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Biomedical subjects

K Fent

Publications and source records attributed to K Fent.

13 recordsLinked to original sources
PubMed2001

Cytochrome P450 induction by nitrated polycyclic aromatic hydrocarbons, azaarenes, and binary mixtures in fish hepatoma cell line PLHC-1.

Nitrated polycyclic aromatic hydrocarbons (NPAHs) and N-heterocyclic aromatic hydrocarbons (azaarenes) are as ubiquitous in the environment as their parent PAH compounds, although occurring at lower concentrations. The toxicological importance of NPAHs and azaarenes is based on their mutagenic and carcinogenic potential. Azaarenes possess a higher solubility and mobility in the environment than PAHs. However, very little is known about the toxicity and cytochrome P450 (CYP)1A induction potencies of NPAHs and azaarenes in fish. Here we report on the cytotoxicities and relative CYP1A induction potencies of 12 NPAHs, 12 azaarenes, and 11 PAHs, determined as neutral red uptake and ethoxyresorufin-O-deethylase (EROD) activity, respectively, in fish hepatoma PLHC-1 cells. Additionally, CYP1A enzyme protein was determined by ELISA for two NPAHs, azaarenes, PAHs, and binary mixtures. Compared with the structurally analogous PAHs, 2-nitronaphthalene, 3-nitrofluoranthene, 2-aza- and 7-azafluoranthene, 1,6-dinitropyrene, benzo[a]acridine and benzo[h]quinoline revealed higher induction potencies, whereas the other compounds showed similar or less activity. The induction potency was highly dependent on the compounds structural properties, reflected by significant correlations between the half-maximal EROD induction (-log EC50) and the molecular descriptors lipophilicity (log Kow) and maximal molecular length (Lmax). Binary mixtures of 6-nitrochrysene + benzo[a]anthracene, 6-nitrochrysene + benzo[a]acridine, and benzo[a]acridine + benzo[a]anthracene showed an additive interaction. The CYP1A induction potencies of NPAHs and azaarenes, demonstrated here for the first time in fish hepatoma cells, suggest that their contribution to the overall CYP1A induction potencies in PAH-contaminated environmental samples have to be taken into account.

Animals↗
PubMed2000

Phenyl- and butyltin analysis in small biological samples by cold methanolic digestion and GC/MS.

A very efficient technique for the analysis of six butyl- and phenyltin compounds in biota samples has been developed. No special equipment is needed for sample preparation, which is based on cold methanolic digestion with subsequent aqueous ethylation and liquid-liquid extraction. For samples of only 40 mg of biological materials, method detection limits ranging from 4 to 52 ng/g were achieved using gas chromatography/mass spectrometry. Relative recoveries for the individual butyl- and phenyltins, referring to perdeuterated organotin analogues as internal standards, ranged from 96 to 107%. Organotin concentrations in insect larvae (Chironomus riparius) and a reference mussel tissue (CRM 477) were determined with excellent precision (RSD <5%), and the measured butyltins in CRM 477 were in good agreement with the certified values. Comparison with accelerated solvent extraction confirmed high accuracy, and application for a bioconcentration experiment with phenyltins demonstrated the robustness and suitability of the method for routine analyses. The procedure allows fast, reliable, and simple determination of organotin compounds in low-size biological samples, which was demonstrated for bioconcentration experiments.

Gas Chromatography-Mass Spectrometry↗
PubMed2000

Cytochrome P4501A induction and porphyrin accumulation in PLHC-1 fish cells exposed to sediment and oil shale extracts.

The present study describes the use of a fish hepatoma cell line (PLHC-1) in monitoring the biological effects of sediments collected from recipient waters of the oil shale industry. Sampling sites were located in River Purtse and River Kohtla in northeast Estonia. The effects of pure oil shale on the PLHC-1 cells were also studied. The cells were exposed to n-hexane-extracted samples in 48-well plates for 24 h, and 7-ethoxyresorufin O-deethylase (EROD) activity, total protein, and porphyrin content were measured in the exposed cells. Polycyclic aromatic hydrocarbon (PAH) contents in the samples were measured by high-performance liquid chromatography (HPLC). All the sediment and oil shale samples induced CYP1A activity and led to porphyrin accumulation in the cells. The most potent inducers were the sediments collected near the oil shale processing plants (site Lüganuse in River Purtse and Kohtla in River Kohtla), as well as those at the most downstream site in River Purtse (Purtse). These samples possessed high total PAH contents, ranging from 4,270 to nearly 150,000 microg/kg dry sediment. The presence of other lipophilic organic contaminants in the samples was not determined in this study. Both EROD activity and porphyrin content exhibited biphasic induction curves, and the ED(50)(1) values for EROD activity were lower than the ED(50)s for porphyrin content. 2,3,7, 8-Tetrachlorodibenzo-p-dioxin induction equivalents (TCDD-EQs) calculated from EROD induction potencies correlated well with total PAHs (r(2) = 0.827 and p = 0.003 for log-transformed data) and also with individual PAHs. TCDD-EQs for porphyrin content did not correlate significantly with total PAHs (log-log r(2) = 0.785, p = 0. 116). The biological potency and PAH contamination of the samples showed the same rank order, except at Lüganuse, where sediment extracts induced CYP1A and porphyrins more than could have been expected based on PAH contents. Bioassay-derived induction EQs (normalized to dibenz(a,h)anthracene) were 20- to 3,200-fold greater than EQs calculated from the concentrations of five PAHs, suggesting important contributions from other compounds or nonadditive effects. The PLHC-1 cells proved to be a sensitive bioanalytical tool for sediments contaminated with PAH-type pollutants in the oil shale processing area. We suggest further use of this bioassay in screening and monitoring waters with similar background of pollution as in northeast Estonia.

Animals↗
PubMed1998

Effects of triphenyltin and other organotins on hepatic monooxygenase system in fish.

The interaction of the organotin fungicide triphenyltin chloride (TPT) with fish microsomal monooxygenase systems has been studied in vitro and in vivo in the marine fish scup (Stenotomus chrysops). In vitro incubation of fish liver microsomes with TPT resulted in the conversion of about 40% of the native total spectral P450 to P420. In addition, a strong concentration-related inhibition of ethoxyresorufin O-deethylase (EROD) activity was observed, with a complete loss at 1.0 mM TPT. Pentoxyresorufin-O-dealkylase (PROD) activity was inhibited only at the highest concentration tested. This suggests either some specificity for the EROD catalyst CYP1A1, or a loss of reductant NADPH cytochrome c reductase as the cause. Further in vitro incubations showed that NADPH, but not NADH, cytochrome c reductase was strongly inhibited at 100 microM TPT and higher. To further investigate this effect, fish were injected with single doses of 5, 25 and 50 microM TPT (1.9, 9.6 and 19.3 mg kg-1 TPT), and 24 and 48 h later, hepatic microsomes were analyzed for total P450 content, EROD activity, NAD(P)H cytochrome c reductase, and the content of three CYP forms. EROD activity tended to be decreased in TPT-treated scup, with the response being stronger after 48 than 24 h. No significant conversion of spectrally determined P450 to cytochrome P420 was found, and cytochrome b5 was not affected. However, both NAD(P)H cytochrome c reductases were significantly inhibited at all concentrations. Immunoblot analysis showed reduction of CYP1A1 content at all doses, being significant at 25 mM after 48 h, but no decrease in CYP3A-like protein, the dominant catalyst of testosterone 6 beta-hydroxylation, nor CYP2B-like protein, the major contributor to indicates significant effects of TPT at high concentrations on fish hepatic CYP1A1 protein, EROD activity and the reductases. TPT seems to act more specifically on CYP1A1 than on other CYP forms. These findings combined with those of our previous studies (Brüschweiler BJ, Würgler FE, Fent K. Environ Toxicol Chem 1996;15:827-735; Fent K, Bucheli TD. Aquat Toxicol 1994;28:107-126; Fent K, Stegeman JJ. Aquat Toxicol 1991;20:159-168; Fent K, Stegeman JJ. Aquat Toxicol 1993;24:219-240) indicate a general degenerative effect of organotins on the fish microsomal monooxygenase system, although some differences are seen between the organotins, and between species. We conclude that these effects of organotins have consequences for use of CYP1A as a biomarker and endocrine disruption.

Animals↗
PubMed1996

Inhibitory effects of heavy metals on cytochrome P4501A induction in permanent fish hepatoma cells.

The interactions in vitro of heavy metals Cd(II), Co(II), Cu(II), Ni(II), Pb(II), and Zn(II) with cytochrome P4501A (CYP1A) induction response and enzyme activity were studied in fish hepatoma cells PLHC-1. Cells were simultaneously exposed to heavy metals and to 3-methylcholanthrene (3-MC), an inducer of CYP1A. Heavy metals were added to the cells in different concentrations. Cytotoxicity were measured in the neutral red (NR) assay, relative CYP1A protein contents in an enzyme-linked immunosorbent assay (ELISA), and CYP1A activities in the ethoxyresorufin-O-deethylase (EROD) assay. All metals had a more pronounced effect on EROD activity than on CYP1A protein content and cytotoxicity. For the most active metal Cd(II), a 50% inhibition of EROD activity was observed at significantly lower concentrations (2.2 x 10(-5) M) than a 50% reduction of CYP1A protein (5.3 x 10(-5) M), and a 50% cytotoxicity (1.4 x 10(-4) M). The inhibitory potency of the metals had the following order: Cd(II) > Ni(II) > Cu(II) > Co(II) = Zn(II) > Pb(II). In a second set of experiments, lysates of 3-MC-induced cells were exposed to heavy metals. Cd(II) and Cu(II) caused a 50% inhibition of EROD activity at significantly lower concentrations than in the experiments with living cells, at 8.2 x 10(-6) M and 1.3 x 10(-5) M, respectively, whereas the effect by Co(II) occurred at a significantly higher concentration (8.2 x 10(-4) M). The results indicate that Cd(II) and Cu(II) in particular may affect the CYP1A system of the liver of fish at low concentrations through direct inhibition of the CYP1A enzyme activity. CYP1A induction response in fish liver is increasingly being used in biomonitoring programs. In the environment, interactions of CYP1A-inducing and CYP1A-inhibiting components (such as heavy metals) can be expected and must be taken into consideration.

Animals↗
PubMed1996

Ecotoxicology of organotin compounds.

Organotin compounds are ubiquitous contaminants in the environment. The high biological activity of some compounds toward aquatic organisms lead to deleterious impacts in aquatic ecosystems. Here, the aquatic ecotoxicology of organotins is reviewed based on a multidisciplinary approach involving environmental chemical, toxicological, and ecological aspects. Basic results were obtained both with field and laboratory studies, and some of the most important recent results and conclusions are critically reviewed. The contamination of and fate in aquatic systems is reported and linked with effects at different levels of biological organization. Major emphasis is placed on the development of a concept of ecotoxicology that encompasses not only effect assessment alone, but also integrates environmental chemistry with aquatic toxicology. Thereby, the influence of speciation for bioavailability, basic modes of toxic action, and aquatic toxicity are discussed. This case study on organotins allows to a certain extent generalizations to ecotoxicology in general.

Animals↗
PubMed1994

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 degrees C, and larval exposure at 16 degrees C were compared. In the embryonic-larval exposure at 16 degrees C, hatching was delayed and hatching success decreased at 15.9 micrograms/L. Mortality increased at > or = 3.9 micrograms/L TPT, and complete mortality occurred after 7 and 9 days at 15.9 and 5.1 micrograms/L, respectively. Mortality was higher at 21 degrees C that at 16 degrees C. Triphenyltin was more toxic to fish in larval stages. The induced effects were dose related, mortality increased at 1.8 microgram/L after 3 days, and was total after 5 days at 10.6 micrograms/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.

Animals↗
PubMed1992

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 micrograms/L for 3 to 10 days at 16 degrees C and 21 degrees 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 micrograms/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 micrograms/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 micrograms/L TBT resulted in alterations in skin, muscle and kidney, with greater effects occurring at 21 degrees C than at 16 degrees 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.

Abnormalities, Drug-Induced↗
PubMed1988

Nephrotoxicity screening in rats; general approach and establishment of test criteria.

The concept of a nephrotoxicity screening test that is based on quantitative assessment of urine collected under standardized conditions for 15.5 h is presented. One to eight urine collections were performed in large numbers of untreated female Sprague-Dawley rats. Normal values for water consumption, urine volume, pH, and excretion of protein, gamma-glutamyltranspeptidase, malate dehydrogenase, electrolytes, glucose, amino acids, leukocytes, erythrocytes, epithelia, unspecified cells and cylinders were determined. Test criteria were established based on the statistical distribution of these measurements. In rats repeatedly placed in metabolism cages, a statistically significant decrease in leukocyte excretion and an increase in excretion of epithelia and unspecified cells were observed. All other variables did not change with time.

Animals↗
PubMed1988

Nephrotoxicity screening in rats: a validation study.

A validation of our non-invasive screening test for the detection of renal damage (Zbinden et al. 1988) is presented. The test is based on repetitive, quantitative urine analysis in groups of six female Sprague-Dawley rats treated on 5 consecutive days with low doses of test substances. Higher doses were administered in the following weeks until nephrotoxic effects or signs of general toxicity were observed. Thirteen reference substances (hexachloro-1:3-butadiene [HCBD], cisplatin, carboplatin, suramin, chloroform, neomycin, rifampicin, phenacetin, phenylbutazone, methicilline, sodium oxalate, ethylene glycol and furosemide) were used. The percentage of rats reaching the test criteria, i.e., pathologic values defined on the basis of measured control values, was determined. In the controls, the overall percentage of rats reaching or exceeding the test criteria was 4.48%, a value that is close to the expected 5%. Evidence of nephrotoxicity was found with all reference compounds. Elevated excretion of cells and occurrence of cylinders were the most sensitive indicators of renal damage. Hematuria was the most frequent finding. Of the other urine constituents measured the enzyme malate dehydrogenase (MDH) was frequently increased. Water consumption, urine volume, pH and specific gravity were occasionally, and protein, glucose, electrolytes, amino acids and gamma-glutamyl-transpeptidase (GGT) were only rarely changed. It is concluded that the screening which is based on quantitative and repeated urine analysis is a useful procedure to detect nephrotoxic chemicals acting by a variety of mechanisms. The histopathological examination of the kidneys contributed useful information of the nature of the toxic effects, but as a screening tool it is less sensitive than quantitative urine analysis.

Animals↗
PubMedDate not supplied

Fish cell lines as versatile tools in ecotoxicology: assessment of cytotoxicity, cytochrome P4501A induction potential and estrogenic activity of chemicals and environmental samples.

In vitro systems such as primary cells and cell lines are of growing importance in ecotoxicology. Cells from different tissues and species of fish are used for the assessment of toxic action of chemicals and evaluation of environmental samples. For organotins and substituted phenols, we have found that the in vitro cytotoxicity is positively correlated with the acute toxicity in vivo, and therefore cytotoxicity assays may serve as an alternative for acute fish toxicity testing. We have been using the hepatocellular carcinoma (PLHC-1) cell line for the assessment of the cytochrome P4501A (CYP1A) induction potential of polyaromatic hydrocarbons (PAHs), nitro-PAHs and azaarenes. For these compounds, the CYP1A induction potential is found to be related to the molecular structure and lipophilicity. In mixtures, CYP1A induction of individual compounds is additive. Based on the comparative investigation of the induction potential we derived an induction equivalency (IEQ) concept that can be applied for the evaluation of environmental samples such as landfill leachates, sediments and motorway runoffs. Fish cell lines are also valuable, rapid and cost-effective tools for the assessment of estrogenic activity of chemicals and environmental samples. We have developed an estrogen-responsive reporter gene system using the rainbow trout gonad cell line RTG-2, in which an estrogen receptor beta form is expressed at very low levels, but is not inducible. As the estrogenic activity is dependent on the cellular level of estrogen receptor (ER), ER has to be co-transfected in transient transfections in addition to an estrogen-responsive reporter gene. Using a dual luciferase system, the estrogenic activity of 12 compounds including alkylphenols, DDT-isomers and its metabolites have been assessed. Our system shows a high sensitivity with a detection limit of 0.05 nM estradiol and is therefore more sensitive than many other mammalian or yeast systems. The relative estrogenic activity (e.g. o,p'-DDT) and other toxicological effects may differ from those in mammalian systems, indicating that a risk evaluation for fish could only be meaningfully assessed in fish-specific systems. This paper illustrates the versatility and high potential of fish cell lines in ecotoxicology.

Animal Testing Alternatives↗