Promotion mechanism of phenobarbital and partial hepatectomy in DENA hepatocarcinogenesis cell kinetics effect

Diethylnitrosamine (DENA, 10 mg kg-1 per day) was fed to rats for 2, 4 and 6 weeks. One week after the cessation of DENA, animals were submitted either to partial hepatectomy or to phenobarbital administration. Partial hepatectomy did not promote neoplastic transformation, except after a 6-week DENA treatment. A minimum of phenobarbital was required to reach a significant promoting effect in DENA carcinogenesis. A too-limited treatment was ineffectual but could be compensated for by prolonged DENA administration. The phenobarbital treatment became unnecessary when neoplastic nodules were present. Phenobarbital continuously given after the carcinogen administration promoted neoplastic transformation even after a subcarcinogenic DENA treatment (2 weeks). It accelerated the pathological evolution and increased the tumour incidence. In these conditions, phenobarbital increased the proliferation advantage of preneoplastic cells over normal cells. In the different experimental modalities, the promoting effect was associated with the induction of chronic cell proliferation, the inhibition of the rapid response to the 2/3 partial hepatectomy and the mitotic circadian rhythm normally present during liver regeneration. It is concluded that the promotion mechanism could consist in disturbing the mitotic control in order to maintain, for a long time, a chronic low level of cell proliferation permitting the selective growth of preneoplastic cells and their subsequent transformation.     

Activation of a ribosomal S6 protein kinase in rapidly emerging diethylnitrosamine-induced gamma-glutamyltranspeptidase-positive hyperplastic liver lesions of the rat

The gamma-glutamyl transpeptidase (GGT)-positive hyperplastic liver lesions which developed in the Fisher 344 rat 7 and 60 days following a single carcinogenic dose of diethylnitrosamine (DENA, 200 mg/kg body weight), short-term dietary exposure to 0.02% 2-acetylaminofluorene (AAF) to suppress the growth of normal hepatocytes, and partial hepatectomy to actuate rapid growth of DENA altered hepatocytes not suppressed by AFF, showed an increased activity of a kinase which specifically phosphorylates the ribosomal S6 protein in vitro. Sham-operated animals showed, on the contrary, no GGT-positive cells and low S6 kinase activity, under the same conditions. After partial hepatectomy, activation of S6 kinase and elevated levels of phosphorylated S6 protein in vitro were detected in the early phases of "normal" hepatocyte proliferation, during liver regeneration, in DENA-treated, GGT-negative preparations, when the "selection" agent AAF was omitted from the diet. The observed activation of S6 kinase in GGT-positive hepatocytes and/or liver nodules could represent an early manifestation of the enhanced proliferation of altered hepatocytes during tumor induction and/or promotion under these conditions.     

Immunohistochemical detection of c-Ha-ras oncogene p21 product in pre-neoplastic and neoplastic lesions during hepatocarcinogenesis in rats

An immunohistochemical study of c-Ha-ras expression was performed on preneoplastic and neoplastic stages of diethylnitrosamine (DENA)-induced hepatocarcinogenesis in rats, using an antibody raised against a peptide sequence of the Ha-ras p21 product. Moderate to high immunostaining intensity was observed in the following hepatocytic lesions: (1) hepatocellular carcinomas (14/14) and associated neoplastic nodules (8/8) and foci of phenotypic alterations (35/40) (after 13-20 months of treatment); (2) neoplastic nodules (6/6) and associated foci (42/50) (after 5-9 months); (3) foci (10/10) (after 2 months); and (4) small, slowly growing foci (26/40) found 9 months after treatment with DENA without prior partial hepatectomy, resulting in low number of nodules and no tumor even after 15 months. No c-Ha-ras p21 was detected immunohistochemically in normal nor in regenerating rat liver. Our results indicate that increased Ha-ras expression is an early and stable event in liver lesions associated with hepatocarcinogenesis. They also imply that increased Ha-ras expression is insufficient (if at all implicated) for inducing fully malignant hepatocyte transformation. It might be indicative of cell populations at an increased transformation risk.     

Expression of a hepatocyte membrane antigen during hepatocarcinogenesis and in the developing liver of the rat

Changes in the expression of a cell membrane antigen during hepatocarcinogenesis and in the developing liver were analyzed by HAM.4, a monoclonal antibody (MAb) against a membrane glycoprotein of normal rat hepatocyte. Of the precancerous lesions observed during hepatocarcinogenesis induced by diethylnitrosamine, 2-acetylaminofluorene and partial hepatectomy, early neoplastic foci were uniformly stained by HAM.4. In contrast, some cells in the neoplastic nodules at the late stage did not express HAM.4 antigen on the cell surface. Of the cancer tissues, well-differentiated hepatocellular carcinomas were stained by HAM.4 whereas poorly differentiated carcinomas did not bind HAM.4 In developing rat liver, HAM.4 antigen was first expressed on fetal hepatocytes at the 18th day of gestation. It gradually increased until 4 weeks after birth when the intensity of the stain was almost the same as in adult rat liver. These results suggest that the expression of a membrane antigen defined by HAM.4 is closely associated with the differentiation of bile canalicular face and that HAM.4 might be useful in characterizing differentiation of cells during malignant transformation of hepatocytes.     

Effects of strain,sex, route of administration and partial hepatectomy on the induction by chemical carcinogens of gamma-glutamyltranspeptidase foci in rat liver

The incidence of gamma-glutamyltranspeptidase (GGT)-positive foci induced by 0.3 mmol/kg diethylnitrosamine (DENA) followed by promotion with 500 ppm sodium phenobarbital in drinking water and was the same in Fischer 344, Sprague-Dawley and Wistar-Lewis rats. There was no difference in the level of GGT-foci initiated by DENA, 7,12-dimethylbenz[a]anthracene (DMBA), or 1,2-dimethylhydrazine (DMH) followed by promotion with phenobarbital with respect to sex or route of administration including gavage and intraperitoneal injection. Maximal stimulation by partial hepatectomy of DENA initiation of GGT-foci occurred when the DENA was administered 18 h after the operation. Our results indicate that the optimal protocol for the rat liver foci assay consists of using partial hepatectomized rats of 1 of the 3 strains and of either sex. The test substance should be administered by either gavage or intraperitoneal injection so that maximal DNA binding coincides with the maximal rate of DNA replication resulting from partial hepatectomy.     

Enzymic retrodifferentiation during hepatocarcinogenesis and liver regeneration in rats in vivo.

The work presented here has concerned the study of early, as well as late, enzymic changes occurring during diethylnitrosamine-induced hepatocarcinogenesis and liver regeneration after partial hepatectomy in comparison with normal liver differentiation. Rank correlation analysis of the enzyme data suggested a step-wise retrodifferentiation i.e. that the liver during carcinogenesis first assumed a neonatal enzymic pattern before attaining a foetal enzymic state. Similar enzymic changes were observed in regenerating liver after partial hepatectomy; again there was a step-wise retrodifferentiation of enzymic pattern and at 3 days post hepatectomy the liver had an enzymic pattern similar to both foetal and neoplastic liver. However, in contrast to liver undergoing neoplastic change, the regenerating liver retained the capacity to undergo redifferentiation towards a normal adult biochemical pattern.     

Promotion by polychlorinated biphenyls of enzyme-altered foci in rat liver

Aroclor 1254 is a complex mixture of polychlorinated biphenyls (PCB) that upon prolonged administration has been reported to produce hepatic tumors in mice and rats. The ability of Aroclor 1254 to promote enzyme-altered foci was determined in an initiation/promotion bioassay in rat liver. Initiation was accomplished in rats that received a 2/3 partial hepatectomy followed in 24 h by diethylnitrosamine (DENA). Aroclor 1254 was administered to each rat 7, 28 and 49 days after the DENA and some of the rats were killed 21 days after each dose of Aroclor. The liver of rats that received Aroclor 1254 on either day 7 or on day 7 and 28 contained an increased incidence of gamma-glutamyltranspeptidase (GGTase)-positive foci compared to partial hepatectomized and DENA treated rats given tricaprylin (the solvent for Aroclor 1254). Therefore, Aroclor 1254 was demonstrated to enhance the appearance of enzyme-altered foci after only a single oral dose.     

Black currant phytoconstituents exert chemoprevention of diethylnitrosamine‐initiated hepatocarcinogenesis by suppression of the inflammatory response

Black currant fruits containing high amounts of anthocyanins are known to possess potent antioxidant and anti‐inflammatory properties. We have previously reported that anthocyanin‐rich black currant skin extract (BCSE) inhibits diethylnitrosamine (DENA)‐initiated hepatocarcinogenesis in rats although the underlying mechanisms are not fully understood. Our present study investigates the anti‐inflammatory mechanisms of BCSE during DENA rat liver carcinogenesis. Dietary BCSE (100 or 500 mg/kg) treatment for 22 wk afforded a striking inhibition of DENA‐induced hepatic gamma‐glutamyl transpeptidase‐positive preneoplastic foci in a dose‐responsive fashion. There was a significant increase in hepatic expression of heat shock proteins (HSP70 and HSP90), cyclooxygenase‐2, and nuclear factor‐κB (NF‐κB) in DENA‐exposed rat livers. Dietary BCSE dose‐dependently abrogated all these elevated inflammatory markers. The possible cardiotoxicity of BCSE was assessed by monitoring cardiac functions using transthoracic echocardiography. BCSE‐mediated anti‐inflammatory effects during rat liver carcinogenesis have been achieved without any cardiotoxicity. Our results provide convincing evidence, for the very first time, that suppression of the inflammatory cascade through modulation of the NF‐κB signaling pathway could be implicated, at least in part, in the chemopreventive effects of black currant bioactive phytoconstituents against experimental hepatocarcinogenesis. These results coupled with an excellent safety profile of BCSE support the development of black currant phytochemicals for the chemoprevention of inflammation‐driven hepatocellular cancer. © 2011 Wiley Periodicals, Inc.     

Metabolism and activation of N-nitrosodimethylamine by hamster and rat microsomes: comparative study with weanling and adult animals

It has been reported that hamster liver preparations are more effective for the metabolic activation of N-nitrosodimethylamine (NDMA) to a mutagen than rat liver preparations. The enzymatic basis for this phenomenon, however, has not been clearly elucidated. The present study was undertaken to examine the enzymology of NDMA metabolism by different hepatic subcellular fractions prepared from hamsters and rats of two different ages, and to investigate the correlation between the metabolism and the activation of NDMA to a mutagen for Chinese hamster V79 cells. The content of cytochrome P-450 was approximately 1.5-fold higher in hamster microsomes than in rat microsomes from both ages (1.19-1.38 versus 0.73-0.83 nmol P-450/mg protein). Weanling hamster microsomes exhibited multiple apparent Km values for NDMA metabolism as did weanling rat microsomes. The apparent Km I value of NDMA demethylase (NDMAd) in hamster microsomes was about one-half that in rat microsomes (36 versus 83 microM) with corresponding Vmax values of 2.09 and 2.57 nmol/min/nmol P-450. The Km I values for denitrosation did not differ from the corresponding values for NDMAd with Vmax values of 0.17 and 0.22 nmol/min/nmol P-450 for hamster and rat microsomes, respectively. These apparent Km values were affected neither by sonication nor by the presence of cytosolic proteins in S9 fractions. Adult rat liver microsomes showed less than one-half the NDMAd activity in weanling rat liver microsomes, whereas such age difference was not observed in hamster liver microsomes. This result was confirmed by Western blotting showing the levels of P-450ac (an acetone-inducible form of P-450) of these microsomes at comparable levels to their NDMAd activities. NDMAd was highly correlated to the metabolic activation of NDMA to a mutagen for V79 cells in an activation system mediated by microsomes prepared from hamsters and rats of different ages. The results from this study clearly demonstrate the enzymatic basis for the more effective metabolism of NDMA in adult hamsters than in adult rats.     

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     

Changes in aldehyde dehydrogenase activity during diethylnitrosamine-initiated rat hepatocarcinogenesis

Diethylnitrosamine following partial hepatectomy followed byphenobarbital promotion was used to study changes in aldehydedehydrogenase (ALDH) activity during rat hepatocarcinogenesis.Over a period of 350 days, animals were killed at intervalsand the ALDH phenotype of normal liver and any lesions was characterizedby histochemkal analysis, total activity assays and gel electrophoresisusing propionaldehyde and NAD⁺ to detect normal liver ALDH activities,and benzaldehyde and NADP⁺ for tumor-associated ALDH. In contrastto previously tested protocols, no significant changes in ALDHactivity were demonstrable by histochemistry or total activityassays in preneoplastic livers. However, nine of 16 (56%) ofthe hepatoceflular carcinomas examined expressed the tumor-associatedALDH phenotype. The present results are integrated with previousobservations as a hypothesis explaining the roles of initiationand promotion in expression of the tumor-associated aldehydedehydrogenase phenotype.     

Changes in carboxylesterase isoenzymes of rat liver microsomes during hepatocarcinogenesis

Among the three major carboxylesterase isoenzymes, RH1, RL1 and RL2, present in microsomes from normal rat liver, RL2 shows hydrolyzing activity towards 12-O-tetradecanoylphorbol-13-acetate and 1-oleoy1-2-acetyl-rac-glycerol, both activators of protein kinase C. Since protein kinase C has been suggested to be involved in carcinogenesis and cell proliferation, alterations in hepatic microsomal carboxylesterase isoenzymes including RL2 were studied during hepatocarcinogenesis induced by the Solt-Farber model. Alteration of RL2 was determined by measuring acetanilide-hydrolyzing activity, by quantifying the protein amount using the single radial immunodiffusion method, and by activity staining following electrophoresis of liver microsomes. The isoenzyme composition of hepatic microsomal carboxylesterase was changed after partial hepatectomy, and marked decreases in RL2 activity and protein content were observed at 4 weeks, at the time of preneoplastic foci induction. Partial hepatectomy alone also resulted in decreased RL2 activity. These findings suggest that RL2 may be involved in regulation of protein kinase C activity by metabolizing its activators at an early stage of hepatocarcinogenesis in rats.     

Changes in Carboxylesterase Isoenzymes of Rat Liver Microsomes during Hepatocarcinogenesis

Among the three major Carboxylesterase isoenzymes, RH1, RL1 and RL2, present in microsomes from normal rat liver, RL2 shows hydrolyzing activity towards 12-O-tetradecanoylphorboI-13-acetate and l-oleoyl-2-acetyl- rac -glycerol, both activators of protein kinase C. Since protein kinase C has been suggested to be involved in carcinogencsis and cell proliferation, alterations in hepatic micro-somal Carboxylesterase isoenzymes including RL2 were studied during hepatocarcinogenesis induced by the Solt-Farber model. Alteration of RL2 was determined by measuring acetanilide-hydrolyzing activity, by quantifying the protein amount using the single radial immunodiffusion method, and by activity staining following electrophoresis of liver microsomes. The isoenzyme composition of hepatic microsomal Carboxylesterase was changed after partial hepatectomy, and marked decreases in RL2 activity and protein content were observed at 4 weeks, at the time of preneoplastic foci induction. Partial hepatectomy alone also resulted in decreased RL2 activity. These findings suggest that RL2 may be involved in regulation of protein kinase C activity by metabolizing its activators at an early stage of hepatocarcinogenesis in rats.     

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.     

Studies on the uptake of 2-deoxy-d-glucose in normal and malignant rat epithelial liver cells in culture.

The rate of uptake of 2-deoxyglucose in normal rat hepatic cells and chemically induced hepatoma cells in culture was studied. The hepatoma cells possessed a higher permeability to the sugar at all stages of culture. However, a decrease in the uptake of 2-deoxyglucose at confluency was observed in cells which exhibited density-dependent inhibition of growth, whether normal or malignant. The normal cells in mitosis showed an increased permeability to the sugar, whereas no such change was observed in the hepatoma cells. The kinetic studies of 2-deoxyglucose transport in normal and transformed rat liver showed a positive correlation between the increase in Vmax and the transformed state of the cells, whether they were transformed in vitro or in vivo. No changes in the apparent Km were found, indicating that there are no qualitative changes in the transport sites. The results suggest that an increase in the number of sites involved in glucose transport is a characteristic of chemically transformed rat liver epithelial cells.     

Immunocytochemical detection of the onco-developmental protein oncomodulin in pre-neoplastic and neoplastic hepatocellular lesions during hepatocarcinogenesis in rats

Oncomodulin is a calcium-binding protein, detectable in extra-embryonic human and rat placental cells and in a wide variety of tumors, but not in any normal embryonic or adult rodent or human tissues. It is also absent from proliferatively active fetal or regenerating adult rat liver. The presence of this oncodevelopmental marker was investigated in pre-neoplastic and neoplastic liver lesions during hepatocarcinogenesis induced in rats by DENA treatment, using an antibody raised against purified oncomodulin. Positive immunostaining was observed in foci of altered hepatocytes, in neoplastic nodules and in HC, but not in the histologically normal surrounding liver parenchyma. The proportion of oncomodulin-positive foci gradually rose from 20-25% at 2-3 months after DENA treatment, to about 88% at 6 months and later. The proportion of positive neoplastic nodules increased from 50% at 5 months to about 73% (range 36-100) at 9 months and later; 88% of the HC found 10 to 20 months after DENA treatment were also positive. That early neoplastic nodules are oncomodulin-positive in a proportion (50%) similar to that of foci after the same duration of treatment is consistent with a lineage relationship between them but makes it unlikely that oncomodulin expression conditions the focus-nodule transition. The role, if any, of oncomodulin in malignant progression remains to be elucidated. It seems out of the question that it is a simple correlate of proliferative activity.     

Perturbations of endogenous levels of orotic acid and carcinogenesis: effect of an arginine-deficient diet and carbamyl aspartate on hepatocarcinogenesis in the rat and the mouse

Feeding excess orotic acid (OA) In the diet promotes the carcinogenicprocess in different organs Including the liver. A number ofmetabolic and genetic disorders are associated with Increasedsynthesis of endogenous OA and some of these disorders appearto pose an Increased risk of liver cancer development. Thisstudy therefore examines whether excess OA of endogenous originalso exerts a promoting effect on hepatocarcinogenesis in themouse and the rat. Increased endogenous synthesis of OA wasachieved by (i) feeding a diet deficient in arginine (AD) and(ii) feedIng excess dietary carbamylaspartate (CA), a precursorfor the synthesis of OA. A single dose of diethylnitrosamine(DENA) was given i.p. to male Fischer 344 rats (200 mg/kg) orto male DBA/2 mice (90 mg/kg). One week later they were placedon either AD diet or the same diet supplemented with 1.3% arginine(AS) for a total of weeks. Two-thirds partial hepatectomy (PH)was performed at the end of the second week. All animals werethen transferred to a control semisynthetic basal diet for atotal of 20 weeks before they were killed. The results indicatedthat AD diet increased the incidence of hepatic nodules in bothrats (percentage area occupied by nodules was 4.7 ± 0.4in the AD group compared to a control value of 0.7 ±0.5) and mice (4/10 mice had nodules >5 mm diameter in theAD group while none in the AS group had such large nodules).In another experiment male Fischer 344 rats similarly initiatedwIth DENA were exposed to either basal diet or basal diet containing2% CA for 4 weeks coupled with PH performed at the end of thesecond week. This regimen was followed by 20 weeks of feedingbasal diet to both groups. Rats given CA developed larger hepatlcfoci and nodules (0.84 ± 0.56 mm³) compared to the controlgroup, which was fed basal diet throughout the experiment (0.07± 0.03 mm³) Further, both AD diet and dietary CA, likedietary OA, induced an increase In hepatic uridine nucleotides.Taken together, these results suggest that Increased levelsof endogenously synthesized OA, like exo genously supplied excessOA, can induce an Imbalance in hepatic nucleotide pools andcan exert a promoting effect on hepatocarcinogenesis.     

Ability of partial hepatectomy to induce gamma-glutamyltranspeptidase in regenerated and transplanted hepatocytes of Fischer 344 and Wistar-Furth rats

The purpose of this study was to investigate the effect of a two-thirds partial hepatectomy on the expression of gamma-glutamyltranspeptidase (GGT) in parenchymal hepatocytes. Two to 3-month-old Fischer 344 (F344), Wistar-Furth (WF), and WF X F344 F1 rats of both sexes were used in this investigation. Partial hepatectomy in the F344 female rat significantly increased the percent of liver area positive for GGT from 1.3 to 20.4%, a 15-fold increase. In contrast, GGT was not induced by partial hepatectomy in either the female WF or WF X F344 F1 hybrid rat or in the male animals of these three strains of rats. This phenomenon, which was only observed in the F344 female rat, was blocked by oophorectomy 2 weeks prior to partial hepatectomy, indicating that the induction of GGT is in part dependent upon female sex hormones. By transplanting both F344 and WF hepatocytes into the axillary fat pads of isogeneic and WF X F344 F1 rats, we showed that the observed strain difference did not result from a variation in the animal hormonal environment but rather was due to the hepatocyte from the F344 rat being intrinsically more susceptible to GGT induction than that from the WF rat. Further, the extent of the enzyme induction was significantly greater when the F344 liver cells were transplanted into female recipient animals. Since hepatectomy is often used in hepatocarcinogenesis studies, it is important to note that in the F344 female animal, the surgical procedure itself increases hepatic GGT levels in both transplanted hepatocytes and the liver in situ. Thus, the F344 female rat should be used with caution in investigations where the carcinogenic potential of chemicals is based upon the formation of GGT-positive hyperplastic nodules in the liver.     

Induction of gamma-glutamyltransferase-positive and nonspecific esterase-positive foci in rat liver by partial hepatectomy following single injection of N-nitrosodiethylamine

The effect of partial hepatectomy (PH) on alteration of liver foci induced by N-nitrosodiethylamine (DENA) was studied in inbred F344 male rats. As early as 2 weeks after PH was performed 6 hours after an injection of 100 mg DENA/kg, gamma-glutamyltransferase-positive hepatocellular foci were induced, whereas DENA alone induced no foci until 12 weeks after PH. The focus counts of the group with PH performed 6 hours after an injection of 100 mg DENA/kg were consistently greater than those of a group with PH performed at 24 hours following DENA injection. At 3 and 6 weeks after PH was done at 12 weeks following treatment with 100 or 200 mg DENA/kg, the focus count was significantly increased compared with that in nonhepatectomized groups. The results indicate that increased liver cell proliferation resulting from PH enhances the conversion of persisting DNA damage to a permanent alteration in DNA. The effect at 12 weeks after exposure supports the concept that DNA damage in hepatocytes is highly persistent.     

Presence of a no-observed effect level for enhancing effects of development of the alpha-isomer of benzene hexachloride (alpha-BHC) on diethylnitrosamine-initiated hepatic foci in rats

The dose dependence of the promoting effects of the alpha-isomer of benzene hexachloride (alpha-BHC) on hepatocarcinogenesis was investigated in a medium-term rat liver bioassay (Ito test). A total of 195 F344 male rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at the start of the experiment and subjected to two-thirds partial hepatectomy at week 3. Two weeks after the administration of DEN, alpha-BHC were fed to rats at doses of 0, 0.01, 0.1, 0.5, 1, 2, 4, 7.5, 15, 30, 60, 125 and 500 ppm in diet for 6 weeks. All surviving animals were killed at week 8, and their livers were examined immunohistochemically for detection of glutathione S-transferase placental form (GST-P)-positive foci, surrogate preneoplastic lesions. Quantitative values for numbers and areas were dose-dependently increased in rats given alpha-BHC at 0.5-500 ppm. However, those for groups treated with 0.01 and 0.1 ppm were decreased, albeit not significantly in comparison to the controls. Cytochrome P450 3A2 (CYP3A2) protein levels and activities showed a good correlation to the number and area of GST-P-positive foci. These results support evidence of hormesis and indicate a no-observed effect level for alpha-BHC promoting potentials may exist regarding rat liver carcinogenesis, which correlates with expression of CYP3A2 in the liver.