Elsevier

Chemosphere

Volume 108, August 2014, Pages 334-342
Chemosphere

Cytotoxicity of binary mixtures of human pharmaceuticals in a fish cell line: Approaches for non-monotonic concentration–response relationships

https://doi.org/10.1016/j.chemosphere.2014.01.077Get rights and content

Highlights

  • The in vitro cytotoxicity of ten pharmaceuticals was investigated using RTG-2 cells.

  • Biphasic concentration–response relationships were observed for some compounds.

  • Cytotoxicity of binary mixtures could be predicted using concentration addition.

  • The in vitro mode of toxic action for the pharmaceuticals tested was non-specific.

Abstract

Predicting the effects of mixtures of environmental micropollutants is a priority research area. In this study, the cytotoxicity of ten pharmaceuticals to the rainbow trout cell line RTG-2 was determined using the neutral red uptake assay. Fluoxetine (FL), propranolol (PPN), and diclofenac (DCF) were selected for further study as binary mixtures. Biphasic concentration–response relationships were observed in cells exposed to FL and PPN. In the case of PPN, microscopic examination revealed lysosomal swelling indicative of direct uptake and accumulation of the compound. Three equations describing non-monotonic concentration–response relationships were evaluated and one was found to consistently provide more accurate estimates of the median and 10% effect concentrations compared with a sigmoidal concentration–response model. Predictive modeling of the effects of binary mixtures of FL, PPN, and DCF was undertaken using an implementation of the concentration addition (CA) conceptual model incorporating non-monotonic concentration–response relationships. The cytotoxicity of the all three binary combinations could be adequately predicted using CA, suggesting that the toxic mode of action in RTG-2 cells is unrelated to the therapeutic mode of action of these compounds. The approach presented here is widely applicable to the study of mixture toxicity in cases where non-monotonic concentration–response relationships are observed.

Introduction

A large number of studies have reported the occurrence of pharmaceuticals in treated wastewater destined for release to the environment (Kasprzyk-Hordern et al., 2009, Miège et al., 2009, Behera et al., 2011, Jelic et al., 2011, Martín et al., 2012, Ratola et al., 2012, Yu et al., 2013). Active pharmaceutical ingredients designed to modulate a specific molecular target in the human body also have the potential to modulate structurally related targets in aquatic animals, particularly vertebrates such as fish (Fent et al., 2006). Reported concentrations of most pharmaceuticals found in municipal wastewater treatment plant (WWTP) effluents and receiving waters are generally below the lowest concentrations known to cause acute toxicity in fish when applied singly (Corcoran et al., 2010), although bioaccumulation of some pharmaceuticals has been observed in fish sampled from effluent-dominated water courses (Brooks et al., 2005, Ramirez et al., 2009) and during laboratory exposures (Schwaiger et al., 2004), indicating that tissue concentrations can reach higher levels than concentrations measured in environmental samples. Furthermore, some compounds such as synthetic steroid contraceptives are known to affect the reproductive success of fish at environmentally relevant concentrations (Kidd et al., 2007). Investigating the possible ecological effects of pharmaceutical mixtures has been identified as a priority research area (EEA, 2010, Boxall et al., 2012). However, predicting the effects of complex mixtures of low concentrations of pharmaceuticals remains a challenge, particularly when possible adverse interactions have not been characterized in susceptible aquatic organisms.

Fish cell lines have long been used for toxicity screening of individual chemicals and complex mixtures, for ranking relative toxicity, and for establishing structure–toxicity relationships (Segner, 1998). Cultured cells represent a convenient model system for examining mixture toxicity prior to undertaking whole-organism studies as they allow large numbers of compounds and combinations to be tested rapidly and cost-effectively.

In this study, we investigated the in vitro toxicity of selected pharmaceuticals in RTG-2 cells (Wolf and Quimby, 1962), a fibroblast-like cell line derived from mixed male and female gonad tissue of rainbow trout (Oncorhynchus mykiss). Cytotoxicity was determined using the neutral red uptake (NRU) assay (Repetto et al., 2008) a cell viability assay widely utilized for in vitro toxicity studies conducted using cultured animal cells. Compounds were selected on the basis of their reported occurrence in WWTP effluents and surface waters, and included four β-adrenergic receptor blockers (β-blockers; atenolol [ATL], metoprolol [MP], pindolol [PD] and propranolol [PPN]), two selective serotonin reuptake inhibitors (fluoxetine [FL] and venlafaxine [VFX]), a non-steroidal anti-inflammatory drug (diclofenac [DCF]), an anti-convulsive (carbamazepine [CBZ]), an anti-rheumatic chemotherapeutic (methotrexate [MTX]), and a synthetic steroidal contraceptive (17α-ethinylestradiol [EE2]). For compounds exhibiting cytotoxicity, cells were exposed to binary mixtures and the resulting concentration–response profiles were compared to responses predicted using mixture modeling approaches.

Section snippets

Chemicals

All chemicals and solvents were purchased from Sigma–Aldrich Pty. Ltd., Australia, unless otherwise specified.

Cell culture and treatments

RTG-2 cells (ECACC 90102529) were obtained from Sigma–Aldrich Pty. Ltd., Australia. Cells were maintained in Liebovitz’ L-15 (Gibco) supplemented with 50 μg mL−1 streptomycin (Gibco), 50 U mL−1 penicillin (Gibco), 1 × MEM non-essential amino acids (Gibco), 15 mM HEPES buffer (pH 7.4; Gibco), and 10% fetal bovine serum (FBS; Gibco). This medium is referred to hereafter as ‘standard growth

Cytotoxicity of individual pharmaceuticals

The aim of this study was to investigate the in vitro cytotoxicity of binary combinations of pharmaceuticals in a fish cell line and to establish whether the toxicity of combined exposures was additive or otherwise. To select candidates from a range of pharmaceutical classes for further study as binary mixtures, cytotoxicity screening of individual compounds in RTG-2 cells was undertaken (Table 1). This showed that FL was the most cytotoxic of the pharmaceuticals tested (72 h EC of 35 μM or 12.1 mg

Conclusions

Reports of uptake, metabolism and elimination of pharmaceuticals by fish in aquatic environments receiving WWTP effluent are limited, but the available data suggest that bioaccumulation of the compounds studied in the present report can occur. Carbamazepine, fluoxetine and norfluoxetine (the major metabolite of fluoxetine) have been detected in the ng g−1 range in liver, muscle and brain tissues of fish sampled from locations in close proximity to WWTP effluent discharges (Brooks et al., 2005,

Acknowledgements

We thank Niels Zaagman for technical assistance. This work was funded by the New South Wales Environmental Trust and CSIRO (Project R-0538-01).

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