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Vitamin A deficiency (VAD) caused by malnutrition and certain intestinal diseases induces visual impairments, including night blindness and photoreceptor cell dysfunction as indicated by reduced a‑ and b‑waves in an electroretinogram (ERG). The effects of VAD on the inner retinal layer cells, including amacrine and ganglion cells, remain to be elucidated. The functions of these cells are reflected in oscillatory potentials (OPs), another component of the ERG. The present study investigated inner retinal layer cell function in VAD rats by analyzing OPs. In the present study, VAD was induced by feeding Brown Norway rats a vitamin A deficient diet for 10 weeks. A reduced body weight and peri‑papillary opacification indicative of papilledema without histopathological alterations were observed, which are considered early symptoms of VAD. At this stage, the ERG revealed reduced OPs as well as a‑ and b‑waves at various intensities of light stimulation. Further analysis indicated that the ratio of the alterations in OPs was more significant than those of a‑ and b‑waves. After 5 weeks of recovery, these changes returned to control levels. These results suggest that OPs are the most sensitive and early marker of VAD‑associated visual impairment in the ERG.

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., (© 2014 by The Author(s).)

E2012, a gamma secretase modulator without affecting Notch processing, aimed at Alzheimer's 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.

The neutral and one-electron oxidized Cu(II) six-membered chelate 1,3-Salcn (1,3-Salcn = N,N'-bis(3,5-di-tert-butylsalicylidene)-1,3-cyclohexanediamine) complexes have been investigated and compared with the five-membered chelate 1,2-Salcn (N,N'-bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexane-(1R,2R)-diamine) complexes. Cyclic voltammetry of Cu(1,3-Salcn) showed two reversible redox waves at 0.48 and 0.68 V, which are only 0.03 V higher than those of Cu(1,2-Salcn). Reaction of Cu(1,3-Salcn) with 1 equiv of AgSbF6 afforded the oxidized complex which exists as a ligand-based radical species in solution and in the solid state. The X-ray crystal structure of the oxidized complex, [Cu(1,3-Salcn)]SbF6, exhibited an asymmetric metal binding environment with a longer Cu-O bond and quinoid distortion in the phenolate moiety on one side, demonstrating at least partial ligand radical localization in the solid state. The ligand oxidation is also supported by XPS and temperature dependent magnetic susceptibility. The electronic structure of the [Cu(1,3-Salcn)](+) complex was further probed by UV-vis-NIR, resonance Raman, and electron paramagnetic resonance (EPR) measurements, and by theoretical calculations, indicating that the phenoxyl radical electron is relatively localized on one phenolate moiety in the molecule. The reactivity of [Cu(1,3-Salcn)](+) with benzyl alcohol was also studied. Quantitative conversion of benzyl alcohol to benzaldehyde was observed, with a faster reaction rate in comparison with [Cu(1,2-Salcn)](+). The kinetic isotope effect (KIE = k(H)/k(D)) of benzyl alcohol oxidation by [Cu(1,3-Salcn)](+) was estimated to be 13, which is smaller than the value reported for [Cu(1,2-Salcn)](+). The activation energy difference between [Cu(1,2-Salcn)](+) and [Cu(1,3-Salcn)](+) was in good agreement with the energy calculated from KIE. This correlation suggests that the Cu(II)-phenoxyl radical species, characterized for [Cu(1,2-salcn)](+) is more reactive for hydrogen abstraction from benzyl alcohol in comparison to the 1:1 mixture of Cu(III)-phenolate and Cu(II)-phenoxyl radical species, [Cu(1,2-Salcn)](+). Thus, the Cu(II)-phenoxyl radical species accelerates benzyl alcohol oxidation in comparison with the Cu(III)-phenolate ground state complex, in spite of the similar activated intermediate and oxidation pathway.

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.

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)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

Spatiotemporal control of surface properties under physiological conditions such as those found in culture media is an important technique in fundamental cell biology, tissue engineering, and cell-based bioelectronics. To this end, we have developed a mild, wet cellular micropatterning technique. The principle of the technique is based on the fact that the cell-repellant property of the albumin-coated substrate rapidly switches to cell-adhesive upon exposure to the reactive oxidizing agent, electrochemically generated hypobromous acid. Herein, we report the effect of the hypobromous acid on serum albumin physisorbed on a hydrophobic substrate. It was found that albumin molecules detach from the substrate by application of the oxidizing agent, resulting in exposure of the underlying hydrophobic surface to the liquid phase. The adsorption of extracellular matrix proteins such as fibronectin onto the hydrophobic surface induces cell adhesion and growth.

We describe an electrochemical method to direct the growth and migration of mammalian cells on a substrate during cultivation in situ. Exposing the albumin-coated substrate to an oxidizing agent, hypobromous acid, electrochemically generated at the tip of the scanning microelectrode, locally switched the substrate from cytophobic to cell-adhesive. This transformation generated the formation of cellular micropatterns. Since the concentration of the oxidizing agent required for the surface processing did not cause significant damage to the cell cultures, we were able to direct in situ cellular proliferation and migration by drawing adhesive micropatterns over the preexisting cellular pattern.

The present paper describes the role of conjugating enzymes in the development of hepatotoxicity after administration of repeated doses of a novel monoamine oxidase type-A (MAO-A) inhibitor, (5R)-3-[2-(( 1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidinone (E2011). The effects of pretreatment with three kinds of conjugating enzyme inhibitors on hepatic lesions induced by E2011 were evaluated in female Sprague-Dawley rats. The inhibitors used were 2,6-dichloro-4-nitrophenol (DCNP; inhibitor of sulfotransferase (ST)), pentachlorophenol (PCP; inhibitor of both ST and acetyltransferase (AT)) or ranitidine (inhibitor of UDP-glucuronosyltransferase (UDP-GT)). Two weeks treatment of E2011 alone at an oral dosage of 150 mg/kg induced hepatocellular changes characterized by nuclear enlargement. Daily pretreatment with DCNP (10 mg/kg, i.p.) enhanced the E2011-induced hepatocellular changes accompanied by single cell necrosis. On the other hand, the hepatotoxicity was clearly diminished by PCP (5 mg/kg, i.p.). Ranitidine pretreatment had no effect. Protection by PCP was attributed to the inhibitory effects of AT in addition to ST; it was considered that the hepatocellular changes caused by E2011 were largely dependent on the formation of acetyl conjugate(s).

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).

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.

(5R)-3-[2-((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5- methoxymethyl-2-oxazolidinone (E2011) is a novel monoamine oxidase type-A (MAO-A) inhibitor. In order to assess toxicological profiles of E2011, doses of 0 (as controls), 30, 100 mg/kg of E2011 were administered to male and female Sprague-Dawley rats once a day for 13 weeks orally by gavage. No mortality or any toxic signs except salivation occurred due to E2011 treatment. Decreased body weight gain and food consumption, increases of alkaline phosphatase and increases of liver weight were the major treatment-related findings observed predominantly in the 100 mg/kg group. Histological examination revealed nuclear enlargement of hepatocytes with appearance of altered cell foci in some cases, and acinar atrophy in Harderian glands in the 100 mg/kg group. Since the histopathological findings in the liver were indicative of an ongoing carcinogenic process, glutathione S-transferase placental form (GST-P) positive hepatic foci were identified immunohistochemically and examined morphometrically. Although GST-P positive hepatic foci were detected in all groups including controls, the number and area of GST-P positive hepatic foci were significantly higher in female rats treated with 100 mg/kg than those in controls. In this paper, possible mechanisms of specific lesions in the liver and Harderian glands will be discussed.

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., (Copyright 1999 Elsevier Science B.V.)

Abstract 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.

Effects of ER-40133, an inhibitor of angiotensin converting enzyme (ACE), on weight gain and sodium and potassium balance were studied in growing SD male rats. Thirty-two animals (seven weeks of age) were divided into two groups; one received a standard diet containing 0.227% sodium and the other a low (0.065%) sodium diet. They were divided into four subgroups; one control group and three treated groups receiving 3, 10 or 30 mg/kg of ER-40133, by gavage, once a day for five consecutive days. Body weight gain (average of the standard and low sodium diet groups) was -32% in the 3 mg/kg group,-74% in 10 mg/kg group and -99% in 30 mg/kg group, when compared with the control group. There was a highly linear correlation between suppression of body weight gain and reduction in sodium and potassium retention for both groups of animals given the standard and low sodium diet. The reduced sodium retention, the primary effect of ACE inhibitors, accounted for about 30% of suppressed weight gain, and the reduced potassium retention, the secondary effect of sodium deficiency, could account for the rest about 70% of weight suppression by ER-40133.

The toxicity profile of benzo[a]pyrene (BP) was examined in the MutaMouse. The transgenic mouse integrated with lambda gt10 lacZ vectors is used worldwide as an experimental animal in in vivo mutagenesis testing systems. There are few toxicity studies including carcinogenicity in the MutaMouse, and so far only a few carcinogenicity studies of BP accompanied with hematological and plasma biochemical examinations have been conducted even in generic mice. Accordingly, male mice were orally administered BP at doses of 75 and 125 mg/kg/day for 5 consecutive days, and complete autopsy was conducted together with pathological, hematological, and plasma biochemical examinations and measurement of organ weights 41 weeks after the last treatment. Squamous cell papilloma and hyperplasia in the forestomach were induced at incidences of 25 and 50%, respectively and were induced 26 weeks after the final treatment without any significant alterations in t he hematological and plasma biochemical parameters in mice of the 125 mg/kg/day BP-treated satellite group. Fourty-one weeks after the final treatments, 75 and 125 mg/kg/day BP induced squamous cell carcinoma, papilloma, and hyperplasia in the forestomach at incidences of 18 and 18%, 36 and 45%, and 91 and 91%, respectively, and anemia possibly due to continuous hemorrhage from tumors in the forestomach. BP (125 mg/kg/day) also produced malignant lymphoma with an incidence of 18%, accompanied by a marked increase in leukocyte count and decrease in erythrocyte count and by a remarkable decrease in body weights 26 and 39 weeks after the last treatment. Moreover, administration of 75 and 125 mg/kg/day BP induced bronchiolar-alveolar hyperplasia in the lung at incidences of 18 and 9%, respectively. Slight increases were also observed in the weight of the liver and in the levels of urea nitrogen, creatinine, and potassium ion in the plasma biochemical examinations, although no significant pathological alterations were found in the liver and kidney. This study provides new information about BP toxicity including carcinogenicity in the MutaMouse developed for in vivo mutational analysis.

We investigated the mechanism of lethal injury following the disruption of microtubules in cultured hepatocytes treated with vinblastine (VBL) or colchicine (COL). These agents kill hepatocytes by a process readily distinguished from two well-known pathways that lead to a loss of viability, namely, oxidative stress and inhibition of mitochondrial electron transport. Cell killing with VBL and COL was accompanied by fragmentation of DNA. Both the loss of viability and the fragmentation of DNA were prevented by the inhibition of protein synthesis within 6 hours following exposure to VBL or COL. Cell death and the fragmentation of DNA were also prevented when Ca2+ was removed from the culture medium. By contrast, the inhibition of protein kinase C prevented cell killing by VBL or COL, but did not alter the extent of DNA fragmentation. The requirements here for protein synthesis, extracellular Ca2+, and protein kinase C activity define a model of apoptosis, or programmed cell death, that seems to involve mechanisms that can be dissociated from the fragmentation of DNA.

The effects of the two inhibitors of poly(ADP-ribose) polymerase, 3-aminobenzamide (ABA) and benzamide (BA), on the oxidative killing of L929 mouse fibroblasts and primary cultures of rat hepatocytes were studied. The killing of L929 cells by tert-butyl hydroperoxide (TBHP) occurred by two mechanisms, one sensitive and the other insensitive to the antioxidant N,N'-diphenylphenylene diamine (DPPD). Cell killing by either mechanism was prevented by the ferric iron chelator deferoxamine. ABA and BA prevented the killing of L929 cells that occurred in the presence, but not in the absence, of DPPD. ABA and BA inhibited the activity of poly(ADP-ribose) polymerase by 85%. Protection was accompanied by the sparing of the depletion of both NAD and ATP, but there was no effect of either ABA or BA on the iron-dependent appearance of single-strand breaks in DNA. Depletion of ATP by treating the fibroblasts with 2-deoxyglucose and sodium azide did not result in any loss of viability. H2O2 similarly killed the L929 cells by a mechanism that depended on a source of ferric iron. However, DPPD had no effect on the cell killing, and ABA and BA completely protected the cells in the presence or absence of DPPD. H2O2 caused the appearance of single-strand breaks that were prevented by deferoxamine, but again not by ABA or BA. ABA and deferoxamine reduced, but did not prevent, the depletion of both NAD and ATP occurring with H2O2. With the cultured hepatocytes, ABA and BA inhibited poly(ADP-ribose) polymerase at concentrations that were without effect on either the extent of cell killing or the depletion of NAD occurring with either TBHP, H2O2, or menadione. These data indicate that the relationship between oxidative DNA damage and the genesis of lethal injury is very different in the two types of cells. In the fibroblasts, the appearance of single strand breaks in DNA was accompanied by depletion of NAD and ATP and subsequently by the death of the cells. These events were mediated by the activity of poly(ADP-ribose) polymerase, as inhibition of the enzyme prevented their development. In the hepatocytes, inhibition of poly(ADP-ribose) polymerase was without effect on the oxidative death of the cells.

Intravenous injection of lipopolysaccharide and D-galactosamine, at doses of 0.2 micrograms/kg and 800 mg/kg, respectively, elicited massive hepatic necrosis within 24 hr in C3H/HeN mice. The plasma L-alanine aminotransferase (ALT, E.C. 2.6.1.2) or L-aspartate aminotransferase (AST, E.C. 2.6.1.1) activities at this point reached more than 2,000 IU/L. However, overt hepatic injury as evaluated by the plasma aminotransferase activities did not develop in mice in which only lipopolysaccharide or only D-galactosamine was injected. No tumor necrosis factor-like activities could be detected in the plasma of galactosamine- and lipopolysaccharide-injected mice as determined by the assay of cytotoxicity to highly tumor necrosis factor-sensitive L-P3 cells through the experimental period of 24 hr. However, passive immunization against mouse tumor necrosis factor-alpha with polyvalent rabbit anti-mouse tumor necrosis factor-alpha antiserum, which was able to neutralize the cytotoxic effects of recombinant mouse tumor necrosis factor-alpha on L-P3 cells, could protect the mice from the development of hepatic injury in a dose-dependent manner. Simultaneous injection of recombinant human tumor necrosis factor-alpha, instead of lipopolysaccharide, with 800 mg/kg of D-galactosamine in lipopolysaccharide-resistant C3H/HeJ mice sensitized the animals more than one thousand-fold to the development of hepatic injury. The livers appeared to be morphologically similar to those of galactosamine- and lipopolysaccharide-injected C3H/HeN mice.

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 micrograms 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 micrograms 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.

Induction of cytochrome P-450 PCN (P-450 PCN) by non-steroidal compound, M79193 (cyclohexane spiro-2,6-chloro-1'-(3-dimethyl-aminopropyl) spiro(chroman-4,4'-imidazolidine)-2',5'-dione], was investigated in both sexes of rats. The immunohistochemical localization of P-450 PCN was also studied in liver lobules of untreated and M79193-treated male rats. Immunoblot analysis of rat liver microsomes with anti-P-450 PCN indicated that the amount of P-450 PCN increased 5- to 7-fold by the treatment with M79193, but no increase was observed in P-450 PB-1 (P450IIB1) or P-450 MC-1 (P-450IA1). P-450 PCN was uniformly distributed in liver lobule of untreated rats, and was significantly increased by the treatment with M79193 in both the periportal and pericentral regions of the lobules.

The localization of sex-specific cytochrome P-450, P-450 (M-1), was investigated immunohistochemically in the liver of untreated- and Aztreonam-treated male rats. P-450 (M-1) was uniformly distributed in the liver lobule of untreated rats. By treatment with Aztreonam, P-450 (M-1) was decreased more in the periportal region rather than the pericentral region of the lobule. In addition, oxidation of testosterone and 1 alpha-hydroxycholecalciferol catalyzed by P-450 (M-1) was also decreased by Aztreonam administration.

The influence of fixed fibroblasts on the glycosaminoglycan (GAG) synthesis of gastric carcinoma cells was examined by incubation along with [3H]glucosamine. In well-differentiated adenocarcinoma cells, the amount of 3H-GAG in the interface material between the carcinoma cells and the fixed fibroblasts was much larger (about twenty times) than in the interface between the carcinoma cells and the bare culture plates, and 3H-GAG consisted mainly of heparan sulfate, with a small amount of dermatan sulfate and chondroitin sulfate. On the other hand, in poorly differentiated carcinoma cells, the amount of 3H-GAG in the interface material produced by the carcinoma cells on the fibroblast was almost the same as on the bare culture dish. In a conventional monolayer culture, well-differentiated adenocarcinoma cells produced a much greater amount of GAG, consisting mainly of dermatan sulfate, chondroitin sulfate and heparan sulfate, than poorly differentiated carcinoma cells. Almost the same amount of hyaluronic acid was secreted into the medium by both types of carcinoma cells.

Pleomorphic adenoma of rat salivary gland is extremely rare, and culture of cells obtained from rat salivary gland tumors has not been reported. We have established a long-term cell line from a pleomorphic adenoma of a Sprague-Dawley rat submaxillary gland. The pleomorphic adenoma was composed of oval or spindle-shaped cells occasionally forming a small duct. Alcian blue-positive intercellular matrices, consisting mainly of glycosaminoglycans, were abundant. The cultured cells showed characteristics similar to those of the original tumor. This cell line should be useful for biological and biochemical studies of glycosaminoglycan-synthesis of pleomorphic adenoma cells.

Hyperplastic liver nodules were induced in rats by administration of an initiator (diethylnitrosamine or 3'-methyl-4-dimethylaminoazobenzene) and/or a promoter (phenobarbital) by the method reported by Tatematsu et al. (1983, Carcinogenesis 4, 381-386). The dolichol content in the liver and liver microsomes of the rats treated with the initiator were approx. 1.5-times higher than that of the control and rats treated with only the promoter. However, the composition of dolichols was not changed. The time course of the dolichyl phosphate concentration in the rat liver treated with both initiator and promoter showed a pattern different from that in the control liver, the initiator-treated liver or the promoter-treated liver. The main component of dolichyl phosphate in liver treated with both the initiator and promoter changed from that with 18 isoprene units to that with 19. It is suggested that the changes in liver dolichols and dolichyl phosphates may be related to the formation of hyperplastic liver nodules.

We encountered a case of a 21-year-old man who had developed acute lymphoblastic leukemia (ALL) shortly after the cessation of approximately six years' human growth hormone treatment for pituitary dwarfism. The patient had also been treated with thyroid preparations and methylandrostanolone, a kind of anabolic steroid. Factors relating to the onset of ALL in this case are discussed.

In 41 salivary gland tumors, the characteristics of the intercellular components and vascular endothelial cells were surveyed by immunohistochemical staining for laminin and factor VIII-related antigen (VIII R:Ag), and by mucopolysaccharidase-digestion for glycosaminoglycan (GAG). In myxomatous areas of pleomorphic adenomas, small vessels (diameter 6.5 +/- 0.11 micron) were frequent and found to be negative or weakly positive by VIIIR:Ag staining although endothelial cells were clearly positive for VIIIR:Ag in capsule surrounding the tumor tissues. Alcian blue stainability was diminished by treatment with both Streptomyces hyaluronidase and chondroitinase. By laminin staining, a vascular pattern was clearly detected, but the majority of tumor cells were not stained. In adenomatous areas, the basement membrane-like linear laminin-staining reaction was observed to be weak and inconsistent around some tumor cell nests. However, in adenoid cystic carcinomas, laminin-positivity was much more intense than in other tumors such as pleomorphic adenoma, mucoepidermoid tumor and adenocarcinoma. In cylindromatous areas, the inner luminal surface in the pseudocysts was markedly positive for laminin, and there was weak positivity around tumor cell nests having a trabecular pattern. By immunoelectron microscopy, a juxtacellular network of replicated basal lamina of tumor cells which lined the inner surface of pseudocysts was positive for laminin. Alcian blue-positivity in the pseudocyst was abolished with heparitinase and chondroitinase, but not with hyaluronidase.

1. Pretreatment of rats with 6-(3-picolyl)amino-2,2,5,8-tetramethylchromane (PATC) for 7 days resulted in a significant increase in the activities of benzphetamine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes prepared 24 h after the last treatment. 2. Analysis by Western blot showed that PATC induces cytochrome P-450 b, P-450 c and P-450 d, which are the major forms of cytochrome P-450 in liver microsomes of rats when pretreated with phenobarbital and 3-methylcholanthrene. 3. Exposure of liver sections to the antibodies to cytochrome P-450 b and P-450 c resulted in intense immunostaining within the centrilobular regions, but produced staining of considerably weaker intensity in the perilobular region. Semiquantitative immunochemical analysis, by image analyser, of cytochrome P-450 b and P-450 c showed that centrilobular hepatocytes were stained more intensively than perilobular hepatocytes. 4. These results indicate that PATC induces cytochromes P-450 b and P-450 c, in the centrilobular hepatocytes to a greater degree than those in the perilobular hepatocytes. 5. Co-administration of PATC with pentobarbital caused a significant increase in pentobarbital sleeping time. Furthermore, PATC was found to cause a decrease in the activity of benzphetamine N-demethylase in liver microsomes prepared 30 min after treatment with the drug.

Choroid plexus papilloma (CPP) was observed by light and electron microscopy, using surgically excised tissues in 7 cases and cultivated cells. CPP cells had numerous microvillous processes showing balloon-like features, lysosomes containing haemosiderin, and highly electron-dense irregular granules about 150-300 nm in diameter. Interstitial cells with highly electron-dense cytoplasm inserted their long and thin processes into the invagination of basal plasmalemma of CPP cells, occasionally breaking down the basal lamina. Many of them were located in the intercellular space among CPP cells, sometimes adhering to the ventricular surface of CPP cells. Ruthenium red stain was positive on the surface of CPP cells and was especially intense on the surface of microvilli and cilia. In culture, CPP cells and interstitial cells migrating from the CPP cell mass showed a phagocytic activity after treatment with Latex.

Immunohistochemical localization of P-450 HFLa, a form of cytochrome P-450 in human fetal livers was investigated in human hepatocellular carcinoma. The cytoplasm of carcinoma cells positively reacted with anti-P-450 HFLa antibodies. It was found that the carcinoma cells showing a pseudoglandular pattern or poorly differentiated appearance exhibited a weaker reactivity with anti-P-450 HFLa antibodies than did relatively differentiated carcinoma cells. In the case of hepatoblastoma, the polygonal or round-shaped tumor cells which differentiated into epithelial structure exhibited a positive reaction with anti-P-450 HFLa antibodies, whereas the spindle-shaped tumor cells which showed a sarcomatous appearance did not react with the antibodies.

Dog liver cytosolic glutathione S-transferases (GSTs) were investigated to characterize their properties in comparison with rat liver transferases. Dog liver GSTs after the glutathione affinity column chromatography showed three subunit bands on SDS-polyacrylamide gel electrophoresis. These three subunits, designated as Yd1 (mol.wt 26,000), Yd2 (mol.wt 27,000) and Yd3 (mol.wt 28,000), were distinctly different from rat liver GST subunits, i.e. Ya(1) (mol.wt 26,500), Yb1(3)/Yb2(4) (mol.wt 27,500) and Yc(2) (mol.wt 28,500). Western blot analysis revealed that Yd1, Yd2 and Yd3 were immunoreacted with anti-rat GST 7-7, 1-1 and 3-3 antibodies, respectively. Four transferase activity fractions, I (pH greater than 7.63), II (pH 6.92), III (pH 5.80) and IV (pH 5.65), were obtained from affinity purified GSTs by chromatofocusing. Each fraction exhibited a characteristic substrate specificity. GST-II, III and IV were all strongly immunoreacted with anti-rat GST 7-7 antibody by immunoblotting, thus suggesting the occurrence of the heterogeneity of transferases immunologically related to rat GST subunit 7 in dog liver. Immunohistochemical examination showed that transferases immunoreacted with anti-GST 7-7 antibody have diffusely distributed throughout the lobule, while enzymes related to subunit 3 have been localized in a narrow range of cells around the central vein. These data suggest that GSTs immunologically associated with rat transferase subunit 7 may be major forms in dog liver.

In order to clarify the biological characteristics of rat mammary tumors induced by 7,12-dimethylbenz-[a]-anthracene (DMBA), histochemical and immunohistochemical studies were performed. Two types of luminal spaces were observed within the tumor. In one type, the lumen was surrounded by eosinophilic columnar cells which were strongly reactive for soybean agglutinin (SBA) but weakly stained with keratin antibodies. In the luminal spaces, substances positive for PAS, dialyzed iron ferrocyanide or alcian blue and resistant to mucopolysaccharidase were occasionally observed. Ultrastructurally, the luminal surface was characterized by the presence of microvilli and tight junctions. In the other type, the lumen was often found in highly cellular foci and surrounded by pale, polygonal or elongated cells which were weakly stained with keratin antibodies but not SBA. The luminal spaces presented a peculiar structure filled mainly with mucoid substances sensitive to hyaluronidase, chondroitinase ABC and heparitinase, and the inner surface of the spaces was surrounded by basement membrane components: laminin, fibronectin and type IV collagen. The results of the present study therefore showed that DMBA-induced mammary tumor consists, partly, of a structure resembling human adenoid cystic carcinoma.