Dietary cadmium and benzo(a)pyrene increased intestinal metallothionein expression in the fish Fundulus heteroclitus.

Fish were individually fed food pellets containing cadmium, benzo(a)pyrene, or a combination of the two, then analyzed for metallothionein mRNA expression in the intestine, liver, and gill using real-time RT-qPCR. An initial experiment using only cadmium showed that ingestion of pellets varied in individual fish, and estimates of cadmium dose from the numbers of ingested pellets indicated considerable individual variability in cadmium dose. Induction of intestinal metallothionein mRNA was apparent, however, and a linear dose-response relationship was observed for metallothionein expression and cadmium dose in the intestine, but not the other organs, which showed no induction. In a second experiment, the entire daily cadmium dose was provided in a single contaminated pellet that was consumed by all treated fish, effectively eliminating the effect of variable ingestion rates on dose, and the interaction between cadmium and benzo(a)pyrene was also investigated. The intestine was again the primary organ for metallothionein induction by cadmium. When benzo(a)pyrene was administered together with cadmium, induction of metallothionein was potentiated by the presence of benzo(a)pyrene, with the main effect seen in the intestine, where already high levels of induction by cadmium alone increased by 1.74-fold when benzo(a)pyrene was present.

Route-specific cellular expression of cytochrome P4501A (CYP1A) in fish (Fundulus heteroclitus) following exposure to aqueous and dietary benzo[a]pyrene.

Mummichog (Fundulus heteroclitus), an estuarine, teleost fish, were exposed for 456 hr to environmentally relevant concentrations of aqueous (10 micrograms/liter) and dietary (10 micrograms/g) benzo[a]pyrene (BP) in static renewal aquaria. Cellular expression of BP-inducible cytochrome P4501A (CYP1A) was evaluated several times during exposure by immunohistochemistry in longitudinal histologic sections of whole fish. CYP1A-associated staining intensities in tissues were scored by a subjective rating system similar to that used previously for qualitative information. Exposure to aqueous BP resulted in high levels of CYP1A-associated immunohistochemical staining in gill pillar cells, heart endothelium, and vascular endothelium. Exposure to dietary BP resulted in only mild to moderate staining in these tissues but high-intensity staining in gut mucosal epithelium. CYP1A induction in hepatocytes appeared most sensitive to aqueous exposure. Route-specific patterns of CYP1A expression were also observed in other cells including gill epithelia, pseudobranch, and skin. Expression of CYP1A in renal tubules and interrenal tissues was not affected by either treatment. Coexposure to both aqueous and dietary BP resulted in a pattern of induction reflecting both routes of exposure. In addition to the subjective rating system for scoring CYP1A expression, we developed a photometric approach that was used to obtain quantitative data on CYP1A-associated staining intensity. Photometric values of CYP1A staining intensity revealed patterns essentially the same as those observed during subjective ranking but were amenable to statistical analysis. The results of this study support the use of tissue-specific patterns of CYP1A expression in identification of target sites and exposure routes for polycyclic aromatic hydrocarbons and other compounds.

Cytochrome P450IA1 in hepatic lesions of a teleost fish (Fundulus heteroclitus) collected from a polycyclic aromatic hydrocarbon-contaminated site.

The expression of cytochrome P450IA1 was examined in hepatic lesions of mummichog (Fundulus heteroclitus), a small, non-migratory teleost fish collected from a site in the Elizabeth River, VA, heavily contaminated with polycyclic aromatic hydrocarbons (PAH) of creosote origin. Immunoblot ('Western' blot) analysis using monoclonal antibody (MAb 1-12-3) to P450IA1 of the marine fish Stenotomus chrysops indicated that cytochrome P450IA1 levels in hepatocellular carcinoma and in foci of cellular alteration were 28-85% lower than those of adjacent non-neoplastic tissue. P450IA1-dependent monooxygenase activity, measured as ethoxyresorufin O-deethylase (EROD), exhibited a similar trend with EROD activity in lesions being 15-77% lower than activity in non-neoplastic tissue. Immunohistochemical examination of liver sections revealed general low intensity P450IA1-associated staining in hepatocellular carcinoma, exocrine pancreatic tissue, bile ducts and cholangiocellular proliferative lesions. Staining intensity of non-neoplastic hepatic parenchyma varied considerably and was focally distributed. In one case intense staining was observed in an altered hepatocellular focus (putative preneoplastic lesion). The results indicate important similarities in the expression of P450IA1 in neoplasms of fish and mammals and suggest an adaptive response of a wild population to carcinogen exposure.

Glutathione S-transferase in intestine, liver and hepatic lesions of mummichog (Fundulus heteroclitus) from a creosote-contaminated environment.

Cytosolic glutathione S-transferase (GSH transferase) activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was elevated approximately three to four-fold in intestine and liver of mummichog (Fundulus heteroclitus) collected from a creosote-contaminated site in the Elizabeth River, Virginia. Intestinal GSH transferase activity at the most heavily contaminated site, at a moderately contaminated site and at a relatively clean site averaged 3.64, 2.83 and 1.11µmoles/min/mg respectively, while values for liver at these sites averaged 2.84, 1.75 and 0.93µmoles/min/mg. In addition, densitometric tracings of sodium dodecylsulfate-polyacrylamide gels of intestine and liver cytosol revealed a similar trend in the staining intensity of a 25.8 kD protein band, which lies within the molecular weight range of GSH transferase subunits. Activity in putative preneoplastic and neoplastic hepatic lesions of fish collected from the creosote-contaminated site was not significantly different from that of adjacent normal tissue. In the laboratory, dietary betanaphthoflavone (ßNF) treatment resulted in a three-fold increase in intestinal GSH transferase. Hepatic GSH transferase activity in the same fish was not affected by dietary ßNF although hepatic monooxygenase activity, measured as ethoxyresorufin O-deethylase (EROD), was. The results of this study indicate a response of the intestinal detoxification system to environmental contaminants and supports previous studies on the importance of intestinal metabolism of foreign compounds. Further, our results indicate the trend towards elevated GSH transferase in liver of feral fish could not be attributed to a cancerous disease state in these fish but indicates chemical induction in this organ as well.

Hepatic neoplasms in the mummichog Fundulus heteroclitus from a creosote-contaminated site.

High prevalences of idiopathic hepatic lesions were found in mummichog, Fundulus heteroclitus, from a site in the southern branch of the Elizabeth River, VA, contaminated with polycyclic aromatic hydrocarbons. Grossly visible hepatic lesions occurred in a total of 93% of the individuals from this site and 33% of these fish had hepatocellular carcinomas. Hepatic lesions were not detected in fish from two less contaminated sites. Lesions included foci of cellular alteration, hepatocellular adenoma, early and advanced hepatocellular carcinomas, and cholangiocellular proliferative lesions. Advanced carcinomas exhibited several distinct cellular patterns and some livers contained multiple neoplasms occupying up to 80% of the hepatic parenchyma. Sediments from the contaminated site contained extremely high concentrations (2200 mg/kg dry sediment) of polycyclic aromatic hydrocarbons, which are believed to originate from an adjacent wood treatment facility that has used creosote. Concentrations were 730- and 35-fold higher than those at the two other sites. These findings indicate a strong positive association between exposure to creosote-contaminated sediments and the high prevalence of hepatic neoplasms in a feral population of mummichog and support the putative role of polycyclic aromatic hydrocarbons in fish hepatocarcinogenesis. Additionally, they suggest that the mummichog may be a useful indicator of exposure to carcinogens in aquatic environments.

Induction of monooxygenase activity in the intestine of spot (Leiostomus xanthurus), a marine teleost, by dietary polycyclic aromatic hydrocarbons.

The response of intestinal monooxygenases to dietary polycyclic aromatic hydrocarbon (PAH) exposure was evaluated in spot (Leiostomus xanthurus), a marine teleost fish. Ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) activities were highest in the pyloric caeca and in the proximal half of the intestine. Intestinal microsomes from fish given control diets had very low levels of EROD and AHH activities relative to those in liver. After exposure to a diet containing 10 mg of 3-methylcholanthrene/kg of food, the levels of intestinal EROD and AHH activities increased 36-fold and 17-fold, respectively, such that intestinal monooxygenase activity exceeded that of the liver, which was not induced by this treatment. A significant increase in intestinal monooxygenase activity occurred in fish receiving dietary benzo[a]pyrene (BP) at concentrations as low as 10 micrograms of BP/kg food. A 5-fold increase in intestinal AHH and EROD activities was observed within 3 hr after administration of dietary BP. A plateau in gut monooxygenase activity occurred after approximately 3 days of PAH exposure; these activities decreased to control levels within 3 days after replacing the PAH diet with the control diet. Starvation resulted in disappearance of detectable monooxygenase activity. Monoclonal antibody (MAB 1-12-3) against the major PAH-inducible cytochrome P-450 (P-450E) in the liver of the marine teleost (Stenotomus chrysops) [Park et al. Arch. Biochem. Biophys. 249, 399 (1986)] recognized a single protein band in intestinal microsomes, with Mr near 54,000, which we conclude is the spot counterpart to cytochrome P-450E.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of preexposure to dietary benzo[a]pyrene (BP) on the first-pass metabolism of BP by the intestine of toadfish (Opsanus tau): in vivo studies using portal vein-catheterized fish.

The effect of preexposure of fish to dietary benzo[a]pyrene (BP) on the intestinal metabolism of BP was examined in toadfish (Opsanus tau). The portal veins of toadfish were cannulated following administration of radiolabeled BP to the intestinal lumen. Because these fish lack a lymphatic vessel system, the portal vein is the sole route by which BP and its metabolites enter the circulation. In fish preexposed to dietary BP (10 mg BP/kg food), the radioactivity entering the portal vein was almost entirely (ca. 90%) BP metabolites. In fish fed a laboratory control diet, a smaller percentage (ca. 60%) of the radioactivity entering the portal vein was in the form of BP metabolites. The enhanced efficiency of the intestines of preexposed fish in metabolizing BP appears to be a result of induction of intestinal aryl hydrocarbon hydroxylase (AHH) activity. Intestinal microsomal AHH activities in control and preexposed fish were 0.033 +/- 0.032 and 0.320 +/- 0.060 nmol.min-1.mg-1, respectively. Gel filtration of portal vein plasma indicated differences in the roles of plasma proteins in transporting BP and BP metabolites. Native BP was associated primarily with the high density lipoproteins, whereas organic-soluble BP metabolites were associated primarily with serum albumin fractions. A large percentage of BP metabolites was recovered as water-soluble conjugates. These studies indicate that in fish, the intestine can be an important organ involved in dietary BP metabolism.

Dietary fat inhibits the intestinal metabolism of the carcinogen benzo[a]pyrene in fish.

Following the intestinal absorption of dietary benzo[a]pyrene (BP) by the killifish, this compound becomes incorporated along with dietary triglycerides into membrane-bound fat vacuoles within the intestinal epithelial cell (1985. J. Lipid Res. 26: 428-434). These vacuoles, arising from the smooth endoplasmic reticulum, are important transient structures involved in both the uptake and metabolism of dietary BP and, presumably, other lipophilic toxicants as well. In the present study we used subcellular fractions isolated from the intestines of spot (Leiostomus xanthurus), a teleost fish, to study factors that influence the metabolism of BP in a fat vacuole/microsomal system. Triglyceride-solubilized BP is capable of diffusion from fat vacuoles to microsomal enzymes. Increases in the concentration of fat vacuoles decrease the availability of BP to microsomal BP hydroxylase. The effect of fat vacuoles on the activity of BP hydroxylase becomes more pronounced as the concentration of BP in our test system decreases. Addition of cytosolic glutathione transferases to the fat vacuole/microsomal system enhances the activity of BP hydroxylase. Examination of binding of 3H-labeled BP to killifish (Fundulus heteroclitus) intestinal cytosolic proteins in vivo indicated that a large fraction of the radioactivity was associated within glutathione transferase. These results suggest that dietary fat inhibits metabolism of low levels of BP in the intestine. A consequence of this would be greater exposure of peripheral tissues to dietary carcinogens.

Comparison of p-Nitrophenol Biodegradation in Field and Laboratory Test Systems.

Acclimation of microbial communities exposed to p-nitrophenol (PNP) was measured in laboratory test systems and in a freshwater pond. Laboratory tests were conducted in shake flasks with water, shake flasks with water and sediment, eco-cores, and two sizes of microcosm. The sediment and water samples used in the laboratory experiments were obtained from the pond. After a 6-day acclimation period, PNP was biodegraded rapidly in the pond. When the pond was treated with PNP a second time, biodegradation began immediately. The acclimation periods in laboratory test systems that contained sediment were similar to that in the pond. The acclimation period was threefold longer in shake flasks without sediment. PNP was biodegraded more slowly by microbial communities acclimated in the laboratory than it was in the pond, and the rate of biodegradation varied with the type of test. The number of bacteria able to mineralize PNP increased by 3 orders of magnitude in the pond during the acclimation period. Similar increases accompanied acclimation in the laboratory systems.

Adaptation of natural microbial communities to degradation of xenobiotic compounds: effects of concentration, exposure time, inoculum, and chemical structure.

Adaptation of microbial communities to faster degradation of xenobiotic compounds after exposure to the compound was studied in ecocores. Radiolabeled test compounds were added to cores that contained natural water and sediment. Adaptation was detected by comparing mineralization rates or disappearance of a parent compound in preexposed and unexposed cores. Microbial communities in preexposed cores from a number of freshwater sampling sites adapted to degrade p-nitrophenol faster; communities from estuarine or marine sites did not show any increase in rates of degradation as a result of preexposure. Adaptation was maximal after 2 weeks and was not detectable after 6 weeks. A threshold concentration of 10 ppb (10 ng/ml) was observed; below this concentration no adaptation was detected. With concentrations of 20 to 100 ppb (20 to 100 ng/ml), the biodegradation rates in preexposed cores were much higher than the rates in control cores and were proportional to the concentration of the test compound. In addition, trifluralin, 2,4-dichlorophenoxyacetic acid, and p-cresol were tested to determine whether preexposure affected subsequent biodegradation. Microbial communities did not adapt to trifluralin. Adaptation to 2,4-dichlorophenoxyacetic acid was similar to adaptation to nitrophenol. p-Cresol was mineralized rapidly in both preexposed and unexposed communities.