Keishibukuryogan is not carcinogenic in Sprague-Dawley rats.

Keishibukuryogan is a traditional Japanese medicine widely administered to patients with menopausal symptoms. Because humans use it on a long-term basis, we believed that a carcinogenicity study was warranted. We orally administered keishibukuryogan (TJ-25) extract powder to 6-week-old Sprague-Dawley rats [Crl:CD(SD)], which were divided into four dosage groups-0 (water for injection), 100, 500 and 2,500 mg/kg/day for 24 months. We found that TJ-25 did not affect the survival rate of either sex. Furthermore, it did not affect the clinical condition of the rats, number of superficial tumors found by palpation, body weight, food consumption, hematology, or gross pathological findings. The severity of degeneration of muscle fiber in the femoral skeletal muscle increased slightly in males and females in the 2,500 mg/kg/day group, but TJ-25 did not increase the number of tumors found on histopathological examination. In our study, oral administration of TJ-25 extract powder in rats for 24 months was not associated with an increased incidence of tumors.

Changes in blood parameters and the expression of coagulation-related genes in lactating Sprague-Dawley rats.

This study measured blood parameters, particularly those related to coagulation, and alterations in the expression levels of blood-coagulation-related genes in lactating Sprague-Dawley rats. The day of delivery was designated as lactation day 0 (LD 0). On the day after delivery (LD 1), prothrombin time and overall activity of vitamin-K-dependent coagulation factors were decreased, whereas fibrinogen contents, platelet counts and antithrombin III concentrations were increased as compared with those in nonpregnant rats. In addition, hepatic expression of blood-coagulation-related genes in the liver was increased at LD 0 as compared with that in nonpregnant rats. These changes may be physiologic responses to prevent prolonged bleeding at delivery. Except for fibrinogen content, which remained elevated, the described changes returned to baseline on and after LD 7. Activities of AST, ALT, and ALP were increased on LD 7, 14, and 21 as compared with nonpregnant rats. In contrast, total protein, albumin, Cl, and Ca were consistently lower on LD 7, 14, or 21 as compared with levels in nonpregnant rats. These results provide background data for evaluation of nursing rats.

Spontaneous Malignant T-cell Lymphoma in a Young Adult Crl:CD (SD) Rat.

We investigated a case of spontaneous malignant T-cell lymphoma observed in a 19-week-old male Crl:CD (SD) rat. The rat showed paralysis beginning 1 week before euthanasia. Hematological examination revealed marked lymphocytosis without distinct atypia. Macroscopically, hepatosplenomegaly and partial atrophy of the thoracic spinal cord were observed. Microscopically, neoplastic cells infiltrated into the liver, splenic red pulp, bone marrow and epidural space of the thoracic spinal cord, while no neoplastic cells were observed in the thymus and lymph nodes. Moreover, the spinal cord showed focal degeneration due to compression by marked infiltration of neoplastic cells in the subdural space. The neoplastic cells were generally small-sized round cells that had a round nucleus with/without a single nucleolus and scanty cytoplasm. Immunohistochemically, the neoplastic cells were positive for CD3 and CD8 and negative for CD79α. Judging from these results, the present tumor in this young adult rat was diagnosed as malignant T-cell lymphoma.

Changes in blood coagulation-related parameters in phenobarbital-treated rabbits.

The effects of repeated administration of phenobarbital (PB) on blood coagulation time were examined using male Japanese white SPF rabbits, which are widely used for toxicological studies. PB was administered to the rabbits by oral gavage for 2 weeks, at dose levels of 0, 12.5, 25 and 50 mg/kg/day. Blood was collected on Days 8 and 14 after each day's dosing to perform blood coagulation examination. The liver was excised, weighed and examined histopathologically. Activated partial thromboplastin time (APTT) was prolonged at dose levels of 12.5 mg/kg/day or more and Thrombotest (TBT) was prolonged at 50 mg/kg/day on Day 8. APTT was prolonged at dose levels of 12.5 mg/kg/day or more, TBT was prolonged at 25 mg/kg/day or more and factor IX activity decreased at 50 mg/kg/day on Day 14. At pathological examination, liver weight increased at dose levels of 25 mg/kg/day or more, and a ground-glass appearance of the hepatocytes was observed in the central and middle parts of lobules at 12.5 mg/kg/day or more. However, changes in factor VII or X activity or prolongation of prothrombin time (PT) were not observed. Therefore, prolongation of blood coagulation time by PB administration in rabbits was considered to be due to PB's effect on the endogenous pathway alone. Moreover, an increase in anti-thrombin III (ATIII) concentration was noted at 50 mg/kg/day; however, no change was noted at dose levels of 25 mg/kg/day or less. This suggests that the contribution of ATIII to the PB-induced prolongation of coagulation time in rabbits was small.

Carbon tetrachloride-induced hepatotoxicity in pregnant and lactating rats.

Carbon tetrachloride (CCl4) is well known to induce hepatotoxicity after being metabolized to trichloromethyl free radical ((.)CCl3) by CYP2E1. In the present study, the hepatotoxicity induced by a single oral dose (2,000 mg/kg) of CCl4 was compared between pregnant (gestation days (GD) 13 and 19) or postpartum (postpartum days (PPD) 1, 13 and 27) and non-pregnant rats. Hepatotoxicity in CCl4-treated pregnant rats evaluated by blood chemistry (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities) and histopathological finding (area of damaged hepatocytes) was minimal on GD19, being weaker than that in non-pregnant rats. CYP2E1 expression in non-treated pregnant rats decreased as pregnancy progressed and reached minimum level on GD19. Thus, the degree of CCl4-induced hepatotoxicity roughly corresponded to CYP2E1 levels during pregnancy. After delivery, hepatotoxicity in CCl4-treated lactating rats was maximal on PPD13, being stronger than that in non-pregnant rats, and then it decreased slightly on PPD27. The CYP2E1 level in the non-treated lactating rats tended to increase but remained at lower levels until PPD13 compared with that in non-pregnant rats. Thus, the degree of CCl4-induced hepatotoxicity did not correspond to CYP2E1 levels during lactation. This suggests that during lactation, there may be certain factors other than CYP2E1 expression responsible for the degree of CCl4-induced hepatotoxicity.

Renal Tubular Cyst Formation in Newborn Rats Treated with p-Cumylphenol.

In this study, we investigated the sequential changes in the development of renal tubular cysts in newborn rats treated with p-cumylphenol (PCP). Fifteen animals per sex were treated orally with 300 mg/kg/day of PCP for up to 18 days from postnatal day (PND) 4 and were sacrificed on PNDs 8, 12, 19 and 22 and after a 7 day recovery period. On PNDs 8 and 12, slight dilatation of the collecting ducts was frequently observed in the medulla and slight papillary necrosis was also noted in some cases. These dilated collecting ducts were lined with slightly hyperplastic epithelial cells. On PNDs 19 and 22, multiple large cystic changes arising from the collecting ducts in the outer medulla were seen. These cystically dilated ducts were also lined with hyperplastic epithelial cells. During the dosing period, the labeling index of proliferating cell nuclear antigen in the collecting duct epithelium was higher in the PCP-treated group than in the control group at all time points. After a 7 day recovery period, the cystic change still remained, although the cell density was decreased and the epithelial cells became flattened. On the other hand, basophilic tubules with peritubular lymphoid cell infiltration were multifocally observed in the cortex. In conclusion, PCP induced multiple renal cysts that developed in the collecting ducts of the outer medulla in neonatal rats, and it is suggested that epithelial cell proliferation may play some roles in the induction of cystic lesions.

Highly invasive intracranial malignant schwannoma in a rat.

A highly invasive intracranial malignant schwannoma containing several masses was detected in a 28-week-old male Crl:CD(SD) rat. Macroscopically, 3 masses were noted in the cranial cavity; one was present at the bottom of the cranial cavity and involved the trigeminal nerve, and the other two were in the parietal bone. Histologically, each mass consisted of fusiform cells with interlacing fascicular, wavy and nuclear pseudopalisading arrangements and round cells with cystic lesions. The tumor cells invaded not only the brain but also the parietal bone. In the brain, the tumor cells infiltrated diffusely into the leptomeningeal and perivascular spaces and parenchyma, in which the tumor cell morphology and invasive pattern closely resembled those of malignant astrocytoma and malignant reticulosis. Immunohistochemically, the tumor cells in the masses showed positive reactions for both S-100 protein and GFAP, while those in the cerebral invasion sites were negative for GFAP and less positive for S-100 protein. Electron microscopically, a single basal lamina layer and short intricate cell processes were confirmed in the tumor cells. From these results, the present tumor was diagnosed as a malignant schwannoma arising in the cranial cavity, probably originating from the trigeminal nerve. The present tumor is considered to be a relatively unique malignant schwannoma based on its growth and invasion patterns.