Role of Atp7b gene in spontaneous and N-diethylnitrosamine-induced carcinogenesis in a new congenic strain, WKAH.C-Atp7b rats.

To examine whether Long-Evans Cinnamon (LEC) rats, a mutant rat model of Wilson's disease, have a susceptibility gene(s) to hepatocarcinogenesis in addition to the causative gene, Atp7b, we established a new congenic strain, WKAH.C-Atp7b rats, in which the Atp7b gene of the LEC rats is inserted into the normal Wistar-King Aptekman Hokkaido (WKAH) background. Hepatocellular tumors developed spontaneously in both sexes of WKAH.C-Atp7b rats, their incidence being slightly lower than that in LEC rats. Incidences of spontaneous liver tumors in LEC, WKAH.C-Atp7b and WKAH rats correlated with hepatic copper and iron concentrations. Medium-term liver bioassay showed that LEC rats were more susceptible to the induction of glutathione S-transferase placental form-positive preneoplastic foci than WKAH.C-Atp7b rats, and WKAH.C-Atp7b rats were more susceptible than WKAH rats. In an N-diethylnitrosamine (DEN)-induced long-term carcinogenicity study, 1) LEC rats were similarly or rather less susceptible to hepatocellular tumors than WKAH.C-Atp7b and WKAH rats, indicating that the progression of the preneoplastic foci to liver cancer in LEC rats was worse than that in WKAH.C-Atp7b and WKAH rats, 2) the incidences of kidney tumors in LEC and WKAH.C-Atp7b rats were higher than that in WKAH rats and high copper concentrations in the kidneys were observed in LEC and WKAH.C-Atp7b rats, 3) LEC rats were resistant to lung carcinogenesis. These data indicate that the susceptibility of LEC rats to liver and kidney carcinogenesis could be explained by Atp7b gene mutation and that the susceptibility to lung carcinogenesis is controlled by gene(s) other than Atp7b.     

Role of Atp7b Gene in Spontaneous and N-Diethylnitrosamine-induced Carcinogenesis in a New Congenic Strain, WKAH.C-Atp7b Rats

To examine whether Long-Evans Cinnamon (LEC) rats, a mutant rat model of Wilson's disease, have a susceptibility gene(s) to hepatocarcinogenesis in addition to the causative gene, Atp7b , we established a new congenic strain, WKAH.C-Atp7b rats, in which the Atp7b gene of the LEG rats is inserted into the normal Wistar-King Aptekman Hokkaido (WKAH) background. Hepatocellular tumors developed spontaneously in both sexes of WKAH.C-Atp7b rats, their incidence being slightly lower than that in LEG rats. Incidences of spontaneous liver tumors in LEG, WKAH.C- Atp7b and WKAH rats correlated with hepatic copper and iron concentrations. Medium-term liver bioassay showed that LEG rats were more susceptible to the induction of glutathione S-transferase placental form-positive preneoplastic foci than WKAH.C -Atp7b rats, and WKAH . C -Atp7b rats were more susceptible than WKAH rats. In an N -diethylnitrosamine (DEN)-induced long-term carcinogenicity study, 1) LEC rats were similarly or rather less susceptible to hepatocellular tumors than WKAH.C-Atp7b and WKAH rats, indicating that the progression of the preneoplastic foci to liver cancer in LEG rats was worse than that in WKAH.C-Atp7b and WKAH rats, 2) the incidences of kidney tumors in LEG and WKAH.C-Atp7b rats were higher than that in WKAH rats and high copper concentrations in the kidneys were observed in LEG and WKAH.C- Atp7b rats, 3) LEC rats were resistant to lung carcinogenesis. These data indicate that the susceptibility of LEG rats to liver and kidney carcinogenesis could be explained by Atp7b gene mutation and that the susceptibility to lung carcinogenesis is controlled by gene(s) other than Atp7b.     

Formation of DNA adducts and induction of lacI mutations in Big Blue Rat-2 cells treated with temozolomide: implications for the treatment of low-grade adult and pediatric brain tumors.

Temozolomide (TMZ) is a chemotherapeutic agent used in the treatment of high-grade brain tumors. Treatment of patients with alkylating chemotherapeutic agents has been established to increase their risk for acute myelogenous leukemia. The formation of DNA adducts and induction of mutations are likely to play a role in the etiology of therapy-related acute myeloid leukemia. To evaluate this issue for TMZ, we have measured the formation of DNA adducts and induction of lacI mutations in Big Blue Rat-2 cells treated with TMZ. Treatment of Big Blue Rat-2 cells with either 0, 0.5, or 1 mM TMZ resulted in lacI mutant frequencies of 9.1 +/- 2.9 x 10(-5), 48.9 +/- 12 x 10(-5), and 89.7 +/- 40.3 x 10(-5), respectively. Comparison of the mutant frequencies demonstrated that 0.5 and 1 mM TMZ treatments increased the mutant frequencies by 5.3- and 9.8-fold and that this increase was significant (P < 0.001). Sequence analysis of the lacI mutants from the TMZ treatment group demonstrated that they were GC-->AT transitions at non-CpG sites, which is significantly different from the mutation spectrum observed in the control treatment group. Treatment of Big Blue Rat-2 cells with various concentrations of TMZ produced a linear increase in the levels of N7-methylguanine and O(6)-methylguanine. The lacI mutation spectrum induced by TMZ treatment is consistent with these mutations being produced by O(6)-MeG. This study establishes TMZ has significant mutagenic potential and suggests that careful consideration in the use of TMZ for the treatment of low-grade adult and pediatric brain tumors should be given.     

Hepatocellular Carcinoma Induction in LEC Rats by a Low Dose of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline

A food-borne heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5- f ]qninoxaline (MeIQx), induces hepatocellular carcinomas (HCCs) in F344 male rats at an incidence of 95%, when fed in the diet at 400 ppm for 61 weeks. In this study, the effect of a low dose of MeIQx was examined in Long-Evans with cinnamon-like coat color (LEC) rats, which have a mutation in Atp7b and suffer from hereditary hepatitis and HCCs, with high levels of copper accumulation in the liver. Rats of the LEC and Long-Evans with agouti coat color (LEA) sibling lines were given a diet containing 40 ppm MeIQx from the age of 23 weeks to 63 weeks, for a total administration period of 40 weeks. In LEC rats, HCCs were observed in 8/8 animals administered MeIQx, and 2/8 rats receiving a normal diet. The number of HCCs per rat (mean±SD) was 2.8±2.0 and 0.3±0.5, respectively. In the LEA rats, however, no tumors were induced by administration of MeIQx. These results indicate that damaged liver associated with compensatory cell proliferation is much more susceptible to chemical hepatocarcinogens, including MeIQx, than the normal liver.     

DNA adduct formation and molecular analysis of in vivo lacI mutations in the mammary tissue of Big Blue rats treated with 7, 12-dimethylbenz[a]anthracene

Recently we compared the lacI and Hprt mutant frequencies (MFs) and types of mutations in lymphocytes of Big Blue((R)) (BB) rats exposed to 7,12-dimethylbenz[a]anthracene (DMBA) under conditions that result in mammary gland tumors. In this study, we have examined the target mammary tissue for DMBA-induced DNA adducts, lacI MF and types of lacI mutations. Seven-week-old female BB rats were given single doses of 0, 20 or 130 mg/kg DMBA by gavage and the DNA adducts and lacI MFs in the mammary tissue were measured over a period of 14 days and 18 weeks, respectively, following treatment. The lacI MF in the mammary tissue increased for 10 weeks and then remained relatively constant; 130 mg/kg DMBA produced a 14-fold increase in the MF (255 +/- 50 x 10(-6) p.f.u.) over control MF (18. 3 +/- 4 x 10(-6) p.f.u.). (32)P-post-labeling analysis of DNA from mammary tissue and splenic lymphocytes of treated rats revealed two major adducts. Comparison of these adducts with DMBA standards indicated that the adducts formed by DMBA involved both G:C and A:T base pairs. DNA sequencing revealed that the majority of DMBA-induced lacI mutations were base pair substitutions and that A:T-->T:A (44% of the independent mutations) and G:C-->T:A (24% of the independent mutations) transversions were the predominant types. Furthermore, the mutational results revealed a 'hotspot' for a G-->T mutation in codon 95 (GTG-->TTG) of the lacI gene in mammary tissue. These results suggest that DMBA is highly mutagenic to lacI in mammary tissue and that adducts with both G:C and A:T base pairs participate in forming mutations in DMBA-treated BB rats.     

Mutations induced by alpha-hydroxytamoxifen in the lacI and cII genes of Big Blue transgenic rats

The antiestrogen tamoxifen is widely used for the treatment of breast cancer and more recently for the prevention of breast cancer. A concern over the use of tamoxifen as a chemopreventive agent is its carcinogenicity in rat liver, through a genotoxic mechanism involving alpha-hydroxylation, esterification, and DNA adduct formation, primarily by reaction with dG. In a recent study [Gamboa da Costa et al., Cancer Lett., 176, 37-45 (2002)], we demonstrated a significant increase in the mutant frequency in the lacI gene of Big Blue rats treated with tamoxifen, and a further increase in rats administered alpha-hydroxytamoxifen. In the present study, we have assessed mutation induction by tamoxifen and alpha-hydroxytamoxifen in the liver cII gene of Big Blue rats and have characterized the types of mutations induced by alpha-hydroxytamoxifen in the liver lacI and cII genes. The mutant frequencies in the liver cII gene were 80 +/- 13 x 10(-6) in the control, 112 +/- 13 x 10(-6) in the tamoxifen-treated group (P < 0.01 vs. control), and 942 +/- 114 x 10(-6) in the alpha-hydroxytamoxifen-treated animals (P < 0.001 vs. control; P < 0.001 vs. tamoxifen). Molecular analysis of the mutants indicated that the alpha-hydroxytamoxifen-induced mutational spectrum differed significantly from the control spectrum, but was very similar to the spectrum induced by tamoxifen for both the lacI and cII genes [Davies et al., ENVIRON: Mol. Mutagen., 28, 430-433 (1996); Davies et al., Carcinogenesis, 20, 1351-1356 (1999)]. G:C --> T:A transversion was the major type of mutation induced by alpha-hydroxytamoxifen and tamoxifen, while G:C --> A:T transition was the main type of mutation in the control. These results support the hypothesis that alpha-hydroxytamoxifen is a major proximate tamoxifen metabolite causing the initiation of tumors in the liver of rats treated with tamoxifen.     

Mutational analysis of the liver, colon and kidney of Big Blue rats treated with 2-amino-3-methylimidazo[4,5-f]quinoline

The heterocyclic aromatic amine (HAA) 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ) induces intestinal tumours and hepatocellular carcinomas in rats, but no tumourigenic effects have been identified in the kidney. The tissue-specific mutagenicity of IQ was studied at the lacI locus in the liver, colon and kidney of Big Blue transgenic rats. At the highest dosing regime of IQ (20 mg/kg for 5 consecutive days) the mean mutant frequencies were significantly increased above background (P < 0.05) and were highest in the liver (12.9 +/- 6.2 x 10(-5)), followed by colon (7.4 +/- 1.4 x 10(-5)) and kidney (5.9 +/- 0.8 x 10(-5)). The mutational spectra from the livers of IQ-treated rats was statistically significantly different to that from the livers of control rats (P < 0.01). The lacI mutation spectra of the liver, colon and kidney from IQ-treated rats were similar. These were characterized by an increase in GC transversions in the liver and colon and an increase in the proportion of 1 bp G:C deletions in the liver and kidney. A single G deletion in the sequence 5'-CGGGA-3', characteristic of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine exposure, was detected in the liver and colon. A 2 bp GC deletion was identified at an identical position in the liver, colon and kidney. The colon was the only organ to contain two larger deletions of 13 and 33 bp. A preference was observed for base substitution mutations at guanine in the sequence 5'-CGC/T-3' and for 1 bp deletions at the guanine doublet in the sequence 5'-CGGA-3', especially in the liver and colon. Using the lacI gene as marker in the Big Blue rat model, the mutations identified in the IQ spectra have similarities to those identified for other HAAs studied in the same experimental system, but not to mutations identified in IQ-induced tumours.     

Mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the mammary gland of Big Blue rats on high-and low-fat diets

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a food-borne mutagen and mammary gland carcinogen in female rats. A high-fat diet has been shown to increase the incidence of PhIP-induced mammary gland tumors. The current study used Big Blue rats harboring the lambda lacI mutational reporter transgene, to address whether the promotional effect of a high-fat diet is mediated via modulation in mammary gland mutagenesis. Big Blue rats were given 10 doses of PhIP (75 mg/kg, p.o.) and placed on defined low-fat (5% corn oil) or high-fat (23.5% corn oil) diet for 6 weeks prior to collecting mammary glands. The lacI mutant frequency (mean +/- standard error, n = 3 rats) was 231 +/- 15 (x10(-6)) and 193 +/- 12 (x10(-6)) in the low-and high-fat group, respectively. Values were increased 12-fold over control but were not significantly different between the two diets. In a parallel study, diet did not alter the mutant frequency induced by 7,12-dimethylbenz[a]anthracene (DMBA) (125 mg/kg, p.o.) in the mammary gland. The findings suggest that the promotion by the high-fat diet is not mediated via an increase in mutations. Consistent with the high potency of DMBA as a mammary carcinogen, the mutant frequency was 20-30% higher with DMBA than with PhIP. Sixty-nine and 56 PhIP-induced lacI mutants were sequenced from the low-and high-fat diet groups, respectively. While the percentage of various types of mutations was identical between the diet groups, some difference in the distribution of mutations along the lacI gene was observed. The mutation spectrum in the mammary gland from rats on both diets was consistent with the formation of PhIP-guanine adducts which were detected by a (32)P-post-labeling assay. Guanine base substitutions accounted for approximately 85% of all mutations irrespective of diet. Single base pair deletions at guanine occurred in 11-17% of mutants. G:C to T:A transversions were the predominant base substitution mutation accounting for 35-43% of all mutations. The majority of all guanine mutations (74%) occurred at guanine bases adjacent to another G:C pair. Five out of 125 (4%) mutations involved a guanine deletion in the 5'-GGGA-3' sequence, a PhIP signature mutation reported previously. Twelve out of 125 (10%) mutations involved the guanine base in the sequence 5'-CAG(Purine)-3' (Pu). The findings from these studies suggest that 5'-CAG(Pu)-3' is an additional characteristic target site for PhIP-guanine adduct-induced mutations in vivo in the mammary gland.     

Preferential induction of guanine deletion at 5'-GGGA-3' in rat mammary glands by 2-amino- 1-methyl-6-phenylimidazo[4,5-b]pyridine.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is known to induce a characteristic mutation, G deletion at the 5'-GGGA-3' site, preferentially in the lacI transgene of the colonic mucosa of Big Blue((R)) rats (BBR) and mice and specifically in the Apc gene of rat colon tumors. In this study, lacI mutations of the mammary glands in PhIP-treated rats were investigated. Six-week-old female (BBRxSprague-Dawley)F(1) rats were administered 10 gavages of 65 mg/kg/day PhIP. Mammary ducts were collected from the macroscopically normal mammary tissue of PhIP-treated and untreated rats at 56-69 weeks of age by collagenase treatment. The mutant frequencies were 25 +/- 2.1x10(-6) in control rats and 323 +/- 44x10(-6) in the PhIP-treated rats. By sequencing 40 and 177 mutants in the control and PhIP-treated groups, respectively, 34 and 149 mutations were considered independent mutations. In the control group, G:C-->A:T transitions at CpG sites dominated and no G:C deletions were detected. In the PhIP-treated group, G:C-->T:A transversions were most frequent (43%), followed by single base pair deletions of G:C (21%). A total of nine deletions were at 5'-GGGA-3' sites, accounting for 29% of the G:C deletions and 6% of the 149 total mutations. Clusters of more than three mutations at one nucleotide position were observed at 12 positions and two were G deletions at 5'-GGGA-3' sites. Comparison of the PhIP-induced mutations in the mammary glands with those previously reported in the colon revealed that G:C-->T:A transversions occurred at a significantly higher frequency in the mammary glands and that G:C deletions occurred at a significantly lower frequency. However, the signature mutation, G deletion at the 5'-GGGA-3' site, was commonly observed in both tissues.     

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.     

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.     

Tumors arising in DNA mismatch repair-deficient mice show a wide variation in mutation frequency as assessed by a transgenic reporter gene

We reported previously that thymic lymphomas arising in mice lacking the DNA mismatch repair (MMR) gene, Msh2(-/-), exhibited striking elevations in the mutation frequency of a transgenic lacI reporter gene when compared with normal Msh2(-/-) tissues. To investigate whether hypermutation was a feature of all tumors arising in MMR-deficient mice, lacI transgene mutation frequencies were obtained from several different mouse tumors deficient for PMS2 and/or MSH2. While lacI gene hypermutation was again clearly evident in Msh2 +/- ms2(-/-) and Msh2(-/-)Pms2(-/-) thymic lymphomas, three non-thymic MSH2-deficient tumors failed to show lacI gene mutation frequency elevations when compared with a normal tissue of MMR-deficient mice. The elevated mutation frequencies in the lymphoid tumors, and the finding of multiple clustered mutations in lacI genes rescued from these tumors, suggest that they are possibly generated by a lymphoma-specific hypermutational mechanism.     

Single nucleotide instability without microsatellite instability in rat mammary carcinomas

Mutation frequencies (MnFs) of the lacI transgene and mutation rates (MRs) of the endogenous hprt gene were analyzed in two mammary carcinoma cell lines that we established from mammary carcinomas that had been induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in female lacI-transgenic rats. Using the lacI transgene, corrected MnF, which is the number of independent lacI mutations that occurred while 102 cells expanded into 10(7) cells and which reflect the dynamic increase of point mutations, was measured. The corrected MnFs in the two mammary carcinoma cell lines (59 x 10(-6) and 72 x 10(-6) mutations) were significantly higher than that in the primary culture of normal mammary epithelium (4.7 x 10(-6)). MRs of the hprt gene in the two mammary carcinoma cell lines (8.2 x 10(-7) and 11 x 10(-7) mutations/hprt/cell division) were also higher than the same control (1.4 x 10(-7)). A:T to C:G transversion was observed at significantly higher frequencies in the two cell lines (6 of 24 and 6 of 25 for lacI; 10 of 67 and 19 of 92 for hprt) than in the control (0 of 6 for lacI; 0 of 4 for hprt). Taking advantage of the lacI transgene, high frequencies of A:T to C:G transversion (6 of 38 and 8 of 33, respectively) was also confirmed in the primary carcinomas of the two cell lines, which indicated the presence of a common abnormality in the cell lines and in the primary carcinomas. Both the established cell lines and their primary carcinomas were negative for microsatellite instability, which is known to be caused mainly by mismatch repair insufficiency and to increase point mutations, and for p53 mutations. These findings showed that the two cell lines, and possibly their primary carcinomas, had increases in the MRs of point mutations attributable to a mechanism(s) different from mismatch repair insufficiency, and we would suggest that such a state be designated as single nucleotide instability (SNI).     

DNA damage triggers imbalance of proliferation and apoptosis during development of preneoplastic foci in the liver of Long-Evans Cinnamon rats

The mutant strain Long-Evans Cinnamon (LEC) rat accumulates copper, resulting in spontaneous hepatitis and subsequent development of hepatocellular carcinomas (HCCs) in the liver, providing a promising model for investigation of the relationship between hepatitis induced by oxidative stress and hepatocarcinogenesis. We examined DNA strand breaks in peripheral blood cells and p53 expression in livers during acute and chronic hepatitis in LEC rats, along with preneoplastic lesions, and cell proliferation and apoptosis in non-cancerous portions of livers from LEC rats aged 7-115 weeks. Immunohistochemistry using antibodies against glutathione S-transferase placental-form (GST-P), proliferating cell nuclear antigen (PCNA), and in situ DNA nick labeling (TUNEL) were used. Long-Evans Agouti (LEA) rats, a sibling line of the LEC strain, were used as controls. In the LEC rats, DNA strand breaks and expression of p53 were significantly higher than that of LEA rats at 24 weeks of age. The number of GST-P-positive (GST-P+) foci/cm2 increased and peaked at 48 weeks old, and the areas rapidly expanded thereafter. The level of cell proliferation increased with the development of hepatitis and was highest at about 48 weeks old. The induction of apoptosis in LEC rats was transiently higher than that in LEA rats during the period from 24 to 34 weeks of age. However, the ratio of PCNA-positive cells to the apoptotic index showed a growth imbalance in favor of cell proliferation, supporting sustained net growth in LEC rats. These findings suggest that DNA damage, reflected in DNA strand breaks, plays a critical role in the development of hepatocellular preneoplastic foci, with an imbalance between high proliferation and relatively low apoptosis.     

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.     

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.     

Induction of lacI mutations in Big Blue rats treated with tamoxifen and alpha-hydroxytamoxifen.

The antiestrogen tamoxifen is carcinogenic in the liver and uterus of rats. Liver tumors appear to result from sequential hydroxylation and esterification of the alpha-carbon of tamoxifen followed by DNA adduct formation. The mechanism for the induction of uterine tumors is not known. Big Blue rats were treated by intraperitoneal injection with 21 daily doses of 54 micromol/kg tamoxifen or its proximate carcinogenic metabolite alpha-hydroxytamoxifen. One month after the last treatment, the mutant frequency in the lacI transgene was determined in the liver and uterus. For comparison, the mutant frequency in the hypoxanthine phosphoribosyl transferase (Hprt) gene of spleen lymphocytes was also measured. In the liver, tamoxifen (32+/-18 mutants/10(6) plaques; mean+/-SD) and alpha-hydroxytamoxifen (770+/-270 mutants/10(6) plaques) caused a significant increase in the mutant frequency of the lacI gene compared to solvent treated controls (10+/-10 mutants/10(6) plaques). 32P-Postlabeling analyses of liver DNA indicated three DNA adducts, one each from tamoxifen, N-desmethyltamoxifen, and N,N-didesmethyltamoxifen. Neither tamoxifen nor alpha-hydroxytamoxifen caused an increase in the mutant frequency in the lacI gene of the uterus or in the Hprt gene of spleen lymphocytes. These results suggest that induction of endometrial tumors in rats is not due to the genotoxicity of tamoxifen.     

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.     

The DNA damaging drug cyproterone acetate causes gene mutations and induces glutathione-S-transferase P in the liver of female Big Blue transgenic F344 rats [published erratum appears in Carcinogenesis 1998 Apr;19(4):707]

The gestagenic and antiandrogenic drug cyproterone acetate (CPA) is mitogenic, tumorigenic and induces DNA-adducts and DNA-repair synthesis in rat liver. Thus CPA is expected to be mutagenic. However in vitro mutagenicity test systems were negative. To examine whether CPA induces mutations in rat liver, the in vivo mutation assay based on Big Blue transgenic F344 rats was employed. Single oral doses of 25, 50, 75, 100 and 200 mg CPA/kg b.w. respectively were administered to female Big Blue rats. Six weeks after treatment, liver DNA was assayed for mutations. At the highest dose, 200 mg CPA/kg b.w., the frequency of (17 +/- 4) x 10(-6) spontaneous mutations was increased to a maximum of (80 +/- 8) x 10(-6) mutations. One-hundred and 75 mg CPA/kg b.w. resulted in mutation frequencies of (35 +/- 5) and (27 +/- 5) x 10(-6), respectively. The mutation frequency at doses of 50 and 25 mg CPA/kg b.w. was similar to that of vehicle treated controls. Statistical analysis of the dose-effect relationship revealed that it was not possible to decide whether a threshold dose exists or not. DNA adducts were analyzed by the 32P-postlabelling technique. The total level of the major and the two minor adducts observed in the autoradiograms increased between doses of 25 to 75 mg CPA/kg b.w. to a maximum of approximately 12,000 +/- 3000 adducts per 10(9) nucleotides. The level did not further increase significantly with 100 and 200 mg CPA/kg b.w. After CPA treatment no preneoplastic liver foci were observed. However, single glutathione-S-transferase placental form (GST-P) positive hepatocytes were observed and the frequency was dependent on the dose. These cells are not supposed to represent initiated cells, since they occurred only transiently after 6 weeks and disappeared thereafter completely. In conclusion, our results demonstrate that CPA is mutagenic in vivo. The mutation frequency increased at high CPA doses, when the increase of the DNA adduct formation had already ceased. This suggests that the mitogenic activity of CPA is required to express the mutations.