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In mammals the cytochrome P450 3A (CYP3A) subfamily isoforms are primarily expressed in liver and intestines with lesser amounts found in other tissues. The aim of this study was to examine the Cellular localization and the expression pattern of CYP3A27 in the gastrointestinal tract (GI tract) of a freshwater teleost species, the rainbow trout (Oncorhynchus mykiss), A fish model used extensively for toxicological and carcinogenesis research. Using an avidin biotinylated enzyme complex and 3,3'-diaminobenzidine staining, strong cytoplasmic immunohistochemical staining was observed for CYP3A27 protein in hepatocytes and in enterocytes of the intestinal ceca and the proximal descending intestine when probed with a polyclonal antibody raised against rainbow trout P450 LMC5, a CYP3A protein. The intensity of epithelial staining decreased distally along the GI tract with faint staining observed in the epithelial cells examined near the vent. Western blot analysis was supportive of the immunohistochemistry results. Northern blot analysis also demonstrated that CYP3A27 mRNA was expressed along the entire GI tract. The major area of CYP3A27 mRNA expression was in the intestinal ceca, followed,by the proximal, descending intestine, at levels that were about three- to five-fold and two- to four-fold, respectively, greater than seen in the liver of the fish studied. Monooxygenase activities of intestinal ceca microsomes against testosterone and progesterone confirmed the presence of active CYP3A enzyme in this tissue. These results suggest that the intestine of rainbow trout may possesses substantial capacity for first-pass metabolism of xenobiotics by CYP3A27, which makes it an excellent model in which to study the consequence of such metabolism. (C) 2001 Elsevier Science.

We have previously demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the induction of the costimulatory molecule CD86 (B7-2) on B220+ and Mac-1+ spleen cells following the injection of allogeneic P815 tumor cells. In this study, TCDD exposure was shown to suppress CD54 and major histocompatibility complex (MHC) class II expression on B220+, Mac-1+, and CD11c+ splenic antigen presenting cells (APC). Furthermore, interleukin-12 (IL-12) production by spleen cells from P815-immunized mice was significantly decreased following exposure to TCDD. To determine if exogenous costimulation could enhance the activation of APC, vehicle- and TCDD-treated mice were injected with an agonistic antibody to murine CD40. Stimulation with anti-CD40 increased the expression of CD86, CD54, and MHC class II on splenic APC and greatly enhanced the production of interleukin-12. TCDD treatment had minimal effects on the anti-CD40-induced expression of accessory molecules on splenic APC. TCDD exposure had no effect on anti-CD40-induced IL-12 in the plasma but suppressed its production from cultured spleen cells. Surprisingly, although stimulation via CD40 increased the activation of APC, allograft effector functions were not restored in TCDD-treated mice, perhaps due to persistent defects in antigen processing and presentation, cytokine production, T cell function, or CD40-independent pathways of APC activation.

Basic fibroblast growth factor (FGF) regulation of developmental markers in cell cultures derived from early zebrafish embryos was examined with the goal of in vitro culture of zebrafish embryonic stem cells and gaining an understanding of extracellular influences on early embryonic development. Markers were stem/primordial germ cell markers pou-2 and vas, neural markers zp-50, pax[zf-a], en-3, and wnt-1, and mesodermal markers gsc and myoD. Previously we had shown that FGF prevents the development of zebrafish pigment cells in vitro. In our culture system, FGF reduced expression of neural-specific markers, possibly implicating the FGF family in suppression of early neural cell development. Exposure to FGF for 24 hours at the time of seeding the cells was sufficient to suppress neural marker expression for a subsequent 4 days of culture, while absence of FGF for the first 24 hours of culture nullified the effect of FGF added subsequently. FGF predictably increased expression of gsc and myoD. Vas expression was unaffected, while pou-2 expression decreased with time in culture in the presence or absence of FGF. However, in situ hybridization identified a subpopulation of cells expressing pou-2, suggesting the possible continued existence of undifferentiated stem cells in the cultures.

γ-Glutamyltranspeptidase (GGT)-deficient mice (GGT−/−) display chronic glutathione (GSH) deficiency, growth retardation, and die at a young age (

The metazoan parasitic blood flukes, Schistosoma spp., infect over 200 million people worldwide and cause extensive human morbidity and mortality. Research strategies for development of anti-schistosomal agents are impeded by the organism’s complex molluscan–mammalian life cycle, which limits experimental approaches and availability of material. We derived long-term continuously proliferative cultures of Schistosoma mansoni sporocysts capable of generating cercariae in vitro . Cultured organisms retained the ability to parasitize the host, and they exhibited developmental regulation of candidate stage-specific genes in the host-free culture system. Evidence for expression of a reverse transcriptase also was found in the cultured organisms, pointing to this activity as a possible mechanistic contributor to the dynamic relationship between the parasite and its hosts. Continuous in vitro propagation of the asexual sporocyst stage allows isolation of clonally derived parasite populations and provides a means to study schistosomal molecular genetics, metabolism, and evasion of host defenses.

Pretreatment of mice with chlordecone (CD) reduced hepatic accumulation of a subsequent dose of [14C]CD without significantly changing [14C]CD biotransformation. To determine if CD-induced changes in hepatic [14C]CD accumulation were coincident with altered cell composition, we examined the effects of CD on hepatic protein and lipid content, on fatty acid profiles of liver and kidney, and on the ultrastructure of hepatocytes. SDS-polyacrylamide gel electrophoresis detected an apparent CD dose-related increase in a microsomal protein with a molecular weight of about 23 kDa. Total liver or kidney lipid contents were not altered by CD but relative amounts of several hepatic fatty acids were changed. CD caused marked hepatic mitochondrial swelling, increased amounts of endoplasmic reticulum, apparently increased numbers of peroxisome-like structures, and decreased numbers of lipid droplets in cytoplasm of hepatocytes. Numbers of lipid droplets were not decreased in perisinusoidal fat storage cells. In addition, the numbers of cytoplasmic lipoprotein vesicles were apparently increased in some hepatocytes. Overall these changes indicated an increased hepatocyte secretory activity and suggested that CD changed hepatocellular lipid transport, storage, and metabolism pathways.

Megaesophagus was diagnosed in 15 llamas on the basis of survey and contrast radiography. Age of onset ranged from 13 months to 9.5 years. Clinical signs varied, with salivation being the most common; regurgitation or dysphagia were noted in only 33% of the cases. Duration of the disorder ranged from 1 week to 5 years. Organophosphate toxicity was the cause of megaesophagus in 1 llama. While most cases were of unknown etiology, the 3 with histological abnormalities included 1 with vagal neuropathy and 2 with a degenerative myopathy of esophageal muscles. The causes in most were undetermined.

The effects of vitamin B12 status on growth and tissue selenium distribution were studied in Sprague-Dawley rats chronically exposed to subtoxic levels of selenite. Vitamin B12 status was monitored by urinary methylmalonic acid excretion and by liver and plasma vitamin B12 levels. Selenite absorption was unaffected by dietary level of vitamin B12. A significant (P < 0.05) interaction of vitamin B12 and selenium was found on growth of rats fed vitamin B12 deficient or control diets. In vitamin B12 depleted rats, there were significant histologic changes in the liver that were characterized by micronodules and regeneration, bile duct reduplication, mild cirrhosis, necrosis of individual hepatocytes and other minor histologic changes. There was no gross or histologic evidence of liver toxicity in rats supplemented with vitamin B12. Rats pair-fed 9 mg/kg selenium with vitamin B12 had significantly lower liver and kidney selenium levels and significantly higher blood selenium levels compared to rats fed the diet without vitamin B12. These results are consistent with the hypothesis that vitamin B12 deficiency limits selenium methylation and excretion, resulting in higher tissue selenium levels and subsequent toxicity.

The sensitivity of a biomarker for environmental contamination with persistent organochlorines and PAHs (cytochrome P450-1A1 induction in feral fish) was evaluated at six sites along 314 km of the Willamette River, Oregon. Whole-body contamination of Northern squaw-fish with approximately 2 pg/g of TCDD and 20 pg/g of TCDF in Portland Harbor was significantly higher than upstream sites. Carp muscle TCDF and hepatic biomarker response were also elevated at this site, as were sediment TCDD, TCDF, and PAHs concentrations. In July, cutthroat trout TCDD concentrations above and below a bleached kraft pulp mill were similar. In October cutthroat trout below the mill contained about 2-fold more TCDD than fish upstream

5. Hsu SM, Raine L, Farger H: 1981, Use of avidin-biotin-peroxidase-complex (ABC) in immunoperoxidase techniques: a comparison between the ABC and unlabeled antibody (PAP) procedure. J Histochem Cytochem 29:577-580. 6. Neer TM, Reavis DU: 1983, Craniopharyngioma and associated central diabetes insipidus and hypothyroidism in a dog. J Am Vet Med Assoc 182:519-520. 7. Patnaik AK, Nafe LA: 1980, Intracranial teratocarcinoma in a dog. Vet Pathol 17:764-769. 8. Ribas JL, Carpenter JL, Mena H, et al.: 1991, Central system meningioma in the dog: a review of 50 cases. J Neuropathol Exp Neurol 50:373. 9. Saunders LZ, Richard CG: 1952, Craniopharyngioma in a dog with apparent adiposogenital syndrome and diabetes insipidus. Cornell Vet 42:490-495. 10. Schulman FY, Carpenter JL, Ribas JL, Brum DE: 1992, Cystic papillary meningioma in the sella turcica of a dog. J Am Vet Med Assoc 200:67-69. 11. Valentine BA, Summers BA, de Lahunta A, et al.: 1988, Suprasellar germ cell tumors in the dog: a report of five cases and review of the literature. Acta Neuropathol 76:94-100. 12. Walsh JW: 1985, Suprasellar germinomas. In: Neurosurgery, ed. Wilkins WH, Rengachary SS, pp. 921-925. McGraw-Hill, New York, NY.

Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, are proastroblasts that are acutely dependent on epidermal growth factor (EGF) for survival. Ultrastructurally, an early change found in SFME cells deprived of EGF was a loss of polysomes which sedimentation analysis confirmed to be a shift from polysomes to monosomes. The ribosomal shift was not accompanied by decreased steady-state level of cytoplasmic actin mRNA examined as an indicator of cellular mRNA level. With time the cells became small and severely degenerate and exhibited nuclear morphology characteristic of apoptosis. Genomic DNA isolated from cultures undergoing EGF deprivation-dependent cell death exhibited a pattern of fragmentation resulting from endonuclease activation characteristic of cells undergoing apoptosis or programmed cell death. Flow cytometric analysis indicated that cultures in the absence of EGF contained almost exclusively G1-phase cells. Some of the phenomena associated with EGF deprivation of SFME cells are similar to those observed upon NGF deprivation of nerve cells in culture, suggesting that these neuroectodermal-derived cell types share common mechanisms of proliferative control involving peptide growth factor-dependent survival.

Indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) are naturally occurring dietary components found in cruciferous vegetables. In the stomach, I3C forms condensation products including DIM. I3C and DIM are marketed as dietary supplements, but little is known about the safety of long-term exposure. Rats were fed either control diet, 1 or 10x the current human dose of absorption-enhanced DIM or 5-7x the maximal recommended dose of I3C. Experimental diets were fed continuously for 3 or 12 months or 2 months followed by control diet for 1 month. Results at 3 or 12 months were similar in most respects. No significant differences between groups were found in blood chemistry. A general decrease in serum enzyme levels in male rats was observed, perhaps indicative of a protective effect. Males fed I3C exhibited higher serum levels of 25-hydroxy-vitamin D3 (25OH-D3). There were no observable differences grossly or histologically between groups, although a high number of hyaline casts were found throughout the kidneys of all animals. In both sexes total hepatic CYP levels were significantly induced by I3C, but not by either dose of DIM. Induction of CYP1A1 and CYP1A2 in liver and CYP1A1 in colon was detected for both sexes fed I3C and the high dose of DIM. CYP3A2 was induced in females fed I3C or the high dose of DIM; males were induced with I3C, but not DIM. No induction of CYP1B1 in the colon was observed in either sex. Long-term exposure to DIM produced no observable toxicity, and comparison to I3C indicates that DIM is a markedly less efficacious inducer of CYP in the rat at doses relevant to human supplementation.

A blue roan Percheron foal was born with poorly circumscribed patchy alopecia of the trunk and legs. Teeth and hoof development were normal. Alopecia was progressive, becoming almost complete by 1 year of age. Histopathological findings in a skin biopsy obtained at 7 months of age were consistent with severe follicular hypoplasia. Sebaceous glands, epitrichial sweat glands and arrector pilae muscles were normal. The horse is alive and otherwise well at 6 years of age, although adult stature is considered small for this breed. The clinical history and histopathological findings are most consistent with a form of congenital hypotrichosis.

Human exposure to polycyclic aromatic hydrocarbons (PAH) occurs through complex mixtures such as coal tar. The effect of complex PAH mixtures on the activation of carcinogenic PAH to DNA-binding derivatives and carcinogenesis were investigated in mice treated topically with NIST (National Institute of Standards and Technology) Standard Reference Material 1597 (SRM), a complex mixture of PAH extracted from coal tar, and either additional benzo[a]pyrene (B[a]P) or dibenzo[a,l]pyrene (DB[a,l]P). In an initiation-promotion study using 12-O-tetradecanoylphorbol-13-acetate as the promoter for 25 weeks, the SRM and B[a]P co-treated mice had a similar incidence of papillomas per mouse compared with the group exposed to B[a]P alone as the initiator. PAH-DNA adduct analysis of epidermal DNA by 33P-post-labeling and reversed-phase high-performance liquid chromatography found the SRM co-treatment led to a significant decrease in the total level of DNA adducts and B[a]P-DNA adducts to less than that observed in mice treated with B[a]P alone at 6, 12 and 72 h exposure. After 24 and 48 h exposure, there was no significant difference in the levels of adducts between these groups. In the DB[a,l]P initiation-promotion study, the co-treated group had significantly fewer papillomas per mouse than mice treated with DB[a,l]P alone as initiator. Averaging over the times of exposure gave strong evidence that mice co-treated with SRM and DB[a,l]P had a significantly lower level of PAH-DNA adducts than mice treated with DB[a,l]P alone. Western immunoblots showed that both cytochrome P450 (CYP) 1A1 and 1B1 were induced by the SRM. These results are consistent with the hypothesis that two major factors determining the carcinogenic activity of PAH within a complex mixture are (i) the persistence of certain PAH-DNA adducts as well as total adduct levels, and (ii) the ability of the components present in the mixture to inhibit the activation of carcinogenic PAH by the induced CYP enzymes.

A 5-year-old Hereford cow was donated to Oregon State University, College of Veterinary Medicine. Two weeks before presentation the cow was treated by a left-flank laparotomy to correct a uterine torsion that revealed an excessive amount of abdominal fluid and a full term dead fetus. Ultrasonographic evaluation of the right cranioventral thoracic cavity was performed owing to physical examination (muffled heart sounds, areas of silence) and thoracocentesis findings suggestive of pleuritis. Ultrasonography revealed hypoechoic pleural effusion and a mass of mixed echogenicity visible within the right atrial lumen extending into the cranial vena cava. A diagnosis of cranial vena cava thrombosis was demonstrated at necropsy. The clinical, ultrasonographic, and pathological features of this less common condition (cranial vena cava thrombosis) in cattle are discussed.

A 31-kg mediastinal mass from a 24-year-old horse is described. Cytologic imprints contained numerous spindle cells with indistinct cytoplasm and multiple foci of palisading nuclei. Histologic sections had these same characteristics and areas of central fibrillar material between palisading nuclei (Verocay bodies). Within histologic sections, spindle cell cytoplasm had positive immunoreactivity for vimentin and S-100, whereas immunoreactivity was not detected for desmin and cytokeratin. Cytologic, histologic, and immunochemical evaluation confirmed a diagnosis of solitary schwannoma.

Three factorial experiments were conducted to determine if high dietary fluoride (F) would inhibit selenite toxicity in rats. Initially, three levels of selenite (0.05, 3, and 5 mg/kg diet) were matched against three levels of F (2, 75, and 150 mg/kg diet). Fluoride failed to prevent the depressive effect of selenite on 8-wk food intake and body wt gain. Selenium (Se) concentration of plasma and kidney and enzymatic activity of whole blood glutathione peroxidase (GSH-Px) were also unaffected by F. Liver Se concentration, however, was slightly (12%) but significantly (p

A spectrum of related malacic disorders called Simmental multifocal symmetrical encephalopathy have been reported in Simmental and Simmental crossbred cattle in Canada, Australia, and New Zealand. 3 The cause of this condition is unknown. We describe multifocal polioencephalomyelomalacia in Simmental calves reared in Oregon, USA, with morphologic lesions similar to those described in Simmental multifocal symmetrical encephalopathy. These calves also had elevated tissue concentrations of aluminum and deficient hepatic copper and manganese concentrations, which may be related to this condition. Four 7-month-old bull calves from a herd of 135 Simmental cows developed paresis, ataxia, and knuckling over of both hind limbs that progressed over a 2-month period to inability to rise. During the previous 2 years, 3 calves similarly affected died. Affected calves remained bright and alert and maintained the ability to suckle. Antibiotic therapy was not beneficial, and complete blood count and serum chemistry profiles were normal in the 3 calves evaluated. Two of the calves were presented to the Oregon State Veterinary Diagnostic Laboratory for postmortem examination. Significant lesions were limited to the central nervous system, with secondary mild symmetrical atrophy of the rear limb muscles. Grossly, oblong to circular foci of malacia of various sizes (up to 1 cm diameter) were seen throughout the gray matter of the brain. Similar gross lesions were within the gray matter of the lumbar spinal cord in 1 of the calves. Microscopically, these lesions were cavitated and were composed almost entirely of necrotic debris (Fig. 1). Scattered viable neuroglial cells were within these foci but neurons were not observed. Microgliosis, astrocytosis (including gemistocytes), active neuronal necrosis, ischemic neuronal changes, and vasculitis were not observed. Vessels were prominent within the lesions, principally because of hypertrophy of endothelial nuclei. Neurofibrillary tangles and/or extensive demyelination were not observed in multiple Bodain- and Luxol fast blue-stained histologic sections. The histopathologic changes were markedly uniform throughout the affected foci. The distribution of brain lesions was essentially identical in the 2 affected calves. The gray matter lesions of the brain involved numerous nuclei but invariably extended beyond nuclear boundaries. Individual lesions often involved more than 1 nucleus with some extension into the adjacent white

ABSTRAcr: Tissue disruption methods were developed and serum-free cell culture media formulated for the maintenance in vitro of cells from juvenile worms (day 18 after infection) of Schistosoma mansoni. Cultures maintained viability for up to 6 mo when plated on a feeder layer of irradiated rat liver cells and survived primarily as clusters of small (2.5-4 tm diameter) cells with a high nuclear-to-cytoplasmic ratio and relatively few organelles identified by electron microscopy. Cultures synthesized a protein profile similar to that of intact worms, and the cell clusters maintained a time- and concentration-dependent contractile response to serotonin. Cells synthesizing DNA were detected by precursor incorporation and flow cytometry in cultures initially and also after several weeks in vitro, although the percentage of cells synthesizing DNA decreased with time. Efforts to identify peptide growth factor-responsive tyrosine phosphorylation were negative, and the overall amount of S. mansoni phosphotyrosine-containing proteins identified by western blot with anti-phosphotyrosine monoclonal antibody was much less than that found in a peptide growth factor-responsive mouse cell line.

Mature, untreated rainbow trout display a marked sex difference in the level and activity of the P-450-dependent mixed-function oxidase system with males demonstrating higher levels than females. This difference is accentuated in the trunk kidney, is specific for endogenous substrates such as lauric acid and sex steroids, and is correlated with greater levels of the constitutive cytochrome P-450 isozyme, LM2. Using an immunohistochemical technique, the cellular localization of P-450 LM2 and P-450 LM4b, the major beta-naphthoflavone inducible isozyme, in the head and trunk kidney of male, female, and juvenile trout has been demonstrated. Immunostaining for P-450 LM2 was observed in the cytoplasm of cells in the second portion (P2) of the proximal tubules of both male and female trout. In male fish, staining appeared to be more intense and involved most of the P2 segments in a tissue section. In female fish, P2 staining was moderate and involved fewer segments. Low staining intensity for P-450 LM4b was observed in the cytoplasm of cells both in the first portion of the proximal tubules and in P2 of male and female fish. Sexual dimorphism was not apparent. The finding of greater amounts of P-450 LM2 in males and low levels of P-450 LM4b in untreated male and female trout confirms previous biochemical studies. Localization of P-450 isozymes in the proximal tubules is consistent with findings in mammalian species. Moderate immunostaining by anti-P-450 LM4b IgG also was observed in the interrenal cells of the head kidney of male, female, and juvenile trout.

An immunohistochemical technique was used to localize the major constitutive cytochrome P450 isozyme, P450 LM2, and the major beta-naphthoflavone-inducible isozyme, P450 LM4b, in the livers of untreated and aflatoxin B1 (AFB1)-initiated, tumor-bearing rainbow trout. In hepatic tissue sections from untreated trout, no regular anatomical pattern within the hepatic parenchymal cells could be discerned for either isozyme. Immunostaining was observed for P450 LM2 along the sinusoidal border of some of the parenchymal cells, there was moderate staining within the cytoplasm of most cells, and there were focal areas of increased staining. There was intense, uniform immunostaining for P450 LM2 within the cytoplasm of the bile duct cells, in the endothelial lining of arterioles, and along the epithelial surface of the gall bladder. Staining for P450 LM4b in livers from untreated trout was barely detectable. In liver tissue sections from AFB1-treated tumor-bearing fish, P450 LM2 appeared to be reduced and P450 LM4b was absent in the hepatocellular carcinoma nodules. An apparent increase in immunostaining for P450 LM4b was observed in nonneoplastic cells juxtaposed next to neoplastic cells as well as in areas distant to the tumors. These results may indicate that the pattern of P450 isozymes is altered in nonneoplastic cells of tumor-bearing trout livers.

The Veterinary Diagnostic Laboratory at Oregon State University received 172 aborted ovine fetuses during the 1985–1987 lambing seasons; from 120 of these, body fluids were evaluated for IgG levels. Fifty-two (43%) of the fetal fluids had immunoglobulin G (IgG) levels greater than 15 mg/dl. Forty-five (87%) of the fluids with elevated IgG levels were confirmed or presumed toxoplasma or Chlamydia abortions. A mean fetal fluid IgG concentration of 111.5 ± 78 mg/dl was found for the 26 toxoplasma abortions; for the 19 Chlamydia abortions, a mean IgG concentration of 109 ± 91 mg/dl was found. Antibody titers equal to or greater than 1:40 against Toxoplasma gondii were detected in 23 fetal fluids. Fetal fluid IgG concentration less than 15 mg/dl was found to be associated with bacterial organisms (i.e., Campylobacter sp.) as the confirmed or presumed cause of abortion. These results suggest that measurement of fetal fluid IgG concentration is a useful, supportive diagnostic test in determining the cause of ovine abortion, and should be included as a routine laboratory procedure for ovine abortion diagnosis.

The immunotoxic properties of two experimental antiallergic drugs, CI-949 and CI-959, were investigated. Wistar rats were gavaged once (CI-949) or twice (CI-959) daily for 21 days with the drugs. Immunotoxicity was assessed using the enzyme-linked immunoabsorbant assay (ELISA) for humoral immunity, a delayed-type hypersensitivity (DTH) procedure for cell-mediated immunity, and natural killer cell (NKC) activity to evaluate spontaneous cytotoxicity. Ratios of body weight to spleen, thymus, liver and kidney weights were determined. Routine histopathology was performed on lymphoid tissue and other body organs. Although 100 mg/kg/day of CI-949 had some stimulating effect on antibody production and NKC cytotoxicity, no consistent immunomodulation was apparent. Except for a significant increase in liver weight at the 100 mg/kg dose of CI-949, no other toxic effects were observed. In contrast to CI-949, CI-959 significantly (P