Two-year carcinogenicity study of 6-mercaptopurine in F344 rats

Summary The carcinogenicity of 6-mercaptopurine (6-MP), an anticancer drug, was examined in F344 rats of both sexes, administered the chemical at dietary levels of 0 (control), 25 ppm or 50 ppm for 2 years. Many tumors developed in all groups including the control group, the organ distribution and histological types being similar to those reported for spontaneous lesions. In males, there was no significant increase in the incidence of any tumor in the treated groups over that in the control group. In females, however, positive trends were noted in the occurrence of C-cell tumors, pheochromocytomas, uterine adenocarcinomas and gliomas, and the incidences of C-cell tumors and pheochromocytomas in the 50 ppm group were significantly higher than the values in the respective control group. In addition, the total numbers of malignant tumors increased significantly in the female 50 ppm group. However, most of the tumors demonstrating increase are frequently observed spontaneous lesions in this strain of rats, and their incidences in the present female control group were lower than in our historical data. In addition, there were no significant differences in the incidences of preneoplastic changes and induction times for the above-listed tumors between the female control and the 50 ppm groups. These results thus indicated that while the carcinogenic potential of 6-MP can not be precluded, it can be only very weak or marginal, after continuous administration in the diet at the 50 ppm level for 2 years. The leukemogenic action of 6-MP was negative under the present experimental conditions.Abbreviation 6-MP 6-mercaptopurine This study was supported by grants-in-aid for cancer research from the Ministry of Health and Welfare of Japan. Part of this work was presented at the 47th Annual Meeting of the Japanese Cancer Association in Tokyo, September 1988     

Glial glucocorticoid receptors in aged Fisher 344 (F344) and F344/Brown Norway rats

Glucocorticoid receptors (GR) regulate glial function, and changes in astrocyte gene expression are implicated in age-related pathology. We evaluated changes in astroglial GR expression in two strains of rats – Fisher 344 (F344; 4, 12 and 24 months) and F344/Brown Norway strain (F344/BN; 4, 12 and 30 months). In both strains basal levels of corticosterone were higher in the oldest groups of rats. Age-related increases in GR (+) astrocytes but not the percent of astrocytes expressing GR were observed in the hippocampus CA1 region in F344 rats. Age-related decreases in CA1 GR (+) astrocytes and the percentage of GR (+) astrocytes were observed in the F344/BN strain only. Similar strain-specific changes were observed in the dentate gyrus. In the hypothalamic paraventricular nucleus: (1) F344 rats exhibited significant decreases in the overall number of glial profiles with age, (2) F344/BN rats exhibited decreases in the numbers of GR (+) astrocytes with aging and (3) the proportion of GR (+) astrocytes decreased in older F344/BN, but not F344 rats. Overall, the data demonstrate age- and strain-related alterations in GR astrocytic expression that may explain unique phenotypic differences in brain function observed in both strains.     

Aging change of mandibular condyle in female F344/N rat

To ascertain whether laboratory rats represent an adequate animal model for aging oral cavity research, this study focused on the morphology of the mandibular condyle. Aging changes of cartilaginous conformation and shape of the mandibular condyles were analyzed in female F344/N rats. In the condylar cartilage, articular, proliferative cell and hypertrophic cell layers were observed in 1-month-old (M) rat, whereas flattened cell layer was notable at 2 M. A mature cell layer was observed in the condylar cartilage of rats at 7.7 M and over. Deranged cartilaginous layers and thinning articular layer were observed in 30.9 M rat. The sagittal length of the condyle decreased, whereas the frontal one increased with aging and/or age. There were three phases in the transition patterns of the size of the condyle, which seemed to correspond to the respective growing, aging and senescence phases in the rat. The results suggested that degenerative change of condylar cartilage in rat was similar to that in human, whereas change of the shape of the condyle was different between rat and human, caused by a different pattern of mastication.     

Aging changes in the periodontal bone of F344/N rat

The aim of this research was to determine whether a rat was an adequate laboratory animal model for periodontal research on elderly humans. Thirty-two F344/NSlc female rats ranged between 30 and 1000 days of age were used. The alveolar bone loss around the molars was assessed by a morphometric method. A significant correlation was found between age and the amount of alveolar bone loss. For further analysis, the rats were grouped into four by age; 30–60 days, 220–430 days, 640–850 days, and more than 850 days. The means of alveolar bone loss were compared between age groups. It was found that the resorption of the alveolar bone around the molars of the rats continued until they were 1000-days-old, and this trend was stronger in the mandible than the maxilla. It was suggested that rats could be used as adequate laboratory animals for periodontal research.     

Induction of digestive-tract tumors in F344 rats by continuous oral administration of N-butyl-N-nitrosourea

Summary Male and female F344/DuCrj rats were administered N-butyl-N-nitrosourea at a concentration of 400 ppm in their drinking water. By the 50th week of the experiment, the cumulative incidence of upperdigestive-tract tumors was as high as 35/39 (90%) and 34/39 (87%) in male and female rats, respectively. Among these, esophageal and forestomach tumors occurred most frequently. Except one female rat with fibroma, upper-digestive-tract neoplasms were of the epithelial type—papilloma, squamous-cell carcinoma or adenocarcinoma. In female rats, vaginal tumors were induced in 16 (41%) animals. Ear-duct tumors and hematopoietic neoplasms were also induced in both sexes of rats, with incidence of less than 21%.This study was supported in part by a grant-in-aid for Cancer Research from the Ministry of Education, Science, and Culture, Japan     

Carcinogenicity studies of some analogs of the carcinogen methapyrilene in F344 rats

Summary Four antihistaminic drugs similar in structure to the rat liver carcinogen methapyrilene were administered to comparable groups of male and female F344 rats in their drinking water for most of their lifetime (80–108 weeks). The concentrations were 0.1% or 0.05% and the total doses received by the animals were comparable with that of methapyrilene which induced 100% incidence of liver neoplasms. No increase in incidence of liver neoplasms was observed after treatment with any of the four compounds, thenyldiamine, chlorothen, methafurylene, or methaphenilene, although each differed structurally from methapyrilene only in one atom or one position of substitution. There were a few animals with neoplasms not usually found in untreated F344 rats, but none of these was found in statistically significant numbers. These results suggest that none of the four analogs of methapyrilene was carcinogenic under the conditions of this study, and that the property of inducing liver neoplasms in rats was confined to the intact methapyrilene molecule.Research sponsored by the National Cancer Institute, DHHS, under contract No. NO1-CO-23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Service, nor does mention of trade names, commercial products or organizations imply endorsement by the U.S. GovernmentProgram Resources Inc., NCI-Frederick Cancer Research Facility P.O. Box B, Frederick, MD 21701, USA     

Establishment of an Aging Farm of F344/N Rats and C57BL/6 Mice at the National Institute for Longevity Sciences (NILS)

In 1996 the National Institute for Longevity Sciences (NILS) started an aging/aged farm (Aging Farm), an animal farm for producing aging/aged laboratory rodents on inbred strains, F344/N rats and C57BL/6 mice at its Experimental Animal Facility Wing, based on plans prepared by the Laboratory Animal Research Facilities (LARF). The NILS Aging Farm, being well established, began internal supply of aging/aged laboratory rodents in 1999 to promote both aging and longevity science. This report describes development of the NILS Aging Farm under NILS Aging Farm Guide and the effectiveness of the Guide.     

Dose-response study of the carcinogenicity of tobacco-specific N-nitrosamines in F344 rats

Summary Tobacco and tobacco smoke contain relatively high amounts of four tobacco-specific N-nitrosamines. Of these, N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosoanatabine (NAT) were bioassayed at three dose levels by subcutaneous injections into male and female F344 rats in 60 subdoses amounting in total to 9, 3, and 1 mmol/kg. Compared with the solvent control group (trioctanoin), both NNN and NNK induced significant numbers of tumors of the nasal cavity (P<0.01) at all three dose levels in both male and female rats. Significant numbers of tumors were also induced by NNK in the lung at all three dose levels and in the liver at the highest dose level (P<0.05). In addition to nasal tumors NNN also induced esophageal tumors at a significant rate in male rats at the high and medium dose levels and in female rats at the high level (P<0.05); NAT was inactive at the three doses tested. Bioassays at lower dose levels as well as biochemical studies are strongly indicated for NNN and NNK since these nitrosamines occur in relatively high amounts in both chewing tobacco and tobacco smoke.Abbreviations HPLC high performance liquid chromatography - NAB N-nitrosoanabasinc - NAT N-nitrosoanatabine - NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone - NNN N-nitrosonornicotine Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthdayThis is No. XXVII of A Study of Tobacco Carcinogenesis. The study is supported by U.S. National Cancer Institute Grant CA-29580We thank Ms. Maria Nicholais and Mr. Joel Reinhardt from the Research Animal Facility for their excellent technical assistance     

Induction of lung tumors and peritoneal mesotheliomas in F344 rats given intragastric N-propyl-N-nitrosourea and histochemical,immunohistochemical, and ultrastructural characteristics of induced mesotheliomas

Summary N-Propyl-N-nitrosourea is a strong leukemogen that induces myelogenic leukemia in Donryu rats and thymic lymphoma in F344 rats when administered in drinking water. In the present study, a single or multiple doses of PNU (total 500 mg/kg body weight) was given to young male and female F344 rats via a stomach tube. The results demonstrated that the percentage of tumor-bearing rats was 100% in all PNU-treated male groups, while that of the control group was 46%. Predominant tumors induced by PNU in male rats were lung adenoma/adenocarcinoma followed by peritoneal mesothelioma, and forestomach papilloma. In females, the tumor incidence of PNU-treated groups varied between 58% and 92% while that of the control group was 42%. Although pituitary tumor was the most frequent tumor in PNU-treated female rats, it was thought to be spontaneous since its incidence in each experimental group was not statistically different from that of the control group. Lung tumors and forestomach papillomas were also induced by PNU in female rats. No thymic lymphoma, however, was found in any of the PNU-treated groups of either sex. Lung tumors developed in almost all PNU-treated male rats and in about one-third of PNU-treated female rats. Mesothelioma was induced only in male rats, and its incidence depended on the treatment schedule. Induced mesotheliomas were extensively examined histologically, histochemically, immunohistochemically, and electron microscopically.Abbreviations used PNU N-propyl-N-nitrosourea - DMSO dimethyl sulfoxide - PAP peroxidase-anti-peroxidase     

Carcinogenicity and organ specificity of N-trimethylsilylmethyl-N-nitrosourea (TMS-MNU), N-neopentyl-N-nitrosourea (neoPNU), and N-methyl-N-nitrosourea (MNU) in rats

Summary The carcinogenicity and organ specificity of TMS-MNU and neoPNU, a carbon-analogue of TMS-MNU, in rats were investigated and compared with those of MNU. Compounds were dissolved in olive oil and rats in the experimental groups received 20 weekly intragastric intubations of 10 mg/kg of MNU or equimolar amounts of TMS-MNU or neoPNU in the same manner. The experiment was terminated when the survivors were sacrificed at the 52nd week after the final adminstration. In the TMS-MNU and MNU groups, tumors of the forestomach were induced and the incidence was 100% in the groups of both sexes. In addition, tumors of the glandular stomach, nervous system, kidney, and lung were also observed in these groups. Neurogenic tumors were found more frequently in the MNU group than in the TMS-MNU group. The incidence of lung tumors, however, was higher in the TMS-MNU group than in the MNU group. On the other hand, in the control and neoPNU groups, no tumor was found in these organs except the lung, and all tumors observed in these two groups were histologically similar to spontaneous ones in this strain of rats. These results indicate that the carcinogenicity of N-alkyl-N-nitrosoureas is dependent on the chemical structure of their alkyl chain. The result of the present study coincides with the previous result that the species of TMS-MNU in the alkylating step is the same as that of MNU, but different from neoPNU. The difference in the organ specificity between TMS-MNU and MNU demonstrates that the organ specificity is dominantly dependent on the distribution of the chemicals, since TMS-MNU may possibly be distributed differently from MNU because of its different partition property.Abbreviations TMS-MNU N-trimethylsilylmethyl-N-nitrosourea - neoPNU N-neopentyl-N-nitrosourea - MNU N-methyl-N-nitrosourea Part of this work was presented at the 46th Annual Meeting of the Japanese Cancer Association in Tokyo September, 1987     

Morphological and immunohistochemical characteristics of dimethylnitrosamine-induced malignant mesenchymal renal tumor in F-344 rats

Summary Mesenchymal renal tumors in F-344 newborn rats were induced by a single dose of dimethylnitrosamine. The induced tumors were successfully transplanted into adult rats under the renal capsule. Neither the primary nor the transplanted neoplasms from various generations of grafts changed their morphological features during the tumor passage, having the same cellularity with high mitotic activity and the tendency to invade the host kidney rapidly. On the basis of lectin histochemistry and immunohistology, the tumor proved to be a mesenchymal neoplasm without any obvious capacity of the proliferating cells to differentiate into any wellknown organoid element normally found in mature renal parenchyma. However, the proliferating neoplastic cells were found to have a strong vimentin positivity with desmin expression. Ultrastructurally, myofilaments with attachment bodies characteristic of smooth muscle cells were generally present in various amounts in many tumor cells. In addition, on the basis of the physiological data and on kidney/tumor renin activity obtained, it is interesting to note that the tumor-graft-invaded kidneys retained their enzyme activity, despite the obvious loss of renal tissue including glomeruli. However, the immunohistochemical findings with anti-renin antibody have clearly shown that this is not due to a renin-producing tumor but rather to the surviving (probably) non-neoplastic arterioles retaining the capacity to produce renin. Although these arterioles have mostly been found next to necrotic areas, commonly occurring in dimethylnitrosamine-induced transplantable renal tumors, the question of a possible physiological role of renin in tumor necrosis or in angiogenesis has remained open.Abbrevations DMN dimethylnitrosamine - F-344 Fischer-344 - WGA wheat germ agglutinin - PNA Arachis hypogaea (peanut) agglutinin - LTA Lotus tetragonolobus agglutinin     

Carcinogenicity of N-alkyl-N-(acetoxymethyl)nitrosamines after subcutaneous injections in F-344 rats

Summary As model compounds for metabolically activated N,N-dialkylnitrosamines, five N-alkyl-N-(acetoxymethyl)nitrosamines were synthesized and their carcinogenicy was testet in F-344 rats of both sexes. Compounds used in this study are N-methyl-(MAMN), N-ethyl-(EAMN), N-propyl-(PAMN), N-butyl-(BAMN), and N-isobutyl-N-(acetoxymethyl)nitrosamines (i-BAMN). All chemicals were dissolved in olive oil and rats received 10 weekly subcutaneous injections of these chemicals (10×5 mg MAMN or equimolar amounts of other chemicals) at the interscapular region. Subcutaneous tumors were detected in many rats of all groups treated with the chemicals, although no tumor was detected in the control group. Lung and/or thyroid tumors were also observed in many rats in the experimental groups. The incidence of subcutaneous tumors was highest in EAMN, followed in order by MAMN, PAMN, BAMN, and i-BAMN. On the contrary, the incidence of lung and thyroid tumors was highest in MAMN and decreased as the length of the alkyl chain of the chemicals increased. Histologically, almost all subcutaneous tumors were malignant fibrous histiocytomas. The results indicate that the chemicals possess systemic as well as local carcinogenicity in F-344 rats. The potent carcinogenic effects at the injection site of the -acetoxy nitrosamines, coupled with their direct mutagenic activity reported previously, support the notion that these derivatives are useful as models for the ultimate form in the metabolic activation of N,N-dialkylnitrosamines.Supported by a grant-in-aid for cancer research from the Ministry of Education, Science, and Culture. Part of this study was presented at the 7th International Meeting on N-Nitroso compounds —Occurrence and Biological Effects, Tokyo, September–October, 1981 -and also at the 40th Annual Meeting of the Japanese Cancer Association, Sapporo, held in October, 1981     

Difference in average survival between F344/Du and F344/N rats is not due to genetic contamination

The average survival was found to be different between substrains of rats, F344/Du and F344/N. One of the causes of this difference could have been genetic contamination by crossbreeding with other strain(s) after the divergence of substrains. These two strains were examined by PCR-SSLP (simple sequence length polymorphism) procedure using microsatellite (Mit) markers to identify the contamination. All 288 Mit markers distributed throughout the genome exhibited no size polymorphisms between these two strains. This indicated that there was no genetic contamination between these two strains. The phenotypic difference in average survival, thus, is not due to genetic contamination.     

The diagnostic significance of left auricular thrombus in F344/N rats

In the process of assessing the main cause of death in F344/N rats from the aging farm of our institute, we have often found left auricular thrombus in autopsy cases of moribund animals. In 319 autopsy cases, 45 were of left auricular thrombus and 44 were accompanied by hematopoietic neoplasms, including overt leukemia and a pre-leukemic condition of leukemia. In cases without splenomegaly, this lesion was found in 13 of 21 animals (61.9%) whereas, in cases with splenomegaly, 31 of 239 were positive for this lesion (13.0%). Thus, left auricular thrombus may be an important macroscopic diagnostic criteria of hematopoietic neoplasms, especially when splenomegaly is absent. Furthermore, this lesion tended to arise in aged animals despite the presence of splenomegaly. These results would therefore greatly contribute to aging science by confirming the health condition of experimental rats and the accuracy of subsequent results.     

Age-related changes in the acoustic startle reflex in Fischer 344 and Long Evans rats

The behavioral consequences of age-related changes in the auditory system were studied in Fischer 344 (F344) rats as a model of fast aging and in Long Evans (LE) rats as a model of normal aging. Hearing thresholds, the strength of the acoustic startle responses (ASRs) to noise and tonal stimuli, and the efficiency of the prepulse inhibition (PPI) of ASR were assessed in young-adult, middle-aged, and aged rats of both strains. Compared with LE rats, F344 rats showed larger age-related hearing threshold shifts, and the amplitudes of their startle responses were mostly lower. Both rat strains demonstrated a significant decrease of startle reactivity during aging. For tonal stimuli, this decrease occurred at an earlier age in the F344 rats: middle-aged F344 animals expressed similar startle reactivity as aged F344 animals, whereas middle-aged LE animals had similar startle reactivity as young-adult LE animals. For noise stimuli, on the other hand, a similar progression of age-related ASR changes was found in both strains. No significant relationship between the hearing thresholds and the ASR amplitudes was found within any age group. Auditory PPI was less efficient in F344 rats than in LE rats. An age-related reduction of the PPI of ASR was observed in rats of both strains; however, a significant reduction of PPI occurred only in aged rats. The results indicate that the ASR may serve as an indicator of central presbycusis.     

Changes in hepatic polyamine catabolism in elderly rats

Abstract: Aims/Background: Given the important role of polyamines (putrescine, spermidine, spermine) in the modulation of macromolecular syntheses, gene expression and proteolysis, alterations in their metabolic pathways could be relevant during senescence. Since the few existing data address mainly polyamine biosynthesis, we studied the oxidative catabolism of polyamines in the liver of rats 3–36 months of age. Methods: Polyamine oxidase activity was fluorimetrically measured using N¹-acetyl-spermine as substrate. Spermidine/spermine N¹-acetyltransferase and diamine oxidase were measured by radiochemical methods using labeled acetyl-coenzyme A and putrescine, respectively, as substrate. Polyamines were separated by HPLC and fluorimetrically quantified after post-column derivatization with o-phthaldialdehyde. Results: Spermidine/spermine N¹-acetyltransferase activity increased in 36-month-old rats and polyamine oxidase activity in 24- and 36-month-old rats. A decline in spermine and increases in spermidine and putrescine in elderly rats suggested an activation of the interconversion pathway of higher into lower polyamines. The activity of diamine oxidase, which degrades putrescine, was enhanced starting from 12 months of age. Conclusion: In the liver of aged rats, an increase in the catabolic enzymes leads to a reconversion of the higher polyamines to putrescine. This increased catabolism may represent an important age-related change and may contribute to impairment of the expression of growth-related genes in senescence.     

Age-related change and its sex differences in histoarchitecture of the hypothalamic suprachiasmatic nucleus of F344/N rats

The present study examined the effects of aging and sex differences on the suprachiasmatic nucleus (SCN) of F344/N rats. In juveniles (1.6-1.9 months of age), adults (11.7-16.3 months of age), and old (29.2-34 months of age) rats, the volume, size of neuronal nucleus and neuronal cell number of the SCN were determined with cresyl fast violet-stained sections. In addition, immunohistochemical analysis was performed for glial fibrillary acidic protein (GFAP). There was no significant effect of aging and sex differences on the SCN volume. The number of neurons in the SCN gradually decreased from juvenile to old age in females. However, in males, the number was significantly decreased in adult and old age rats. The size of neuronal nuclei in the SCN was significantly decreased by increasing age in both sexes, except for the ventrolateral part of the SCN of males. In the dorsomedial part of the SCN of females, the density of GFAP-immunoreactive components was significantly higher in adult age rats than in rats of other ages. However, there was no significant increase in the density of the SCN in adult males. These results suggest that morphological changes in neuronal and astroglial cells occur in the SCN with aging in a sex-specific manner.     

Effect of TRC and F/TRC Strengthening on the Cracking Behaviour of RC Beams in Bending

The increasing demand on the performance of existing structures, together with their degradation, is among the main drivers towards the development of innovative strengthening solutions. While such solutions are generally aimed at increasing the load-bearing capacity of structural elements, serviceability limit states also play an important role in ensuring the performance and durability of the structure. An experimental campaign was performed to assess the cracking behaviour of reinforced concrete beams strengthened with different typologies of Textile-Reinforced Concrete. The specimens were monitored using Digital Image Correlation (DIC) technology in order to obtain a quantitative evaluation of the evolution of the crack pattern throughout the whole test. Results show the beneficial effects of this retrofitting strategy both at ultimate limit states and serviceability limit states, provide detailed insights on the progression of damage in the specimens and highlight how different parameters impact the cracking behaviour of the tested elements.     

Dose-response study of urinary bladder carcinogenesis in rats by N-butyl-N-(4-hydroxybutyl)nitrosamine

Summary A dose-response study was carried out in male F344 rats with a specific urinary bladder carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), which was administered as a solution in drinking water for up to 112 weeks. BBN was given in four different concentrations, 50, 10, 5, and 1 ppm, to groups of 30 rats. The highest dose induced urinary bladder cancer in all rats treated by week 91, decreasing yields being associated with lower dose levels: an incidence of 76.7% was observed with 10 ppm, followed by 20.0% with 5 ppm. The lowest dose (1 ppm) only induced two cases of papilloma (6.9%).Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthdaySupported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan, the Japan Tobacco and Salt Public Corporation, and the Experimental Pathological Research Association