Time course of the incidence/multiplicity and histopathological features of murine colonic dysplasia, adenoma and adenocarcinoma induced by benzo[a]pyrene and dextran sulfate sodium.

Benzo[a]pyrene (BP) is mutagenic but noncarcinogenic in the murine colon. Recently, we reported rapid induction of colonic tumors by treatment of CD2F1 mice with BP (125 mg/kg for 5 days) followed by a colitis inducer, dextran sulfate sodium (DSS) (4% in drinking water for 1 or 2 weeks). However, there are no reports on detailed time course and histopathological features of colonic proliferative lesions in this model. Here, we show the detailed time course of colonic dysplasia, adenoma and adenocarcinoma induced by treatment with BP, DSS, and a combination of the two (BP/DSS). In the colon of mice exposed to BP/DSS, 14.6 dysplastic foci per mouse were present one week after DSS treatment (week 4). The number of dysplastic foci decreased with time to 3.1 at week 9 and thereafter remained almost constant. At week 4, 1.5 adenocarcinomas were also observed, with a marked increase in numbers with time, reaching 29.3 at week 14. In contrast, the number of dysplastic foci induced by DSS alone showed a time course similar to that following BP/DSS treatment; however, only a few tumors appeared. Neither dysplastic foci nor neoplastic lesions were induced by BP only. In mice exposed to BP/DSS, ß-catenin was demonstrated immunohistochemically in the nucleus and/or cytoplasm of the tumor cells, and this translocation from the cell membrane was evident in subsets of dysplastic foci. In dysplastic foci induced by DSS alone, ß-catenin was absent in the nucleus/cytoplasm. These finding suggest that aberrant ß-catenin accumulation in dysplastic foci is associated with tumor progression in this BP/DSS model.

Brunner's gland lesions in rats induced by a vascular endothelial growth factor receptor inhibitor.

Vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitors are reported to cause reversible mucosal hyperplasia (adenosis) in the duodenum of rats; however, the pathogenesis is not fully elucidated. Using lenvatinib, a VEGF RTK inhibitor, we characterized the histologic time course of this duodenal change in rats. At 4 weeks, there was degeneration and necrosis of Brunner's gland epithelium accompanied by neutrophil infiltration around the affected glands. At 13 weeks, the inflammation was more extensive, and Brunner's gland epithelium was attenuated and flattened and was accompanied by reactive hyperplasia of duodenal epithelium. At 26 weeks, the changes became more severe and chronic and characterized by marked cystic dilation, which extended to the external muscular layer. These dilated glands exhibited morphological characteristics of duodenal crypt epithelium, suggestive of replacement of disappeared Brunner's glands by regenerative duodenal crypt epithelial cells. Similar changes were not present in similar time course studies in dog and monkey studies, suggesting that this is a rodent- or species-specific change. Based on the temporal progression of Brunner's gland lesion, we identify degeneration and necrosis of the Brunner's glands as the primary change leading to inflammation, cystic dilatation, and regeneration with cells that are morphologically suggestive of duodenal crypt epithelium.

Granulomatous nephritis consistent with malakoplakia in a cynomolgus monkey.

Malakoplakia is a rare form of chronic granulomatous inflammation in mammals, and usually affects the urinary tract in humans. In this report, we present a case of granulomatous nephritis consistent with malakoplakia in a 4-year-old male cynomolgus monkey. Gross examination showed that the kidney was markedly enlarged and adhered to the surrounding organs. Histology showed that there was diffuse interstitial infiltration of histiocytes with abundant foamy eosinophilic cytoplasm resembling von Hansemann cells, PAS-positive granular cytoplasm and occasional PAS- and iron-positive intracellular small inclusion bodies. Electron microscopy showed that these histiocytes contained abundant lysosomes and phagolysosomes but no obvious Michaelis-Gutmann bodies. Based on these findings, a diagnosis of granulomatous nephritis consistent with early malakoplakia was made. This is the first report in a monkey of a renal lesion consistent with malakoplakia.

DNA polymerase κ suppresses inflammation and inflammation-induced mutagenesis and carcinogenic potential in the colon of mice.

BACKGROUND: Chronic inflammation induces DNA damage and promotes cell proliferation, thereby increasing the risk of cancer. DNA polymerase κ (Pol κ), involved in translesion DNA synthesis, counteracts mutagenesis induced by inflammation in the colon of mice. In the present study, we examined whether Pol κ suppressed inflammation-induced colon tumorigenesis by treating inactivated Polk knock-in (Polk-/-) mice with dextran sulfate sodium (DSS), an inducer of colon inflammation. RESULTS: Male and female Polk-/- and Polk+/+ mice were administered 2% DSS in drinking water for six consecutive days, succeeded via a recovery period of 16 days, followed by 2% DSS for another two days. DSS treatment strongly induced colitis, and the severity of colitis was higher in Polk-/- mice than in Polk+/+ mice. The mice were sacrificed after 19 weeks from the initiation of the first DSS treatment and subjected to pathological examination and mutation analysis. DSS treatment induced colonic dysplasia, and the multiplicity of dysplasia was higher in Polk-/- mice than in Polk+/+mice. Some of the dysplasias in Polk-/- mice exhibited ß-catenin-stained nucleus and/or cytoplasm. Mutation frequencies in the gpt reporter gene were increased by DSS treatment in Polk-/- mice, and were higher than those in Polk+/+ mice. CONCLUSIONS: Pol κ suppresses inflammation and inflammation-induced dysplasia as well as inflammation-induced mutagenesis. The possible mechanisms by which Pol κ suppresses colitis- and colitis-induced dysplasia are discussed.

o-Aminoazotoluene, 7,12-dimethylbenz[a]anthracene, and N-ethyl-N-nitrosourea, which are mutagenic but not carcinogenic in the colon, rapidly induce colonic tumors in mice with dextran sulfate sodium-induced colitis.

BACKGROUND: Several rodent models with chemically induced colon cancer have been developed. Among these models, dextran sulfate sodium (DSS), a colitis inducer, combined with azoxymethane as a colon mutagenic carcinogen, is commonly used. We previously reported that although benzo [a] pyrene (BP) is mutagenic but not carcinogenic in the colon, it rapidly develops colon tumors at a high incidence/multiplicity after treatment with DSS. In the present study, we examined whether other colon-mutagenic non-carcinogens (CMNCs) induced colon tumors after treatment with DSS. RESULTS: o-Aminoazotoluene, 7,12-dimethylbenz[a]anthracene, and N-ethyl-N-nitrosourea were selected as CMNCs. Male CD2F1 mice were orally administered CMNC for 5 consecutive days. After a 9-day dose-free period, mice were treated with 4% DSS in drinking water for 1 week. Three months after DSS treatment, colon samples were collected for histopathology and ß-catenin immunohistochemistry analyses. All CMNCs in combination with DSS induced colonic adenocarcinomas at a high incidence/multiplicity in the distal and middle parts of the colon, coinciding with the location of colitis. Unlike in normal cells where ß-catenin is exclusively located on the cell membrane, in adenocarcinoma cells, it was translocated to both the nucleus and cytoplasm or only to cytoplasm. The translocation of ß-catenin is closely associated with colon carcinogenesis in rodents and humans. No colonic tumors or dysplastic lesions were found after exposure to either CMNC or DSS alone. CONCLUSION: We provided further evidence clearly showing that CMNCs can rapidly induce colonic tumors in mice with DSS-induced colitis, even if they are not colonic carcinogens.

Efficacy of oral E1210, a new broad-spectrum antifungal with a novel mechanism of action, in murine models of candidiasis, aspergillosis, and fusariosis.

E1210 is a first-in-class, broad-spectrum antifungal with a novel mechanism of action-inhibition of fungal glycosylphosphatidylinositol biosynthesis. In this study, the efficacies of E1210 and reference antifungals were evaluated in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. Oral E1210 demonstrated dose-dependent efficacy in infections caused by Candida species, Aspergillus spp., and Fusarium solani. In the treatment of oropharyngeal candidiasis, E1210 and fluconazole each caused a significantly greater reduction in the number of oral CFU than the control treatment (P < 0.05). In the disseminated candidiasis model, mice treated with E1210, fluconazole, caspofungin, or liposomal amphotericin B showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also highly effective in treating disseminated candidiasis caused by azole-resistant Candida albicans or Candida tropicalis. A 24-h delay in treatment onset minimally affected the efficacy outcome of E1210 in the treatment of disseminated candidiasis. In the Aspergillus flavus pulmonary aspergillosis model, mice treated with E1210, voriconazole, or caspofungin showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also effective in the treatment of Aspergillus fumigatus pulmonary aspergillosis. In contrast to many antifungals, E1210 was also effective against disseminated fusariosis caused by F. solani. In conclusion, E1210 demonstrated consistent efficacy in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. These data suggest that further studies to determine E1210's potential for the treatment of disseminated fungal infections are indicated.

Histopathological characteristics of luteal hypertrophy induced by ethylene glycol monomethyl ether with a comparison to normal luteal morphology in rats.

Ethylene glycol monomethyl ether (EGME) is a known reproductive toxicant that induces luteal hypertrophy in rat ovaries. In this study, we characterized the histopathological features of corpora lutea (CL) from EGME-treated rats and compared them with normal CL formation and regression. Normally cycling female Sprague-Dawley rats were treated with 5-bromo-2'-deoxyuridine (BrdU) intraperitoneally on the morning of estrus and their ovaries were examined 1 (metestrus), 4 (estrus), 8 (estrus), or 12 (estrus) days later to observe the transition of BrdU-labeled cells within in the CL. CL at each time point of estrus stage were classified into 4 types: Type I (newly formed CL), Type II (mature CL), Type III (regressing CL), and Type IV (residual CL). CL almost fully regressed within 4 estrus cycles. In contrast, in female rats given EGME orally (30, 100, or 300 mg/kg for 2 or 4 weeks), luteal cells were hypertrophic with abundant cytoplasm. Although the size of CL varied, all CL in EGME-treated rats had histological features similar to Type II CL, but they were more hypertrophic with less apoptosis. These results suggest that EGME has a luteal hypertrophic effect on all CL phases, including regression.

DNA polymerase kappa counteracts inflammation-induced mutagenesis in multiple organs of mice.

In vitro studies indicate that DNA polymerase kappa (Polκ) is able to accurately and efficiently perform DNA synthesis using templates containing various types of DNA damage, including benzo[a]pyrene (BP)-induced N2 -deoxyguanosine adducts. In this study, we examined sensitivity of inactivated Polk knock-in (Polk-/- ) mice to BP carcinogenicity in the colon by administering an oral dose of BP plus dextran sulfate sodium (DSS), an inflammation causing promoter of carcinogenesis. Although colon cancer was successfully induced by BP plus DSS, there was no significant difference in tumor incidence or multiplicity between Polk-/- and Polk+/+ mice. Malignant lymphoma was induced in thymus by the treatment only in Polk-/- mice, but it lacked statistical significance. Mutant frequencies (MFs) in the gpt reporter gene were strongly enhanced in colon; almost to the same extent in both types of mice. Micronucleus formation in bone marrow at the high dose of BP and DNA adducts in colon and lung was not significantly different between two types of mice. Surprisingly, however, Polk-/- mice exhibited significantly higher MFs in colon and lung than did Polk+/+ mice when they were treated with DSS alone. The most prominent mutation induced by DSS treatment was G:C to C:G transversion, whose specific MF in proximal colon was 30 times higher in Polk-/- than in Polk+/+ mice. DSS alone did not enhance MF at all in Polk+/+ mice. The results indicate that Polκ does not suppress BP-induced mutagenesis and carcinogenesis in the colon, but counteracts inflammation-induced mutagenesis in multiple organs. Environ. Mol. Mutagen. 60:320-330, 2019. © 2019 Wiley Periodicals, Inc.

Wif1 and Ifitm3 gene expression preferentially altered in the colon mucosa of benzo[a]pyrene pre-treated mice following exposure to dextran sulfate sodium.

Benzo[a]pyrene (BP) is highly mutagenic and yet does not lead to tumor development in the murine colon. We recently reported the generation of colonic tumors one week after treatment with BP followed by dextran sulfate sodium (DSS), a colitis-inducer. In this BP/DSS model, male CD2F1 mice were treated orally with BP at 125 mg/kg/day for 5 days, followed by 4% DSS in drinking water for one week. There has been no report so far on the molecular mechanisms involved in tumor development in this model. In the present study, we performed global gene expression analysis on the colonic mucosae obtained from BP-exposed mice one week after treatment with DSS and those treated with the vehicle, BP, or DSS alone. Global gene expression analysis revealed that there were 563 genes preferentially altered (≥2-fold vs vehicle group) in the colonic mucosae exposed to both BP and DSS. Furthermore, comparative gene expression analysis combined with Ingenuity Pathway Analysis™ identified 2 genes associated with Wnt/ß-catenin signaling pathway that were preferentially up-regulated (≥2-fold vs vehicle group) when BP and DSS were treated in combination in the distal part (site of predilection for tumor induction) of the colonic mucosae, especially in colonic tumors: WNT inhibitory factor 1 (Wif1; 14.6-fold increase) and interferon induced membrane protein 3 (Ifitm3; 5.7-fold increase). In colonic tumors, expression of Wif1 and Ifitm3 proteins were both confirmed by western blot analysis. These findings suggest that these genes are associated with rapid induction of colonic tumors in mice after exposure to BP/DSS, providing insights into the mechanisms of the BP/DSS short-term colon carcinogenesis.

Prothrombin activation during carbon tetrachloride-induced acute liver injury in mice.

Serine protease thrombin is a central factor for blood coagulation and is an integral part of inflammatory reactions. Prothrombin activation of the liver homogenates during carbon tetrachloride-induced liver injury was investigated. At 72 h after administration of carbon tetrachloride, the prothrombin activation activity reached the maximal value, and then decreased to the basal level at 168 h. The alanine transaminase (ALT) activity, an index of tissue injury, was dramatically elevated at 36 h after treatment, and almost recovered to normal levels at 72 h. Histochemical analysis revealed that the proliferation of hepatocytes and remarkable phagocytosis by macrophages was observed at 72 h, in contrast to severe tissue damage at 36 h. Finally, we showed that prothrombinase activity, composed of factor Xa and factor Va, was involved in the injury-associated prothrombin activation. Taken together, these results strongly suggest that prothrombin activation by the prothrombinase complex occurred following tissue destruction in carbon tetrachloride-induced liver injury.

Spontaneous gingivitis related to hair penetration in rats.

Maxillary gingivae from male and female Crl:CD(SD) rats at 12, 16, 21, and 34 weeks of age were examined histologically. The incidence of gingivitis was approximately 40%, with no age or sex predilection, and was most frequent between the first and second molar. Lesions were characterized by acute focal neutrophilic infiltration into the gingival mucosa, occasionally with inflammatory exudate. In severe cases, inflammation extended to the periodontal ligament with abscess formation, and adjacent alveolar bone destruction/resorption. The most characteristic finding was the presence of hair shafts associated with the lesion, which was observed in approximately 80% of the rats with gingivitis. These findings suggest that molar gingivitis occurs in rats from an early age and persists thereafter, and that the main cause of gingivitis in rats is hair penetration into the gingiva. It would be prudent to keep these background lesions in mind as potential modifiers in toxicity studies.

Rapid induction of colonic adenocarcinoma in mice exposed to benzo[a]pyrene and dextran sulfate sodium.

Previously, we reported that the mutation frequency was markedly increased in the colon after the oral treatment of mice with an environmental mutagen/carcinogen, benzo[a]pyrene (BP); however this was not followed by tumor development. The reasons for this are as yet unresolved. The purpose of the present study is to explore the mechanisms why a high frequency of mutations induced by BP in the colon is not associated with subsequent tumor development. We show in this study that oral administration of BP to CD2F(1) mice at 125 mg/kg/day for 5 days can lead to adenocarcinomas in the mouse colon both at Weeks 4 (5/8 mice) and 11 (100% of mice), but only in the presence of inflammation induced by 4% dextran sulfate sodium (DSS) in the drinking water for up to 2 weeks. These data indicate that, in this DSS model, BP induced mutagenic events lead to tumors in the mouse colon, a tissue which is not a BP target organ. DSS-induced inflammation in a tissue primed with mutagenic risk is a key to the induction of tumors in this model. This study provides a novel, rapid and useful colon carcinogenesis model (BP/DSS model).

Collaborative work on evaluation of ovarian toxicity. 11) Two- or four-week repeated-dose studies and fertility study of ethylene glycol monomethyl ether in female rats.

The objective of this study was to determine the optimal period of administration for detection of ovarian toxicity in rat repeated-dose toxicity studies. A well-known ovarian toxicant, ethylene glycol monomethyl ether (EGME), was administered to female rats at dose levels of 0, 30, 100, or 300 mg/kg for 2 or 4 weeks (repeated-dose toxicity studies). The same doses were administered to female rats for 2 weeks prior to mating, during mating, and until Day 6 of pregnancy (fertility study). In the repeated-dose toxicity studies, continuous diestrus was observed at > or = 100 mg/kg regardless of period of administration. The alterations of ovarian morphology observed at > or = 100 mg/kg after 2 or 4 weeks of administration were characterized by hypertrophy of the corpora lutea with decreased cellular debris indicating apoptosis, and increased proliferating cell nuclear antigen (PCNA)-negative large atretic follicles. The finding that newly-formed basophilic corpora lutea were scarce in affected animals exhibiting continuous diestrus suggested suppression of ovulation due to hypertrophic corpora lutea. In the fertility study, irregular estrous cycles, prolonged mating periods, lower pregnancy rates and decreased corpora lutea of pregnancy were observed at > or = 100 mg/kg. The irregularities of estrous cycle observed in some animals at 30 mg/kg were minimal. The ovarian histopathological changes in repeated-dose toxicity studies correlated well with impairment of female fertility found in the fertility study. It is concluded that a repeated-dose toxicity study with a treatment period for 2 weeks or longer is sufficient for evaluation of ovarian toxicity induced by EGME.

Podocyte injury promotes progressive nephropathy in zucker diabetic fatty rats.

The zucker diabetic fatty (ZDF-fa/fa) rat is one of the attractive models for type II diabetes based on impaired glucose tolerance caused by the inherited insulin-resistance gene fa. Characterization of nephropathy in this model may provide useful insights into the mechanism of the progression of diabetic nephropathy. The present study analyzed the pathophysiology of diabetes and nephropathy, including the process of glomerulosclerosis in this model by biochemical and morphometric analyses. In addition, we conducted studies in podocytes in culture to examine the direct effects of high glucose on podocytes. ZDF-fa/fa rats showed overt diabetes despite hyperinsulinemia as early as 3 months of age. Blood glucose levels increased further with a considerable decrease of insulin levels at 5 months. Glomerular filtration rate (GFR) was significantly elevated until 3 months, but fell to the level seen in lean rats by 7 months. Proteinuria started to rise during the period of increased GFR, and increased further after GFR had fallen to within the normal range. Renal fibronectin, collagen iv, and vascular endothelial growth factor mRNA levels were increased at 7 months. Glomerulosclerosis commenced as early as 5 months of age, and was associated with glomerular hypertrophy and mild mesangial expansion with evidence of accentuated podocyte injury, as revealed by increased expression of desmin. Electron microscopy suggested that degeneration of podocytes and the development of tuft adhesions were responsible for the glomerular sclerosis in this model. In addition, glomeruli from the diabetic rats showed up-regulation of the cyclin kinase inhibitors, p21 and p27. Further studies suggested that the increase in p27 expression was predominantly caused by podocytes, because predominant immunolocalization of p27 in podocytes in diabetic rats and high glucose medium induced cell hypertrophy accompanied by p27 up-regulation in differentiated podocyte cell lines. In conclusion, progressive diabetic nephropathy in ZDF-fa/fa rats is associated with evidence of podocyte injury. High concentrations of ambient glucose induced podocyte hypertrophy and stress in vitro, suggesting that the podocyte is a likely target of the diabetic milieu.