United States Environmental Protection Agency Environmental Research Laboratory Duluth MN 55804 Research and Development EPA-600/S3-83-076 Nov. 1983 Project Summary Detection of Mutagenic/ Carcinogenic Alteration in Fish David E. Hinton, James E. Klaunig, Michael M. Lipsky, Rhona M. Jack, and Benjamin F. Trump The feasibility of using fish as bioassay organisms to detect mutagenic/carcino- genic substances in the aquatic environ- ment was tested in species not common- ly employed in chemical carcinogenesis bioassay. Microsomal fractions from livers of channel catfish (Ictalurus punctatus). fathead minnow (Pimephales promelas). bluegill sunfish (Lepomis macrochirus), brown bullhead (Ictalurus nebulosus). and mummichogs (Fundulus heteroclitus) were used. Data obtained from these species were compared to those obtained in rainbow trout (Salmo gairdneri) - a more commonly employed fish - and mammalian species. The presence and relative amount/activities of microsomal protein, cytochromes P- 450 and bs, NADPH cytochrome C reductase, aminopyrine demethylase, and aryl hydrocarbon hydroxylase were determined. The effects, both morpholog- ic and biochemical, in exposure to the known MFOS inducing agents, PCBs, benzo(a)pyrene, and 3-methylcholanth- rene were studied. Exposure caused induction of enzymes and proliferation of endoplasmic reticulum membranes of hepatocytes. High pressure liquid chromatographic analysis of benzo(a)py- rene (BP) metabolism in catfish and trout were performed. Both species produced the following metabolites of BP: 9,10-diol BP; 4,5-diol BP; 7,8-diol BP. 9-OH BP; 3-OH BP; and quinones. Catfish postmitochondrial supernatant converted BP and 2-acetylaminofluorene (AAF) into mutagenic intermediates in a microbial mutagen system. Catfish liver cells were isolated and maintained with high viability (98%) for 10 days. When incubated with H-BP, these cells showed preferential accumulation of label over nuclei. Subsequent liquid scintillation analysis of cell fractions obtained by cesium chloride centrifuga- tion revealed radioactivity in DNA fractions. Foci of hepatocellular altera- tion, hyperptastic areas and bile ductular hyperplasia were seen in channel catfish chronically exposed to the chemical carcinogen AAF. These data indicate the suitability of conducting further studies on this ubiquitous species designed to determine dose- response characteristics to various chemical carcinogens. On the basis of microsomal metabolism and cellular response, it appears feasible to use fish tissue to test for mutagenic/carcinogenic compounds in the aquatic environment and to develop bioassay methodology for testing possible carcinogenic proper- ties of new chemical formulations prior to their introduction into the aquatic environment. This Project Summary was developed by EPA's Environmental Research Laboratory, Duluth, MN. to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report ordering information at back). Introduction A majority of human cancers are due to chemical carcinogens in the environ- ment. Since the oceans ultimately be- come the reservoir for every pollutant en- tering the biosphere, the importance of the aquatic environment in considera- tions of carcinogenic effects of chemical pollutants cannot be overly emphasized. ------- Correlated Biochemical and Morphologic Studies of Effects of Xenobiotics on Fish Liver To characterize the response of certain microsomal components in channel catfish, we administered a commercial PCB mixture (Aroclor 1254*) as seven daily intraperitoneal injections of 50 mg/kg b.w. Via correlated biochemical studies on hepatic microsomal mixed function oxidative system (MFCS)enzymes and ultrastructural studies of hepatocytes we analyzed acute and subacute responses. A similar approach was used with rainbow trout exposed to 3-MC (50 mg/kg i.p. x 4 days). Our results on hepatic MFOS components of the channel catfish and rainbow trout and their response to Aroclor 1254 and 3-MC exposure are summarized in the full re- port. Acute exposure (7 days) resulted in a moderate increase in some MFOS com- ponents. Cytochrome P-450 increased approximately 33% in channel catfish. Specific activity of NADPH-cyt-c-reductase was increased to 1.6 times control values. The greatest change was observed in amount of cytochrome b5, which increased 2.5-fold. Aminopyrine deme- thylase activity, microsomal protein and liver to body weight ratios did not change after acute exposure. In contrast, rat MFOS components were markedly increased after an identical concentration and duration of exposure. Subacute (21 days) of PCB treatment resulted in a large increase in catfish MFOS components with respect to controls and acutely exposed fish. Amounts of cytochromes P-450 and bs increased three and two times over controls respectively. NADPH-cyt-c- reductase was elevated to a level similar to that seen after the acute exposure. Aminopyrine demethylase, unchanged by acute treatment, was increased three- fold over controls after subacute treatment. Again, microsomal protein and liver to body weight ratios were unchanged. Thus, the response of the catfish MFOS to acute PCB treatment is a moderate induction, much less than is seen in the rat. Subacute exposure resulted in a greater degree of induction. When compared to control morphology, changes were also seen in hepatocyte ultrastructure of channel catfish after acute PCB exposure. Dilated cisternae and meandering tracts of endoplasmic reticulum (ER) were seen. Minimal •Mention of trade names or commercial products does not constitute endorsement or recommendation for use. increases in agranular ER profiles were noted. These appeared as focal, discrete aggregations. Increased lipid droplets also characterized hepatocytes of acutely exposed channel catfish. Subacute exposure of catfish resulted in alterations in agranular ER not seen in acutely exposed fish liver. Tubular and vesicular profiles, nearly absent in control catfish hepatocytes, were increased in number and often accumulated in large aggregates. In addition, parallel stacks of agranular ER in continuity with granular ER were observed. Often these patterns contributed to large whorls of ER membranes in affected cells. Biochemical levels of induction follow- ing acute and subacute Aroclor 1254 exposure in the catfish correlated well with ER content and morphology. Higher levels of MFOS induction were accompanied by apparent increases in agranular ER content and variations of ER morphology. Acute 3-MC exposure caused significant induction of rainbow trout MFOS com- ponents. Cytochrome P-450 increased to over three times control amounts. NADPH-cyt-c-reductase activity was elevated two times over controls. As in the catfish, neither microsomal protein nor liver to body weight ratios of trout were altered by 3-MC treatment. Morphologically, acute MFOS induction correlated with apparent increases in trout liver endoplasmic reticulum. Ultra- structural alterations caused by acute PCB and 3-MC exposure involved granular ER content and morphology, although focal increases in agranular membranes were noted. The major response after 21 days of exposure to Aroclor 1254 was extensive proliferation of ER. ARYL Hydrocarbon (Benzo-a-Pyrene) Hydroxylase (AHH) in Channel Catfish Liver Polycyclic aromatic hydrocarbons (PAH), an important class of environmen- tal pollutants which cause tumors in experimental animals including fish and have been implicated in human carcino- genesis, are activated by the NADPH- dependent MFOS. That portion of the MFOS responsible for this metabolic activation is the AHH system. AHH from livers of control fish and from livers of fish exposed to 3-MC or to Aroclor 1254 was characterized as to pH, temperature, and protein concentration optima. Linearity of reaction over time was established. With respect to temperature, an optimum of 30°C was found for control catfish liver microsomes. This corresponds to the temperature optima reported for rainbow trout, Finnish lake trout, and various marine species. A shift to a higher temperature range (30°C - 40°C)followed treatment with 3-MC or Aroclor 1254 in the catfish. The induced system seems to have quite a broad temperature range with no sharp drop at higher temperatures such as that found in the trout. The effect of in vitro addition of benzoflavone (BF) on AHH was studied in control, Aroclor 1254 and 3-MC-pretreated fish. Inhibition of activity was seen in 3- MC-pretreated samples at all ranges of BF tested (Figure 1). Inhibition ranged from 12% at .001 mM BF to 76% at 1 mM BF. PCB treatment left AHH less sensitive to BF inhibition at most concentrations than did 3-MC treatment. In control samples, .001 mM BF had no effect on AHH levels; however, at concentrations of .01 mM, up to 200% enhancement of activity was seen. Enhancement decreased with increasing BF concentrations, until at 1 mM BF, 22% inhibition was seen. No enchancement was seen in samples from either treated group at all concentrations tested. Studies thus far have demonstrated that two forms of AHH can be distinguished, one in control fish liver microsomes and the other in microsomes from 3-MC or PCB-pretreated fish. The enzymes are distinguished by the differential effect of BF on AHH. Both 3-MC and Aroclor 1254 treatment in vivo cause the behavior of the AHH enzyme to change. Characteris- tically, the induced enzyme is similar to the control enzyme with respect to pH optimum but differs in temperature optimum and BF sensitivity (at the same concentration). Benzo-a-pyrene, administered as a single i.p. injection of 100 mg/kg b.w. in corn oil, induced its own in vitro metabo- lism within 48 hr. in livers of three of four channel catfish (Figure 2-A). Differences in response of exposed individuals was apparent with AHH values ranging from 120 to 350% of control values. Taken as a group, the mean value for exposed fish was 1.90± 1.41 compared to 0.50 ±0.04 for controls (p<.025). When BP was administered as six daily i.p. doses of 25 mg/kg (Figure 2-B), induction of AHH was seen and individual variation among fish was reduced. The mean AHH value in exposed fish was 2.67 ± 0.97. This is, on the average, a 513% increase over the control mean of 0.52 ±0.01. These values were significant- ly different (p<0.01). When BPwas given as a single gastric intubation, induction of AHH occurred in 80% of treated fish (Figure 2-C). Interindividual variation was apparent. The mean for exposed fish ------- O Microsomes from control liver • Microsomes from 3-MC-treated livers A Microsomes from PCB-pretreated livers 200 - /50 - c o CJ 700 .0007 .007 .07 .7 mM 7,8 Benzoflavone Figure 1. Effect of 7,8 benzoflavone concentration on in vitro A HH activity. Control equals the same sample without BF. Data illustrated are from a single representative experiment. Triplicate assays were performed with variation between replicates less than 10%. a 5-° o 1 40 AHH WBP/20 m \> Co b b o nmole 3- b 0 4 r+f & n rf 1 T 1 © 3 f3 rf rt •4- -i- © V 5 T + •h * Figure 2. Effects of BP on hepatic AHH activity in channel catfish. Slashed bars represent the average of control fish ±S.D. between animals. Numbers over the bars represent the number of fish in the experiment. Clear bars represent individual treated fish ± S.D. for triplicate assays on each animal. Treatment: (A) one i.p. injection of 100 mg < BP/kg b. w.. sacrificed after 48 hours; (B) six daily injections of 25 mg BP/kg b. w.. sacrificed after 24 hrs; (C) one gastric intubation of 100 mg BP/kg b. w., sacrificed after 14 days. (1.73 ± 0.95) was 208% of control values and was significantly different (p <.025). 3-MC Induction of Channel Catfish AHH The effects of five daily i.p. injections of 20 mg 3-MC/kg body weight upon channel catfish liver AHH are shown (Figure 3). All fish exposed inthis manner exhibited induction with the individual response ranging from 10 to 25 times control values. The experimental mean of 4.20 ±1.73 was statistically different from the control mean of 0.27 + 0.12 (p <.01). When constant dose (25 mg 3-MC/kg b.w.) was maintained for 4 days and fish were then killed daily up to one week after exposure, it was possible to deter- mine the duration of effect. These data are presented in the full report. AHH was increased dramatically by one day after the cessation of exposure. At day 4 following cessation of exposure, the AHH activity was 24 times the control mean, and by day 7 after treatment, AHH was 19-fold the control mean. The deviation between individual fish decreased markedly as the time of exposure length- ened. The highest values for NADPH- cytochrome-c-reductase and cytochrome P-450 were seen at day 4. At this time, the mean cytochrome P-450 estimation was 2.2 times over the control value. By day 7, cytochrome P-450 values were only slightly higher than controls. Microsomal protein concentration in catfish appeared to increase in the fish killed 4 and 7 days after treatment ended; however, no clear cut correlation between induction and protein concentration was seen. The mechanism for induction in fish has not yet been explained. New enzyme synthesis or enzyme modification is one possibility; however, changes in the membrane composition or conforma- tion should also be considered. Metabolism of Benzo-a-Pyrene by Microsomal Fraction of Fish Liver Metabolites formed by the reaction of fish liver microsomal fractions with 80- 100 nmoles of 3H-BP (specific activity 150-200 fiC\/umo\e were analyzed by high pressure liquid chromatography (HPLC). Each ml of assay volume included 0.1 M phosphate buffer, pH 7.0-7.2, 3.0 mM MgCI, O.1 mM EDTA, 0.4 mM NADP, 10 mM glucose-6-phosphate, 15-20/uCi BP, and 1.5 Units of glucose-6-phosphate dehydrogenase as the NADPH generating system. BF (0.1 mM) was added in 20/ul of acetone in some experiments. Protein ------- 6.0 .c r ?! •S o I 4.0 2.0 Figure 3. Effect of 3-MC on hepatic AHH activity in catfish. Five fish were injected on five consecutive days with 20 mg 3-MC/kg b. w.. and sacrificed after 24 hrs. The slashed bar represents the mean of three control fish ± 1 S.D. between animals. Clear bars represent individual treated fish ± 1 S.D. for triplicate assays. concentration was maintained at 0.1 mg per assay. After incubation in a shaking water bath at 30°C for 20 minutes, the reaction was stopped by the addition of 1 ml acetone. The contents of 10 assay tubes were combined, and metabolites were extracted three times with ethyl acetate. Radioactivity remaining in the aqueous phase was less than 0.1 % of the total. The ethyl acetate extracts were pooled, flash evaporated, and the meta- bolites were resuspended in 0.1 ml of glass distilled methanol. Metabolite separation was performed using a Varian high pressure liquid chromatograph with a 25 cm Whatman Partisil column (PXS10/250DS) of inside diameter, 4.6mm. Column temperature was ambient. For analysis, 10-20//I of the methanolic extract was injected into the HPLC. A methanol-water gradient was used to elute metabolites. Two solvents designated 'a' and 'b' were used. Solvent 'a' was 30:70 methanol: water, and solvent 'b' was 70:30 methanol water. Initially, concentration of 'b' in 'a' was 25%. This was increased linearly at 3%/min and at a flow rate of 1.2 ml/min to 100% 'b'. This reverse phase chroma- tography, proceeding from a more to less polar solvent, elutes polar components first. Approximately 200 fractions were collected (0.4 min/fraction) in order to elute all metabolites and unmetabolized parent BP which, due to its nonpolar nature, is eluted last. All samples were co-chromatographed with 14C-labeled BP metabolites from rat liver microsomes as an internal standard or with pure individual metabo- lites to validate identification of peaks (absorbance monitored at 254 nm). Since the rat liver BP metabolite pattern is well characterized, it was used as a biological standard. Peaks were also identified by retention times established with pure metabolite standards. Radioactivity of eluted fractions was determined by liquid scintillation counting. The 3H counts were computed to obtain specific activity for each metabolite, and expressed as pmoles BP metabolized/min/ mg protein. The 14C counts were used only for identification purposes. The sum of counts from fractions collected prior to thefinal BPpeakrepresentedtotal BPme- tabolized. Individual metabolites were ex- pressed as a percent of this total. In this manner, quantitative differences in acti- vity of various metabolites were com- pared in control and treated fish to estab- lish effect of in vivo exposure of xenobi- otics upon in vitro BP metabolism. Table 1 provides a summary of results in terms of percent metabolism of BP into the various metabolites. Three different control and 3-MC-pretreated catfish were used to obtain these data. The 3-MC- pretreated catfish showed an apparent increase in activity toward the formation of all metabolites. Control and 3-MC val- ues for 9,10-diol BP and 7,8-diol BP were significantly different by the T test (p<0.05). Of the total radioactivity added per assay, 3-MC-pretreated microsomes metabolized 12-28% (3.75-4.8 fjC\) and control microsomes metabolized 14-22% (3.0-5.6 A»Ci). The PCB-pretreated catfish also show an increase in 9,10-diol BPand 7,8-diol BP, although total metabolism of BP was only 3% (0.43 /uCi). 3-MC-pre- treated trout also showed increases in 9,10-diol BP and 7,8-diol BP over control. Control trout metabolized only 3% of the labeled BP (0.64 //Ci), while 3-MC-pre- treated trout metabolized 13% (2.78//CJ). The in vitro addition of 0.1 nM BF to microsomes from untreated catfish caused an increase inall metabolites over the straight control preparations, plus a 28.8% metabolism of BP (97.9 //Ci) corresponding to the enhancement of AHH discussed earlier. The presence of in vitro BF in 3-MC-pretreated microsomal 4 ------- Table 1. Summary of BP Metabolite Formation as % of Metabolism Metabolite Catfish Trout Control +BF 3-MC +BF PCB Control 3-MC BP-9,10-diol BP - 4,5-diol BP - 7.8-diol 9-OH - BP 3- OH - BP quinones total BP metabolized, % AHH nmoles/20 min/mg protein 1.97±15 3.77 ±.93 7.47±1.27 14.5±2.3 13.17±3.17 12.5 ±1.32 14-22 0.6 3.9 14.0 20.0 33.0 22.0 28.8 1.0 3.77±1.07 6.03±4.03 1.25±1.09 19.2 ±2.85 13.6 ±4.07 16.4 ±3.38 12-28 6.08 4.0 1.9 5.4 5.7 12.0 10 0.7 9.5 2.6 12.6 19.6 5.5 13.8 3 2.71 1.7 1.8 3.3 13.0 8.5 45.0 3 0.54 5.9 1.0 14.0 10.6 12.6 22.0 13 7.37 Data from three individual fish are given for control and 3-MC-pretreated catfish (50 mg/kg b. w. x 4 days). The concentration of BF when added was 0.1 nM. Data for trout represent "pooled" microsomal fractions from 4 control and 3-MC treated fish (50 mg/kg b. w.). preparations markedly inhibited formation of most metabolites and AHH activity. Total metabolism was only 10% (2.76 yuCi). A great deal of variation was seen in BP metabolism among fish; however, treated fish generally metabolized more BP. Some 9,10-diol BP and 7,8-diol BP formation may be at the expense of 4,5- diol BP, which decreased in 3-MC- pretreated trout, PCB-pretreated catfish, and one 3-MC-pretreated catfish. This study has demonstrated that in vivo exposure of catfish and trout to xenobiotics affects the in vitro metabolism of BP. BF reduced metabolism of BP significant- ly in samples from 3-MC-pretreated fish and enhanced metabolism in control samples. This correlates well with characterization in which AHH was enhanced in control samples by addition of 0.1 mM BF. AHH, however, was inhibited in microsomal fractions from 3- MC-pretreated fish after the in vitro addition of 0.1 mM BF. Since the formation of every metabolite is affected, the inhibition must take place at the oxidase level. Microbial Mutagen Assays These followed standard methods using Salmonella typhimurium tester stains TA-1535, TA-1537, TA-98, and TA-100. In order to incubate NADPH generating system, S-9 and test compound directly on petri dishes with bacterial tester strains, an incubation temperature of 37°C was used. In previous experiments with catfish liver AHH, 70% of the activity at temperature optimum (30°C) remained when assay temperature was maintained at 37°C. Following incubation, the number of revertant colonies was estab- lished by direct counting. For a biological control, S-9 from livers of rats previously exposed to Aroclor 1254 was used in companion trials with fish S-9. The results of the microbial mutagen assay using S-9 from fish with the four tester strains of Salmonella typhimurium and AAF, BP and MNNG are given (Table 2). AAF and BP, when incubated with S-9 and cofactors of the MFCS, were positive in all four tester strains. MNNG showed a variable effect (i.e., positive in TA-100 and TA-1535 but negative in TA-98 and minimal in TA-1537). The effect of fish protein (S-9 concentra- tion upon the number of revertant col- onies observed when concentration of test compound is kept constant (20 fjg/ plate) is shown (Figure 4). When AAF and BP were used, in- creases in S-9 concentration were asso- ciated with increases in numbers of re- vertant colonies. This response was near- ly linear between S-9 protein concentra- tions of 0.17 and 0.35 mg/plate but flat- tened at higher protein concentrations. By contrast, changes in fish S-9 protein did not affect the number of MNNG-in- duced revertants. This compound does not require microsomal activation to act as a mutagen in the Ames assay. The microbial mutagen assay results show that postmitochondrial supernatant (S-9) from channel catfish liver converted AAF and BP into mutation-causing metabolites. Culture of Isolated Fish Hepatocytes The liver is the major site of experimen- tally-induced teleost tumors, and as such, would provide an ideal subject for in vitro assay. The liver also possesses an active and inducible MFOS and has been shown to be sensitive to a broad spectrum of procarcinogens. The epithelial nature of the liver also qualifies it as a model for epithelial carcinogensis in general. For these reasons, the culture of isolated liver cells was attempted. Optimal methods for isolation of catfish liver cells were developed. Liver pieces were trypsinized at 25°C for variable periods of time. The results of this study are shown in Table 3. Eight hours trypsinization gave the greatest yield of viable liver cells as established via trypan blue staining and direct counting with a hemocytometer. The yield of viable cells was 3.25 x 106 per gram body weight (2.8 x106/g liver). An average viability (trypan blue exclusion) of 98% was obtained. Following isolation, liver cells were plated on to 90 mm2 culture dishes and allowed to stabilize for 24 hrs. Represen- tative plates were sampled for viability by phase contrast microscopy and trypan blue staining after 5 and 10 days. Cells were also sampled for electron microscopy. The ultrastructural characteristics of the isolated liver cells were indistinguish- Table 2. Induction of Mutations Using Salmonella typhimurium Strains and S-9 Fraction from Catfish Liver Tester Strain TA-98 TA-100 TA-1535 TA-1537 Compound* AAF BP MNNG AAF BP MNNG AAF BP MNNG AAF BP MNNG Revertants/ ug CMPD/mg Protein*, 72.7±21.5 155.1 ±85. 8 No response 110.7±36.1 112.0±68.0 43.2+8.3 86.0±38.0 68.5±22.3 56.6+6.8 41.8±14.8 32.9±13.3 1.3+0.4 *Compounds: AAF (2-acetylaminofluorene); BP (Benzo(aipyrene); MNNG (N-methyl-N'-nitro-N-nitrosoguanidine). fFive concentrations of S-9 protein (0.17 - 2.51 mg/plate) used per each of six concentrations (5-30 ug/'plate) test compound. Values represent the number of revertants per jjg of compound tested per mg protein ± S. D. sub- sequent to the subtraction of the number of spontaneous revertants (background level). ------- AAF = 2-acetylaminofluorene 20 fig/plate; BP = benzo(a)pyrene 20 fig/plate; MNNG = N-methyl-N'-nitro-N-nitrosoguanidine 20 fjg/plate. Table 3. Effect of Duration of Trypsinization on Catfish Liver Cell Yield s 5 8 •S> 6 o I 2 s tL Q: MNNG x- Figure 4. 0.17 0.34 0.84 Protein Concentration Effect offish S-9 protein concen- tration upon the number of revertant colonies (TA-1535). able from those of the intact liver. After 5 and 10 days of culture, increases in glycogen content were noted. Cells during these periods began to aggregate andjunctional complexes were frequently noted. These cultures were used to determine uptake and intracellular localization of 3H-BP. When cells were exposed to 1.25 fjg 3H-BP (32 //Ci/ml of medium) for 24 hrs. and autoradiograms prepared, grains were concentrated over nuclei (Table 4). Net nuclear grains (nuclear number less cytoplasm and background) averaged 10 (Table 4). Radioactivity of DNA isolated from liver cells following 24hrs incubation with 3H-BP averaged 490 dpm//yg. One limitation of studies restricted to subcellular fractions is the occurrence of artifacts inherent in preparation which may damage key enzymes and/or disrupt compartmentation of others. The use of intact cells provides a system more akin to that of the whole organ or tissue from which the cell type of interest has been taken. To date, the results have been most encouraging. High yields of viable cells have been routinely obtained. Initial experiments employing autoradiographic localization techniques after incubation of cultures with 3H-BP showed preferen- tial localization over liver cell nuclei. This finding, coupled with recovery of radioac- tivity in DMA fractions suggests interaction of BP with cellular components. Additional studies are needed to determine whether binding to cellular macromolecules has occurred and, if so, to elucidate the Duration hrs 2 4 8 12 Total Number of Cells Isolated x10e 36.8 98.3 243.5 7.3 % Viable 96 99 98 75 Total Number of Cells Per g bw x/06 0.43 1.31 3.25 0.08 Total Number of Viable Cells Per g bw x/06 0.47 1.30 3.18 0.06 Temperature of trypsinization was 25°C. Table 4. Localization and Quantification of 3H-BP in Subcellular Components of Cultured Primary Fish Liver Cells Experiment Number Autoradiographic Analysis* (grain counts over equal areas) Liquid Scintillation f (dpm/fjg DNA) Nuclei Cytoplasm Intercellular (space background) 1 2 3 33.4+14.7 30.8±11.8 37.8±12.8 18.4±6.3 19.5±7.1 15.2±5.4 6.2+1.7 6.4+1.2 5.8±1.4 480 380 630 *1xW6 cells exposed to 1.25 3H-BP (32 ud/ml of medium). Grain counts = mean ± S.D. of 100 nuclei, 100 randomly-selected cytoplasmic and 100 randomly-selected intercellular spaces of equivalent area/experiment. \5x1Oe cell as above. Each experiment was performed on a separate cellular isolate from individual channel catfish. nature of such binding. Since the metabolism of BP varies between species and between organs in the same species as well as with the type of preparation used (i.e., whole cells, microsomal fractions, or nuclear fractions), further studies on metabolism, mutagenesis and in vitro carcinogenesis using intact epithelial cells from fish are needed. Morphologic Findings in Chronic Carcinogen Exposures Chronic dietary exposure of channel catfish to AAF or FBPA for up to 14 months produced no grossly observable tumors. Since major emphasis on the morphologic alterations following carcin- ogen exposure was placed on liver, extensive analysis of this organ was performed. Control channel catfish liver of this study closely resembled earlier published descriptions. Exocrine pancreas was found in adventitia of portal veins. Cords of cells existing as a dual-plated muralium were seen. Some regions of hepatocyte cytoplasm were opaque while other areas were nearly transparent in H&E stained preparations. The latter corresponded to regions where glycogen was present. At later time periods (12 and 14 months), regions of control liver showed alterations in architectural pattern. In these areas hepatocyte margins were indistinct and areas were interpreted as necrotic. In addition, control liver at 14 months showed occasional foci of round cell accumulation. These were interpreted as inflammatory foci and were comprised primarily of mononuclear cells. The above were located in portal and in some instances midzonal regions of hepatic lobules. High resolution light microscopic analysis (HRLM) (toluidine blue-stained sections of Epon-embedded material) revealed cytologic properties of control hepatocytes. In these, dark staining material was arranged as a perinuclear cuff with extensions to cell periphery. The hepato- cytes contained one nucleus and were cuboidal to pyramidal in shape. After 9 months of exposure to AAF, focal sites of necrosis with vascular congestion were encountered in eight of nine fish. Fat vacuoles were present in centrolobularand midzonal regions of the hepatic lobules. In two-thirds of the animals, peribiliary fibrosis was seen. A common finding in all fish chronically exposed to carcinogens was the biliary epithelial response. This included cyto- plasmic vacuolization and pyknosis of nuclei in bile ductular and ductal epithe- lium. In addition, diffuse inflammation throughout the hepatic lobule was noted in one of nine fish after 9 months of exposure. After 12 months of exposure to AAF, one of 12 livers showed a hyperplastic focus in which hepatocytes contained. ------- increased eosinophilia and basophilia, and existed as trabeculae some 8-10cells in thickness. Mitotic figures were seen in some hepatocytes of the foci. Evidence of a chronic necrotizing process was seen in 7 of the 12 animals studied. Necrotic cells were seen throughout entire regions of the liver. In 3 of the 12 animals, necrotic areas were associated with localized vascular congestion. Two-thirds of the animals at this time showed evidence of peribiliary fibrosis and 7 of 12 animals showed diffuse inflammatory foci through- out liver lobes. Fatty change was encoun- tered in a single animal at this time. Nonspecific tubular epithelial changes were seen in some kidneys. All other organs were nonremarkable. Morphologic alteration occurred in livers of all 27 fish surviving 14 months exposure to AAF. Microscopic findings in other organs were nonremarkable. In general, hepatic architecture was maintained in all fish. Foci and areas of cellular alterations were encountered in 14 of 27 fish. These lesions were typically comprised of cells which showed increased acidophilia and basophilia. They were larger than those seen at 12 months. In addition, these clumps of cells showed numerous mitotic figures. No tumors were encountered in any of the fish studied at this time. Hepatocyte nuclei in foci of necrotic hepatocytes was observed. Two of the five fish showed small foci of inflammatory cells in liver parenchyma. Cytologic features of individual hepatocytes were identical to that described in control fish. Four fish were killed after 2 months of exposure to FBPA and were processed for HRLM. In these individuals, normal liver architectural pattern was seen. In two of the four animals, small areas were observed in which cells showed a disorganized pattern of dark staining material and it was difficult to distinguish between individual cells in these areas. These foci were interpreted as necrotic. No fat was observed. Bile ductular proliferation was not seen. Peribiliary fibrosis occurred in two of the four animals studied. All other features were identical to controls. After 3 months of exposure to the compound, the only alterations observed were small sites of necrosis similar to areas described above. Cytologic features of individual hepatocytes were identical to controls. After 6 months of exposure to FBPA, a normal liver architectural pattern was seen. In one of three treated fish, dark staining regions within cytoplasm were irregularlyarranged; however, the response was focal. No necrosis, fat, bile duct proliferation, •eribiliary fibrosis or inflammation was observed. Cytology of individual hepato- cytes revealed dispersal of dark staining material peripherally with central portion of cells containing light staining material. This pattern of cytoplasmic rearrangement is similar to that seen in hepatocytes in which smooth endoplasmic reticulum has proliferated. Livers from six treated and six control fish were studied by routine light microscopy after 9 months of exposure to FBPA. In general, liver architecture was well-maintained. How- ever, in two of the six fish, areas were seen in which sinusoids were not apparent. Cytoplasm of cells in these regions revealed a hyalinized appearance. Necrosis was not found at this time. Fat was seen as clear vacuoles within hepatocytes in one of the six animals. In addition, the same liver showed diffuse small foci of inflammatory cells. After 12 months of exposure to FBPA, five of six livers observed showed normal architec- tural pattern. In the other, the outer margins of the liver revealed numerous bulges and alternating constrictions resulting in a "scalloped" appearance. Necrosis was seen in two of the six livers examined at this time. Sites of necrosis and pyknosis were common. In three of the six livers, areas were seen in which cytoplasm of hepatocytes revealed in- creased opacity. In addition, these areas were sites of vascular congestion. Fat was observed in two of the six animals as clear vacuoles within hepatocytes of centrolobular and midzonal regions. Bile duct proliferation, not seen in animals exposed for shorter periods of time to FBPA, now appeared in four of the six animals. Bile stasis was indicated by expanded, perfectly rounded lumina of ductules and ducts and thin lining epithelial cells suggested increase intra- luminal pressure. Five of the six animals studied showed inflammation particularly pericholangitis. Numerous mitotic figures were seen in bile ductular epithelium. In livers of three fish, bile ductal epithelial hyperplasia was apparent. One of these had progressed to papillarly projections within the lumen. This configuration with numerous mitotic figures is consistent with a diagnosis of cholangioma. When compared to controls maintained for identical periods of time, livers of channel catfish exposed to FBPA for 14 months revealed changes. Both control and treated livers showed necrotic areas although these were by far greater in treated fish. Areas of necrosis in both groups were associated with fibrosis. Granulomas were seen in the liver of one control fish but not in treatedfish. Hepatic architecture was similar in both groups. However, cytoplasm of hepatocytes from treated fish contained more stamable area—often completely filling the cell. Controls apparently contained abundant glycogen which, by H&E, did not stain, making these cells nearly transparent. Cytologic features of hepatocytes from the 14-month FBPA group included abundant acidophilic inclusions which were frequently as large as nuclei. In basophilic regions of cytoplasm, a fine vacuolization was seen. Nuclei, generally one per cell, were lucent, rounded and had a single prominent nucleolus. Treated livers revealed oval cell prolifera- tion in 15 of 32 fish surviving the 14- month exposure. Peribiliary fibrosis, percholangitis and melanomacrophage centers were common. In 8 of 32 livers, areas were seen in which cytoplasm appeared hyalinized and gave a general basophilic hue with H&E. Nuclei in these regions were more opaque and generally uniform. However, some nuclei were enlarged, oval in shape and indented. Cells in these regions existed as con- tinuous sheets and it was difficult to visualize sinusoids. In one fish, the above described region was seen as a nodule which compressed adjacent hepatocytes. This led to a diagnosis of minimal deviation hepatocellular carcinoma. Mito- tic figures were seen in the above. In the interpretation of liver lesions, previous reports in various teleost species were reviewed. The response of the channel catfish liver to AAF and FBPA was encouraging. However, the latency period was long. In light of the amount of water required to maintain large numbers of this size fish and the problem of disposal of large amounts of contaminated water, use of smaller species such as small aquarium fishes may be advisable. Further study of carcinogenesis in the catfish could be extended to egg, embryo- larval exposures in which concentrated dosages of carcinogen are followed by rinsing and subsequent culture under routine conditions. These procedures may constitute a feasible alternative to the above-noted problems. Egg, embryo- larval exposures need to be extended to other species in an effort to determine an ubiquitous, sensitive, indicator organism for aquatic carcinogen bioassay. In light of the projected increasing national reliance on coal-based energy production, regional contributions of environmental PAH from high point source emission associated with coal combustion, coking and conversion processes require surveillance of environ- mental quality. The response of the channel catfish to BP and 3-MC coupled ------- with its ubiquitous occurrence in streams and lakes gives added importance to continued studies with this species including: varying routes of exposure, bioaccumulation, acute and chronic morphologic alterations and possible tumorigenesis. Such would help to determine the suitability of this species to serve as a sentinel organism for aquatic- borne PAH in fresh water habitats. Conclusions Based upon the data obtained from this study, the following conclusions have been made. • All fish species studied were found to contain a hepatic mixed-function oxidative system (MFOS) qualitatively similar to that of other teleosts and rodents. • Enzyme activity of the fish MFOS was increased by exposure to polychlorin- ated biphenyls and polycyclic aromatic hydrocarbons, benzo(a)pyrene (BP) and 3-methylcholanthrene (3-MC). • Increased enzyme activity correlated with proliferation of endoplasmic reticulum of hepatocytes. • Channel catfish and rainbow trout liver microsomal fractions metabolized BP into diols, hydroxy derivatives, and quinones. • Pretreatment of catfish and trout with 3-MC increased formation of 7,8- and 9,10-diols of BP. Since diol formation proceeds via epoxide formation, evidence indicates fish MFOS activated BP to a reactive carcinogen. • Catfish MFOS formed mutagenic intermediates of BP and AAF in a microbial mutagen system. • Isolated primary hepatocytes of chan- nel catfish can be maintained with high viability for 10 days and permit detailed analysis of cellular events in chemical carcinogenesis as weft" as direct interspecies comparison. The presence of foci and areas of hepatocellular alteration, nodular lesions and bile ductular hyperplasia suggest neoplastic responses to chronic carcinogen exposure in chan- nel catfish liver. It is feasible to use fish to test for the presence of mutagenic/carcinogenic substances in the aquatic environment and to determine whether new chem- ical formulations, proposed for wide- spread usage, would have mutagenic/ carcinogenic potential in aquatic species. The use of fish species with relatively large body size coupled with the chronic nature of laboratory exposures designed to demonstrate carcinogeni- city of compounds results in sizeable quantities of contaminated water which require decontamination prior to discharge. The volume of contamin- ated water can be diminished by use of closed system exposures but remains a problem to be considered in projects of this nature. The establishment of regional centers of excellence to provide a safe environment for testing of potentially carcinogenic substances under con- trolled conditions is strongly recom- mended. Such would provide opportun- ity for collaboration between govern- ment, industrial and university person- nel to establish necessary prerequisites for aquatic carcinogenesis bioassay. Defined nutritional requirements of these species used in long term assay are needed. D. E, Hinton is with West Virginia University. Morgantown, WV 26506; J. E. Klaunig is with the Medical College of Ohio. Toledo, OH 43699; M. M. Lipsky. R. M. Jack, and B. F. Trump are with the University of Maryland School of Medicine. Baltimore, MD 21201. Gary E. Glass is the EPA Project Officer (see below). The complete report, entitled "Detection of Mutagenic/Carcinogenic Alteration in Fish." (Order No. PB 83-253 559; Cost: $ 13.00, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Environmental Research Laboratory U.S. Environmental Protection Agency 6201 Congdon Blvd. Duluth, MN. 55804 United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Official Business Penalty for Private Use $300 ------- |