Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells.

Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.

Effects of gut microbiota on in vivo metabolism and tissue accumulation of cytochrome P450 3A metabolized drug: Midazolam.

The link between drug-metabolizing enzymes and gut microbiota is well established. In particular, hepatic cytochrome P450 (CYP) 3A activities are presumed to be affected by gut microbiota. However, there is no direct evidence that the gut microbiota affects CYP3A metabolism or the clearance of clinically relevant drugs in vivo. Our purpose was to evaluate the effects of gut microbiota on in vitro and in vivo drug metabolism and on the clearance of midazolam, which is a standard CYP3A metabolized drug. Hepatic Cyp3a activity and in vitro midazolam hydroxylase activity were compared using specific pathogen-free (SPF) and germ-free (GF) mice. In a pharmacokinetics (PK) study, SPF and GF mice were intraperitoneally injected with 60 mg/kg of midazolam, and plasma and tissue concentrations were measured. Hepatic Cyp3a activity and midazolam hydroxylase activity were significantly lower in GF mice than in SPF mice. Notably, in the PK study, the area under the plasma concentration-time curve from time zero to infinity and the elimination half-life were approximately four-fold higher in GF mice compared with SPF mice. Furthermore, the concentration of midazolam in the brain 180 min after administration was about 14-fold higher in GF mice compared with SPF mice. Together, our results demonstrated that the gut microbiota altered the metabolic ability of Cyp3a and the tissue accumulation of midazolam.

Histopathological and functional changes in a single-dose model of combretastatin A4 disodium phosphate-induced myocardial damage in rats.

Cardiotoxicity is a concern in the development of microtubule-disassembling agents (MDAs) as vascular-disrupting agents of tumors. This study investigated cardiotoxicity in rats induced by a single-dose of combretastatin A4 disodium phosphate (CA4DP), an MDA and discussed the use of this rat model in nonclinical studies of MDAs. First, CA4DP (120 mg/kg) was administered to rats intravenously, and cardiac histopathology and blood biomarkers were examined after 0.5, 24, and 72 h. Next, CA4DP (120 mg/kg) was administered to rats intravenously, and the electrocardiography and echocardiography results were analyzed. The results showed that at 0.5 h after dosing, plasma creatine kinase (CK), CK-muscle/brain (CK-MB), and fatty acid binding protein 3 levels increased. At 24 h, lactate dehydrogenase (LDH)-1, CK, and CK-MB levels increased, and multifocal vacuolar degeneration of myocardial cells was observed in the apical inner layer. At 72 h, LDH-1 levels were increased, and multifocal myocardial necrosis was observed in the interventricular septum and inner layer of the apex of left ventricular wall. Furthermore, at 0.5 h, heart rate (HR), ejection fraction (EF), and cardiac output (CO) decreased. At 24 h, CO decreased. Finally, at 72 h, HR, EF, and CO decreased, and depression of the T-wave amplitude was observed. In conclusion, myocardial injury, bradycardia, and depressed cardiac function were induced in rats by a single-dose of CA4DP. The lesion distribution and electrocardiographic features suggested that myocardial injury was induced by ischemia. These findings are similar to MDA-induced cardiotoxicity in humans, and this rat model will prove useful in studies of the cardiotoxicity in humans.

Combretastatin A4 disodium phosphate-induced myocardial injury.

Histopathological and electrocardiographic features of myocardial lesions induced by combretastatin A4 disodium phosphate (CA4DP) were evaluated, and the relation between myocardial lesions and vascular changes and the direct toxic effect of CA4DP on cardiomyocytes were discussed. We induced myocardial lesions by administration of CA4DP to rats and evaluated myocardial damage by histopathologic examination and electrocardiography. We evaluated blood pressure (BP) of CA4DP-treated rats and effects of CA4DP on cellular impedance-based contractility of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). The results revealed multifocal myocardial necrosis with a predilection for the interventricular septum and subendocardial regions of the apex of the left ventricular wall, injury of capillaries, morphological change of the ST junction, and QT interval prolongation. The histopathological profile of myocardial lesions suggested that CA4DP induced a lack of myocardial blood flow. CA4DP increased the diastolic BP and showed direct effects on hiPS-CMs. These results suggest that CA4DP induces dysfunction of small arteries and capillaries and has direct toxicity in cardiomyocytes. Therefore, it is thought that CA4DP induced capillary and myocardial injury due to collapse of the microcirculation in the myocardium. Moreover, the direct toxic effect of CA4DP on cardiomyocytes induced myocardial lesions in a coordinated manner.

Cardiotoxic changes of colchicine intoxication in rats: electrocardiographic, histopathological and blood chemical analysis.

The microtubule inhibitor colchicine is cardiotoxic and is suggested to impair impulse formation and conduction. However, little is known about the electrocardiographic (ECG) changes induced by colchicine in experimental animals and the detailed pathogenesis of its cardiotoxicity. Therefore, we analyzed cardiotoxicity in colchicine-treated rats using electrocardiographic, histopathological and blood-chemistry approaches. A telemetry device for transmitting ECG data was implanted into male Crl:CD(SD) rats, and ECG tracings were obtained. At 6 weeks of age, 1.25 mg/kg colchicine was injected intravenously once daily for 2 consecutive days, and ECG waveforms and heart rate variability were analyzed. Furthermore, 1.25 mg/kg colchicine or vehicle was injected for 1 or 2 consecutive days in other rats at 6 weeks of age. One day after the final dosing, heart and blood samples were taken for histopathological and bloodchemical examination. ECG analysis revealed a prolonged RR interval, QRS duration, PR interval and QT interval. Heart rate variability analysis showed an increase in high frequency (HF) components as an index of parasympathetic nervous activity. In blood chemical examinations, colchicine induced high levels of parameters of cardiac injury and low levels and/or variations in Ca, inorganic phosphorus, potassium and chloride. Histopathologically, colchicine-treated rats showed eosinophilic granular degeneration and cytoplasmic vacuolation of ventricular myocardial cells but no remarkable change in the atrioventricular node. Not only blood chemical and histopathological changes but also ECG changes were induced in colchicine-treated rats, which indicated a decrease in myocardium excitability and conductivity, and these changes might be related to increased parasympathetic nervous activity and low blood Ca levels.

Lactobacillus casei Shirota enhances the preventive efficacy of soymilk in chemically induced breast cancer.

Soy foods are known to be effective for breast cancer prevention. The habitual consumption of soy isoflavones in combination with the probiotic Lactobacillus casei Shirota (LcS) was shown to decrease the risk of breast cancer occurrence in our previous population-based case-controlled study among Japanese women. The present study aimed to elucidate the cooperative prevention mechanism of soymilk and LcS using an animal carcinogenic model. Female Sprague-Dawley rats received a high-fat, AIN-76A diet containing soymilk, LcS, both soymilk and LcS, or none and were orally exposed to 2-amino-1-methyl-6-penylimidazo[4,5-b]pyridine at a dose of 85 mg/kg bodyweight eight times for 2 weeks. The development of palpable mammary tumors was monitored for 17 weeks. Tumor tissues were immunohistochemically examined for estrogen receptor (ER)-α, Ki-67 and CD34. Compared with the control group, the incidence and multiplicity of mammary tumors were reduced by soymilk alone and soymilk in combination with LcS, while tumor volume was decreased by LcS alone and LcS in combination with soymilk. An immunohistochemical analysis revealed that soymilk in combination with LcS more effectively reduced the numbers of ER-α-positive and Ki-67-positive cells in tumors than soymilk alone and that both soymilk and LcS inhibited tumor angiogenesis. These results demonstrated that soymilk prevents the development of mammary tumors and that LcS suppresses tumor growth, potentially enhancing the preventive efficacy of soymilk. The habitual consumption of LcS in combination with soymilk might be a beneficial dietary style for breast cancer prevention.

A high-fat diet and multiple administration of carbon tetrachloride induces liver injury and pathological features associated with non-alcoholic steatohepatitis in mice.

The aim of the present study was to establish a progressive steatohepatitis mouse model because few reported animal models of non-alcoholic steatohepatitis (NASH) show the progression from fatty liver to steatohepatitis. C57BL/6N mice were fed a high-fat diet (HFD) to develop obesity and were either administered carbon tetrachloride (CCl4 ) eight times (0.05 mL/kg, s.c., once, followed by 0.1 mL/kg, s.c., seven times) or not. Serum parameters and hepatic histopathology were examined. In a separate experiment, CCl4 was administered subcutaneously from 0 to eight times to HFD-fed obese mice to investigate progressive changes. Markers of oxidative stress, inflammation and apoptosis, as well as histopathological changes in the liver, were analysed. The HFD-fed obese mice showed fatty liver but not steatohepatitis. In contrast, HFD-fed mice administered CCl4 eight times showed histopathological features of steatohepatitis (fatty liver, inflammation, hepatocellular ballooning and fibrosis) and increased serum alanine aminotransferase levels. However, the multiple administration of CCl4 to obese mice reduced the ratio of reduced glutathione to oxidized glutathione, superoxide dismutase activity and mitochondrial DNA copy number, leading to the development of chronic oxidative stress, increased numbers of apoptotic cells and increased levels of both tumour necrosis factor-α and transforming growth factor-ß mRNA. The resulting inflammation led to increased hydroxyproline content in the liver and fibrosis. The present study demonstrates that multiple administration of CCl4 to HFD-fed obese mice induces chronic oxidative stress that triggers inflammation and apoptosis and leads to the development of fibrosis in the liver, resulting in progression from fatty liver to steatohepatitis. This murine model will be useful in the research of hepatic disorders.

Essential roles of IL-6 trans-signaling in colonic epithelial cells, induced by the IL-6/soluble-IL-6 receptor derived from lamina propria macrophages, on the development of colitis-associated premalignant cancer in a murine model.

Activation of the IL-6/Stat3 via IL-6 trans-signaling plays an important role in the pathogenesis of inflammatory bowel disease. Colitis-associated cancer (CAC) is a large bowel cancer and occurs with long-standing inflammatory bowel disease. The role of the IL-6/Stat3 in the development of CAC has not been fully understood. We investigate whether IL-6 trans-signaling contributes to the development of CAC using a mouse colitis-associated premalignant cancer (CApC) model. Chronic colitis (CC) was induced in BALB/c mice using dextran sodium sulfate. CApC was induced by dextran sodium sulfate treatment to CC-affected mice. IL-6 expression was determined by quantitative RT-PCR and immunofluorescence staining in colon. Phospho-Stat3 expression was examined by Western blotting and immunofluorescence analysis. The expression of IL-6 receptors (i.e., the IL-6R alpha-chain and gp130) and tumor necrosis factor-alpha converting enzyme in the colon was examined by laser-capture microdissection and immunofluorescence staining. Soluble IL-6R alpha (sIL-6R alpha) was examined by Western blotting of epithelial cell-depleted colonic tissues. We also investigated whether a soluble gp130-Fc fusion protein could prevent CApC. IL-6 expression was increased in the colon of CC- and CApC-affected mice and was restricted to lamina propria-macrophages. The expression of IL-6R alpha and tumor necrosis factor-alpha converting enzyme was increased in the lamina propria CD11b-macrophages of CC-affected mice. sIL-6R alpha expression was also increased in these tissues. Reduced levels of IL-6R alpha generation were observed in the colonic epithelial cells of CC- and CApC-affected mice and were associated with the increased expression of gp130 and phospho-Stat3. Treatment with soluble gp130Fc significantly reduced the CApC. IL-6 trans-signaling in epithelial cells induced by macrophage-derived IL-6/sIL-6R alpha plays a crucial role in the development of CAC.

Development of incident severity classification for laboratory animals.

An incident reporting system (IRS) prevents possible adverse events by collecting and analyzing incidents that occur. However, few studies are available regarding IRSs in the laboratory animal field. This study aimed to develop an incident severity classification for laboratory animals (ISCLA) to evaluate the usefulness of the IRS in laboratory animal facilities. Twenty-three incidents reported from March 2019 to February 2020 on our IRS were retrospectively reviewed. Three of the 23 incidents failed to obtain some experimental data. Two of these incidents were harmless to animals, but the other caused the animals moderate distress. In addition, two of the three incidents made animals unsuitable for experiments. Since the inconsistent impact of incidents on animals and experiments prevented the comparison of the severity of individual incidents, we developed the ISCLA. According to the ISCLA, the above three incidents were classified into Category 3b and 4a. The others were classified into Category 0 (n=5), 1 (n=6), 2 (n=3), and 3a (n=6) in ascending order of severity. No incident was classified into Category 4b and 5. Furthermore, incidents occurring in the animal housing area were more severe than those occurring in the supporting area (P=0.002). This study showed that incident occurrences had characteristics that were not visible from individual incidents alone. Moreover, the ISCLA was considered useful when conducting the IRS and taking improvement measures in laboratory animal facilities.

Normal variation of the gut microbiota affects hepatic cytochrome P450 activity in mice.

Several studies revealed that substantial artificial changes in the gut microbiota resulted in modification of hepatic cytochrome P450 3a (Cyp3a) in mice. Consequently, we hypothesized that "normal" variation of the gut microbiota might also alter hepatic Cyp activity and lead to individual differences in drug metabolism. Therefore, this study investigated the effects of normal gut microbiota variation on hepatic Cyp activity under the same genetic and environmental conditions using ex-germ-free mice. Using the feces of three breeder BALB/c mice (Jcl, Slc, and Crj), germ-free BALB/cYit mice were conventionalized (Yit-Jcl, Yit-Slc, and Yit-Crj). The gut microbiota composition and hepatic Cyp activity of these donors and recipients were evaluated. 16S rRNA sequencing revealed clear differences of the gut microbiota among donors and among recipients. Cyp3a activity was significantly higher in Slc mice than in Jcl and Crj mice. Notably, among recipients, Cyp3a activity was significantly higher in Yit-Slc and Yit-Crj mice than in Yit-Jcl mice. Cyp2b activity was significantly higher in Slc mice than in Jcl and Crj mice. Cyp2b activity was significantly higher in Yit-Slc mice than in Yit-Jcl mice. Additionally, in correlation analysis, some genera displayed significant positive or negative correlations with Cyp activity, particular the strong positive correlation between Clostridium sensu stricto 1 with Cyp3a activity. In conclusion, this study demonstrated that normal variation of the gut microbiota affected hepatic Cyp3a and Cyp2b activity, which might result in individual differences of drug metabolism.

Perifosine, a Bioavailable Alkylphospholipid Akt Inhibitor, Exhibits Antitumor Activity in Murine Models of Cancer Brain Metastasis Through Favorable Tumor Exposure.

Metastatic brain tumors are regarded as the most advanced stage of certain types of cancer; however, chemotherapy has played a limited role in the treatment of brain metastases. Here, we established murine models of brain metastasis using cell lines derived from human brain metastatic tumors, and aimed to explore the antitumor efficacy of perifosine, an orally active allosteric Akt inhibitor. We evaluated the effectiveness of perifosine by using it as a single agent in ectopic and orthotopic models created by injecting the DU 145 and NCI-H1915 cell lines into mice. Initially, the injected cells formed distant multifocal lesions in the brains of NCI-H1915 mice, making surgical resection impractical in clinical settings. We determined that perifosine could distribute into the brain and remain localized in that region for a long period. Perifosine significantly prolonged the survival of DU 145 and NCI-H1915 orthotopic brain tumor mice; additionally, complete tumor regression was observed in the NCI-H1915 model. Perifosine also elicited much stronger antitumor responses against subcutaneous NCI-H1915 growth; a similar trend of sensitivity to perifosine was also observed in the orthotopic models. Moreover, the degree of suppression of NCI-H1915 tumor growth was associated with long-term exposure to a high level of perifosine at the tumor site and the resultant blockage of the PI3K/Akt signaling pathway, a decrease in tumor cell proliferation, and increased apoptosis. The results presented here provide a promising approach for the future treatment of patients with metastatic brain cancers and emphasize the importance of enriching a patient population that has a higher probability of responding to perifosine.

Spontaneous Cardiac Hypertrophy in a Crl:CD(SD) Rat.

Cardiac hypertrophy was observed in a 9-week-old Crl:CD(SD) rat that died unexpectedly. The animal was allocated to the control group of a toxicity study, and no abnormalities in its general conditions, body weight or food intake were observed. Necropsy revealed an increase in heart weight. Gross examination indicated cardiac enlargement with thickening of the right and left ventricular walls. Histopathological examination revealed hypertrophy of the cardiomyocytes in the right and left ventricular walls and the interventricular septum. Electron microscopic examination indicated bizarre nuclei and accumulation of an increased number of various sizes of mitochondria in the perinuclear region of the hypertrophied myocytes. Hypertrophied myocytes connected by intensely folded intercalated disks were also observed. Based on these findings, the animal was diagnosed with cardiac hypertrophy. This is the first case report of cardiac hypertrophy in this strain.

Evaluation of a 28-day repeated-dose micronucleus test in rat glandular stomach, colon, and liver using gastrointestinal tract-targeted genotoxic-carcinogens and non-carcinogens.

The usefulness of the rat repeated-dose liver and gastrointestinal (GI) tract micronucleus (MN) tests to detect site-specific carcinogens has been shown previously using 22 chemicals in a study conducted by the Mammalian Mutagenicity Study group in the Japanese Environmental Mutagen Society. However, in the 6th International Workshop on Genotoxicity Testing, the need for further data to identify the sensitivity and specificity of the GI tract MN test and the specificity of the liver MN test, for the purpose of regulatory use, was mentioned. In the present study, we conducted additional studies to validate the performance of the 28-day repeated-dose GI tract and liver MN tests using genotoxic stomach carcinogens, N-nitroso-N-methylurea (MNU) and N-methyl-N-nitrosourethane (NMUT); genotoxic colon carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP), and non-carcinogens, sodium chloride, sucrose, and amaranth. Male Crl:CD (SD) rats were administered with each chemical by oral gavage for 28 days and the micronucleated cell frequencies in the glandular stomach, colon, and liver were monitored. MNU and NMUT showed positive results in the glandular stomach, and PhIP did so in the colon. These carcinogens showed negative results in the liver, which is not a target organ for these chemicals. Negative results were obtained for all three non-carcinogens in the glandular stomach, colon, and liver. Therefore, it was shown that the glandular stomach and colon MN tests with 28-day repeated-dose regimen have a good sensitivity for detecting genotoxic GI tract carcinogens as positive and have a good specificity to determined non-carcinogens as negative. The negative results with these six chemicals in the liver provide additional evidence supporting the good specificity of the 28-day repeated-dose liver MN test.

Imbalance in intestinal microflora constitution could be involved in the pathogenesis of inflammatory bowel disease.

Since genetically engineered animal models of inflammatory bowel disease (IBD) do not develop colitis under germ-free conditions, the intestinal microflora is thought to be one of the most important environmental factors associated with IBD. To understand the involvement of intestinal microflora in the pathogenesis of IBD, we analyzed the constituents of intestinal microflora in IBD. Faecal samples from 73 patients with ulcerative colitis (UC) and 23 patients with Crohn's disease (CD) were analyzed by quantitative PCR using 16S rRNA gene-targeted group-specific primers for Bacteroides fragilis group, Bifidobacterium, Clostridium coccoides groups, Clostridium leptum subgroup, Atopobium cluster, and seven species of Bacteroides. We analyzed the distribution of the predominant microflora by fluorescence in situ hybridization (FISH) using group-specific probes. We also examined the concentration of faecal organic acids produced by intestinal microflora. Contrary to previous reports, we found that the B. fragilis group was significantly decreased in the faeces of patients with IBD. Moreover, B. vulgatus was the predominant microflora in healthy controls and relatively decreased among IBD patients. Most of the microflora adhering to the colonic mucosa surrounding the mucus layer comprised C. coccoides group and Bifidobacterium. B. fragilis group mainly inhabited the faeces, but did not adhere to or invade the mucosa. The concentrations of propionic and butyric acids in the faeces were significantly decreased in patients with IBD. These findings indicate that IBD is not caused by a specific intestinal bacterial cluster or species and that disordered intestinal microflora could be involved in the pathogenesis of IBD.

Involvement of UDP-glucuronosyltransferase activity in irinotecan-induced delayed-onset diarrhea in rats.

We assessed the involvement of UDP-glucuronosyltransferase (UGT) activity in episodes of irinotecan hydrochloride (CPT-11)-induced delayed-onset diarrhea using a mutant rat strain with an inherited deficiency of UGT1A (Gunn rats). Gunn rats exhibited severe diarrhea after the intravenous administration of CPT-11 at a dose of 20 mg/kg, whereas Wistar rats did not. In the epithelium of the small intestine and cecum in Gunn rats, the shortening of villi, degeneration of crypts, and destruction of the nucleus were observed. The AUC, MRT, and t (1/2) of CPT-11, and the AUC of 7-ethyl-10-hydroxycamptothecin (SN-38) in plasma were, respectively, 1.6-fold, 1.5-fold, 1.7-fold, and 6.5-fold higher, and the cumulative biliary excretion rate of SN-38 was 2.3-fold higher, in Gunn rats than Wistar rats. SN-38 glucuronide excreted via bile in Wistar rats was not de-conjugated in the small intestinal lumen. The SN-38 AUC values in small intestinal tissues were also 5.0 to 5.8-fold higher in Gunn rats than Wistar rats. In conclusion, Gunn rats developed severe delayed-onset diarrhea after i.v. administration of CPT-11 at a much lower dose. Severe intestinal impairments would be induced in Gunn rats through exposure to SN-38 at high levels for a long period mainly via the intestinal lumen and partially via the bloodstream. These results clarified that the deficiency of UGT activity contributed greatly to the induction of the CPT-11-induced delayed-onset diarrhea and epithelial impairment in the intestine. In the clinic, great care is needed when using chemotherapy with CPT-11 in patients with poor UGT activity.

Microscale spatial analysis provides evidence for adhesive monopolization of dietary nutrients by specific intestinal bacteria.

Each species of intestinal bacteria requires a nutritional source to maintain its population in the intestine. Dietary factors are considered to be major nutrients; however, evidence directly explaining the in situ utilization of dietary factors is limited. Microscale bacterial distribution would provide clues to understand bacterial lifestyle and nutrient utilization. However, the detailed bacterial localization around dietary factors in the intestine remains uninvestigated. Therefore, we explored microscale habitats in the murine intestine by using histology and fluorescent in situ hybridization, focusing on dietary factors. This approach successfully revealed several types of bacterial colonization. In particular, bifidobacterial colonization and adhesion on granular starch was frequently and commonly observed in the jejunum and distal colon. To identify the bacterial composition of areas around starch granules and areas without starch, laser microdissection and next-generation sequencing-based 16S rRNA microbial profiling was performed. It was found that Bifidobacteriaceae were significantly enriched by 4.7 fold in peri-starch areas compared to ex-starch areas. This family solely consisted of Bifidobacterium pseudolongum. In contrast, there was no significant enrichment among the other major families. This murine intestinal B. pseudolongum had starch-degrading activity, confirmed by isolation from the mouse feces and in vitro analysis. Collectively, our results demonstrate the significance of starch granules as a major habitat and potential nutritional niche for murine intestinal B. pseudolongum. Moreover, our results suggest that colonizing bifidobacteria effectively utilize starch from the closest location and maintain the location. This may be a bacterial strategy to monopolize solid dietary nutrients. We believe that our analytical approach could possibly be applied to other nutritional factors, and can be a powerful tool to investigate in vivo relationships between bacteria and environmental factors in the intestine.

Evaluation of repeated dose micronucleus assays of the liver and gastrointestinal tract using potassium bromate: a report of the collaborative study by CSGMT/JEMS.MMS.

The food additive potassium bromate (KBrO3) is known as a renal carcinogen and causes chromosomal aberrations in vitro without metabolic activation and in vivo in hematopoietic and renal cells. As a part of a collaborative study by the Mammalian Mutagenicity Study group, which is a subgroup of the Japanese Environmental Mutagen Society, we administered KBrO3 to rats orally for 4, 14, and 28 days and examined the micronucleated (MNed) cell frequency in the liver, glandular stomach, colon, and bone marrow to confirm whether the genotoxic carcinogen targeting other than liver and gastrointestinal (GI) tract was detected by the repeated dose liver and GI tract micronucleus (MN) assays. In our study, animals treated with KBrO3 showed some signs of toxicity in the kidney and/or stomach. KBrO3 did not increase the frequency of MNed cells in the liver and colon in any of the repeated dose studies. However, KBrO3 increased the frequency of MNed cells in the glandular stomach and bone marrow. Additionally, the MNed cell frequency in the glandular stomach was not significantly affected by the difference in the length of the administration period. These results suggest that performing the MN assay using the glandular stomach, which is the first tissue to contact agents after oral ingestion, is useful for evaluating the genotoxic potential of chemicals and that the glandular stomach MN assay could be integrated into general toxicity studies.

Alleviation of side effects induced by irinotecan hydrochloride (CPT-11) in rats by intravenous infusion.

PURPOSE: Irinotecan hydrochloride (CPT-11) is a potent topoisomerase I inhibitor and is established and used widely as an antitumor agent. However, it sometimes causes severe side effects such as myelosuppression and diarrhea. These dose-limiting toxicities prevent the adoption of CPT-11 in aggressive chemotherapy. Thus we sought to determine in a rat model whether extending the period of infusion of CPT-11 would ameliorate the adverse reactions. METHODS: CPT-11 was administered intravenously (i.v.) to rats at a dose of 60 mg/kg per day for four consecutive days as a bolus injection or as 3-, 8- or 24-h infusions, and then blood cell counts and the incidence of acute and delayed-onset diarrhea were monitored. RESULTS: Serious acute and delayed-onset diarrhea and marked decreases in the number of neutrophils and lymphocytes were observed in the bolus injection group. These symptoms were alleviated in the infusion groups with the degree of alleviation dependent on infusion time. In the bolus injection group, mucosal impairment of the cecal epithelium including wall thickening, edema, a decrease in the number and size of crypts, and the formation of a pseudomembrane-like substance was observed, whereas these changes were less severe in the infusion groups. Areas under the plasma concentration-time curves (AUCpla) of CPT-11 and its metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), differed little among the bolus injection group, and the 3-h and 8-h infusion groups. However, the AUCpla values of CPT-11 and SN-38 were significantly decreased and increased, respectively, in the 24-h infusion group. The maximum plasma concentrations (Cmax) of CPT-11 decreased with increasing infusion time, but those of SN-38 did not. CONCLUSIONS: It was confirmed that the side effects of CPT-11 were alleviated by extending the infusion time. The pharmacokinetic parameters suggested that the Cmax of CPT-11 is closely related to the incidence and severity of adverse reactions such as myelosuppression and acute and delayed-onset diarrhea.

[Intestinal microflora and cancer prevention].

Based on the results of ecological studies of intestinal microflora, we have been studying from various perspectives, the relationship between health and intestinal microflora. Concerning intestinal microflora and cancer, we reported in Cancer Research (61: 2395-2398, 2001) that spontaneous colon cancers that developed in Tcr beta/p53-double knockout mice did not develop in germfree conditions. This study, catching the attention of researchers, was reviewed in the 'News Feature' column of Nature (415: 8-9, 2002), which introduces recent studies attracting significant attention. Cancers are diseases of genes. However, in this study, which Nature found to be an as intriguing transformation (development of colon cancer in this case), did not occur only by genetic changes, unless accompanied by environmental factors (intestinal flora in this case). The fact that intestinal microflora are involved in the development of colon cancer suggests, on the other hand, that cancers can be prevented by controlling intestinal microflora. Here, data will be presented to show the relationship between intestinal microflora and colon cancers, mainly from our studies, and the mechanism involved will be further discussed.

Histopathological studies of microtubule disassembling agent-induced myocardial lesions in rats.

Microtubule disassembling agents (MDAs) such as colchicine (COL) and vincristine sulfate (VCR) are known to be cardiotoxic. However, few attempts have been made to histopathologically examine cardiac lesions induced by MDAs. In this study, we endeavored to induce myocardial injury in rats by administering MDAs and to clarify the morphological features of these myocardial lesions. Male rats were intravenously administered COL (1.00 or 1.25mg/kg for 2 days at single daily doses) or VCR (0.50 or 0.75 mg/kg for 2 days at single daily doses). The day after administration, hearts were excised and examined histopathologically, immunohistochemically and electron microscopically. Degeneration and necrosis of myocardial cells with vacuolation were observed in rats administered COL at 1.25mg/kg or VCR at 0.75 mg/kg. Electron microscopic examination revealed vacuoles in swollen mitochondria. Moreover, there were cells showing pyknosis and karyorrhexis in the interstitium. TUNEL and immunohistochemical staining for endothelial cells and electron microscopic examination identified the apoptotic cells in the interstitium to be vascular endothelial cells. These vascular endothelial lesions were induced by lower doses of MDAs than were myocardial lesions. Furthermore, common sites of cardiac lesions induced by MDAs had almost the same distribution as areas positive for pimonidazole, a marker of hypoxia. These findings indicate that MDAs occasionally damage mitochondria in myocardial cells, and suggest that these changes involve microcirculatory dysfunction induced by endothelial cell injury.