Takanori Maejima

Interlaboratory Comparison of Short-Term Carcinogenicity Studies Using CB6F1-rasH2 Transgenic Mice

In order to evaluate a short-term carcinogenicity testing system using CB6F1-Tg rasH2 ( rasH2-Tg) mice carrying a human prototype c-Ha-ras gene, 26-week studies were conducted in 12 different facilities as a part of an International Life Science Institute Health and Environmental Science Institute (ILSI HESI) international collaborative project. In each study N-methyl-N-nitrosourea (MNU) was administered to a separate group of rasH2-Tg mice by single intraperitoneal injection (75 mg/kg) as a positive control. We herein have summarized the mortality, body weight change, and neoplastic and nonneoplastic lesions detected in these positive control groups as representative historical positive control data. Also, we performed an interlaboratory comparison of the response of rasH2-Tg mice to MNU based on the data of 11 positive control groups from these studies. Although the body weight of rasH2-Tg mice showed lower values than that of non-Tg mice during the experimental period, body weight gain in the rasH2-Tg mice was similar to that in non-Tg mice. The mortality of rasH2-Tg mice during the study period was very low, the same as for the non-Tg mice. Incidences of spontaneous alveolar/bronchiolar adenomas and splenic hemangiomas/hemangiosarcomas were also low in the rasH2-Tg mice. Nonneoplastic lesions detected in the rasH2-Tg mice were similar to those in non-Tg mice, excluding the incidence of myopathy. There were interlaboratory differences in mortality and incidence of some lesions in the MNU-treated groups. However, the causes of death were common among the 11 laboratories and almost all the MNU-treated rasH2-Tg mice developed forestomach squamous cell papillomas/carcinomas or malignant lymphomas. This suggests that there is no appreciable difference in the response of the rasH2-Tg mouse to MNU used as a positive control. Therefore, it is concluded that MNU would be an adequate positive control compound in this testing system.

Retinal function and morphology in monkeys with ethambutol-induced optic neuropathy.

PURPOSE Ethambutol-induced optic neuropathy is a well recognized adverse ocular event. However, abnormalities of the retina in this optic neuropathy are not fully understood. Therefore, the purpose of the present study was to investigate both functional and morphological alterations of the retina induced by ethambutol in monkeys. METHODS Ethambutol was orally administered to three cynomolgus monkeys, initially at 400 mg/kg/day followed by 800 mg/kg/day, for a maximum of 39 weeks. Full-field electroretinograms (ERGs) were recorded at intervals of approximately one month. The protocol included standard ERG responses to white flashes obtained under dark-adapted conditions (rod, combined rod-cone, oscillatory potentials) or with a white background (single-flash cone, 30 Hz flicker). In addition, we measured the ERG elicited with red flashes under blue background light (single-flash cone response [R/B]). All the ethambutol-treated monkeys were euthanized, and the retinae and various other nervous system tissues were examined histopathologically. RESULTS No obvious changes were observed in the standard full-field ERGs. On the other hand, selective attenuation of the photopic negative response (PhNR) of the single-flash cone response (R/B) was observed in two out of three ethambutol-treated monkeys at week 22 or 28. Histopathology of these two monkeys revealed single cell necrosis of the retinal ganglion cells (RGCs), decreased RGCs in the parafovea and increased microglial cells in the nerve fiber layer in the retina, in addition to demyelination and glial reaction in the optic nerve, chiasm and tracts. CONCLUSIONS The attenuated PhNR and histopathology of the retina indicated that RGCs were markedly damaged, both functionally and morphologically in monkeys with ethambutol-induced optic neuropathy. These results implied that RGCs are predominantly affected in the retina of patients with ethambutol-induced optic neuropathy.

Role of connexin 32 in acetaminophen toxicity in a knockout mice model

Gap junctional intercellular communication (GJIC), by which glutathione (GSH) and inorganic ions are transmitted to neighboring cells, is recognized as being largely involved in toxic processes of chemicals. We examined acetaminophen (APAP)-induced hepatotoxicity clinicopathologically using male wild-type mice and mice lacking the gene for connexin32, a major gap junction protein in the liver [knockout (Cx32KO) mice]. When APAP was intraperitoneally administered at doses of 100, 200, or 300mg/kg, hepatic centrilobular necrosis with elevated plasma aminotransferase activities was observed in wild-type mice receiving 300mg/kg, and in Cx32KO mice given 100mg/kg or more. At 200mg/kg or more, hepatic GSH and GSSG contents decreased significantly and the effect was more severe in wild-type mice than in Cx32KO mice. On the other hand, markedly decreased GSH staining was observed in the hepatic centrilobular zones of Cx32KO mice compared to that of wild-type mice. These results demonstrate that Cx32KO mice are more susceptible to APAP hepatotoxicity than wild-type mice, and indicate that the distribution of GSH of the centrilobular zones in the hepatic lobules, rather than GSH and GSSG contents in the liver, is important in APAP hepatotoxicity. In conclusion, Cx32 protects against APAP-induced hepatic centrilobular necrosis in mice, which may be through the GSH transmission to neighboring hepatocytes by GJIC.

Methemoglobinemia Induced by 1,2-Dichloro-4-nitrobenzene in Mice with a Disrupted Glutathione S-Transferase Mu 1 Gene

A specific substrate to Mu class glutathione S-transferase (GST), 1,2-dichloro-4-nitrobenzene (DCNB), was administered to mice with a disrupted GST Mu 1 gene (Gstm1-null mice) to investigate the in vivo role of murine Gstm1 in toxicological responses to DCNB. A single oral administration of DCNB at doses of 500 and 1000 mg/kg demonstrated a marked increase in blood methemoglobin (MetHB) in Gstm1-null mice but not in wild-type mice. Therefore, Gstm1-null mice were considered to be more predisposed to methemoglobinemia induced by a single dosing of DCNB. In contrast, 14-day repeated-dose studies of DCNB at doses up to 600 mg/kg demonstrated a marked increase in blood MetHB in both wild-type and Gstm1-null mice. However, marked increases in the blood reticulocyte count, relative spleen weight, and extramedullary hematopoiesis in the spleen were observed in Gstm1-null mice compared with wild-type mice. In addition, microarray and quantitative reverse transcription-polymerase chain reaction analyses in the spleen showed exclusive up-regulation of hematopoiesis-related genes in Gstm1-null mice. These changes were considered to be adaptive responses to methemoglobinemia and attenuated the higher predisposition to methemoglobinemia observed in Gstm1-null mice in the single-dose study. In toxicokinetics monitoring, DCNB concentrations in plasma and blood cells were higher in Gstm1-null mice than those in wild-type mice, resulting from the Gstm1 disruption. In conclusion, it is suggested that the higher exposure to DCNB due to Gstm1 disruption was reflected in methemoglobinemia in the single-dose study and in adaptive responses in the 14-day repeated-dose study.

N-Methyl-N-Nitrosourea-Induced Acute Alteration of Retinal Function and Morphology in Monkeys.

PURPOSE The purpose of this study was to investigate both functional and morphologic alteration of the retina acutely induced by N-methyl-N-nitrosourea (MNU) in monkeys. METHODS The MNU was administered intravenously at a single dose of 40 mg/kg to six cynomolgus monkeys, and standard full-field electroretinograms (ERGs) were recorded 1, 3, and 7 days after dosing. In addition, the rod and cone a-waves in response to high-intensity flashes were analyzed by the a-wave fitting model (a-wave analysis). The photopic negative response (PhNR) was also recorded at the same time points. Furthermore, the retinas of two animals each were examined histopathologically 1, 3, or 7 days after dosing. RESULTS The MNU attenuated all the standard full-field ERGs including the rod-driven and cone-driven responses; in the combined rod-cone response, the b-wave was more affected than the a-wave. In the a-wave analysis, the sensitivity parameters (S) of the rod and cone a-waves had decreased on the day after dosing and remained unchanged thereafter. The maximum response parameter (Rmax) of the rod a-wave gradually decreased. On the other hand, the Rmax in the cone a-wave transiently increased on the day after dosing and decreased thereafter; the PhNR amplitude showed a similar time course change. Histopathologically, the retinal lesion on the day after dosing mainly consisted of pyknosis and karyorrhexis in the photoreceptor nucleus. Depletion of some photoreceptor nuclei, and shortening and disorientation of the photoreceptor segments became prominent at 3 and 7 days after dosing. Localization of degenerated photoreceptors was consistent with that of rhodopsin-positive photoreceptors, resulting in a well-preserved central fovea. CONCLUSIONS Our results indicated that MNU acutely induced rod-dominant photoreceptor degeneration in monkey retinas, but the photoreceptor function was impaired in both the rods and cones. Functional involvement of the postreceptoral components was also indicated.

Mixed Type of Malignant Mesothelioma in an Aged Male ICR Mouse.

Multiple whitish nodules in the thoracic cavity at the site of the thymus were observed in a 101-week-old male ICR mouse. In a histopathological examination, the neoplastic cells were predominantly fusiform in shape and proliferated in sarcomatoid growth patterns. Some neoplastic cells showed epithelial growth patterns, such as the ductal structures. Mitotic figures were frequently seen, and small necrotic foci and invasion to adjacent thoracic organs were noted. In Alcian blue staining, bluish materials were observed between fusiform-shaped cells and in some of the lumens of the ductal structures. In immunohistochemistry, both fusiform-shaped and ductal structure-forming cells were positive for vimentin and weakly positive to positive for cytokeratin. Based on the aforementioned findings, the thoracic nodules were diagnosed as a mixed type of malignant mesothelioma. This case was thought to be rare because of the very low occurrence of spontaneous mesothelioma in mice.

Twenty-six-Week carcinogenicity study of chloroform in CB6F1 rasH2-transgenic mice.

The carcinogenic potential of chloroform was evaluated in a short-term carcinogenicity testing system using CB6F1 rasH2-Tg (rasH2-Tg) mice. Chloroform was administered to rasH2-Tg males at doses of 28, 90, or 140 mg/kg and rasH2-Tg females at 24, 90, or 240 mg/kg by oral gavage for 26 weeks. Wild-type (non-Tg) male and female mice received doses of 140 mg/kg and 240 mg/kg, respectively. N-methyl-N-nitrosourea was administered to rasH2-Tg mice by single intraperitoneal injection (75 mg/kg) as a positive control. In both the rasH2-Tg and non-Tg mice, there was no signifi cant increase in the incidence of neoplastic lesions by chloroform treatment. The incidence of hepatocellular foci in the rasH2- and non-Tg females receiving 240 mg/kg was increased. Forestomach tumors and malignant tumors occurred in most of the rasH2-mice in the positive control group. Swelling or vacuolation of hepatocytes, a toxic change induced by chloroform, occurred in both the rasH2-Tg and non-Tg mice. It is concluded that chloroform, a putative human noncarcinogen, did not show evidence of carcinogenic potential in the present study using rasH2-Tg mice. This study suggests that the rasH2-Tg mouse model may not be appropriate for detecting nongenotoxi c carcinogens. However, the sensitivity of rasH2-Tg mice to nongenotoxi c carcinogens should be assessed with consideration of the results from the other ILSI-HESI project studies.

A Spontaneous Ganglioneuroma in the Adrenal Medulla of a Young Wistar Hannover Rat

A nodule was observed in the adrenal medulla of a twenty-week-old male Wistar Hannover rat. The nodule was predominantly (over 80%) composed of neural components, with ganglion cells scattered in sparse supporting tissue containing nerve fibers and Schwann cells. In the peripheral area of the tumor, atypical chromaffin cells were also observed. Accumulation of eosinophilic serous fluid was also noted in the stromal tissue. There were neither mitotic figures in the ganglion cells nor necrotic foci. In immunohistochemistry, the ganglion cells were positive for neuronal nuclei (NeuN), and negative for proliferating cell nuclear antigen, S-100, and chromogranin A. There were some NeuN-positive small cells in the peripheral area of the tumor. These findings indicate that this tumor was a ganglioneuroma. This seems to be an extremely rare case, as the spontaneous occurrence of ganglioneuroma in rats is very low, even in two-year carcinogenicity studies.