The effect of long-term feeding of orotic acid on the incidence of foci of enzyme-altered hepatocytes and hepatic nodules in Fischer 344 rats

The present study was designed to determine the long-term effectsof orotic acid (OA), a multi-organ tumor promoter, in rats notexposed to any carcinogen. Male Fischer 344 rats (130–150g) were divided into two groups and given either a semisyntheticbasal diet (BD) or the same diet containing 1% OA. Animals fromboth groups were killed after 1 or 2 years of treatment. Fociof placental glutathione-S-transferase (GST 7–7) positivehepatocytes were observed in the livers of both BD and OA fedrats killed after 1 year. However, they were more in numberin animals receiving OA (156±21 versus 51±11/cm³).After 2 years, hepatic nodules were seen in almost all the animalsgiven OA and in 30% of the rats given BD. The nodules were oftwo main types: (i) a reddish-brown type, present in 85% ofrats exposed to OA and in 27% of rats given BD, and (ii) a greyish-whitetype, found in 50% of animals fed OA and in none of the animalsfed BD. These two types of lesions were also histologicallydifferent. Reddish-brown nodules were composed of slightly enlargedhepatocytes resembling normal surrounding tissue, while greyish-whitenodules were similar in structure and are indistinguishablefrom hepatic nodules induced by genotoxic chemical carcinogens.The results are interpreted to suggest that the foci/nodulesseen in OA-fed rats are due to a promoting effect of OA on spontaneouslyarising and/or diet-induced altered cells.     

A subnecrogenic dose of diethylnitrosamine is able to initiate hepatocarcinogenesis in the rat when coupled with fasting/refeeding

Caloric restriction causes a generalized decrease in growthrate and has been repeatedly associated with an inhibitory effecton cancer development in several systems. In contrast, exposureto complete fasting followed by refeeding is as metabolic conditionassociated with increased cell turnover in different organs,including the liver. The present study examines whether suchcondition is able to sustain the induction of initiated hepatocytesfollowing a subnecrogenic dose of diethylnitrosamine (DENA).Male Fisher-344 rats were fasted for 4 days and 1 day afterrefeeding they were given a single dose of DENA (20 or 200 mg/kgbody wt, i.p.). Negative and positive control groups were fedad libitum and injected with 20 and 200k mg/kg of DENA, respectively.One week later all animals were subjected to the resistant hepatocytemodel for the selection of hepato-cyte nodules and they werekilled 2 weeks thereafter. Results indicated the presence ofgamma-glutamyltransfer-ase (GGT) positive foci and nodules (38± 7/cm²) in rats regularly fed and given 200 mg/kg ofDENA, while virtually no focal lesions (<1/cm²) were foundin the group receiving 20 mg/kg of DENA and fed throughtoutthe experiment. However, a significant number of GGT positivefoci/nodules (14 ± 7) also developed in rats exposedto fasting and given 20 mg/kg of DENA 24 h after refeeding.No evidence of helpatocellular necrosis was found in the lattergroup follwing DENA administration. No effect of fasting wasobserved when rats received 200 mg/kg of DENA. It is concludedthat fasting/refeeding provides conditions which are able tosustain initiation in rat liver by a subnecrogenic dose of acarcinogen. These findings are in contrast with the commonlyreported inhibitory effect of chronic food restriction on variousstages of carcinogenesis, including initiation.     

Effect of vitamin E on the induction and evolution of enzyme-altered foci in the liver of rats treated with diethylnitrosamine

The effects of dietary vitamin E (VE) on the steps of hepatocarcinogenesis,the induction and growth of -glutamyltranspeptidase (GGT)-positivefoci and their evolution into persistent nodules, were analyzedin the liver of rats treated with diethylnitrosamine (DEN).The induction of GGT-positive foci was inhibited by a diet containing0.36–1.5% VE given after initiation with 200 mg/kg bodyweight (b.w.) DEN for 6 weeks with partial hepatectomy (PH)on week 3. The numbers and areas of GGT-positive foci were enhancedby diets containing 036 and 0.72% VE, given for 1 week afterinitiation with 10 mg/kg b.w. DEN and PH, followed by selectionby 0.02/ 2-acetylaminofluorene (AAF) and carbon tetrachloride(CCl⁴), but these were not enhanced by a diet containing 1.5%VE. Remodeling of hyperplastic nodules was not affected by thediet containing 0.72% VE given after initiation with DEN andselection for 12 weeks. The staining characteristics of GGTwere different between remodeling and persistent nodules, exceptfor those of the glutathione-S-transferase placental form (GST-P).The results obtained suggest that VE could prevent the veryearly events during hepatocarcinogenesis, the induction of phenotypicallyaltered foci, but could no longer affect the later stages, theevolution of foci into persistent nodules.     

Inhibitory effect of sinigrin and indole-3-carbinol on diethytnitrosamine-induced hepatocarcinogenesis in male ACI/N rats

The modifying effects of sinigrin (Sin) and indole-3-carbinol(I3C) on the hepatocarcinogenesis induced by diethyl-nitrosamine(DEN) were investigated in male ACI/N rats. Rats were dividedinto six groups: group 1 was given DEN (40 p.p.m.) in the drinkingwater for 5 weeks, starting at 7 weeks of age; group 2 was treatedwith DEN and diet containing 1200 p.p.m. Sin; group 3 receivedDEN and diet containing 1000 p.p.m. I3C; group 4 was given Sindiet alone; group 5 was given I3C diet alone; and group 6 servedas controls. The diet containing Sin or I3C was fed to the ratsstarting at 6 weeks of age until 1 week after the carcinogenexposure. At termination of the experiment (week 29), the incidencesof iron-excluding altered foci (11.22±3.22/cm²) and livercell tumors (6/12, 50%) and the tumor multiplicity (0.9/rat)in rats of group 2 were significantly smaller than those ofgroup 1 (foci incidence, 48.33±6.34/cm², tumor incidence,10/10, 100% multiplicity, 9.5/rat) (P < 0.02). Similarly,the incidence of iron-excluding hepatocellular foci (17.65±4.67/cm2)and tumor multiplicity (2.4/rat) with a slight reduction oftumor incidence (9/12, 75%) in rats of group 3 were significantlylower than those of group 1 (P < 0.001). There were no livercell neoplasms in rats of groups 4, 5 and 6. Thus, Sin and I3Cinhibited the hepatocarcinogenesis induced by DEN when theywere administered concurrently with the carcinogen.     

Effect of chronic phenobarbital administration on the gamma-glutamyl transpeptidase activity of hyperplastic liver lesions induced in rats by the Solt/Farber initiation: selection process of hepatocacinogenesis

A chronic 8 to 11 week administration of the hepatic tumor promoterphenobarbital (0.05% in drinking water) to rats previously subjectedto the initiation:selection process of Solt and Farber was foundto further increase the gamma-glutamyl transpeptidase activityof individual hyperplastic liver nodules of 4.0–10.0 mmin diameter over comparably sized nodules form control livers.Those rats which received 11 weeks of the chronic phenobarbitaltreatment also showed a significant increase in their liverwet weights. In addition, random tissue samples of non-nodularliver taken from the 11 week phenobarbital-treated rats exhibiteda gamma-glutamyl transpeptidase mean specific activity whichwas {small tilde}3 times higher than that of control non-nodularliver samples. In contrast, there was a 1.9-fold increase inthe mean % gamma-glutamyl transpeptidase-positive area (cm²),as determined histochemically, in cryostat sections made fromnon-nodular samples of the 11 week phenobarbital-treated ratswhen compared with that of control liver sections. Interruptionof the chronic phenobarbital administration at 8 weeks followedby 3 weeks of control treatment resulted in a reversal of thegammaglutamyl transpeptidase activity response shown by thehyperplastic liver nodules and non-nodular liver tissue samples.Thus, phenobarbital can quantitatively modulate gamma-glutamyltranspeptidase activity in carcinogeninduced hyperplastic liverlesions in the rat during the early stages of hepatocarcinogenesis.     

Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid

Chemoprevention of liver carcinogenesis by S-adenosyl-Lmethionine(SAM) was studied in F344 male rats. The rats were given 1,2-dimethylhydrazine(1,ZDMH) 2 HCl (100 mg/kg, i.p.) 18 h after two-thirds hepatectomy.One week later they were fed a semisynthetic basal diet containing1% orotic acid (OA) for 29 weeks. At this time the rats weretransferred to the basal semisynthetic diet and were killed3 weeks later. SAM treatment (384 µmol/kg/ day, im.),was started 1 week after 1,2-DMH and was continued up to theend of the experiment. Controls received solvent alone. SAMexerted an inhibitory effect on the induction of preneoplasticand neoplastic lesions. For example, nodules with diametersof 1-2 and 2-6 mm exhibited a decrease in both incidence andnumber per liver, while no such inhibitory effect was seen inthe category of larger nodules. Furthermore, hepatocellularcarcinoma (HCC) also exhibited a decrease in the SAM-treatedgroup. The numberniver and incidence were 0.04 and 4.8% respectivelyin the SAM-treated group, compared to 0.38 and 37.8% in thecontrol group. Microscopic examination showed the presence ofwell-differentiated carcinomas and atypical nodules in controlrats, while only one small, well-differentiated tumor and onenodule with patterns of initial transformation were seen inSAM-treated rats. No patchy staining of glutathione-S-transferase,indicative of remodeling, was observed in nodules of both SAM-treatedand control rats. Nodules and HCCs developing in SAM-treatedrats exhibited a relatively high number of apoptotic bodies.Apoptotic bodies count showed 2.8-and 1.8-fold increases innodules and HCCs of SAM-treated rats with respect to controls.These results indicate that SAM exerts a chemopreventive effecton hepatocarcinogenesis induced by the OA model. SAM seems tobe more effective in inhibiting nodule to HCC progression thanon the growth of nodule per se. The inhibitory effect is associatedwith an increase in cell loss by apoptosis in nodules and HCC.     

The development of hepatocellular carcinoma in initiated rat liver after a brief exposure to orotic acid coupled with partial hepatectomy

Previous work from this laboratory has revealed that a minimumof 10–20 weeks of continuous exposure to 1% dietary oroticacid (OA) is necessary for this regimen to exert a significantpromoting effect on the carcinogenic process in rat liver. Thepresent study investigates the effect of partial hepatectomy(PH), given during a short-term exposure (4 weeks) to OA, onthe development of hepatocyte nodules (HN) and hepatocellularcarcinoma (HCC) initiated by diethyl-nitrosamine (DEN). MaleFischer 344 rats (130–150 g) were given a single doseof DEN (200mg/kg body wt i.p.)- Starting a week later they werefed either a semisynthetic basal diet (BD) or the same dietcontaining 1% OA for 2 weeks; two-thirds PH was then performedfollowed by another 2 weeks of BD or OA diet respectively. Atthe end of this treatment some animals from both groups werekilled while the rest were continued on BD and killed at 20or 56 weeks thereafter. The results showed no difference betweenthe two groups in the incidence of     

Effect of orotic acid on in vivo DNA synthesis in hepatocytes of normal rat liver and in hepatic foci/nodules

One of the proposed mechanisms by which orotic acid (OA) promotesliver carcinogenesis is by differentially mito-inhibiting thenormal hepatocytes while permitting the initiated ones to respondto growth stimuli to form foci/nodules. In an attempt to examinethis hypothesis, the present study was designed to determine(i) whether OA inhibits DNA synthesis in normal hepatocytesin vivo, and (ii) whether hepatocytes from hepatic foci/nodulesare relatively resistant to the mito-inhibitory effects of OA.The results of this study indicate that OA given i.p. as a tabletof 300 mg at the time of partial hepatectomy (PH) almost completelyinhibited liver DNA synthesis. Three days later—a timeperiod by which the implanted tablet disappeared—the hepatocytesresumed DNA synthesis. Exposure to OA results in an accumulationof uridine nucleotides and a decrease in adenosine nucleotides.Creation of such an imbalance in nucleotide pools appears tobe important for OA to inhibit DNA synthesis. Adenine (a tabletof 300 mg), an agent that inhibits the metabolism of OA to uridinenucleotides, counteracted the mito-inhibitory effects of OA.To determine whether the hepatocytes in foci/nodules are resistantto the mito-inhibitory effects of OA, rats were initiated withdiethylnitrosamine (DENA; 150 mg/kg) and promoted by the resistant-hepatocytemodel. Fourteen weeks after the administration of DENA, therats were subjected to PH in the presence or absence of OA (300mg tablet). The results indicated that, in contrast to hepatocytesin normal or surrounding non-nodular liver, a subpopulationof hepatocyte foci/nodules appear to be relatively resistantto the mito-inhibitory effects of OA. These findings supportthe hypothesis that differential mito-inhibition is a possiblecomponent in the promoting effect of OA. However, whether thisis the mechanism by which OA promotes liver carcinogenesis needsto be further investigated.     

Resistance of hepatic nodules to orotic acid-induced accumulation of uridine nucleotides

It has been hypothesized that orotic acid (OA) promotes ratliver carcinogenesis by a differential mitoinhibitory mode.Consistent with this hypothesis, hepatic nodules are relativelyresistant to OA-induced mitoinhibition. OA-induced mitoinhibitionis dependent on the metabolism of OA to uridine nucleotides.The present studies investigate the uptake and metabolic pathwayof OA, both in vivo and in vitro, as a possible basis for theresistance of hepatic nodules to OA-induced mitoinhibition.Rats bearing hepatic nodules exposed to 1% dietary OA exhibitedincreased levels of uridine nucleotides in the surrounding non-nodularliver (from 0.44 to 0.70 mg/g liver) but not in the hepaticnodules. Further, following administration of [³H]OA i.p., noduleshave significantly lower levels of acid-soluble radioactivitycompared to the non-nodular surrounding tissue. Furthermore,most of the acid-soluble radioactivity was present as uridinenucleotides, suggesting that the OA taken up was converted touridine nucleotides. Similarly, hepatocytes from nodules inprimary culture incubated with radiolabeled OA, have significantlylower levels (46–60%) of acid-soluble radioactivity. Theseresults suggest that the decreased uptake of OA by hepatic nodulesmay be a factor contributing to the observed resistance of hepaticnodules to the mitoinhibitory effects of OA.     

The enhancing effect of fasting/refeeding on the growth of nodules selectable by the resistant hepatocyte model in rat liver

Caloric restriction causes a generalized decrease in growthrate and has been shown to delay the development of both spontaneousand induced neoplasia. In contrast to chronic food restriction,the extreme condition of fasting/refeeding is associated withan overall increase in cell turnover in several organs, includingliver, compared with regular feeding. The present study wastherefore designed to investigate the effect of complete foodwithdrawal followed by refeeding on the growth of hepatocytenodules in initiated rat liver. Male Fischer 344 rats were givena single dose of diethylnitrosamine (DEN, 200 mg/kg i.p.) andthen, starting 1 wk later, they were exposed to one or threecycles of fasting (3 days) followed by refeeding (11 days).The control group was fed continuously. Seven weeks after DENadministration all rats were subjected to the resistant hepatocytemodel (2-acetylaminofluorene coupled with CCl₄) and 2 weekslater 2/3 partial hepatectomy (PH) was performed. All animalswere killed 2 weeks after surgery. At PH rats given one cycleof fasting/refeeding had significantly larger glutathione S-transferase7–7-positive hepatic lesions compared with controls (meanarea 0.73 ± 0.04 versus 050 ± 0.05 mm², P <0.025; mean percent area 25.6 ± 3.2 versus 12.4 ±0.9, P < 0.005), while no significant change was observedin their number. The observed differences were more pronouncedwith three cycles of fasting/ refeeding. A similar pattern ofresults was obtained at the time of killing. It is concludedthat fasting/refeeding can exert a positive effect on the growthof rat hepatocyte foci and nodules, in contrast to the generalinhibitory effect on carcinogenesis caused by food restriction.     

Evaluation of the tumor-promoting activity of two {beta}-adrenoreceptor blocking agents, propranolol and atenolol, in liver of Fischer 344 rats

The tumor-promoting activity of two ß-adrenoreceptorblocking agents, propranolol and atenolol, was tested in a two-stageprotocol of hepatocarcinogenesis in male and female Fischer344 rats. Propranolol is a lipophlllc non- selective ß-blockermainly eliminated via the liver; atenolol is a hydrophilic ß¹-selectiveblocking agent, mainly eliniin ated via the kidney. Animalswere initiated with a single dose of diethylnitrosamine (DEN,200 mg/kg, i.p.) and, after 17 days of recovery, were continuouslytreated with propranolol (75–100 mg/kg) or atenolol (300mg/kg) by gavage for up to 21 months. Rats given phenobarbital(0.05% in the diet) were used as positive controls. After 2,4 and 8 months of promotion, preneoplastic lesions were quantifiedby staining sections of liver for     

贞芪扶正胶囊对大鼠实验性肝癌发生发展的影响

目的 :观察贞芪扶正胶囊在化学致癌剂二乙基亚硝胺 (diethylnitrosamine ,DENA)诱发大鼠肝癌过程中 ,对肝癌发生、发展的影响。方法 :实验分为 4组 ,除单纯致癌组 (D组 )外 ,其余各组大鼠均在给予致癌剂前 (A组 )、同时 (B组 )或后 (C组 )加给贞芪扶正胶囊。在给予致癌剂 2 0周后取肝脏作肉眼、光镜和电镜检查。结果 :单纯致癌组大鼠均形成肝癌。服用贞芪扶正胶囊的各组情况则有较大差异 ,A组大鼠显示致癌剂对肝脏毒性损伤明显低于其他各组 ,并且当其他各组动物均已形成肝癌甚至已有转移时 ,此组中仅有少数大鼠形成肿瘤 ,多数大鼠则仅出现DENA毒性刺激而形成的细胞增生结节。结论 :贞芪扶正胶囊有预防或延缓肝癌发生的作用     

Inhibitory effect of 4,4'-diaminodiphenylmethane on liver, kidney and bladder carcinogenesis in rats ingesting N-ethyl-N-hydroxyethylnitrosamine or N-butyl-N-(4-hydroxybutyl)nitros-amine

The effects of 4,4'-diaminodiphenylmethane (DDPM) administrationin the ‘post-initiation’ stage of liver, kidneyand bladder carcinogenesis were examined in male F344 rats.In experiment I, rats were given drinking water containing 0.1%N-ethyl-N-hydroxyethyl-nitrosamine for 2 weeks then diet containing0.1% DDPM for 32 weeks. In week 3, the right kidney was removed.The incidence of hepatocellular carcinoma was significantlyless in rats given DDPM than in controls. DDPM decreased theincidence and average number/cm² of neoplastic nodules and renalcell tumors of the kidney. In experiment II, rats were given0.01% N-butyl-N-(4-hydroxybutyl)nitrosamine for 4 weeks andthen 0.1% DDPM for 34 weeks in their drinking water. DDPM inhibitedthe induction of papillomas in the bladder. These results indicatethat DDPM administration in the ‘post-initiation’stage inhibited liver, kidney and bladder carcinogenesis inrats.     

Pomegranate-mediated chemoprevention of experimental hepatocarcinogenesis involves Nrf2-regulated antioxidant mechanisms

Hepatocellular carcinoma (HCC), one of the most prevalent and lethal cancers, has shown an alarming rise in the USA. Without effective therapy for HCC, novel chemopreventive strategies may effectively circumvent the current morbidity and mortality. Oxidative stress predisposes to hepatocarcinogenesis and is the major driving force of HCC. Pomegranate, an ancient fruit, is gaining tremendous attention due to its powerful antioxidant properties. Here, we examined mechanism-based chemopreventive potential of a pomegranate emulsion (PE) against dietary carcinogen diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis that mimics human HCC. PE treatment (1 or 10 g/kg), started 4 weeks prior to the DENA challenge and continued for 18 weeks thereafter, showed striking chemopreventive activity demonstrated by reduced incidence, number, multiplicity, size and volume of hepatic nodules, precursors of HCC. Both doses of PE significantly attenuated the number and area of γ-glutamyl transpeptidase-positive hepatic foci compared with the DENA control. PE also attenuated DENA-induced hepatic lipid peroxidation and protein oxidation. Mechanistic studies revealed that PE elevated gene expression of an array of hepatic antioxidant and carcinogen detoxifying enzymes in DENA-exposed animals. PE elevated protein and messenger RNA expression of the hepatic nuclear factor E2-related factor 2 (Nrf2). Our results provide substantial evidence, for the first time, that pomegranate constituents afford chemoprevention of hepatocarcinogenesis possibly through potent antioxidant activity achieved by upregulation of several housekeeping genes under the control of Nrf2 without toxicity. The outcome of this study strongly supports the development of pomegranate-derived products in the prevention and treatment of human HCC, which remains a devastating disease.     

In vitro and in vivo response of hepatocytes from hepatic nodules to the mitoinhibitory effects of phenobarbital

One of the proposed mechanisms by which phenobarbital (PB) promoteshepatocarcinogenesis in the rat is by differential mitoinhibition.However, our earlier studies indicated that PB inhibited DNAsynthesis in vitro in hepatocytes isolated from both surroundingnon-nodular liver and hepatic nodules promoted by orotic acid(OA). Since nodules generated by one promoter need not necessarilybe resistant to another promoter, the present study was undertakento determine whether foci/nodules promoted by PB itself areresistant to the mitoinhibitory effects of PB. Accordingly,rats were initiated with diethylnitrosamine (DENA, 200 mg/kgi.p) and promoted with PB (0.07% of PB as its sodium salt) intheir drinking water for 16 or 33 weeks. In vitro studies indicatedthat PB (3–5 mM) inhibited DNA synthesis induced by epidermalgrowth factor (EGF) in hepatocytes from surrounding non-nodularliver as well as from nodules promoted by PB for 33 weeks. Inanother experiment, initiated rats exposed to PB for 33 weekswere subjected to either two-thirds partial hepatectomy (PH)or sham hepatectomy. Hepatocytes were labelled with tritiatedthymidine in vivo for 48 h. Autoradiographic analysis indicatedthat in the presence of PB, the hepatocytes from both foci/nodulesand the surrounding non-nodular liver responded to PH to thesame extent. In addition, they both responded to PH less efficientlyas compared to the corresponding controls. Further, initiatedrats exposed to PB for 16 weeks when subjected to PH and killed4 weeks thereafter, the percentage area occupied by     

Vanadium-mediated suppression of diethylnitrosamine-induced chromosomal aberrations in rat hepatocytes and its correlation with induction of hepatic glutathione and glutathione S-transferase

Vanadium, a dietary micronutrient, has recently been found to possess a potent antitumor activity during chemically induced rat liver carcinogenesis. In the present study, attempts have been made to understand the basic mechanism of the antitumor response of vanadium by monitoring its effect on chromosomal aberrations (CA) in rat liver cells during the early preneoplastic steps of diethylnitrosamine (DENA)-induced hepatocarcinogenesis. Supplementary vanadium at 0.5 ppm was found to afford a unique protection against DENA-evoked CA 96 h after DENA injection. Concurrent administration of glutathione (GSH) at 200 mg/kg 2 h before DENA treatment potentiated the suppressive effect of vanadium against CA when the rats were sacrificed 96 h after the carcinogenic insult. Pretreatment nf rate with buthionine sulfoximine (890 mg/kg) and/or diethylmaleate (600 mg/kg) 0.5 or 2 h prior to DENA injection resulted in a significant inhibition of vanadium-mediated protection of CA with a concomitant fall in hepatic GSH level. Rats given injection of bromosulfophthalein (250 mg/kg), a substrate inhibitor of glutathione S-transferase (GST), 0.5 h before DENA treatment displayed a prominent suppression of the protective effect of vanadium on DENA-induced CA. Long-term supplementation of vanadium also triggered protective effect against the induction of CA 15, 30 or 45 days following DENA treatment which was maximally observed on structural aberrations followed by numerical aberrations. At these time points, vanadium was found to lower the mitotic index of hepatic cells which was otherwise elevated with DENA alone. Vanadium restored DENA-dependent decrement in the ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) in rat liver cells. The DENA-induced increased frequency of micronucleated PCE as well as NCE was also attenuated following vanadium supplementation. The anticlastogenic effect of vanadium was found to be parallel to its ability to induce the activity of hepatic GST with a concurrent induction of hepatic GSH pool which were rather decreased in DENA control group. The results of this study, thus, provide evidence that vanadium-dependent induction of GSH-mediated GST-catalyzed detoxificational capacity of the host is presumably related to its suppressive effect against CA. This may explain, in part, the antitumor efficacy of this trace element.     

Promoting effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene in mouse hepatocarcinogenesis

The effects of phenobarbital (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene(TCPOBOP) on liver hyperplasia, induction of microsomal enzymeactivities, and two-stage hepatocarcinogenesis were evaluatedin B6C3F1 female mice. For 4 weeks four groups of mice receivedPB (500 p.p.m. in the drinking water), TCPOBOP (3 mg/kg i.p.once every week), PB together with TCPOBOP or corn oil vehiclei.p. TCPOBOP induced liver hyperplasia and hypertrophy and increasedp-nitroanisole-O-demethylase and aminopyrine-N-demethylase morethan PB. Neither chemical changed UDPG-transferase activity.The association of PB and TCPOBOP gave the same effects as TCPOBOPalone. Other four groups of mice were treated with N-nitroso-N-diethylamineat 7 days of age and then, starting from 8 weeks of age, receivedthe above specified weekly treatments for 20 weeks and werethen sacrificed. Hepatocellular nodules > 150 µm werefound in all animals of all groups. Due to increased size ofthe liver compared to controls, the number of nodules/cm³ decreasedafter PB and TCPOBOP treatments given alone or together; howeverthe mean volume of nodules and the percentage of liver volumeoccupied by nodules increased after TCPOBOP but not after BPtreatment, and the association of PB and TCPOBOP was even moreeffective than TCPOBOP alone. Hepatocellular adenomas >2.4mm in diameter were observed in 5 of 10 TCPOBOP-treated mice(total of 11 nodules) and in 5 of 11 mice that received PB plusTCPOBOP (total of 15 nodules). Hepatocellular carcinomas wereseen in one mouse treated with PB and in three mice given PBand TCPOBOP.     

Prevention by acetylsailcylic acid of liver cirrhosis and carcinogenesis as well as generations of 8-hydroxydeoxyguanosine and thiobarbituric acid-reactive substances caused by a choline-deficient, L-amino acid-defined diet in rats

Effects of acetylsailcylic acid (ASA) (aspirin) on the pathogenesisof fatty liver, cirrhosis and hepatocarcinogenesis caused bya choline-deficient L-amino acid-defined (CDAA) diet were examinedin male Fischer 344 rats fed a CDAA diet supplemented with 0,0.1, 0.2, 0.4 or 0.8% ASA for 30 weeks. ASA at concentrationsof >0.2% prevented the development of both cirrhosis andpreneoplastic and neoplastic nodules, but without any directlyassociated prevention of fatty changes. ASA also prevented hepatocyteproliferation and the generation of thiobarbituric acid-reactivesubstances and 8-hydroxydeoxyguanosine caused by feeding theCDAA diet, analyzed, respectively, after 1, 12 and 12 weeks.The results clearly indicate that the anti-inflammatory drugASA, which is not a lipotropic factor, can prevent the pathogenesisof cirrhosis and hepatocarcinogenesis caused by a CDAA diet,which is possibly partly associated with the prevention of reactiveoxygen species production.     

Inhibition of aflatoxin B1 -hepatocarcinogenesis in rats by {beta}-naphthoflavone

Effects of ß-naphthonflavoe (ßNF) on theactivity of hepatic microsomal aflatoxin B₁ (AFB₁)-4-hydroxylase- the cytochrome P-450-dependent enzyme system which catalyzesthe metabolism of AFB₁ to AFM₁ - and on AFB₁ induced in vivohepatocarcinogenesis were investigated in weanling male Fischerrats. A single i.p. injection of ßNF in doses of 20mg/kg and 150 mg/kg induced AFB₁ -4-hydroxylase 3- and 4-fold,respectively, 48 h post injection. Feeding of diet containing0.01% ßNF for a period of 9-weeks induced AFB₁ 2-fold.AFB₁, given by intubatlon in a dose of 25 µg five times/weekfor 8 weeks, produced 42 weeks later a 100% incidence of liverlesions (neoplastic foci, nodules or tumors), but feeding ßNFin diet at a con centration of 0.015% for one week prior toand during the 8 weeks of AFB treatment inhibited AFB₁ hepatocarcinogenesis by -7%. These results are in accord with the suggestionthat AFB₁ induction may be associated with the inhibition ofAFB₁ carcinogenesis, possibly occurring as a consequence ofaccelerated detoxi-fication of AFB₁ via its conversion to AFM₁     

Spontaneous and 2-nitropropane induced levels of 8-hydroxy-2'-deoxyguanosine in liver DNA of rats fed iron-deficient or manganese- and copper-deficient diets

Rats (Wistar, female, 4 weeks old) were fed iron-deficient (Fe^–;2.2 µg Fe/g) or manganese- and copper-deficient (Mn.Cu^–;0.3 µg Mn/g, 0.4 ug Cu/g) diets for 8 weeks to determinethe oxidative damage of DNA by element deficiency. After feedingof the diets, 2-nitropropane (2-NP, 80 mg/kg body weight) wasadministered i. p. as an inducer of 8-hydroxy-2'-deoxyguanosine(8-OH-dG) to the element-deficient rats. The hemoglobin concentrationof rats in the Fe- group showed an induction of severe anemia(8.4 g/100 ml whole blood). In the Mn.Cu^– group, Mn-super-oxidedismutase (SOD) activities of plasma and Cu.Zn-SOD activitieswere significantly lower than that of the normal diet group.However, total SOD activities of plasma were not depressed severelyin contrast to that of the liver in the Mn.Cu^– group.Background (spontaneous) levels of 8-OH-dG in normal diet groupwere 0.96 + 0.37/105 deoxyguanosine (dG), however, significantlyhigher levels were detected in the Fe^– group (1.56±0.19,P < 0.01). Conversely, a lower (but not significant) levelof 8-OH-dG than the normal diet group were detected in the Mn.Cu^–group (0.78 ±0.08). Six hours after 2-NP treatment,8-OH-dG levels in liver DNA were significantly induced to 1.44+ 0.24 in the normal diet fed group 1.89±0.22 in theFe^– and 1.08±0.12 in the Mn.Cu^– groups. Comparedto the normal diet group, these induced levels of 8-OH-dG inthe Fe-group were significantly higher (P < 0.05), and thatin Mn. Cu^– group were significantly lower (P < 0.05).The high levels of 8-OH-dG in severe iron deficiency might bethe results of (i) an increase of hydroxyl radical generationby accumulated copper in hepatocytes; or (ii) a depression ofenzymatic activity for removing 8-hydroxy-2' -deoxyguanosinein DNA, which is dependent on divalent cations. On the otherhand, the low level of 8-OH-dG in manganese and copper deficiencymight be the result of a decrease of lipid peroxidation whichhas been suggested to be an intermediator from active oxygenspecies to hydroxyl radical.