Differential expression of two rainbow trout CYP1A genes was measured in vivo and in vitro in response to treatment with the model CYP1A inducers beta-naphthoflavone (BNF), 3-methylcholanthrene (3-MC), isosafrole (ISF), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, only in vitro). Originally described by Berndtson and Chen (Arch. Biochem. Biophys. 310, 187-195, 1994) as CYP1A1 and CYP1A2, these genes were renamed CYP1A3 and CYP1A1, respectively, by the P450 nomenclature committee. A significant, differential, inducer-dependent induction of the two CYP1A mRNAs, as measured by RNase protection assay, was observed in vivo. CYP1A3 and CYP1A1 mRNA levels in liver were significantly induced 50- and 18-fold, respectively, following ip injection with BNF. Conversely, CYP1A3 and CYP1A1 mRNA levels were significantly induced 5- and 66-fold, respectively, following ip injection with 3-MC. Isosafrole had no significant effect on in vivo induction of CYP1A mRNA levels. In primary cultures of hepatocytes, BNF, 3-MC, ISF, as well as TCDD all significantly induced CYP1A3 and CYP1A1 mRNA levels compared to controls. The differential induction of the two CYP1A genes was not as evident in vitro as in vivo. In addition, reanalysis and sequence comparison of the these two trout CYP1A genes with the first trout CYP1A cDNA described by Heilmann et al. (DNA 7, 379-387, 1988) indicate that the Heilmann cDNA is a hybrid of the two trout genes. The 5' portion of the cDNA sequence (212 bp) was determined by sequencing of a genomic clone and is 100% identical to the trout CYP1A3 gene. The majority of the cDNA sequence (2377 bp), however, was sequenced from a partial cDNA clone and is 99.2% identical to trout CYP1A1. Although the nomenclature of these two trout CYP1A genes is undergoing revision, these results demonstrate a differential, inducer-dependent response to model mammalian CYP1A inducers.

The toxicity of two chlorinated hydrocarbons, carbon tetrachloride (CCl4) and chloroform (CHCl3) was studied in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. The aim was to study the cytotoxicity of these two chemicals in vitro in fish hepatocytes and to evaluate the sensitivity of the model system in comparison to mammalian systems. Both chemicals showed a steep dose response in this system with CCl4 being five times more toxic than CHCl3 measured as LDH release. Glutathione levels were rapidly depleted in the presence of CCl4 and CHCl3. There was a difference, however, in the pattern of GSH depletion. CCl4-induced GSH depletion followed the release of LDH, while CHCl3 caused a significant decrease in the GSH content prior to LDH release. The cytochrome P450 inhibitor, SKF-525A (12 μM) decreased the toxicity of 5 mM CCl4 and of doses ranging from 10 to 25 mM CHCl3. The addition of the antioxidant DPPD (20 μM) decreased the toxicity of both CCl4 and CHCl3. The protection was more pronounced with CHCl3 than with CCl4. When the time course of GSH depletion with EC50 concentrations of each chemical was studied, DPPD effectively protected the cells from CHCl3-induced GSH depletion, while no significant maintenance of GSH was seen in cells treated with DPPD and CCl4. The relationship between DNA single strandbreaks (SSB) and cytotoxicity may provide some insight into potential carcinogenicity of non-genotoxic chemicals. In cells treated with CHCl3, DNA SSB were seen only concommitant with high toxicity. With CCl4, however, DNA SSB were seen in the absence of LDH release at 2.5 mM. This study indicates that the toxicity of CCl4 and CHCl3 in trout hepatocytes is due to metabolism and the formation of free radicals at doses causing low toxicity and to a combination of metabolism dependency and solvent effect at doses causing high toxicity. This study also indicates that there are differences in the mechanisms of toxicity of CHCl3 and CCl4 in rainbow trout hepatocytes. Furthermore, the model system based on primary cultures of rainbow trout hepatocytes is a sensitive tool in toxicological studies and is comparable to model systems based on mammalian hepatocytes.

Human liver tissue slices, homogenates, organ cultures, subcellular fractions and primary hepatocyte cultures represent valuable tools for analysis of metabolism, toxicity and efficacy of xenobiotics. However, the limited access to human tissue and the high test variability with different tissue isolates underline the significance of having standardized and defined human liver cell models. This report describes the establishment of replicative cultures of human liver epithelial cells and their immortalization in serum-free medium. Highly efficient immortalization is achieved by the infection of primary liver cells with a retroviral recombinant carrying the simian virus 40 (SV40) large T antigen gene. These cultures have an indefinite life-span, are non-tumorigenic and express differentiation markers and metabolic functions of normal primary hepatocytes. Therefore, these cells are useful for various pharmaco-toxicological applications.

The uptake of the bile acids cholate (CHA) and taurocholate (TCHA) was studied in isolated hepatocytes from rainbow trout (Oncorhynchus mykiss). Both CHA and TCHA were taken up in a concentration- and temperature-dependent manner with optimum temperature at 15 degrees C and a strikingly efficient uptake even at low temperatures (0-5 degrees C). The total uptake was a combination of a saturable [Michaelis-Menten constant (Km) for CHA, 20 microM; Km for TCHA, 19 microM] and a nonsaturable component. The maximal uptake rate of the saturable component was 416 and 805 pmol.mg protein-1.min-1 for CHA and TCHA, respectively. The uptake of both bile acids was shown to be energy dependent, since it was inhibited by the metabolic inhibitors antimycin A, oligomycin and carbonyl cyanide m-chlorophenylhydrazone. The uptake was clearly Na+ independent, since isosmotic replacement of extracellular Na+ by Li+, choline, or K+ did not inhibit the uptake. Furthermore, it seemed to be independent of the presence of extracellular Cl-, since it was not inhibited by replacement of Cl- with sodium gluconate. On the whole, our results show that the hepatocellular uptake of bile acids in rainbow trout is mediated by a Na(+)-independent carrier system, with characteristics resembling the corresponding transport component in mammalian hepatocytes, but with high efficiency even at low temperatures.

A method is described for establishing continuous cell lines of liver epithelial cells by transformation of cultured hepatocytes isolated by collagenase/dispase of adult human liver tissue. Between 2 to 5×105 hepatocytes are inoculated into a 60 mm culture dish. The cells are incubated in a serum-free medium. Once the cells begin to divide, they are transformed by transfection with a plasmid containing SV40 early region genes.

It is thought that the extensive industrial use of arsenic, gallium and indium, which have applications as the materials for III-V semiconductors, will increase human exposure to these compounds in the near future. We have undertaken the development of new biological indicators for assessing exposure to these elements. Element-specific alterations in protein synthesis patterns were expected to occur following exposure to arsenic compounds. We examined alterations in protein synthesis in primary cultures of rat kidney proximal tubule epithelial cells by sodium arsenite, gallium chloride and indium chloride, utilizing two-dimensional gel electrophoresis. After incubation with the chemicals for 20 h, newly synthesized proteins were labeled with [S-35]methionine. A protein with a molecular weight (M(r)) of 30 000 was markedly induced on exposure to 10 muM arsenite or 300 muM gallium chloride, and synthesis of proteins with M(r) values of 85 000, 71 000, 65 000, 51 000, 38 000 and 28 000 were also increased by exposure to arsenite and gallium chloride. No significant changes were observed upon exposure to indium. Some of these increased proteins could be heat-shock proteins.

Studies were conducted to address glucuronidation of 3-trifluoromethyl-4-nitrophenol (TFM) in sea lampreys Petromyzon marinus, channel catfish Ictalurus punctatus, rainbow trout Oncorhynchus mykiss, and bluegills Lepomis macrochirus. The ability of these species to biotransform TFM was investigated by determining the kinetics of UDP-glucuronyltransferase (UDPGT; also known as glucuronosyltransferase) in vitro from hepatic microsomal preparations. Maximal velocity (V(max) nmol/min.mg) for UDPGT activity toward TFM was significantly greater (P < 0.05) in bluegills (1.52), rainbow trout (1.82), and channel catfish (1.46) than in sea lampreys (0.68). Binding affinities (K(m)) of UDPGT for TFM varied significantly among species in the following order: bluegill (58 muM) > rainbow trout (97 muM) > channel catfish (172 muM) > sea lamprey (261 muM). Analysis of V(max)/K(m) ratios, a measure of enzyme efficiency (nmol/min.mg.muM TFM), indicated that the efficiency of UDPGT activities in all species examined was influenced more by binding affinity (K(m)) than by the V(max) of the reaction. These calculated ratios were progressively lower for species that were previously reported to be more sensitive to aqueous TFM (i.e., to have lower LC50s, TFM concentrations lethal to half the test fish). Sea lampreys appear to have relatively low UDPGT activity and binding affinity for phenolic substrates. This, in part, may account for the sensitivity of the sea lamprey to aqueous TFM.

Environmental exposure of rainbow trout, Oncorhynchus mykiss, to tetrachloroethylene was associated with massive mortalities (about 50%) with acute hepatic and renal toxicity. The liver changes consisted of diffuse hepatocellular necrosis. The kidney changes were manifested as degeneration and necrosis of epithelial cells lining the first segment of the proximal tubule. Intensely basophilic newly developing nephrons were observed in surviving fish sampled 10 days to 3 wk following exposure. The presence of developing nephrons in adult fish may serve as a biomarker for nephrotoxic pollutant exposure.

Effects of TFM (3-trifluoromethyl-4-nitrophenol) were observed in bullfrog (Rana catesbeiana) larvae and adults in vivo by aqueous and intraperitoneal (i.p.) exposures. UDP-glucuronyltransferase (UDPGT) kinetics were examined in vitro from larval and adult hepatic microsomes. The effect of TFM on bullfrog egg development was also examined.Bullfrog larvae and adults showed similar sensitivities to TFM by i.p. exposure (LD50s = 11.62 and 15.35 mg/kg, respectively), however, larvae were more sensitive than adults in aqueous exposures (LC50s = 0.95 and 12.99 mg/l, respectively). Bullfrog egg development was arrested at the blastula stage at greater-than-or-equal-to 1.0 mg/l TFM. No microscopic lesions were observed in tissues from TFM-exposed larvae.TFM-UDPGT kinetics in bullfrog larvae and adults differed significantly (P < 0.05). V(max) for TFM-UDPGT in bullfrog larvae and adults was calculated to be 1.05 and 2.45 nmol/min . mg, respectively; the apparent K(m) for bullfrog larvae and adults was 38 and 64 muM, respectively. However, the enzyme V(max)/K(m) ratio between the two life stages was not significantly different. Based on data from this and other studies, greater sensitivity to aqueous TFM in gill-respiring larvae (compared with adults) does not primarily appear to be a function of differences in UDPGT-mediated metabolism, but rather may reflect differences in other processes such as uptake.

A survey was conducted to evaluate the effect of tadpole infestations on warmwater fish pond culture. The survey was sent to 174 pond culturists in Florida and 30 in Arkansas with a 21% overall response rate. Most respondents believed: (1) tadpole infestation was a serious problem, primarily due to competition between tadpoles and fish for food; (2) tadpoles caused harvest problems and impaired post-harvest sorting and grading; and (3) their tadpole control efforts were less-than-or-equal-to 30% effective. It was estimated that an average of 117 worker-hours of labor per surveyed facility were expended annually to contend with tadpole-related problems. Financial losses due to tadpoles were $1,000-8,000 per year for most farmers surveyed (54%) and $10,000-30,000 per year for others (36%). Annual losses of revenue from ornamental fish production in Florida (based on survey) and baitfish production in Arkansas (U.S. Fish and Wildlife Service) due to tadpole infestations were estimated at $1.78 million and $6.2 million, respectively.

New nephrons developed in goldfish, Carassius auratus, several weeks following hexachlorobutadieneinduced (HCBD) nephrotoxicity. Basophilic clusters of presumptive nephrogenic cells incorporated 5-bromo, 2'deoxyuridine (BrdU) one week after HCBD injection, indicating initiation of DNA synthesis. These clusters, like renal vesicles in the developing kidney, elongated, fused with collecting ducts and developed into immature nephrons during the next 2 weeks. Stereologic quantification showed the volume percent of the kidney occupied by the developing nephrons was greater in HCBD-treated fish 2, 3, 4, and 10 weeks after injection than in the control fish. The presence of large numbers of developing nephrons may provide a marker for renal injury in fish from contaminated waterways.

Regeneration of the epithelium following injury is a vital process in many organs of both mammals and fish.An undcrstanding of this response is essential when evaluating the impact of xenobiotics on tissues such as the kidney.The present studles demonstrate regeneration of the goldfish Carassius auratus renal proximal tubular ep~thelium follo\vlng toxicant induced injury (hexachlorobutadiene, HCBD 500 mg k g ' ) .HCRD-induced vacuolation and necrosis of the proximal tubular epithelium were observed within 24 h and continued for 10 wk following the s ~n g l e intraperitoneal injection.A flattened, basophilic rcgeneratlng ep~thelium was observed along the intact basement membrane 1 wk post HC'BD-treatment.In the follow~ng weeks, as the regenerating epithelial cells differentiated, they developed vactioles and became necrotic.In addition. 2wk after exposure, newly formed nephrons developed from basophilic cell clusters adjacent to collecting ducts.Ten weeks following the slngle HCBD injection, flbrotic glomeruli and large dilated proximal segments with altered morphology were present.Thc rate of cell turnover in normal and injured kidneys was determined using bromodeoxyuridine (RrdlJ) pulse and repetitive labeling.BrdU labeling indices were elevated in the regenerating tubules, distal segments, collecting ducts, basophilic cell clusters and developing nephrons of HCBD-treated fish.Intense labeling occurred in the collecting ducts 4 d after HCRD-administration. BrdU incorporated into cells on Day 4 was noted in the regenerating epithelia] cells on Day 7, indicating that thesc cells are probably derived from cells in the lower portions ot the nephron.Labeling indices remained elevated in proximal segments 10 wk following toxicant adn~inistriltion.

Two cases of retinoblastoma in fish are described. The neoplasms occurred in a porkfish (Anisotremus virginicus, Linnaeus) and in a brown bullhead (Ictalurus nebulosus, Lesueur).

Journal of Fish DiseasesVolume 12, Issue 1 p. 57-62 Fusarium solani isolated from newborn bonnethead sharks, Sphyrna tiburo (L.) A. G. MUHVICH, Corresponding Author A. G. MUHVICH Department of Pathology, Environmental Pathobiology and Aquatic Toxicology Laboratory, University of Maryland School of Medicine, Baltimore, USADepartment of Pathology, School of Medicine, University of Maryland, Room 7-11 MSTF, 10 S. Pine Street, Baltimore, Maryland 21201, USA.Search for more papers by this authorR. REIMSCHUESSEL, R. REIMSCHUESSEL Department of Pathology, Environmental Pathobiology and Aquatic Toxicology Laboratory, University of Maryland School of Medicine, Baltimore, USASearch for more papers by this authorM. M. LIPSKY, M. M. LIPSKY Department of Pathology, Environmental Pathobiology and Aquatic Toxicology Laboratory, University of Maryland School of Medicine, Baltimore, USASearch for more papers by this authorR. O. BENNETT, R. O. BENNETT Department of Pathology, Environmental Pathobiology and Aquatic Toxicology Laboratory, University of Maryland School of Medicine, Baltimore, USASearch for more papers by this author A. G. MUHVICH, Corresponding Author A. G. MUHVICH Department of Pathology, Environmental Pathobiology and Aquatic Toxicology Laboratory, University of Maryland School of Medicine, Baltimore, USADepartment of Pathology, School of Medicine, University of Maryland, Room 7-11 MSTF, 10 S. Pine Street, Baltimore, Maryland 21201, USA.Search for more papers by this authorR. REIMSCHUESSEL, R. REIMSCHUESSEL Department of Pathology, Environmental Pathobiology and Aquatic Toxicology Laboratory, University of Maryland School of Medicine, Baltimore, USASearch for more papers by this authorM. M. LIPSKY, M. M. LIPSKY Department of Pathology, Environmental Pathobiology and Aquatic Toxicology Laboratory, University of Maryland School of Medicine, Baltimore, USASearch for more papers by this authorR. O. BENNETT, R. O. BENNETT Department of Pathology, Environmental Pathobiology and Aquatic Toxicology Laboratory, University of Maryland School of Medicine, Baltimore, USASearch for more papers by this author First published: January 1989 https://doi.org/10.1111/j.1365-2761.1989.tb01291.xCitations: 19AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume12, Issue1January 1989Pages 57-62 RelatedInformation

White perch (Morone americana) sampled from 15 estuaries of the Chesapeake Bay contained a variety of hyperplastic and neoplastic alterations in their livers. The lesions were derived from bile ductular epithelium and/or hepatocytes. The biliary lesions consisted of hyperplasias and adenomas. The hepatocellular lesions consisted of focal populations of altered cells and neoplasms, of clear-cell and basophilic morphologic appearance. The hepatocellular lesions were conspicuous in the absence of the decreased amount of copper accumulation. In this respect, the cells comprising these lesions are reminiscent of cells from hepatocellular neoplasia in mice and rats resistant to accumulation of iron.

Journal of Fish DiseasesVolume 10, Issue 4 p. 319-322 Eosinophilic granular cell response to a microsporidian infection in a sergeant major fish, Abudefduf saxatilis (L.) R. REIMSCHUESSEL, Corresponding Author R. REIMSCHUESSEL Department of Pathology, University of Maryland at Baltimore, Maryland, USADr R. Reimschuessel, Department of Pathology, University of Maryland at Baltimore, Baltimore, Maryland 21201, USA.Search for more papers by this authorR. O. BENNETT, R. O. BENNETT Department of Pathology, University of Maryland at Baltimore, Maryland, USASearch for more papers by this authorE. B. MAY, E. B. MAY Department of Pathology, University of Maryland at Baltimore, Maryland, USASearch for more papers by this authorM. M. LIPSKY, M. M. LIPSKY Department of Pathology, University of Maryland at Baltimore, Maryland, USASearch for more papers by this author R. REIMSCHUESSEL, Corresponding Author R. REIMSCHUESSEL Department of Pathology, University of Maryland at Baltimore, Maryland, USADr R. Reimschuessel, Department of Pathology, University of Maryland at Baltimore, Baltimore, Maryland 21201, USA.Search for more papers by this authorR. O. BENNETT, R. O. BENNETT Department of Pathology, University of Maryland at Baltimore, Maryland, USASearch for more papers by this authorE. B. MAY, E. B. MAY Department of Pathology, University of Maryland at Baltimore, Maryland, USASearch for more papers by this authorM. M. LIPSKY, M. M. LIPSKY Department of Pathology, University of Maryland at Baltimore, Maryland, USASearch for more papers by this author First published: July 1987 https://doi.org/10.1111/j.1365-2761.1987.tb01077.xCitations: 30AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume10, Issue4July 1987Pages 319-322 RelatedInformation

Non-inflation of the swim bladder is a common anomaly associated with cultured larval striped bass Morone saxatdjs.Histological examination of tank-cultured larvae revealed 30 1.0 had noninflated swim bladders.Two distinct morphological types of abnormal swim bladders were observed: glandular with marked fibrosis, and degenerative with severe fibrosis.Histological observations suggest that non-inflation of the swim bladder is irreversible.