Lymphocyte Subsets in the Adrenal Glands of Dogs With Primary Hypoadrenocorticism.

Primary hypoadrenocorticism, or Addison's disease, is an autoimmune condition common in certain dog breeds that leads to the destruction of the adrenal cortex and a clinical syndrome involving anorexia, gastrointestinal upset, and electrolyte imbalances. Previous studies have demonstrated that this destruction is strongly associated with lymphocytic-plasmacytic inflammation and that the lymphocytes are primarily T cells. In this study, we used both immunohistochemistry and in situ hybridization to characterize the T-cell subtypes involved. We collected postmortem specimens of 5 dogs with primary hypoadrenocorticism and 2 control dogs and, using the aforementioned techniques, showed that the lymphocytes are primarily CD4+ rather than CD8+. These findings have important implications for improving our understanding of the pathogenesis and in searching for the underlying causative genetic polymorphisms.

Disruption of Trim9 function abrogates macrophage motility in vivo.

The vertebrate immune response comprises multiple molecular and cellular components that interface to provide defense against pathogens. Because of the dynamic complexity of the immune system and its interdependent innate and adaptive functionality, an understanding of the whole-organism response to pathogen exposure remains unresolved. Zebrafish larvae provide a unique model for overcoming this obstacle, because larvae are protected against pathogens while lacking a functional adaptive immune system during the first few weeks of life. Zebrafish larvae were exposed to immune agonists for various lengths of time, and a microarray transcriptome analysis was executed. This strategy identified known immune response genes, as well as genes with unknown immune function, including the E3 ubiquitin ligase tripartite motif-9 (Trim9). Although trim9 expression was originally described as "brain specific," its expression has been reported in stimulated human Mϕs. In this study, we found elevated levels of trim9 transcripts in vivo in zebrafish Mϕs after immune stimulation. Trim9 has been implicated in axonal migration, and we therefore investigated the impact of Trim9 disruption on Mϕ motility and found that Mϕ chemotaxis and cellular architecture are subsequently impaired in vivo. These results demonstrate that Trim9 mediates cellular movement and migration in Mϕs as well as neurons.

Assessment of geographical variation in the respiratory toxicity of desert dust particles.

The health consequences of sand particle inhalation are incompletely understood. This project evaluated the respiratory toxicity of sand particles collected at military bases near Fort Irwin USA, in Iraq (Camp Victory, Taji and Talil), and Khost Afghanistan. Our primary focus was on assessing the role of soluble metals in the respiratory toxicity of the sand particles using in vitro and in vivo methods. Replicating rat type II alveolar cell cultures (RLE-6TN) were exposed to sand extracts or vehicle control in serum-free media for ≤24 h. Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and assessment of lactate dehydrogenase leakage. The relative in vitro cytotoxicity of the sand extracts was Taji ≈ Talil > Afghanistan > Camp Victory ≈ Fort Irwin. We also assessed extracts of Camp Victory, Afghanistan, and Taji sand for acute and delayed pulmonary toxicity in rats following intratracheal administration. Assessments included biochemical analysis of bronchoalveolar lavage fluid (BALF) and lung histopathology. The in vitro cytotoxicity assay results were partially predictive of in vivo responses. The more cytotoxic Taji sand extract induced an acute irritant response in rats following intratracheal administration. Rats given the less cytotoxic Camp Victory sand extract had minimal biochemical or cytological BALF changes whereas rats given either the Afghanistan or Taji sand extracts demonstrated BALF changes that were suggestive of mild lung inflammation. Unexpectedly, we observed similar lung pathology in all extract-exposed rats. The results of our study can be used to prioritize future particle inhalation studies or guide epidemiological study design.

Repeated exposure of goldfish (Carassius auratus) to tricaine methanesulfonate (MS-222).

Goldfish that have been repeatedly exposed to tricaine methanesulfonate (MS-222) require greater concentration of the drug to attain equivalent planes of anesthesia, but the mechanism for this increased anesthetic need is unknown. Minimum anesthetic concentration (MAC) is a commonly used method with which to compare anesthetics. It was hypothesized that fish exposed to MS-222 daily would have an increased MAC. It was also hypothesized that fish exposed daily to MS-222 would develop histomorphologic changes to their gills to explain the increasing demand. Forty-nine Serasa comet goldfish were enrolled and were divided into three populations (n = 15, n = 15, and n = 19). In trial 1, using an up-down method, MAC was determined daily after 4 min of exposure to MS-222 for which the starting concentration was 160 mg/L. In trial 2, MAC was determined following 2 min of exposure to MS-222 for which the starting concentration was 260 mg/L. In trial 3, four naive fish were euthanatized and gills collected for histology and electron microscopy (EM). The remaining fish were exposed to MS-222 daily for 4 wk. Four fish were euthanatized and their gills submitted for similar examination at 2 wk and 4 wk. MAC for fish exposed to MS-222 for 4 min increased from 120 to 160 mg/L. The regression line had a slope of 1.51 +/- 0.26 (R2 = 0.65; P < 0.0001). MAC for fish exposed to MS-222 for 2 min increased from 210 pmm to 220 mg/L; the regression line had a slope of 0.52 +/- 0.38 (R2 = 0.12; P = 0.2). Histologic and EM examination of gills did not show morphologic changes indicative of a reaction to MS-222. Goldfish in this study had an increased requirement for MS-222 following daily exposure for 4 min but not following daily exposure for 2 min at a higher concentration. The cause of this increased anesthetic need is not related to morphologic changes to the gills.

Clinical and pathological effects of the polyopisthocotylean monogenean, Gamacallum macroura in white bass.

An aquaculture research facility experienced high mortality rates in white bass Morone chrysops associated with a monogenean infestation of the gills, but not in striped bass Morone saxatilis in the same facility. All mortalities had pale gills. Monogeneans, identified as Gamacallum macroura (MacCallum and MacCallum 1913) Unnithan 1971, were found on the gills. Pale-gilled and healthy white bass were selected with no particular attention to condition for venipuncture and euthanasia for postmortem examination, including parasite counts from gills. The median packed cell volume (PCV) of fish with gill pallor was 12.5% (range 9-37%) while PVC of fish with more normal color was 30% (27-33%). Association between the PCV and gill pallor score was statistically significant, as was the association between PCV and the number of monogeneans found on the gills of each fish. Median estimated white blood cell count of fish with gill pallor, at 12.05 × 10(3/)µL (range 3.8-24.7), was significantly lower than of apparently healthy fish: 24.7 × 10(3)/µL (17.3-31.5). Histopathology of the gill arches of pale-gilled fish revealed multifocal moderate to severe branchitis, focal areas of dilated hyperplastic lamellae occluded by fibrin, and monogeneans attached to the lamellae. Fish that were apparently healthy had grossly similar histologic lesions, but at lower frequency and severity.

Two myristoylated alanine-rich C-kinase substrate (MARCKS) paralogs are required for normal development in zebrafish.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin binding protein substrate of protein kinase C (PKC) and critical for mouse and Xenopus development. Herein two MARCKS paralogs, marcksa and marcksb, are identified in zebrafish and the role of these genes in zebrafish development is evaluated. Morpholino-based targeting of either MARCKS protein resulted in increased mortality and a range of gross phenotypic abnormalities. Phenotypic abnormalities were classified as mild, moderate or severe, which is characterized by a slight curve of a full-length tail, a severe curve or twist of a full-length tail and a truncated tail, respectively. All three phenotypes displayed abnormal neural architecture. Histopathology of Marcks targeted embryos revealed abnormalities in retinal layering, gill formation and skeletal muscle morphology. These results demonstrate that Marcksa and Marcksb are required for normal zebrafish development and suggest that zebrafish are a suitable model to further study MARCKS function.

Cellular proliferation, cell death, and liver histology in Gambusia affinis after dietary exposure to benzidine and 2-aminofluorene.

Chronic exposure to arylamines through diet and/or smoking has been associated with genetic changes and tumorigenesis. Cellular proliferation, apoptosis, and histological changes in liver tissue were investigated in Gambusia affinis (G affinis) after chronic dietary exposure to 6.9 mM and 0.069 mM concentrations of benzidine (BZ), 2-aminofluorene (2AF), and their combination for 4, 8, and 12 weeks, respectively. The proliferation assay indicated non-dose-dependent increases in cellular proliferation over the controls for all treatment groups at 4 and 12 weeks but not at 8 weeks except for the low dose of 2AF. The apoptosis assay showed effects in the low-dose group of 2AF and BZ at 4 weeks only. Hematoxylin/eosin staining of liver tissue revealed an increase in oval/spindle cell proliferation and altered foci formation in the treated groups compared with controls. These results demonstrate a mammalian-like response to 2AF and BZ in G affinis liver.

Gross and histologic evaluation of 5 suture materials in the skin and subcutaneous tissue of the California sea hare (Aplysia californica).

Invertebrates are increasing in their importance to both the public and private aquarium trade and play a vital role in biomedical research. Surgical techniques have become an important approach to obtaining data and maintaining good health in both of these areas. However, studies examining tissue reaction to suture material in invertebrates are lacking. The current study evaluated the gross and histologic reaction of Aplysia californica to 5 commonly used suture materials, including polydioxanone, black braided silk, polyglactin 910, monofilament nylon, and monofilament poliglecaprone. Histologic samples were graded on the amount of edema (score, 1 to 4), inflammation (1 to 4), and granuloma formation (1 to 4) present, and a final overall histology score (1 to 6) was assigned to each sample. Compared with untreated control tissue, all suture materials caused significantly increased tissue reaction, but the overall histology score did not differ among the suture materials. Silk was the only suture that did not have a significantly increased granuloma score when compared with the control. Although none of the suture materials evaluated seemed clearly superior for use in Aplysia, we recommend silk because of its less robust granuloma induction, which is favorable in a clinical and research setting.

Exposure to the synthetic FXR agonist GW4064 causes alterations in gene expression and sublethal hepatotoxicity in eleutheroembryo medaka (Oryzias latipes).

The small freshwater teleost, medaka (Oryzias latipes), has a history of usage in studies of chronic toxicity of liver and biliary system. Recent progress with this model has focused on defining the medaka hepatobiliary system. Here we investigate critical liver function and toxicity by examining the in vivo role and function of the farnesoid X receptor alpha (FXRalpha, NR1H4), a member of the nuclear receptor superfamily that plays an essential role in the regulation of bile acid homeostasis. Quantitative mRNA analysis of medaka FXRalpha demonstrates differential expression of two FXRalpha isoforms designated Fxralpha1 and Fxralpha2, in both free swimming medaka embryos with remaining yolk (eleutheroembryos, EEs) and adults. Activation of medaka Fxralpha in vivo with GW4064 (a strong FXRalpha agonist) resulted in modification of gene expression for defined FXRalpha gene targets including the bile salt export protein, small heterodimer partner, and cytochrome P450 7A1. Histological examination of medaka liver subsequent to GW4064 exposure demonstrated significant lipid accumulation, cellular and organelle alterations in both hepatocytes and biliary epithelial cells of the liver. This report of hepatobiliary injury following GW4064 exposure extends previous investigations of the intrahepatic biliary system in medaka, reveals sensitivity to toxicant exposure, and illustrates the need for added resolution in detection and interpretation of toxic responses in this vertebrate.

Characterization of eversion syndrome in captive Scyphomedusa jellyfish.

OBJECTIVE: To determine whether Scyphomedusa jellyfish with eversion syndrome had alterations in husbandry conditions, elemental content, or histologic appearance, compared with unaffected jellyfish. ANIMALS: 123 jellyfish (44 with eversion syndrome and 79 without) at 6 institutions. PROCEDURES: Elemental analyses were performed on 24 jellyfish with eversion syndrome and 49 without, and histologic examinations were performed on 20 jellyfish with eversion syndrome and 30 without. A questionnaire distributed to 39 institutions with Scyphomedusa jellyfish was used to gather information about husbandry, environmental conditions, and prevalence of eversion syndrome. RESULTS: For the 39 institutions that responded to the questionnaire, prevalence of eversion syndrome ranged from 0% to 30%. For Aurelia aurita, eversion was more common at institutions with only captive-raised and no wild-caught jellyfish. Eversion was most common among young (approx 1- to 2-month-old) growing jellyfish and older (> 6-month-old) jellyfish. Elemental analysis revealed only minor differences between affected and unaffected jellyfish, with great variation among jellyfish from the same institution and among jellyfish from different institutions. Striated muscle degeneration and necrosis and extracellular matrix (mesoglea) degeneration were evident on histologic examination of affected jellyfish. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that eversion syndrome is a complex phenomenon associated with degenerative changes of the bell matrix.

Enhanced transcription of complement and coagulation genes in the absence of adaptive immunity.

A recessive nonsense mutation in the zebrafish recombination activating gene 1 (rag1) gene results in defective V(D)J recombination; however, animals homozygous for this mutation (rag1(-/-)) are reportedly viable and fertile in standard, nonsterile aquarium conditions but display increased mortality after intraperitoneal injection with mycobacteria. Based on their survival in nonsterile environments, we hypothesized that the rag1(-/-) zebrafish may possess an "enhanced" innate immune response to compensate for the lack of an adaptive immune system. To test this hypothesis, microarray analyses were used to compare the expression profiles of the intestines and hematopoietic kidneys of rag1 deficient zebrafish to the expression profiles of control (heterozygous) siblings. The expression levels of 12 genes were significantly altered in the rag1(-/-) kidney including the up regulation of a putative interferon stimulated gene, and the down regulation of genes encoding fatty acid binding protein 10, keratin 5 and multiple heat shock proteins. The expression levels of 87 genes were shown to be significantly altered in the rag1(-/-) intestine; the majority of these differences reflect increased expression of innate immune genes, including those of the coagulation and complement pathways. Subsequent analyses of orthologous coagulation and complement genes in Rag1(-/-) mice indicate increased transcription of the complement C4 gene in the Rag1(-/-) intestine.

Toward a molecular equivalent dose: use of the medaka model in comparative risk assessment.

Recent changes in the risk assessment landscape underscore the need to be able to compare the results of toxicity and dose-response testing between a growing list of animal models and, quite possibly, an array of in vitro screening assays. How do we compare test results for a given compound between vastly different species? For example, what dose level in the ambient water of a small fish model would be equivalent to 10 ppm of a given compound in the rat's drinking water? Where do we begin? To initially address these questions, and in order to compare dose-response tests in a standard rodent model with a fish model, we used the concept of molecular dose. Assays that quantify types of DNA damage that are directly relevant to carcinogenesis integrate the factors such as chemical exposure, uptake, distribution, metabolism, etc. that tend to vary so widely between different phyletic levels. We performed parallel exposures in F344 rats and Japanese medaka (Oryzias latipes) to the alkylating hepatocarcinogen, dimethylnitrosamine (DMN). In both models, we measured the DNA adducts 8-hydroxyguanine, N(7)-methylguanine and O(6)-methylguanine in the liver; mutation frequency using lambda cII transgenic medaka and lambda cII transgenic (Big Blue(R)) rats; and early morphological changes in the livers of both models using histopathology and immunohistochemistry. Pulse dose levels in fish were 0, 10, 25, 50, or 100 ppm DMN in the ambient water for 14 days. Since rats are reported to be especially sensitive to DMN, they received 0, 0.1, 1, 5, 10, or 25 ppm DMN in the drinking water for the same time period. While liver DNA adduct concentrations were similar in magnitude, mutant frequencies in the DMN-exposed medaka were up to 20 times higher than in the Big Blue rats. Future work with other compounds will generate a more complete picture of comparative dose response between different phyletic levels and will help guide risk assessors using "alternative" models.

A quantitative real-time RT-PCR assay to measure TGF-beta mRNA and its correlation with hematologic, plasma chemistry and organo-somatic indices responses in triamcinolone-treated Atlantic menhaden, Brevoortia tyrannus.

A quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assay was developed to measure transforming growth factor-beta (TGF-beta) in Atlantic menhaden (Brevoortia tyrannus), an estuarine-dependent species plagued by ulcerative skin lesions in the estuaries along the eastern United States. Atlantic menhaden were acclimated in a closed system for two weeks prior to initiation of the study. The synthetic glucocorticoid, triamcinolone acetonide (10mg/kg body weight) was administered by intracoelomic injection and its effect on the splenic mononuclear cell TGF-beta mRNA transcription, liver-somatic index, spleno-somatic index, hematology, and plasma chemistry were compared to untreated fish at 48 and 96h post-treatment. Triamcinolone-treated Atlantic menhaden showed suppression of TGF-beta mRNA production, neutrophilia, monocytosis, lymphopenia, and an increase in blood glucose concentrations. The health indices used in this study may help us interpret some of the changes observed during the development of ulcerative skin lesions in wild-caught menhaden.

Dynamic gene expression changes precede dioxin-induced liver pathogenesis in medaka fish.

A major challenge for environmental genomics is linking gene expression to cellular toxicity and morphological alteration. Herein, we address complexities related to hepatic gene expression responses after a single injection of the aryl hydrocarbon receptor (AHR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) and illustrate an initial stress response followed by cytologic and adaptive changes in the teleost fish medaka. Using a custom 175-gene array, we find that overall hepatic gene expression and histological changes are strongly dependent on dose and time. The most pronounced dioxin-induced gene expression changes occurred early and preceded morphologic alteration in the liver. Following a systematic search for putative Ah response elements (AHREs) (5'-CACGCA-3') within 2000 bp upstream of the predicted transcriptional start site, the majority (87%) of genes screened in this study did not contain an AHRE, suggesting that gene expression was not solely dependent on AHRE-mediated transcription. Moreover, in the highest dosage, we observed gene expression changes associated with adaptation that persisted for almost two weeks, including induction of a gene putatively identified as ependymin that may function in hepatic injury repair. These data suggest that the cellular response to dioxin involves both AHRE- and non-AHRE-mediated transcription, and that coupling gene expression profiling with analysis of morphologic pathogenesis is essential for establishing temporal relationships between transcriptional changes, toxicity, and adaptation to hepatic injury.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces organ- specific differential gene expression in male Japanese medaka (Oryzias latipes).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental contaminant with well-known adverse effects in fish. In this study, we initially exploited suppression subtractive hybridization (SSH) as a screening tool to assess qualitative gene expression changes in whole brain, liver, and testis of adult male Japanese medaka (Oryzias latipes) exposed for 48 h to a single intraperitoneal-injected dose of TCDD (10 microg TCDD/kg body weight). Across these three organs, SSH identified a total of 335 unique genes. Each set of forward- and reverse-subtracted organ cDNA libraries consisted of a distinct gene list and corresponding distribution of biological processes, suggesting that transcript profiles of these libraries were highly organ-specific. Based on sequence match significance and frequencies within each set of organ libraries, genes hypothesized to be strongly responsive (42 total) within male medaka brain, liver, or testis were semi-quantitatively screened with replicate cDNA nylon membrane arrays. In addition, TCDD-treated male medaka were surveyed for gross histological analysis of brain, liver, and testis. In general, adverse histopathological changes were not observed in the brain, and glycogen depletion was observed only in the liver. However, significant histological changes occurred in the testis, and included disorganization of spermatogenesis at the testis periphery, disruption of the interstitium, Leydig cell swelling, and Sertoli cell vacuolation. Of the 42 genes screened by cDNA array analysis, cytochrome P450 1A (CYP1A) mRNA was the only transcript significantly higher in TCDD-exposed brain, whereas 12 transcripts (including CYP1A) were significantly higher in TCDD-exposed liver, and 34 transcripts were significantly lower in TCDD-exposed testis. Therefore, the degree of TCDD-induced alterations observed in each organ at a gross histological level corresponded well with the number and ontology of gene transcripts affected on the array. Based on real-time reverse transcription polymerase chain reaction (RT-PCR), relative CYP1A (but not AHR1) transcript levels were confirmed to be significantly higher in TCDD-treated brain and liver. However, CYP1A was not significantly induced in TCDD-exposed testis, suggesting that gene expression and histopathological responses observed in the testis at 48 h may be CYP1A-independent. Based on these data, unique liver-specific and testis-specific mRNA-level targets in male medaka were identified as promising biomarkers of acute TCDD-induced toxicity.

Issues related to the use of fish models in toxicologic pathology: session introduction.

Ready or not, fish models are "here to stay." No longer are fish confined to a few specialized laboratories, nor are they exclusively the purview of zoologists or environmental toxicologists. In fact, the institution that does not house at least 1 fish facility is probably not at the forefront of cutting edge research. In toxicologic pathology, fish models are increasingly being used to provide high animal numbers at relatively low cost in carcinogenicity testing and developmental research, and to provide mechanistic information on fundamental cellular processes. In this session, we attempt to provide some perspective for the pathologist that is faced with planning or performing experiments or testing protocols using fish models, or with reading or interpreting fish studies. First, we cover how to approach fish studies from the contract laboratory standpoint, including sectioning, quality control, and GLP considerations. Then, we discuss specifics on the use of the rainbow trout, zebrafish, and Japanese medaka models. The rainbow trout has a rich history in carcinogenicity and mechanistic cancer research. Similarly, the 2 workhorses in the small fish category, zebrafish and medaka, have found their way into many laboratories doing developmental biology and genomics research as well as carcinogenicity testing. Some fascinating genetically altered fish models have been developed with both of these species. This manuscript provides a session overview of the use of small fish models in toxicologic pathology, along with some historical perspective on how these models have played a role in the current state of the science.

Evaluation of the tissue reactions in the skin and body wall of koi (Cyprinus carpio) to five suture materials.

Five different suture materials (silk, monofilament nylon, polyglyconate, polyglactin 910, and chromic gut) were placed in the skin and body wall of 10 Doitsu (scaleless) koi (Cyprinus carpio). After seven days the sutures were retrieved from five of the fish in 5 mm and 6 mm punch biopsies, and after 14 days they were retrieved in the same way from the other five. The tissue reactions were evaluated by gross visual inspection and by histological examination. The total inflammatory reaction was graded on a scale from 0 (no inflammation) to 5 (severe inflammation). The synthetic suture materials generally induced a moderate inflammatory reaction that decreased after seven days. After 14 days the superficial reaction to monofilament nylon was substantial, and the tissue reactions to the organic suture materials were slightly greater than the reactions to the synthetics. The inflammatory response to silk was greater after 14 days than after seven, and chromic gut induced a moderately severe inflammatory response after seven days; the chromic gut sutures fell out before the biopsies were taken after 14 days. The organic materials induced intense inflammatory reactions which did not subside if the suture remained in the tissue.