Tsuneo Masui

Placental Glutathione S-Transferase (GST-P) as a New Marker for Hepatocarcinogenesis: In Vivo Short-Term Screening for Hepatocarcinogens

Our laboratory has developed an in vivo short-term screening test for hepatocarcinogens based on quantitation of γ-glutamyl transpeptidase (γ-GT) positive foci. However, γ-GT positive hepatocytes appear in periportal areas under a variety of circumstances apparently unrelated to hepatocarcinogenesis. Glutathione S-transferase placental type (GST-P), which is hardly detectable in normal rat liver, was recently demonstrated as a new marker protein for preneoplastic liver foci. In experiment I, rats were initially given a single dose (200 mg/kg) of diethylnitrosamine intraperitoneally and 2 weeks later were treated with test compounds for 6 weeks. All rats were subjected to partial hepatectomy at week 3. The long-term development of preneoplastic lesions was followed in rats for 50 weeks. The immunohistochemical investigation of GST-P binding and the histochemical demonstration of γ-GT in serial sections revealed that almost all γ-GT foci were GST-P positive, but 5–10% of GST-P foci could not be detected by γ-GT staining. From week 8, many γ-GT foci partially lost γ-GT activity. However, no comparable disappearance of GST-P was evident in the lesions. All hepatocellular carcinomas (HC) found at week 50 consisted of GST-P positive HC cells. In contrast, 37.9% (11/29) of HC were negative for γ-GT. In experiment II (in vivo short-term screening test for hepatocarcinogens), rats were treated in the same manner as for experiment I and killed at week 8. Fifty-eight chemicals were investigated for their potential to modify GST-P positive foci development. All hepatocarcinogens and hepatopromoters clearly enhanced the induction of GST-P foci, whereas non-hepatocarcinogens and non-hepatopromoters did not. BHA and acetaminophen inhibited the development of foci. These results suggest the adoption of GST-P as a new and more accurate marker enzyme for rat liver carcinogenesis.

Induction of Glandular Stomach Cancers in C3H Mice Treated with N‐Methyl‐N‐nitrosourea in the Drinking Water

Establishment of an animal model of stomach carcinogenesis in mice was attempted using N‐methyl‐N‐nitrosourea (MNU) in the drinking water. One hundred and forty‐eight male 6‐week‐old C3H mice were given MNU in their drinking water at a concentration of 120 ppm (group 1), 60 ppm (group 2), 30 ppm (group 3) or 0 ppm (group 4) for 30 weeks. At the end of this time, dose‐related induction of adenomatous hyperplasias was found. From weeks 31 to 54 adenocarcinomas developed in a dose‐dependent manner in groups 1, 2 and 3. In total, 6 well differentiated and 5 poorly differentiated adenocarcinomas as well as 6 signet ring cell carcinomas arose in 15 stomach cancer‐bearing animals in group 1, 4 well differentiated and 2 poorly differentiated adenocarcinomas with one signet ring cell carcinoma in 5 mice of group 2 and one well differentiated adenocarcinoma in group 3. In the forestomach, only one squamous cell carcinoma was found at week 54 in group 1 along with a single well differentiated adenocarcinoma in the duodenum. Thus, MNU in the drinking water selectively induced neoplastic lesions in the glandular stomach epithelium of mice.

Clonal analysis of glandular stomach carcinogenesis in C3H HeN ↔ BALB c chimeric mice treated with N-methyl-N-nitrosourea

The clonal growth of gastric carcinomas was investigated immunohistochemically in C3H<==>BALB/c chimeras using a strain specific antibody. C3H, BALB/c and chimeric mice were given N-methyl-N-nitrosourea 0.5 mg/mice once a week for a total of 10 times by intragastric intubation and observed until week 50. In normal gastric mucosa of the chimeras, each gland was composed entirely of C3H strain specific antigen (CSA)-positive or -negative cells and no mixed glands were found. Cells of all adenomatous hyperplasias and adenocarcinomas in chimeric mice were, in each case, homogeneous for one or other of the parental types, while comprising both surface mucous cell and pyloric gland cell forms. The results clearly suggest that individual cancers are derived from single cells with multi-potential activities and that cellular differentiation of gastric cancer cells occurs secondarily.

Correlative histochemical studies on preneoplastic and neoplastic lesions in the kidney of rats treated with nitrosamines.

SummaryRenal tubular lesions induced in male rats by two different carcinogens, N-nitrosomorpholine (NNM) and N-ethyl-N-hydroxyethylnitrosamine (EHEN), using a limited exposure “stop” protocol were investigated histochemically to demonstrate phenotypic cellular changes. The parameters measured included basophilia, glycogen content and the activity of the enzymes glucose-6-phosphatase (G6PASE), glycogen synthetase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase (SDH), alkaline phosphatase (ALP), acid phosphatase (ACP) and γ-glutamyl transpeptidase (γ-GT). The lesions observed were predominantly of either basophilic or oncocytic types. In each case, tubular lesions (altered tubules) appeared to give rise to epithelial tumors (epitheliomas) with the same cellular phenotype. Basophilic tubules and epitheliomas proved to be strongly positive for GAPDH and G6PDH while demonstrating a reduction or loss of G6PASE, ALP, ACP, γ-GT, and SDH compared with controls and the surrounding proximal or distal tubules. In addition, large basophilic epitheliomas demonstrated an increase in both SYN and PHO activities. In contrast, most oncocytic tubules and oncocytomas characterized by bundant densely granular cytoplasm showed a reduction in the activity of G6PDH, but were intensely positive for SDH. However, a few oncocytic lesions demonstrated a decrease in both SDH and G6PDH activity. Rarely, decreased SDH and elevated G6PDH activities were observed in altered tubules resembling oncocytic tubules. It remains to be clarified whether these tubules represent a variation of the oncocytic lesions or, perhaps, another type of tubular lesion. The results indicate that basophilic and oncocytic epithelial tumors differ in their cytochemical pattern and histogenesis. In line with earlier suggestions, the basophilic tumors apparently originate from the proximal renal tubules, while the oncocytomas develop from the distal parts of the nephron. The basophilic tumors are characterized by an increased pentose phosphate pathway and glycolysis, with a corresponding reduction in mitochondrial respiration. However, the majority of the oncocytomas show an increased activity of the mitochondrial enzyme SDH, and a marked decrease in the activity of the key enzyme of the pentose phosphate pathway.

Dose-dependent Induction of Mammary Carcinomas in Female Sprague-Dawley Rats with 2-Amino-l-methyl-6-phenylimidazo[4,5-b]pyridine

The dose‐dependence of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) induction of mammary carcinomas was investigated in female Sprague‐Dawley (SD) rats given PhIP in the diet for 48 weeks at concentrations of 0, 25, 100 and 200 ppm in experiment 1, and 0, 12.5, 50 in experiment 2. Yields of ductular lesions, including intraductal papillomas and carcinomas, as well as papillo‐tubular and solid‐tubular carcinomas, showed dependence on the dose, with the respective total incidences being 0, 4.8, 25, 72.2 and 0, 10 and 35%. There was thus no apparent carcinogen exposure threshold. The present results confirmed the carcinogenicity demonstrated in a previous study using F344 rats and revealed the SD rat strain to be more susceptible.

Promoting effect of sodium chloride in 2-stage urinary bladder carcinogenesis in rats initiated by N-Butyl-N-(4-hydroxybutyl)-nitrosamine

SummaryThe promoting effect of sodium chloride (NaCl) in 2-stage urinary bladder carcinogenesis in F344 rats initiated by 2 doses of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was investigated. The incidences of PN hyperplasia were significantly higher in rats initiated with 0.01 or 0.05% BBN when they were given diet containing 10% NaCl for 32 weeks than when they were given control diet. The incidence of papilloma in rats given 0.05% BBN followed by diet containing 10% or 5% NaCl tended to be higher than that in control rats. The urine of rats given diet containing NaCl was larger in volume and had lower osmolality than that of controls. The total urinary sodium and chloride contents were also increased, whereas those of potassium and phosphorus were decreased. No calculus formation or crystalluria was observed. These data suggest that excess intake of sodium as NaCl has a weak promoting effect in 2-stage urinary bladder carcinogenesis.

Highly metastatic hepatocellular carcinomas induced in male F344 rats treated with N-nitrosomorpholine in combination with other hepatocarcinogens show a high incidence of p53 gene mutations along with altered mRNA expression of tumor-related genes

The carcinogenic and metastatic processes are thought to consist of a sequence of steps, and animal models featuring highly metastatic lesions are clearly necessary to allow analysis of the whole process of transformation from preneoplastic changes to high grade metastatic tumors, and to access effectiveness of therapeutic treatments of advanced cancers in vivo. The purpose of the present study was to establish a model and to screen for reported genetic alterations in induced lesions. In the present study, it was confirmed that lung metastasis of hepatocellular carcinomas (HCCs) induced in male F344 rats by N-nitrosomorpholine (NNM), given in the drinking water at a dose of 120 ppm for 24 weeks, was significantly enhanced by additional carcinogenic pretreatments and that a single i.p. injection of 100 mg/kg body weight N-diethylnitrosamine (DEN) alone was sufficient for that purpose. Molecular biological analyses of the induced lesions revealed point mutations in the p53 gene in 60.9% of HCCs, and elevated expression of mRNAs for p53, c-myc, c-fos, TGF-alpha, TGF-beta1, alpha-fetoprotein, GST-P, and GGT, and decreased mRNA expression of EGF and EGFR in HCCs when compared to controls. No obvious association of gene alterations with metastatic potential of primary tumors was found except for an increase in the incidence of p53 mutations. Since the process of metastasis is thought to be sequential and selective, further comparative analysis of metastatic and primary lesions should clarify the mechanisms involved in the multi-step process of metastasis.

Primary monoclonal and secondary polyclonal growth of colon neoplastic lesions in C3H/HeN→BALB/c chimeric mice treated with 1,2‐dimethylhydrazine: Immunohistochemical detection of C3H strain‐specific antigen and simple sequence length polymorphism analysis of DNA

To determine the clonality and cellular origin of colon pre‐neoplastic and neoplastic lesions, C3H/HeN→BALB/c chimeric mice treated with 1,2‐dimethylhydrazine (DMH) were investigated immunohistochemically using a specific antibody to C3H strain‐specific antigen (CSA) enabling immunohistochemical discrimination of C3H cells in histological sections of chimeric mouse tissues. To confirm the results of immunostaining, simple sequence length polymorphism (SSLP) analysis was performed on DNA samples extracted from histological sections of adenocarcinomas. C3H/HeN→BALB/c chimeric mice were produced by an aggregation procedure and together with BALB/c and C3H/HeN animals were given weekly s.c. injections of 20 mg/kg body weight DMH for up to 20 weeks. At weeks 20 and 35 animals were killed and autopsied. In normal colonic mucosa of the chimeras, each gland was composed entirely of either CSA‐positive or ‐negative cells and no mixed glands were found. Cells of all focal atypias in chimeric mice were, in each case, homogeneous for one or another of the parental types. Of 91 adenomas in chimeric mice, only one comprised both types of cell. Among 119 adenocarcinomas, 12 contained cells of both parental types. In these tumors, however, the 2 phenotypes were not mixed together at random but arranged in discrete areas, with intermingling limited to the junctions. SSLP analysis demonstrated DNAs extracted from CSA‐positive and ‐negative tumors to exhibit the polymorphic patterns of C3H and BALB/c, respectively, while mixed CSA‐positive and ‐negative tumors showed mixtures of both polymorphic DNA types.

Enhancing Effect of Cadmium on Rat Ventral Prostate Carcinogenesis Induced by 3,2′‐Dimethyl‐4‐aminobiphenyl

The effects of cadmium given at different stages during 3,2′‐dimethyl‐4‐aminobiphenyl (DMAB)‐induced rat prostate carcinogenesis were investigated using male F344 rats. Animals were given 10 subcutaneous injections of 50 mg/kg body weight of DMAB or the corn oil vehicle at two‐week intervals. In addition, cadmium was administered at doses of 0, 10, or 30 /μmol/kg body weight as single intramuscular injection on the 1st day of the experiment or one day after the last injection of DMAB at week 20. Two further groups were subjected to administration of cadmium at 10 μmol/kg at week 20 and then 5 μmol/kg at week 40, or 10 μmol/kg at week 20 and then 5 μmol/kg at weeks 30, 40 and 50. At the termination, 60 weeks after the beginning of the experiment, the incidences and multiplicity of ventral prostate carcinomas in the groups given cadmium plus DMAB demonstrated a consistent tendency for increase over control values (groups receiving DMAB or cadmium alone). The numbers of carcinomas per rat and per unit area of prostate section were significantly elevated in the two groups given low doses of cadmium after cessation of DMAB administration. Cadmium alone also induced a few prostate carcinomas. The influence on development of prostate tumors did not appear to be a result of the induced severe testicular atrophy because serum testosterone levels were not affected. The results indicate that cadmium and DMAB can act synergistically to cause rat prostate carcinogenesis.

Promoting effects of various agents in rat urinary bladder carcinogenesis initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine

The effects of various chemicals on the development of neoplastic lesions in the urinary bladder were investigated in male F344 rats given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) as an initiator in their drinking water for 4 weeks. The compounds tested, indomethacin, acemetacin, epsilon-aminocaproic acid (EACA), diphenyl, allopurinol and acetaminophen (AAP), were added to the diet or drinking water for 32 weeks, and all animals were killed at the end of week 36. Of the chemicals tested, only diphenyl significantly increased the incidences and average numbers (per 10 cm basement membrane) of papillary or nodular hyperplasias (PN hyperplasia), papillomas and carcinomas of the urinary bladder over those in animals treated with BBN alone. These findings show that diphenyl is a promoter of urinary bladder carcinogenesis in male F344 rats.