Butylated hydroxytoluene lacks the activity of phenobarbital in enhancing diethylnitrosamine-induced mouse liver carcinogenesis

The effect of the food additive butylated hydroxytoluene (BHT) as an enhancer of liver carcinogenesis in mice was investigated. Liver carcinogenesis was initiated by intraperitoneal injection of diethylnitrosamine (DEN) in male B6C3F1 mice at 100 or 200 mumol/kg body weight once a week for 10 weeks (total exposure 1000 or 2000 mumol/kg body weight). After an exposure-free recovery interval of 4 weeks, groups of mice were fed either basal diet or diets containing either 5000 ppm BHT or 500 ppm phenobarbital (PB), as a positive control, for 24 weeks. Exposure to the initiating doses of DEN alone induced no liver foci at 10 weeks or at 14 weeks after the recovery period, but at termination at 38 weeks, foci and adenomas were present in a dose-related incidence. In the groups given BHT after DEN/recovery, the incidence and the multiplicity of liver foci and adenomas were not different from those in mice given only DEN/recovery, whereas, in the groups given PB after DEN, liver lesions were increased by 1.7-3.0-fold. In conclusion, BHT had no promoting or syncarcinogenic effect on DEN-induced mouse liver carcinogenesis, whereas under the same conditions, PB acted as an enhancer.     

Natural killer activity in a medium-term multi-organ bioassay for carcinogenesis.

Natural killer (NK) cell activity was evaluated after the initiation and promotion steps in a medium-term multi-organ bioassay for carcinogenesis. NK cell activity was assessed in vitro by Cr51 release assay at the 4th and 30th weeks of the experiment. Male Wistar rats were sequentially initiated with N-diethylnitrosamine (DEN i.p.), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN drinking water), N-methyl-N-nitrosourea (MNU i.p.), dihydroxy-di-N-propylnitrosamine (DHPN drinking water) and N,N'-dimethylhydrazine (DMH s.c.) at subcarcinogenic doses for 4 weeks (DMBDD initiation). One group was evaluated at the 4th week and the other was maintained without any further treatment until the 30th week. Two initiated groups were exposed through the diet to 2-acetylaminofluorene (2-AAF) or phenobarbital (PB), from the 6th until the 30th week. Five additional groups were studied to evaluate the effects of each initiator on NK activity. All groups submitted to initiation only, initiation plus promotion, or promotion only, developed significantly more preneoplastic lesions than the untreated control group. The main target organs for tumor development in the initiated animals were the liver and the colon, irrespective of treatment with 2-AAF or PB. NK cell activity was not affected by exposure to genotoxic carcinogens after initiation, at the 4th week. Treatments only with PB or 2-AAF did not change NK cell activity. However, decreased NK cell activity was registered in the group only initiated with DMBDD and in the group given DMBDD+2-AAF. This late depression of NK cell activity at the 30th week could be related to the production of suppressing molecules by the tumor cells.     

Modification by sodium L-ascorbate, butylated hydroxytoluene, phenobarbital and pepleomycin of lesion development in a wide-spectrum initiation rat model

Rats were treated for 1 week each with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), 0.2% N-bis(2-hydroxypropyl)-nitrosamine (DHPN) and 0.2% N-ethyl-N-hydroxyethylnitrosamine (EHEN) in the drinking water, and then administered diet containing 5% sodium L-ascorbate (Na-AsA), 1% butylated hydroxytoluene (BHT) or 0.05% phenobarbital (PB), or weekly intraperitoneal injections of 2 mg of pepleomycin per kg body weight until week 36. Histopathological examination revealed that all exerted significant modulation effects on tumor development in the various target organs. Na-AsA was found to inhibit liver but promote renal pelvis and bladder carcinogenesis. BHT similarly decreased liver and enhanced bladder lesion development. PB, in contrast promoted hepatocarcinogenesis. However both PB and BHT were associated with increased incidences of adenomas and adenocarcinomas of the thyroid. Thus the wide-spectrum initiation model allowed confirmation of site-specific modification potential and in addition demonstrated potentiation of kidney and bladder carcinogenesis promotion by pepleomycin.     

Pentachlorophenol (PCP) produces liver oxidative stress and promotes but does not initiate hepatocarcinogenesis in B6C3F1 mice.

To elucidate the mechanism of hepatocarcinogenesis of pentachlorophenol (PCP) in mice, critical effects related to carcinogenicity were studied in the livers of B6C3F1 male mice administered PCP at concentrations of 600 and 1200 p.p.m. in the diet for 8 weeks. Oxidative stress was assessed by measurements of 8-oxodeoxyguanosine (8-oxodG) in the liver nuclear DNA and hepatocyte cell proliferation was quantified by bromodeoxyuridine incorporation. Also, initiation and promotion were assessed in a two-stage hepatocarcinogenesis model in which one group of mice was given PCP at concentrations of 600 and 1200 p.p.m. as initiator for the first 13 weeks with subsequent administration of phenobarbital (PB) as promoter at a concentration of 500 p.p.m. in the drinking water for 29 weeks. A second group was initiated with diethylnitrosamine (DEN) at 20 p.p.m. in the drinking water for the first 13 weeks followed after a 4 week recovery interval by PCP at concentrations of 300 and 600 p.p.m. in the diet for 25 weeks. Significant elevations in 8-oxodG levels and cell proliferation were observed in a dose-dependent manner. Incidences and multiplicities of hepatocellular tumors in mice treated with PCP after DEN initiation were increased compared with those in mice given initiation only. In contrast, in mice given PCP as initiator followed by PB no enhancement of neoplastic lesions occurred. These findings are interpreted to demonstrate that PCP exerts a promoting action, but not an initiating effect on liver carcinogenesis and that the promoting action is related to oxidative stress and compensatory hepatocellular proliferation.     

Natural Killer Activity in a Medium-term Multi-organ Bioassay for Carcinogenesis

Natural killer (NK) cell activity was evaluated after the initiation and promotion steps in a medium-term multi-organ bioassay for carcinogenesis. NK cell activity was assessed in vitro by Cr⁵¹ release assay at the 4th and 30th weeks of the experiment. Male Wistar rats were sequentially initiated with N-diethylnitrosamine (DEN i.p.), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN drinking water), N-methyl-N-nitrosourea (MNU i.p.), dihydroxy-di-N-propylnitrosamine (DHPN drinking water) and N, N'-dimethylhydrazine (DMH s.c.) at subcarcinogenic doses for 4 weeks (DMBDD initiation). One group was evaluated at the 4th week and the other was maintained without any further treatment until the 30th week. Two initiated groups were exposed through the diet to 2-acetylaminofluorene (2-AAF) or phenobarbital (PB), from the 6th until the 30th week. Five additional groups were studied to evaluate the effects of each initiator on NK activity. All groups submitted to initiation only, initiation plus promotion, or promotion only, developed significantly more preneoplastic lesions than the untreated control group. The main target organs for tumor development in the initiated animals were the liver and the colon, irrespective of treatment with 2-AAF or PB. NK cell activity was not affected by exposure to genotoxic carcinogens after initiation, at the 4th week. Treatments only with PB or 2-AAF did not change NK cell activity. However, decreased NK cell activity was registered in the group only initiated with DMBDD and in the group given DMBDD+2-AAF. This late depression of NK cell activity at the 30th week could be related to the production of suppressing molecules by the tumor cells.     

Lack of Promoting Effects of Phenobarbital at Low Dose on Diethyl-nitrosamine-induced Hepatocarcinogenesis in TGF-a Transgenic Mice

Phenobarbital (PB), a rodent non-genotoxic carcinogen, showed hormesis, biphasic effects on rat livercarcinogenesis. To test the hypothesis that the hormesis earlier observed for PB induced hepatocarcinogenesis mightalso exist in the TGF-α transgenic mice model, one which is highly susceptible to carcinogenesis, the carcinogenic orpromotion effects of a wide range of phenobarbital (PB) concentrations were investigated. Two weeks after a singlei.p. dose of 5 mg /kg bw of diethylnitrosamine (DEN) to 15 day old mice, animals were treated with diet containingPB at doses of 0, 2, 15 or 500 ppm. The incidence and multiplicity of tumors, including hepatocellular adenomas andcarcinomas, were significantly increased by the high dose of PB, but no significant difference among the groupsreceiving 2 and 15 ppm for liver tumors when compared to DEN alone group. The proliferating cell nuclear antigenindices for liver tumors and surrounding hepatocytes in high dose PB treated mice were significantly increased, butno change was noted at the lower doses. The total cytochrome P450 content in the liver was also elevated by 500 ppmof PB, while hepatic 8-OHdG levels demonstrated no significant change. In conclusion, PB at high dose enhancesDEN-induced hepatocarcinogenesis in TGF-α transgenic mice, but low doses lack any significant effects. One possiblemechanism of phenobarbital carcinogenicity might be influenced by cytochrome P450 system exhibiting a strongpromoting activity for liver of mice.     

Comparative study of diethylnitrosamine-initiated two-stage hepatocarcinogenesis in C3H, C57BL and BALB mice promoted by various hepatopromoters

The comparative inducibility of enzyme-altered islands (EAIs) by diethylnitrosamine (DEN) and their responsiveness to hepatopromoters belonging to different classes were studied in C3H/HeN (C3H), C57BL/6N (C57) and BALB/cA (BALB) mice. Male mice were given an i.p. injection of DEN (20 micrograms/g body weight) after partial hepatectomy at 6 weeks of age and then fed either basal diet or diet containing phenobarbital (PB) (500 p.p.m.), clofibrate (CF) (1000 p.p.m.) or ethynyl estradiol (EE) (10 p.p.m.). The numbers and size distribution of EAIs were assessed after the mice were killed at week 20, utilizing stereological methods. In the groups receiving DEN but no promoter, the number and mean size of the lesions were far larger in the C3H mice than in the other strains. Under the promoting pressure of PB, the growth of EAIs in C3H and BALB were accelerated remarkably, but those of C57 mice only slightly. Interestingly, in BALB the number of EAIs was much fewer than those of C3H in spite of their good sensitivity to PB, suggesting that BALB was refractory to the initiation process by DEN. A promoting effect for CF could only be demonstrated for the C3H strain and EE as the dose used inhibited the development of EAIs in all the strains. The experimental data thus indicate that interstrain differences in two-stage hepatocarcinogenesis among mice with different genetical backgrounds may exist, either in initiation or promotion, or in both processes.     

The natural history and dose-response characteristics of enzyme-altered foci in rat liver following phenobarbital and diethylnitrosamine administration

Enzyme-altered foci (EAF) were induced in the Liver of femalerats by 70? partial hepatectomy (PH), followed by a single intragastricadministration of diethylnitrosamine (DEN) at a dose of 10 mg/kg.The stability and response of these foci to various doses ofthe hepatic promoting agent, phenobarbital (PB), were studied.The number of yglutamyltranspeptidasepositive (GGT⁺) EAF resultingfrom PH/DEN followed by PB (0.05%) administration for 1 week,2 weeks, 1 month, 2 months, 3 months or 4 months did not significantlychange when the administration of the promoting agent was followedby a 6-month period of a diet containing no PB. These data demonstratethe stability of the fwi induced by the PH/DEN/PB regimen andindicate that the increased number of foci resulting from PBpromotion in the absence of overt hepatic necrosis are not reversibleon removal of the promoting stimulus. Chronic administrationof dose levels of PB below 0.001% in the diet failed to demonstratean increase in the number of EAF over the number in the controlanimals not promoted with PB. A linear increase in the numberof EAF was observed when rats were chronically fed doses ofPB ranging between 0.001% and 0.05% in the diet, whereas dietconcentrations of PB > 0.05% did not result in any furtherincrease in the number of EM. The number of EAF resulting fromPH/DEN followed by 0.05% PB in the diet increased during thefirst 3–4 months of promotion. Thereafter, the numberof foci did not change despite the continued administrationof PB for as long as 8 months. These data suggest the presenceof an apparent threshold (no effect level) for promotion byPB and demonstrate the presence of a maximal response of EAFto this promoting agent after initiation by a single dose ofDEN.     

The carcinogenic effect of methapyrilene combined with nitrosodiethylamine given to rats in low doses.

The carcinogenic effects of combinations of methapyrilene hydrochloride (MP), nitrosodiethylamine (NDEA), and phenobarbital (PB) or partial hepatectomy (PH) were examined following sequential treatment of rats. MP is a generally non-genotoxic liver carcinogen of moderate potency, NDEA is a genotoxic liver carcinogen, PB is primarily a liver tumor promoter and PH induces cell proliferation. The dose of each carcinogen was chosen to be below that causing significant liver tumor incidence when given singly. There were 12 protocols involving groups of 28 female rats each. Short treatments with NDEA and MP were followed by 60 weeks of PB promotion or by partial hepatectomy. Each treatment was given separately or in double combination as controls. Several animals of each group were killed at intervals during the experiment for examination of toxic effects and the presence of altered hepatic foci. In only 3 of 12 groups was there a significant incidence of rats with liver neoplasms: the two groups given three treatments: NDEA, MP and PB (86% tumors) or NDEA, MP and PH (33%), and the group receiving NDEA and MP without promotion (46%). The results clearly indicated a co-carcinogenic effect between NDEA and MP. Continuous PB potentiated tumor development, while PH did not. There was no evidence of liver toxicity from any of the treatments, but clear cell foci observed in three groups at weeks 13 and 33 correlated with the later development of liver neoplasms.     

Promoting effects of phenobarbital and 3'-methyl-4-dimethylaminoazobenzene on the appearance of gamma-glutamyltranspeptidase positive foci in rat liver pretreated with varying doses of diethylnitrosamine

The promotion potential of phenobarbital (PB) and 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) on liver carcinogenesis after initiation with various doses of diethylnitrosamine (DEN) was assessed using an in vivo short term system. Male F344 rats were pretreated with a single intraperitoneal injection of varying doses of DEN (0, 6, 12, 25, 50, 100 or 200 mg/kg body wt), and 2 weeks later were treated with 0.05% PB or 0.06% 3'-Me-DAB for 6 weeks. All animals were subjected to partial hepatectomy 3 weeks after the DEN treatment. Quantitation of gamma-glutamyltranspeptidase-positive (gamma-GT+) foci revealed a DEN dose-dependent response. Magnitude of promotion by PB and more pronounced by 3'-Me-DAB was, in contrast, strongest at the lower doses of DEN. The results suggest that quantitative differences with regard to initiation level may exist, influencing the promotability of initiated cells.     

Unique patterns of gene expression changes in liver after treatment of mice for 2 weeks with different known carcinogens and non-carcinogens

Previously we demonstrated that the mouse liver tumor response to the non-genotoxic carcinogens oxazepam and Wyeth-14,643 involved more differences than similarities in changes in early gene expression. In this study we used quantitative real-time PCR and oligonucleotide microarray analysis to identify genes that were up- or down-regulated in mouse liver early after treatment with different known carcinogens, including oxazepam (125 and 2500 p.p.m.), o-nitrotoluene (1250 and 5000 p.p.m.) and methyleugenol (75 mg/kg/day), or the non-carcinogens p-nitrotoluene (5000 p.p.m.), eugenol (75 mg/kg/day) and acetaminophen (6000 p.p.m.). Starting at 6 weeks of age, mice were treated with the different compounds for 2 weeks in the diet, at which time the livers were collected. First, expression of 12 genes found previously to be altered in liver after 2 weeks treatment with oxazepam and/or Wyeth-14,643 was examined in livers from the various chemical treatment groups. These gene expression changes were confirmed for the livers from the oxazepam-treated mice in the present study, but were not good early markers for all the carcinogens in this study. In addition, expression of 20 842 genes was assessed by oligonucleotide microarray [n = 4 livers/group, 2 hybridizations/liver (with fluor reversals)] and the results were analyzed using the Rosetta Resolver System and GeneSpring software. The analyses revealed that several cancer-related genes, including Fhit, Wwox, Tsc-22 and Gadd45b, were induced or repressed in unique patterns for specific carcinogens and not altered by the non-carcinogens. The data indicate that even if the tumor response, including molecular alterations, is similar, such as for oxazepam and methyleugenol, early gene expression changes appear to be carcinogen specific and seem to involve apoptosis and cell cycle-related genes.     

Enhancing effect of malachite green on the development of hepatic pre-neoplastic lesions induced by N-nitrosodiethylamine in rats.

The effects of malachite green (MG) and phenobarbitone (PB) were compared on the development of pre-neoplastic lesions during N-nitrosodiethylamine(DEN)-induced hepatocarcinogenesis in male Wistar rats. Rats were administered 200 p.p.m. DEN in drinking water for a period of 1 month. After an interval of 2 weeks the animals were given either MG (25 p.p.m.) or PB (500 p.p.m.) in drinking water for 2.5 months. The effects were monitored on the basis of the morphological appearance of the liver, histological pattern, gamma-glutamyltranspeptidase (GGT)-positive foci, total GGT activity and the induction of glycogen-deficient islands. Both MG and PB were found to enhance liver carcinogenesis to a significant extent when compared with either their corresponding controls or animals given DEN alone. The enhancing effect of MG at 25 p.p.m. is comparable with PB at 500 p.p.m. An enhancing effect of MG on DEN-induced hepatocarcinogenesis in the rats was demonstrated.     

Inactivation of p27Kip1 promotes chemical mouse liver tumorigenesis in the resistant strain C57BL/6J

The biochemical function of p27Kip1 as an inhibitor of cyclin-dependent kinases is well-established, but the role of p27 as a tumor suppressor depends on specific cellular contexts. Previous studies using p27 knockout mice on mixed C57BL/6J x 129/Sv strain background did not find a tumor suppressor role of p27 in the liver. An important feature of mouse liver tumorigenesis is strain-dependent tumor susceptibility. Here, we determined the role of p27 in liver tumorigenesis in C57BL/6J mice, a liver tumor resistant strain, in response to a diethylnitrosamine (DEN) and phenolbarbital (PB) two-stage carcinogenesis protocol. At 6 mo of age, while livers of DEN-PB treated p27+/+ and p27-/- C57BL/6J mice appeared morphologically normal, p27-/- livers, but not p27+/+ livers, contained readily detectable glucose-6-phosphatase (G6Pase)-deficient foci. At the 9-mo time point, p27-/- mice developed significantly enhanced liver tumor phenotypes than p27+/+ mice as demonstrated by increased numbers and sizes of liver surface nodules, increased liver-to-body weight ratios, and increased numbers of G6Pase-deficient nodules and histologically diagnosed foci and adenomas in liver sections. Hepatic lesions in p27-/- livers contained more proliferating hepatocytes than lesions in p27+/+ livers, while the numbers of apoptotic cells appeared similar in lesions of both genotypes. Unexpectedly, tumors in p27-/- livers contained only slightly elevated Cdk2 kinase activity compared with normal livers. These results reveal a liver tumor suppressor role of p27 in this resistant mouse strain, and the need to further study the role of Cdk2 kinase in liver tumor promotion by p27 inactivation.     

Suppressive Effects of S-Methyl Methanethiosulfonate on Promotion Stage of Diethylnitrosamine-initiated and Phenobarbital-promoted Hepatocarcinogenesis Model

Modifying effects of S-methyl methanethiosulfonate (MMTS) on diethylnitrosamine (DEN)-initiated and phenobarbital (PB)-promoted hepatocarcinogenesis were examined in rats. Five-week-old male F344 rats were divided into 8 groups. After a week, groups 1–5 were given DEN (100 mg/kg body weight, i.p.) once a week for 3 weeks, whereas groups 6–8 received vehicle treatment. Group 2 was given 100 ppm MMTS containing diet in the initiation phase. From 4 weeks after the start of experiment, groups 3 and 5 were fed MMTS, and groups 1–3 and 7 received drinking water containing 500 ppm PB. Group 6 was given MMTS diet alone throughout the experiment (24 weeks). The incidences of hepatocellular adenoma and total liver tumors were significantly smaller in group 3 than those of group 1. The average numbers of hepatocellular adenoma, carcinoma and total tumors in group 3 were significantly smaller than in group 1. Glutathione, S-transferase placental form-positive foci were also significantly decreased by MMTS treatment in the promotion phase. MMTS treatment in the initiation or promotion phase reduced ornithine decarboxylase activity in the liver of rats given DEN. The antioxidant activity against lipid peroxidation of MMTS was confirmed in tests with rabbit erythrocyte membrane ghosts or rat hepatocytes. These results suggest that MMTS is a promising chemopreventive agent for liver neoplasia when concurrently administered with PB.     

Lack of enhancing effects of fenvalerate and esfenvalerate on induction of preneoplastic glutathione S-transferase placental form positive liver cell foci in rats

The modifying effects of fenvalerate and esfenvalerate administration on liver carcinogenesis were investigated in male F344/DuCrj rats initially treated with N-nitrosodiethylamine (DEN). Two weeks after a single dose of DEN (200 mg/kg, intraperitoneally), rats were given fenvalerate at dietary levels of 1500, 500, 150, 50 and 15 parts per million (ppm), esfenvalerate at 500 ppm, or 2-acetylamino-fluorene (2-AAF) at 200 ppm and sodium phenobarbital (PB) at 500 ppm as positive controls for 6 weeks. At week 3 following DEN administration, all animals were subjected to partial hepatectomy. Prominent neurologic signs and moderate retardation of body weight were observed in the groups given 1500 ppm fenvalerate and 500 ppm esfenvalerate, although no adverse effects on survival were evident. While statistically significant increases in relative liver weights were noted in rats given fenvalerate at doses of 1500 or 500 ppm, no toxic hepatocyte lesions were found. Neither fenvalerate nor esfenvalerate significantly increased the numbers or areas of glutathione S-transferase placental form (GST-P) positive liver cell foci observed after DEN initiation, in clear contrast to the positive controls, 2-AAF and PB. The results thus demonstrated that fenvalerate and esfenvalerate are non-toxic for rat hepatocytes and lack modifying potential for liver carcinogenesis in our medium-term bioassay system.     

Nine-week detection of six genotoxic lung carcinogens using the rasH2/BHT mouse model

A 9-week in vivo rasH2/butylhydroxytoluene (BHT) model for the detection of genotoxic lung carcinogens was validated, using six potent positive test compounds, dimethylnitrosamine (DMN; 15 mg/kg, i.p.), diethylnitrosamine (DEN; 100 mg/kg, i.p.), ethylnitrosourea (ENU; 120 mg/kg, i.p.), 3-methylcholanthrene (MC; 100 mg/kg, i.p.), 7,12-dimethylbenz(a)anthracene (DMBA; 5 mg/kg, i.g.) and benzo(a)pyrene (B(a)P; 80 mg/kg, i.p.), each given to rasH2 mice of both genders by single administration for initiation followed by promoter BHT treatment. Statistically significant increase in the incidence and multiplicity of lung tumors was observed in rasH2 mice treated with BHT following exposure to all of the carcinogens tested. The data overall suggest the rasH2/BHT model to be a powerful screening tool for genotoxic lung carcinogens.     

Inhibitory Effect of Chlorophyllin on Diethylnitrosamine and Phenobarbital-induccd Hepatocarcinogenesis in Male F344 Rats

Modifying effects of chlorophyllin (CHL) on the diethylnitrosamine (DEN)-phenobarbital (PB) hepatocarcinogenesis model were examined in rats. Five-week-old male F344 rats were divided into 8 groups. Groups 1 through 5 were given i.p. injections of DEN (100 mg/kg body weight) once a week for 3 weeks beginning one week after the start of the experiment, while groups 6 through 8 received vehicle treatment. Groups 1, 2, 3 and 7 received drinking water with 500 ppm PB from one week after the end of carcinogen or vehicle treatment. CHL-containing diet (2000 ppm) was given to group 2 during the initiation phase and to groups 3 and 5 during the promotion and the post-initiation phase, respectively. Group 6 was given the experimental diet alone throughout the experiment (24 weeks). Liver neoplasms were present in DEN-treated groups and PB treatment promoted liver tumorigenesis. The incidences of adenoma in groups 2 and 3 were significantly smaller than in group 1 (P<0.05 and P<0.02), although the reductions in the incidences of liver cell cancer were not significant. The average numbers of liver neoplasms/rat in group 2 were significantly smaller than in group 1 (P<0.05-P<0.005), Glutathione S -transferase placental form-positive foci were also significantly decreased by CHL treatment (P<0.05 and P<0.001). DEN and PB exposure increased liver ornithine decarboxylase activity and this increase was significantly inhibited by feeding of CHL during the initiation phase (P<0.001). These results suggest that CHL is a chemopreventive agent for liver neoplasia.     

Cleistocalyx nervosum Extract Ameliorates Chemical-Induced Oxidative Stress in Early Stages of Rat Hepatocarcinogenesis

Purpose: To study the effect of Cleistocalyx nervosum extract (CE) on diethylnitrosamine (DEN) andphenobarbital (PB) induced oxidative stress in early stages of rat hepatocarcinogenesis. Materials and Methods:Male Wistar rats were divided into 4 groups, with Group 1 as a negative control and Group 2 was a positivecontrol receiving DEN injections once a week and PB in drinking water for 6 weeks. Two weeks before DENinitiation and PB treatment, Groups 3 and 4, were fed with 500 and 1000 mg/kg of CEs, respectively, for 8weeks. Results: A number of GST-P-positive foci, preneoplastic lesions, in the liver were markedly increased incarcinogen administered rats, but was comparatively decreased in rats treated with 1000 mg/kg of CE. The CEreduced malondialdehyde in serum and in the livers of rats treated with DEN and PB. Moreover, CE significantlyincreased the activities of glutathione peroxidase and catalase in rat liver. Conclusions: CE appeared to exertits chemopreventive effects by modulating antioxidant status during DEN and PB induced early stages ofhepatocarcinogenesis in rats.     

Hepatocyte expression of tumor associated aldehyde dehydrogenase (ALDH-3) and p21 Ras following diethylnitrosamine (DEN) initiation and chronic exposure to di(2-ethylhexyl)phthalate (DHEP)

Phthalate ester such as di(2-ethylhexyl)phthalate (DEHP) eitherpromote or inhibit rat liver tumorigenesis depending on thecarcinogenesis protocol. In this study, we examined the expressionof two histochemical markers, the tumor associated isozyme ofaldehyde dehydrogenase (ALDH-3) and the oncoprotein p21 Ras,in the livers of male F344 rats. The rats were initiated withDEN and further treated with either DEHP (a known inhibitorof hepatocarcinogenesis), phenobarbital (PB, a known promoterof hepatocarcinogenesis), or a combination of DEHP and PB. Thestudies were designed to examine the expression of these markersin both normal appearing liver and hepatic hyperplastic andneoplastic lesions andto correlate the early expression of themarkers at 26 weeks in the normal appearing liver to later tumorincidence at 52 weeks. The expression of each marker was detectedby immunohistochemical methods on formalin-fixed paraffin embeddedsections of normal appearing liver or liver lesions. We foundthat ALDH-3 and p21 expression were significantly enhanced inrats receiving PB after DEN initiation at 26 weeks and thatthe incidence of hepatocellular carcinomas was likewise increasedcompared to control or DEN only treated animals. DEN initiationfollowed by a combination of PB and either 0.1 or 0.5% DEHPsignificantly reduced ALDH-3 but not p21 Ras expression at 26weeks compared to DEN plus PB only. These treatment regimensalso reduced the incidence of hepatocellular carcinomas at 52weeks. DEN followed by any of the three doses of DEHP withoutPB resulted in ALDH-3 expression similar to DEN alone. However,p21 Ras expression was significantly increased after these treatments.For all treatment groups, both the early (26 weeks) expressionof p21 Ras and ALDH-3 correlated with hepatocellular carcinomaincidence at 52 weeks. However, the correlation between hepatocellularcarcinoma and ALDH-3 expression was better than p21 Ras or theother markers we have studied. We concluded that ALDH-3 expressionis significantly downregulated after DEHP treatment, and thatexpression of the isozyme correlated with later hepatocarcinomaincidence and may indicate a significant relationship betweenALDH-3 expression and epatocarcinogenesis during DEHP treatment.     

CANCER BIOLOGY: Expression of growth factors and growth factor receptors in the liver of C57BL/10J mice following administration of phenobarbitone

Liver enlargement is a common feature of non-genotoxic rodenthepatocarcinogens administered at high doses. In the presentstudy, the expression of growth factors and growth factor receptorswas investigated in the C57BL/10J mouse during liver enlargementinduced by the non-genotoxic rodent hepatocarcinogen, sodiumphenobar-bitone (PB). Male mice were dosed 0–2500 p.p.m.PB in the diet for 1, 4 and 13 weeks. There was a dose and timedependent increase in liver weight Hepatocyte replication, assessedby incorporation of bromodeoxyuridine, was increased in a dose-dependentmanner at week 1 only (18-fold increase at 2000 p.p.m.) andwas predominantly localized in the centrilobular region. Atweek 1, PB (2500 p.p.m.) caused transient increases in transforminggrowth factor