Lee A. Courtney

Cytological changes during progression of neoplasia in selected fish species

Abstract Cytological changes during progression of experimentally induced hepatic neoplasia in fishes were reviewed with emphasis on recent findings in Cyprinodon variegatus and Oryzias latipes. Hepatocytes are particularly sensitive to toxic changes during early phases of response to carcinogens reflecting both lethal and sublethal alterations. In these degenerative lesions, enzyme histochemical studies reveal marked deficiency of glucose-6-phosphate dehydrogenase, glucose-6-phosphatase and adenosine triphosphatase. Surviving hepatocytes are either enlarged, encircled by cells with small nucleus to cytoplasm ratios, and have altered nuclear morphology suggestive of an inability to divide, or are smaller, apparently rapidly dividing, and have basophilic cytoplasm. In both species, development of spongiosis hepatis occurred following cytotoxic phases. This lesion apparently provides abundant space for cellular remodeling during neoplastic progression leading to eventual multinodular change. Foci of altered hepatocytes included basophilic, eosinophilic (both species) and clear cell (Cyprinodon variegatus only). Enzyme alterations preceded other tinctorial, morphologic alterations and were seen in cells composing foci and tumors, suggesting lineage of phenotypic alteration. Cytologic changes within other resident cell populations during neoplastic progression were reviewed.

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...

INTERACTION OF CHEMICAL POLLUTANTS AND VIRUS IN A CRUSTACEAN: A NOVEL BIOASSAY SYSTEM*

A large group of shrimp, 23.3% of which had light patent Baculovirus infections, was divided equally into two groups. One group was exposed to the chemical stressor Aroclor 1254 (a polychlorinated biphenyl) at 0.7 ppb for 35 days in flowing seawater. The other group was maintained as a control group in flowing seawater. Viral prevalence in exposed shrimp samples increased with time at a significantly greater rate than did viral frequency in control shrimp. Viral prevalence in Aroclor-exposed shrimp survivors was 75% after 35 days, whereas in control shrimp, only 45.7% had patent viral infections. This finding suggests an interaction among chemical stressor (Aroclor 1254), host, and virus. The nature or mechanism of this interaction has not been defined, but the shrimp-virus system shows, promise for future bioassays of influence of low concentrations of pollutants on natural pathogenhost interactions.

Autographa californica Nuclear Polyhedrosis Virus Efficiently Enters but Does Not Replicate in Poikilothermic Vertebrate Cells

The host range of the insect virus Autographa californica nuclear polyhedrosis virus (AcMNPV) was examined. AcMNPV could not initiate a productive infection in frog, turtle, trout, or codling moth cell lines. After exposure to AcMNPV, neither viral DNA nor RNA synthesis could be detected in these cell lines as assayed by nucleic acid probe hybridization. Entry of AcMNPV nucleocapsids into the cytoplasm and viral DNA into the nucleus, however, was as efficient in the nonpermissive cell lines as it was in a permissive insect cell line. The data suggest that the block in AcMNPV infection in these nonpermissive cell lines is at a stage subsequent to viral DNA entry into the nucleus.

Comparison of three histological fixatives on the immunoreactivity of mammalian P-glycoprotein antibodies in the sheepshead minnow, Cyprinodon variegatus.

Mammalian P-glycoprotein is a highly conserved integral membrane protein functioning as an energy-dependent efflux pump which decreases the concentration of certain lipophilic aromatic compounds entering the cell by diffusion. Expression of such a transporter in teleost species could play a significant role in conferring resistance to fish populations exposed to xenobiotic stressors and may serve as a potential indicator of species at risk for certain environmental contaminants. In previous studies we demonstrated that a strong correlation existed between corresponding mammalian and teleost tissues showing immunoreactivity to specific mammalian P-glycoprotein antibodies. In the present study, comparisons of staining pattern, intensity, and tissue specificity between tissues treated in Bouins, Dietrichs and Lillies histological fixatives were determined in the sheepshead minnow, Cyprinodon variegatus, using monoclonal antibodies C219, C494, JSB-1 and polyclonal antiserum MDR(Ab-1). Immunoreactivity of these antibodies was found to be fixative-dependent. Results are presented illustrating the differential staining patterns and tissue specificity observed for each tissue type, fixative, and antibody combination. Our data indicate tissue fixation has a significant impact on P-glycoprotein antibody immuno-reactivity in teleost tissues and must be considered in the comparison and interpretation of results.

Histopathological Evidence of Regeneration following Hepatotoxic Effects of the Cyanotoxin Microcystin-LR in the Hardhead Catfish and Gulf Killifish.

The cyanobacterial toxin microcystin-LR (MC-LR) causes liver damage in several freshwater fish species. In the present study, two estuarine species, hardhead catfish Arius felis and gulf killifish Fundulus grandis, were injected intraperitoneally with MC-LR at 45-300 μg/kg and their livers examined histopathologically for up to 23 d postinjection (PI) in the catfish and 5 d PI in the killifish. The livers from both species exhibited extensive, diffuse hepatocellular necrosis by 6 h PI. The necrosis persisted, and by day 2 large numbers of basophilic cells had emerged throughout the liver parenchyma. These cells occurred individually or in small clusters. By 72 h, the basophilic cells appeared to be highly proliferative with numerous mitotic figures and were arranged in cords and tubules similar to mature hepatic parenchyma. Regeneration of the liver parenchyma was noted in gulf killifish at 5 d PI, as tracts of basophilic cells were still evident. In hardhead catfish, there were no signs of necrosis or proliferating basophilic cells by 9 d PI, and the hepatic parenchyma appeared normal except for the vacuolation of many hepatocytes and some areas of hepatic megalocytosis. The degenerative changes in the two species studied were similar to but more pronounced than those observed in freshwater fish species exposed to MC-LR. Evidence of regeneration from the hepatotoxic effects of MC-LR suggests that the toxin can be used to study regenerative processes in fish livers.

Diagnostic criteria for proliferative thyroid lesions in bony fishes

Thyroid proliferative lesions are rather common in bony fishes but disagreement exists in the fish pathology community concerning diagnostic criteria for hyperplastic versus neoplastic lesions. To simplify the diagnosis of proliferative thyroid lesions and to reduce confusion regarding lesion interpretation, we propose specific criteria for distinguishing hyperplastic from neoplastic lesions. Development of these criteria was based on the examination of a large series of proliferative lesions from Japanese medaka (Oryzias latipes), lesions from other small fish species, and a reexamination of the 97 cases of proliferative thyroid lesions from bony fishes deposited in the Registry of Tumors in Lower Animals. Specific diagnostic criteria are provided for all lesion categories including follicular cell hyperplasia (simple, nodular, or ectopic), adenoma (papillary or solid), and carcinoma (well- or poorly differentiated). These criteria should assist fish pathologists in describing and categorizing naturally occurring proliferative lesions from wild fishes, lesions that develop in laboratory fishes due to suboptimal culture practices or water quality, those in fishes used in toxicological assays, and captive aquarium fishes.

DIELDRIN INDUCES CYTOSOLIC [3H]7,12-DIMETHYLBENZ[a]ANTHRACENE BINDING BUT NOT MULTIDRUG RESISTANCE PROTEINS IN RAINBOW TROUT LIVER

Previously it was demonstrated that biliary excretion of a single dose of [14C]dieldrin or [3H]7, 12-dimethylbenz/alanthracene (DMBA) was stimulated up to 700% and 300%, respectively, in rainbow trout fed 0.3-0.4 mg dieldrin/kg/d for 9-12 wk. This was not explained by increased activities of hepatic microsomal xenobiotic-metabolizing enzymes or increased amounts of any of six cytochrome P-450 isozymes quantitated by Western blots. It was hypothesized that stimulated excretion was explained by induction of (1) cytosolic binding proteins that facilitated intracellular trafficking of DMBA to sites of metabolism, or (2) ATP-dependent proteins that transport xenobiotic metabolites from liver to bile. Binding of 15 and 60 nmol [3H]DMBA/mg protein increased about 200% in hepatic cytosol from dieldrin-fed fish. A 50-fold molar excess of unlabeled DMBA reduced binding of 15 nmol [3H]DMBA/mg protein (nonspecific binding) by the same amount in cytosol from control and dieldrin-fed fish, indicating that dieldrin induced specific binding. Liver sections from control and dieldrin-fed fish were treated with multidrug resistance (MDR) protein monoclonal antibodies C494, C219, and JSB-1, and polyclonal antibody MDR Ab-1. There were no marked differences in optical densities of immunohistochemical staining near bile canaliculi of control and dieldrin-fed fish. Induction of xenobiotic binding capacity in cytosol of dieldrin-fed rainbow trout at least partially explained altered DMBA disposition in fish pretreated with this cyclodiene insecticide.Previously it was demonstrated that biliary excretion of a single dose of [14C]dieldrin or [3H]7,12-dimethylbenz[a]anthracene (DMBA) was stimulated up to 700% and 300%, respectively, in rainbow trout fed 0.3?0.4 mg dieldrin/kg/d for 9?12 wk. This was not explained by increased activities of hepatic microsomal xenobiotic-metabolizing enzymes or increased amounts of any of six cytochrome P-450 isozymes quantitated by Western blots. It was hypothesized that stimulated excretion was explained by induction of (1) cytosolic binding proteins that facilitated intracellular trafficking of DMBA to sites of metabolism, or (2) ATP-dependent proteins that transport xenobiotic metabolites from liver to bile. Binding of 15 and 60 nmol [3H]DMBA/mg protein increased about 200% in hepatic cytosol from dieldrin-fed fish. A 50-fold molar excess of unlabeled DMBA reduced binding of 15 nmol [3H]DMBA/mg protein (nonspecific binding) by the same amount in cytosol from control and dieldrin-fed fish, indicating that dieldrin induced specific binding. Liver sections from control and dieldrin-fed fish were treated with multidrug resistance (MDR) protein monoclonal antibodies C494, C219, and JSB-1, and polyclonal antibody MDR Ab-1. There were no marked differences in optical densities of immunohistochemical staining near bile canaliculi of control and dieldrin-fed fish. Induction of xenobiotic binding capacity in cytosol of dieldrin-fed rainbow trout at least partially explained altered DMBA disposition in fish pretreated with this cyclodiene insecticide.

ENVIRONMENTAL SIGNIFICANCE OF BACULOVIRUS INFECTIONS IN ESTUARINE AND MARINE SHRIMP

Certain enveloped, rod-shaped DNA viruses have long been known as pathogens of insects under the descriptive term “nuclear polyhedrosis viruses.” These viruses have been extensively and intensively studied since Berghold’s early reports in 1947. Subsequent to Berghold’s classic early studies, many rod-shaped viruses associated with polyhedral inclusion bodies of a crystalline nature have been described from different species of insects that represent several orders of Insecta. At present, The International Committee on Nomenclature of Viruses places the nuclear polyhedrosis viruses of arthropods in subgroup A under the genus or group name Baculovirus.’ Prior to 1973, there were no reports of viruses that resemble baculoviruses in animals other than insects or mites. In 1973 and 1974, the first reports 3, were made of baculovirus-like particles and associated polyhedral inclusion bodies in a noninsect arthropod host. The new host was the pink shrimp, Penaeus duorurum, from Florida waters of the northern Gulf of Mexico. These reports indicated for the Baculovirus group a host range extension into the arthropod class Crustacea. In regard to specific characterization and identification of the shrimp virus, it is pertinent to report that not all of Koch’s postulates have been satisfied. Koch’s postulates, however, were meant to be used to show specificity of a microorganism as an etiologic agent for a disease condition and not specifically to determine phylogenetic affinity or identity of the microorganism. The latter task (identification) includes determination of biologic, morphologic, chemical, and physical characteristics. Much of our effort has gone into these determinations for the shrimp virus. The first of Koch’s postulates (that of association or presence of a microorganism with a disease condition) has been satisfied for patent virus infections in shrimp; that is, inclusion bodies and virions are present in all patent infections that exhibit cytopathologic characteristics. The second of Koch’s postulates (that of isolation and pure culture of the microorganism) has not been satisfied for the shrimp virus and poses a severe problem because of the lack of continuous cell cultures of crustacean tissues in which to isolate and grow the virus. At present, we are attempting to use established insect cell lines in which to grow the shrimp virus. The baculoviruses have attracted much attention in recent years largely because some microbiologists and entomologists consider these viruses to be

Comparative sensitivity of six scleractinian corals to temperature and solar radiation.

Scleractinian corals were exposed to 6 combinations of temperature and solar radiation to evaluate effects on coral bleaching, survival, and tissue surface area changes during and after exposure. A recirculating coral exposure system was coupled to a solar simulator to allow laboratory testing of 6 species of Caribbean corals (Diploria clivosa, Montastraea faveolata, Porites divaricata, Stephanocoenia intersepta, Siderastrea radians, and Siderastrea siderea). Significant bleaching occurred in all of the corals exposed to high irradiance except S. siderea. Elevated light levels resulted in a decrease in photochemical efficiency for all species during the exposure period, with S. siderea showing the smallest decrease. The most prominent reductions in photochemical efficiency occurred in M. faveolata and S. intersepta, and these species exhibited extensive tissue loss and the highest mortality. In contrast to high irradiance, high temperatures significantly decreased photochemical efficiency for only D. clivosa and did not lead to severe tissue loss for this species. These results demonstrate species-specific responses to solar radiation and temperatures, with M. faveolata and S. intersepta being the most susceptible to bleaching due to high irradiance.