The mode of action (MOA) approach reveals interactive effects of environmental pharmaceuticals on Mytilus galloprovincialis
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)
- et al.
Investigating the environmental transport of human pharmaceuticals to streams in the United Kingdom
Science of the Total Environment
(2004) Multixenobiotic resistance as a cellular defence mechanism in aquatic organisms
Aquatic Toxicology
(2000)- et al.
Aquatic ecotoxicology of fluoxetine
Toxicology Letter
(2003) - et al.
Highly active human pharmaceuticals in aquatic systems: a concept for their identification based on their mode of action
Aquatic Toxicology
(2010) Initial risk assessment for three beta-blockers found in the aquatic environment
Chemosphere
(2005)- et al.
Estrogens disrupt serotonin receptor and cyclooxygenase mRNA expression in the gonads of mussels (Mytilus edulis)
Aquatic Toxicology
(2010) - et al.
Quantitative PCR analysis of two molluscan metallothionein genes unveils differential expression and regulation
Gene
(2005) - et al.
Physiological effects of diclofenac, ibuprofen and propranolol on Baltic Sea blue mussels
Aquatic Toxicology
(2010) - et al.
Ecotoxicology of human pharmaceuticals
Aquatic Toxicology
(2006) - et al.
Exposure of mussels to a polluted environment: insights into the stress syndrome development
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
(2010)