Comparative genotoxic potential of 27 polycyclic aromatic hydrocarbons in three human cell lines.

Polycyclic Aromatic Hydrocarbons (PAHs) form a family of compounds that are generally found in complex mixtures. PAHs can lead to the development of carcinogenesis. The Toxicity Equivalent Factor (TEF) approach has been suggested for estimating the toxicity of PAHs, however, due to the relative weakness of available data, TEF have not been applied for the risk characterization of PAHs as food contaminants in Europe. The determination of new TEFs for a large number of PAHs could overcome some limitations of the current method and improve cancer risk assessment. The present investigation aimed at deriving new TEFs for PAHs, based on their genotoxic effect measured in vitro and analyzed with mathematical models. For this purpose, we used a genotoxicity assay (γH2AX) with three human cell lines to analyze the genotoxic properties of 27 selected PAHs after 24 h treatment. For 11 compounds, we did not detect any genotoxic potential. For the remaining 16 PAHs, the concentration-response for genotoxic effect was modelled with the Hill equation; equivalency between PAHs at low dose was assessed by applying constraints to the model parameters. We developed for each compound, in each cell line, Genotoxic Equivalent Factor (GEF). Calculated GEF for the tested PAHs were similar in all cell lines and generally higher than the TEF usually used. These new equivalent factors for PAHs should improve cancer risk assessment.

Effect of skin metabolism on dermal delivery of testosterone: qualitative assessment using a new short-term skin model.

The skin is a metabolically active organ expressing biotransformation enzymes able to metabolize both endogenous molecules and xenobiotics. We investigated the impact of metabolism on the delivery of testosterone through the skin with an ex vivo pig ear skin system as an alternative model for human skin. Penetration, absorption and metabolic capabilities were investigated up to 72 h after application of [(14)C]-testosterone doses of 50-800 nmol on either fresh or frozen skin, with the latter model being metabolically inactive. Testosterone absorption and metabolite production were monitored by radio-HPLC and gas chromatography-mass spectrometry. Testosterone absorption through frozen skin was much lower, irrespective of the dose of testosterone applied, compared to fresh skin. Using fresh skin samples, >95% of the radioactivity recovered in culture media, as well as the skin itself, corresponded to metabolites. These results were compared with the metabolic data obtained from other in vitro systems (liver and skin microsomes). The present work leads to the conclusion that most of the enzymatic activities expressed in liver fractions are also expressed in pig and human skin. The metabolic activity of the skin can modulate the biological activity of pharmaceuticals (and xenobiotics). Consequently, it can also greatly affect transdermal drug delivery.

Disposition of fipronil in rats.

In the scientific literature, little attention has been paid to the disposition of fipronil, a phenyl pyrazole insecticide. In this study, the tissue distribution, the metabolic fate, and the elimination of fipronil was investigated in rats using radiolabeled fipronil. When a single oral dose of (14)C-fipronil (10 mg kg(-1) b.w.) was given to rats, the proportion of dose eliminated in urine and feces 72 h after dosing was ca 4% for each route. At the end of the experiment the highest levels of radioactivity were found in adipose tissue and adrenals. The main part of the radioactivity present in investigated tissues (adipose tissue, adrenals, liver, kidney, testes) was due to fipronil-sulfone. Five additional metabolites, isolated from urine were characterized by LC-MS/MS. Most of them are formed by the loss of the trifluoromethylsulphinyl group and subsequent hydroxylation and/or conjugation to glucuronic acid or sulfate. In conclusion, the retention of the metabolite fipronil sulfone in tissues following fipronil administration raises the question of the potential toxicity of this insecticide.

The PERICLES research program: an integrated approach to characterize the combined effects of mixtures of pesticide residues to which the French population is exposed.

Due to the broad spectrum of pesticide usages, consumers are exposed to mixtures of residues, which may have combined effects on human health. The PERICLES research program aims to test the potential combined effects of pesticide mixtures, which are likely to occur through dietary exposure. The co-exposure of the French general population to 79 pesticide residues present in the diet was first assessed. A Bayesian nonparametric model was then applied to define the main mixtures to which the French general population is simultaneously and most heavily exposed. Seven mixtures made of two to six pesticides were identified from the exposure assessment. An in vitro approach was used for investigating the toxicological effects of these mixtures and their corresponding individual compounds, using a panel of cellular models, i.e. primary rat and human hepatocytes, liver, intestine, kidney, colon and brain human cell lines. A set of cell functions and corresponding end-points were monitored such as cytotoxicity, real-time cell impedance, genotoxicity, oxidative stress, apoptosis and PXR nuclear receptor transactivation. The mixtures were tested in equimolar concentrations. Among the seven mixtures, two appeared highly cytotoxic, five activated PXR and depending on the assay one or two were genotoxic. In some experiments, the mixture effect was quantitatively different from the effect expected from the addition concept. The PERICLES program shows that, for the most pesticides mixtures to which the French general population is exposed, the toxic effects observed on human cells cannot be easily predicted based on the toxic potential of each compound. Consequently, additional studies should be carried on in order to more accurately define the mixtures of chemicals to which the consumers are exposed, as well as to improve the investigation, prediction and monitoring of their potential human health effects.

Effect of mono-(2-ethylhexyl) phthalate on human and mouse fetal testis: In vitro and in vivo approaches.

The present study was conducted to determine whether exposure to the mono-(2-ethylhexyl) phthalate (MEHP) represents a genuine threat to male human reproductive function. To this aim, we investigated the effects on human male fetal germ cells of a 10⁻5 M exposure. This dose is slightly above the mean concentrations found in human fetal cord blood samples by biomonitoring studies. The in vitro experimental approach was further validated for phthalate toxicity assessment by comparing the effects of in vitro and in vivo exposure in mouse testes. Human fetal testes were recovered during the first trimester (7-12 weeks) of gestation and cultured in the presence or not of 10⁻5 M MEHP for three days. Apoptosis was quantified by measuring the percentage of Caspase-3 positive germ cells. The concentration of phthalate reaching the fetal gonads was determined by radioactivity measurements, after incubations with ¹4C-MEHP. A 10⁻5 M exposure significantly increased the rate of apoptosis in human male fetal germ cells. The intratesticular MEHP concentration measured corresponded to the concentration added in vitro to the culture medium. Furthermore, a comparable effect on germ cell apoptosis in mouse fetal testes was induced both in vitro and in vivo. This study suggests that this 10⁻5 M exposure is sufficient to induce changes to the in vivo development of the human fetal male germ cells.

Comparative potency approach based on H2AX assay for estimating the genotoxicity of polycyclic aromatic hydrocarbons.

Polycyclic Aromatic Hydrocarbons (PAHs) constitute a family of over one hundred compounds and can generally be found in complex mixtures. PAHs metabolites cause DNA damage which can lead to the development of carcinogenesis. Toxicity assessment of PAH complex mixtures is currently expressed in terms of toxic equivalents, based on Toxicity Equivalent Factors (TEFs). However, the definition of new TEFs for a large number of PAH could overcome some limitations of the current method and improve cancer risk assessment. The current investigation aimed at deriving the relative potency factors of PAHs, based on their genotoxic effect measured in vitro and analyzed with mathematical models. For this purpose, we used a new genotoxic assay (γH2AX) with two human cell lines (HepG2 and LS-174T) to analyze the genotoxic properties of 13 selected PAHs at low doses after 24h treatment. The dose-response for genotoxic effects was modeled with a Hill model; equivalency between PAHs at low dose was assessed by applying constraints to the model parameters. In the two cell lines tested, we observed a clear dose-response for genotoxic effects for 11 tested compounds. LS-174T was on average ten times more sensitive than HepG2 towards PAHs regarding genotoxicity. We developed new TEFs, which we named Genotoxic Equivalent Factor (GEF). Calculated GEF for the tested PAHs were generally higher than the TEF usually used. Our study proposed a new in vitro based method for the establishment of relevant TEFs for PAHs to improve cancer risk assessment.

Use of the γH2AX assay for assessing the genotoxicity of bisphenol A and bisphenol F in human cell lines.

Bisphenol A (BPA) and bisphenol F (BPF) are widely used to manufacture plastics and epoxy resins. Both compounds have been shown to be present in the environment and are food contaminants, with, as a result, a low but chronic exposure of humans. However, the fate and possible bioactivation of these compounds at the level of human cell lines was not completely elucidated yet. In this study, we investigated the ability of human cells (intestinal cell line: LS174T, hepatoma cell line: HepG2, and renal cell line: ACHN) to biotransform BPA and BPF, and focused on the cytotoxicity and genotoxicity of these two bisphenols, through the use of a novel and efficient genotoxic assay based on the detection of histone H2AX phosphorylation. BPA and BPF were extensively metabolized in HepG2 and LS174T cell lines, with stronger biotransformation capabilities in intestinal cells than observed in liver cells. Both cell lines produced the glucuronide as well as the sulfate conjugates of BPA. Conversely, the ACHN cell line was found to be devoid of any metabolic capabilities for the two examined bisphenols. Cytotoxicity was tested for BPA, BPF, as well as one metabolite of BPF produced in vivo in rat, namely dihydroxybenzophenone (DHB). In the three cell lines used, we observed similar ranges of toxicity, with DHB being weakly cytotoxic, BPF exhibiting an intermediary cytotoxicity, and BPA being the most cytotoxic compound tested. BPA and DHB were not found to be genotoxic, whatever the cell line examined. BPF was clearly genotoxic in HepG2 cells. These results demonstrate that some human cell lines extensively metabolize bisphenols and establish the genotoxic potential of bisphenol F.

Use of the γH2AX assay for assessing the genotoxicity of polycyclic aromatic hydrocarbons in human cell lines.

The development of in vitro genotoxic assays as an alternative method to animal experimentation is of growing interest in the context of the implementation of new regulations on chemicals. However, extrapolation of toxicity data from in vitro systems to in vivo models is hampered by the fact that in vitro systems vary in their capability to metabolize chemicals, and that biotransformation can greatly influence the experimental results. Therefore, much attention has to be paid to the cellular models used and experimental conditions. Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic ubiquitous pollutants. Human exposure to PAHs is mainly from food origin. In this study, a detailed analysis of the biotransformation capabilities of three human cell lines commonly used for in vitro testing (HepG2, ACHN and Caco-2) was undertaken using 3 model PAHs (benzo(a)pyrene [B(a)P], fluoranthene [FLA] and 3-methylcholanthrene [3-MC]). Concomitantly the genotoxicity of these PAHs was investigated in different cell lines, using a new genotoxic assay (H2AX) in 96-well plates. The metabolic rates of B(a)P, FLA and 3-MC were similar in HepG2 and Caco-2 cell lines, respectively, though with the production of different metabolites. The ACHN cell line was shown to express very limited metabolic capabilities. We demonstrated that the PAHs having a high metabolic rate (B(a)P and 3-MC) were genotoxic from 10(-7) molar in both HepG2 and Caco-2 cells. The present study shows that H2AX measurement in human cell lines competent for the metabolism, is an efficient and sensitive genotoxic assay requiring less cells and time than other currently available tests.

Characterisation of bioactive compounds in infant formulas using immobilised recombinant estrogen receptor-alpha affinity columns.

In this study, the use of recombinant estrogen receptor alpha (ERalpha)-based affinity columns was reported, for the isolation and the identification of estrogenic substances present in complex matrices, focusing on bioactive compounds present in foodstuff. The capability of affinity columns to trap high, but also low-affinity radio-labelled ligands (17beta-estradiol, genistein and bisphenol A) was demonstrated. Three pooled samples of infant formulas (milk-based, hypoallergenic and soy-based formulas for infants aged 0-4 months) from a EU market basket were prepared by the CASCADE Network of Excellence. After determining the estrogenic activity of these food samples, human recombinant ERalpha ligand binding domain (LBD) based affinity columns combined with suitable analytical methods (high resolution LC-MS/MS) were used to identify the bioactive compounds present in the soy-based formula extract, namely phytoestrogens (genistein and daidzein) involved in the agonistic activity measured. Incubations of genistein with liver microsomes were carried out and the extracts analysed following the same protocol, demonstrating that hERalpha affinity columns can also be used for trapping active metabolites. This approach combining bioluminescent cell lines with this useful tool based on hERalpha-LBD affinity columns thus allowed the purification and the concentration of both known and unknown estrogenic ligands prior to investigation of their structure using LC-MS.

Biotransformation of genistein and bisphenol A in cell lines used for screening endocrine disruptors.

In vitro assays provide the opportunity for generating alerts for chemicals which interact with hormone receptors and are also valuable tools for mechanistic research. However, the limited capabilities of in vitro models to metabolically activate or inactivate xenobiotics may lead to misinterpretation of the in vitro data if such information is not taken into account. The aim of this study was to investigate the metabolic capabilities of human HepG2, human MCF7 and mouse HC11 cell lines used for testing endocrine disruptors (EDs) toward radiolabelled bisphenol A and genistein, two estrogenic compounds for which metabolic pathways in vivo as in vitro are well known. Incubations were performed during 12-48 h with 250.10(3) cells in 12 wells plates and 5-25 microM of substrates. The kinetics of formation of the metabolites were studied. Rat liver slices were used as reference for comparison with the metabolic capabilities of the cell lines. HC11 cells did not show any biotransformation capability while the major biotransformation pathways in HepG2 and MCF7 cells were conjugation to sulfate and to a lesser extent to glucuronic acid. We detected no phase I metabolite, even in rat liver slices. These results suggest that HC11 cells should be a valuable cellular system to study the intrinsic estrogenic activity of the tested compound, while HepG2 and MCF7 cells can help to take into account part of the metabolic fate of the tested compound that occur in vivo. However, since phase I enzymes are poorly or not at all expressed in these systems, their use in endocrine disruptor testing may result in false negative for compounds for which bioactivation is a prerequisite.

Proteomic and phosphoproteomic analysis of cellular responses in medaka fish (Oryzias latipes) following oral gavage with microcystin-LR.

Chronic and subchronic toxicity resulting from exposure to microcystins (MCs) receives increasing attention due to the risk of bioaccumulation of these toxins by aquatic animals, including fish. The mechanisms of action of MCs that target the liver, involve modifications of protein phosphorylation resulting from phosphatases 1 and 2A inhibition. Therefore, studying phosphoprotein modifications by using a specific phosphoprotein stain Pro-Q Diamond in fish liver contaminated with MC-leucine-arginine (MC-LR), the most toxic MC, should help dissecting disturbed signaling and metabolic networks. We have recently used this technology to identify several proteins that are modulated either in expression or phosphorylation in the liver of medaka following short-term exposure to MC-LR by balneation. In the present study, we have decided to use an alternative way of introducing the toxin into fish; that is by gavage (force-feeding). This was first achieved using tritiated MC-LR and allowed us to quantify the quantity of toxin incorporated into fish and to demonstrate that the toxin is mainly accumulated in liver. Afterwards a proteomics study limited to liver cytosolic proteins of contaminated animals showed that several proteins were up or down regulated either in quantity or in phosphorylation or both. Some of them had been previously detected as modified in balneation experiments but new molecules were identified as involved in signal transduction pathways activated by the toxin. In addition, in the conditions used (5 microg toxin/g body weight) anatomopathological studies supported a process of apoptonecrosis established after 24h, which was suggested to proceed by the evolution of some of the proteins after 2h contamination.

Occupational exposure to pesticides and risk of hematopoietic cancers: meta-analysis of case-control studies.

OBJECTIVE: In this study we conducted a meta-analysis of 13 case-control studies that examined the occurrence of hematopoietic cancers in pesticide related occupations in order to undertake a qualitative and quantitative evaluation of a possible relationship. METHODS: Pubmed databases were searched for case-control studies published between 1990 and 2005 investigating the relation between hematopoietic cancers and occupational exposure to pesticides. Fixed and random effect meta-analysis models were used depending on the presence of heterogeneity between studies. RESULTS: The overall meta-odds ratio obtained after pooling 44 ORs from 13 studies was 1.3 (95% CI: 1.3-1.5). We realized stratified analysis on three different types of hematopoietic cancers (non-Hodgkin lymphoma (NHL), leukemia and multiple myeloma). A significant increased risk of NHL was found (OR = 1.35; 95% CI = 1.2-1.5). Moreover, increased risks of Leukemia (OR = 1.35; 95% CI = 0.9-2) and multiple myeloma (OR = 1.16; 95% CI = 0.99-1.36) were also detected but these results were not statistically significant. Significant heterogeneity existed among the different studies and a publication bias was detected. Therefore, a meta-regression was carried out. Our results showed that a long period of exposure (more than 10 years) provided an increase in the risk of all hematopoietic cancers and for NHL by fractions of 2.18 (95% CI = 1.43-3.35) and 1.65 (95% CI = 1.08-2.51), respectively. CONCLUSIONS: The overall meta-odds ratio suggests that there is a significantly positive association between occupational exposure to pesticides and all hematopoietic cancers as well as NHL. A major limitation of our meta-analysis is the lack of sufficient data about exposure information and other risk factors for hematopoietic cancer (genetic predisposition, ethnic origin, immunodepression...). In addition, data concerning specific subtypes of hematopoietic cancers are often confusing. Thus, future epidemiological studies should undertake a major effort to assess the identity and the level of pesticides exposure and should control for the most likely potential confounders.

Disposition of genistein in rainbow trout (Oncorhynchus mykiss) and siberian sturgeon (Acipenser baeri).

Genistein (G) is a xenoestrogen from soy present in fish diet. In vivo, a 50-fold difference in sensitivity to genistein on vitellogenin (VTG) synthesis was found when comparing trout and sturgeon. This difference was not linked to the estrogen receptor affinity nor to the sensitivity of induction of the VTG pathway. The study was performed to check if differences in the G disposition in the two species could explain their difference of sensitivity to G. A pharmacokinetic analysis of radiolabeled G was performed to determine its bioavailability and metabolism in both species. G was used at levels corresponding to fish farm exposure. G plasma levels after chronic ingestion were found to be 15.6 times higher in sturgeon than in trout. Sturgeon primarily produces sulfate conjugates after G ingestion whereas trout mainly produces glucuronides. Sturgeon was able to excrete orobol glucuronide in bile. An important first pass effect was suggested in both species. No accumulation of G or its metabolites was observed in the two species. Trout muscles accounted only for 0.14 of radioactivity 48 h post-ingestion similarly to sturgeon. Trout viscera accounted for 15% of the radioactivity 48 h post-ingestion. In sturgeon, 48 h post-ingestion, viscera accounted for 21.5% of the radioactivity. These rates decreased rapidly thereafter. The study partly explains the difference in sensitivity to G, previously recorded between the two species. In addition, it shows that human exposure to G through farmed fish consumption is negligible.

Removal of bisphenol A by a nanofiltration membrane in view of drinking water production.

The efficiency with which a nanofiltration membrane (Desal 5 DK) removes bisphenol A (BPA) was investigated, together with the mechanisms involved. Whereas high retention (>90%) was obtained at the beginning of the filtration, the observed retention coefficient (R(obs)) decreased to around 50% when the membrane became saturated, due to adsorption of BPA onto the membrane structure. The presence of ions (Na+, Cl-) affects the R(obs), this effect being attributed to a change in BPA hydrodynamic radius. Moreover, in our operating conditions, the presence of natural organic matter (1mg/L) in the feed solution does not lead to variation in BPA retention at steady state.

Dichloroaniline retention by nanofiltration membranes.

This study evaluates the performance of two nanofiltration membranes in removing a herbicide: dichloroaniline. The membranes, one polyamide and one cellulose acetate, have a cut-off in the range 150-300 g/mol (manufacturers' data). The experiments were carried out with solutions of dichloroaniline in demineralized water, with concentrations from 1 to 10 ppb. For each membrane, the amount of herbicide retained and adsorbed by the membrane was determined as a function of feed concentration and transmembrane pressure. The two membranes, made of different materials but having the same nominal cut-off, retained dichloroaniline to very different extents and by different mechanisms.

Metabolic fate of [14C]-2,4-dichlorophenol in macrophytes.

The metabolic fate of 2,4-dichlorophenol (DCP) was investigated in six macrophytes representing different life forms. Salvinia natans and Lemna minor were chosen as surface-floating plants, Glyceria maxima and Mentha aquatica as emergent species and Myriophyllum spicatum and Hippuris vulgaris as submerged aquatic plants. After uptake of a [U-phenyl-14C]-DCP solution followed by a 48 h water chase, whole plants (L. minor, S. natans) or excised shoots were harvested and aqueous extracts were analysed by high performance liquid chromatography (HPLC). Metabolites were then isolated, submitted to enzymatic or chemical hydrolyses and characterised by electrospray ionisation-mass spectrometric analyses. Whereas DCP monoglucosides or more complex monoglucoside esters, either malonyl or acetyl, were found in most species, an unusual glucosyl-pentose conjugate was identified as the DCP major metabolite in L. minor and G. maxima. Our results showed for the first time the ability of five macrophytes to uptake and metabolise DCP and the characterisation of their metabolic pathways of DCP biotransformation.

Regio-specific hydroxylation of nonylphenol and the involvement of CYP2K- and CYP2M-like iso-enzymes in Atlantic salmon (Salmo salar).

Previously, it has been demonstrated that nonylphenol (NP) has a dual function in regulating reproductive hormones by: (1) increasing the activity of steroid metabolizing enzymes at low concentration, that does not induce vitellogenin (Vtg) and zona radiata proteins and (2) decreasing the activity of these enzymes at higher concentration [Environ. Health Perspect. 105 (1997) 109; Environ. Toxicol Chem. 16 (1997) 2576]. To more precisely understand the estrogenic effects of NP in fish and a possible interference with steroid hormone metabolism, we investigated in the Atlantic salmon the identity of the cytochrome P450 enzymes involved in NP hydroxylation. Up to 9 metabolites were separated by radio-HPLC when [R-(14)C]-4n-NP was incubated with hepatic microsomes from juvenile salmon. In control fish the major metabolites were identified as monohydroxylated products at omega-, (omega-1)- and (omega-2)-positions of the alkyl chain of 4n-NP. Salmon hepatic microsomes formed omega-, (omega-1)- and (omega-2)-lauric acid (LA) hydroxylation products. The potency of alpha-naphthoflavone, ketoconazole and ethynylestradiol (non-specific, but strong inhibitors of CYP1A, 2K and 3A, respectively) on LA and NP hydroxylations were assessed in the present study. Ketoconazole inhibited omega-, (omega-1)- and (omega-2)-hydroxylations of LA and 4n-NP and was the only inhibitor of omega-hydroxylation of both substrates. Ethynylestradiol specifically inhibited (omega-1)- and (omega-2)-hydroxylations of LA as well as 4n-NP. Interestingly, the lowest NP dose (1 mg/kg) was the most potent inducer of NP-metabolites formation. These results suggest the involvement of CYP2M- and 2K-like enzymes in terminal and subterminal hydroxylations of 4n-NP respectively, and was confirmed by the competitive inhibition between LA and 4n-NP. The production of one unidentified 4n-NP metabolite was not affected by any of the chemicals used, suggesting a possible ring hydroxylation with involvement of another cytochrome P450 isoform. Our data reveal a novel aspect of CYP isozymes involvement in NP metabolism that may complicate the assessment of its endocrine effects. Hence, the regio-selective hydroxylation of endocrine disruptors, such as NP, by CYP isozymes is revealed as a possible new marker of estrogenicity.

Identification of the major metabolites of prochloraz in rainbow trout by liquid chromatography and tandem mass spectrometry.

The metabolic pattern of the imidazole fungicide prochloraz [N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]imidazole-1-carboximide] was investigated in rainbow trout (Oncorhynchus mykiss). Following a single oral administration of [(14)C]prochloraz, levels 4.3 +/- 4.1 and 3.9 +/- 1.8% of the dose were excreted in the bile after 48 h in male and female animals, respectively. Urinary radioactivity accounted for 1.3 +/- 0.4 and 2.4 +/- 1.1% of the dose over the same period in males and females. Metabolites from both matrices were separated by reversed-phase HPLC with radioactive detection and analyzed by positive and/or negative electrospray ionization mass spectrometry. No unchanged prochloraz was detected in the analyzed excreta. The major biotransformation products in bile were the aldehyde corresponding to the cleavage of the imidazole ring, N-2-(2,4,6-trichlorophenoxy)ethylurea, and the glucuronide conjugate of 2,4,6-trichlorophenoxyethanol. In urine, the major metabolite was 2,4,6-trichlorophenoxyacetic acid. On the basis of enzymatic hydrolysis by beta-glucuronidase and LC-MS analyses, this study demonstrates that rainbow trout are able to biotransform prochloraz, mainly as glucuronide conjugates.

Metabolic activity in primary cultures of fish hepatocytes.

In aquatic toxicology, isolated liver cells from fish can be used as a tool to generate initial information on the hepatic metabolism of xenobiotics, and on the mechanisms of xenobiotic activation or deactivation. This isolation of teleost liver cells is achieved by enzymic dissociation, and monolayer cultures of fish hepatocytes in serum-free medium maintain good viability for 3-8 days. During in vitro culture, fish liver cells express stable levels of phase I and phase II enzymes, such as cytochrome P4501A or glutathione S-transferase, and the cells show an induction of biotransformation enzymes after exposure to xenobiotics. The xenobiotic metabolite pattern produced by fish hepatocytes in vitro is generally similar to that observed in vivo. Limitations to more-intensive application of cultured fish hepatocytes as a screen in aquatic hazard assessment are partly due to the rather limited scope of existing studies, i.e. the focus on one particular species (rainbow trout), and on one particular biotransformation enzyme (cytochrome P4501A), as well as a lack of comparative in vitro/in vivo studies.