Study of the separate and combined effects of the non-planar 2,5,2',5'- and the planar 3,4,3',4'-tetrachlorobiphenyl in liver and lymphocytes in vivo.

Polychlorinated biphenyls (PCBs) are a group of industrial chemicals that are widely distributed in the environment. Because these compounds occur as mixtures, studies of their possible interactive effects are essential for an understanding of the mechanism of the toxicity of these mixtures. For the determination of a possible interaction of the effects in vivo of 2,5,2',5'-tetrachlorobiphenyl (TCB) and 3,4,3',4'-TCB, rats were exposed to a single dose of diethylnitrosamine (DEN) and subsequently to 0.1 p.p.m. 3,4,3',4'-TCB and/or 10 p.p.m. 2,5,2',5'-TCB in the feed for 1 year. The two major targets of PCB toxicity, the liver and the peripheral blood, were examined after these treatments. TCB treatment after DEN exposure caused a predominance of increased placental glutathione S-transferase (PGST) and deficiencies of ATPase as preneoplastic markers in focal hepatic lesions. When 0.05% phenobarbital (PB) was administered after DEN exposure, the distribution of markers in altered hepatic foci (AHF) was essentially equal for increased PGST and gamma-glutamyltranspeptidase (GGT) and for ATPase deficiency. Many of these AHF also exhibited increased P450 b/e expression. Our results demonstrated that the two PCB congeners interacted in vivo to produce an increase in AHF that were PGST positive and ATPase negative. PGST-positive and ATPase-negative AHF correlated best with focal areas of P450 b/e expression. The combination of the two PCBs caused a greater than additive decrease in the total number of lymphocytes and antibody-producing B-cells. Also the thymocyte-dependent T-helper cells isolated from the animals receiving the combination of TCBs demonstrated a morphologically abnormal subpopulation. The results indicate that the interaction of 2,5,2',5'-TCB and 3,4,3',4'-TCB in vivo induced much greater toxicity and mutagenicity in peripheral lymphocytes and hepatocytes than treatment with either congener alone.     

Effect of dietary retinyl palmitate on the promotion of altered hepatic foci by 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'- hexachiorobiphenyl in rats initiated with diethylnitrosamine

The purpose of this study was to determine the effects of dietaryvitamin A on the tumor promoting effect of 3,3',4,4'-TCB and2,2',4,4',5,5'-HCB in a two-stage rat hepatocarcinogeneis modelwith diethylnitrosamine (DEN, 150 mg/kg) as the initiator. Twoweeks after DEN injection rats were fed a purified diet containingeither 2000 or 100 000 IU of vitamin A in the form of retinylpalmitate. Rats received four biweekly injections of 3,3',4,4'-TCB,2,2',4,4',5,5'-HCB (300 µmol/kg), or both (150 µmol/kgeach) in corn oil (10 ml/kg) for 8 weeks. Control animals receivedvehicle only. Six rats in each group that received no DEN treatmentwere used as additional control animals. Ten days after thelast injection the rats were killed. In rats fed the low retinylpalmitate diet, treatment with 3,3',4,4'-TCB, 2,2',4,4',5,5'-HCBor both compounds lowered hepatic retinyl palmitate content.This effect was prevented by high dietary retinyl palmitatesupplementation in rats treated with 2,2',4,4',5,5'-HCB, butnot 3,3',4,4'-TCB or both com pounds together. Histopathologicalexamination of the liver showed that high dietary retinyl palmitatelessened the severity of hepatocellular necrosis and fatty changesinduced by 3,3',4,4'-TCB alone or in combination with 2,2',4,4',5,5'-HCB.The latter did not cause significant pathological lesions tothe liver. However, high dietary retinyl palmitate was not ableto prevent thymic involution caused by 3,3',4,4'-TCB. The numberand volume of altered hepatic foci were increased by 2,2',4,4',5,5'-HCBand particularly 3,3',4,4'-TCB; no synergistic effect was seen.Supplementation with high dietary retinyl palmitate diminishedthe number and volume of foci. These results show that supplementationwith high dietary retinyl palmit ate protects against hepatocellularnecrosis, fatty changes, and preneoplastic changes induced by3,3',4,4'-TCB as well as against preneoplastic changes inducedby 2,2',4,4',5,5'-HCB. In addition, these two agents did notsynergistically induce preneoplastic changes in DEN-inducedrats.     

Quantitative stereologic study of the effects of varying the time between initiation and promotion on four histochemical markers in rat liver during hepatocarcinogenesis

The effects of varying the interval of time between initiation with diethylnitrosamine (DEN) and promotion by phenobarbital (PB) on the development of altered hepatic foci (AHF) and hepatomas in female Fischer 344 rats was investigated. The intervals between DEN initiation after a 70% partial hepatectomy and a subsequent 6 month period of promotion by feeding of PB were 1 day, 1 week, 1 month, 2 months, 6 months and 11 months. The number and volume percentage occupied by AHF were determined by quantitative stereologic methods on serial frozen sections stained for the markers gamma-glutamyltranspeptidase (GGT), canalicular adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6Pase) and the placental form of glutathione S-transferase (GST-pi). The number of AHF was greatest when the initiation-promotion interval was only 1 day, and there was a tendency for the number of AHF to decrease as the interval between initiation with DEN and the start of PB promotion was extended. An 11 month delay between initiation and promotion resulted in only 20% fewer AHF than when promotion was begun 1 day after initiation. On the other hand, the volume percentage fraction of AHF did not change when the initiation-promotion interval was increased from 1 day to 2 months. An interval of 6 months roughly doubled the volume percentage fraction, but an interval of 11 months led to a 7- to 8-fold increase in the volume percentage of AHF over that from a 1 day interval. The phenotypic distribution of AHF was significantly lower in relation to certain markers, especially GGT and GST-pi, in those animals only initiated with DEN compared with those initiated with DEN and promoted with PB. When no exogenous promotion was given, there was still a nearly linear increase in both the number and volume percentage occupied by AHF in the liver of rats initiated with DEN. On the other hand, rats subjected to a 1 week interval between DEN initiation and PB promotion exhibited the greatest number of hepatocellular carcinomas 14 months after initiation, compared with other groups. These studies demonstrated a gradually decreasing effectiveness of PB as a promoting agent to stimulate the growth of all AHF initiated by DEN as the interval between initiation and promotion was extended.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of phenobarbital and dietary choline deficiency promoted preneoplastic lesions in rat liver by environmental contaminant di(2-ethylhexyl)phthalate

The effect of di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer and environmental contaminant, on the emergence of gamma-glutamyltranspeptidase positive (GGT+) preneoplastic foci in the liver of rats fed promoting diets was studied. GGT+ foci were initiated in the liver of Sprague--Dawley male rats with a single dose of diethylnitrosamine (DEN) following partial hepatectomy. One series of control rats received saline vehicle alone. Promotion of foci was commenced by feeding: (1) a choline-deficient diet (CD); (2) a choline-supplemented diet (CS) containing 0.06% phenobarbital (CS + PHB); or (3) a CD diet containing 0.06% phenobarbital (CD + PHB). In the absence of initiation by DEN, dietary treatments did not increase the number of GGT+ foci. In rats receiving DEN, each promoting regimen effectively increased the number of GGT+ foci above levels in control rats fed only the choline-supplemented diet. Inclusion of the plasticizer at a level of 2% in each of the dietary promotion treatments, however, effectively inhibited the appearance of the foci.     

Occurrence of progressive DNA damage coincident with the appearance of foci of altered hepatocytes

The technique of alkaline elution was used to evaluate alterationsin nuclear DNA obtained from livers of rats that had receiveda single 6.6 mg/kg dose of diethylnitrosamine (DEN) during liverregeneration and subsequent exposure (7 days after partial hepatectomy)to phenobarbital (PB). DNA from normal and regenerating liverdemonstrated a significant increase in the rate of elution followingDEN administration. In those DEN-treated groups that did notreceive PB, the rate of DNA elution decreased slowly but failedto return to normal by 44 weeks. Exposure to PB hastened recoveryof a normal DNA elution profile in normal liver following DENtreatment, and by 44 weeks, DNA from these rats eluted at anormal rate. However, in rats treated with DEN during liverregeneration, the rate of DNA elution began to increase at 28weeks of PB exposure and became progressively more rapid throughthe 36th and 44th weeks. This latter group also demonstratedfoci of -ghitamyl-transferase (GGT)positive hepatocytes at 28weeks of PB exposure that increased in number and size concomitantlywith the increasing rate of DNA etution. At 44 weeks, one ormore primary hepatocellular carcinomas were present in 73% ofthe rats in this group; none was seen in any other group. Fociof GGT positive hepatocytes, an increasing rate of DNA elutionand eventual primary hepatocellular carcinoma were seen in agroup of rats that was begun on PB as late as 85 days afterpartial hepatectomy and DEN treatment.     

Focal and non-focal hepatic expression of placental glutathione S-transferase in carcinogen-treated rats

Carcinogenesis develops in stages that have been operationallydefined as initiation, promotion and progression. Although morphologicalend points have been described for detection and quantitationof these stages, to date initiation has been assessed only inthe context of clonal growth in response to certain promotingagents. Initiated cells are morphologicatly indistinguishablefrom surrounding cells and early changes at the cellular levelduring initiation have not been clarified. One commonly usedend point for the detection of preneoplastic hepatic lesionsis their aberrant expression of the placental isozyme of glutathioneS-transferase (PGST). Because single hepato cytes expressingPGST have been detected in aged rats and in those administeredhepatocarcinogens, it has been suggested that such cells constitutea population of putat ively initiated hepatocytes. In orderto further elucidate the characteristics of single PGST-positivehepatocytes, we analyzed the number of these cells 2 and 18weeks after various doses (0–100 mg/kg) of diethylnitrosamine(DEN) and of dimethylbenz[a]anthracene (DMBA). When determined14 days after carcinogen administration, the number of singlehepatocytes expressing PGST was greater after DEN administration(ranging from 0.8±0.3 per cm² transection of liver at1 mg/kg to 33.0±4.7 at 100 mg/kg) than after DMBA administration(ranging from 0.25±0.14 at 10 mg/kg to 3.03±0.5at 100 mg/kg); none were detected in control rats of the sameage. Additional rats were maintained on a basal diet or a basaldiet plus phenobarbital for a further 4 month period. Whereasindividual PGST-positive hepatocytes were only sporadicallydetected in rats treated with DMBA and maintained on a basaldiet for 18 weeks, those rats placed on phenobarbital for 16weeks had an even higher number of such PGST-positive hepatocytes than at 2 weeks after DMBA administration. In contrast,the dose-response curve observed for DEN- treated rats 18 weeksafter carcinogen administration was similar to that observed2 weeks after carcinogen treatment for both phenobarbital- andnon-phenobarbital-treated rats. In addition, the number of singlePGST-positive hepatocytes detected at 2 weeks was directly parallelto the number of altered hepatic foci expressing PGST 18 weeksafter DEN administration. The dose-dependent induction of PGST-positivesingle hepatocytes after treatment with two hepatocarcinogens,the dose-dependent growth of altered hepatic foci (AHF) expressingPGST with phenobarbital administration and the parallel dose-responsecurve of single hepatocytes expressing PGST and later of AHFexpressing PGST argue strongly for a precursor role of singlePGST-positive cells in the development of AHF expressing PGST.     

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.     

The quantitative analysis and stability of histochemical markers of altered hepatic foci in rat liver following initiation by diethylnitrosamine administration and promotion with phenobarbital

The stability and response of histochemical phenotypes of altered hepatic foci (AHF) were studied both in the presence and following the withdrawal of 0.05% phenobarbital (PB) treatment in rats previously given a single dose of diethylnitrosamine (DEN) 20-24 h following partial hepatectomy (PH). AHF were scored by their expression of three biochemical markers: gamma-glutamyl transpeptidase (GGT), adenosine triphosphatase and glucose-6-phosphatase (G6P). AHF demonstrated significant heterogeneity with respect to the marker alterations. The use of three markers in the present study confirmed the findings of our earlier study, which showed the maximal response of GGT+ AHF to PB administration following PH/DEN initiation and the stability of GGT+/AHF induced by the PH/DEN/PB regimen after the withdrawal of PB. In the regimen employed, the GGT marker alone scored the great majority of the AHF detected by all three markers. The frequency distribution of histochemical phenotypes remained relatively constant in AHF during continuous PB administration and in AHF promoted by PB followed by a 6-month period of feeding a diet containing no PB. These findings suggest that individual AHF remain phenotypically stable throughout the PB promotion phase, i.e., do not progress from one phenotype to another. In every marker class, the mean volume of AHF increased during continuous PB administration. These data illustrate the enhancing effect of PB on the growth of the AHF. The size of AHF continued to increase following the withdrawal of PB in the 3-month PB treatment group, but not in the animals treated for 4 months. A mechanism that may account for the differences in these two treatment groups is discussed.     

Induction of major chromosome aberrations in Chinese hamster ovary cells by alpha-difluoromethylornithine

DL-alpha-Difluoromethylornithine (DFMO) is a specific irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17) and has antitumor effects. In this paper, we show that DFMO inhibits the growth of and causes severe chromosomal damage in Chinese hamster ovary cell strain A7 which grows without serum but has deficient arginase activity and therefore requires ornithine or polyamines for continuous replication. In ornithine-containing medium, the A7 cells had very few chromosome aberrations, but incubation of these cells with 0.5 mM DFMO for 7 days induced chromosome aberrations in 12 to 46% of the mitoses. Depletion of polyamines by omitting ornithine from the medium also caused chromosome aberrations. The chromosomal damage found after DFMO treatment alone and ornithine deprivation alone were of similar nature. In addition to chromosome breaks, there were chromosome fragmentation and structurally changed chromosomes including rings, chromatid exchange configurations, and chromosome elongations. A phenomenon resembling premature chromosome condensation was also seen. Double-minute chromosomes were visible in some mitoses, and the chromosome elongations sometimes gave an impression of homogeneously staining regions.     

Correlation of O4-ethyldeoxythymidine accumulation, hepatic initiation and hepatocellular carcinoma induction in rats continuously administered diethylnitrosamine

Recent experiments have demonstrated that O⁶-ethyldeoxy-guanosine(O⁶-EtdG) is efficiently repaired while O⁴-ethyl-deoxythymidine(O⁴-EtdT) accumulates in hepatocyte DNA of 8-week-old F-344rats during continuous diethylnitrosamine (DEN) administration.To determine if O⁴-EtdT accumulation correlates with hepaticinitiation, we have quantitated O⁴-EtdT concentrations, andthe incidence of-glutamyl transferase positive (GGT+) foci andhepatocellular carcinoma induced by increasing duration of exposureto DEN in the drinking water (40 p.p.m.). In 8-week-old F-344rats the number of GGT + foci increased non-linearly with durationof exposure and reached a maximum of 500 foci/cm³ after 10 weeks.Administration of DEN to 8-week-old F-344 rats for 6 weeks followedby a 15-week administration of 0.05% phenobarbital (PB) in thediet did not result in the induction of hepatocellular carcinoma.Exposure of 4-week-old F-344 rats to DEN for up to 10 weeksproduced an O₄-EtdT steady-state concentration (7–10 x10^–6 mol O⁴-EtdT/mol dT) similar to that previously observedafter administration of DEN to 8-week-old F-344 rats. However,the maximal concentration of O⁴-EtdT was detectable after ashorter period of DEN administration in the younger rats. Theincidence of GGT+ foci also increased more rapidly in 4-week-oldrats, but again plateaued at 500 foci/cm³ after 4, 6 or 8 weeksof DEN administration. A 100% incidence of hepatocellular carcinomaoccurred in 4-week-old rats administered DEN for 6, 8 or 10weeks, followed by promotion with 0.05% PB in the diet untilweek 22 of the study. Lower incidences of bepato-cellular carcinoma(89 and 6%) were observed following PB-promotion of rats administeredDEN for 4 and 2 weeks, respectively. The influence of age onDEN-induced hepatic initiation was examined further by quantitatingGGT + foci induced by 4 weeks of DEN administration in groupsof rats which were 4–14 weeks old at the start of thecarcinogen exposure. The results demonstrated that the youngerrats were 15-fold more susceptible than the older rats to theinitiating effects of DEN. This growth-dependent effect on hepaticinitiation in the presence of nearly equivalent amounts of pro-mutagenicDNA damage further implicates the necessity of replication forhepatic initiation.     

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.     

Effects of 10alpha,25-dihydroxyvitamin D3 on doxorubicin-induced chromosomal aberrations in rat bone marrow cells

The present study was carried out to evaluate the effects of 1alpha,25-dihydroxyvitamin D3 (VD) on chromosomal aberrations induced by doxorubicin (DXR). Wistar rats were divided into eight experimental groups of five animals each. Control group animals were treated with i.p. distilled water. The animals in three VD groups were given only VD for 4, 6 or 8 weeks. In the DXR groups the animals were given only DXR. In the combination groups VD doses were given for 4, 6 or 8 weeks for each group and DXR was injected 24 h before sacrificing the rats. DXR (50 mg/100 g b.w.) was injected intraperitoneally and VD by gavage 3 microg/kg/day twice weekly. Animals treated with both VD and DXR showed a low frequency of chromosomal aberrations and abnormal metaphases when compared with animals treated with DXR alone (p < 0.0001). The numbers of both chromosomal aberrations and abnormal metaphases were similar in weeks 6 and 8 (p > 0.05) and lower than those in week 4 for the VD groups (p < 0.0001). Under the present experimental conditions, the efficiency of VD in protecting cells against DXR-induced chromosome damage was found to be dose dependent. The protective effects of VD on chromosome aberrations induced by DXR are discussed in the light of literature data.     

Non-linearity of neoplastic conversion induced in rat liver by low exposures to diethylnitrosamine

Neoplastic conversion induced in rat liver by diethylnitrosamine(DEN) was quantified by measuring preneoplastic and neoplasticlesions over a 34 week period in the beginning of which thecarcinogen was given at three dose levels and two dose ratesfor the first 10 weeks, after which animals were maintainedfor 24 weeks with either no further exposure or were fed phenobarbital(PB) to promote neoplastic development of cells converted byDEN. DEN was injected s.c. inmale F344 rats at weekly or biweeklyintervals for total doses of 1, 2 or 4 mmol/kg body wt and thenthe rats were maintained on basal diet alone or diet containing0.05% PB. At the end of exposure, DEN had produced a dose-relateddecrease in centrilobular glutamine synthetase-expressing (GS⁺)hepatocytes which is indicative of mild cytotoxicity. All dosesinduced foci that were     

Beta-carotene inhibits rat liver chromosomal aberrations and DNA chain break after a single injection of diethylnitrosamine.

Beta-carotene (BC) has recently been found to possess potent anti-tumour activity in chemically induced rat liver carcinogenesis. In the present study, attempts have been made to understand the basic cytogenetic and molecular mechanism of the anti-tumour effect of BC by monitoring its effect on rat liver chromosomal aberrations (CAs) and DNA chain breaks during the early preneoplastic stage of diethylnitrosamine (DEN)-induced hepatocarcinogenesis in male rats. DNA chain breaks, however, can be detected with great sensitivity by exposing crude cell lysates to alkaline solutions and monitoring the rate of strand unwinding so that one strand break per chromosome can easily be detected. Supplementary BC, in basal diet (120 mg kg[-1]), was given to rats 15 days before carcinogenic threat with DEN. Under these experimental conditions, BC was found to afford a unique protection against DEN-induced CAs 96 h after DEN injection. Long-term treatment with BC also triggered a protective effect on induction of CAs 15, 30 or 45 days after DEN treatment, which was maximal on structural aberrations followed by numerical and physiological types. BC treatment for 15 days before DEN injection was found to offer a significant (P < 0.001) protection in the generation of single-strand breaks compared with DEN control. Thus, BC ranks as a potential chemopreventive agent for the future so far as chemical rat liver carcinogenesis is concerned.     

Transplacental effect of N-diethylnitrosamine on chromosomal aberrations in fetal tracheal cells of the Syrian hamster: comparison between epithelial and mesenchymal cells

Chromosomal aberrations were investigated in tracheal cellsof fetal Syrian hamsters after transplacental administrationof N-diethylnitrosamine (DEN). On the 15th day of pregnancy,Syrian hamsters were injected s.c. with a tumorigenic dose ofDEN (50,100 and 200 mg/kg body weight). Two hours later, thefetal tracheae were isolated, the epithelial tissue was separatedfrom the mesenchymal tissue by collagenasetreatment, and theneach cell population was transferred to cell culture after Dispasetreatment. At 24, 48 and 72 h after the cell cultivation, chromosomaldamage was examined. The results clearly showed that a highincidence of chromosomal aberrations, especially chromatid-typeexchanges, was seen in the epithelial cells of DEN-treated groups.However, significant induction of chromosomal aberrations wasnot observed in the mesenchymal cells from DEN-treated groups.     

Quantitative stereological studies of a 'selection' protocol of hepatocarcinogenesis following initiation in neonatal male and female rats

A modified initiation-selection procedure for neonatal male and female rat hepatocarcinogenesis were examined utilizing the methods of quantitative stereology. In this study, diethylnitrosamine (10 mg DEN/kg) was given a few days after birth. At weaning, the rats were fed 0.02% 2-acetylaminofluorene (AAF) for 2 weeks with a mitotic stimulus [70% partial hepatectomy (PH)] after 1 week on the diet. Quantitative stereological analyses in conjunction with the use of several enzyme markers were used to determine the number and volume of altered hepatic foci (AHF) detected at 1 week, 3 months and 7 months after the selection procedure. This format resulted in an equivalent number of AHF in male and female rats. The AHF were three times larger in males than in females 1 week after discontinuation of AAF administration. Three months after the selection procedure, the number of AHF had decreased by at least a third and their volume percentage was the same in male and female rats. After 7 months, the number and volume fraction of detectable AHF in females were comparable to those which had been observed at 1 week after selection. In the male, the number but not the volume fraction were similar at 7 months compared with 1 week after selection. Both initiation with DEN and selection with AAF/PH contribute independently to the total population of AHF in male and female rats. At least half of the AHF detected 7 months after the selection protocol were due to DEN administration alone. Rats receiving only the AAF/PH selection exhibited one third of the number of AHF observed with the complete protocol. Administration of a non-necrogenic dose of DEN to neonatal rats when coupled with the AAF/PH selection procedure resulted in a significant promotion of the growth of initiated hepatocytes at 1 week, 3 months or 7 months after the selection procedure. These studies demonstrated that (i) the number of AHF detected after a non-necrogenic dose of DEN during the first week of life with subsequent AAF/PH selection after weaning decreases within the first 3 months after the selection procedure, but can re-develop with a promotion stimulus; (ii) the AAF/PH selection procedure itself may initiate hepatocytes in the absence of DEN administration; (iii) the AAF/PH selection procedure is equally effective with respect to the number of AHF observed after phenobarbital promotion in weaning male and female rats initiated near birth.      

Synergistic effect of a choline-devoid diet and phenobarbital in promoting the emergence of foci of gamma-glutamyltranspeptidase-positive hepatocytes in the liver of carcinogen-treated rats

The effect of feeding phenobarbital (PHB) with a choline-devoid (CD) diet on the emergence of foci of gamma-glutamyltranspeptidase (GGT)-positive hepatocytes in the liver of carcinogen-treated rats was investigated. Male Sprague-Dawley rats were given a single dose of diethylnitrosamine (50 mg/kg) 18 hr after a partial hepatectomy and 10 days later were placed on a plain choline-supplemented (CS) diet, a plain CD diet, or the CS and CD diets containing 0.06% PHB. Groups of rats were killed after 5 and 7 weeks of feeding each of the four diets, the livers were taken, and the number and size of foci of GGT-positive hepatocytes were determined. In rats fed the CS + PHB diet, the number of foci per sq cm of liver section was greater than that in rats fed the plain CS diet but smaller than that in rats fed the plain CD diet. Addition of PHB to the CD diet resulted in twice as many foci as in the plain CD diet and foci larger than those resulting from the plain CD diet. THe hepatocytes in the foci of rats fed th CD and CD + PHB diets showed, uniformly, not only GGT positively but also a relative absence of fatty change. The results indicate that PHB and a CD diet, when combined, have a synergistic effect in promoting the evolution of liver cells, initiated by a chemical carcinogen, to foci of altered GGT-positive hepatocytes. This promoting regimen may become useful in studies concerned with the initiation and promotion stages of liver carcinogenesis.     

Association between growth stimulation by phenobarbital and expression of cytochromes P450 1A1, 1A2, 2B1/2 and 3A1 in hepatic hyperplastic nodules in male F344 rats.

This study was conducted to examine relationships between phenobarbital (PB) treatment, specific cytochrome P450 gene expression patterns and growth rates of hepatic hyperplastic nodules. Nodules were induced in 8 week old male F344 rats by a Solt-Farber resistance protocol. Six weeks after diethylnitrosamine (DEN) initiation, subgroups of rats were either kept on control chow diet or transferred to a chow diet containing 0.05% PB, then killed 2 weeks later. [3H]Thymidine was delivered continuously via osmotic minipump during the final 3 days of the experimental to label dividing cells. PB treatment resulted in a 89% increase in the number of persistent gamma-glutamyl transpeptidase (GTT) nodules per cm2 section, a 278% increase in the area of persistent GGT nodules per cm2 section, and a 116% increase in the average area per persistent nodule. PB increased the number of [3H]thymidine-labeled persistent GGT nodules but did not significantly change the labeling index (LI) distribution pattern or the average LI. A slight but uniform increase in CYP1A2 expression (relative to surrounding, non-nodular tissue) was observed in 50% (23/46) and 59% (60/102) of persistent nodules in control and PB-treated animals respectively. In contrast, for nodules undergoing remodeling, CYP1A2 expression was elevated in only 9% (2/22) and 0% (0/24) in control and PB groups respectively. In the PB group, CYP2B1/2 was underexpressed in 53% (54/102) of persistent GGT nodules and in 0% (0/24) of the remodeling nodules. Comparing LI among the persistent GGT nodules, those that displayed simultaneous increases in CYP1A2 and decreases in CYP2B1/2 had the highest LI, and were followed in level by those expressing either increases in CYP1A2 or decreases in CYP2B1/2. Nodules that expressed both CYP1A2 and 2B1/2 in a manner similar to the surrounding tissue had the lowest LI. Thus, these data suggest that expression of specific forms of cytochrome P450 may be an important factor in determining other phenotypic characteristics, e.g. rate of cell proliferation and GGT expression, within specific nodules.     

Role of diethylnitrosamine, 2-acetylaminofluorene and partial hepatectomy in the expression of glutathione-S-transferase-P and gamma-glutamyltranspeptidase in the early steps of rat liver carcinogenesis.

Three factors involved in the Solt and Farber model of rat liver carcinogenesis were studied alone and in various combinations: diethylnitrosamine (DEN) initiating dose, 2-acetylaminofluorene (2-AAF) feeding and partial hepatectomy. The administration of DEN alone (200 mg/kg) was able to switch on glutathione-S-transferase, placental type (GST-P) expression 3 weeks later at a low level (85 U/micrograms protein) which was stable for 10 weeks in the absence of histopathological lesions. During the same time, gamma-glutamyl transpeptidase (GGT) activity presented 2 waves of increase. The feeding of 0.03% 2-AAF for 2 weeks appeared as a determinant factor in the expression of GST-P protein as well as GGT induction (15- and 7-fold versus DEN alone, respectively). The addition of partial hepatectomy enhanced again GST-P expression (1.5-fold) and GGT induction (2-fold). However, GST-P foci increased in size, not in number while GGT foci increased both in size and in number. These data indicated that 2-AAF was a crucial component of the selection procedure since partial hepatectomy alone, with or without DEN initiation was inefficient in promoting GST-P expression. Therefore, 2-AAF would be able to promote the growth of GST-P-positive cells initiated by DEN, a mechanism likely responsible for its tumor-promoting effect.     

Sequential analysis of DNA damage and repair during the development of carcinogen-induced rat liver hyperplastic lesions

The level of DNA fragmentation, as evaluated by alkaline elution, and of unscheduled DNA synthesis (UDS), as measured by autoradiography, was determined in the parenchymal cells from the entire liver during the development of hyperplastic lesions induced in the rat by the following treatment: diethylnitrosamine (DEN) (200 mg/kg i.p.) on Day 0; CCl4 (2 ml/kg intragastrically) on Day 21; dietary administration of 0.02% 2-acetylaminofluorene during the third and the fourth wk; and of 0.05% phenobarbital from the sixth wk. Both DNA fragmentation and UDS were constantly detected, concomitantly with the presence of gamma-glutamyltransferase (gamma-GT)-positive hepatocytes, in the primary cultures derived from the liver of rats of this experimental group sacrificed at 4, 5, 6, and 7 wk after DEN injection, their amount being approximately the same at the fourth and at seventh wk. Moreover, evidence of DNA alterations was still present, albeit diminished, 22 wk after the beginning of treatment. In contrast, DNA fragmentation and UDS did not persist past the fifth wk, and gamma-GT-positive hepatocytes were very few or totally absent in hepatocyte primary cultures from control rats treated with DEN alone, 2-acetylaminofluorene alone, or 2-acetylaminofluorene:CCl4. CCl4 alone, and phenobarbital alone caused only a modest, albeit statistically significant, increase in DNA elution rate and UDS, respectively. In a comparison performed on hepatocyte primary cultures obtained from rats of the experimental group sacrificed at the fifth wk after the injection of DEN, the level of UDS was higher in gamma-GT-positive than in gamma-GT-negative hepatocytes. These results indicate that the regimen used to induce the selective proliferation of initiated hepatocytes actually produces extensive DNA lesions which can give rise to additional carcinogenic initiations.