Effects of fibrates, anti-inflammatory drugs and antidepressants in the fish hepatoma cell line PLHC-1: Cytotoxicity and interactions with cytochrome P450 1A
Introduction
Human pharmaceuticals enter the aquatic environment via discharges from sewage treatment plants where they reach considerable concentrations (Daughton and Ternes, 1999, Thomas and Hilton, 2004, Wiegel et al., 2004). In surface waters, pharmaceuticals generally occur at low concentrations (ng–μg/L) and do not exert acute toxicity for the aquatic fauna (Halling-Sørensen et al., 1998, Daughton and Ternes, 1999, Kolpin et al., 2002). Nevertheless, low-concentration and long-term effects of pharmaceuticals in non-target organisms are largely unknown and remain to be investigated.
In humans, pharmaceuticals are metabolized mainly by members of cytochrome P450 (CYP) families 1–4 (Tredger and Stoll, 2002). Moreover, pharmaceuticals have the potential to cause side effects in both target and non-target organisms through inhibition or induction of CYP isoforms. The study of interactions between pharmaceuticals and CYPs is of critical importance to better assess the environmental risk of these compounds, with a special attention to CYP1A isoforms. Indeed, CYP1A enzymes are involved in the activation of carcinogenic xenobiotics such as polycyclic aromatic hydrocarbons to electrophilic reactive metabolites, leading to toxicity and cancer, which may be an issue in long-lived animals such as fish (Lin, 2006, Ma and Lu, 2007). In fish, many environmental pollutants such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans act as CYP1A inducers, and CYP1A has been recognized as biomarker for the assessment of aquatic pollution (Whyte et al., 2000). Additionally, CYP1A induction is closely related to detrimental effects such as apoptosis and embryonic mortality in exposed fish (Cantrell et al., 1996, Dong et al., 2002). Thus, the interaction of pharmaceutical compounds with CYP1A enzymes is likely to have a significant toxicological relevance in fish.
In a previous work we demonstrated that widely used pharmaceuticals belonging to various therapeutic classes were able to inhibit the catalytic activity of different cytochrome P450 enzymes in carp liver (Thibaut et al., 2006). Among them, CYP1A-catalyzed activity (ethoxyresorufin O-deethylase EROD) was strongly inhibited by fibrate and anti-depressive drugs.
Thus, the present work aimed at further assessing the effects of pharmaceutical compounds in fish by looking at in vitro cytotoxicity as well as the interaction with CYP1A enzymes and the potential consequences in terms of cell toxicity. The PLHC-1 cell line was selected for the study because it contains the aryl hydrocarbon receptor and an inducible CYP1A system (Hahn et al., 1993, Fent, 2001). The study was carried out on 11 pharmaceuticals belonging to three therapeutic classes: lipid regulators from the fibrate group (clofibrate, bezafibrate, fenofibrate and gemfibrozil), non-steroidal anti-inflammatory drugs (ibuprofen, diclofenac, naproxen and ketoprofen) and anti-depressives (fluoxetine, fluvoxamine and paroxetine). Fibrates are one of the several classes of blood lipid regulators commonly prescribed to reduce hypercholesterolemia and to prevent coronary heart disease. They are highly used, ubiquitous and persistent (Daughton and Ternes, 1999). Clofibrate, bezafibrate, fenofibrate and gemfibrozil have been detected in river water, ground water and sea water at nanogram to microgram per litre level. Non-steroidal anti-inflammatory drugs are commonly used for the treatment of fever, pain and inflammation. Concentrations of ibuprofen, diclofenac, naproxen and ketoprofen raising microgram per litre were reported in the aquatic environment (Daughton and Ternes, 1999, Heberer, 2002, Fent et al., 2006). Fluoxetine, fluvoxamine and paroxetine are selective serotonin reuptake inhibitors, a major class of widely prescribed antidepressants that includes Prozac, Luvox and Paxil. Fluoxetine has been detected in municipal sewage effluents and surface waters (Kolpin et al., 2002, Metclafe et al., 2003) and recently found in fish tissues at a concentration greater than 0.1 ng/g (Brooks et al., 2005). Most of the selected pharmaceuticals have been shown to interact with CYP enzymes, including CYP1A isoforms, both in humans and rat. Indeed, fibrates are well-known inducers of CYP4A isoforms (Amacher et al., 1997, Raucy et al., 2004); ibuprofen and ketoprofen were reported to decrease CYP1A activity in rat liver (Pappas et al., 1998), and antidepressants from selective serotonin reuptake inhibitors class are potent inhibitors of CYP1A2, CYP2C19 and CYP2D6 (Hemeryck and Belpaire, 2002).
Section snippets
Chemicals and solutions
Eagle’s Minimum Essential Medium, foetal bovine serum, l-glutamine, sodium pyruvate, nonessential amino acids, penicillin G, streptomycin, phosphate buffered saline (PBS) and trypsin-EDTA were from Gibco BRL Life Technologies (Paisley, Scotland, UK). Reduced glutathione (GSH), vitamin E (d-α-tocopherol) succinate, 7-ethoxyresorufin, 7-hydroxyresorufin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), α-naphthoflavone (ANF), β-naphthoflavone (BNF), ethynylestradiol (EE2) and
Cytotoxicity
A small but significant decrease in cell viability was observed in cells incubated with 20 μM gemfibrozil, naproxen, ketoprofen or fluvoxamine for 6 h (9–21%), however, cell viability returned to control levels with longer exposure times (Fig. 1). Among the tested compounds (20 μM), fluoxetine and paroxetine had the strongest cytotoxic effect: cell viability was of 82% and 70% after 6 h exposure and further decreased to 52% and 6% after 24 h.
Cytochrome P450 inhibitors (PBO, ANF and EE2) were not
Cytotoxic effects of pharmaceuticals
Among the tested pharmaceuticals, fluoxetine and paroxetine were cytotoxic on PLHC-1 cells; cytotoxicity was relatively weak (18–30%) after 6 h of incubation but it increased with exposure length. The cytotoxicity of fluoxetine has been previously reported in fish cell cultures; the concentration of drug required to reach 50% of the maximal cytotoxic effect (EC50) was of 5 μM in PLHC-1 cells exposed for 24 h (Laville et al., 2004), and of 66 μM in primary rainbow trout hepatocytes (Laville et al.,
Conclusion
Fluoxetine and paroxetine (20 μM) exerted cytotoxic effects in PLHC-1 cells; the metabolism of these compounds can induce reactive metabolites formation and/or oxidative stress, damage of cell membranes and lead to cell death. Cytotoxicity of pharmaceuticals was modulated by cytochrome P450 inhibitors, suggesting that P450- and mainly CYP1A-dependent metabolism reduces cell toxicity. Clofibrate, bezafibrate, ibuprofen, naproxen and antidepressants (1–10 μM) also induced CYP1A activity. These
Acknowledgements
This study was supported by the Spanish Ministry of Science and Education under Project Ref. CGL2005-02846. Dr. Rémi Thibaut acknowledges an I3P contract from the Spanish Government.
References (58)
- et al.
Hepatic microsomal enzyme induction, β-oxidation, and cell proliferation following administration of clofibrate, gemfibrozil, or bezafibrate in the CD rat
Toxicology and Applied Pharmacology
(1997) - et al.
Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death
Brain Research Review
(1997) - et al.
Cytotoxicity of pharmaceuticals found in aquatic systems: comparison of PLHC-1 and RTG-2 fish cell lines
Aquatic Toxicology
(2006) - et al.
Embryotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): the embryonic vasculature is a physiological target for TCDD-induced DNA damage and apoptotic cell death in medaka (Orizias latipes)
Toxicology and Applied Pharmacology
(1996) - et al.
Enhanced antioxidant and cytoprotective abilities of vitamin E succinate is associated with a rapid uptake advantage in rat hepatocytes and mitochondria
Free Radical Biology and Medicine
(2001) Fish cell lines as versatile tools in ecotoxicology: assessment of cytotoxicity, cytochrome P4501A induction potential and estrogenic activity of chemicals and environmental samples
Toxicology in Vitro
(2001)- et al.
Ecotoxicology of human pharmaceuticals
Aquatic Toxicology
(2006) - et al.
Occurrence of pharmaceutical products in a municipal effluent and toxicity to rainbow trout (Oncorhynchus mykiss) hepatocytes
Ecotoxicology and Environmental Safety
(2006) - et al.
Cytochrome P4501A induction and inhibition by 3,3′,4,4′-tetrachlorobiphenyl in an Ah receptor-containing fish hepatoma cell line (PLHC-1)
Aquatic Toxicology
(1993) - et al.
Occurrence, fate and effects of pharmaceutical substances in the environment – a review
Chemosphere
(1998)
Tracking persistent pharmaceutical residues from municipal sewage to drinking water
Journal of Hydrology
Induction of drug metabolizing enzymes: a survey of in vitro methodologies and interpretations used in the pharmaceutical industry – do they comply with FDA recommendations?
Chemico-Biological Interactions
Analysis of the effects diclofenac has on Japanese medaka (Oryzias latipes) using real-time PCR
Chemosphere
Effects of human pharmaceuticals on cytotoxicity, EROD activity and ROS production in fish hepatocytes
Toxicology
Fibrates and their newly synthesized glycinate or glycinate-methylester derivatives: comparison of their interactions with liver cytochrome P450 dependent monooxygenase- and oxidase-functions in vitro
Experimental and Toxicologic Pathology
Characterization of the hepatic responses to the short-term administration of ciprofibrate in several rat strains. Co-induction of microsomal cytochrome P450IVA1 and peroxisome proliferation
Biochemical Pharmacology
Glutathione deficiency produced by inhibition of its synthesis, and its reversal; applications in research and therapy
Pharmacology and Therapeutics
Evaluation of chemicals as inhibitors of trout cytochrome P450s
Toxicology and Applied Pharmacology
Antioxidant defense against antidepressants in C6 and 1321N1 cells
Chemico-Biological Interactions
Inhibition of in-vitro aflatoxin b-1-DNA binding in rainbow trout by CYP1A inhibitors – alpha-naphthoflavone, beta-naphthoflavone, and trout CYP1A peptide antibody
Comparative Biochemistry and Physiology C
The occurrence of selected human pharmaceutical compounds in UK estuaries
Marine Pollution Bulletin
Glutathione depletion and the production of reactive oxygen species in isolated hepatocyte suspensions
Chemico-Biological Interactions
Pharmaceuticals in the river Elbe and its tributaries
Chemosphere
Vitamin E succinate protects hepatocytes against the toxic effect of reactive oxygen species generated at mitochondrial complexes I and III by alkylating agents
Chemico-Biological Interactions
Toxicological effects of the lipid regulator gemfibrozil in four aquatic systems
Aquatic Toxicology
The conduct of in vitro and in vivo drug–drug interaction studies: a pharmaceutical research and manufacturers of America (PhRMA) perspective
Drug Metabolism and Disposition
The role of cytochrome P4502D6 in the metabolism of paroxetine by human liver microsomes
British Journal of Clinical Pharmacology
Evidence that 4-allyl-o-quinones spontaneously rearrange to their more electrophilic quinone methods: potential bioactivation mechanism for the hepatocarcinogen safrole
Chemical Research in Toxicology
Determination of select antidepressants in fish from an effluent-dominated stream
Environmental Toxicology and Chemistry
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