increase (8- to 10-fold) in the activity of peroxisomal beta-oxidation enzymes. Feeding ciprofibrate at 0.025% concentration for 15 months followed by a 0.0125% for 6 months led to the development of hepatic adenomas in 8/14 (57%) and hepatocellular carcinomas (HCC) in 3/14 (21%) mice. In mice given 0.0125% ciprofibrate for 18 months 5 of 8 (62%) and 3 of 8 (37%) developed adenomas and HCC respectively. Similar to the findings observed in rats, both the adenomas and HCC were negative for gamma-glutamyltranspeptidase. These results in C57BL/6N mice of hepatocarcinogenic effect of ciprofibrate, a non-genotoxic chemical, indicate that peroxisome proliferation can be used as a reliable parameter to evaluate the carcinogenicity of hypolipidaemic compounds.     

Induction of altered hepatic foci in rats by the administration of hypolipidemic peroxisome proliferators alone or following a single dose of diethylnitrosamine

The effect of feeding hypolipidemic peroxisome proliferators on the induction of altered hepatic foci (AHF) in Fischer rats was studied in order to determine whether such agents can induce or promote the development of AHF. In the first study, rats were fed ciprofibrate (10 mg/kg/day) for 1 yr. AHF, neoplastic nodules, and hepatocellular carcinomas were induced. The presence of putative gamma-glutamyltranspeptidase (GGT) activity was numerically the most common marker, although it was absent in larger foci and nodules. A deficiency in canalicular ATPase and glucose-6-phosphatase provided the best markers for the larger foci and nodules. In the second study, rats were subjected to partial hepatectomy, and half of the animals were then intubated with diethylnitrosamine (10 mg/kg). One wk later, rats were fed Wy-14,643 at concentrations of 0, 0.05, and 0.1% in the diet for 6 mo. At 6 mo, the number and volume of foci were increased by the feeding of Wy-14,643 after partial hepatectomy alone and were greatly increased when Wy-14,643 was fed after partial hepatectomy/diethylnitrosamine administration. Canalicular adenosine triphosphatase and glucose-6-phosphatase deficiencies were the most common markers of AHF, and AHF of these phenotypes occupied practically all of the focal volume. The larger AHF did not express GGT, and those foci exhibiting GGT were much less common and occupied very little volume. The absence of the GGT protein itself, as opposed to an inhibition of GGT activity, was verified by immunohistochemical staining using an antibody to GGT. These studies show that hypolipidemic peroxisome proliferators can stimulate an increase in AHF following a single dose of diethylnitrosamine and a mitotic stimulus, and they thus can act as promoters in two-stage liver carcinogenesis. GGT is a poor marker for identifying AHF induced by peroxisome proliferators during the early, premalignant phase of hepatocarcinogenesis.     

Inhibition of ciprofibrate-induced hepatocarcinogenesis in the rat by dimethylthiourea, a scavenger of hydroxyl radical.

DNA damage caused by oxidative stress is considered to play an important role in peroxisome proliferator-induced hepatocarcinogenesis in rats and mice. In this study, we investigated the effect of dimethylthiourea (DMTU), a known hydroxyl radical scavenger, on ciprofibrate-induced hepatocarcinogenesis. Male F-344 rats were fed a diet containing 0.025% ciprofibrate and given daily intraperitoneal injections of DMTU (5 days a week) at a dose of 50 or 250 mg/kg body weight for 60 weeks at which time the study was terminated. Livers from all animals were analyzed grossly and microscopically for incidence, number and type of tumors. All rats given ciprofibrate alone developed altered areas, neoplastic nodules (NN) and hepatocellular carcinomas (HCC). Combined administration of ciprofibrate and DMTU resulted in inhibition of tumor development. In the group given higher doses of DMTU the incidence of NN was 100% and HCC 0%. The number of tumors per liver also significantly decreased (p<0.001). At lower dose levels DMTU caused significant reduction in the number of tumors per liver (p<0. 05) and a slight reduction (29%) in the incidence of HCC. The inhibitory effect of DMTU on ciprofibrate-induced hepatocarcinogenesis could be explained by hydroxyl radical scavenging properties of DMTU, resulting in decreased free radical induced DNA damage.     

Absence of gamma-glutamyltranspeptidase activity in lung metastasis in rats with hepatocellular carcinomas induced by ciprofibrate, a peroxisome proliferator

The incidence of lung metastasis in rats with hepatocellular carcinomas (HCC) induced by ciprofibrate, a peroxisome proliferator and the expression of gammaglutamyl transeptidase (GGT) in the metastatic lesions was studied. HCC were induced in 75 male F-334 rats by chronic dietary administration of ciprofibrate (0.025% w/w) for 16-22 months. The incidence of lung metastasis was 25% in rats killed between 14 and 16 months which increased to 56% in rats killed between 20 and 22 months. The lung metastases were multifocal and present in both the blood vessels and parenchyma. All the metastatic foci examined for the expression of GGT by histochemical stain were devoid of this enzyme. The results of this study clearly demonstrate the malignant behavior of ciprofibrate-induced liver tumors and the absence of GGT expression in metastatic lesions a phenotypic property that is peculiar to the primary HCC induced by several peroxisome proliferators.     

Activity of the anorectic agent 1,3-bis[2-cyano-5-(trifluoromethyl)-phenyl]triazene in in vitro and in vivo liver promoting assays

A substituted 1,3-diaryltriazene, 1,3-bis[2-cyano-5-(trifluoromethyl)phenyl]triazene (BPT), was studied for promoting activity in vitro and in vivo. BPT inhibited intercellular molecular exchange between cultured hepatocytes and rat liver epithelial cells, although the effect was not consistent. For the in vivo assay, male F344 rats were first exposed to N-2-fluorenylacetamide (FAA) for 8 weeks to induce liver altered foci, after which those maintained on control diet for an additional 12 weeks developed a 33% incidence of liver neoplasms. In rats given 0.02% BPT in the diet as a second exposure, the final incidence of liver neoplasms was 92%, which was comparable to the enhancement by phenobarbital (PB), a known liver neoplasm promoter. In the rats given BPT after FAA, the area occupied by gamma-glutamyltranspeptidase (GGT)-positive preneoplastic and neoplastic lesions was significantly higher than in the rats exposed to FAA only. Feeding of BPT alone for 12 weeks did not induce either liver altered foci or neoplasms and it was non-genotoxic in the hepatocyte DNA repair test. Therefore, although additional studies are needed to firmly establish the basis for the enhancement of liver carcinogenesis, BPT is suggested to be a new type of liver neoplasm promoter.     

The effect of pre- and post-treatment with phenobarbital on the extent of gamma-glutamyl transpeptidase positive foci induced in rat liver by N-nitrosomorpholine

Male Sprague-Dawley rats were treated with a combination of N-nitrosomorpholine (NNM) and phenobarbital and the extent of foci positive for gamma-glutamyl transpeptidase (gamma-GT), as demonstrated histochemically in liver, was quantitated. Phenobarbital (0.05% in the diet) was given either alone, before, or after a 7-week period of treatment with NNM (80 ppm in the drinking water). When given after the carcinogen treatment, phenobarbital enhanced the formation of gamma-GT-positive foci in liver compared with rats treated with NNM alone. No such increase, however, was observed when phenobarbital was administered before treatment with NNM. Phenobarbital alone did not produce any apparent amount of foci in liver. These data suggest that the enhancement of carcinogenic response mediated by phenobarbital as observed under the present experimental conditions is only operative in the presence of pre-existing initiated hepatocytes.