Induction and origin of hepatocytes in rat pancreas.

2-[4(2,2- Dichlorocyclopropyl )phenoxy]2-methyl propionic acid (ciprofibrate), a peroxisome proliferator , induced hepatocytes in the pancreas of adult male F-344 rats when added to their diet at a dosage of 10 mg/kg body weight for 60-72 wk. These cells are morphologically indistinguishable from hepatic hepatocytes and were usually localized adjacent to islets of Langerhans with extensions into surrounding acinar tissue. A significant increase in the volume density of peroxisomes, together with immunochemically detectable amounts of two peroxisome-associated enzymes, was observed in pancreas with hepatocytes of rats maintained on ciprofibrate. Uricase-containing crystalloid nucleoids, specific for rat hepatocyte peroxisomes, were present in pancreatic hepatocytes. These structures facilitated the identification of cells with hybrid cytoplasmic features characteristic of pancreatic acinar and endocrine cells and hepatocytes. Such cells are presumed to represent a transitional state in which pancreas specific genes are being repressed while liver specific ones are simultaneously expressed. The presence of exocrine and/or endocrine secretory granules in transitional cells indicates that acinar/intermediate cells represent the precursor cell from which pancreatic hepatocytes are derived.

Sequential histologic study of rat liver during peroxisome proliferator [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]-acetic acid (Wy-14,643)-induced carcinogenesis.

The livers of male inbred F344 rats fed Wy-14,643 [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (CAS: 50892-23-4) in the diet at a concentration of 0.1% (wt/wt) were examined sequentially at 5, 10, 17, 21, 26, 30, 35, 40, 52, 60, and 70 weeks. At 5 weeks the livers were markedly enlarged and histologically showed markedly enlarged hepatocytes with prominent nucleoli. At 21 weeks altered acidophilic areas were seen in 2 of 3 animals that were killed. Between 26 and 52 weeks neoplastic nodules were noted of 1-8 mm containing cells with morphologic features similar to those observed in altered areas. Hepatocellular carcinomas (HCC) were observed in 1 animal killed at 30 weeks and in all the animals sacrificed at 60 weeks and later. [3H]thymidine nuclear labeling studies showed marked proliferative activity of cells in altered areas, neoplastic nodules, and HCC. Altered areas, neoplastic nodules, and HCC were consistently negative for gamma-glutamyltransferase and showed decreased ATPase activity. Glucose-6-phosphatase (Glc-6-Pase) activity was decreased in altered areas and neoplastic nodules. However, some of the HCC showed a strong positive reaction for Glc-6-Pase.

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.

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.

Effects of inositol hexaphosphate on growth and differentiation in K-562 erythroleukemia cell line.

Inositol hexaphosphate (InsP6) has recently been shown to inhibit experimental cancers in vivo. Since the lower phosphorylated forms of InsP6 are important in cell growth in a wide variety of mammalian cells, we tested the efficacy of InsP6 in growth reduction of K-562 human erythroleukemia cells in vitro. We report that InsP6 decreases the K-562 cell population by 19-36% (P less than 0.001) concomitant to an increased differentiation as evidenced by ultrastructural morphology and increased hemoglobin synthesis. Pilot experiments to study the mechanism of action of InsP6 show that following treatment with InsP6, the concentration of intracellular [Ca2+] ([Ca2+]i) is increased by 57% (P less than 0.02). Likewise, a 41% increase (P less than 0.05) in InsP3 and a 26% decrease (P less than 0.02) in InsP2 were noted 1 h following treatment with InsP6. Contrary to the dogma that cell division is associated with increased [Ca2+]i, our data show that reduced cell growth and enhanced differentiation is associated with increased [Ca2+]i and increased InsP3 in the presence of InsP6.

Induction of fatty acid beta-oxidation and peroxisome proliferation in the liver of rhesus monkeys by DL-040, a new hypolipidemic agent.

Many structurally unrelated hypolipidemic agents and certain phthalate-ester plasticizers induce hepatomegaly and proliferation of peroxisomes in liver parenchymal cells of rodents, but there is relatively limited evidence regarding the ability of such compounds to induce peroxisome proliferation in the livers of nonrodent species including man. The present study was designed to determine if DL-040 (4-(((1,3-benzodioxol)-5-yl)methyl)amino-benzoic acid), a newly developed hypolipidemic agent, induces peroxisome proliferation in the liver of adult rhesus monkeys. Feeding of DL-040 (300 mg/kg body wt for 1 week; and 400 mg/kg body wt for 10 weeks) caused a significant increase in peroxisome population as determined by ultrastructural and morphometric analyses. The DL-040-induced peroxisome proliferation was accompanied by increases in the levels of catalase, carnitine acetyltransferase and the peroxisomal fatty acid beta-oxidation system. As expected, DL-040 caused a significant reduction of serum cholesterol and low density lipoprotein content. These data suggest that hepatic peroxisome proliferation is inducible in nonhuman primates at dose levels that exceed therapeutic levels.

Phthalate esters as peroxisome proliferator carcinogens.

The phthalate ester di(2-ethylhexyl) phthalate is both a peroxisome proliferator and a hepatic carcinogen. Peroxisome proliferators as a class are hepatocarcinogenic in rodent species. However, none of the peroxisome proliferators tested to date including the phthalate esters and related alcohol and acid analogs have demonstrated mutagenic or DNA-damaging activity in the in vitro Salmonella typhimurium/microsomal or the lymphocyte 3H-thymidine assays. A working hypothesis is proposed that peroxisome proliferation itself initiates neoplastic transformation of hepatic parenchymal cells by increasing intracellular rates of DNA-damaging reactive oxygen production. Evidence which supports such a hypothesis includes increased fatty acid beta-oxidation, elevated H2O2 levels, accumulation of peroxidized lipofuscin, disproportionately small increase in catalase, and elevated peroxisomal uricase activity which accompany peroxisome proliferation in hepatocytes. Direct testing of this hypothesis will provide insight into mechanisms of phthalate ester carcinogenicity and cytotoxicity.

Comparison of hepatic peroxisome proliferative effect and its implication for hepatocarcinogenicity of phthalate esters, di(2-ethylhexyl) phthalate, and di(2-ethylhexyl) adipate with a hypolipidemic drug.

Peroxisome proliferation is inducible in hepatocytes of rodent and nonrodent species by structurally dissimilar hypolipidemic drugs and certain phthalate ester plasticizers. The induction of peroxisome proliferation appears to be a tissue specific response limited largely to the hepatocyte. Peroxisome proliferation is associated with increases in the activity of the H2O2-generating peroxisomal fatty acid beta-oxidation system and in the amount of peroxisome proliferation-associated 80,000 MW polypeptide (PPA-80). Chronic administration of these non-DNA damaging and nonmutagenic peroxisome proliferators to rats and mice results in the development of hepatocellular carcinomas. Comparative morphometric and biochemical data from rats treated with varying dose levels of ciprofibrate, a hypolipidemic drug, and di(2-ethylhexyl) phthalate, and di(2-ethylhexyl) adipate, the widely used plasticizers, indicate that the hepatocarcinogenic potency of these agents is correlatable with their ability to induce peroxisome proliferation, peroxisomal beta-oxidation and PPA-80. Available evidence strongly favors the role of peroxisome proliferation-associated oxidative stress in the induction of liver tumors by peroxisome proliferators.

Lack of covalent binding of peroxisome proliferators nafenopin and Wy-14 643 to DNA in vivo and in vitro.

[3H][2-methyl-2-p-(1,2,3,4-tetrahydro-naphthyl)phenoxy] propionic acid (nafenopin), a hepatocarcinogenic peroxisome proliferator, when administered p.o. to normal intact and partially hepatectomized male F344 rats did not show any significant binding to DNA and RNA, but bound to proteins. The in vitro incubation of [3H]nafenopin and [3H]4-chloro-[6-(2,3-xylidino)pyrimidinylthio]acetic acid (Wy-14643), another peroxisome proliferator, with hepatic microsomes and calf thymus DNA also showed no significant binding of these chemicals to DNA.

Immunofluorescent and confocal laser cytometric analyses of centromeres in V79 cells.

Previously, a modified anticentromere antibody (ACA) technique was established in the V79 Chinese hamster lung cells to simultaneously analyze chromosome damage and aneuploidy induced by various agents. Using this method, cyclophosphamide (CP) was evaluated further in the presence and absence of S9 activation for micronucleus/aneuploidy induction. The specific binding nature of ACA to the centromeric region was also analyzed using a confocal scanning laser cytometry. The results indicated that CP was primarily a clastogen and S9 activation was required for its activity. Vinblastine, the positive control for aneuploidy, produced predominantly centromere containing micronuclei and the addition of S9 was not required for its activity. X-radiation, the positive control for clastogenicity, predominantly produced centromere negative micronuclei confirming its clastogenicity. An evaluation of centromeric region under the standard fluorescence microscope indicated that ACA generally binds to most centromeric regions in a cell. However, by confocal imaging it was found that ACA binds to the central core proteins of the centromere region and not to the peripheral proteins.

Simultaneous evaluation of dexamethasone-induced apoptosis and micronuclei in rat primary spleen cell cultures.

Apoptosis or programmed cell death is a biological event that is biochemically and morphologically distinct from cellular necrosis. Nonetheless, its relationship has not been studied in terms of a cytogenetic endpoint such as micronucleus formation. In the present study, based on cytological observations, the incidence of dexamethasone-induced apoptotic cells was related to the frequency of micronucleated cells in vitro. Rat primary spleen cells were grown in 6-well plates with RPMI 1640 media using concanavalin A and lipopolysaccharide as mitogens. At culture initiation, the test agent dexamethasone (10, 20 or 40 microM) and a cytokinesis inhibitor cytochalasin B (3 micrograms/ml) were added. Cultures were harvested 18 h and 40 h later. Slides were prepared and stained with Diff-Quik stain. Frequencies of apoptotic cells and micronucleated binucleate cells were enumerated cytologically based on 500 cells per treatment from the same slides. The results showed a dose-dependent increase in the number of apoptotic cells in rat spleen cultures treated with dexamethasone. At 18 h, the percentages of apoptotic cells were 0.8, 1.6, 3.4 and 4.4 with 0, 10, 20 and 40 microM dexamethasone, respectively. The corresponding percentages of apoptotic cells at 40 h were: 2.8, 2.6, 5.6 and 10.4. However, at the same concentrations of dexamethasone, the micronucleus frequency in binucleate cells remained relatively unchanged. The phenomenon of apoptosis induced by dexamethasone was confirmed biochemically based on a characteristic DNA 'ladder' pattern by gel electrophoresis. These data suggest that dexamethasone at the concentrations which induced apoptosis did not produce cytogenetic damage. Also, these findings indicate that micronucleus formation and nuclear changes leading to apoptosis are separate events and these endpoints may not be closely correlated for dexamethasone.

Absence of Ki-ras mutations in exocrine pancreatic tumors from male rats chronically exposed to gabapentin.

Human pancreatic malignancies originating from duct cells most frequently demonstrate activation of Ki-ras gene by G-to-A transition at codons 12 and 13. Rat pancreatic exocrine tumors more frequently and almost exclusively derive from acinar cells and thus differ morphologically from human pancreatic neoplasms. Male Wistar rats fed with 2% gabapentin (1-(aminomethyl)cyclohexane acetic acid) in diet for 2 years developed pancreatic exocrine adenomas and adenocarcinomas. To study the mutations in Ki-ras gene, rat pancreatic proliferative lesions induced by gabapentin were retrospectively analyzed by PCR amplification of DNA isolated from paraffin sections of formalin-fixed rat pancreatic adenomas and adenocarcinomas, using specific primers for regions encoding exon 1 (codon 12/13) and exon 2 (codon 61). The amplified 110-bp fragments of exon 1 and exon 2 were analyzed for mutations at codon 12/13 and 61. The results showed Ki-ras mutations at codon 12 in human pancreatic carcinomas. Novel mutations GGT-to-TGT and GGT-to-CGT were detected at codon 12 in 1/5 and 2/5 human pancreatic tumors. Rat adenomas or carcinomas induced by gabapentin expressed wild type sequences at codons 12, 13 and 61. These findings were confirmed by allele-specific oligonucleotide hybridization, single-strand confirmation polymorphism of exon 1 and direct sequencing of exon 1 and exon 2. The absence of mutations in these rat pancreatic tumors suggests that these tumors do not correspond to the human tumors, and that the pathogenesis of this rodent tumor formation may follow different molecular mechanisms.

Miscellaneous tumour-like lesions of the ovary: cross-sectional imaging review.

Miscellaneous tumour-like ovarian lesions are histobiologically diverse, and are often mistaken for the more common ovarian cancers, leading to aggressive management. Knowledge of characteristic clinical, laboratory and imaging findings of these select non-neoplastic ovarian entities allows correct diagnoses and permits optimal management.