John W. Fournie

Diagnostic Criteria for Degenerative, Inflammatory, Proliferative Nonneoplastic and Neoplastic Liver Lesions in Medaka (Oryzias latipes): Consensus of a National Toxicology Program Pathology Working Group

Diagnostic criteria are presented for degenerative, inflammatory, nonneoplastic proliferative, and neoplastic lesions in the liver of medaka (Oryzias latipes), a small fish species frequently used in carcinogenesis studies. The criteria are the consensus of a Pathology Working Group (PWG) convened by the National Toxicology Program. The material examined by the PWG was from medaka exposed to N-nitrosodiethylamine for 28 days, removed to clean water, and sacrificed 4, 6, or 9 mo after initiation of exposure. Degenerative lesions included hepatocellular intracytoplasmic vacuolation, hepatocellular necrosis, spongiosis hepatis, hepatic cysts, and hepatocellular hyalinization. Inflammatory lesions consisted of granulomas, chronic inflammation, macrophage aggregates, and focal lymphocytic infiltration. Nonneoplastic proliferative lesions comprised foci of cellular alteration (basophilic focus, eosinophilic focus, vacuolated focus, and clear cell focus) and bile duct hyperplasia. Neoplastic lesions included hepatocellular adenoma, hepatocellular carcinoma, cholangioma, and cholangiocarcinoma. Two lesions composed mainly of spindle cells were noted, hemangiopericytoma and spindle cell proliferation. Rather than being an exhaustive treatment of medaka liver lesions, this report draws from the published literature on carcinogen-induced liver lesions in medaka and other fish species and attempts to consolidate lesion criteria into a simplified scheme that might be useful to pathologists and other researchers using medaka lesions for risk assessment or regulatory purposes.

Utility of Splenic Macrophage Aggregates as an Indicator of Fish Exposure to Degraded Environments

Abstract The utility of splenic macrophage aggregates (MAs) as an indicator of fish exposure to degraded environments was evaluated in several species of estuarine fishes as part of the Environmental Protection Agencys Environmental Monitoring and Assessment Program–Estuaries (EMAP-E). Using image analysis, we measured the number and mean size of MAs per square millimeter on tissue sections of spleen from 983 fishes representing seven species from 266 stations scattered across coastal estuaries of the Gulf of Mexico. At 16 stations, at least one fish exhibited a high density of MAs (>40 MAs/mm2). Densities of MAs that exceeded 40/mm2 correlated with exposure to either hypoxic conditions or sediment contamination. Fishers exact test showed that the observed frequencies of joint occurrence between high numbers of MAs and both high sediment contaminants and low dissolved oxygen were significantly greater than the expected background frequencies. For all 16 sites where MAs were greater than 40/mm2, sediment...

Use of the Japanese medaka (Oryzias latipes) and guppy (Poecilia reticulata) in carcinogenesis testing under national toxicology program protocols.

A need exists for whole animal toxicity, mutagenesis, and carcinogenesis models that are alternative to the traditional rodent test models and that are economical, sensitive, and scientifically acceptable. Among small fish models, the Japanese medaka (Oryzias latipes) is preeminent for investigating effects of carcinogenic and/or toxic waterborne hazards to humans. The guppy (Poecilia reticulata ), although less widely used, is valuable as a comparison species. Both species are easy to maintain and handle in the laboratory and there is a large body of background information on their responsiveness to a range of classes of carcinogens. There are considerable data on the occurrence of background diseases and on spontaneous neoplastic lesions, both of which occur relatively rarely. With few modifications, the medaka and guppy are amenable to carcinogenicity testing under the rigid standards established by the National Toxicology Program (NTP) for rodent tests. The advantages of the small fish in carcinogenesis studies are best realized in long-term studies that involve environmentally realistic exposures. Studies to identify chronic effects can be conducted in about 12 months, near the life span of medaka in our laboratory. Practically, 9-month studies are optimal but shorter study cycles and a variety of exposure/growout and initiation/promotion scenarios are available. Studies on 3 compounds tested in medaka under NTP protocols are under review and preliminary analysis indicates that chronic carcinogenicity bioassays with medaka, guppy, and potentially with other small fish species are feasible and scientifically valid.

Prevalence of gross pathological abnormalities in estuarine fishes

Abstract Gross external pathological abnormalities are frequently used as indicators of anthropogenic influence in estuarine and marine waters, but little baseline information is available to identify what constitutes normal prevalences (percentages of animals affected). To establish this baseline, trawl samples were collected from 120 randomly located estuarine sites in the Virginian biogeographic province (mid-Atlantic) in 1990 and from 220 randomly located sites in the Louisianian biogeographic province (Gulf Coast) in 1991 and 1992. Sediment contaminant concentrations were measured at each site. In all, 24,291 fish representing 143 species were examined for gross pathological abnormalities, and background prevalences were estimated to be 0.5% in midAtlantic and 0.7% in Gulf Coast estuaries. Fifteen types of gross abnormalities were noted. Skin lesions were the most prevalent abnormalities in both provinces, followed by ocular abnormalities (e.g., exophthalmia, keratitis) in the Virginian Province and ...

Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.

Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated. The objectives of this article are to illustrate a number of specific morphologic findings in commonly examined fish tissues (e.g., gills, liver, kidney, and gonads) that are frequently either misdiagnosed or underdiagnosed, and to address related issues involving the interpretation of histopathologic data. To enhance the utility of this article as a guide, photomicrographs of normal and abnormal specimens are presented. General recommendations for generating and publishing results from histopathology studies are additionally provided. It is hoped that the furnished information will be a useful resource for manuscript generation, by helping authors, reviewers, and readers to critically assess fish histopathologic data.

Exocrine Pancreatic Neoplasms in the Mummichog (Fundulus heteroclitus) from a Creosote-Contaminated Site

A high prevalence of exocrine pancreatic neoplasms occurred in mummichog, Fundulus heteroclitus, from a creosote-contaminated site in the Elizabeth River, Virginia. A total of 20 neoplasms were found in a group of about 1,300 fish obtained at this site over a 2-yr period. Of 240 fish collected during October 1991, 3.3% had pancreatic neoplasms. Adjusted total lesion prevalence for large adult fish (Size Class III: total length = 75–85 mm; Size Class IV: total length > 85 mm) was 6.7%. Pancreatic neoplasms were not observed in 234 fish collected at this site during May 1991, nor were they found in 420 fish obtained during fall 1991 from 1 uncontaminated and 6 moderately contaminated localities. Lesions involved both mesenteric and intrahepatic exocrine pancreas and ranged from well-differentiated acinar cell adenomas to poorly differentiated acinar cell carcinomas. One fish had an atypical acinar cell focus. All specimens with pancreatic neoplasms also had hepatocellular lesions. This epizootic of exocrine pancreatic neoplasia is the first to be reported in a wild fish population. Based on chemical characterization of the site and limited experimental data on chemically induced pancreatic carcinogenesis in other small fish species, the neoplasms were probably caused by exposure of the mummichog to chemical carcinogens in their environment.

Multigenerational exposure of the estuarine sheepshead minnow (Cyprinodon variegatus) to 17β-estradiol. I. Organism-level effects over three generations.

A 280-d study examined the effects of 17β-estradiol (E2) on reproduction and development of the sheepshead minnow (Cyprinodon variegatus) exposed from the parental (F0) through three subsequent (F1, F2, and F3) generations and evaluated the need for multigenerational assessments of the risks of endocrine-disrupting chemicals. This first three-generation study exposed adult F0 and F1 fish to measured concentrations of 0.01, 0.04, 0.08, 0.2, and 0.3 μg E2/L; the F2 and F3 generations were exposed to 0.2 μg E2/L or less. The cumulative 21-d production of normal embryos was significantly reduced in the F0 generation at 0.3 μg E2/L and in the F1 and F2 generations at 0.08 μg E2/L or more. The daily reproductive rate was significantly reduced in all three generations at 0.08 μg E2/L or more during spawning days 8 to 14 and 15 to 21. The proportion of infertile eggs from F1 fish was significantly increased above that of the solvent controls at 0.04 and 0.2 μg E2/L and from F2 fish at 0.04 μg E2/L or more. Changes in liver, kidney, and gonadal tissues were seen in the F0 and F1 generations exposed to 0.2 μg E2/L or more. The female gonadosomatic index was significantly decreased at 0.3 μg E2/L in the F0 and F1 generations. Estradiol affected the hepatosomatic index only in female F1 fish, but not in a dose-dependent manner. All F1 fish in 0.3 μg E2/L appeared to be phenotypically female. Our results indicate that life-cycle exposure to E2 significantly decreased embryo production by F1 and F2 fish at concentrations lower than those affecting the F0 generation, and they emphasize the importance of evaluating the impact of an estrogenic chemical on reproduction through a minimum of two (F0 and F1) generations.

Fixation of Fish Tissues

Publisher Summary Fixation has been applied to animal tissues mainly for histological or pathological studies. This chapter provides information dealing with the principal facets of fixing fish tissues; it discusses the purpose of fixation and the basic elements of the chemistry of fixation. The chapter describes the various types of fixatives available, provides recipes for the most commonly used fixative mixtures, and discusses the advantages and disadvantages of the fixatives. The choice of fixatives for specific procedures such as preparation of liver tissue for light microscopy, the preparation of whole small fish for histological evaluation, and the use of molecular and immunohistochemical techniques are also discussed. Fixatives are chemical substances that act as coagulants or non-coagulants of proteins in cells and tissues. Several criteria should be considered in choosing a fixative including: (i) the kind of tissue; (ii) the purpose of the study; (iii) the type of lesion; (iv) whether fish tissues or whole fish are being prepared; and (v) the staining or special techniques to be employed. Generally, it appears that protocols developed for immunohistochemical studies using mammalian models are suitable for fish. The process of fixation obviously involves cell death and therefore substances used in the process must be handled carefully.

Comparison of estrogen-responsive plasma protein biomarkers and reproductive endpoints in sheepshead minnows exposed to 17β-trenbolone

Protein profiling can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In previous studies, mass spectral analysis revealed four peptides (2950.5, 2972.5, 3003.4, 3025.5m/z) in the plasma of estrogen agonist-treated male and gravid female sheepshead minnows (Cyprinodon variegatus, SHM), which served as distinct estrogenic biomarkers. In this study, a 21-day reproductive assay with adult SHM was conducted to investigate possible dose-related effects of the synthetic androgen, 17beta-trenbolone, on expression of these four estrogen-responsive peptides. In addition, the response of the peptide biomarkers were compared to traditional reproductive endpoints of fecundity, histopathology, secondary sex characteristics, length, weight, hepatosomatic index, female gonadosomatic index and plasma vitellogenin (VTG) levels. Fish were continuously exposed to 0.005, 0.05, and 5.0 microg/l, a solvent control (triethylene glycol, TEG), and a seawater control (SW) using an intermittent flow-through dosing system. Plasma was analyzed for the presence of the four peptide biomarkers by MALDI-TOF MS and VTG protein by quantitative ELISA. Male fish from the trenbolone treatments and controls showed no expression of the four peptide biomarkers or measurable levels of VTG. The estrogen-responsive biomarkers and plasma VTG were constitutively expressed in females from the SW, TEG, 0.005 and 0.05 microg/l exposures. All four peptide biomarkers were significantly reduced (p<0.0002 to p<0.005) at the 5.0 microg/l treatment level which corresponded with significant reductions in fecundity and changes in ovarian morphology. A distinct but non-significant reduction in VTG was also observed in female fish from the 5.0 microg/l treatment. Results of this study suggest application of these estrogen-responsive protein biomarkers may be a cost effective alternative to fecundity measures which are labor intensive and expensive to conduct.

Macrophage Aggregates: Biomarker for Immune Function in Fishes?

Macrophage aggregates (MAs) are believed to be functional equivalents of germinal centers, active in storage of exogenous and endogenous waste products, the immune response, and iron storage and recycling. Numerous studies have shown an increase in their number, size or hemosiderin content in fish collected at contaminated sites. For this reason, MAs have been suggested as potentially sensitive biomarkers of contaminant exposure. Although they are structures observed histologically, it has also been suggested they may be immunotoxicologic biomarkers. To determine possible relationships between MA formation and macrophage function, we examined data from two field studies and one laboratory study. We found a significant correlation between a decreased chemotaxic response of macrophages and the formation of more numerous, smaller MAs in mummichog from a contaminated site. In laboratory-exposures to arsenic, macrophage function appeared to be a more sensitive indicator at the lower levels of dietary arsenic. However, MA appeared to provide a more dose-dependent and comprehensive indicator of toxicity.