Kazuo Tsukidate

Involvement of tumor necrosis factor-α in the pathogenesis of activated macrophage-mediated hepatitis in mice

The possible involvement of tumor necrosis factor-alpha in the pathogenesis of an experimentally induced hepatitis was investigated. Balb/c mice were primed with Propionibacterium acnes to induce the infiltration of mononuclear cells into the liver. Immunohistochemical study showed that most of the accumulated mononuclear cells at 7 days were Mac-2 positive, suggesting that they were activated macrophages. An injection of lipopolysaccharide resulted in massive hepatic necrosis and high mortality in the mice within 24 hours. Plasma tumor necrosis factor-alpha activity initially rose sharply and then declined over 3 hours. The increase in plasma aminotransferase activity correlated well with the elevation of plasma tumor necrosis factor-alpha activity. Pretreatment with dexamethasone or 16,16-dimethyl-prostaglandin E2 attenuated not only the elevation of plasma tumor necrosis factor-alpha activity but also the increase in plasma aminotransferase activity and improved the survival rate. Passive immunization against tumor necrosis factor-alpha showed protective effects. These findings suggest that tumor necrosis factor-alpha released from activated macrophages may play a crucial role in the pathogenesis of this murine hepatitis.

Comparison of the mutational spectra of the lacZ transgene in four organs of the Muta™Mouse treated with benzo[a]pyrene: target organ specificity

We recently demonstrated that not all organs with a high rate of induction of mutation in the lacZ transgene develop tumors in the lambdalacZ transgenic mice (MutaMouse) used for a long-term carcinogenicity study with benzo[a]pyrene (BP). To better understand the role of chemical-induced in vivo mutations in carcinogenesis, we compared the mutational spectra of the lacZ transgene in four organs of the MutaMouse obtained 2 weeks after five daily consecutive oral treatments with 125 mg/kg/day BP. lacZ transgenes were analyzed in two target organs (forestomach and spleen) and two non-target organs (colon and glandular stomach) for BP-induced carcinogenesis in MutaMouse, and all of these organs were highly mutated in the lacZ transgene. The sequence data showed similar mutational spectra of the lacZ transgene between the two target organs; the predominant mutations were G:C-->T:A transversions (55% and 50% for forestomach and spleen, respectively), followed by deletions (20% and 21% for forestomach and spleen, respectively) mainly at G:C site. The frequent G:C-->T:A transversions are consistent with reports of the mutational spectra produced in the p53 gene in tumors generated in rats and mice exposed to BP. In contrast, the mutational spectra of the lacZ transgene in the two non-target organs are different from those in the target organs, and are also suggested to differ from one another. These findings suggest an organ/tissue-specific mechanism of mutagenesis.

Multiple organ mutation in the lacZ transgenic mouse (Muta mouse) 6 months after oral treatment (5 days) with benzo[a]pyrene.

We have recently demonstrated that not all organs with high rates of mutation in the lacZ transgene develop tumors using the Muta Mouse. To better understand the role of in vivo mutation in carcinogenesis, we examined the mutant frequencies (MF) of the lacZ transgene in tumor-bearing and non tumor-bearing organs. MF, recovered after 2 weeks (the data taken from our previous study) and after 26 weeks following oral doses of 125 mg kg-1 day-1 benzo[a]pyrene (BP) for five days were compared. The organs examined included the target organs (forestomach, spleen, and lung) and non-target organs (colon, glandular stomach, and liver) for BP carcinogenesis. The data indicated that lacZ MF were markedly increased over spontaneous frequencies in the organs examined and that the organ which showed the highest MF was the colon, followed by the forestomach>spleen>glandular stomach, liver, and lung in that order. These findings indicate that the MF of the lacZ transgene in each organ, even 26 weeks after the start of the treatment does not fully correlate with the known target organs of BP. Furthermore, the lacZ MF in a non-papilloma region of a forestomach with a papilloma was equivalent to the two highest MF observed in the healthy colon (non-target organ) of mice at 26 weeks. These observations also indicate that the generation of tumors requires the induction of mutations as well as other factor(s) specific to the target organs. These results clearly suggest that highly mutated organs do not always progress to tumors in the transgenic mouse.

E2012-Induced Cataract and Its Predictive Biomarkers

E2012, a gamma secretase modulator without affecting Notch processing, aimed at Alzheimers disease by reduction of amyloid β-42, induced cataract following repeated doses in the rat. Cataract appeared first at week 10-11 of treatment as a posterior subcapsular area with granular/punctate opaque or shiny dots along the suture line, characterized histologically as lenticular fiber degeneration, which eventually coalesced to form a triangular or circular opacity. It was associated with prolonged and sustained elevation of lenticular desmosterol (24-dehydrocholesterol), the final precursor of cholesterol, and decrease in lenticular cholesterol. In vitro studies to investigate the effect of E2012 on cholesterol metabolism demonstrated that E2012 inhibits 3β-hydroxysterol Δ24-reductase (DHCR24) at the final step in the cholesterol biosynthesis. In vivo lenticular concentration of E2012 after 13-week repeated dose with cataract was well above those where inhibition was observed in vitro. There was no cataract formation at doses where desmosterol did not accumulate in the lens. The elevation of desmosterol and decreased cholesterol levels were also seen in the liver and plasma and preceded those in the lens. These results demonstrate that E2012 induces cataract in the rat by inhibiting DHCR24 at the final step of cholesterol synthesis with associated elevation in desmosterol within the lens, preceded by desmosterol changes that would serve as a predictive safety biomarker for lenticular opacity.

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).

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.

Prophylactic activity of dihydro-heptaprenol, a synthetic polyprenol derivative, against Sendai virus infection in mice

Abstract The effect of a chemically synthesized polyprenol derivative, dihydroheptaprenol (DHP), on the non-specific resistance of mice to Sendai virus infection was investigated. The mice that received 200 μg of DHP intranasally twice, at 3 days and 1 day before the infection, showed a significant protection against Sendai virus infection. Treatment of mice twice even with as much as 2000 μg of DHP through the subcutaneous route, however, had no protective effect against infection. Excess interferon and tumour necrosis factor production in intranasally DHP-treated mice was seen 1 day after the infection when compared with Sendai virus alone controls or with DHP alone controls. Variance analysis of these findings indicates a prophylactic activity of DHP in pulmonary viral infections.

Assessment of potential mutagenic activities of a novel benzothiazole MAO-A inhibitor E2011 using Salmonella typhimurium YG1029.

The potential initiation activities of a novel monoamine oxidase type-A (MAO-A) inhibitor E2011, which induced preneoplastic foci in the rat liver, were investigated by comparing the mutagenic activity of E2011, 6-aminobenzothiazole (6-ABT, a structural scaffold of E2011) and its derivatives, which are suggested primary reactive metabolites for E2011-induced hepatotoxicity in the rats in vivo, in the Ames assay system employing two Salmonella tester strains, TA100 and YG1029, a bacterial O-acetyltransferase-overproducing strain of TA100. E2011, a tertiary amine, showed no mutagenic activity both in the Salmonella typhimurium TA100 and YG1029 with and without S9 mix. On the other hand, a secondary aromatic amine ER-174238-00, a typical decarbonated metabolite of E2011, showed weak but significant mutagenicity in YG1029 in the presence of S9 mix, and a primary aromatic amine ER-174237-00, an N-dealkylated derivative of ER-174238-00, exhibited S9-dependent potent mutagenicity in YG1029. Thus, it appears that primary and secondary amino moieties of benzothiazole derivatives at C(6)-position are the specific structures contributing to their mutagenic activity. In addition, the alkyl group at C(2)-position of E2011, ER-174237-00 and ER-174238-00 is suggested to intensify the mutagenic activity, since the mutagenicity of ER-174237-00 is approximately two-fold higher than that of 6-ABT, which has hydrogen at C(2)-position in the place of the alkyl group. These results strongly suggest that E2011 has potential initiation activities in the rat liver in vivo after undergoing decarbonation, one of the metabolic pathways, at the carbonyl moiety of oxazolidinone ring to form mutagenic amine(s).

A new method for preparing electron microscopic specimens of Helicobacter pylori.

A new method for preparing electron microscopic specimens of Helicobacter pylori was developed and used to examine the ultrastructure of this bacterium. We have also investigated the morphological changes in the bacterium when exposed to amoxicillin using our new method. Bacterial specimens for electron microscopy are usually prepared by collecting the bacteria by centrifugation during the fixation and dehydration processes. In our new method the bacteria are filtered through and adsorbed onto a filter before fixation, and the entire filter containing the adhered bacteria is fixed and dehydrated. Using this method we were able to obtain electron photomicrographs in which the external appearances or internal structures of the bacteria were well conserved. The advantages of this method are that it uses only a small amount of bacterial suspension, shortens the time required for the dehydration procedure, and keeps the artifacts to the minimum. Amoxicillin treatment resulted in coccoid form with blebs in the bacterial surface and the appearance of vacuoles, granules, and an area of low electron density in the cytoplasm at one and four minimum inhibitory concentrations. These changes were consistent with results previously reported in the literature.

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.