Osamu Sawamoto

The suitability of rat peripheral blood in suchronic studies for the micronucleus assay

To examine the suitability of using rat peripheral blood from animals used in subchronic toxicity studies for micronucleus analysis, we orally administered phenacetin or 6-mercaptopurine for 14 days to groups of six rats and compared their micronucleus frequencies to the bone marrow micronucleus frequencies of rats similarly treated for only 2 days. In the 14-day test, phenacetin significantly increased the frequency of micronucleated reticulocytes in peripheral blood at 500 mg/kg starting from day 9, and at 750 and 1500 mg/kg starting from day 6; 6-mercaptopurine gave a positive response at 20 mg/kg starting from day 6. Positive responses in the bone marrow assay were obtained at the same dose levels. In the 2-day test, micronucleated polychromatic erythrocyte frequencies increased significantly at 1000 and 2000 mg/kg for phenacetin, and at 50, 100, and 200 mg/kg for 6-mercaptopurine. These results suggest that micronucleus assays using peripheral blood from rats in subchronic animal studies of phenacetin and 6-mercaptopurine are feasible and at least as sensitive for the assessment of micronuclei as an acute bone marrow micronucleus test.

Immunohistochemical Characterization of Macrophages and Myofibroblasts in α-Naphthylisothiocyanate (ANIT)–Induced Bile Duct Injury and Subsequent Fibrogenesis in Rats

To investigate pathogenesis of post–bile duct (BD) injury fibrosis, interlobular BD epithelial injury was induced in male F344 rats by a single IP injection of α-naphthylisothiocyanate (75 mg/kg body weight) and rats were observed for 12 days. On days 1 to 2, cholangiocytes were injured and desquamated. On days 3 to 5, the affected BD began to regenerate, showing positive staining for CK19 and vimentin. On days 5 to 9, fibrotic areas gradually developed around regenerating BD in Glisson’s sheath. These consisted of cells positive for vimentin, desmin, and α-SMA; vimentin- and desmin-positive cells were increased in early stage (days 1–3), whereas α-SMA-positive cells appeared in mid (days 4–7) and late stages (days 8–12), although there were cells coexpressing these cytoskeletons. On day 12, BD regeneration almost completed, with reduced fibrosis. Macrophages positive for ED2 (CD163) increased transiently in early stage, whereas those reacting to ED1 (CD68), OX6 (MHC II), and SRA-E5 (CD204) showed a consistent increase throughout the experiment. Interestingly, OX6-positive cells were limited to Glisson’s sheath, whereas SRA-E5-positive cells were seen exclusively along sinusoids of hepatic lobules. MCP-1 mRNA increased significantly in early stage. This study shows that macrophages exhibiting different immunophenotypes and distributions participate in post-BD injury fibrosis associated with myofibroblasts expressing various mesenchymal cytoskeletons.

Development of Sperm Granulomas in the Epididymides of L-Cysteine-Treated Rats

We investigated the histopathological effects of excess L-cysteine on the male rat reproductive tract during sexual maturation. Male 6-week-old Sprague—Dawley rats were injected intraperitoneally daily with L-cysteine, 1,000 mg/kg body weight, for 1, 2, 3, and 4 weeks. L-Cysteine-treated rats developed sperm granulomas in the epididymides at an incidence of 0% (0/6), 50% (3/6), 83% (5/6), and 100% (6/6) in rats examined at study weeks 1, 2, 3, and 4, respectively. These sperm granulomas were unilateral or bilateral, and most frequently involved the proximal cauda region of the epididymides. Interestingly, small ducts, indicative of immaturity, were seen frequently in L-cysteine-treated rats. These findings suggest that the maturation of epididymides in L-cysteine-treated rats might be delayed. Additionally, dilated ducts and interstitial edema, suggestive of an increase in intraluminal pressure, were seen often in the epididymides of L-cysteine-treated rats. Labeling spermatozoa and epithelial cells with monobromobimane indicated no influence of the thiol—disulfide status of L-cysteine to the epididymides. The testes and prostate glands also showed no effects, suggesting that inhibited epididymis maturation was not a result of hormonal deficiencies. We speculate that defective development of the ducts might result in aberrant fluid flow, leading to ductal rupture in the epididymides. In that case, sperm granulomas might form around leaked spermatozoa.

Cisplatin-Induced Renal Interstitial Fibrosis in Neonatal Rats, Developing as Solitary Nephron Unit Lesions

Cisplatin (CDDP)-induced renal lesions in rats prove a useful model for analysis of the pathogenesis of post-tubular injury-renal interstitial fibrosis. This study investigated the histopathological changes in 10-day-old neonatal rats induced by a single injection of CDDP (4.5 mg/kg). Compared with age-matched controls, on postinjection (PI) days 1 to 6, the number of apoptotic cells, demonstrable with TUNEL method, was significantly increased in CDDP-treated neonates, and there was no marked epithelial necrosis nor fibrotic lesions. Fibrotic lesions began to be developed solitarily around some nephrons with dilated ducts in the corticomedullary junction on PI day 10 and the lesions became more prominent until PI day 20. The α-SMA-positive myofibroblastic cells were seen exclusively in the fibrotic lesions. Additionally, the numbers of macrophages reacting with ED1 (specific for exudate macrophages), ED2 (for resident macrophages), and OX6 (recognizing MHC class II antigens expressed in antigen-presenting macrophages/dendritic cells) were significantly increased around the affected renal tubules. A greater immunoreaction for TGF-β1 was seen mostly in the renal epithelial cells of CDDP-treated neonates. These findings indicated that macrophage populations and myofibrolastic cells as well as TGF-β1 may be responsible for the production of neonatal renal interstitial fibrosis. Compared with CDDP-injected adult rats that develop extensive interstitial fibrosis (Yamate et al., J Comp Pathol, 1995), the formation of fibrotic lesions was delayed, and the lesions were limited to the area around the affected nephrons; this could be attributable to differences in renal morphology between neonates and mature kidney of adult rats.

Slowly progressive cholangiofibrosis induced in rats by α-naphthylisothiocyanate (ANIT), with particular references to characteristics of macrophages and myofibroblasts.

A progressive cholangiofibrosis was developed as an animal model in 6-week-old male F344 rats by repeated intraperitoneal injections of α-naphthylisothiocyanate (ANIT) for 19 weeks; liver samples were examined at post-first injection (PFI) weeks 3, 7, 10, 13, 16 and 19, focusing on characteristics of macrophages and myofibroblasts by immunohistochemical analyses. In the affected Glissons sheath consisting of inflammatory cell infiltrates, bile duct proliferation and advancing fibrosis, the number of macrophages reacting to OX6 (recognizing MHC class II) increased consistently (PFI weeks 3-19), suggesting a central role of antigen presenting cells in the biliary fibrosis; macrophages reacting to ED1 (CD68, reflecting phagocytic activity) and ED2 (CD163, relating to proinflammatory factor production) showed a significantly increased number at PFI weeks 7-19 and PFI weeks 13-19, respectively. Interestingly, macrophages positive for SRA-E5 (CD204, reflecting lipid metabolism) increased at PFI weeks 7-19, and the appearance was limited in the sinusoids around the affected Glissons sheath. Myofibroblasts appearing in the affected Glissons sheath reacted to vimentin and desmin at early (PFI weeks 3-7) and mid (PFI weeks 10-13) stages, and then they came to strongly express α-smooth muscle actin at late stage (PFI weeks 16-19). This study shows that macrophages exhibit heterogeneous properties depending on stages and locations; in association with such macrophage populations, myofibroblasts expressing various cytoskeletons participate in cholangiofibrosis. These characteristics would be useful in evaluating the pathogenesis of possible cholangio-toxicants.

Relationship of interstitial edema with L-cysteine-induced sperm granulomas in the pubertal rat epididymis.

Although the pathogenesis of sperm granulomas is complicated, the leakage of spermatozoa into extraluminal tissues is regarded as a crucial event. It has been previously shown that pubertal rats injected with L-cysteine develop interstitial edema followed by sperm granulomas in the epididymis. In this study we investigated the relationships between these two lesions in 6-week old rats given daily intraperitoneal injections of L-cysteine (1,000 mg/kg body weight) for 4 weeks. Rats were examined during weeks 0, 1, 2, 3 and 4 after the first injection. Interstitial edema (moderate or severe) and sperm granulomas were seen in the corpus and cauda epididymis of L-cysteine-treated rats in study weeks 2, 3, and 4. There was no marked alteration of basement membrane of the epididymal ducts in the edematous tissues as shown by immunohistochemistry with an antilaminin antibody. However, the extravasation of Evans blue dye given I hour before necropsy suggested that the severe interstitial edema was due to increased vascular permeability. In addition, a small number of neutrophils were seen in the edematous tissues, suggesting that they might play a role in the increased vascular permeability and leakage of epididymal fluid. Interestingly, slight interstitial edema was observed in the caput epididymis in both control and L-cysteine-treated rats in early study weeks 0, 1, and 2. It is speculated that this change was related to the leakage of epididymal fluid due to increased intraluminal pressure depending on rat epididymal maturation. Taken together, these findings suggest that the severe interstitial edema results from increased vascular permeability. This, along with increased intraluminal pressure, might be the trigger for duct rupture, the prerequisite for sperm granuloma formation associated with excessive doses of L-cysteine.

Participation of bone morphogenetic protein (BMP)-6 and osteopontin in cisplatin (CDDP)-induced rat renal fibrosis

The significance of bone morphogenetic protein (BMP)-6 and osteopontin (OPN) in renal fibrosis is poorly understood. We investigated the expression of BMP-6 and OPN in cisplatin (CDDP; 6mg/kg, once, i.p.)-induced renal fibrosis in F344 rats, and their effects on kidney fibroblast (NRK-49F), mesenchymal pericyte (MT-9) and renal epithelial cell (NRK-52E) lines. Histopathologically, the CDDP injection showed desquamation of renal tubular epithelial cells at the cortico-medullary junction on days 1-3 that followed insufficient regeneration on days 5-9 and progressive interstitial fibrosis by day 35. In addition to TGF-β1 (the most powerful fibrogenic factor), increase in mRNAs of BMP-6 and OPN was seen consistently after the injection. Immunohistochemically, BMP-6 was expressed both in the damaged renal epithelial cells and spindle-shaped myofibroblasts (expressing α-smooth muscle action [α-SMA]) in the fibrotic areas; OPN expression was seen exclusively in the injured renal epithelial cells. Treatment of BMP-6 or OPN increased α-SMA mRNA in MT-9 cells, similar to effects of TGF-β1 on MT-9 and NRK-49F cells. Interestingly, TGF-β1 addition increased BMP-6 and OPN mRNAs in NRK-52E cells. Collectively, it was found that BMP-6 and OPN considerably participate in progressive renal fibrosis through development of myofibroblasts, in relation with TGF-β1.

Establishment of a Transplantable Rat Pulmonary Carcinoma-Derived Cell Line (IP-B12) as a New Model of Humoral Hypercalcemia of Malignancy and Bone Metastasis

A cloned cell line (IP-B12) derived from a transplantable rat pulmonary carcinoma (IP), of which neoplastic cells produce parathyroid hormone-related protein (PTHrP), was established. Tumors induced in syngeneic F344 rats by intraperitoneal injection of IP-B12 cells had features of pulmonary adenocarcinomas, consisting of neoplastic cells immunopositive to PTHrP. The IP-B12 tumor-bearing rats developed severe emaciation and hypercalcemia, with a marked elevation of plasma PTHrP level; there was an increase in osteoclastic areas of the femur and calcium depositions in systemic organs, indicating progression to humoral hypercalcemia of malignancy (HHM) in the tumor-bearing rats. In addition, the injection of IP-B12 cells into the left cardiac ventricle of syngeneic rats resulted in osteolytic skeletal metastases in the long bones and vertebrae. In the metastatic lesions, histologically, neoplastic cells showed an immunopositive reaction to PTHrP, and a prominent osteoclastic activity was seen; bone lesions, including osteolysis, fracture, and nerve compression as well as replacement of bone marrow cells by proliferated tumor cells were similar to those reported in human cancer patients with bone metastases. IP-B12 is a new animal model for HHM and osteolytic bone metastases, and will become a useful tool for studies on the pathogenesis and therapeutic strategies for such conditions.

Macrophage Populations in l-Cysteine-Induced Rat Sperm Granulomas

Histopathologically, sperm granulomas consist of a central mass of degenerating spermatozoa surrounded by many epithelioid macrophages and lymphocytes. Using monoclonal antibodies (ED1, ED2, and OX6), the authors investigated immunohistochemically the participation of different macrophage populations in epididymal sperm granulomas induced in pubertal rats by repeated injection of L-cysteine. Monocyte-like and epithelioid macrophages expressed the ED1 antigen found on activated lysosomal membranes in rat blood monocytes and exudate macrophages, but did not express the ED2 antigen found on the membrane antigens of rat resident macrophages. Cells expressing MHC class II antigens (as detected by the OX6 antibody) were present in the granulomas in moderate numbers, particularly in the early stages. Ultrastructurally, fragmented spermatozoa were observed in the cytoplasm of epithelioid macrophages. These findings suggest that macrophages appearing in rat sperm granulomas originate mainly from blood monocytes, and that they have a high phagocytic activity and a potential for antigen presentation.

Characteristics of nitrogen metabolism in rats with thioacetamide-induced liver cirrhosis.

Female Sprague-Dawley rats were given 0.03% thioacetamide (TAA) in their drinking water daily for 4 or 12 weeks, and were then given normal water for 4 weeks after the end of a 12-week TAA treatment to investigate amino acid metabolism. In the malnourished precirrhotic stage (stage 1) and the malnourished cirrhotic stage (stage 2), the aromatic amino acids (AAA), Glu, Asp, Orn, Arg and Cit increased, and the branched-chain amino acids (BCAA) decreased slightly. Because these changes normalized in the well-nourished cirrhotic stage (stage 3), they might have resulted from impairment of hepatocytes and malnutrition. The net uptake of BCAA into the liver increased in stage 2, but the AAA uptake did not exceed that in normal controls. Portal venous plasma AAA increased to the same level as arterial plasma AAA. These results suggest that the decrease in BCAA was partially due to liver uptake and that the increase in AAA was induced by reduction of liver uptake and overproduction in extrahepatic tissues. The liver contents of BCAA and AAA were unchanged in all stages, so were fully utilized in the impaired liver. The increases in Glu, Asp, Orn and Cit might have resulted from overproduction in the liver, because these contents of the liver increased in stage 2. In conclusion, the changes in amino acid metabolism in rats with cirrhosis induced by TAA closely resemble those seen in human liver cirrhosis.