Karl Fent

Induction of cytochrome P450 as a biomarker for environmental contamination in aquatic ecosystems

Abstract Representing toxicant‐induced changes in biological systems, biomarkers can serve as linkers between contamination (cause) and biological effects, and therefore provide unique information on ecosystem health. Hence, they are increasingly used for assessing the exposure of organisms to environmental contamination. Here, application of the induction of cytochrome P4501A (CYP1A), certainly the best studied biomarker, in field trials with freshwater and marine fish is reviewed. Moreover, the use of a number of other biomarkers, alone or in concert with CYP1A, is evaluated. Determination of CYP1A in field studies with fish has to consider several influencing factors, such as species, age, reproductive stage, temperature, and possible inhibitors. The overview shows that CYP1A induction is significantly related to contaminant levels in the environment in 93% of the investigated field studies. It is apparent that only certain classes of xenobiotica act as inducers of CYP1A (e.g., PAHs, coplanar PCBs, pol...

Concentrations of the antifouling compound Irgarol 1051 and of organotins in water and sediments of German North and Baltic Sea marinas

Abstract A survey of 2-methylthio-4-tert-butylamino-6-cyclopropylamino-s-triazine (Irgarol 1051) and organotins, used as biocides in antifouling paints, was carried out in German marinas of the North and Baltic Sea. Highest levels of the s-triazine herbicide were found in Baltic Sea marinas where water exchange was restricted (water up to 440 ng/l; sediment about 220 ng/g dry weight), while in the North Sea marinas with higher water exchange rates lower concentrations were encountered (water 11–170 ng/l; sediment 3–25 ng/g dry weight). Although the application of the antifouling agent tributyltin (TBT) was banned for small vessels (

Effects of tributyltin in vivo on hepatic cytochrome P450 forms in marine fish

The interaction of tributyltin chloride (TBT) in vivo with different forms of hepatic microsomal cytochrome P450 and ethoxyresorfin O-deethylase (EROD) activity was studied in scup (Stenotomus chrysops). Fish were injected with single doses of 3.3, 8.1 and 16.3 mg/kg TBT. Hepatic microsomes were analyzed 24 h later for total cytochrome P450 content, and three P450 forms by immunoblotting: scup P450E, the major polyaromatic hydrocarbon-inducible form (CYPlAl), scup P450A, the major contributor to microsomal testosterone 6β-hydroxylase activity, and scup P450B, which oxidizes testosterone at several different sites including the 15α position. Spectrally determined conversion of P450 to its degraded form cytochrome P420 occurred at all TBT doses, the conversion being considerable only at the highest dose with more P420 (65%) than P450 (35%). EROD activity tended to be decreased by TBT in all doses. with a significant reduction at 16.3 mg/kg. Microsomal protein degradation as revealed by SDS-polyacrylamide gel electrophoresis occurred at the highest dose. Immunoblot analysis with a monoclonal antibody to P450E (CYPlAl) showed a decrease of CYPlA protein content at all TBT doses, with a significant loss at 16.3 mg/kg, similar to the EROD activity pattern. Immunoblot analysis with polyclonal antibodies to P450A and P450B showed decreases in P450A and P450B protein content only at the highest dose, the decrease being significant for P450A only. Cytochrome b5 content was unaffected. In the liver, metabolites of TBT, dibutyltin and monobutyltin, were identified at all doses, with a decreased content, however, in fish given the highest dose. This study indicates important biochemical effects of TBT in fish liver, and suggests that exposure to TBT may alter both cytochrome P450 dependent metabolism, and induction response to other environmental pollutants.

Inhibition of hepatic microsomal monooxygenase system by organotins in vitro in freshwater fish

Abstract The interaction in vitro of organotins tributyltin (TBT) and triphenyltin (TPT) with the hepatic microsomal monooxygenase systems in the freshwater fish, rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla), and bullhead (Cottus gobio) was studied. Hepatic microsomes were incubated in vitro with TBT and TPT and various components analyzed. Ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by TBT and TPT in a concentration-dependent manner in all fish. Rainbow trout microsomes were more sensitive than were eel or bullhead microsomes. Total inhibition of EROD activity occurred both at 0.5 mM TBT and TPT in rainbow trout, whereas I mM decreased EROD activity to 15% (TBT) or 3% (TPT) in eel, and 35% (TBT) or 18% (TPT) in bullhead, respectively. As this effect may be caused by inhibition of different components of the microsomal electron transport system, different enzymes were studied separately. In all fish, both organotins led to a time- and concentration-dependent decrease in spectral total microsomal P450 content, and formation of cytochrome P420. TPT led to a greater inactivation of P450 enzyme than TBT, and induced a 50% loss in all fish at 0.08 mM TPT, whereas in case of TBT a 50% loss occurred at 0.18 mM in rainbow trout, 0.30 mM in bullhead, and 0.83 mM in eel. Cytochrome b5 content was not affected, but both organotins led to an almost selective inhibition of either NADH or NADPH cytochrome c reductase activity in trout and eel, or of both in bullhead. Whereas TBT inhibited NADH cytochrome c reductase, TPT acted strongly on NADPH cytochrome e reductase. The study demonstrates species-related significant and selective effects of TBT and TPT on different components of the microsomal monooxygenase system in freshwater fish. This leads to inactivation of native enzymes and inhibition of enzyme activities.

Tributyltin-induced effects on early life stages of minnows Phoxinus phoxinus

Toxicity and histopathological effects of tributyltin chloride (TBT) were studied in early life stages of minnows Phoxinus phoxinus. Eggs and yolk sac fry (newly hatched larvae) were exposed in a static-renewal procedure to aqueous TBT concentrations ranging from 0.82 to 19.51 μg/L for 3 to 10 days at 16°C and 21°C, respectively. Aqueous TBT concentrations were determined by capillary GC-FPD and revealed a concentration decrease during the static phase. TBT exposure led to mortality, behavioral, gross morphological and histopathological effects. In larvae, increased mortality, deformation of body axis, paralysis and opaque eyes occurred at 4.26 μg/L TBT and higher both in the embryonic-larval and larval exposure. Histological changes were evident at initial TBT concentrations of 0.82 up to 19.51 μg/L, and were more pronounced after embryonic-larval exposure than after larval exposure. Degenerative alterations occurred in skin, skeletal muscle, kidney, corneal epithelium, lens, pigment layer of the retina and choroid, retina, and CNS including spinal cord. Hydropic vacuolation of the cytoplasm and, in more pronounced cases, irreversible nuclear alterations such as pycnosis, karyorrhexis and karyolysis were also evident. Exposure to 0.82 μg/L TBT resulted in alterations in skin, muscle and kidney, with greater effects occurring at 21°C than at 16°C. Toxicity was significantly reduced in the presence of sediment. The observed histopathological effects suggest that early life stages of fish may be negatively affected in environments that are considerably polluted by TBT.

Effects of triphenyltin on fish early life stages

Using a static-renewal procedure, effects of triphenyltin chloride (TPT) on hatching, survival, and morphology were assessed in early life stages of European minnows Phoxinus phoxinus. Embryonic-larval exposure at 16 and 21°C, and larval exposure at 16°C were compared. In the embryoniclarval exposure at 16°C, hatching was delayed and hatching success decreased at 15.9 μg/L. Mortality increased at ≥3.9 μg/L TPT, and complete mortality occurred after 7 and 9 days at 15.9 and 5.1 μg/L, respectively. Mortality was higher at 21°C that at 16°C. Triphenyltin was more toxic to fish in larval stages. The induced effects were dose related, mortality increased at 1.8 μg/L after 3 days, and was total after 5 days at 10.6 μg/L. In all high TPT exposures, larvae developed skeletal malformations (bent tails), showed impaired swimming behavior or paralysis, and eyes became opaque. Marked histopathological alterations were found. Degenerative hydropic vacuolation of the cytoplasm were evident in skeletal muscles, skin, kidneys, corneal epithelium, lens, pigment layer of the retina and choroid, retina, and CNS including spinal cord. In severe cases, nuclear changes including pycnosis and karyorrhexis occurred. The observed toxicity of TPT was similar to that of tributyltin, but TPT acted more selectively on the lens and CNS, whereas other tissues were less affected. The study indicates that Phoxinus phoxinus larvae are negatively affected at peak TPT concentrations found in polluted environments.

Bioaccumulation and bioavailability of tributyltin chloride: Influence of pH and humic acids

Abstract The influence of pH and humic acids on bioaccumulation and bioavailability of tributyltin chloride (TBT) was studied in Daphnia and yolk sac larvae of fish Thymallus thymallus. In both organisms, bioaccumulation followed a first-order uptake process, and metabolism of TBT was slow. Bioconcentration factors in Daphnia and Thymallus were 198 and 2015, respectively. Uptake rates and bioaccumulation of TBT in Daphnia were significantly higher at pH 8.0, where TBT predominates as neutral TBTOH species than at pH 6.0, where it predominates as cation. Humic acids led to a decrease in TBT bioaccumulation at ≥ 1.8 mg·l−1 dissolved organic carbon (DOC) in Daphnia and ≥ 4.0 mg·l−1 DOC in Thymallus larvae. Bioaccumulation of TBT decreased with increasing concentrations of humic acids. Hence, chemical speciation of TBT is a determinant factor for the bioavailability, and thus bioconcentration and toxicity of this organotin compound.

Effects of tributyltin chloride in vitro on the hepatic microsomal monooxygenase system in the fish Stenotomus chrysops

Abstract Tributyltin (TBT) has been shown to be highly toxic to a number of aquatic species, but the mode(s) of action on a biochemical level remain(s) to be elucidated. Here we investigate the interaction in vitro of TBT with hepatic microsomal cytochrome P-450 and associated enzyme activity in scup (Stenotomus chrysops). Hepatic microsomes from β-naphthoflavone (βNF) induced scup were incubated in vitro with TBT and various components analyzed. TBT led to a time- and concentration-dependent decrease in total microsoinal P-450 content. This decrease was accompanied by the formation of cytochrome P-420. A complete loss of P-450 occurred with 1 mM TBT after 30 min incubation. Ethoxyresorufin-O-deethylase (EROD) activity was strongly inhibited by TBT in a concentration-dependent manner, with a complete inhibition at 0.3 mM TBT after 15 min incubation. Other components of the microsomal electron transport system were also investigated. Neither cytochrome b5 content, nor NADPH-cytochrome c reductase activity were affected by TBT at concentrations up to 0.5 mM TBT. NADH-cytochrome c reductase activity, however, showed a concentration-dependent increase, with more than a doubling of activity at 0.5 mM TBT. This indicates that TBT has different effects on these reductases. In conclusion, TBT can strongly interact with hepatic microsomal P-450 in fish leading to destruction of native enzyme and inhibition of enzyme activity. Microsomal monooxigenase system; Cytochrome P-450; Tributyltin chloride: Interaction; Scup (Stenotomus chrysops)

Cytotoxicity in vitro of organotin compounds to fish hepatoma cells PLHC-1 (Poeciliopsis lucida)

Abstract Cytotoxicity in vitro to fish hepatoma cells PLHC-1 has been analyzed for a series of 21 organotin compounds consisting of all degrees of alkylation and arylation. The sensitivity of the neutral red (NR) assay and the tetrazolium salt reduction (MTT) assay was similar for most of the organotin compounds. Cytotoxic effects were found at concentrations between 10 −8 M and 10 −2 M. For various trisubstituted organotin compounds, including tributyltin and triphenyltin, which are used in antifouling paints and as pesticides, respectively, cytotoxic concentrations in the range of 10 −8 M to 10 −6 M were observed. Based on the concentration reducing NR uptake by 50% (NR 50 ), the sequence of Cytotoxicity amongst butyltins was tributyltin ⪢ bis(tributyl)-tin ⪢ dibutyltin ⪢ tetrabutyltin ⪢ butyltin ⪢ tin(IV). Tributyltin induced effects on cell functions quickly, as a reduction in NR uptake by 30% was recorded after 30 min exposure to 4 · 10 −7 M. The ranking order in Cytotoxicity in the MTT assay of phenylated organotins was triphenyltin ⪢ diphenyltin ⪢ phenyltin ⪢ tin(IV). Cytotoxic concentrations of tributyltin and triphenyltin measured in the bromodeoxyuridine (BrdU) assay and with the crystal violet (CV) staining method do not differ significantly from those determined in the NR and MTT assay. Tri-and disubstituted organotin compounds exhibit a significant correlation between the noctanol/water partition coefficient (logK ow ) and the NR 50 ( n = 10, r = 0.86, P = 0.001) and the MTT 50 values ( n = 11, r = 0.85, P = 0.001), respectively. In addition, good qualitative correlations between in vitro Cytotoxicity data and in vivo fish toxicity data were found (for NR assay n = 8, r = 0.86, P = 0.001; for MTT assay n = 9, r = 0.80, P = 0.001). he results indicate that the in vitro Cytotoxicity assays using PLHC-1 cells are useful tools for the estimation of the acute toxicity to fish of organotins and possibly other compounds.

Bioconcentration and elimination of tributyltin chloride by embryos and larvae of minnows Phoxinus phoxinus

Abstract Accumulation, elimination and possible transformation of tributyltin chloride (TBT) were studied in embryos and yolk sac larvae of minnows Phoxinus phoxinus . Embryonic uptake of TBT was compared to larval uptake at aqueous TBT concentrations of nominal 8.9 μg/1. TBT, dibutyltin (DBT) and monobutyltin (MBT), were analyzed by GC-FPD in the water and in whole-body samples. Embryonic and larval uptake of TBT from water was rapid, being considerably lower in the embryonic, than in the larval phase. Whole body tissue concentrations did not reach plateaus, being 4.65 μ/g after embryonic-larval exposure for 164 h, and 4.35 μg/g after larval exposure for 96 h to 8.9 μg TBT/1. Calculated bioconcentration factors (wet wt./water) were 107 in embryos after 100 h exposure, and 538 in larvae after 96 h. Uptake and subsequent elimination were assessed after larval exposure to 4.5 μg/l. TBT concentrations in larvae reached 1.59 μg/g after 93 h exposure, and decreased to 1.48 μg/g after 75 h of depuration, indicating a slow elimination of this compound. The occurrence of only low and relatively constant concentrations of metabolites DBT and MBT indicate an absence, or an extremely slow catabolism of TBT in embryos and yolk sac larvae. These findings indicate a considerable potential of TBT for bioconcentration in fish early life stages.