Threshold dose dependence in phenobarbital promotion of rat hepatocarcinogenesis initiated by diethylnitrosamine.

The dose-response of phenobarbital (PB) promotion of hepatocarcinogenesis in rats was investigated. Male F344 rats were given 1, 4, 16, 75, 300 or 1200 p.p.m. PB solutions given ad libitum as their drinking water for 39 weeks following initiation with a single i.p. injection of diethylnitrosamine (DEN) (100 mg/kg). At week 40, the incidence of hepatic tumors was increased clearly in the DEN + PB groups given 300 p.p.m. PB or above, as compared to that in the group given DEN only. Linear dose-response curves for numbers and sizes of enzyme-altered hepatic foci (gamma-glutamyl-transpeptidase or placental glutathione S-transferase positive foci) were obtained in the dose range 16-1200 p.p.m. PB. The minimum promoting dose level of PB for enzyme-altered foci, estimated from dose-response curves by the Logit model, was calculated to be 15-23 p.p.m. Thus while dose dependence was demonstrated over a large range, a threshold was evident at low doses.     

Effect of fasting/refeeding on the incidence of chemically induced hepatocellular carcinoma in the rat.

Caloric restriction has been associated with a delay in the development of both spontaneous and induced neoplasia. In contrast, cycles of fasting/refeeding were shown by us and others to enhance the incidence of early lesions during chemical carcinogenesis in rat liver. The present, long-term study was undertaken to establish whether such a diffential effect would also extend to the later phases of cancer development, until the overt appearance of neoplasia. Male Fischer 344 rats were initiated with a single dose of diethylnitrosamine (DENA, 200 mg/kg i.p.) and starting 1 week later they were either exposed to three cycles of fasting (3 days) followed by refeeding (11 days) or were fed continuously. Seven weeks after DENA administration the rats were exposed to the resistant hepatocyte model of the liver tumor promotion protocol. All animals were killed 1 year after initiation. Incidence of hepatocellular carcinoma was 2-fold higher in the fasted/refed group compared with the controls (72 versus 36%). In addition, cancers were also larger and of higher histological grade in the former group, with one animal showing metastases to the lungs, while no metastases developed in control animals. Fasting caused a decrease in total liver DNA (from 25.2 +/- 1.1 to 16.5 +/- 1.1 mg after 3 days) which was associated with a decrease in hepatocyte labeling index and mitotic activity and high levels of single cell death (apoptosis). In contrast, a sharp increase in hepatocyte proliferation was observed on day 2 of refeeding and this was more pronounced in glutathione S-transferase 7-7 positive foci compared with surrounding liver (10.2 +/- 2.3 versus 4.6 +/- 0.8%). Such a proliferative wave was associated with a sharp decline in the incidence of cell death. It is concluded that fasting/refeeding performed early after initiation accelerates the development of chemically induced hepatocellular carcinoma in the rat.     

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.     

Enhancement of diethylnitrosamine hepatocarcinogenesis in rats by exposure to polychlorinated biphenyls or phenobarbital.

Diethylnitrosamine (DENA) hepatocarcinogenesis was enhanced by sequential exposure with polychlorinated biphenyls (PCBs) and with phenobarbital. This was shown by the earlier appearance of tumors and a significant increase in the number of tumors. However, there was no evidence suggesting an acceleration of tumor growth.     

Mouse strain susceptibility to diethylnitrosamine induced hepatocarcinogenesis is cell autonomous whereas sex-susceptibility Is due to the micro-environment: analysis with C3H <--> BALB / c sexually chimeric mice.

In man, liver cancer is on the increase, especially in males. Sex differences also exist in rodent models. To elucidate the mechanisms, chimeric mice were produced by amalgamation of early embryos from high and low hepatocarcinogen-susceptible strains, C3H and BALB / c. Tumor formation was initiated with 10 mg / kg of diethylnitrosamine at the ages of 7 and 14 days and mice were sacrificed at 30 and 45 weeks. The chimeras were classified into XY <--> XY, XY <--> XX, XX <--> XY, and XX <--> XX in terms of sex chromosomes by means of polymerase chain reaction-simple sequence length polymorphism analysis (SSLP) using Y chromosome-specific Sry primers in combination with the D3Mit21 marker. Liver lesions were analyzed histopathologically, by immunostaining using a C3H strain-specific antibody and by DNA in situ hybridization with the Y chromosome-specific digoxigenin-labeled Y353 / B probe. Sex and strain genotyping by SSLP analysis matched histological observations, confirming the reliability of our system. The strain differences in liver tumor numbers of each strain type in XY <--> XY and XX <--> XX subtypes of C3H <--> BALB / c chimeras were retained well (P < 0. 0001 and P < 0.001, respectively), indicating a minimum influence of the C3H or BALB / c surrounding milieu on development of individual lesions. On the other hand, significant promotion of XX cell tumors was evident in phenotypically male sexually chimeric XY <--> XX and XX <--> XY chimeras for both C3H (P < 0.02) and BALB / c (P < 0.01) lesions compared to the XX <--> XX case. The results suggest the presence of hormonal or micro-environmental factors specific for males, which are not caused cell-autonomously. Basic strain differences, however, are determined by intrinsic genetic factors rather than the strain-dependent micro-environment.     

Anti-carcinogenic action of phenobarbital given simultaneously with diethylnitrosamine in the rat

The present work has been planned in order to elucidate the effect of phenobarbital (PB: 15 mg per rat of ingested dose) on carcinogenesis when it is administered simultaneously with diethylnitrosamine (DEN: 10 mg/kg/day). Wistar rats (180 g) were treated by DEN alone or by DEN + PB during 2, 4 and 6 weeks according to our schedule for hepatocarcinogenesis. After the end of the treatment, the number and the size of induced PAS positive preneoplastic foci was significantly reduced when PB was given simultaneously with DEN for 4 and 6 weeks. The mitotic inhibition and the production of micronuclei normally observed after partial hepatectomy in DEN treated rats were also significantly decreased in DEN + PB treated rats. When the treatment last only 2 weeks, the presence of PB did not change significantly the last parameters. In DEN + PB treated rats, the survival was prolonged and the tumor incidence decreased as compared with the results obtained by DEN alone. It is concluded that PB, which promotes carcinogenesis when administered after the DEN treatment, reduces the carcinogen effect when given simultaneously with DEN. This ‘anti-carcinogen’ effect acts on the initiation as well as on the promotion of the precancerous lesions. Biochemical investigations are in progress to obtain more information about this ‘paradoxical’ PB effect.     

Persistent effect of a low dose of preadministered diethylnitrosamine on the induction of enzyme-altered foci in rat liver

The effect of a low dose of preadministered diethylnitrosamine(DEN) on the induction of enzyme-altered foci in the liversof male full-grown Fischer 344 rats was studied. As a pretreatment,DEN at a dose of 10 mg/kg body wt was injected i.p. At varioustimes after DEN pretreatment a complete initiation, consistingof administration of the same dose of DEN by the same routein rats subjected to partial hepatectomy (PH), was performed,followed by application of selection pressure. Enzyme-alteredfoci stained with -glutamyltrans-peptidase (-GTP) and glutathioneS-transferase placental form (GST-P) were then assayed. Decreasesin the numbers and areas of foci in the rats which receivedsaline + PH 14 or 28 days after DEN pretreatment were observedin comparison with rats which received saline + PH immediatelyafter DEN. On the other hand, the numbers and areas of fociwere not decreased in rats which received the complete initiation,consisting of DEN + PH, at various times after DEN pretreatmentwhen compared with rats which received these at the same timeas the DEN pretreatment. This persistent effect of DEN pretreatmenton the complete initiation lasted up to 182 days after the timeof DEN pretreatment. In this experiment, GST-P was found tobe a more sensitive marker for the detection of putative preneoplasticliver-cell foci than -GTP.     

Effect of dietary fat on the initiation of hepatocarcinogenesis by diethylnitrosamine or 2-acetylaminofluorene in rats.

The purpose of this study was to determine if increasing dietary fat, either as saturated fat or polyunsaturated fat, would alter initiation of hepatocarcinogenesis by diethylnitrosamine (DEN) or 2-acetylaminofluorene (AAF). Rats were fed one of three purified diets: a low-fat (LF) diet (containing 5% of calories as safflower oil), a high saturated fat (HSF) diet (containing 48% of calories as palm oil) and a high polyunsaturated fat (HPUF) diet (containing 48% of calories as safflower oil). Four weeks later, all rats were subjected to partial hepatectomy (PH). Rats were then divided into four groups and received no carcinogen, DEN (10 mg/kg, p.o., 24 h after PH) or AAF (25 or 100 mg/kg, p.o., 12 h after PH). Five days later, all rats were fed an unrefined diet, and 9 weeks later, all rats were fed phenobarbital in the diet for 26 weeks as a tumor promoter. In rats initiated with DEN, the number of gamma-glutamyl transpeptidase-positive and ATPase-negative foci was higher in the rats fed the HPUF diet, but not the HSF diet, as compared to rats fed the LF diet. The incidence of neoplastic nodules, the mean focal volume and the volume fraction, however, were not significantly altered by dietary fat in DEN-injected rats. The dietary fat content of the diet did not affect the induction of altered hepatic foci or neoplastic nodules in rats initiated with AAF or receiving no initiation. This study shows that initiation of hepatocarcinogenesis can be influenced by dietary fat, but that the effect may be carcinogen-specific.     

High sensitivity of the LEC rat liver to the carcinogenic effect of diethylnitrosamine

The initiation sensitivity of the liver of the LEC (Long-Evans with a cinnamon-like coat color) rat, a new mutant strain with a high incidence of spontaneous liver tumors, was studied by treatment with low doses of diethylnitrosamine (DEN) coupled with modified Solt and Farber's selection. LEC and control LEA (Long-Evans with an agouti coat color) rats received i.p. injections of 10 mg/kg of DEN, then selected by feeding with a diet containing 0.02% 2-acetylaminofluorene (AAF) for 7 days combined with partial hepatectomy (PH). The numbers of placental glutathione S-transferase (GST-P)-positive foci in the livers of LEC rats were 10 times higher than those in LEA rats. These results suggested a high sensitivity of the LEC rat liver to the carcinogenic effect of DEN. The association between initiation sensitivity and spontaneous liver-tumor development and the possible usefulness of the LEC rat for in vivo short-term tests of hepatocarcinogens are discussed.     

A novel pharmacodynamically based approach to dose optimization of carboplatin when used in combination with etoposide

Thrombocytopenia, the dose-limiting toxicity of carboplatin, is manageable and predictable with the dosing equation: Dose (mg/m2) = (0.091) (creatinine clearance)/(body surface area) (desired percentage change in platelet count) + 86. We used this equation to dose patients receiving carboplatin (day 1) and etoposide (80 mg/m2 days 1 to 3). An initial cohort of 14 patients with non-small-cell lung cancer (NSCLC) were treated with 75% of the calculated dose of carboplatin (29 evaluable courses) as a precaution against added myelosuppression from combination chemotherapy. The observed reduction in platelets was essentially equal to the reduction in platelets predicted if patients had received carboplatin alone. Subsequently, a second cohort of 20 evaluable patients with NSCLC received the full calculated dose of carboplatin and etoposide (51 evaluable courses). There was a linear relationship between observed (o) and predicted (p) reductions in platelets. With full-dose carboplatin in combination with etoposide, there was a significantly greater carboplatin dosage administered, greater reduction in platelets, and lower platelet nadir. Among 34 evaluable patients (80 courses) there was one complete response (CR) and four partial responses (PRs) for an overall response rate of 15% (90% confidence +/- 9%). The median duration of survival for responders was 336+ days and for nonresponders was 204+ days. Therapy was well tolerated. This study, in addition, supports our concept of individualized dosing of carboplatin and the underlying pharmacokinetic/pharmacodynamic relationships and represents an interesting pharmacodynamic and quantitative approach to studying potential drug-drug interactions and defining appropriate dosages for combination chemotherapy.     

Serine 727 phosphorylation of STAT3: An early change in mouse hepatocarcinogenesis induced by neonatal treatment with diethylnitrosamine

STAT3 activation is involved in development and progression of hepatocellular carcinoma (HCC). We investigated STAT3 activation during hepatocarcinogenesis induced by neonatal diethylnitrosamine (DEN) treatment in mice. Nuclear accumulation and phosphorylation of STAT3 were detected in altered hepatocyte foci in the early stages as well as adenomas and HCCs in the late stages. Although total STAT3 levels were the same between the hepatic lesions and normal livers, S727‐phosphorylated STAT3 was enhanced in adenomas and HCCs, whereas Y705‐phosphorylated STAT3 was detected mainly in HCCs. In mouse HCC cell lines, although both S727 and Y705 remained un‐ or hypophosphorylated under serum‐free conditions, fetal bovine serum (FBS) induced strong S727/weak Y705 phosphorylation, STAT3 nuclear accumulation and cell proliferation, whereas IL‐6 treatment without FBS caused Y705 phosphorylation without S727 phosphorylation, STAT3 nuclear accumulation or cell proliferation. When HCCs were simultaneously treated with FBS/IL‐6, selective suppression of S727 phosphorylation by an MEK inhibitor prevented STAT3 nuclear accumulation and cell proliferation. Furthermore, an S727 phosphorylation‐deficient STAT3 mutant (S727A) had a diminished capacity to accumulate in the nucleus when compared with wild‐type (WT) or the phosphorylation‐mimic mutant (S727D) following treatment with FBS/IL‐6. After treatment with FBS/IL‐6, the cells expressing the S727A mutant proliferated more slowly than those expressing WT or S727D mutant. In contrast, suppression of Y705 phosphorylation by a JAK inhibitor in the FBS/IL‐6 treated cells did not affect STAT3 nuclear accumulation or cell proliferation. Taken together, these data demonstrate that STAT3 activation, mainly through S727 phosphorylation, contributes to the DEN‐induced hepatocarcinogenesis at the earliest stages. © 2013 Wiley Periodicals, Inc.     

Effect of simultaneous administration of clofibrate with diethylnitrosamine on hepatic tumorigenesis in the rat

Effect of clofibrate administered simultaneously with diethylnitrosamine (DEN) on hepatocarcinogenesis in F344 male rats was investigated. DEN was given in drinking water at a concentration of 40 ppm for 5 weeks. Rats were fed the diet containing clofibrate at concentrations of 0.1% and 0.25% during DEN-treatment. All rats were killed 25 weeks after the beginning of DEN- treatment, and hepatic tumors over 1 mm in diameter were counted.Hepatic tumors in the rats given clofibrate at both concentrations were twice as many as those in rats given DEN alone. The significant increase in number of hepatic tumors was observed mainly in the tumors under 10 mm in diameter. Thus, the enhancing effect of clofibrate on hepatocarcinogenesis was evident even when fed simultaneously with the carcinogen, in contrast with phenobarbital (PB).     

Phenobarbital as a promoter in the initiation/selection process of experimental rat hepatocarcinogenesis

Supplementary introduction of a phenobarbital (PB) promotionstep after the Solt and Farber procedure dramatically increasesthe number of phenotypically-altered hepatocytes. These hepatocytesoccupy {small tilde}40% of the liver volume after one week ofPB treatment. These areas constitute a relatively uniform cellularpopulation with altered histological pheno-type and with distincthistochemicai markers used by other authors for the detectionof premalignancy. This procedure leads to the appearance ofnumerous hepatocellular carcinomas at approximately the 36 weeksstage. It was suggested that the early hepatocellular alterationsafter the initiation/selection procedure followed by PB mightcorrespond to the hyperplasia of phenotypically-altered epidermalcells at the conversion step of mouse skin tumor promotion.     

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     

Altered cobalamin distribution in rat hepatomas and in the livers of rats treated with diethylnitrosamine.

The distribution of cobalamin cofactors was investigated in the livers and tumors of rats bearing transplanted Morris 7777 or 7800 hepatomas, in the livers of rats treated with the hepatocarcinogen diethylnitrosamine, and in normal rats. There was a significant increase in the proportion of methylcobalamin both in livers and tumors from rat bearing the hepatomas 7777 and 7800 compared to the proportion of methylcobalamin in the livers of normal rats. The total cobalamin content of the hepatomas was significantly lower than that of host or control livers. Similarly, the total cobalamin content of the livers from the tumor-bearing rats was less than that in control animals. The administration to rats of an acute dose of diethylnitrosamine led to an 84% increase in the hepatic concentration of methylcobalamin. Chronic administration of diethylnitrosamine slightly increased the hepatic methylcobalamin concentration, but this was not statistically significant. Liver weight was reduced, and the hepatic content of total cobalamin fell to 55% of that in control animals.