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Acetaminophen (APAP) administration (600 mg/kg, ip) to 18-hr fasted, 3-month-old male CD-1 mice results in necrosis of the convoluted renal proximal tubules with a corresponding elevation of plasma urea nitrogen (BUN). Administration of the gamma-glutamyl transpeptidase inhibitor, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydroxy- 5-isoxazoleacetic acid (AT-125) (50 mg/kg, ip), to mice 30 min before APAP significantly diminished the APAP-induced histopathologic damage and BUN elevation. Administration of the organic-anion transport inhibitor, probenecid (150 mg/kg, ip), 30 min before APAP challenge also protected against the APAP-induced elevation of BUN and detectable histopathologic changes. By contrast, pretreatment of mice with the cysteine conjugate beta-lyase inhibitor, (aminooxy)acetic acid (100 mg/kg, ip), 1 hr before APAP did not alter nephrotoxicity. None of the pretreatments altered the APAP-induced elevation of plasma sorbitol dehydrogenase activity, nor were there any detectable changes in liver histopathology after APAP challenge. Despite the protective effects of both probenecid and AT-125 against nephrotoxicity, they did not affect either the level of immunochemically detectable covalent binding to protein or the depletion of renal glutathione at 4 hr after APAP. Thus, the protection appears independent of effects on renal APAP uptake or activation and indirectly suggests that an APAP-glutathione conjugate may contribute to the observed nephrotoxicity.

Administration of hepatotoxic doses of acetaminophen (APAP) to mice results in necrosis, not only of liver cells but of renal proximal tubules and bronchiolar and olfactory epithelium. In the liver, covalent binding is localized to the centrilobular hepatocytes which later undergo necrosis. This study was undertaken to compare the cellular distribution of bound APAP in all four major target tissues with that of cytochrome P4502E1 (a P450 isoenzyme commonly associated with APAP bioactivation), with emphasis on the cell types which later undergo necrosis. Tissues were collected from mice at selected times after APAP administration (600 mg/kg, po) and fixed by microwave irradiation for immunohistochemistry, or in formalin for histopathological study. Immunohistochemical localization of bound APAP was performed on 5-microns paraffin sections using an affinity-purified anti-APAP antibody. Similar tissues from naive mice were used for immunohistochemical localization of cytochrome P4502E1 (using a polyclonal sheep anti-P4502E1 antibody). Positive staining with both the anti-APAP and the anti-P4502E1 antibodies was similar in distribution, being present in the cell types which become damaged by APAP in all four target tissues. These results demonstrate that covalent binding and subsequent necrosis are localized in common with cytochrome P4502E1, suggesting that, as in the liver, toxicity in extrahepatic targets is also related to the ability of these tissues to activate APAP in situ.

Acetaminophen (APAP) administration (600 mg/kg, ip) to 18-hr-fasted, 3-month-old male CD-1 mice results in necrosis of the renal convoluted proximal tubules. To selectively inhibit APAP activation in the kidney but not in the liver, 10-week-old male CD-1 mice were castrated under ether anesthesia and allowed to recover for a minimum of 2 weeks before use. Microsomal activation of APAP in vitro was monitored by trapping the APAP-derived electrophile as an N-acetylcysteine conjugate. Production of the conjugate was significantly decreased in renal microsomes from castrated mice. By contrast, hepatic microsomal production of the APAP conjugate was not changed by castration. Castration also did not alter APAP covalent binding in liver or APAP hepatotoxicity. Also, the overall metabolism of APAP was not altered by castration, as there were no significant differences in the 24-hr excretion of APAP urinary metabolites between castrated and sham-operated mice. However, castration did significantly protect mice against APAP nephrotoxicity, as evidenced by decreases in plasma urea nitrogen levels and in the severity of histopathologic damage assessed 12 hr after APAP. Castration also reduced the level of selective covalent binding of APAP metabolites to renal proteins as detected on Western blots with affinity-purified anti-APAP antibodies. These findings suggest that, for the mouse, intrarenal biotransformation of APAP to a reactive electrophile significantly contributes to the APAP covalent binding within the kidneys and the subsequent nephrotoxicity.

Acetaminophen (APAP) administration (600 mg/kg, po) results in proximal tubular necrosis in 18-hr fasted, 3-month-old male CD-1 mice. This study was undertaken to determine if deacetylation of APAP to p-aminophenol (PAP) is a prerequisite to nephrotoxicity in the mouse, as it is in the Fischer rat. Administration of either APAP or PAP to mice resulted in significant elevations of plasma urea nitrogen and marked proximal tubular necrosis at 12 hr after dosing. Prior inhibition of APAP deacetylation by the carboxylesterase inhibitors bis(p-nitrophenyl) phosphate or tri-o-tolyl-phosphate did not alter APAP hepatotoxicity or nephrotoxicity. By contrast, pretreatment with the MFO inhibitor piperonyl butoxide decreased APAP nephrotoxicity but not that of PAP. Immunochemical analysis of kidneys from APAP-treated mice demonstrated covalently bound APAP but no binding was detected after mice were treated with a nephrotoxic dose of PAP. Since the antibody used has been characterized as being directed primarily against the N-acetyl moiety of bound APAP metabolite and since it did not react with kidney proteins of mice given a nephrotoxic dose of PAP, it is unlikely that APAP deacetylation preceded binding or that acetylation of bound PAP occurred. Taken together, these findings indicate that in the CD-1 mouse, APAP-induced nephrotoxicity differs from that previously described for the Fischer rat and likely involves cytochrome P450-dependent activation and subsequent covalent binding of a metabolite without prior deacetylation.

This study was undertaken to evaluate the early ultrastructural changes during the development of acetaminophen hepatotoxicity. Doses at or near the threshold for hepatotoxicity were selected to permit comparison of early reversible effects to those which ultimately progressed to necrosis in the absence of early agonal effects or drug-induced mortality. Both 300- and 600-mg/kg doses resulted in similar declines in hepatic glutathione levels to 14 and 22% of control values, respectively, by 2 hours, with more rapid recovery after the low dose. Plasma sorbitol dehydrogenase activity was elevated after 600 mg/kg but not after 300 mg/kg. During the first 2 hours after acetaminophen there was cytomegaly with rapid progression to necrosis after 600 mg/kg but minimal progression after 300 mg/kg. Ultrastructurally, vesiculation, vacuolation and mitochondrial and plasma membrane degeneration culminated in scattered single cell death by 4 hours and widespread centrilobular necrosis by 8 hours after 600 mg/kg. The time course of lesion development was slower after 300 mg/kg with damage restricted to the first two to three rows of centrilobular cells and limited numbers of isolated necrotic cells by 8 hours. By 18 to 24 hours livers of mice given 300 mg/kg appeared normal. Results are consistent with the endoplasmic reticulum being the site of acetaminophen activation and initial attack. However, early ultrastructural changes in mitochondria and plasma membrane observed after the high dose were not prominent after the low dose. This suggests that early acetaminophen damage to these organelles may play a critical role in acetaminophen hepatotoxicity.

Pen-raised North American wild turkeys (Meleagris gallopavo L.) were experimentally infected with marble spleen disease (MSD) to determine their susceptibility to this disease. Gross and microscopic lesions were consistent with experimental MSD in pheasants and domestic turkeys: an enlarged mottled spleen, intranuclear inclusion bodies, and absence of pulmonary edema and hemorrhage. Detectable levels of viral antigen were not demonstrable in sera of turkeys using the agar gell precipitin test.

Marble spleen disease (MSD) of ring-necked pheasants (Phasianus colchicus L.) was transmitted in the laboratory to pheasants and turkeys (Meleagris gallopavo L.) by oral, colonic, and intravenous routes of inoculation, using cell-free supernatant fluids of splenic suspension from birds with naturally occurring MSD. Gross lesions consisted of large mottled spleens, a lesion more prominent in infected pheasants than in turkeys. The only microscopic lesion and also the criterion of infection were the presence of typical intranuclear inclusions of MSD. Similar to microscopic observations in naturally infected birds was the presence of inclusions in cells of spleen, bone marrow, liver, lung, bursa of Fabricius, and intestine-associated lymphoid tisse (IALT) OF EXPERIMENTALLY INFECTED BIRDS. Ultrastructural examination of splenic cells with intranuclear inclusions from turkey and pheasant with experimentally transmitted disease revealed viral particles and inclusions morphologically indistinguishable from those observed in naturally infected birds. Results of viral isolation procedures in turkey embryo chorioallantoic membrane (CAM) and yolk sac, as well as in turkey embryo fibroblast (TEF) and turkey kidney cell (TKC) cultures, were negative. Serologic assay by agar gel immunodiffusion tests indicated cross-reactivity between splenic MSD antigen and spleinic hemorrhagic enteritis (HE) of turkey antigen. Lines of fusion were formed between the splenic antigen from naturally occurring MSD in pheasants, experimentally transmitted MSD in turkeys, and HE of turkeys, using the homologous serum antibody to each of the 3 antigens.

Acetaminophen in acute overdose is primarily recognized as potentially hepatotoxic with few descriptions of extrahepatic lesions other than nephrotoxicity. Fasted adult, male mice, both standard and germ-free, were given acetaminophen orally and killed at selected times, from 30 minutes to 48 hours after treatment. In addition to the expected hepatic effects after 600 mg acetaminophen/kg, degenerative and necrotic changes were found in four non-hepatic tissues. Nephrosis developed 2 to 4 hours after treatment and paralleled the course of hepatic damage. Necrosis of bronchiolar epithelium in the absence of inflammation was evident 4 to 6 hours after acetaminophen administration as was onset of testicular changes. Spermatidic degeneration with early development of spermatidic multinucleated giant cells were characteristic features. Areas of lymphoid necrosis were also visible in splenic follicles and Peyer's patches 18 to 24 hours after treatment. These observations have demonstrated that other tissues in addition to liver and kidney are damaged by acetaminophen toxicity and should be considered in cases of acetaminophen overdosage.

A purification procedure, using chloroform or fluorocarbon extraction and centrifugation on a cushion of cesium chloride (CsCL), was designed to isolate the causative virus of marble spleen disease. Virus was purified, inoculated into turkeys, and subsequently reisolated by purification from the spleen of inoculated turkeys, thus fulfilling Koch's postulates. Splenic antigen was detected by the agar gel precipitin test, and viral inclusions with viral particles were observed by light and electron microscopy. Results of further studies indicated splenic lymphoreticulum cell hyperplasia was a sensitive indicator of marble spleen disease virus (MSDV) infection. Direct fluorescent antibody staining revealed nuclear fluorescence in virus-infected splenic cells from turkeys inoculated with purified MSDV. With negative stain electron microscopy, MSDV was observed to be 90 nm across with an icosahedral capsid composed of 252 capsomeres. This morphologic feature was consistent with that of an avian adenovirus. Serologic evidence for classification of MSDV as an adenovirus was the cross reaction of MSDV antigen with antiserum to turkey adenovirus serotypes TA-1 and TA-2 in the agar gel precipitin test.

Intraarticular inoculation of Mycoplasma bovis into the joints of six-week-old calves caused severe arthritis in five inoculates and mild arthritis in a sixth. Intraarticular inoculation of killed M. bovis did not cause arthritis. Arthritic calves had fever, joint swelling, lameness, neutrophilia, and intercurrent pneumonia from which M. bovis could not be recovered. Gross lesions were massive fibrinosuppurative synovitis and tenosynovitis, erosion of cartilage, and its replacement by polypoid granulation tissue. Histologic lesions were extensive ulceration of synovial membranes, leukocytic infiltration of the subsynovium, congestion, hyperemia, and thrombosis of the subsynovial vessels. Cartilage erosion was accompanied by chronic osteomyelitis and formation of pannus tissue. The presence of thrombi and platelet aggregates suggests that the inflammatory process in the synovium may arise from the interaction of M. bovis with the vasculature and the coagulation process of the host.

Diets containing 25,000 (2.5%) or 50,000 ppm (5.0%) agar, guar gum, gum arabic, locust-bean gum or tara gum were fed to groups of 50 male and 50 female F344 rats and B6C3F1 mice for 103 wk. Separate groups of 50 rats and 50 mice of each sex served as controls for each study. There were no significant differences in survival between any of the dosed groups of rats or mice and their respective control groups. Depressions in body-weight gain greater than 10% for dosed groups relative to their respective control groups were observed for male (low dose only) and female mice fed diets containing agar, female mice fed diets containing guar gum (high dose only), male mice fed diets containing locust-bean gum (high dose only) and male and female mice fed diets containing tara gum (high dose only). Depressions in body-weight gain greater than 5% were observed for female rats fed diets containing agar, guar gum or gum arabic. There were no histopathological effects associated with the administration of the test materials. Under the conditions of these bioassays, none of the five polysaccharides was carcinogenic for F344 rats or B6C3F1 mice of either sex.

A white-tailed deer (Odocoileus virginianus), shot during the 1978-79 New Jersey hunting season, was presented with an enlarged, multinodular liver and numerous skin growths. The skin lesions were found to be fibromas and the liver tumor was identified as a hepatocellular adenocarcinoma, a rare neoplasm, not only in deer but all wild animals.

To determine if the rabbit can act as an intermediate host for ascarids from certain carnivorous animals (skunks, pigs, dogs), 12 rabbits were infected with Ascaris columnaris, Ascaris suum, or Toxocara canis. Clinical signs, microscopic lesions, and morphologic features of ascarids with naturally occurring and experimentally induced A columnaris infections were identical. Of the 3 induced parasitic infections, A columnaris caused the most severe lesions in the central nervous system (CNS). It appears that rabbits may potentially act as intermediate hosts for A columnaris.

A tissue cyst-forming coccidian morphologically resembling the known species Hammondia has a mink-muskrat life cycle. Cysts are found in skeletal muscle of muskrats (Ondatra zibetheca). Mink (Mustela vison) fed infected muskrat carcasses shed oocysts for 4 to 6 days after a prepatent period of 6 to 8 days. The oocysts, 99% of which are unsporulated in mink feces, measure 11.5 to 12 microns X 10 to 11 microns. Sporulated oocysts have 2 sporocysts, each with 4 sporozoites. The present work was insufficient to establish whether this Hammondia-like parasite is identical to the known Hammondia spp. or is a new parasite, although the evidence gathered supports the hypothesis that this parasite is a new member of the genus Hammondia.

Male CD-1 mice 1, 1.5, 2, and 3 months old were given 600 mg of acetaminophen (APAP)/kg, po, and liver damage was assessed 12 hr later. The most severe hepatotoxicity was in 3-month-old mice, while the other age groups exhibited little damage. The onset of susceptibility to APAP hepatotoxicity did not correlate with the level of activity of the mixed-function oxidase system as assessed in vitro, since drug metabolizing capability was similar between 2- and 3-month-old mice. Through 4 hr after administration of APAP to 2- and 3-month-old mice in vivo, glutathione (GSH) depletion and both plasma and liver APAP concentrations were similar between ages. Additionally, 24 hr after dosing, 3-month-old mice excreted marginally more APAP-glucuronide conjugate and parent compound in urine than 2-month-old animals, while both age groups excreted similar amounts of the APAP-sulfate and GSH-derived conjugates. Even though the extent of binding of radioactive APAP to macromolecules at 4 hr was similar between 2- and 3-month-old animals, the pattern of immunochemically targetted cytosolic and microsomal proteins was different. Thus, in APAP exposure the extent of binding to specific proteins rather than the overall amount of covalent binding may be the critical determinant of the hepatotoxic response. In the present study, the age-related differences in susceptibility to APAP-induced hepatotoxicity were related to the differences in selective protein arylation.