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Background data of 2-year-old male and female F344 gpt delta rats

Although gpt delta rats, as reporter gene-transgenic rats, were originally developed for in vivo mutation assays, they have also been used to evaluate chemical carcinogenesis and comprehensive toxicity. Therefore, it is necessary to accumulate background data on carcinogenicity and general toxicity in gpt delta rats. Here, we investigated the background data of 110-week-old male and female F344 gpt delta rats and wild-type rats. There was no effect of reporter gene transfection on animal survival rates and body weights during the experiment. The relative weight of male gpt delta rat adrenals was significantly higher than that of wild-type rats, possibly due to the higher incidence of pheochromocytoma. There were no intergenotype differences in the incidence of nonneoplastic lesions in both sexes, including chronic progressive nephropathy and focus of cellular alteration in the liver, which had a higher incidence in both genotypes. Additionally, the significantly higher incidence of adrenal pheochromocytoma in male gpt delta rats than that in wild-type rats was likely incidental because of the lack of differences in the incidences of preneoplastic (male and female) and neoplastic (female) adrenal lesions in both genotypes. Other neoplastic lesions in both sexes showed no intergenotype differences in incidence rates, although large granular lymphocytic leukemia in the spleen and Leydig cell tumors in the testes of males showed higher incidence rates. Overall, there were no effects of reporter gene transfection on the spectrum of spontaneous lesions in F344 gpt delta rats, thus supporting their applicability in evaluating chemical toxicity and carcinogenicity.


Introduction
Although various genotoxicity tests have been developed, in vivo mutation assays using reporter gene transgenic rodents are promising tools to evaluate chemical mutagenicity owing to the involvement of in vivo metabolism and target organs. The gpt delta rodents are an established reporter gene-transgenic animal model used for in vivo mutation assays and were originally developed by Nohmi et al. 1 . These gpt delta rats are transfected with 5-10 copies of lambda EG10, including a reporter gene, which insert themselves into chromosome 4 of the somatic and germ cells of systemic organs [2][3][4] . The F344, Sprague-Dawley (SD), and Wistar rat strains have been established as background strains for gpt delta rats. In particular, reporter gene mutation assays using gpt delta rodents have many advantages. For example, these rats can be used for the detection of point and deletion mutations via gpt and Spiassays, respectively 5 .
Detailed information on the types of mutations that are induced by chemicals has enabled us to utilize gpt delta rats to investigate the underlying mechanisms of carcinogenesis. In fact, combined assays for evaluation of DNA modification and/or the microenvironmental status of cells with reporter gene mutation assays have been used to provide evidence of the mechanisms of chemical carcinogenesis [6][7][8][9][10][11] . In addition, we have developed a medium-term animal model that is capable of simultaneously detecting in vivo mutagenicity and tumor-promoting activities in the liver and kidney 12,13 . However, these applications of gpt delta rats require knowledge of the sensitivity to chemical carcinogens of the background strain. Our previous studies have demonstrated that medium-term exposure of gpt delta rats to various hepatocarcinogens induced the development of glutathione Stransferase placental form (GST-P) foci to the same degree as those observed in wild-type (WT) rats [12][13][14][15] . As it is impossible to examine the carcinogenic sensitivity of the gpt delta rats to all chemical carcinogens, evaluation of spon-taneous tumor spectra could provide valuable information.
Notably, gpt delta rodents are able to provide data on the general toxicity of test chemicals and have enabled us to evaluate comprehensive toxicity, including general toxicity and in vivo genotoxicity, in a single study, conforming to the 3R's principle of animal use in laboratory experiments 16,17 . In a previous study of medium-term administration of some chemicals, toxicological responses in gpt delta rats were found to be identical to those in WT rats with the same background 18 . However, no reports have described spontaneous age-related lesions and their effects on chronic toxic responses.
In the current study, we aimed to obtain background data required to expand the applications of gpt delta rats by evaluating parameters related to general toxicity and carcinogenicity in gpt delta rats and WT rats of the same background strain (F344 rats).

Experimental animals and housing conditions
The protocol was approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences (Approval number: 208). Male and female 5-weekold specific pathogen-free F344/NSlc rats and F344/NSlc-Tg (gpt delta) rats carrying approximately five tandem copies of the transgene lambda EG10 per haploid genome were obtained from Japan SLC, Inc. (Shizuoka, Japan) and acclimated for 1 week prior to testing. The rats were housed in polycarbonate cages with hardwood chips for bedding in a conventional animal facility. Three or four rats were housed in each cage. Animals were maintained under controlled temperature (23 ± 2°C), relative humidity (55 ± 5%), air changes (12 times/h), and lighting (12-h light-dark cycle) conditions with free access to a basal diet (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water. Male and female 6-week-old F344 and F344 gpt delta rats were maintained for 104 weeks (n = 75). At the end of each experiment, the rats were anesthetized using isoflurane, then euthanized by exsanguination via transection of the abdominal aorta.

Organ weights and histopathological assessment
Complete necropsies were performed for all animals, and their internal organs, i.e., brain, thymus, lungs, heart, spleen, liver, adrenal glands, kidneys, and gonads, were weighed. These organs and additional tissues (skin, mammary gland, sternum with marrow, femur with marrow, mandibular and mesenteric lymph nodes, salivary glands, aorta, trachea, tongue, esophagus, stomach, small and large intestines, pancreas, urinary bladder, epididymis, seminal vesicles, prostate gland, bulbourethral glands, uterus, vagina, pituitary gland, thyroid glands, parathyroid glands, spinal cord with vertebrae, trigeminal nerve, sciatic nerve, harderian glands, femoral skeletal muscle, and nasal cavity) were fixed in 10% neutral buffer formalin, and paraffin-embedded sections were routinely prepared and stained with hematoxylin and eosin for histopathological examination.
Bony tissues, including the nasal cavity, vertebrae, sternum, and femur, were decalcified with a mixture of 10% formic acid and 10% buffered formalin for up to 2 weeks. As previously reported, histopathological grading for non-proliferative lesions was performed as follows: (1) minimal, (2) mild, (3) moderate, (4) marked, and (5) severe 19 . Number of lesions of focal medullary hyperplasia and benign pheochromocytoma in adrenals of male WT and gpt delta rats were counted and their calculated multiplicity per affected rat.

Statistical analysis
Variations in body weights, organ weights (both absolute and relative weights), and multiplicity of focal medullary hyperplasia and benign pheochromocytoma lesions in male WT and gpt delta rats were analyzed by assessing the variance for homogeneity using F-tests. Student's t-tests and Welch's t-tests were used to assess the homogeneous and heterogeneous data, respectively. For comparison of histopathological changes between gpt delta and WT rats, incidences were analyzed with Fisher's exact probability test.

Survival rates and body weights
There were no intergenotype differences in the survival rates of both male and female rats during the experiment (Fig. 1A). Thirty-seven male WT, 42 male gpt delta, 20 female WT, and 14 female gpt delta rats were found dead or were euthanized when moribund. Figure 1B shows the body weight curves. Body weights of male gpt delta rats were significantly higher than those of corresponding WT rats from the start of the experiment until week 4. However, body weights of male gpt delta rats were significantly lower than those of WT rats from 10 weeks onwards until the end of the experiment. There were no intergenotype differences in body weights in female rats during the experiment.

Organ weights
The results of organ weight assessments are shown in Table 1. The data from moribund-sacrificed or found-dead animals were excluded. The absolute weights of the brains of male and female F344 gpt delta rats were significantly lower than those of WT rats. Relative weights of the brains, lungs, hearts, adrenals, and kidneys of male gpt delta rats were significantly higher than those of male WT rats. Absolute and relative weights of the lungs and livers of female gpt delta rats were significantly higher than those of female WT rats.

Histopathological examination
Some organs could not be evaluated owing to the loss of materials during processing or specimen preparation. The missing organs and their numbers are listed in Supplementary Table 1.
Histopathological findings of nonneoplastic lesions are shown in Table 2. The incidence rates of all nonneoplastic lesions did not show intergenotype differences. Histopatho-logical findings of neoplastic lesions are listed in Table 3. The incidence of benign pheochromocytoma in male gpt delta rats was significantly higher than that in male WT rats. The multiplicity of focal medullary hyperplasia and benign pheochromocytoma in affected male rats showed no intergenotype differences ( Table 4). The incidence rates of other tumors showed no intergenotype differences. Representative photographs of nonneoplastic and neoplastic lesions are shown in Fig. 2 and 3, respectively.

Discussion
As background data could be informative for investigating the toxicity and carcinogenicity of environmental chemicals, a comparison of the survival rates, body and organ weights, and the incidence of spontaneous lesions in reporter gene-transgenic rats and WT rats was performed. There are known to be strain differences in the spectrum of spontaneous lesions in rats 20,21 . Moreover, F344 rats have     3  3  0  0  Tumor, basal cell, benign  2  0  0  0  Carcinoma, basal cell  1  0  0  0  Carcinoma, squamous cell  1  1  0  1  Adenoma, sebaceous cell  2  0  0  0  Tumor, hair follicle, benign  1  1  0  0  Subcutis  Fibroma  5  3  1  3  Fibrosarcoma  0  0  1  2  Fibrosarcoma, pleomorphic  0  1  0  0  Lipoma  0  1  1  0  Hemangioma  0  0  0 1 Adenoma, preputial/clitoral gland  been used in long-term toxicological and carcinogenicity studies for many years, and abundant background data have been accumulated. Therefore, in the current study, F344 gpt delta rats were selected among the three background strains of gpt delta rats. For both sexes, there were no intergenotype differences  in survival rates throughout the experimental period. Body weights of male gpt delta rats were significantly higher than those of WT rats from the start of the experiment until week 4 and were significantly lower from week 10 onwards, until the end of the experiment. These outcomes were inconsistent with those in a previous validation study 18 wherein the body weights of F344 and SD gpt delta rats were found to be lower than those of corresponding WT rats at the beginning of the experiment (at 6 weeks of age). As the differences observed in the current study were smaller, despite the observed statistical significance, the differences in growth curves observed in male gpt delta rats seemed to be incidental.
There were significant differences in the absolute and/ or relative weights of various organs, including the brain, lungs, heart, adrenal glands, and kidneys of male gpt delta rats and the brain, lungs, and liver in female gpt delta rats. However, the incidence and severity of spontaneous lesions in all organs, except the adrenal glands, were almost identical between the two genotypes, as described below. Thus, it is unlikely that these fluctuations had a biological significance. However, increases in the relative weights of the adrenal glands in male gpt delta rats were thought to be associated with increases in the incidence of benign pheochromocytoma, as described below.
Several spontaneous nonneoplastic lesions, such as chronic progressive nephropathy in the kidney, bile duct hyperplasia, focus of cellular alteration in the liver, and eosinophilic globules in the nasal cavity, frequently occur in aged F344 rats [22][23][24][25][26] . In the current study, a higher incidence of spontaneous nonneoplastic lesions reported in F344 rats were also observed in gpt delta rats, and there were no intergenotype differences in their incidence. Additionally, the incidence rates of rare nonneoplastic lesions in F344 rats also showed no intergenotype differences.
The histopathological analysis of neoplastic lesions revealed that the incidence of benign pheochromocytoma in male gpt delta rats was significantly higher than that in male WT rats. Historical control data from the National Toxicology Program (NTP) showed that control incidence of benign pheochromocytoma in male F344 rats was 98/696 (14.1%) with an inter-study range of 8-22% 27 . In this study, the incidence of pheochromocytoma in male gpt delta rats was 30/75 (40%), which was higher than that of control data obtained from NTP. However, the incidence of benign pheochromocytoma in female gpt delta rats did not differ from that in female WT rats. In addition, adrenal focal medullary hyperplasia, a preneoplastic lesion of pheochromocytoma 28 , showed no intergenotype differences in male and female gpt delta rats. In addition, multiplicity of focal medullary hyperplasia and benign pheochromocytoma in affected animals showed no intergenotype differences. These results indicated that the increased incidence of benign pheochromocytoma in male gpt delta rats may be incidental, rather than an effect of transfection of the reporter gene. Various spontaneous tumors have higher incidence rates in aged F344 rats, e.g., large granular lymphocyte leukemia, Leydig cell adenoma in the testis, anterior lobe adenoma in the pituitary gland, and fibroadenoma in the female mammary gland [29][30][31][32] . In the current study, these tumors were observed in gpt delta rats of both sexes to some extent. Notably, no intergenotype differences were observed. Additionally, the incidence of infrequent neoplastic lesions in F344 rats showed no intergenotype differences.
In conclusion, spontaneous lesions discovered in gpt delta rats with the F344 background strain seemed to be identical to those in WT rats. These findings supported the applicability of gpt delta rats as appropriate tools to evaluate the toxicity and carcinogenicity of environmental chemicals.

Disclosure of Potential Conflicts of Interest:
The authors declare no potential, real, or perceived conflicts of interest with respect to the research, authorship, and/or publication of this article. Values are mean ± SD.