Bioconcentration of xenobiotics in trout bile: a proposed monitoring aid for some waterborne chemicals.

A technique is proposed for the monitoring of certain xenobiotic pollutants in suspect aquatic enviornments by fish bile analysis. Bile removed from rainbow trout (Salmo gairdneri) exposed to nine different radioactive compounds in vivo contained concentrations of radioactivity greater than those in the surrounding water. Bile-to-water radioactivity ratios as high as 10,000: 1 were found after 24-hour exposures. The results of these experiments suggest that analysis of bile of wild or caged fish from a suspect site may be useful as a qualitative monitoring aid for certain types of xenobiotics in water.

Biotransformation and induction: implications for toxicity, bioaccumulation and monitoring of environmental xenobiotics in fish.

Biotransformation of xenobiotics in fish occurs by many of the same reactions as in mammals. These reactions have been shown to affect the bioaccumulation, persistence, residue dynamics, and toxicity of select chemicals in fish. P-450-dependent monooxygenase activity of fish can be induced by polycyclic aromatic hydrocarbons, but phenobarbital-type agents induce poorly, if at all. Fish monooxygenase activity exhibits ideal temperature compensation and sex-related variation. Induction of monooxygenase activity by polycyclic aromatic hydrocarbons can result in qualitative as well as quantitative changes in the metabolic profile of a chemical. Induction can also alter toxicity. In addition, multiple P-450 isozymes have been described for several fish species. The biotransformation products of certain chemicals have been related to specific P-450 isozymes, and the formation of these products can be influenced by induction. Exposure of fish to low levels of certain environmental contaminants has resulted in induction of specific monooxygenase activities and monitoring of such activities has been suggested as a means of identifying areas of pollutant exposure in the wild.

Effects of the mosquito larvicide GB-1111 on red-winged blackbird embryos.

Golden Bear Oil (GB-1111; legal trade name for GB-1313) is a petroleum distillate that is used in the United States and other countries as a larvicide for mosquito suppression. As part of a multi-species evaluation of the potential effects of GB-1111 on birds, red-winged blackbird eggs were collected, artificially incubated, and treated with one of five amounts of GB-1111 varying from 0 to 10 times the expected exposure from a spray application of the maximum recommended amount (X=47 l/ha, 5 gal/ac). The application of 10 X caused a significant reduction in hatching success. A dose-related reduction of hepatic microsomal monooxygenase activity (EROD) was detected. Among body weights, skeletal measurements, and age at death, only crownrump length was different among experimental groups. Overall, the potential hazard to embryos of a representative wetland passerine appears minimal until the application rate exceeds 3 X.

Effects of the mosquito larvicide GB-1111 on bird eggs.

Golden Bear Oil (GB-1111; legal trade name for GB-1313) is a petroleum distillate used in the United States and other countries as a mosquito larvicide. As part of an evaluation of the potential effects of GB-1111 on birds, fertile eggs of mallards (Anas platyrhynchos) and bobwhite (Colinus virginianus) were incubated in the laboratory, and treated on day 4 of incubation with external applications equivalent to either 0, 1/3, 1, 3 or 10 times the maximum rate (X) of 47 l/ha (5 gal/A) of field application of GB-1111. Hatching success was significantly reduced in mallards treated at 3 and 10 times the maximum field application, with a calculated approximate LD50 of 1.9 times the maximum field application. Most mortality occurred within a week of treatment. Hepatic P450-associated monooxygenase activity (ethoxyresorufin-O-dealkylase; EROD) was negatively related to dose. In the 3X group there was a significant increase in the concentration of hepatic reduced glutathione (GSH) but a decrease in protein-bound thiols (PBSH). Hatching success of bobwhite was marginally reduced at the highest level of treatment (10X). Other effects at this level in bobwhite included a significant increase in incidence of abnormal embryos or hatchlings, lower body and liver weights, and a two-fold increase in hepatic microsomal EROD activity in hatchlings. The recommended maximum rate of field application of GB-1111 is unlikely to impair the survival or development of bobwhite embryos but is potentially toxic to mallard embryos under conditions of larvicide drift or spray overlap.

Identification of in vitro cytochrome P450 modulators to detect induction by prototype inducers in the mallard duckling (Anas platyrhynchos).

Seven modulators of mammalian monooxygenase activity were screened for their ability to selectively stimulate or inhibit in vitro monooxygenase activities of hepatic microsomes from mallard ducklings treated with phenobarbital, beta-naphthoflavone, 3,3',4,4',5-pentachlorobiphenyl or vehicle. Microsomes were assayed fluorometrically for four monooxygenases: benzyloxy-, ethoxy-, methoxy-, and pentoxyresorufin-O-dealkylase, in combination with each of the seven modulators. Four combinations: alpha-naphthoflavone and 2-methylbenzimidazole with benzyloxyresorufin, and Proadifen with methoxy- and ethoxyresorufin, respectively, were evaluated further. beta-Naphthoflavone-treated groups were clearly distinguished from the corn oil vehicle control group by all of the assays and by the effects of the modulators in three of the four assay/modulator combinations. Enzyme activities of the phenobarbital and saline groups were statistically similar (P > or = 0.05) when assayed without modulator added, but each assay/modulator combination distinguished between these groups. The PCB-treated group was distinguished from the corn oil vehicle control group only for BROD activity, with or without the presence of modulator. Graphing of per cent modulation of BROD activity versus initial BROD activity provided the clearest distinction between all of the study groups. Identification of these selective in vitro modulators may improve detection and measurement of low level cytochrome P450 induction in avian species. Also, both the monooxygenase activities induced and the impacts of the modulators indicated differences between mammalian and avian cytochromes P450.

Polycyclic aromatic hydrocarbons, aliphatic hydrocarbons, trace elements, and monooxygenase activity in birds nesting on the North Platte River, Casper, Wyoming, USA.

Tree swallow (Tachycineta bicolor) and house wren (Troglodytes aedon) eggs and chicks were collected near a refinery site on the North Platte River, Casper. Wyoming, USA and at a reference site 10 km upstream. Total polycyclic aromatic hydrocarbon (PAH) concentrations in swallow and wren chicks were higher at the refinery site than at the reference site. Polycyclic aromatic hydrocarbon concentrations in sediment and chick dietary samples were consistent with these findings. The general lack of methylated PAHs in sediment, diet, and bird carcasses suggested that the PAHs were derived from combustion and not from petroleum. The predominance of odd-numbered aliphatic hydrocarbons and the low ratios (< or =0.25) of pristane:n-C17 and phytane:n-C18 in chick and diet samples also suggested that swallow and wren chicks were not being chronically exposed to petroleum. Mean ethoxyresorufin-O-dealkylase and benzyloxyresorufin-O-dealkylase activities in tree swallow livers averaged nine times higher at the refinery site than at the reference site and were probably induced by exposure to PAHs. Trace element concentrations in eggs and livers of swallows and wrens were similar or greater at the reference site than at the refinery site. Selenium, strontium, and boron concentrations were elevated in eggs and livers of swallows and wrens at both the refinery and reference sites.

Tumor prevalence and biomarkers of exposure in brown bullheads (Ameiurus nebulosus) from the tidal Potomac River, USA, watershed.

Associations between contaminant exposure and liver and skin tumor prevalence were evaluated in brown bullheads (Ameiurus nebulosus) from the tidal Potomac River, USA, watershed. Thirty bullheads (> or = age 3) were collected from Quantico embayment, near a Superfund site that released organochlorine contaminants; Neabsco Creek, a tributary with petroleum inputs from runoff and marinas; and Anacostia River (spring and fall), an urban tributary designated as a Chesapeake Bay region of concern, that was contaminated with polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and organochlorine pesticides. Fish were collected from the Tuckahoe River, as a reference. Cytochrome P450 activity, bile PAH metabolites, and muscle organochlorine pesticide and PCB concentrations were measured in randomly selected individuals and sediment contaminants were analyzed. We found statistically significant differences in liver tumor prevalences: Anacostia (spring), 50%; Anacostia (fall), 60%; Neabsco, 17%; Quantico, 7%; and Tuckahoe, 10%. Skin tumor prevalences were significantly different: Anacostia (spring), 37%; Anacostia (fall), 10%; Neabsco, 3%; Quantico, 3%; and Tuckahoe, 0%. Tumor prevalence in Anacostia bullheads warrants concern and was similar to those at highly contaminated sites in the Great Lakes. Evidence was found of higher PAH exposure in Anacostia fish but a cause-effect linkage could not be established. Fish tumor surveys, with histopathologic examination of internal and external organs, are recommended for monitoring the status of regions of concern.

Metabolism of [14C]2-methylnaphthalene by rainbow trout (Salmo gairdneri) in vivo.

Rainbow trout were exposed to aqueous [14C]2-methylnaphthalene and their bile was examined for metabolites of [14C]2-methylnaphthalene. Trout which were pretreated with the monooxygenase inducer beta-naphthoflavone exhibited greater levels of total metabolites, glucuronide conjugates and dihydrodiol metabolites of [14C]2-methylnaphthalene in bile as compared to non-induced trout. The ratio of 2-hydroxymethyl-naphthalene to dihydrodiols found was greater in non-induced than in induced trout. These results are consistent with previously published studies on the metabolism of [14C]2-methylnaphthalene by rat and trout hepatic microsomes in vitro.

Distribution and elimination of naphthalene and 2-methylnaphthalene in rainbow trout during short- and long-term exposures.

The accumulation and elimination of 14C in rainbow trout tissues following short- and long-term exposures to aqueous 14C-naphthalene or 14C-2-methylnaphthalene were studied. After exposure for eight hr to 0.005 mg/L or 0.023 mg/L of 14C-naphthalene most tissues of fingerling rainbow trout studied contained 14C at 20 to 100 times the water levels while fat and bile contained 14C at several hundred times water levels. The half-lives for elimination of 14C from all tissues except fat were less than 24 hr. Exposure of fingerling rainbow trout to 14C-naphthalene or 14C-2-methylnaphthalene for four weeks in a continuous-flow delivery system resulted in maximum tissue levels of these chemicals of from 40 to 300 times the water concentration. Maximum bile 14C levels were 13,000 and 23,500 times the water concentration for 14C-naphthalene and 14C-2-methylnaphthalene exposure, respectively. Elimination of 14C accumulated from 14C-naphthalene in this long-term exposure was much slower than after short-term exposures, while elimination of 14C accumulated from 14C-2-methylnaphthalene was biphasic. The presence of parent compounds and metabolites in acetone extracts of muscles was determined by TLC. The data suggest that the biphasic release of 14C from muscle of trout exposed to 14C-2-methylnaphthalene may be due to differential elimination of parent compound and metabolites.

Uptake metabolism, and elimination of 14C-labeled 1,2,4-trichlorobenzene in rainbow trout and carp.

Fingerling rainbow trout (Salmo gairdneri) were exposed to 14C-labeled 1,2,4-trichlorobenzene (TCB) for 8 h in a static exposure (0.018 mg/l) or for 35 d in a continuous-flow exposure (0.020 mg/l) followed by a subsequent elimination period. For the 2 d after the 8-h exposure, the half-time (t 1/2) of elimination of 14C from muscle and liver was 0.4 d, while after the 35-d exposure an early rapid elimination of 14C from these tissues (t 1/2 = 0.4 d) was followed by a slower elimination (t 1/2 = 50 d) during d 4-36. The maximum bioconcentration factors for 14C in muscle and liver were 51 and 102 after the 8-h exposure and 89 and 389 during the 35-d exposure. The values for bile were much greater, reaching 240 after the 8-h exposure and 1400 during the 35-d exposure. When larger trout and carp (Cyprinus carpio) were exposed to [14C] TCB (0.2-0.4 mg/l) the bioconcentration factor for bile 14C to water 14C was less than 100. Pretreatment of trout with beta-naphthoflavone, an inducer of hepatic mixed-function oxidase, increased this bioconcentration factor for bile to several hundred. Solvent partitioning and thin-layer chromatography (TLC) indicated that about 60% of the 14C in bile of control trout or carp was present as highly polar biotransformation products, while for induced trout the value was more than 90%. TLC in two solvent systems suggested that at least two such products were present in bile from control fish and at least three in bile from induced fish. About half of the 14C in bile from induced trout was more polar than the 14C in bile from normal trout.

Structural requirements for the inhibition of phthalate ester hydrolysis in rainbow trout by methylenedioxyphenyl compounds.

1. The ability of a number of methylenedioxyphenyl compounds and paraoxon to inhibit the hydrolysis and oxidation of di-2-ethylhexyl phthalate by rainbow trout tissue preparations was examined. 2. In addition to paraoxon, only the methylenedioxyphenyl compounds with long side-chains (piperonyl butoxide and tropital) inhibited the hydrolysis of di-2-ethylhexyl phthalate by trout liver subcellular fractions and trout serum. 3. Paraoxon decreased the production of the major metabolite of di-2-ethylhexyl phthalate by trout liver microsomes (+ NADPH) suggesting that this metabolite arises via further metabolism of mono-2-ethylhexyl phthalate.

Distribution and biliary excretion products of di-2-ethylhexyl phthalate in rainbow trout.

After 24-hr exposures of rainbow trout to 0.5 ppm of di-2-ethylhexyl [14C]phthalate, one-half of the radioactivity present in the fish was localized in the bile. The bile was pooled and fractionated by selective solvent extraction before and after beta-glucuronidase hydrolysis. Individual radioactive compounds were further separated and characterized by thin-layer chromatography. Gas chromatography-mass spectrometry was used to confirm the results of thin-layer chromatographic analysis. These procedures demonstrated that bile contained a number of DEHP metabolites, but only about 1% of unchanged DEHP. The major metabolite, mono-2-ethylhexyl phthalate glucuronide accounted for 72% of the total bile radioactivity. The remaining bile radioactivity was found to be present as phthalic acid glucuronide, mono-2-ethylhexyl phthalate and two partially characterized polar metabolites.

Metabolism of di-2-ethylhexyl phthalate by subcellular fractions from rainbow trout liver.

Trout liver homogenates metabolized di-2-ethylhexyl phthalate (DEHP) to monoethylhexyl phthalate (MEHP) without added NADPH and to MEHP and more polar metabolites with added NADPH. Both hydrolysis and oxidative metabolism of DEHP were inhibited by piperonyl butoxide. The 10,000g pellet, 100,000g pellet and 100,000g supernatant fraction from liver homogenates all catalyzed the hydrolysis of DEHP and all but the 100,000g supernatant fraction showed the shift to more polar metabolites with added NADPH; serum also catalyzed the hydrolysis of DEHP. Measurement of the microsomal marker, glucose 6-phosphatase, and the mitochondrial marker, succinic dehydrogenase, revealed that DEHP-hydrolytic activity was associated with microsomes and the 100,000g supernatant fraction, whereas DEHP oxidation was associated only with microsomes.

Effect of piperonyl butoxide on disposition of di-2-ethylhexyl phthalate by rainbow trout.

1. Piperonyl butoxide in vitro inhibits the oxidation and hydrolysis of di-2-ethylhexyl phthalate, and the hydrolysis of the butyl ester of 2,4-dichloro-phenoxyacetic acid by liver homogenate fractions and serum from rainbow trout. 2. The rates of oxidation and hydrolysis of di-2-ethylhexyl phthalate by liver homogenates from rainbow trout pre-exposed to piperonyl butoxide (1 mg/l) were considerably lower than those by liver homogenates from control trout. 3. Disposition of di-2-ethylhexyl [14C]phthalate in rainbow trout in vivo was modified by pre-exposure to piperonyl butoxide. The piperonyl butoxide-treated trout had lower levels of 14C in bile and higher levels of 14C in blood and muscle than control trout. 4. Muscle of control and piperonyl butoxide-exposed trout showed similar concentrations of mono-2-ethylhexyl phthalate but the concentration of di-2-ethylhexyl phthalate in muscle from piperonyl butoxide-exposed trout was three times the control value.

Metabolism of 2-methylnaphthalene in the rat in vivo. I. Identification of 2-naphthoylglycine.

Urine was collected from four female rats for 3 days after two subcutaneous injections with 0.3 mg of 2-methyl[8-14C]naphthalene per kg. Of the 14C injected, 55% was found in the urine. The urine was solvent-fractionated into a toluene fraction (4.9% of urine 14C), a chloroform fraction (11.7%), two ethyl acetate fractions (41.7%), and a methanol fraction (37.8%). Only about 3-5% of urine 14C appeared to be unchanged 2-methylnaphthalene. A major radioactive peak which was isolated from the chloroform fraction was further subdivided into three peaks by HPLC. These metabolites, which may be three isomeric dihydrodiols of 2-methylnaphthalene, represented 6-8% of urine 14C. The more polar fractions contained several radioactive peaks when examined by TLC. One of these peaks was hydrolyzed with base to glycine and a 14C-labeled material which was similar to 2-naphthoic acid by TLC and by HPLC. Mass spectrometry of this latter material and of the unhydrolyzed metabolite confirmed the presence of 2-naphthoic acid and 2-naphthoylglycine, respectively. In total, 2-naphthoylglycine accounted for 30-35% of urine 14C.

Tumor prevalence and biomarkers of exposure in brown bullhead (Ameiurus nebulosus) from Back River, Furnace Creek, and Tuckahoe River, Maryland.

Brown bullheads (Ameiurus nebulosus) were collected from 2 locations near Baltimore, Maryland, Back River and Furnace Creek, and 1 (reference) location, Tuckahoe River, to compare the prevalence of tumors (liver and skin) and visible skin lesions (fin erosion and abnormal barbels). Cytochrome P450 activity measured as ethoxyresorufin-O-deethylase, biliary PAH-like fluorescent metabolites, and fillet contaminant concentrations were determined as indicators of exposure in a randomly selected subset of the fish. There were no significant differences in liver tumor prevalence: Back River = 8% (4/50), Furnace Creek = 0% (0/50), and Tuckahoe River = 2.6% (1/39; p = 0.20, extension of Fisher's exact test). Skin tumor prevalence was as follows: Furnace Creek = 12% (6/50), Back River = 8% (4/50), and Tuckahoe River = 0% (0/39; p = 0.063). In the Back River fish, there was a 40% (20/50) prevalence of fin erosion and a 28% (14/50) prevalence of abnormal (shortened, clubbed, or missing) barbels. Fin erosion was not observed in the other collections, and only 10% (5/50) of the Furnace Creek fish had abnormal barbels (p < 0.001 for both lesions). There were statistically significant differences in mean EROD activity, with levels in Furnace Creek and Back River fish approximately twice that found in Tuckahoe River fish. There were also significant differences in mean benzo(a)pyrene-like bile metabolite concentrations: the lowest mean was in the Tuckahoe River fish, 8 times higher in Furnace Creek fish, and 13 times higher in Back River fish. Of the 3 groups, the Back River bullheads appear to be most adversely affected by contaminant exposure because they had the highest prevalence of liver tumors, fin erosion, and abnormal barbels.

Metabolism of 2-methylnaphthalene by rat and rainbow trout hepatic microsomes and purified cytochromes P-450.

The metabolism of 14C-2-methylnaphthalene by hepatic microsomes and purified cytochromes P-450 from rat and rainbow trout was studied using HPLC and radioactivity counting. The effects of pretreatment with beta-naphthoflavone and phenobarbital on the formation of three dihydrodiol metabolites and 2-hydroxymethylnaphthalene by microsomes and purified cytochromes P-450 were compared. The metabolism observed with microsomes could not adequately be accounted for by the combined metabolism observed with the purified cytochromes P-450. Differences in the microsome suspension buffers used when preparing microsomes for cytochrome purification as compared to preparing microsomes for direct use appeared to be a significant factor in these differences.

Lack of developmental and reproductive toxicity of 2,3,3',4, 4'-pentachlorobiphenyl (PCB 105) in ring-necked pheasants.

Mono-ortho PCBs are global contaminants of wildlife with the potential to produce toxicity by an aryl hydrocarbon receptor (AhR)-mediated mechanism. To determine the potency of 2,3,3',4, 4'-pentachlorobiphenyl (PCB 105) for producing reproductive and developmental toxicity, adult ring-necked pheasant hens (Phasianus colchicus) were orally dosed with 0, 0.06, 0.6, or 6 mg PCB 105/kg hen/week for 10 weeks to achieve cumulative doses of 0, 0.6, 6, or 60 mg PCB 105/kg hen after which hens were bred with untreated roosters once per week for 8 weeks. Except at week 6 of the egg-laying period when cumulative egg production in the 6 mg PCB 105/kg hen group was greater than controls, fertilized egg production was not significantly different between treatment groups. Embryo mortality and chick mortality were not significantly different between treatment groups. Total body and heart weights of all chicks 1 day posthatch (dph) were not different between groups, however, liver weights of chicks from the 60 mg/kg treatment group were greater than controls at 1 dph. The first chick to hatch from each hen was reared to 21 dph and among these birds, the total body, liver, and heart weights were not different between groups. There were no dose-related malformations of the beak or limbs, and no signs of subcutaneous edema, ascites, or pericardial edema in chicks at 1 or 21 dph. Hepatic microsomal monooxygenase activities [ethoxyresorufin-O-dealkylase (EROD), benzyloxyresorufin-O-dealkylase (BROD), and methyloxyresorufin-O-dealkylase (MROD)] were significantly elevated in chicks at 1 dph from hens given a cumulative PCB 105 dose of 6 mg/kg and in chicks at 21 dph from hens given a cumulative PCB dose of 60 mg/kg. These results indicate that a cumulative PCB 105 dose up to 60 mg/kg hen does not decrease the production of fertilized eggs or increase embryo or chick mortality in ring-necked pheasants, but does increase chick hepatic monooxygenase activity.