Elsevier

Aquatic Toxicology

Volumes 140–141, 15 September 2013, Pages 249-256
Aquatic Toxicology

The mode of action (MOA) approach reveals interactive effects of environmental pharmaceuticals on Mytilus galloprovincialis

https://doi.org/10.1016/j.aquatox.2013.06.005Get rights and content

Highlights

  • The mode of action approach guided the analysis of fluoxetine (FX) and propranolol (PROP) effects in Mediterranean mussels.

  • Interactive effects on cAMP/PKA system agree with a direct (PROP) and indirect (FX) action on specific molecular targets.

  • mRNA levels of a mussel 5-HT1 receptor were up-regulated by FX, while unchanged by PROP or FX + PROP.

  • mRNA levels of a ABCB gene encoding the P-glycoprotein was also affected, since this gene product is under a PKA regulation.

  • The mode of action approach suggests an evolutional conservation of biological targets between human and mussels.

Abstract

Aquatic organisms are unintentionally exposed to a large number of pharmaceutical residues in their natural habitats. Ecotoxicological studies have agreed that these compounds are not harmful to aquatic organisms, as their environmental concentrations are typically too low. However, recent reports have shown biological effects at such low concentrations when biological endpoints related to the therapeutic effects are assessed. Therefore, conservation of molecular targets is now addressed as a key aspect for the development of more efficient test strategies for pharmaceutical environmental risk assessment, providing the rationale for the mode of action (MOA) approach. In the present study the MOA approach was used to investigate the interactive effects of fluoxetine (FX) and propranolol (PROP) on the Mediterranean mussels (Mytilus galloprovincialis). Indeed, organisms in the environment are exposed to pharmaceutical mixtures throughout their lifetime, and particular combinations may be of concern. The antidepressant FX increases serotonin (5-HT) levels in the synaptic cleft by inhibiting 5-HT reuptake. PROP, a prototypical β-adrenoceptor antagonist, also blocks 5-HT1 receptors, which are negatively coupled to cAMP-mediated signaling. Cell signaling alterations potentially triggered by 5-HT1 receptor occupation were therefore assessed after a 7-day mussel exposure to FX or PROP, alone or in combination, each at 0.3 ng/L concentration. FX decreased cAMP levels and PKA activities in digestive gland and mantle/gonads, in agreement with an increased occupation of 5-HT1 receptors. PROP caused a decrease in cAMP levels and PKA activities in digestive gland and an increase in cAMP levels in mantle/gonads, consistent with a differential expression of adrenergic and 5-HT receptors in the two tissues. Co-exposure to FX and PROP provides significant indications for antagonistic effects of the pharmaceuticals, consistent with a direct (PROP) and indirect (FX) action on the same molecular target. Interestingly, FX induced over-expression of a 5-HT1 gene product, and PROP counteracted such increase when the mixture was administered, while having per se no effect. Finally, mRNA expression of the ABCB gene encoding the MXR-related transporter P-glycoprotein was reduced by both pharmaceuticals in the digestive gland, while decreased by FX, increased by PROP, and not affected by the mixture in mantle/gonads. Since transcription of this gene product is under cAMP/PKA modulation, the impairment of regulatory pathways triggered by low concentrations of pharmaceuticals have the potential to affect the ability of animals to elaborate strategies of defense or adaptation toward further stress factors. In this specific case, the pharmaceutical mixture limits the detrimental effects of the single compounds.

Introduction

A range of organisms is unintentionally exposed to a large number of pharmaceutical residues in their natural habitats (Boxall and Long, 2005, Heberer, 2002, Kolpin et al., 2002, Larsson et al., 2007). Ecotoxicological studies agree that these compounds generally do not cause acute toxicity to aquatic organisms, as their environmental concentrations are typically too low (reviewed in Fent et al., 2006, Santos et al., 2010). Although chronic studies continue to be in the minority, there is evidence for negative impacts of these contaminants on living organisms and ecosystems (Fent et al., 2006, Santos et al., 2010). Indeed, pharmaceuticals behave quite differently from conventional pollutants, being designed to affect specific molecular targets at relatively low doses; therefore, low concentrations do not represent per se a safety factor.

According to the current guidelines for environmental risk assessment (ERA) of human (EMEA/CHMP, 2006) and veterinary (VICH, 2000, VICH, 2005) pharmaceuticals, the ERA process consists of an initial exposure assessment (Phase I) followed by a fate and effect analysis (Phase II), which is required only when exposure-based threshold values, or action limits, are exceeded. Little information is available for evaluating whether the actual action limits do represent reliable safety factors or whether they may prevent environmentally hazardous substances from being assessed. Indeed, to date most studies on ecotoxicity of pharmaceuticals deal with acute effects, whereas data on long-term toxicity are scarce and often limited to laboratory test species (reviewed in Schmitt et al., 2010).

A literature review on acute and chronic effects of pharmaceuticals on different experimental models (Schmitt et al., 2010) highlights that when the spectrum of test organisms employed is extended to environmentally relevant species, and biological endpoints related to the therapeutic actions of the pharmaceutical are assessed, significant effects of pharmaceuticals at concentrations below or close to the action limits can be detected. It is possible to hypothesize that these compounds can be pharmacologically active in organisms in which the drug targets are expressed and functional; therefore, the evolutionary conservation of molecular targets in a given species could potentially increase the risk of ecotoxicological effects (Gunnarsson et al., 2008, Schmitt et al., 2010).

Recently, a conceptual model defined “mode-of-action” (MOA) approach was developed to address the issue of environmental risk posed by pharmaceuticals (ECETOC, 2007, Christen et al., 2010, Schmitt et al., 2010). The suggested approach assumes that all the relevant information about a pharmaceutical's therapeutic effect and toxicity in mammals must be taken into account and integrated with the knowledge of the physiology of the species used as the experimental model. In particular, the degree of evolutionary and functional conservation of drug targets between mammals and the model species should be assessed, together with the physiological pathways potentially affected (Gunnarsson et al., 2008, Christen et al., 2010). This background knowledge is then used to make predictions on the MOA and toxicity of a pharmaceutical in the non-target species showing a conserved drug target, and to tailor the experimental design of the ecotoxicity tests, for example by selecting specific endpoints that are relevant to the MOA, or by choosing an appropriate duration of exposure to the drug (Ankley et al., 2007, Christen et al., 2010).

In the present study the MOA approach was used to investigate the interactive effects of fluoxetine (FX) and propranolol (PROP) on the Mediterranean mussels (Mytilus galloprovincialis). M. galloprovincialis is acknowledged as a suitable indicator species to assess pollution impact on coastal environments; these are gaining increasing attention as to the issue of environmental contamination by pharmaceuticals, given that a large part of the world population lives in close proximity to the seacoast, and in many cases waste products directly enter coastal marine environments through sewage effluent discharge.

Mussels live at the sediment/water interface and filter large volumes of water, including suspended materials and colloids (Gagnè et al., 2007), and are known to efficiently bioaccumulate toxic compounds.

FX has received considerable attention in the framework of risk assessment investigations with emerging contaminants due to its frequent detection in aquatic environments (Oakes et al., 2010); moreover, it is recognized as one of the human pharmaceuticals with the highest acute toxicity toward some non-target organisms (Oakes et al., 2010). FX is the active ingredient of the antidepressant Prozac®, the most widely prescribed psychoactive drug on the market; it acts as a selective serotonin reuptake inhibitor (SSRI) in the treatment of depression and other mood disorders by increasing the serotonin levels in neuron synaptic space (De Vane, 1999, Fent et al., 2006, Hiemke and Härtter, 2000). Serotonin (5-hydroxytryptamine, 5-HT) is involved in hormonal and neuronal mechanisms and plays a key role in regulating food intake, metabolism and reproductive success in invertebrates (Fabbri and Capuzzo, 2010, Tierney, 2001). By interfering with serotoninergic regulation, FX has, therefore, the potential to impair relevant physiological functions in invertebrates.

PROP is a β adrenergic receptor antagonist used in human therapies to counteract cardiovascular pathologies (Weir, 2009), but it also acts as a 5-HT1 receptor antagonist (Tierney, 2001). PROP is widely detected in aquatic environments (Ashton et al., 2004, Thomas and Hilton, 2004, Wille et al., 2011). The drug was recently reported to bioconcentrate up to about 360 μg/g w.w. in mussel tissues (Ericson et al., 2010), and also to affect cAMP signaling and ABCB mRNA expression (Franzellitti et al., 2011a).

Section snippets

Experimental animals and holding conditions

Specimens of M. galloprovincialis (5–7 cm in length) were collected from the northwestern Adriatic Sea coast by fisherman of the “Cooperativa Copr.al.mo” (Cesenatico, Italy), and transferred to the laboratory in seawater tanks with continuous aeration. Animals (30 per aquarium) were acclimated for 3 days in aquaria containing 60 L of aerated artificial 35 psu seawater at 16 °C, under a natural photoperiod. Mussels were fed once a day with an algal slurry (Koral filtrator, Xaqua, Italy). Fifteen

Effects of FX, PROP and their mixture on cAMP levels and PKA activity

Cyclic AMP content and PKA activities in digestive gland and mantle/gonads of mussels exposed to either FX or PROP or their mixture are reported in Fig. 1, Fig. 2. Basal levels measured in control mussels were 255.97 ± 10.28 pmol cAMP/g tissue w.w. (digestive gland) and 178.71 ± 19.41 pmol cAMP/g tissue w.w. (mantle/gonads), in agreement with values reported previously (Franzellitti et al., 2011a, Martin-Diaz et al., 2009). Levels of cAMP in digestive gland were significantly decreased by FX or PROP

Discussion

The application of the “mode of action” (MOA) approach was employed in this study to evaluate the effects of FX, PROP and their mixture on Mediterranean mussels. Mammalian data guided the selection of relevant endpoints based of the read-across hypothesis that both pharmaceuticals could have the same effects in human and mussels by acting through conserved MOAs. The therapeutic action of FX as a SSRI is to increase serotonergic neurotransmission at mammalian synapses by blocking 5-HT reuptake

Conclusion

The MOA approach successfully guided the evaluation of FX and PROP effects on Mediterranean mussels and showed that exposure to low environmental concentrations of the pharmaceuticals affected cell signaling mediators in mussels, including cAMP contents, PKA activity, and mRNA levels for a 5-HT1 receptor. Such mediators are specifically involved in the therapeutic actions of the compounds suggesting an evolutionary conservation of the biological targets between humans and mussels. The effects

Acknowledgements

The research has been funded by MEECE EU-FP7 Project, and Italian Ministry of University and Research to E.F.

References (60)

  • S. Franzellitti et al.

    The β-blocker propranolol affects cAMP-dependent signaling and induces the stress response in Mediterranean mussels, Mytilus galloprovincialis

    Aquatic Toxicology

    (2011)
  • S. Franzellitti et al.

    Interactive effects of nickel and chlorpyrifos on Mediterranean mussel cAMP-mediated cell signaling and MXR-related gene expressions

    Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology

    (2011)
  • F. Gagnè et al.

    Neuroendocrine disruption and health effects in Elliptio complanata mussels exposed to aeration lagoons for wastewater treatment

    Chemosphere

    (2007)
  • M. Gros et al.

    Development of a multi-residue analytical methodology based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for screening and trace level determination of pharmaceuticals in surface and wastewaters

    Talanta

    (2006)
  • J. Hannon et al.

    Molecular biology of 5-HT receptors

    Behavioural Brain Research

    (2008)
  • T. Heberer

    Occurrence, fate, and removal of pharmaceutical residues in the aquatic environment: a review of recent research data

    Toxicology Letter

    (2002)
  • C. Hiemke et al.

    Pharmacokinetics of selective serotonin reuptake inhibitors

    Pharmacology & Therapeutics

    (2000)
  • J.W. Kim et al.

    Acute toxicity of pharmaceutical and personal care products on freshwater crustacean (Thamnocephalus platyurus) and fish (Oryzias latipes)

    Journal of Toxicological Sciences

    (2009)
  • D.G. Larsson et al.

    Effluent from drug manufactures contains extremely high levels of pharmaceuticals

    Journal of Hazardous Materials

    (2007)
  • O.H. Lowry et al.

    Protein measurement with the Folin phenol reagent

    Journal of Biological Chemistry

    (1951)
  • Y. Nakamura et al.

    The effects of pH on fluoxetine in Japanese medaka (Oryzias latipes): acute toxicity in fish larvae and bioaccumulation in juvenile fish

    Chemosphere

    (2008)
  • S.F. Owen et al.

    Uptake of propranolol, a cardiovascular pharmaceutical, from water into fish plasma and its effects on growth and organ biometry

    Aquatic Toxicology

    (2009)
  • L.H. Santos et al.

    Ecotoxicological aspects related to the presence of pharmaceuticals in the aquatic environment

    Journal of Hazardous Materials

    (2010)
  • K.V. Thomas et al.

    The occurrence of selected human pharmaceutical compounds in UK estuaries

    Marine Pollution Bulletin

    (2004)
  • A.J. Tierney

    Structure and function of invertebrate 5-HT receptors: a review

    Comparative Biochemistry and Physiology - Part A

    (2001)
  • T. Vasskog et al.

    Occurrence of selective serotonin reuptake inhibitors in sewage and receiving waters at Spitsbergen and in Norway

    Journal of Chromatography A

    (2008)
  • K. Wille et al.

    Rapid quantification of pharmaceuticals and pesticides in passive samplers using ultra high performance liquid chromatography coupled to high resolution mass spectrometry

    Journal of Chromatography A

    (2011)
  • B.S. Alexander et al.

    Stereoselective blockade of central [3H]5-hydroxytryptamine binding to multiple sites (5-HT1A, 5-HT1B and 5-HT1C) by mianserin and propranolol

    Journal of Pharmacy and Pharmacology

    (1987)
  • G.T. Ankley et al.

    Repeating history: pharmaceuticals in the environment

    Environmental Science & Technology

    (2007)
  • M. Banni et al.

    Gene expression rhythms in the mussel Mytilus galloprovincialis (Lam.) across an annual cycle

    PLoS ONE

    (2011)
  • Cited by (0)

    View full text