Elsevier

Toxicology in Vitro

Volume 22, Issue 5, August 2008, Pages 1128-1135
Toxicology in Vitro

Effects of fibrates, anti-inflammatory drugs and antidepressants in the fish hepatoma cell line PLHC-1: Cytotoxicity and interactions with cytochrome P450 1A

https://doi.org/10.1016/j.tiv.2008.02.020Get rights and content

Abstract

Effects of 11 pharmaceuticals belonging to three therapeutic classes (lipid regulators from the fibrate group, non-steroidal anti-inflammatory drugs and anti-depressives from the selective serotonin reuptake inhibitors group) were assessed in the fish hepatoma cell line (PLHC-1) by looking at cytotoxicity and interactions with cytochrome P450 1A (CYP1A) function. Among the tested pharmaceuticals, fluoxetine and paroxetine exerted cytotoxic effects, cell viability decreased to 52% and 6% after 24 h of exposure to 20 μM fluoxetine and paroxetine, respectively. The cytotoxicity of both compounds was modulated by cytochrome P450 inhibitors and was dramatically reduced when culture medium was supplemented with reduced glutathione and vitamin E succinate. Additionally, exposure of PLHC-1 cells to some pharmaceuticals led to an early and transient induction of ethoxyresorufin O-deethylase (EROD) activity: bezafibrate and antidepressants induced EROD activity at a concentration of 1 μM whereas clofibrate, ibuprofen and naproxen acted as inducers at a higher concentration (10 μM). These effects might be of toxicological concern since alterations of CYP1A may affect xenobiotic metabolism and toxicity.

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.

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