Expression of TRPM-2 during involution and regeneration of the rat liver.

Increased message levels of testosterone-repressed prostate message-2 (TRPM-2) have been associated with programmed cell death in many tissues. To study its involvement in the apoptotic elimination of hepatocytes during liver involution and regeneration, levels of TRPM-2 message were evaluated in situ and by the ribonuclease protection assay. Although significant increases in apoptotic bodies were observed in rats 96 h following treatment with lead nitrate and ethylene dibromide, an increase in TRPM-2 message was not detected. Therefore, the expression of TRPM-2 mRNA may be a poor indicator of the extent to which apoptosis occurs during liver involution.

A 15-year followup of 646 schizophrenic outpatients.

The 15-year hospitalization course of 646 chronic schizophrenic outpatients treated between 1958 and 1963 was determined using records of the research clinic, the Kings County Psychiatric Hospital, and the New York State Department of Mental Hygiene. The cohort was heterogeneous with regard to previous history of hospitalization: 20.6 percent had never been hospitalized, 22.0 percent had experienced only crisis admissions, and 57.4 percent had experienced long-term psychiatric hospitalization. The results indicate that 58.8 percent were hospitalized subsequent to project entry. A relationship was observed between previous history and hospitalization and hospitalization during the followup period. Patients with no previous history of hospitalization was less likely to be hospitalized than patients with crisis admissions only, who in turn were less likely to be hospitalized than patients with a history of hospitalization in a long-term psychiatric treatment facility (39.1 percent vs. 53.5 percent vs. 67.9 percent; p less than .001). The implications of these findings for future followup studies are discussed.

Experimental renal papillary necrosis in the Syrian hamster.

These studies evaluate the susceptibility of the Syrian hamster to the induction of renal papillary lesions after exposure to 2-bromethylamine (2-BEA), mefenamic acid, indomethacin, acetaminophen and phenylbutazone. In most cases there were 25 animals per dose group. Papillary necrosis was produced by single or multiple ip doses of 75 mg 2-BEA/kg and above, and was present within 12 hr of administration of a dose of 500 mg/kg body weight. There were lesions in the renal papilla of hamsters 10 days after a single dose of 500 mg 2-BEA/kg. The severity of papillary lesions increased up to 4 days after exposure in hamsters given a single dose of either 250 or 500 mg 2-BEA/kg. The severity of papillary lesions did not increase with the number of doses in hamsters given multiple doses (2-5) of 100 mg 2-BEA/kg. Renal papillary necrosis was observed in about 40% of hamsters given 100, 200 or 400 mg mefenamic acid/kg. Only a few of the hamsters given indomethacin had renal papillary lesions and none of those given acetaminophen (up to 400 mg/kg) or phenylbutazone (up to 600 mg/kg) developed renal papillary lesions.

Rubratoxin B mycotoxicosis in the Swiss ICR mouse.

The LD50 for rubratoxin B dissolved in dimethylsulphoxide and administered to ICR mice by ip injection was 0.31 (0.22-0.43) mg/kg body weight. Gross alterations consisted of congestion of the liver and spleen and pallor and mottling of the kidneys. The histopathological alterations seen were hepatic and splenic congestion and renal tubular degeneration. The morphopathogenesis of lesions following a single ip LD50 dose was evaluated in a second study. Hepatic lesions were observed in mice killed between 8 and 40 hr after dosing and included diffuse sinusoidal congestion with mild sinusoidal ectasia, leucostasis, multifocal cytoplasmic vacuolation and necrosis of individual hepatocytes. Renal lesions were mild, not time-dependent, and consisted of mild degenerative changes in tubular epithelial cells of the outer stripe of the outer zone of the medulla. The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum were increased 2 hr after dosing, peaked at 4 hr and returned to control values by the end of the test period. In a third study, rubratoxin B was administered ip daily for 1 wk at doses of 25, 50 and 75% of the ip LD50. Toxicity was dose related and cumulative with multiple doses at the highest dose. In a fourth study, rubratoxin B was administered ip at a dose of 75% of the ip LD50 daily for 1 wk. Histopathological alterations included hepatic congestion and mild sinusoidal ectasia, multifocal necrosis of hepatocytes, splenic congestion and mild renal tubular degeneration. Serum activities of AST and ALT were increased after multiple doses of rubratoxin B.

Rubratoxin B mycotoxicosis in the Syrian hamster.

The LD50 for rubratoxin B dissolved in dimethylsulphoxide and administered to Syrian golden hamsters by ip injection was 0.4 (0.2-0.8) mg/kg body weight. The greatest number of deaths occurred 6-24 hr after administration. Gross alterations consisted of congestion of the liver, spleen and kidneys and histopathological alterations involved congestion of the spleen and congestion and mild degenerative changes in hepatocytes. In a second study, rubratoxin B was administered ip daily for 1 wk at doses of 25, 50 and 75% of the ip LD50. Mortality was greatest in the 50 and 75% dose groups. Toxicity was cumulative with multiple doses. Gross alterations were similar to those found in the LD50 study. Histopathological alterations included renal tubular degeneration and necrosis and focal necrosis of hepatocytes. The morphopathogenesis of lesions following a single ip LD50 dose was evaluated in a third study. Histopathological alterations were limited to the kidney and were characterized by renal tubular degeneration and necrosis. Renal lesions were first seen at 2 hr after administration and increased in severity to a maximum at 20 hr. Tubular regeneration was first seen at 24 hr and was found to the end of the test period (72 hr). Serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and serum concentrations of total and indirect bilirubin were increased by 8 hr after dosing and returned to control values by the end of the test period. In a fourth study, rubratoxin B was administered ip daily for 1 wk at a dose of 25% of the ip LD50. Gross alterations were similar to those in the other studies. Histopathological alterations included progressive renal tubular degeneration and necrosis. Serum activities of AST and ALT and concentration of blood urea nitrogen (BUN) were progressively increased with increasing numbers of doses. Urinalysis indicated progressive renal tubular damage.

Rubratoxin B mycotoxicosis in the Mongolian gerbil.

The LD50 for rubratoxin B dissolved in dimethylsulphoxide and administered to Mongolian gerbils by ip injection was 2.0 (2.26-1.77) mg/kg body weight. The gross alterations observed at autopsy were pallor and mottling of the kidneys and liver and congestion of the spleen. The histopathological alterations seen were renal tubular degeneration and necrosis, degenerative changes in hepatocytes, and congestion of the spleen. The morphopathogenesis of lesions after a single ip LD50 dose was evaluated in a second study. The histopathological alterations that were observed were focal degeneration and necrosis of hepatocytes and renal tubular degeneration and necrosis. Hepatic lesions were observed in gerbils killed between 2 and 12 hr after dosing and included multifocal cytoplasmic vacuolation and coagulative necrosis of hepatocytes. The renal lesions were first observed 2 hr after dosing and increased to maximum severity at 40 hr after dosing. Tubular regeneration accompanied ongoing tubular necrosis at the end of the test period. The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum were increased 4 hr after dosing, peaked at 24 hr and remained elevated to the end of the test period. Serum K+ concentration was increased 16 hr after dosing and remained elevated to the end of the test period. In a third study, rubratoxin B was administered ip once daily for 7 days at doses of 25, 50 or 75% of the ip LD50. Toxicity was dose related and cumulative with multiple doses. Histopathological alterations included renal tubular degeneration and necrosis, mild tubular dilation and focal necrosis of hepatocytes. In a fourth study, rubratoxin B was administered ip at a dose of 25% of the ip LD50 once daily for 7 days. Histopathological alterations included renal tubular degeneration, mild renal tubular dilation and focal necrosis of hepatocytes. Activities of AST and ALT in serum were slightly increased after multiple doses of rubratoxin B. Results of urinalysis indicated hepatic and renal tubular damage.

Toxicity of Fusarium moniliforme var. subglutinans for chicks, ducklings, and turkey poults.

Corn-based diets contaminated with various concentrations of a moniliformin-producing isolate of Fusarium moniliforme var. subglutinans were found to be lethal for chicks, ducklings, and turkey poults. Ducklings appeared to be the most sensitive to the lethal effects of the toxic feed. Gross lesions were ascites, hydropericardium, and myocardial pallor. Microscopic lesions were limited to the heart and liver, and they consisted of degeneration and necrosis of the myocardium and degeneration of hepatocytes. Cardiotoxicosis was the apparent cause of death.

Pathology of pentastomid infections (Sebekia mississippiensis) in fish.

Differential pathogenesis was observed in two species of fish naturally infected with the pentastome Sebekia mississippiensis. Mosquitofish (Gambusia affinis) showed a mild inflammatory response to developing nymphs, whereas swordtails (Xiphophorus helleri) had an extensive granulomatous inflammatory reaction with accompanying hemorrhage, myositis, and myodegeneration. This suggested that certain species of tropical fish reared in the southeastern United States may be at risk to potentially harmful infections with this parasite.

Effect of squamous metaplasia on infection of hamster trachea organ cultures with Mycoplasma pneumoniae.

An organ culture system for hamster trachea was developed for maintenance of the ciliated respiratory epithelium during periods of extended cultivation (i.e., greater than 20 days). Evaluation of five serum types showed that horse serum and fetal calf serum were best for the maintenance of epithelial ciliary activity and morphology. Rings that were opened on one side ("split rings") had the best maintenance of the ciliated epithelium as judged by the retention of ciliary activity and normal histological appearance after 3 to 4 weeks in culture. The in vitro induction of squamous metaplasia was achieved by cultivating explants in Waymouth MAB 87/3 (vitamin A-free) medium, without serum. This system allowed a direct comparison of the effects of Mycoplasma pneumoniae infection in two epithelial types, ciliated pseudostratified columnar and keratinizing squamous. Attachment of 14C-labeled mycoplasmas was more than twofold greater in the normal epithelium. Pretreatment of explants with neuraminidase decreased attachment for both squamous and pseudostratified epithelial surfaces to a similar basal level. Recovery of viable organisms from infected tissue of both epithelial types indicated that the organism titer remained essentially constant during the infection period, but was significantly higher for the pseudostratified ciliated epithelium. These results suggest that specific receptor sites for M. pneumoniae are markedly reduced by the induction of squamous metaplasia and, hence, appear to be specific for the normal respiratory surface containing goblet cells and pseudostratified, ciliated epithelial cells.

Fialuridine accumulates in DNA of dogs, monkeys, and rats following long-term oral administration.

Accumulation of the antiviral nucleoside analogue fialuridine (FIAU; 1-(2'-deoxy-2'-fluoro-beta-D-arab-inofuranosyl-5-iodouracil) in genomic DNA was examined with a modified version of a recently developed RIA for FIAU. DNA was obtained from tissues of dogs administered FIAU at 0, 1, 2, or 3 mg/kg of body weight per day for 90 days, monkeys administered FIAU at 0 or 25 mg/kg per day for 30 days, and rats administered FIAU at 0, 255, or 510 mg/kg per day for 70 days. FIAU incorporation was observed in all species. In the rat, FIAU was incorporated into DNA of all tissues examined, with highest concentrations in the liver followed by jejunum, spleen, and heart. FIAU was also incorporated into sperm DNA. Incorporation rates were as high as 11,000 pmol of FIAU per mumol of thymidine or 1 FIAU molecule per 90 thymidine molecules. In dogs and rats, the extent of incorporation was dose-dependent. Across species, FIAU concentrations in DNA were not singly dependent on the total dose administered but also may have been dependent on the duration of exposure. These studies show that FIAU accumulates to high concentrations in genomic DNA of liver as well as other tissues during chronic oral administration and suggest that net accumulation of FIAU in DNA may be a critical step in FIAU-induced toxicity.

Incidence of spontaneous neoplastic and nonneoplastic lesions in Charles River CD-1 mice varies with breeding origin.

Three separate control lifetime studies were conducted with untreated Crl:CD-1 (ICR)BR mice using a total of 400 mice/sex maintained to 21 mo of age. Similar husbandry practices and environmental conditions were used for all 3 studies. It was noted after study initiation that the Charles River breeding facility of origin was different for each study. The aggregate range of survival and incidence of neoplasms for the combined studies was similar to that previously reported. However, these 3 groups of mice had prominent variation in survival and in the incidence of pulmonary adenomas and systemic amyloidosis in males and females, and in the incidence of hepatocellular neoplasms in males. The present studies indicate that consistent procurement of test animals is an additional variable to be considered in the establishment of a valid database within a test facility when using an outbred mouse.

Antimycoplasmal activity of dimethylphenols in a tracheal explant culture system.

Mycoplasma pneumoniae induces pneumonia-like symptoms in hamsters and causes ciliostasis and cytonecrosis in hamster tracheal explants. 2,4-Dimethylphenol and, to a lesser extent, its 2,3-, 2,5-, and 2,6-dimethylphenol isomers protected tracheal explants from these changes after exposure to virulent M. pneumoniae strain PI 1428. The effect was concentration, time, and isomer dependent. At concentrations of 10(-9) M or greater, 2,4-dimethylphenol completely prevented the morphological (loss of ciliated cells) and biochemical (decreased dehydrogenase activity) changes normally observed after exposure to M. pneumoniae. Apparently, 2,4-dimethylphenol interfered with an early event in the infection process. Complete protection required that it be present during the first 2 h of exposure of the explants to the infecting mycoplasmas. These xylenols may prove to be useful tools for helping to define the mechanisms of pathogenesis in certain respiratory infections.

Reduction of hepatic and renal nonprotein sulfhydryl content and increased toxicity of rubratoxin B in the Syrian hamster and Mongolian gerbil.

Studies were undertaken to determine if the toxicity and pathology of rubratoxin B, a nephrotoxic and hepatotoxic mycotoxin, could be altered by increasing or decreasing the tissue concentrations of nonprotein sulfhydryls in the liver and kidneys. Rubratoxin B dissolved in DMSO was administered ip to weanling male Syrian hamsters and Mongolian gerbils at doses of 0.4 mg/kg and 2.0 mg/kg, respectively. Rubratoxin B caused a 70% decrease in hepatic and a 60% decrease in renal cortical nonprotein sulfhydryl (NPSH) concentration, an index of tissue reduced glutathione concentration, in both species. Treatment with cysteine prior to rubratoxin B administration did not greatly alter the decreases in NPSH concentration, but did decrease the severity of renal lesions. Treatment with diethyl maleate prior to rubratoxin B administration caused an 80% reduction in hepatic and a 70% reduction in renal NPSH concentration, which was prolonged by rubratoxin B treatment. The incidence and severity of renal lesions was increased in rubratoxin B-treated hamsters and gerbils given diethyl maleate compared to animals given rubratoxin B alone. Additionally, hepatic lesions were seen in hamsters and were more frequent and severe in gerbils that were treated with diethyl maleate prior to rubratoxin B dosing compared to animals given rubratoxin B alone. Renal and hepatocellular NPSH concentration appears to decrease during rubratoxin B toxicosis in the hamster and gerbil, and appears to be contributory in lesion development, since lesions in the liver and kidneys were more severe in animals in which NPSH concentrations were reduced by treatment with diethyl maleate.

Rubratoxin B mycotoxicosis in the Syrian hamster: ultrastructural alterations.

Rubratoxin B was given to Syrian hamsters as a single intraperitoneal dose of 0.4 mg/kg. Hamsters were killed at 1, 2, 4, and 6 hr after dosing, and the kidneys and liver were fixed by intravascular perfusion. Renal ultrastructural alterations were evident at 1 hr after treatment and were limited to the straight portion of the proximal tubule. The most prominent alterations were brush border disruption, dilation of smooth endoplasmic reticulum, mitochondrial swelling, cytoplasmic rarefaction, myelin figure formation, and swelling of basal interdigitating processes. Renal alterations were suggestive of damage to membrane structure and/or transport functions and interference with cellular bioenergetics. Hepatic alterations were not seen in the rubratoxin B-treated hamsters of this study.

Differences in the attachment of Mycoplasma pneumoniae cells and membranes to tracheal epithelium.

Hamster trachea organ cultures were exposed to isolated membranes of Mycoplasma pneumoniae, PI 1428. Attachment, monitored by the uptake of tritiated membranes, was relatively insensitive to neuraminidase pretreatment, unlike the attachment of viable cells. Membrane attachment was optimal when explants were incubated with 50 to 100 micrograms of membrane protein per ml in minimal essential medium broth while gently being rotated (1 rpm) in a roller apparatus for 90 to 120 min at 37 degrees C. Saturation of the receptor sites with viable cells failed to inhibit subsequent membrane attachment. Induction of squamous metaplasia by extended cultivation of tracheal explants in a vitamin A-free medium reduced the content of ciliated cells without significantly affecting total cell viability, but did not alter the attachment of M. pneumoniae membranes. Collectively, the data indicate that the mechanism of attachment of M. pneumoniae membranes to respiratory epithelium is distinct from the receptor site-mediated attachment of M. pneumoniae cells.

Subchronic toxicity, metabolism, and pharmacokinetics of the aminobenzamide anticonvulsant ameltolide (LY201116) in rhesus monkeys.

Studies were undertaken to define the subchronic toxicologic profile of ameltolide, an aminobenzamide anticonvulsant, in young adult rhesus monkeys. Daily doses of ameltolide, dissolved in 10% aqueous acacia, were administered orally via nasogastric intubation at dosages of 5, 10, 20, 45, and 100 mg/kg. Deaths occurred in two monkeys, one each at 45 and 100 mg/kg, which were directly attributable to the effects of the compound. The exact cause of death in these monkeys was not readily apparent. A third monkey (100 mg/kg) was killed moribund on Day 82 of the study due to conditions not directly related to treatment. Clinical signs in monkeys treated with 100 mg/kg included convulsions, diarrhea, weakness, inappetance, vomition, and ataxia. Plasma concentrations of the N-acetyl metabolite of ameltolide were greater than parent drug concentrations by one to two orders of magnitude. Mean area under the plasma-time curve (AUC) values for ameltolide were larger than expected at doses of 20 mg/kg or greater, while AUC values for the metabolite were less than expected at 45 and 100 mg/kg. These findings suggest a saturation of metabolism and/or excretion at the two higher doses. Similar nonlinearity was seen with mean peak concentrations for both parent and metabolite. No specific target organ toxicity was found on histological evaluation of tissue sections. Methemoglobin concentration was increased in monkeys given 45 or 100 mg ameltolide/kg. This change was not considered to be toxicologically important as there were no corroborative clinical, gross, or histopathological findings. Ameltolide administered by nasogastric intubation at doses up to 20 mg/kg/day for 3 months did not cause any toxicologically important alterations in rhesus monkeys.

H-ras 61st codon activation in archival proliferative hepatic lesions isolated from female B6C3F1 mice exposed to the leukotriene D4-antagonist, LY171883.

LY171883, a peroxisome proliferator and leukotriene D4-antagonist, induced a statistically significant increase in the number of hepatic lesions in B6C3F1 female mice in a 2 year oncogenicity study at dietary doses of 0.0225% and 0.075%. The mutation frequency and spectrum of the 61st codon of H-ras was determined for 64 independent, archived lesions from the LY171883 2 year oncogenicity study using the polymerase chain reaction (PCR), allele specific oligo hybridization (ASO) and DNA sequencing. Results showed 41 (64%) of these lesions had mutations at the 61st codon (16/21 hepatocellular carcinomas, 4/10 hepatocellular adenomas, 19/26 focal hepatocellular hyperplasias and 2/7 focal hepatocellular atypia). These mutations consisted of 18 C-A transversions, 16 A-G transitions and seven A-T transversions. Compared to the mutation frequency for spontaneously occurring archival B6C3F1 hepatic lesions (41%), the frequency of LY171883 lesions (64%) was significantly higher (P < 0.01). The frequencies of H-ras 61st codon mutations among the LY171883 lesion types (hepatocellular carcinomas 76%, hepatocellular adenomas 40%, focal hepatocellular hyperplasias 73% and hepatocellular atypia 29%) were also significantly different (P = 0.035). In contrast, spontaneous lesions showed no statistical difference in the frequencies of mutation among lesion types (P > 0.5). The mutation spectrum of the LY171883 lesions was not significantly different from the spontaneous spectra. It may be concluded that based on the similarity in mutation spectrum and the increase in mutation frequency, LY171883 may selectively promote spontaneous hepatic lesions containing H-ras 61st codon mutations. In addition, the difference in mutation frequency among lesion types does not support a linear progression of all LY171883 lesions through focal atypia, focal hepatocellular hyperplasias, hepatocellular adenomas and hepatocellular carcinomas.

Genetic alterations in the 61st codon of the H-ras oncogene isolated from archival sections of hepatic hyperplasias, adenomas and carcinomas in control groups of B6C3F1 mouse bioassay studies conducted from 1979 to 1986.

In order to better understand the molecular events in murine hepatocarcinogenesis, the frequency and types of mutations in the murine H-ras proto-oncogene isolated from 184 independent, spontaneously occurring hepatic lesions were determined. Hepatocellular foci, hyperplasias, adenomas and carcinomas were obtained from archival samples of control male (134 samples) and female (50 samples) B6C3F1 mice used in oncogenicity studies that were conducted at Lilly Research Laboratories from 1979 to 1986. The 61st codon region of the H-ras oncogene from these sections was amplified using the polymerase chain reaction. Mutation frequencies were determined by restriction fragment length polymorphism analysis. The types of mutations were characterized by allele-specific oligonucleotide hybridization and confirmed by DNA sequencing. Forty-two per cent of the carcinomas, 44% of the adenomas, 42% of the hyperplasias and 29% of the foci contained mutations at the 61 codon. The mutation spectra for the carcinomas, adenomas and hyperplasias consisted of mostly CAA-AAA transversions, followed by CAA-CGA transitions, followed by CAA-CTA transversions. These results demonstrate that: (i) the frequency of spontaneous mutations in the H-ras 61st codon is equivalent in murine hyperplasias, adenomas and carcinomas, and (ii) sex was not a determining factor in either the mutation frequency or mutation spectrum for the spontaneous lesions. If these lesions represent successive stages in the carcinogenic process, then these results suggest that mutations in the 61st codon of H-ras are early events in spontaneous murine hepatocarcinogenesis.