Age-related susceptibility to the carcinogenic effect of the peroxisome proliferator WY-14,643 in rat liver.

The carcinogenicity of the peroxisome proliferator WY-14,643 was compared in young (starting age 2 months) and old (starting age 15 months) rats. Old rats had a 5- to 7-fold higher yield of grossly visible hepatic tumors following 22 weeks of dietary WY-14,643 when compared to young rats. Volume densities of foci with large cells and homogeneously basophilic cytoplasm, cytologically similar to adenomas and carcinomas, were also higher in old rats fed WY-14,643 when compared to young rats. Although peroxisome proliferation and sustained hepatocellular proliferation have been suggested as relevant for the mechanism of WY-14,643 carcinogenicity, neither response was exaggerated in old versus young rats. Since initiation is considered to occur spontaneously and irreversibly, old rats may have a greater accumulation of spontaneously initiated hepatocytes than young rats. If so, these results are consistent with the hypothesis that the carcinogenic mechanism of the peroxisome proliferator WY-14,643 is due to the promotion of spontaneously initiated hepatocytes.     

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.     

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.      

Failure of the peroxisome proliferator WY-14,643 to induce unscheduled DNA synthesis in rat hepatocytes following in vivo treatment

Peroxisome proliferator hepatocarcinogens lack genotoxic activityin numerous in vitro assays using non-target cells which donot respond with peroxisome proliferation. Therefore, the effectof in vivo treatment with WY-14,643 on DNA repair was quantitatedin rat hepatocytes, the target cell for carcinogenesis. PalmitoylCoA oxidase and carnitine acetyltransferase activities in isolatedhepatocytes were elevated by WY-14,643 (50 mg/kg/day by gavagefor up to 5 consecutive days) and by WY-14,643 (0.1%) or di(2-ethylhexyl)phthalate (DEHP) (1.2%) feeding (for up to 28 days), indicatingperoxisome proliferation had occurred. DNA repair as unscheduledDNA synthesis (UDS) was measured autoradiographically as netnuclear grains following thymidine incorporation in primaryhepatocyte cultures. Treatment of rats with WY-14,643 (gavageor feeding) or DEHP (feeding) did not induce UDS. Addition of2-acetylaminofluorene to replicate cultures demonstrated thatWY-14,643 or DEHP treatment did not prevent repair response.Additional cultures were treated with H₂O₂ (0.8 mM H₂O₂ 3 xat 1-h intervals) to evaluate the ability of UDS to detect anyrepair which may be induced by peroxisomal metabolism. H₂O₂did not induce UDS at this concentration, nor did it prevent2-acetylaminofluorene- induced repair. UDS was, however, observedin a separate experiment using a higher concentration of H₂O₂.In summary, a highly carcinogenic peroxisome proliferator didnot induce UDS in the target cell for carcinogenesis in spiteof peroxisome proliferation following in vivo treatment.     

The peroxisome proliferator WY-14,643 promotes hepatocarcinogenesis caused by endogenously generated oxidative DNA base modifications in repair-deficient Csbm/m/Ogg1-/- mice

Basal levels of endogenously generated oxidative DNA modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) are present in apparently all mammalian cells, but their relevance for the generation of spontaneous cancers remains to be established. Both the 8-oxoG levels and the resulting spontaneous mutations are increased in the livers of Csb(m/m)/Ogg1(-/-) mice, which are deficient in the repair of 8-oxoG. In order to determine the consequences of these additional oxidative DNA modifications and mutations and thus assess the tumor initiating potency of this type of endogenous DNA damage, we treated Csb(m/m)/Ogg1(-/-) mice and repair-proficient controls with the peroxisome proliferator WY-14,643 (0.025% ad libitum), a potent inducer of liver cell proliferation. The treatment did not generate any additional oxidative DNA damage; the elevated levels of 8-oxoG in the Csb(m/m)/Ogg1(-/-) mice even decreased. Also, the spontaneous mutation frequencies observed in the lacI gene of BigBlue Csb(m/m)/Ogg1(-/-) mice, which were approximately 3-fold higher than in the repair-proficient mice, declined by 39% under the treatment, whereas the frequencies in the livers of the repair-proficient animals remained unchanged. Preneoplastic lesions (staining positive or negative for glucose-6-phoshatase) developed in the livers of both wild-type and Csb(m/m)/Ogg1(-/-) mice after 30 weeks. Both the numbers and the total volumes of the lesions were approximately 6-fold higher in the repair-deficient mice than in the wild-type mice. The results indicate that spontaneous mutations generated from endogenous oxidative DNA base damage efficiently translate into increased tumorigenesis when cell proliferation is stimulated.     

Enhancement by Wy-14,643, a hepatic peroxisome proliferator, of diethylnitrosamine-initiated hepatic tumorigenesis in the rat.

Diethylnitrosamine (DEN), at a concentration of 100 parts/10(6) in drinking water for 14 days, caused the development, by 48 weeks, of very few liver tumours in 5 of 18 (27%) male F=344 rats fed control diet. When the DEN treatment was followed one week later by continuous feeding of the hypolipidemic hepatic peroxisome proliferator, Wy-14,643, at 0.1% dietary level, all of 28 rats (100%) developed, between 38 and 48 weeks, a significantly higher number of liver tumours. Furthermore, laparotomy at 22 weeks revealed that several rats fed Wy-14,643 after DEN initiation had developed visible liver nodules, suggesting that Wy-14,643 also accelerates the appearance of these tumours. Administration of another peroxisome proliferator, clofibrate, at 0.5% level in the diet after DEN initiation, also caused a substantial enhancement of liver tumorigenesis. The enhancement of liver-tumour development by clofibrate, however, was less than that by Wy-14,643. The marked enhancing effect of Wy-14,643 may be due to its profound hepatomegalic and peroxisome proliferative properties.     

Initiator-specific promotion of hepatocarcinogenesis by WY-14,643 and clofibrate

The possibility that selection of an initiating agent couldhave a significant impact on the ability to detect subsequentpromoting activity of peroxisome proliferators was examined.Initiation was achieved by established methods using 2-acetylaminofluorene(2-AAF: 0.02% in diet for 8 weeks) or diethylnitrosamine (DEN;150 mg/kg body wt by single i.p. injection) in male F344 rats.Following initiation, the peroxisome proliferators WY-14,643or clofibrate were each fed (0.1% of diet) for up to 37 weeks.Both WY-14,643 and clofibrate lacked promoting activity, asmeasured by increases in the volume density of homogeneous basophilicfoci and incidence or multiplicity of hepatocellular neoplasiafollowing 2-AAF initiation compared to non-initiated controls.These negative results sharply contrasted with the observedpromoting activity of dietary WY-14,643 and clofibrate followingDEN initiation. Peroxisome proliferation, measured as inductionof acyl-CoA oxidase activity, was consistently observed in peroxisomeproliferator-fed rats despite prior initiation with 2-AAF orDEN. These results suggest that detection of promoting activityfor peroxisome proliferators depends on selection of the initiatingagent.     

Biological potential of basophilic hepatocellular foci and hepatic adenoma induced by the peroxisome proliferator, Wy-14,643

The biological potential of hepatic foci and tumors inducedby peroxisome proliferators such as Wy-14,643 has been poorlycharacterized. In this study, male F-344 rats (n = 20/group/timepoint) were fed Wy-14,643 (0.1%) for 22, 37 or 52 weeks (‘W-22’,‘W-37’ or ‘W-52’ respectively). At eachtime point some rats were killed and additional Wy-14,643-fedrats were switched to basal diet (Wy-14,643/‘stopped’)for up to 104 weeks (referred to as ‘W-22/S’, ‘W-37/S’and ‘W-52/S’). Homogeneous basophilic foci, butnot clear cell foci, increased in number and size in W-37 andW-52 rats. In W-37/S rats, clear cell foci replaced basophilicfoci as the most frequent phenotype. In serial section overlays,adenosine triphosphatase deficient foci accounted for only 16%of basophillc foci in W-52 rats and 16% of clear cell foci inW-37/S rats at 52 weeks. The replication of basophilic fociof W-37 rats was markedly Increased (focal labeling index, ELI= 61.8% versus non-focal labeling index, LI = 11.4%; controlLI = 0.8%). Clear cell foci from W-37/S rats at 52 weeks hada ELI of 1.6% (non-focal LI = 0.6%). Hepato cellular adenomaswere increased in W-37 (11/20 rats and 0.8 tumors/rat) and W-52groups (19/20 rats and 2.8 tumors/rat). Prevalence of hepatocellularcarcinomas was elevated in W-52 rats (6/20 rats) but not inW-22 or W-37 rats. Following removal of Wy-14,643, prevalenceof animals with malignant, metastatic hepatocellular carcinomasin W-52/S rats was similar to the prevalence in W-52 rats. However,Wy-14,643-induced adenomas completely regressed in W-37/S andW-52/S groups. In summary, significant morphological continuitybetween highly proliferative basophilic foci and hepatocellulartumors was identified, emphasizing the superiority of basophiliaas a marker for lesious leading to development of hepatocellularneoplasia in rats fed Wy-14,643. An important biological distinctionwas noted between regressive hepatic adenomas and progressivehepatocellular carcinomas induced by a peroxisome proliferator.     

Effect on the expression of c-met, c-myc and PPAR-{alpha} in liver and liver tumors from rats chronically exposed to the hepatocarcinogenic peroxisome proliferator WY-14, 643

The induction of rodent hepatic tumors by peroxisome proliferators(PP) appears to depend on focal growth of hepatocytes. Expressionof the oncogenes c-met and c-mycis altered following regenerativestimuli in rat liver, suggesting involvement of their proteinproducts in hepatocyte replication. In addition, increases inc-myc and c-met mRNA expression are observed in multiple typesof human and rodent tumors, including hepatocellular carcinoma.A study was designed to test the hypothesis that developmentof PP-induced hepatic neoplasms occurs as a result of overexpressionof c-met or c-myc. Male F344 rats were exposed to WY-14, 643for 22 or 78 weeks (1000 p.p.m. in the diet). Messenger RNAwas extracted from liver tumors (78 weeks) and surrounding non-lesionliver of exposed rats and non-lesion liver from age-matchedcontrol rats. Levels of mRNA expression were compared usingNorthern analysis. Significant increases in c-met (     

Wy-14,643, a peroxisome proliferator, inhibits compensative cell proliferation and hepatocyte growth factor mRNA expression in the rat liver

Previously, we found that a peroxisome proliferator significantly reduced hepatic and plasma hepatocyte growth factor (HGF) levels in male F-344 rats, and that the growth of preneoplastic or neoplastic cells induced by this peroxisome proliferator was markedly inhibited by HGF. Here, we examined the effects of [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643), a peroxisome proliferator, on cell proliferation and HGF mRNA levels in the liver of rats after stimulation of compensative cell proliferation. After 2 weeks of treatment with Wy-14,643, hepatic DNA synthesis caused by partial hepatectomy was decreased by 50% compared with untreated controls. DNA synthesis was maintained at the same reduced level for up to 10 weeks. During this period, hepatic HGF mRNA level was also much lower in Wy-14,643-treated rats than untreated controls. Therefore Wy-14,643, a peroxisome proliferator, would inhibit the growth of normal hepatocytes, and then produce an advantageous circumstance for the selective growth of neoplastic or preneoplastic cells.     

Role of PPAR alpha in the mechanism of action of the nongenotoxic carcinogen and peroxisome proliferator Wy-14,643

Chronic administration of peroxisome proliferators to mice and rats results in hepatomegaly and ultimately carcinogenesis. The mechanism underlying the carcinogenic effect of nongenotoxic peroxisome proliferators is not well understood. To determine whether nongenotoxic carcinogenesis is receptor mediated, we evaluated the effect of the prototypical peroxisome proliferator Wy-14,643 on replicative DNA synthesis and carcinogenesis in the PPAR alpha-null mouse line. Male mice (F4, Sv/129 ter) of both genotypes (+/+) and (-/-) were fed either a control diet or one containing 0.1% Wy-14,643 for either 1 week, 5 weeks, or 11 months. Wild-type mice fed the Wy-14,643 diet for 1 or 5 weeks showed increased hepatic labeling by bromodeoxyuridine (BrDU) compared to untreated controls. In contrast, there was no increase in hepatic BrDU labeling index in (-/-) mice fed the Wy-14,643 diet for the same time periods compared to controls. After 11 months, 100% of the (+/+) mice fed the Wy-14,643 diet had multiple hepatocellular neoplasms, including adenomas and carcinomas, while the (-/-) mice fed the Wy-14,643 diet were unaffected. This work demonstrates that the effects of Wy-14,643 on replicative DNA synthesis and hepatocarcinogenesis are mediated by PPAR alpha.      

Inhibition of WY-14,643 induced hepatic lesion growth in mice by rotenone

The effect of rotenone treatment on [4-chloro-6-(2,3-xylidino)-2- pyrimidinylthio] acetic acid (WY-14,643) hepatic lesion growth in male B6C3F1 mice was investigated. Following induction of hepatic focal lesions by diethylnitrosamine (DEN) 35 mg/kg twice a week for 8 weeks, mice were placed into one of the four treatment groups: group I, control NIH-07 diet (control diet), group II, rotenone (600 mg/kg diet), group III NIH-07 diet containing WY-14,643 (1000 mg/kg diet), and group IV, NIH-07 diet containing WY-14,643 (1000 mg/kg diet) and rotenone (600 mg/ kg diet). Mice were killed after 30 and 60 days of dietary treatment. The effect of treatment with WY-14,643 and rotenone on hepatic lesion growth was examined by estimating the number of focal lesions per liver and the relative volume of focal lesions. WY-14,643 (group III) increased both the number and the volume of focal lesions. In particular, an increase in number and volume of basophilic lesions was seen. Co-treatment with WY-14,643 and rotenone (group IV) decreased both the number and the volume of the total number of focal lesions and basophilic foci compared with WY-14,643 treatment alone (group II). Alterations in the growth of hepatic focal lesions was further investigated by examining DNA synthesis and apoptosis within individual lesions. WY-14,643 (group III) treatment increased the DNA synthetic labeling index in all foci. Co-treatment of rotenone and WY-14,643 (group IV) decreased focal DNA synthesis and mitosis and increased the incidence of apoptotic hepatocytes. These data suggest that rotenone's ability to inhibit WY-14,643-induced hepatic focal lesion growth was mediated through a decrease in hepatic focal proliferation and an increase in focal apoptosis.      

Malignant tumors in rats fed nafenopin, a hepatic peroxisome proliferator.

Nafenopin (2-methyl-2-[P-(1,2,3,4-tetrahydro-1-naphthyl)phenoxy] propionic acid; Su-13,437), a potent hypolipidemic hepatic peroxisome proliferator, was fed to male F344 rats at a dietary concentration of o.1% until the end of the experiment at 25 months. Between 18 and 25 months, 12 of 15 rats (80%) developed tumors. Approximately 73% (11/15) developed hepatocellular carcinomas, and 10% (3/15) developed pancreatic acinar cell tumors, including 1 metastasizing carcinoma. The hepatocellular carcinomas as well as the acinar cell carcinoma of the pancreas were transplantable successfully through 6 generations.     

Hepatocellular DNA synthesis in rats given peroxisome proliferating agents: comparison of WY-14,643 to clofibric acid, nafenopin and LY171883

The mitogenic effects of peroxisome proliferating agents have been implicated in their carcinogenicity. WY-14,643 stimulates an increase in hepatocellular DNA replication that persists with continued administration, but it is unclear if other peroxisome proliferators share this property. In these studies, WY-14,643 was compared to clofibric acid, nafenopin and LY171883 given to rats in the diet for up to 30 days. DNA replication in the rat liver was quantified by immunohistochemical methods after continuous s.c. infusion of bromodeoxyuridine by osmotic minipump. During the first 7 days of treatment, WY-14,643 (0.1% in diet) and nafenopin (0.05%) increased the percentage of bromodeoxyuridine-labeled hepatocytes to greater than 50%, from 3% in controls. Clofibric acid (0.5%) and LY171883 (0.3%) increased the labeling to approximately 33%. The replicative response to each of the compounds was localized primarily to the periportal region of the liver lobule. The time-course of replication induced by clofibric acid and WY-14,64.3 was examined over 3 day intervals. The peak of replication in response to clofibric acid occurred during days 4-6, whereas the effect of WY-14,643 peaked during days 1-3 and was much greater than clofibric acid. The replicative response to WY-14,643 persisted through 30 days at dietary concentrations of 0.1 and 0.005%. Nafenopin, LY171883 and clofibric acid were without effect on DNA replication on days 28-30 even though the hepatomegaly and induction of peroxisomal beta-oxidation persisted. Thus, under the conditions of these experiments, the persistent replicative effect through 30 days was unique to WY-14,643. Although sustained replication in the general population of hepatocytes may be involved in the carcinogenesis of WY-14,643, it does not appear to be a factor in the hepatocarcinogenesis of the other peroxisome proliferators.     

Characteristics of the hepatocarcinogenesis caused by dehydroepiandrosterone, a peroxisome proliferator, in male F-344 rats

The characteristics of the hepatocarcinogenesis induced by dehydroepiandrosterone(DHEA) were compared with that induced by other peroxisome proliferatorssuch as [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]aceticacid (Wy-14,643) and di(2-ethylhexyl)phthalate (DEHP). MaleF-344 rats were given a diet containing DHEA at 0.5 or 1%, Wy-14,643at 0.1% and DEHP at 2% for up to 78 weeks. In rats fed 0.5 or1% DHEA the incidence of neoplasias was 20% after 52 weeks.At 78 weeks all rats treated with 1% DHEA had numerous grosslyvisible nodules and the incidence of hepatic neoplasia was dose-dependentThe magnitude of hepatocellular tumorigenicity after DHEA treatmentwas less potent than that after Wy-14,643, but more than thatafter DEHP treatment. Peroxisomal [3-oxidation activity increasedthree- or six-fold after a 10 week course of 0.5 or 1% DHEArespectively and this was significantly lower than that inducedin Wy-14,643- or DEHP-fed rats. From 52 to 78 weeks these activitiesincreased 3–9 times over that in controls. In both thegroup of rats treated with Wy-14,643 and those treated withDEHP, peroxisomal p-oxidation constantly increased 11- to 15-foldduring the experiment Catalase activity increased 1.3- to 1.5-foldfor the first 10 weeks of DHEA treatmentand then recovered tothe control level. The activities of glutathione peroxidaseand glutathione S-transferase decreased markedly after 30 weeksin DHEA-treated rats and the decreases were sustained for upto 78 weeks. The profile of changes in enzyme activities inthe rats fed DHEA was not significantly different from thatof those fed Wy-14,643 or DEHP. There were no increases in 8-hydroxydeoxyguanosine, oxidative DNA damage or lipid peroxidelevel in the liver in any of the treated rats at 10 or 30 weeks.Since these results showed that the characteristics of hepatocarcinogenesiscaused by DHEA were basically similar to those caused by Wy-14,643and DEHP, typical peroxisome proliferators, hepatocarcinogenesisinduced by DHEA is probably due to the same mechanisms as thatinduced by general peroxisome proliferators.     

Analysis of activated protooncogenes in B6C3F1 mouse liver tumors induced by ciprofibrate, a potent peroxisome proliferator

Liver tumors from B6C3F1 mice induced by the potent peroxisomeproliferator ciprofibrate, a hypolipidemic drug, were evaluatedfor the presence of transforming genes by the nude mouse tumorigenicityassay. As reported earlier, the tumors were not activated bya point mutation in codon 61 of H-ras. Two of the eight tumorsexamined contained a mutation in codon 13 or an H-ras gene mutatedin codon 117. Screening of another 23 ciprofibrate-induced livertumors by oligonucleotide hybridization analysis and directDNA sequencing resulted in the identification of three tumorDNA samples with point mutations in codon 117 of the H-ras gene.In addition, another tumor sample contained a K-ras gene witha mutation in codon 61. Mutations in these codons have beenseen only rarely in chemically induced liver tumors from thismouse strain. Of 15 spontaneous B6C3F1 liver tumors screenedin the same manner, one exhibited a K-ras gene activated bya mutation in codon 13 and a second contained an H-ras geneactivated by a mutation in codon 117. These ras gene mutationshave not been reported previously from spontaneous liver tumors.The frequency and spectrum of ras oncogene mutations characterizedin ciprofibrate-induced liver tumors differ significantly fromthe frequency and pattern identified in spontaneously occurringliver tumors. The results of this study with a limited numberof samples suggest that ras protooncogene activation or activationof other protooncogenes that can be detected by the nude mousetumorigenicity assay are not frequent events in the mechanismof carcinogenicity of the peroxisome proliferator ciprofibrate.However, the lower frequency and distinct pattern of H-ras mutationsobserved in these tumors disprove the assumption of promotionof spontaneous hepatocarcinogenesis by ciprofibrate.     

Dietary glycine inhibits the growth of B16 melanoma tumors in mice.

Dietary glycine inhibited hepatocyte proliferation in response to the carcinogen WY-14,643. Since increased cell replication is associated with hepatic cancer caused by WY-14,643, glycine may have anti-cancer properties. Therefore, these experiments were designed to test the hypothesis that dietary glycine would inhibit the growth of tumors arising from B16 melanoma cells implanted subcutaneously in mice. C57BL/6 mice were fed diet supplemented with 5% glycine and 15% casein or control diet (20% casein) for 3 days prior to subcutaneous implantation of B16 tumor cells. Tumor volume was estimated from tumor diameter for 14 days. Tumors were excised, weighed and sectioned for histology post-mortem. B16 cells and endothelial cells were cultured in vitro to assess effects of glycine on cell growth. Statistical tests were two-sided and a P-value of 0.05 was defined as a significant difference between groups. Weight gain did not differ between mice fed control and glycine-containing diets. B16 tumors grew rapidly in mice fed control diet; however, in mice fed glycine diet, tumor size was 50-75% less. At the time of death, tumors from glycine-fed mice weighed nearly 65% less than tumors from mice fed control diet (P < 0.05). Glycine (0.01-10 mM) did not effect growth rates of B16 cells in vitro. Moreover, tumor volume and mitotic index of B16 tumors in vivo did not differ 2 days after implantation when tumors were small enough to be independent of vascularization. After 14 days, tumors from mice fed dietary glycine had 70% fewer arteries (P < 0.05). Furthermore, glycine (0.01-10 mM) inhibited the growth of endothelial cells in vitro in a dose-dependent manner (P < 0.05; IC50 = 0.05 mM). These data support the hypothesis that dietary glycine prevents tumor growth in vivo by inhibiting angiogenesis through mechanisms involving inhibition of endothelial cell proliferation.     

Recovery of hyperplastic responsiveness in rat liver after dosing with the peroxisome proliferator methylclofenapate

Methylclofenapate (MCP) was administered daily by gavage (25 mg/kg) for 7 days to groups of adult male rats. Dosing was interrupted for 28, 35, 56, 70 or 84 days and then resumed (25 mg/kg by gavage at 0 and 24 h). During the second period of dosing animals were killed in groups of three at 6, 12, 18, 24, 30, 36, 42 and 48 h after the resumption of dosing. Hepatocytes in S-phase, labelled with bromodeoxy-uridine, were analysed by flow cytometry, cell sorting and microscopy. It was observed that total S-phase activity was just significantly elevated (approximately 20% of maximum) over corn oil controls after an interval of 28 days between initial and subsequent dosing periods. After an interval of 35 days total S-phase activity was approximately 65% of maximum, and full hyperplastic responsiveness, equal to that observed in naive animals given MCP, was detected after interruptions in dosing of 56, 70 and 84 days. The recovery of S-phase responsiveness during the interruptions in dosing was accompanied by an increase in the proportion of 2 X 2N hepatocytes from approximately 10% in animals dosed continuously with MCP, to approximately 11.4% after 28 days interruption, 17% after 35 days and control levels (approximately 20%) after 56, 70 and 84 days. Irrespective of the magnitude of the hyperplasia elicited by the second period of dosing with MCP, the proportion of 2 X 2N cells was reduced to the same levels as those observed in animals dosed continuously with MCP (approximately 10%). Very low S-phase activity (0.05%) was observed in animals dosed continuously with MCP, this level of activity being similar to that in animals given corn oil continuously.     

Tumor promotion by the peroxisome proliferator nafenopin involving a specific subtype of altered foci in rat liver

The involvement of tumor promotion in the hepatocarcinogenic action of peroxisome proliferators has not been generally accepted. We studied the effect of nafenopin (NAF) as a model compound in a two-stage initiation-promotion protocol. Carcinogenesis was initiated by a single dose of aflatoxin B1 (AFB1) in female (AFB1, 5 mg/kg) and male (AFB1, 2 mg/kg) Wistar rats. After recovery NAF was fed via the diet, providing a daily dose of 100 mg/kg body weight. Phenobarbital (PB) (50 mg/kg body weight) was fed to female rats as a positive control. The following results were obtained. (a) At weeks 40, 55, 59, and 70, significantly more and larger liver tumors were present in AFB1-NAF-treated rats than in rats receiving either compound alone, and the effect of the combined treatment was clearly more than additive, in three independent experiments including both sexes. This suggests tumor promotion by NAF. Male rats responded more strongly than females. Similarly, PB enhanced the yield of liver tumors. Histologically, tumors were hepatocellular adenoma or carcinoma. In group AFB1-PB the majority consisted of eosinophilic and glycogenstoring cells. However, adenoma and carcinoma of groups AFB1-NAF and O-NAF consisted of weakly basophilic cells. (b) Phenotypically altered foci were evaluated in hematoxylin- and eosin-stained liver sections from the female rats. NAF treatment after AFB1 had little effect on number and size of eosinophilic-clear cell foci and decreased the number of trigroid foci. However, it led to a dramatic increase (20-fold after 70 weeks of NAF treatment) in number and size of foci of a special phenotype that was extremely rare after AFB1 alone and virtually absent in group AFB1-PB. Hepatocytes in these foci are characterized by weak diffuse basophilia and some eosinophilia, similar to the phenotype in adenoma and carcinoma, and by absence of gamma-glutamyltranspeptidase (GGT) expression. Based on these findings, we propose the hypothesis that NAF promotes the development of liver tumors via a mechanism involving amplification of a specific subtype of altered hepatic foci.