Donryu Strain Rats Research Articles

Heredity mode of genetic polymorphism in aldehyde oxidase activity in Donryu strain rats

Donryu strain rats show genetic polymorphisms in the aldehyde oxidase gene, resulting in the phenotypic expression of ultrarapid metabolizers with homozygous nucleotide sequences (337G, 2604C), extensive metabolizers with heterozygous nucleotide sequences (377G/A, 2604C/T), and poor metabolizers with homozygous nucleotide sequences (377A, 2604T). In the mating experiments the ratio of the number of ultrarapid metabolizers, extensive metabolizers, and poor metabolizers rats in the F1 generation from the heterozygous F0 extensive metabolizers male and female rats was roughly 0.6 : 1.5 : 1, and the ratio converged to approximately 1 : 2 : 1 in the F2 generation from the heterozygous F1 extensive metabolizers male and female rats. On the contrary, all the F2 generation from homozygous F1 ultrarapid metabolizers male and female rats or from homozygous F1 poor metabolizers male and female rats had the ultrarapid metabolizers or the poor metabolizers genotypes and phenotypes. The genotypes completely agreed with the phenotypes in all individuals of F0, F1, and F2 generations. The results indicate that the genetic polymorphism of aldehyde oxidase in Donryu strain rats obeys Mendelian heredity. The reason for a low ratio of the ultrarapid metabolizers rats in the commercially available Donryu strain rats — not more than several per cent — compared with the ratio expected from the Mendelian rule is unknown.

Construction and Expression of Mutant cDNAs Responsible for Genetic Polymorphism in Aldehyde Oxidase in Donryu Strain Rats

We demonstrated the genetic polymorphism of aldehyde oxidase (AO) in Donryu strain rats: the ultrarapid metabolizer (UM) with nucleotide mutation of (377G, 2604C) coding for amino acid substitution of (110Gly, 852Val), extensive metabolizer (EM) with (377G/A, 2604C/T) coding for (110Gly/Ser, 852Val/Ala), and poor metabolizer (PM) with (377A, 2604T) coding for (110Ser, 852Ala), respectively. The results suggested that 377G > A and/or 2604C > T should be responsible for the genetic polymorphism. In this study, we constructed an E. coli expression system of four types of AO cDNA including Mut-1 with (377G, 2604T) and Mut-2 with (377A, 2604C) as well as naturally existing nucleotide sequences of UM and PM in order to clarify which one is responsible for the polymorphism. Mut-1 and Mut-2 showed almost the same high and low activity as that of the UM and PM groups, respectively. Thus, the expression study of mutant AO cDNA directly revealed that the nucleotide substitution of 377G > A, but not that of 2604C > T, will play a critical role in the genetic polymorphism of AO in Donryu strain rats. The reason amino acid substitution will cause genetic polymorphism in AO activity was discussed.

Lack of Formation of Aldehyde Oxidase Dimer Possibly Due to 377G>A Nucleotide Substitution

In addition to the many articles reporting on the marked differences in species and large differences in rat strains in response to aldehyde oxidase (AO), individual differences in some rat strains have also been reported. However, little has been clarified about any related molecular biological mechanisms. We previously revealed that nucleotide substitutions of 377G>A and 2604C>T in the AO gene might be responsible for individual differences in AO activity in Donryu strain rats. By using native polyacrylamide gel electrophoresis/Western blotting in this study, the lack of formation of the AO dimer protein, which is essential for catalytic activity, was shown in poor metabolizer Donryu rats, and this could be a major reason for the individual differences. Rat strain differences were also verified from the same perspectives of nucleotide substitutions and expression levels of a dimer protein. Rat strains with high AO activity showed nucleotide sequences of (377G, 2604C) and a dimer protein. In the case of those with low AO activity, the nucleotide at position 2604 was fixed at T, but varied at position 377, such as G, G/A, and A. An AO dimer was detected in the liver cytosols of rat strains with (377G, 2604T), whereas a monomer was observed in those with (377A, 2604T). These results suggest that the lack of formation of a dimer protein leading to loss of catalytic activity might be due to 377G>A nucleotide substitution. Individual and strain differences in AO activity in rats could be explained by this 377G>A substitution, at least in the rat strains used in this study.

Lipid peroxidation in the liver of carcinogen-resistant rats

Recently, we developed a new strain of rats that exhibit marked resistance to the hepatotoxic and carcinogenic actions of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) and some other carcinogens. In this work, we compared lipid peroxidation in the liver of these carcinogen-resistant (R) rats and the parental Donryu strain rats that are sensitive (S) to hazardous actions of these carcinogens. The liver microsomal fractions of the R group contained less amounts of polyunsaturated fatty acids. Microsomal lipid peroxidation in the presence of exogenous NADPH was much lower in R rats than in S rats. Liver microsomes of R rats were much less active than those of S rats also in producing 4-hydroxynonenal, carbonyl compounds and conjugated diene. The hepatic contents of ascorbic acid, glutathione, α-tocopherol and coenzyme Q in the R rats were similar to those in S rats. The activities of the free radical scavenger enzymes, Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), in the two groups were also similar. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are both thought to function in disposal of these cytotoxic aldehydes. The liver microsomal and mitochondrial ALDH activities of the two groups were similar. The ADH activity of the liver cytosolic fraction of R rats was nearly twice that of S rats, as measured with 4-hydroxynonenal as substrate. The higher ADH activity may explain the decreased lipid peroxidation in R rats at least partly, if this enzyme is involved in lipid peroxidation.

Allergen-Induced Biphasic Bronchoconstriction in Rats

The development of an allergic bronchoconstriction model in rats is described. In actively sensitized Donryu strain rats, there was a remarkable biphasic increase in airway resistance within 10 min after antigen challenge on day 9 to day 21. The increase in airway resistance, correlated with the IgE titer and the dose of antigen, was inhibited by disodium cromoglycate (DSCG) or by aminophylline. This bronchoconstriction was remarkably blocked by methysergide (25 and 100 micrograms/kg) while pyrilamine inhibited it partially at the same dose. Serotonin (greater than 30 micrograms/kg) but not histamine (less than 1,000 micrograms/kg) induced a bronchoconstriction. FPL-55712 (1,10 mg/kg) inhibited it significantly. The content of thromboxane B2 (TxB2) in plasma increased during the bronchoconstriction while the content of peptide-leukotrienes (p-LTs) in plasma did not increase significantly. OKY-046 inhibited not only allergic bronchoconstriction but also the increase in TxB2 levels in plasma. The late phase of the bronchoconstriction was more susceptible to OKY-046. In conclusion, this model seems to be useful for the screening of antiasthma drugs because of a relationship with the dose of antigen, IgE titer and the susceptibility to an antiallergic drug or a bronchodilator. It is demonstrated that the major part of this allergic bronchoconstriction depends on serotonin, and it is also suggested that thromboxane A2 may play an important role in the late phase of the bronchoconstriction.

Inhibitory effect of fumaric acid on 3'-methyl-4-(dimethylamino)azobenzene-induced hepatocarcinogenesis in rats.

Fumaric acid (FA) suppressed the carcinogenesis in the liver of rats fed 3'-methyl-4-(dimethylamino)azobenzene (3'-Me-DAB), and a study was performed to examine the effect of FA on deoxyribonucleic acid (DNA) synthesis and subcellular structures of hepatocytes under the anticarcinogenic regimens. Male Donryu strain rats were given 3'-Me-DAB by being fed a diet containing 0.06% 3'-Me-DAB for 50 d. They were then given a diet containing 1% FA and drinking water containing 0.025% FA for 53 to 69 weeks. Hepatocytes were isolated from the liver by the collagenase perfusion method and placed in culture, and their activity for DNA synthesis was measured in terms of the incorporation of [3H]dThd into DNA. An enhanced DNA synthesis of hepatocytes was noted in the rats given FA, indicating that FA enhanced the proliferation of hepatocytes to counteract the carcinogenic effect of 3'-Me-DAB. An electron microscopic examination indicated that the distribution of subcellular organella was almost normal in the FA-treated hepatocytes.

Effect of the dietary alpha-linolenate/linoleate balance on lipid compositions and learning ability of rats. II. Discrimination process, extinction process, and glycolipid compositions.

Donryu strain rats through two generations were fed semi-purified diets supplemented with safflower seed oil (rich in linoleic acid) or with perilla seed oil (rich in alpha-linolenic acid), or a conventional laboratory chow (normal control diet). Brightness-discrimination learning ability was determined to be the highest in the perilla oil-fed group, followed by the normal group, and then by the safflower group, extending our earlier observation in a different strain of rat that alpha-linolenic acid is a factor in maintaining high learning ability (Yamamoto, N., M. Saitoh, A. Moriuchi, M. Nomura, and H. Okuyama. 1987. J. Lipid Res. 28: 144-151). After the brightness-discrimination learning test was administered, extinction of learning was measured. The time required for extinction was significantly longer in the safflower group than in either the perilla group or the normal diet group. Thus, the dietary alpha-linolenate/linoleate balance affected both the learning and the extinction of learning. The glycolipids of the cerebrum, cerebellum, and olfactory lobe were analyzed. Although the fatty acid compositions of the sulfatide and gangliosides were significantly different in the three parts of the brain, relatively little difference was observed in the fatty acids of glycolipids between the safflower group and the perilla group, suggesting that gross changes in brain glycolipids are not responsible for the differences in learning abilities between these dietary groups.

The different effects of recombinant human tumor necrosis factor on rat fibrosarcoma sublines.

The antitumor effect of recombinant human tumor necrosis factor (rH-TNF) on two clones of rat fibrosarcoma with different metastatic potential to lymph nodes was examined. The colony formation of clone A, which has high metastatic potential, was completely inhibited by continuous exposure to rH-TNF at 50 U/ml. In contrast, colony formation of clone G, which has low metastatic potential, was not inhibited by high concentrations of rH-TNF (10,000 U/ml). The inhibitory effect of rH-TNF on colony formation by clone A was also observed with a 1-h exposure to rH-TNF. This effect was time and concentration dependent, as determined by the colony assay, 3H-thymidine uptake assay, and 51Cr-release assay. 3H-thymidine and 3H-uridine uptake per cell of clone A exposed to rH-TNF was not decreased. This suggests that the mechanisms of the antitumor effect of rH-TNF were not due to inhibition of DNA and RNA synthesis of tumor cells. In vivo growth and lymph node metastases of clone A inoculated i.p. to Donryu strain rats were completely suppressed by 14 consecutive i.p. injections of 10(5) or 10(6) U/kg per day of rH-TNF. On the other hand the growth of clone G was not influenced by rH-TNF administration.

Decreased induction of gamma-glutamyltransferase activity by 3'-methyl-4-dimethylaminoazobenzene in the liver of rats given carcinogen-containing diet for several generations.

gamma-Glutamyltransferase (G-GT) has been widely used as a marker of the preneoplastic stage of chemical carcinogenesis. We obtained male rats of Donryu strain that showed a reduced response to 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) in terms of induction of G-GT activity in the liver, by giving rats of this strain diet containing the carcinogen for several generations. In these animals, hepatic G-GT activity was only slightly higher than normal even after more than 20 weeks of continuous post-weaning exposure to the carcinogen, and it decreased to below the normal level if administration of the carcinogen was discontinued. In normal male rats, administration of diet containing 3'-MeDAB for more than 2 weeks resulted in an increase in hepatic G-GT activity, and the activity was increased further by a single injection of hydrocortisone. This response to glucocorticoid was also lost in "3'-MeDAB-resistant rats." Transient elevation of fetal hepatic G-GT activity occurred at the end of gestation of "resistant rats" as well as normal rats, but the highest activity at birth of the "resistant rats" was significantly lower than that of normal rats. We observed retardation of tumor development in the liver of fourth and fifth generation rats maintained continuously on diet containing 3'-MeDAB.

Effects of streptococcal preparation OK-432 on chemically-induced intestinal carcinoma in rats.

Intestinal tumors were induced by repeated subcutaneous injection of 1,2-dimethylhydrazine in Donryu strain rats for 28 weeks. A streptococcal preparation, OK-432 was administered at different times of the tumor induction_ Between the OK-432 treated and the untreated groups no significant difference was observed in the incidence of intestinal tumors, the total number of tumors per rat, the location of tumors, or the survival time. Though low incidence of extra· intestinal malignancies in the OK-432 treated animals seemed to be the only positive finding, it was not statistically significant.

STUDIES ON THE MORPHOGENESIS OF THE TUMORS EXPERIMENTALLY INDUCED BY ETHYLNITROSOUREA WITH SPECIAL REFERENCE TO THE LIGHT AND ELECTRON MICROSCOPY OF MICROTUMORS

Tumors of the nervous system were induced in rats of Donryu strain by a single injection of 50mg/kg.b.w. of ethylnitrosourea (ENU) through the mothers. A total of 126 tumors were produced in 92 of 117 treated offsprings. The survival time of animals with tumor development ranged from 53 to 607 days. The neural tumors amounted 104, comprising 82.5% of the total number of neoplasms. Besides grossly visible tumors, histological examination of the brain and spinal cord disclosed a total of 166 foci of glial cell hyperplasia or tumors of microscopic size. On histological sections 86 of these lesions measured less than lmm in diameter, 61 between 1 and 2mm, and 19 more than 2mm. The predominant cell type of the smaller lesions was either astrocytic or oligodendroglial. With increasing tumor size, a number of microtumors with a mixed cellular composition became frequent. Sites of predilection for such microtumors were the subcortical white matter of the brain, the hippocampus, the paraventricular areas, the basal ganglia, and the spinal cord. It would seem that grossly visible tumors are formed by expansion and fusion of these lesions of microscopic size and by development of anaplasia of constituent glial cells.Light and electron microscopic studies were also made on the brain and the trigeminal nerve of rats treated transplacentally with ENU at intervals of 10, 35, 80, 120, 150, 200, 250, 300 and 400 days after birth. A total of 48 microtumors were found in the brain of rats which had survived over 120 days. Electron microscopy disclosed these lesions to be largely composed of cells with ultrastructural features of astrocyte and oligodendroglia. The presence of a large amount of extracellular matrix was considered in part to account for the honeycombed structure typical of oligodendroglial microtumors in light microscopy. Observations of the trigeminal nerve disclosed occasional foci of diffuse endoneurial cell proliferation which started to arise from the 80th day. At the ultrastructural level, the proliferating cells were found to retain a typical Schwann cell appearance and relationship to axon. The nerve fiber was often surrounded by numerous cell processes, forming onion bulb-like structure.The third experiment was undertaken to investigate the acute cytotoxic effect of ENU on the nervous system. A single dose of 50 or 100mg ENU was given to rats perinatally and the nervous system was examined with light and electron microscopy at intervals from 6 to 48 hours after injection. A considerable proportion of cells constituting subependymal matrix layer of the lateral ventricle and extragranular layer of the cerebellum were found undergoing degeneration with disappearance of mitotic figures after 6 to 12 hours. Schwann cells in the trigeminal nerve and glial cells of the cerebral white matter were also found similarly involved. Neurons seemed to be less affected.From these observations it was concluded that oligodendroglia, astrocytes and Schwann cells are the most susceptible targets in the brain and nerve to carcinogenic nitrosourea compounds.

Esophageal carcinoma in rats induced by N-amyl-N-methylnitrosamine.

N-Amyl-N-methylnitrosamine (AMN) induced esophageal carcinoma in Donryu strain rats when given in drinking water. The incidence of carcinomas was high when AMN was given as 0.0015% solution for 90 days or 0.003% solution for 60 days. There was no difference in the incidence in males and females. Sequential studies showed that 4-week administration was necessary to induce esophageal papillomas, and 8-week administration to induce esophageal carcinomas. Whole-body autoradiography after intravenous injection of N-amyl-N-methyl[14C]-nitrosamine (14C-AMN) indicated that AMN accumulated preferentially in the esophagus.

Effect of Propionibacterium acnes or BCG on enzyme activities in spleen lymphocytes of Donryu strain rats.

Immunopotentiated rats, which were injected with Propionibacterium acnes or BCG, had the 50% survival twice as long as those in untreated controls after intravenous inoculation of Sato lung carcinoma (SLC) cells. The amount of labeled tumor cells in the lung of the adjuvant-treated rats decreased significantly in the first 20 hr after intravenous injection of 51Cr-labeled tumor cells compared to that of control animals. The elevated activities of ATPase and acid phosphatase in the whole nucleated spleen cells as well as spleen lymphocytes separated by Ficoll-Conray gradient were also demonstrated in adjuvant-treated groups. These data suggested that the elevation of ATPase and acid phosphatase activities in nucleated spleen cells as well as spleen lymphocytes has an important role for the suppression of tumor growth in adjuvant-treated rats.

Immunosupressive activity of the basic protein from tumor tissue.

The basic protein prepared from AH-130 ascites hepatoma cells was tested for its effect on the cell-mediated immune response of Donryu strain rats. The basic protein suppressed the response of peripheral and lymph node lymphocytes to phytohemagglutinin and lymphocyte trapping in the antigen-draining lymph node. These results indicated that the basic protein from tumor tissue possibly had an immunosuppressive activity and affected inadequately the cell-mediated immune response as usually observed in tumor-bearing hosts.

Inhibition of syngeneic tumor growth in rats immunized with allogeneic skin grafts

The growth of a transplantable tumor (KMT-17) in syngeneic Wistar King Aptekman/Mk (WKA) rats was inhibited by preimmunization with allogeneic normal cells from Donryu strain rats. The phenomenon is referred to as allogeneic cell immunity. A slight inhibition was observed in rats immunized with normal liver, spleen, kidney, embryonal cells, whole blood and white blood cells from allogeneic Donryu rats. A strong inhibition was observed in animals which had rejected allogeneic skin grafts, particularly from the Donryu and Kyoto rats. The inhibition was comparatively weak after immunization with skin grafts from the Long Evans, ACI, Buffalo, Ficsher, Sprague Dawley or Tokyo allogeneic rat strains. This immunizing effect was dependent on the viability of the grafts and was easily abrogated by low-dose irradiation. The mechanism of allogeneic cell immunity is considered to be a non-specific stimulation of the immunity against antigenic tumors in the syngeneic host

Effect of sulfated polysaccharides on blood-borne pulmonary metastasis in rats.

The inhibitory effect of sulfated polysaccharides on blood-borne metastasis was examined. As a model of blood-borne metastasis, the ascitic form of hepatoma AH-109A tumor was injected intravenously into Donryu strain rats. Examination of the pulmonary metastatic nodules developed 2 weeks later showed inhibitory effect of the five sulfated polysaccharides tested. Xylan sulfate was the most inhibitory, and exerted its inhibitory effect when the tumor cells were in the pulmonary capillary beds. However, fromthe rapid disappearance of radioactivity from the lungs after injection of 125IUDR-labeled AH-109A cells, tumor cells seemed to be retained in the lungs for only a very short time. Measurement of the anticoagulative and fibrinolytic activities of three sulfated polysaccharides showed that the inhibitory effect of these compounds on blood-borne metastasis was proportional to their anticoagulative and fibrinolytic activities, xylan sulfate showing the highest activities. These results suggest that sulfated polyaccharides may inhibit blood-borne pulmonary metastasis by inhibiting the lodging of tumor cells in the pulmonary capillary beds.

Polychlorinated biphenyl(s) as a promotor in experimental hepatocarcinogenesis in rats

The effects of dietary polychlorinated biphenyls (PCBs) on hepatocarcinogenesis in female rats of Donryu strain receiving 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) were investigated. Oral administration of PCBs after administration of 3'-Me-DAB resulted in a high incidence (64%) of hepatocarcinoma. In contrast, administration of a similar amount of PCBs before, or together with 3'-Me-DAB did not induce hepatic tumors. Administration of a slightly larger amount of PCBs alone did not induce liver tumors, while administration of a slightly larger amount of 3'-Me-DAB alone caused only a low incidence (13%) of hepatocarcinoma. These results strongly suggest that PCBs exert a potent promoting action in experimental azo dye hepatocarcinogenesis.

HISTOPATHOLOGICAL STUDIES OF NERVOUS SYSTEM TUMORS IN RATS INDUCED BY TRANSPLACENTAL ADMINISTRATION OF ETHYLNITROSOUREA

Sixteen pregnant rats of Donryu Strain were given intravenously with a single dose of 50 mg/ kg of Ethylnitrosourea (ENU) on the 15th day (Group I, 9 rats) and the 20th day (Group II, 7 rats) of gestation.A total of 126 tumors (Group I, 61 and Group II, 65) were produced in 92 of 117 treated offspring which had survived into tumor bearing age. The mean survival time of Group I rats with tumor developement was 317 days after brith and that of Group II rats was 276.The sites of the total of 126 tumors were formed as following : 27 were produced in the brain (Group I, 21 and Group II, 6), 21 in the spinal cord, 42 in the cranial nerve and ganglia, 8 in the spinal nerve roots, 6 in the peripheral nerves and 22 in the extraneural organs. The neural tumors amouted 104, comprising 82.5% of the total number of the neoplasm.Significant differences in incidence of the tumors of the brain and those in extraneural organs were demonstrated between Group I and Group II rats.According to the cytology and the pattern of predominant tumor cells, tumors of the brain and spinal cord were classified into the following types : mixed glioma (18), oligodendroglioma (18), anaplastic astrocytoma or anaplastic glioma (6), ependymoma-like tumor (5) and unclassified tumor (1). The histology of mixed glioma and oligodendroglioma were most common. 5 tumors produced in the spinal cord were classified as ependymoma-like tumor and they showed an isomorphic cell appearance with such architectural characteristics as crowns of cell nuclei around vascular vessels and pseudorosettes. No tumors were found with typical ependymal rosettes.Almost all the cranial nerve tumors were located in the trigeminal nerve and Gasserian ganglia, microscopically the majority of neurinomas were highly anaplastic with friquent mitotic figures.Besides grossly visible tumors, histological examinations of the brain and the spinal cord disclosed a total of 138 (Group I, 84 and Group II, 54) foci of glial cell hyperplasia or tumors of microscopic size. Sites of predilection for such foci or small microtumors were the subcortical white matter of the brain, the basal ganglia, the spinal cord, the hippocumpus and the periventricular areas. The predominant cell type of such lesions was either oligodendroglial or astrocytic glia. With increasing tumor size, a number of microtumors with a mixed cellular composition became frequent. There are no focus of the ependymal cell hyperplasia.The transplantation experiment demonstrated that anaplastic neurinomas were highly transplantable and capable of subsequent transfers through a number of generations.This experiment demonstrated that a single transplacental exposure of ENU was capable of inducing neuroectodermal tumors, and probably taget cells of it were the astroglia, the oligodendroglia and Schwann's sheath cells.

Histopathological studies of the nervous system tumors in rats induced by N-nitroso-methyl-urea.

Histopathological examinations were made on tumors of the nervous system induced in rats of Donryu strain by weekly intravenous injections with N-nitroso-methyl-urea (NMU) or by a single administration of NMU through the mothers. A total of 176 neural and nonneural neoplasms were produced in this study. It was suggested that the fetal nervous system of Donryu rats was also highly susceptible to the oncogenic effects of NMU. Of these tumors produced, those of the peripheral nervous system amounted to 121, comprising 68.7% of the total number of the neoplasm. Microscopically, most of the nerve tumors showed the histology corresponding to that of human neurinomas. Many tumors, however, disclosed more or less anaplastic cytological appearance. Fifteen gliomas were produced in the brain and spinal cord. Microscopically, they were classified into mixed glioma, oligondendroglioma and anaplastic astrocytoma. The commonest brain tumors produced in rats from intravenously treated group were periventricular mixed gliomas, while gliomas in rats from transplacentably treated group showed an isomorphic histology with a close resemblance to that of oligodendroglioma.

Effect of oil-attached BCG cell wall on the kinetics of lymphocytes in the tumor-draining node.

The lymphocyte distribution into the tumor-draining node was studied with AH-130 hepatoma cells and Donryu strain rats in relation to the adjuvant activity of the oil-attached BCG whole cell wall. The mixed inoculation of the oil-attached BCG cell wall with tumor cells resulted initially in further augmentation of increased distribution of 51Cr-labeled lymphocytes into the draining-node induced by inoculation of the tumor cells alone, and secondarily in the systemic stimulation of response of the lymph node lymphocytes to phytohemagglutinin. Suppression of the inoculated tumor growth and lymph node metastasis was finally observed. These results were discussed in connection with the therapeutic effect of BCG and its cell wall fraction.