Comparison of three rat liver foci bioassays--incidence of preneoplastic foci initiated by diethylnitrosamine

Three rat liver foci bioassays have been compared with respect to their sensitivity by the histochemical demonstration of preneoplastic foci, and by the biochemical determination of alterations in enzyme activities of serum indicating hepatotoxicity. We studied the initiation/promotion schedules according to Oesterle and Deml (A), and according to Pereira (B, Broad Spectrum Protocol), and the initiation/selection protocol according to Tatematsu et al. (C), with diethylnitrosamine (DEN), given as a single initiating dose of 10 and 30 mg/kg body wt respectively. With all schedules Sprague-Dawley rats, either females, 3 weeks old (A), or males, 6 weeks old (B, C) were used. For promotion polychlorinated biphenyls (A) or phenobarbital (B) were administered. Selection was performed with 2-acetylaminofluorene (C). The rats in schemes (B) and (C) underwent partial hepatectomy one day prior to initiation. The number and total area of foci deficient in adenosine-5'-triphosphatase (ATPase) and positive in gamma-glutamyltranspeptidase (GGTase) was evaluated. In the complete schedule with 30 mg of DEN in system (A) foci incidence exceeded that of the other systems by about 7-fold (ATPase) and 2-fold (GGTase) respectively. The lower dose of DEN and all control experiments resulted in a respective lower foci yield. With scheme (C), but not with schemes (A) and (B), e.g. serum fructose-1.6-bisphosphatase and alkaline phosphatase were increased, suggesting liver cell damage. Thus tested with DEN, scheme (A) is most sensitive and causes a low impairment of animals' welfare.     

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)     

Strain differences in susceptibility to 2-acetylaminofluorene and phenobarbital promotion of rat hepatocarcinogenesis in a medium-term assay system: quantitation of glutathione S-transferase P-positive foci development

Strain differences in susceptibility to promotion by the liver carcinogens 2-acetylaminofluorene (2-AAF) and phenobarbital (PB) were examined in the medium-term bioassay system initially developed in our laboratory using male F344 rats as the test animal and glutathione S-transferase placental form (GST-P)-positive foci as the lesion end-point. Numbers and areas per cm2 of induced GST-P-positive hepatocellular foci were compared in LEW, F344, NAR, SD, WBN, SHR, Wistar and ODS rats initiated with diethylnitrosamine (DEN) and subjected to partial hepatectomy during subsequent administration of 2-AAF or PB. LEW, SD, WBN, and F344 rats were most susceptible to hepatopromotion by both compounds, with a hundred fold increase in lesion area being observed for 2-AAF in the LEW case. NAR and SHR strains demonstrated an intermediate response, while Wistar and, in particular, the related ODS rats demonstrated very low susceptibilities. The obvious strain differences could be expressed in terms of comparative indices of promoting effects of 2-AAF and PB as well as DEN itself regarding each of the 8 strains tested. The use of F344 rats for the bioassay model was validated by the relatively high sensitivity to both DEN and 2-AAF initiation as well as second-stage promotion stimulus exhibited.     

Strain Differences in Susceptibility to 2-Acetylaminofluorene and Phenobarbital Promotion of Rat Hepatocarcinogenesis in a Medium-term Assay System: Quantitation of Glutathione S-Transferase P-positive Foci Development

Strain differences in susceptibility to promotion by the liver carcinogens 2-acetylaminofluorene (2-AAF) and phenobarbital (PB) were examined in the medium-term bioassay system initially developed in our laboratory using male F344 rats as the test animal and glutathione S -transferase placental form (GST-P)-positive foci as the lesion end-point. Numbers and areas per cm² of induced GST-P-positive hepatocellular foci were compared in LEW, F344, NAR, SD, WBN, SHR, Wistar and ODS rats initiated with diethylnitrosamine (DEN) and subjected to partial hepatectomy during subsequent administration of 2-AAF or PB. LEW, SD, WBN, and F344 rats were most susceptible to hepatopromotion by both compounds, with a hundred fold increase in lesion area being observed for 2-AAF in the LEW case. NAR and SHR strains demonstrated an intermediate response, while Wistar and, in particular, the related ODS rats demonstrated very low susceptibilities. The obvious strain differences could be expressed in terms of comparative indices of promoting effects of 2-AAF and PB as well as DEN itself regarding each of the 8 strains tested. The use of F344 rats for the bioassay model was validated by the relatively high sensitivity to both DEN and 2-AAF initiation as well as second-stage promotion stimulus exhibited.     

Cell Proliferation and regulation of negative growth factors in mouse liver foci

Foci of altered hepatocytes are preneoplastic lesions capableof progressing to hepatocellular carcinomas. To Characterizethe growth of preneoplastic hepatic lesions, size of hepaticfoci was analyzed with regard to growth factor regulation andhepatocyte proliferation in focal and non-focal hepatocytes.Twelve-day-old female B6C3F1 mice were initiated with a singledose of the potent mutagen N-nitrosodiethylamine (DEN) (5 mg/kgbody weight). Beginning at 6 weeks of age, mice were exposedfor 16 weeks to 2038 p.p.m. unleaded gasoline (UG) vapor or1 p.p.m. ethinyl estradiol (EE) in the diet. Analysis of hepaticfoci demonstrated that UG significantly increased, but EE significantlydecreased the size of DEN-initiated foci. Hepatic labeling index(LI), as measured by the incorporation of 5-bromo-2'-deoxyuridine,was similar in non-focal hepatocytes at 16 weeks in all groups(0.4–0.8%) and greatly increased in hepatic foci. HepatocyteLI was significantly increased in DEN/UG foci (29%, n = 41)and significantly decreased in DEN/EE foci (6% n=23) relativeto DEN/control focal hepatocytes(18% n=25). The mean LI of focicorrelated with the focal size differences observed in the treatmentgroups. Immunohistochemical analysis with antibodies directedto the negative growth regulator transforming growth factorbetal (TGF-ß1) demonstrated a consistent decreaseof TGF-ß1 in DEN/Ct and DEN/UG hepatic foci relativeto non-lesion hepatocytes. Similar results were seen with mannose6-phosphate/insulin-like growth factor-11 receptor (M6P/IGF-IIR), which facilitates activation of latent TGF-ß1.In contrast, only 50% of DEN/EE foci had decreased levels ofTGF-ß1 and M6P/IGF-II R relative to non-focal hepatocytes.These data suggest that proliferative responses observed inhepatic foci may be correlated with foci size. In contrast,chemically induced proliferative responses in non-focal hepatocytesafter subchronic exposure cannot necessarily be used to predictproliferative effects in preneoplastic cell populations. Furthermore,these studies suggest that hepatic foci may occur by M6P/IGF-IIR enhancing activation of latent TGF-ß1 in non-focalhepatocytes but not in the focal hepatocytes, thereby affordingfocal hepatocytes a selective growth advantage.     

Diethylnitrosamine exposure-responses for DNA damage, centrilobular cytotoxicity, cell proliferation and carcinogenesis in rat liver exhibit some non-linearities

The exposure-responses for several effects of limited exposuresto diethylnitrosamine (DEN) in the livers of male Fischer 344rats were measured and phenobarbital promotion was used to enhanceexpression of initiation of carcino-genesis. Five doses rangingfrom a cumulative total of 0.5 to 4 mmol DEN per kg body weightwere given as weekly i.p. injections for 10 weeks. This wasfollowed by 4 weeks recovery, after which the groups were maintainedon either a basal diet or 0.05% phenobarbital (PB) to promoteliver tumor development. All doses of DEN produced ethylationin liver DNA, which increased with dose. Indicative of toxicity,the centrilobular zone of glutamine synthetase-positive hepatocyteswas reduced in relationship to exposure up to a cumulative exposureof 3 mmol/kg. The two lower exposures to DEN produced no increasein cell proliferation, whereas higher exposures resulted inmarked increases, {small tilde}4-fold between 1.0 and 2.0 mmol/kgcumulative. At the end of the recovery period (14 weeks), hepatocellularaltered foci (HAF), which expressed the placental form of glutathioneS-transferase, were induced by all exposures, with an increaseof {small tilde}4-foId between the exposures of 1.0 and 2.0mmol/kg being the greatest. In rats maintained on basal dietor PB for 24 weeks after exposure, HAF increased further andwith exposures of 2.0 mmol/kg and above, all rats developedhepatocellular carcinomas. With 1.0 mmol/ kg, no liver tumoroccurred in 12 rats without promotion, whereas in those givenPB, two adenomas and two carcinomas were present in 12 rats.At the lowest exposure of 0.5 mmol/ kg, no tumor occurred inrats on basal diet, although HAF increased {small tilde}7-fold.With PB promotion, only one adenoma developed in 12 rats andHAF increased another 2-fold. Thus, the findings document non-linearityfor some of the effects of DEN and a near no-effect level forinitiation of promotable liver neoplasms at the lowest exposurein spite of a substantial induction of HAF.     

Progression of hepatic neoplasia in medaka (Oryzias latipes) exposed to diethylnitrosamine.

Progression of hepatic neoplasia was assessed in medaka (Oryzias latipes) following aqueous exposure to diethylnitrosamine (DEN). Larvae (2 weeks old) were exposed to 350 or 500 p.p.m. DEN for 48 h, while adults (3-6 months old) were exposed to 50 p.p.m. DEN for 5 weeks. Fish were maintained as long as possible to determine malignant potential of resultant neoplasms. A total of 423 medaka with 106 hepatic neoplasms were examined. There were marked differences in tumor prevalence between exposure groups including: (i) higher prevalence of hepatocellular carcinomas in medaka exposed as adults (100% of hepatocellular tumors in adult-exposed medaka were malignant, while only 51.5% of larval hepatocellular tumors were malignant); (ii) higher prevalence of biliary tumors in medaka exposed as larvae (46.4% of all tumors in larval-exposed medaka were biliary versus 8.1% in adult-exposed fish); (iii) higher prevalence of mixed hepato-biliary carcinomas in adult-exposed medaka (24.3%) compared with those exposed as larvae (3%). In addition, a unique hepatocellular lesion termed 'nodular proliferation' was only observed in adult-exposed medaka. The lesion was characterized by small size (50-300 microm), complete loss of normal tubular architecture and variable megalocytosis. Nodular proliferation was distinct from preneoplastic foci of cellular alteration and may represent microcarcinomas. There was a step-wise increase in mean diameter with age (days post-exposure) from nodular proliferation (174 microm, 17 days) to hepatocellular carcinoma (1856 microm, 62 days) and mixed carcinomas (3209 microm, 93 days) in adult-exposed medaka. Metastasis was observed with 19 neoplasms and tumors with the highest metastatic potential were hepatocellular and mixed carcinomas. The most common form of metastasis was trans-coelomic, followed by direct invasion and distant metastasis, presumably via the vascular route. Differences in tumor prevalence between exposure groups were believed to be the result of length of DEN exposure rather than age of fish at the time of exposure. In larval medaka with brief (48 h) DEN exposure, neoplasms are thought to be the result of dedifferentiation of hepatic cells, with slow progression of foci of cellular alteration to benign and then malignant tumors. In contrast, with adult medaka and prolonged (5 week) DEN exposure, neoplasms are believed to result from initiation of committed stem cells and formation of microcarcinomas ('nodular proliferation'), before progressing to larger hepatocellular and then mixed carcinomas.     

Chemopreventive effects of coumaperine from pepper on the initiation stage of chemical hepatocarcinogenesis in the rat.

This study was designed to investigate the chemopreventive action of three natural products, coumaperine, aurapten and an extract from rosemary, against the initiation stage of rat hepato-carcinogenesis. Coumaperine has been isolated from white pepper as a naturally occurring antioxidative agent, but its potential modifying effects on carcinogenesis remain unclear. In experiment 1, a modification of the model developed by Tsuda et al. was applied, with assessment of numbers and areas of induced glutathione S-transferase placental form (GST-P)-positive hepatocellular foci in male F344 rats. Coumaperine, aurapten and the extract from rosemary were administered i.g. at 100 mg / kg / day once daily for 5 days with initiation by diethylnitrosamine (DEN) on day 4 (20 mg / kg, i.p.). Numbers and areas of GST-P-positive foci in each group given test chemicals tended to be decreased as compared to the vehicle control group values, significance being achieved for number with coumaperine. Experiment 2 was planned to investigate the mechanism of the inhibitory effects of coumaperine. Livers at 8 h after initiation by DEN were examined with coumaperine administered at 100 mg / kg / day once daily for 3 days. Proliferating cell nuclear antigen (PCNA)-positive cells tended to be decreased as compared to the vehicle control, but no effects on apoptosis or cytochrome P-450 (CYP) 2E1 expression were apparent. Our results suggest that coumaperine provides protection against initiation of hepatocarcinogenesis, and that this is related to inhibition of cell proliferation.     

Chemopreventive Effects of Coumaperine from Pepper on the Initiation Stage of Chemical Hepatocarcinogenesis in the Rat

This study was designed to investigate the chemopreventive action of three natural products, coumaperine, aurapten and an extract from rosemary, against the initiation stage of rat hepatocarcinogenesis. Coumaperine has been isolated from white pepper as a naturally occurring antioxidative agent, but its potential modifying effects on carcinogenesis remain unclear. In experiment 1, a modification of the model developed by Tsuda et al. was applied, with assessment of numbers and areas of induced glutathione S-transferase placental form (GST-P)-positive hepatocellular foci in male F344 rats. Coumaperine, aurapten and the extract from rosemary were administered i.g. at 100 mg/kg/day once daily for 5 days with initiation by diethylnitrosamine (DEN) on day 4 (20 mg/kg, i.p.). Numbers and areas of GST-P-positive foci in each group given test chemicals tended to be decreased as compared to the vehicle control group values, significance being achieved for number with coumaperine. Experiment 2 was planned to investigate the mechanism of the inhibitory effects of coumaperine. Livers at 8 h after initiation by DEN were examined with coumaperine administered at 100 mg/kg/day once daily for 3 days. Proliferating cell nuclear antigen (PCNA)- positive cells tended to be decreased as compared to the vehicle control, but no effects on apoptosis or cytochrome P-450 (CYP) 2E1 expression were apparent. Our results suggest that coumaperine provides protection against initiation of hepatocarcinogenesis, and that this is related to inhibition of cell proliferation.     

Genetic control of susceptibility to diethylnitrosamine carcinogenesis in inbred ACP (grc+) and R16 (grc) rats

The R16 strain, which carries the major histocompatibility complex-linked growth and reproduction complex (grc), and its normal counterpart, the ACP strain, were initiated at 8 wk of age with a single i.p. dose of diethylnitrosamine (DEN), and 2 wk later they were fed either a choline-deficient (CD) or a choline-supplemented (CS) diet. The rats were sacrificed 2, 4, 6, 10, and 12 mo later; complete autopsies were performed, and all of the tissues were examined histologically. Sections of the liver were also examined histochemically for gamma-glutamyl transpeptidase activity. Shortly after the administration of DEN, the R16 strain showed a significant increase in the number and size of gamma-glutamyl transpeptidase-positive foci and more severe histological changes (disruption of the lobular architecture, bile duct and oval cell proliferation, cellular atypia, and accumulation of fat) compared with the ACP strain. These changes occurred in animals fed either CD or CS diet, but they were much more extensive and severe in the animals on the CD diet. They did not occur in rats of either strain fed the diets alone. The first hepatocellular carcinoma appeared in the R16 rats on the CD diet at 4 mo after administration of the DEN and on the CS diet, at 10 mo. The only hepatocellular carcinoma that occurred in the ACP rats did so at 12 mo in one animal on the CD diet. Combining the data at 10 and 12 mo for the rats on the CD diet, 50% (20 of 40) of the R16 rats had hepatocellular carcinomas, whereas only 3% (one of 30) of ACP rats did. The R16 strain (22%, 9 of 40), but not the ACP strain (0 of 30), also had a variety of other malignancies: squamous cell carcinomas (8%); renal cell carcinomas (8%); lymphomas (5%); and pheochromocytoma (3%). A similar pattern of malignancies also occurred in the R16 rats on the CS diet, and there were no malignancies in the ACP rats. These observations indicate that the grc confers unusual susceptibility to the induction of cancer by the chemical carcinogen DEN and that this genetic susceptibility to cancer of the R16 strain extends beyond the primary target organ of the carcinogen used.     

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.     

Compared promoting potential of D-galactosamine, carbon tetrachloride and partial hepatectomy in rapid induction of preneoplastic liver lesions in the rat

Effectiveness of two different chemically induced stimuli for hepatocellular proliferation was compared with regard to that of commonly used partial hepatectomy (PH), for the purpose of developing short-term protocol for the assay of promoting agents of hepatocarcinogenesis. Enhancing effect of D-galactosamine (DGA) and carbon tetrachloride (CCl4) given during the promotion procedure by 2-acetylaminofluorene (2-AAF) was compared along with PH in rats initiated by diethylnitrosamine (DEN), using preneoplastic glutathione S-transferase positive (GST-P+) hepatocyte foci as an end-point marker lesion. The number of GST-P+ foci per cm2 was largest in the group given CCl4 followed by DGA, no treatment (2-AAF alone) and PH. In contrast, the area (mm2) per cm2 and mean diameter of the focus were largest in the PH group then DGA followed by CCl4 and no treatment. The results indicate that the number of GST-P+ foci were not clearly affected by 3 different treatments whereas area and size of foci which represented the result of promoting effect were clearly influenced by those treatments, indicating they caused differential proliferation of initiated cells. In this respect, even though PH is the most potent procedure, DGA is also efficient and preferred to CCl4 for the non-surgical enhancing method.     

Iron promotes DEN initiated GST-P foci in rat liver

Diethylnitrosamine (DEN) was administered to rats as a single dose, which is known not to give rise to liver tumours without subsequent promotion. Iron dextran (Fe/Dex) was then administered parenterally to the animals, to induce iron overload. At 3 and 6 months after the final Fe/Dex treatments, livers were examined quantitatively for the numbers of the placental form of glutathione-S-transferase (GST-P) expressing foci, the area occupied by these foci and their size distribution. The results demonstrate that iron not only increased the number of foci after DEN initiation in the rat liver, but that the area occupied by these lesions increased significantly between 3 and 6 months after initiation. There is no evidence that iron increased the number of GST- P expressing foci present in rats not exposed to DEN. This indicates that iron did not act as an initiator in this rodent model of liver cancer. The increase in the area of the liver occupied by the foci in iron and DEN treated rats was due to an increase in the size of the foci, as well as to an increase in the number of foci. This is the first demonstration that iron can act as a promoter of DEN initiated hepatocytes. It also demonstrates that fibrogenesis is not an absolute requirement for the promotion, by iron, of liver foci in the rat, and that this could also be the case for iron overload in man. Iron may also act as a promoter of already initiated hepatocytes in the development of human liver cancer, as it does in the rat.      

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

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

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

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

Morphology and morphometric investigation of hepatocellular preneoplastic lesions and neoplasms in connexin32-deficient mice

Gap junctions are composed of protein subunits, called connexins, and provide a pathway for the exchange of ions and small molecules between contacting cells. This transfer of molecules is thought to be an important pathway for direct cell communication, and is involved in tissue homeostasis, growth control and embryonic development. Impairment of gap junctional intercellular communication (GJIC) via different mechanisms may therefore contribute to dysregulated cellular proliferation and subsequent tumor development. We investigated the effect of Connexin32-deficiency on liver histology and the formation of preneoplastic foci and hepatocellular neoplasms in transgenic knockout mice, as Connexin32 (Cx32) is the major gap junction protein in the liver. Loss of Cx32 does not alter the morphology of extrafocal liver tissue. However, after administration of a single dose of diethylnitrosamine (DEN), given 2 weeks after birth, the number and volume fraction of preneoplastic foci showed a 3.3-fold to 12.8-fold increase in the Cx32-deficient mice as compared with the corresponding wildtype groups, regardless of sex and age of the animals. Number and volume fraction of hepatocellular adenomas and carcinomas also increased significantly in these animals. The experimental groups did not differ in the morphology of the different types of preneoplastic foci and neoplasms. On the other hand, Cx32-deficiency without DEN treatment did not lead to an increase in the spontaneous development of any type of preneoplastic hepatic foci or hepatocellular neoplasms in up to 18-month-old Cx32-deficient mice as compared with wildtype controls. In conclusion, our results indicate that impairment of GJIC in mouse liver due to deletion of the Cx32 coding DNA clearly promotes the carcinogenic effect of DEN administration and results in a higher susceptibility to hepatocellular neoplasms, but does not appear to initiate hepatic tumor development.     

Chemoprevention of bicyclol against hepatic preneoplastic lesions

Oral administration of 100 and 200 mg/kg body weight/day of 4,4-dimethoxy-5,6,5', 6'-dimethylene-dioxy-2-hydroxymethyl-2'-carbonyl biphenyl, Bicyclol, inhibited rat hepatic preneoplastic lesions induced by diethylnitrosamine (DEN). Bicyclol reduced densities of number and area of gamma-glutamyltransferase positive foci, indexes for neoplastic hyperplasia; and also suppressed protein expressions for glutathione S transferase P isoform (GST-P) and alpha-fetal protein and mRNA for N-ras, c-myc and PKCalpha genes. With increases of total microsomal P450 and specific CYP2B1 activities in normal rat liver, Bicyclol enhanced particularly the denitrosation of DEN, a low toxic pathway of metabolism. There is a minor effect of Bicyclol on the deethylation of DEN to produce highly mutagenic metabolites. These results suggest that Bicyclol exists the ability of protecting hepatocytes from the mutagenicity of DEN. Such hypothesis was validated by the observation that Bicyclol inhibited DEN-induced unscheduled DNA synthesis, a DNA damage index, in primary cultured rat hepatocytes. More, in virto Bicyclol inhibited two-stages transformation of mice fibroblastic Balb/c 3T3 cells induced by 3-methylcholanthrene and tetradecanoyl-phorbol 13-acetate (TPA), and blocked the anchorage-independent growth of transformed cells in soft agar. Bicyclol also suppressed TPA-stimulated Balb/c 3T3 cell proliferation in both cell number and 3H-thymidine incorporation. Dot blot indicated that Bicyclol inhibited mRNA expressions of H-ras, c-myc and PKCalpha genes by TPA-stimulation. These data demonstrate that Bicyclol prevents carcinogens-induced animal neoplasm and cell malignant transformation via mechanisms at stages of initiation and promotion. It substantiates those evidences that Bicyclol would be used as potential a chemopreventive agent for hepatocarcinogenesis along with its major therapy against chronic anti-hepatitis.     

Sex-differentiated deoxycholic acid promotion of rat liver carcinogenesis is under pituitary control

The influence of continuous growth hormone (GH) infusion and of implantation of ectopic pituitary grafts (PG) to male rats on the sex differences (male greater than female) in efficiency of promotion with dietary deoxycholic acid (DCA; 0.5% w/w) was studied in the livers of diethylnitrosamine (DEN)-initiated Wistar rats. For comparison, liver regeneration after partial hepatectomy (PH) was examined in differently treated animals. The endpoints examined included the number and size of enzyme-altered foci, hepatic c-myc expression and liver weight gain. The area per focus was 2- to 3-fold larger in initiated, DCA-treated males than in the corresponding PG-bearing males, GH-treated males and in females. The expression of the c-myc gene was increased approximately 2-fold in initiated and promoted males, while the increase in females was very small. In both groups of hormone-treated males the expression was at the same level as in females, significantly lower than in the corresponding DEN/DCA-treated males. Liver weight gain in response to PH in initiated as well as uninitiated rats of both sexes was significantly stimulated by DCA. No sex differences or effects of PG on regeneration could be discerned. In conclusion, a sex difference, regulated by a pituitary influence, in focal growth and in c-myc expression was observed during DCA promotion of DEN-initiated rats. This might indicate a correlation between the GH-regulated, proliferation-associated c-myc gene and focal growth during sex differentiated promotion in rat liver. Furthermore, the lack of sex differences in liver weight gain in response to PH during DCA treatment suggests that selective mitoinhibition is not involved, and might in this model indicate hormone-dependent selective stimulation of focal growth as a mechanism for tumor promotion.