The high hepatocarcinogen susceptibility of LEC rats is genetically independent of abnormal copper accumulation in the liver

We previously reported that LEC rats, which show a spontaneousoccurrence of liver injury and hepatocellular carcinoma (HCC),are highly susceptible to chemical carcinogens such as diethylnitrosamine(DEN). Since abnormal copper accumulation in the liver of LECrats was found to be a cause of liver injury, it is necessaryto elucidate whether the carcinogen susceptibility of LEC ratsis related to the accumulation of copper in the liver. In thisstudy we have examined the relationship between the susceptibilityof F1 [LEC x LEA or LEC x Fischer 344 (F344)] and F1 backcrossrats to DEN and hepatic copper concentration, as copper accumulationhas been demonstrated to be inherited as an autosomal recessivetrait. The groups of F1 and F1 backcross rats were given a singleintraperitoneal injection of DEN (20 mg/kg body wt) and subjectedto a modified Solt—Farber protocol for assaying glutathioneS-transferase placental form (GST-P)-positive foci. The hepaticcopper concentration was examined by atomic absorption. Althoughno F1 rats showed a high copper concentration in the liver,the numbers of foci were as high as those in LEC rats whichaccumulate copper. Backcross rats separated into high and lowcopper concentration groups at an almost 1: 1 ratio, but therewas no significant difference in the mean numbers of foci betweenthese two groups. The results clearly indicate that the highsusceptibility of LEC rats to DEN is genetically independentof copper accumulation in the liver. A possible dominant inheritanceof this high carcinogen susceptibility was suggested. Biochemicalmeasurement of cytochromes P450 and b5 in the liver of F1 ratsindicated that alterations in drug metabolizing enzymes maybe partially responsible for the high carcinogen susceptibilityof LEC rats.     

Resistance of Copenhagen rats to chemical induction of glutathione S- transferase 7-7-positive liver foci

Copenhagen (Cop) rats are completely resistant to the chemical induction of mammary adenocarcinomas, but their susceptibility to hepatocarcinogenesis is virtually unknown. Rat liver is a well- characterized and easily manipulated tissue in which to study carcinogenesis. Therefore, if Cop rats are resistant to hepatocarcinogenesis, studies into resistance mechanisms may be feasible. Male Cop and F344 rats, 7-8 weeks old, were initiated using either N-nitrosodiethylamine (DEN) (200 mg/kg, i.p.) or a two-thirds partial hepatectomy (PH) followed by N-methyl-N-nitrosourea (MNU) (60 mg/kg, i.p.). The rats were then promoted using a modified resistant hepatocyte (RH) protocol (a combination of four doses of 2- acetylaminofluorene (2-AAF) and a single dose of CCl4 that provides a selective mitotic stimulus for initiated cells). Six weeks after initiation the rats were killed and liver sections were stained for glutathione S-transferase 7-7 (GST 7-7), a marker for putative preneoplastic hepatocytes. Cop rats were found to be highly resistant, having a approximately 9- and approximately 27-fold smaller percentage of liver area occupied by GST 7-7-positive foci than susceptible F344 rats following initiation by DEN and MNU respectively. Furthermore, gross liver nodules did not form in any of the Cop rats, whereas all F344 rat livers contained nodules. Hepatic necrosis caused by DEN during initiation, and CCl4 during promotion is necessary to stimulate compensatory hepatocyte division. We demonstrated that these agents do indeed increase serum transaminase levels and produce histologic evidence of necrosis in Cop rats. In order for liver foci to grow rapidly in the RH protocol, the surrounding normal hepatocytes must be mito-inhibited by 2-AAF. We found that the degree of mito-inhibition of normal hepatocytes by 2-AAF is the same in Cop and F344 rats. These results show that the Cop rat is highly resistant to the chemical induction of putative preneoplastic liver foci and nodules.      

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.     

Low Susceptibility of Long-Evans Cinnamon Rats to N-Butyl-N-(4-hydroxybutyl)-nitrosamine-induced Urinary Bladder Carcinogenesis and Inhibitory Effect of Urinary Copper

We studied the susceptibilities to N -butyl- N -(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis of male Long-Evans Cinnamon (LEC), F344 and Long-Evans Agouti (LEA) rats. Male rats ( n =21) were given 0.1% BBN in their drinking water from week 6, 8 and 10 for one week, and killed in week 56. The incidences of transitional cell tumors (papillomas plus carcinomas) in BBN-treated LEC and F344 rats were 12% and 76%, respectively ( P < 0.001, experiment 1), and those in LEC and LEA rats were 11% and 95%, respectively ( P < 0.001, experiment 2). When male LEC and F344 rats were given 0.1% BBN in their drinking water for 7 days, the intake of BBN and the urinary concentration of its active metabolite, N -butyl- N -(3-carboxypropyl)nitrosamine (BCPN), were higher in the LEC rats ( P < 0.01). The urinary pHs of untreated LEC and F344 rats were similar between week 6 and 30. The urinary copper concentration was lower in LEC rats before jaundice than in F344 rats, but its concentrations in 28- and 50-week-old LEC rats were 1.7 and 2.3 times those in F344 rats. In a two-stage carcinogenesis study using F344 rats, i.p. injections of cupric nitrilotriacetate increased urinary copper excretion, and inhibited BBN induced bladder carcinogenesis. In a two-stage carcinogenesis study using LEC rats, oral administration of D-penicillamine decreased urinary copper excretion, and increased BBN-induced bladder cancer, although the difference was not significant. These data show that LEC rats are resistant to bladder carcinogenesis and suggest that urinary copper has a significant role in their resistance.     

Role of Copper Accumulation in Spontaneous Renal Carcinogenesis in Long-Evans Cinnamon Rats

Spontaneous renal cell tumors in totals of 223 male and female Long-Evans Cinnamon (LEC) rats of 51–120 weeks old, 157 male F344 rats of 51–120 weeks old, and 14 male Long-Evans Agouti (LEA) rats of 51–70 weeks old were examined histologically. The incidences of renal cell tumors increased with age in male and female LEC rats, but no tumors developed in F344 or LEA rats. Dilated atypical tubules of the kidneys were observed at high incidence in aged LEC rats. Copper staining of LEC rat kidneys showed a positive reaction in proximal tubules of the cortex and the outer stripe of the medulla. The renal copper concentration of LEC rats reached a peak in the period of necrotizing hepatitis with renal tubular necrosis, and was higher than that in F344 rats for up to 106 weeks. In contrast, the renal iron concentration of LEC rats was lower than that in F344 rats except in the period of necrotizing hepatitis. Long-term treatment of LEC rats with d -penicillamine, a copper-chelating agent, inhibited accumulation of copper, but not iron, in the kidneys, and inhibited the development of karyomegaly of proximal tubules and dilated atypical tubules. These results suggest that persistent copper accumulation after toxic necrosis of tubules is the major cause of spontaneous renal carcinogenesis in LEC rats.     

Low susceptibility of Long-Evans Cinnamon rats to N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced urinary bladder carcinogenesis and inhibitory effect of urinary copper.

We studied the susceptibilities to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis of male Long-Evans Cinnamon (LEC), F344 and Long-Evans Agouti (LEA) rats. Male rats (n=21) were given 0.1% BBN in their drinking water from week 6, 8 and 10 for one week, and killed in week 56. The incidences of transitional cell tumors (papillomas plus carcinomas) in BBN-treated LEC and F344 rats were 12% and 76%, respectively (P<0.001, experiment 1), and those in LEC and LEA rats were 11% and 95%, respectively (P<0.001, experiment 2). When male LEC and F344 rats were given 0.1% BBN in their drinking water for 7 days, the intake of BBN and the urinary concentration of its active metabolite, N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), were higher in the LEC rats (P<0.01). The urinary pHs of untreated LEC and F344 rats were similar between week 6 and 30. The urinary copper concentration was lower in LEC rats before jaundice than in F344 rats, but its concentrations in 28- and 50-week-old LEC rats were 1.7 and 2.3 times those in F344 rats. In a two-stage carcinogenesis study using F344 rats, i.p. injections of cupric nitrilotriacetate increased urinary copper excretion, and inhibited BBN-induced bladder carcinogenesis. In a two-stage carcinogenesis study using LEC rats, oral administration of D-penicillamine decreased urinary copper excretion, and increased BBN-induced bladder cancer, although the difference was not significant. These data show that LEC rats are resistant to bladder carcinogenesis and suggest that urinary copper has a significant role in their resistance.     

Role of copper accumulation in spontaneous renal carcinogenesis in Long-Evans Cinnamon rats.

Spontaneous renal cell tumors in totals of 223 male and female Long-Evans Cinnamon (LEC) rats of 51-120 weeks old, 157 male F344 rats of 51-120 weeks old, and 14 male Long-Evans Agouti (LEA) rats of 51-70 weeks old were examined histologically. The incidences of renal cell tumors increased with age in male and female LEC rats, but no tumors developed in F344 or LEA rats. Dilated atypical tubules of the kidneys were observed at high incidence in aged LEC rats. Copper staining of LEC rat kidneys showed a positive reaction in proximal tubules of the cortex and the outer stripe of the medulla. The renal copper concentration of LEC rats reached a peak in the period of necrotizing hepatitis with renal tubular necrosis, and was higher than that in F344 rats for up to 106 weeks. In contrast, the renal iron concentration of LEC rats was lower than that in F344 rats except in the period of necrotizing hepatitis. Long-term treatment of LEC rats with D-penicillamine, a copper-chelating agent, inhibited accumulation of copper, but not iron, in the kidneys, and inhibited the development of karyomegaly of proximal tubules and dilated atypical tubules. These results suggest that persistent copper accumulation after toxic necrosis of tubules is the major cause of spontaneous renal carcinogenesis in LEC rats.     

Modifying effects of benzyl isothiocyanate and benzyl thiocyanate on DNA synthesis in primary cultures of rat hepatocytes

The effects of benzyl isothiocyanate (BITC) and benzyl thiocyanate(BTC) on two types of DNA synthesis were examined in hepatocyteprimary cultures (HPC). Male F344 rats were fed BITC- or BTC-containingdiets at a concentration of 400 p.p.m. Using hepatocytes isolatedfrom these rats, DNA repair was measured by unscheduled DNAsynthesis (UDS) for some genotoxic carcinogens, e.g. 2-acetylaminofluorene(AAF), methylazoxymethanol (MAM) acetate, 9,10-dimethyl-1,2-benzanthracene(DMBA) and diethylnitrotro-samine (DEN), and compared with thatin the hepatocytes from rats without BITC or BTC treatment.Replicative DNA synthesis (RDS) was also evaluated in the hepatocytesof rats with or without thiocyanate treatment. Both BITC andBTC reduced UDS elicited by these carcinogens. The level ofRDS in the hepatocytes of rats exposed to BITC or BTC was markedlylower than in the cells of rats without BITC or BTC exposure.These results indicate that in vivo exposure to BITC and BTCsuppressed carcinogen-induced genotoxicity and cell proliferativeactivity and suggest that this assay may prove useful in detectingchemopreventive agents for cancer and in investigating the propertiesof carcinogenesis modifiers.     

Hereditary low level of plasma ceruloplasmin in LEC rats associated with spontaneous development of hepatitis and liver cancer

Both young (5 weeks old) and old (61-100 weeks old) hereditary hepatitis LEC rats showed a markedly low level of plasma ceruloplasmin (Cp) ferroxidase activity as compared with that of age-matched LEA and BN strain rats. This trait was genetically examined by the use of (BN x LEC) F1 hybrid and (F1 x LEC) backcross rats. The F1 hybrids never developed hepatitis and showed a similar level of Cp to that found in the parental BN rats. Among the backcross rats with about 1:1 segregation rate for hepatitis, affected rats had a remarkably decreased level of Cp, as found in LEC rats, whereas unaffected rats exhibited a similar level of Cp to that of BN, F1 and LEA rats. These results indicate that the low level of Cp is heritable in a single autosomal recessive mode in LEC rats. The observed tight link between the low Cp level and the hepatitis in LEC rats suggests that defective copper metabolism may be associated with the occurrence of hepatitis in LEC rats, since Cp is a copper-binding protein primarily involved in copper transport from the liver.     

Age-dependent induction of preneoplastic liver cell foci by 2-acetylaminofluorene, phenobarbital and acetaminophen in F344 rats initially treated with diethylnitrosamine

Effects of age on the induction of glutathione S-transferase placental form (GST-P)-positive hepatic foci in rats were examined using a medium-term liver bioassay system (for carcinogens). F344 male rats aged 6, 26 and 46 weeks were initially given a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and, beginning 2 weeks later, received 0.02% 2-acetylaminofluorene (2-AAF), 0.05% phenobarbital (PB) or 1.3% acetaminophen (AAP) in the diet for 6 weeks. All animals were subjected to two-thirds hepatectomy 3 weeks after the DEN injection and were killed at week 8. Quantitative analysis of GST-P-positive foci revealed significantly (P less than 0.001) increased induction over control levels in terms of both numbers and areas for 2-AAF at all ages (6, 26 and 46 weeks), but especially in the 6-week-old case. In the PB- and AAP-treated groups, the respective enhancing and inhibitory influences were most pronounced in the animals aged 6 weeks, and were less marked in older rats. Thus, the response of F344 rats to the modifying effects of chemicals was age-dependent, the conclusion being drawn that young rats are more susceptible and therefore more appropriate for assessment of carcinogenic, promoting and inhibitory effects of chemicals.     

Age-dependent Induction of Preneoplastic Liver Cell Foci by 2-Acetylammo-fluorene, Phenobarbital and Acetaminophen in F344 Rats Initially Treated with Diethylnitrosamine

Effects of age on the induction of glutathione S -transferase placental form (GST-P)-positive hepatic foci in rats were examined using a medium-term liver bioassay system (for carcinogens). F344 male rats aged 6, 26 and 46 weeks were initially given a single intraperitoneal injection of diethylnitro-samine (DEN, 200 mg/kg) and, beginning 2 weeks later, received 0.02% 2-acetylaminofluorene (2-AAF), 0.05% phenobarbital (PB) or 1.3% acetaminophen (AAP) in the diet for 6 weeks. All animals were subjected to two-thirds hepatectomy 3 weeks after the DEN injection and were killed at week 8. Quantitative analysis of GST-P-positive foci revealed significantly (P< 0.001) increased induction over control levels in terms of both numbers and areas for 2-AAF at all ages (6, 26 and 46 weeks), but especially in the 6-week-old case. In the PB- and AAP-treated groups, the respective enhancing and inhibitory influences were most pronounced in the animals aged 6 weeks, and were less marked in older rats. Thus, the response of F344 rats to the modifying effects of chemicals was age-dependent, the conclusion being drawn that young rats are more susceptible and therefore more appropriate for assessment of carcinogenic, promoting and inhibitory effects of chemicals.     

High expression of heparin-binding EGF-like growth factor in rat hepatocarcinogenesis

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family and is highly expressed in hepatoma tissues but not in normal liver. However, it is unknown when HB-EGF is induced during hepatocarcinogenesis and what are the mechanisms underlying its high expression in hepatoma. To address this issue, the expression of HB-EGF was investigated during hepatocarcinogenesis in LEC (Long-Evans with a cinnamon-like coat color) rats, which spontaneously develop hepatitis and hepatoma. LEA (Long-Evans with an agouti coat color) rats were used as controls. Furthermore, the induction of HB-EGF mRNA by various agents was investigated in a rat hepatoma cell line and hepatocytes in primary culture. Expression of HB-EGF mRNA in the liver was very low at the stage of acute and chronic hepatitis and markedly increased at the stage of hepatoma in LEC rats. Non-involved tissues adjacent to hepatoma showed low expression of HB-EGF mRNA. Immunochemical studies revealed positive staining in hepatoma tissues. Induction of HB-EGF mRNA by several growth factors was observed in a hepatoma cell line but not in normal hepatocytes. Our results suggest that HB-EGF is associated with the early progression steps of hepatoma. © 1996 Wiley-Liss, Inc.     

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.     

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.     

Genetic linkage between copper accumulation and hepatitis/hepatoma development in LEC rats.

The concentration of copper in the livers of Long-Evans rats with cinnamon-like coat color (LEC), in which hepatitis and then hepatomas develop spontaneously, was recently found to be abnormally high. Therefore, we examined the copper concentrations in the livers of LEC F1 backcrosses (LEC F1 x LEC) to determine the linkage of copper accumulation with development of hepatitis. Consistent with a previously reported ratio of rats with hepatitis to rats without hepatitis of about 1:1, hepatitis developed in 14 of 30 F1 backcrosses. The copper concentrations in the livers of all LEC F1 backcrosses with hepatitis were abnormally high and comparable to those of LEC rats. In contrast, the concentrations in all backcrosses without hepatitis were similar to those in normal Long-Evans with agouti coat color or Brown-Norway rats. Copper accumulation was shown to be closely linked with the development of hepatitis in LEC rats and appeared to be a possible cause of hepatitis. The concentrations of copper in the livers of Fischer 344 rats after carbon tetrachloride treatment were in the range for normal liver, indicating that a high copper concentration in the liver is specific to LEC rats and not a specific characteristic of hepatitis. Furthermore, we found that the size and level of ceruloplasmin mRNA in the livers of LEC rats were the same as those in LEA rats and that the size and level of ceruloplasmin polypeptide in their livers and plasma were almost the same as those in LEA rats. Therefore, these results suggest that the copper accumulation is not due to alteration of expression or to gross alteration of the ceruloplasmin gene.     

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.     

Examination of genotoxicity, toxicity and morphologic alterations in hepatocytes following in vivo or in vitro exposure to methapyrilene

The antihistamine methapyrilene (MP) was widely used as a componentof cold, allergy and sleep-aid medications in the 1970s untilit was identified as a potent rat liver carcinogen. MP doesnot induce positive responses in most short-term genotoxicityassays, which suggests that it is carcinogenic by a non-genotoxicmechanism. We have evaluated the potential of MP to induce unscheduledDNA synthesis (UDS), a genetic end point and S-phase synthesis(SPS), an indicator of cell proliferation, in Fischer-344 (F344)rat and B6C3F1 mouse liver. We also examined the response ofMP in hepatocytes from two species treated in vitro. MP failedto induce UDS in rat or mouse liver following in vivo treatment,or in hepatocytes from rat and adult human treated in vitro.Control rats and mice yielded <0.3% of cells in S-phase (%S).In contrast, MP induced significant elevations in SPS both inmale F344 rat (6.3%S) and female B6C3F1 mice (1.4%S). In themale rat, sorbitol dehydrogenase (SDH), bilirubin, serum glutamicoxaloacetic transaminase (SGOT) and serum glutamic pyruvatetransaminase (SGPT) showed elevations of 9-, 10-. 17- and 28-foldover controls respectively, indicating that significant hepatotoxicitywas induced by MP. This was confirmed by histopathologic examination,which revealed significant periportal and focal necrosis followedby an increased presence of mitotic figures. These results indicatethat MP is not genotoxic in rat liver, but is a potent inducerof hepatic cell proliferation by inducing toxicity and subsequentregeneration, which may be an important mechanism of hepatocarcinogenesis.     

Two-dimensional electrophoretic analysis of hepatitis-associated polypeptides in liver of LEC rats developing spontaneous hepatitis

High-resolution two-dimensional polyacrylamide gel electrophoresis in combination with silver staining was used to analyze between 800 and 1000 cytosolic and particulate polypeptides from age-matched livers of normal male Long-Evans rat with Agouti coat color (LEA) and Long-Evans rat with Cinnamon-like coat color (LEC) rats with hereditary trait of hepatitis at ages long before, immediately prior to, and just after the onset of hepatitis. Although the electrophoretic patterns of polypeptide expression were very similar with respect to the overall spot patterns, a number of polypeptides which differed either qualitatively or quantitatively were noted. Two constitutively expressed cytosolic polypeptides, P29.5 (Mr 29.5 kDa/pI 6.73) and P30 (30 kDa/6.70), were not detected in livers of LEC animals at any age. In the normal LEA rats both P29.5 and P30 were detected as early as one day after birth and both were expressed at similar concentrations at all ages. In the LEC rats P30-C (30 kDa/6.68) was constitutively expressed in close proximity to the expected position of P30, and P30-C was not detected in the LEA rats. By means of non-equilibrium pH gradient electrophoresis two relatively basic polypeptides were detected in the LEC rats. P18ne was detected immediately prior to and P27ne immediately after the clinical manifestation of hepatitis. Experiments in F1 backcross ([LEA x LEC] x LEC) animals, however, failed to demonstrate any genetic link between either the expression or lack of expression of P29.5, P30, P30-C, or P18ne and hepatitis development. P27ne was detected in all backcross animals exhibiting hepatitis, but was never observed in LEC rats prior to the onset of hepatitis. Although we were unable to identify any unique loss of expression of polypeptides which are genetically linked to hepatitis susceptibility in LEC rats, specific subsets of quantitatively modulated polypeptides were detected.     

Evidence of alterations in base excision repair of oxidative DNA damage during spontaneous hepatocarcinogenesis in Long Evans Cinnamon rats

The Long-Evans Cinnamon (LEC) rat, an animal model for Wilson's disease, is an inbred mutant strain, which because of the genetic copper metabolism disorder develops hepatitis approximately 4 months after birth, followed by chronic hepatitis later in life, and eventually all of the surviving animals from liver injury and hepatitis develop spontaneous hepatocellular carcinomas. This animal model also shows that the generation of reactive oxygen species and the accumulation of oxidative damage in the liver DNA has significantly increased over the lifetime of LEC versus the wild-type Long-Evans Agouti (LEA) rats. Thus, the LEC rats having this genetically induced oxidative condition are proved to be very useful model for the study of endogenous DNA lesions and their relation to spontaneous carcinogenesis. In this study, we tested the hypothesis that differences do exist between these two rat strains in respect to their capacity to repair oxidative DNA base modification, which could explain the elevation of endogenous oxidative damage in the LEC rat liver DNA. We found that both the activity and expression at the protein and RNA levels of major DNA glycosylases, endonuclease III and 8-oxoguanine DNA-glycosylase, which initiate the excision and repair of oxidized bases, were significantly altered during the acute (16-18 weeks) and early chronic (24 weeks) phases of hepatitis. Enzyme levels were restored in the later period of chronic hepatitis (week 40) in the LEC rat liver as compared with the age-matched LEA rats. This early reduction in the capacity to repair oxidative DNA base damage could have contributed to the accumulation of mutagenic adducts in liver DNA. These findings show for the first time in an animal model that acute hepatitis impairs the repair of oxidative DNA base damage and strongly suggest that the repair of endogenous DNA adducts plays a critical role in the development of spontaneous hepatocellular carcinoma in LEC rats.     

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.