Aquatic Toxicology 146 (2014) 264–265
Contents lists available at ScienceDirect
Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Commentary
Response to Commentary on “Are some invertebrates exquisitely sensitive to the human pharmaceutical fluoxetine?” S. Franzellitti ∗ , E. Fabbri Department of Biological, Geological, and Environmental Sciences, University of Bologna, Italy
As a physiology group mainly engaged in studying cell signalling and neuroendocrine modulation of cellular responses, the trigger to address our investigation on environmental pharmaceuticals was the lack of attention for the relevant differences in biological effects between pharmaceuticals and more conventional pollutants, such as metals, PAH, etc. Indeed, pharmaceuticals may have side effects, hopefully at high doses, but are designed to interact with specific biological targets and produce therapeutic effects at the lowest possible doses. These interactions are not necessarily revealed by acute or chronic toxicity tests on standard organisms and endpoints, and the recent literature underlines the need for new approaches. In particular, the application of the Mode of Action (MOA) approach (Ankley et al., 2007, 2010; Gunnarsson et al., 2008) reported by us and in other studies (Franzellitti et al., 2013; reviewed in Schmitt et al., 2010) provides a substantial contribution to the understanding of subtle, target-specific effects of pharmaceuticals on non-target organisms, showing that the evolutionary conservation of molecular targets in a given species potentially increases the risk of effects of bioactive compounds. When going deep into MOA-related effects, changes are reported in the environmental concentration range, as in the recent papers addressed by Sumpter and MargiottaCasaluci in their commentary. In Franzellitti et al. (2013) we suggested “fluoxetine has the potential to affect the ability of animals to elaborate strategies of defence or adaptation”. Changes of cAMP levels are crucial as the nucleotide acts as second messenger of hydrophilic hormones and neurotransmitters in all animals including humans. Therefore, in some ways fluoxetine is an endocrine-disrupting chemical. In mussels the increase of cAMP levels also represents a response to stress generated by polluted environments (Dailianis et al., 2003; Raftopoulou et al., 2006). On the other hand, cAMP reduction alters a number of physiological functions at different cell levels (Fabbri and Capuzzo, 2010). In the long term the above changes – due to endogenous or exogenous stimuli – impair organism homeostasis, leading to altered reproduction, metabolism, and growth, if not death. Nevertheless, animals may recover if the event is limited in time.
∗ Corresponding author. Tel.: +39 0544 937311; fax: +39 0544 937411. E-mail address: [email protected] (S. Franzellitti). 0166-445X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.aquatox.2013.11.019
Fluoxetine effects reported by Franzellitti et al. (2013) are per se quite clear, although they were unexpected in vivo at this low concentration. Fluoxetine at 0.3 ng/L decreased cAMP levels, PKA activities and a related mRNA expression in digestive gland and mantle/gonads, putatively caused by increased serotonin (5-HT) levels and occupation of 5-HT1 receptors as observed in haemocytes (Franzellitti and Fabbri, 2013). The work specifically addressed the effects of a single low-dose of fluoxetine. This is of course a limitation. However, we can anticipate that our experiments on fluoxetine went on, and the above results are now corroborated by independent data on molecular, cellular and tissue parameters in a wide range of fluoxetine concentrations, including 0.3 ng/L. 1. Regulatory issues On the whole, our paper and analogous literature highlights some main environmental issues: (a) acute and chronic ecotoxicity tests on standard organisms and endpoints cannot be taken as conclusive proofs of environmental health, when pharmaceuticals or endocrine-disrupting chemicals are present; (b) low concentrations of pharmaceuticals, i.e. of specifically designed bioactive compounds, do not ensure per se environmental safety; (c) xenosteroids are not the only ones possessing the potential to affect wildlife homeostasis and reproduction, so further contaminants with non-steroid MOAs deserve investigations. Many results obtained at environmentally relevant concentrations were indeed surprising, and do have major regulatory implications. Limitedly to fluoxetine, examples of MOA-related effects are those on gamete release reported by Lazzara et al. (2012) at 20–200 ng/L, and it is worth noting that cAMP in bivalves is known to regulate gonad maturations and spawning (Fabbri and Capuzzo, 2010). Moreover, as reported by Mennigen et al. (2010), fluoxetine disrupts energy metabolism in fish at 540 ng/L concentration, serotonin being involved in the specific regulation of carbohydrate metabolism (Lam and Heisler, 2007). We may also cite those on carbamazepine, with effects linked to the pharmaceutical MOA observed already at 100 ng/L (Martin-Diaz et al., 2009). At 236 ng/L carbamazepine yielded a tissue level of 21.4 ng/g in mussels after a 7-days exposure, and caused significant down-regulation of hsp70, SOD, MT and Pgp mRNA expressions (Contardo-Jara et al., 2011).
S. Franzellitti, E. Fabbri / Aquatic Toxicology 146 (2014) 264–265
Needless to say that regulatory constraints are particularly difficult to establish for these substances, as of course the use of pharmaceuticals for human and veterinary health cannot be avoided. However, environmental pharmaceuticals pose a problem that cannot be further neglected. Wildlife displaying conserved molecular targets is definitively affected, and pharmaceutical residues may enter the human diet provoking unwanted phenomena typical for low doses, i.e. synergy in mixtures or resistance (e.g. antibiotics). 2. More information needed Much remains to be done to understand whether fluoxetine is more potent in molluscs than in humans. The observation by Sumpter and Margiotta-Casaluci is absolutely correct and deserves answers. We are aware of the ‘fish plasma model’ proposed by Huggett et al. (2004), based on the comparison between human blood concentrations and those of treated fish. We also acknowledge the paper by Rand-Weaver et al. (2013) and the discussion on pharmacodynamics of fluoxetine. Unfortunately, such comparisons may be not that direct with mussel haemolymph, which operates in an open-circulating system and cells are reached almost one by one by the fluid. Nevertheless, these are taken as suggestions for designing future experiments. As a matter of fact, so far only a few laboratories have had the interest to explore interactions of fluoxetine in invertebrates at the molecular/cellular levels. Despite the facts that serotonin is one of the most active neuromodulators in invertebrates and that it is used in farms to induce spawning, knowledge about its receptors, re-uptake transporters (which type, in which tissues, etc.) is extremely poor. We must admit, however, that only small funds are given to these research-based topics, whereas much more interest is given to wide-scale environmental approaches, which often unfortunately neglect to answer emerging and crucial questions such as specificity of action, mechanisms, and potential for disruption of homeostasis by pharmaceuticals. In conclusion, none of the available reports have demonstrated ecological consequences of environmental pharmaceuticals. However, they have clearly pointed out effects on animal homeostasis. Thus, the regulatory issue has to be considered as an important reason to do research to the highest standards possible; however, the regulatory field must seriously consider MOA-related aspects, and possibly promote, guide and support newly oriented research activities.
265
References Ankley, G.T., Bennett, R.S., Erickson, R.J., Hoff, D.J., Hornung, M.W., Johnson, R.D., Mount, D.R., Nichols, J.W., Russom, C.L., Schmieder, P.K., Serrrano, J.A., Tietge, J.E., Villeneuve, D.L., 2010. Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment. Environ. Toxicol. Chem. 29, 730–741. Ankley, G.T., Brooks, B.W., Huggett, D.B., Sumpter, J.P., 2007. Repeating history: pharmaceuticals in the environment. Environ. Sci. Technol. 41, 8211–8217. Contardo-Jara, V., Lorenz, C., Pflugmacher, S., Nützmann, G., Kloas, W., Wiegand, C., 2011. Exposure to human pharmaceuticals carbamazepine ibuprofen and bezafibrate causes molecular effects in Dreissena polymorpha. Aquat. Toxicol. 105, 428–437. Dailianis, S., Domouhtsidou, G.P., Raftopoulou, E., Kaloyianni, M., Dimitriadis, V.K., 2003. Evaluation of neutral red retention assay, micronucleus test, acetylcholinesterase activity and a signal transduction molecule (cAMP) in tissues of Mytilus galloprovincialis (L.), in pollution monitoring. Mar. Environ. Res. 56, 443–470. Fabbri, E., Capuzzo, A., 2010. Cyclic AMP signaling in bivalve molluscs: an overview. J. Exp. Zool. A 313, 179–200. Franzellitti, S., Buratti, S., Valbonesi, P., Fabbri, E., 2013. The mode of action (MOA) approach reveals interactive effects of environmental pharmaceuticals on Mytilus galloprovincialis. Aquat. Toxicol. 140–141, 249–256. Franzellitti, S., Fabbri, E., 2013. Cyclic-AMP mediated regulation of ABCB mRNA expression in mussel haemocytes. PLoS ONE 8, e61634. Gunnarsson, L., Jauhiainen, A., Kristiansson, E., Nerman, O., Larsson, D.G.J., 2008. Evolutionary conservation of human drug targets in organisms used for environmental risk assessments. Environ. Sci. Technol. 42, 5807–5813. Huggett, D.B., Ericson, J.F., Cook, J.C., Williams, R.T., 2004. Plasma concentrations of human pharmaceuticals as predictors of pharmacological responses in fish. In: Kummerer (Ed.), Pharmaceuticals in the Environment: Sources, Fate, effects and Risks. , 2nd ed. Springer-Verlag, Berlin (Chapter 27). Lam, D.D., Heisler, L.K., 2007. Serotonin and energy balance: molecular mechanisms and implications for type 2 diabetes. Expert Rev. Mol. Med. 9, 1–24. Lazzara, R., Blázquez, M., Porte, C., Barata, C., 2012. Low environmental levels of fluoxetine induce spawning and changes in endogenous estradiol levels in the zebra mussel Dreissena polymorpha. Aquat. Toxicol. 106–107, 123–130. Martin-Diaz, M.L., Franzellitti, S., Buratti, S., Valbonesi, P., Capuzzo, A., Fabbri, E., 2009. Effects of environmental concentrations of the antiepileptic drug carbamazepine on biomarkers and cAMP-mediated cell signaling in the mussel Mytilus galloprovincialis. Aquat. Toxicol. 94, 177–185. Mennigen, J.A., Sassine, J., Trudeau, V.L., Moon, T.W., 2010. Waterborne fluoxetine disrupts feeding and energy metabolism in the goldfish Carassius auratus. Aquat. Toxicol. 100, 128–137. Raftopoulou, E.K., Dailianis, S., Dimitriadis, V.K., Kaloyianni, M., 2006. Introduction of cAMP and establishment of neutral lipids alterations as pollution biomarkers using the mussel Mytilus galloprovincialis. Correlation with a battery of biomarkers. Sci. Total Environ. 368, 597–614. Rand-Weaver, M., Margiotta-Casaluci, L., Patel, A., Panter, G.H., Owen, S.F., Sumpter, J.P., 2013. The read-across hypothesis and environmental risk assessment of pharmaceuticals. Environ. Sci. Technol. 47, 11384–11395. Schmitt, H., Boucard, T., Garric, J., Jensen, J., Parrott, J., Péry, A., Römbke, J., Straub, J.-O., Hutchinson, T.H., Sánchez-Argüello, P., Wennmalm, A., Duis, K., 2010. Recommendations on the environmental risk assessment of pharmaceuticals: effect characterization. Integr. Environ. Assess. Manag. 6, S588–S602.
Contents lists available at ScienceDirect
Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Commentary
Response to Commentary on “Are some invertebrates exquisitely sensitive to the human pharmaceutical fluoxetine?” S. Franzellitti ∗ , E. Fabbri Department of Biological, Geological, and Environmental Sciences, University of Bologna, Italy
As a physiology group mainly engaged in studying cell signalling and neuroendocrine modulation of cellular responses, the trigger to address our investigation on environmental pharmaceuticals was the lack of attention for the relevant differences in biological effects between pharmaceuticals and more conventional pollutants, such as metals, PAH, etc. Indeed, pharmaceuticals may have side effects, hopefully at high doses, but are designed to interact with specific biological targets and produce therapeutic effects at the lowest possible doses. These interactions are not necessarily revealed by acute or chronic toxicity tests on standard organisms and endpoints, and the recent literature underlines the need for new approaches. In particular, the application of the Mode of Action (MOA) approach (Ankley et al., 2007, 2010; Gunnarsson et al., 2008) reported by us and in other studies (Franzellitti et al., 2013; reviewed in Schmitt et al., 2010) provides a substantial contribution to the understanding of subtle, target-specific effects of pharmaceuticals on non-target organisms, showing that the evolutionary conservation of molecular targets in a given species potentially increases the risk of effects of bioactive compounds. When going deep into MOA-related effects, changes are reported in the environmental concentration range, as in the recent papers addressed by Sumpter and MargiottaCasaluci in their commentary. In Franzellitti et al. (2013) we suggested “fluoxetine has the potential to affect the ability of animals to elaborate strategies of defence or adaptation”. Changes of cAMP levels are crucial as the nucleotide acts as second messenger of hydrophilic hormones and neurotransmitters in all animals including humans. Therefore, in some ways fluoxetine is an endocrine-disrupting chemical. In mussels the increase of cAMP levels also represents a response to stress generated by polluted environments (Dailianis et al., 2003; Raftopoulou et al., 2006). On the other hand, cAMP reduction alters a number of physiological functions at different cell levels (Fabbri and Capuzzo, 2010). In the long term the above changes – due to endogenous or exogenous stimuli – impair organism homeostasis, leading to altered reproduction, metabolism, and growth, if not death. Nevertheless, animals may recover if the event is limited in time.
∗ Corresponding author. Tel.: +39 0544 937311; fax: +39 0544 937411. E-mail address: [email protected] (S. Franzellitti). 0166-445X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.aquatox.2013.11.019
Fluoxetine effects reported by Franzellitti et al. (2013) are per se quite clear, although they were unexpected in vivo at this low concentration. Fluoxetine at 0.3 ng/L decreased cAMP levels, PKA activities and a related mRNA expression in digestive gland and mantle/gonads, putatively caused by increased serotonin (5-HT) levels and occupation of 5-HT1 receptors as observed in haemocytes (Franzellitti and Fabbri, 2013). The work specifically addressed the effects of a single low-dose of fluoxetine. This is of course a limitation. However, we can anticipate that our experiments on fluoxetine went on, and the above results are now corroborated by independent data on molecular, cellular and tissue parameters in a wide range of fluoxetine concentrations, including 0.3 ng/L. 1. Regulatory issues On the whole, our paper and analogous literature highlights some main environmental issues: (a) acute and chronic ecotoxicity tests on standard organisms and endpoints cannot be taken as conclusive proofs of environmental health, when pharmaceuticals or endocrine-disrupting chemicals are present; (b) low concentrations of pharmaceuticals, i.e. of specifically designed bioactive compounds, do not ensure per se environmental safety; (c) xenosteroids are not the only ones possessing the potential to affect wildlife homeostasis and reproduction, so further contaminants with non-steroid MOAs deserve investigations. Many results obtained at environmentally relevant concentrations were indeed surprising, and do have major regulatory implications. Limitedly to fluoxetine, examples of MOA-related effects are those on gamete release reported by Lazzara et al. (2012) at 20–200 ng/L, and it is worth noting that cAMP in bivalves is known to regulate gonad maturations and spawning (Fabbri and Capuzzo, 2010). Moreover, as reported by Mennigen et al. (2010), fluoxetine disrupts energy metabolism in fish at 540 ng/L concentration, serotonin being involved in the specific regulation of carbohydrate metabolism (Lam and Heisler, 2007). We may also cite those on carbamazepine, with effects linked to the pharmaceutical MOA observed already at 100 ng/L (Martin-Diaz et al., 2009). At 236 ng/L carbamazepine yielded a tissue level of 21.4 ng/g in mussels after a 7-days exposure, and caused significant down-regulation of hsp70, SOD, MT and Pgp mRNA expressions (Contardo-Jara et al., 2011).
S. Franzellitti, E. Fabbri / Aquatic Toxicology 146 (2014) 264–265
Needless to say that regulatory constraints are particularly difficult to establish for these substances, as of course the use of pharmaceuticals for human and veterinary health cannot be avoided. However, environmental pharmaceuticals pose a problem that cannot be further neglected. Wildlife displaying conserved molecular targets is definitively affected, and pharmaceutical residues may enter the human diet provoking unwanted phenomena typical for low doses, i.e. synergy in mixtures or resistance (e.g. antibiotics). 2. More information needed Much remains to be done to understand whether fluoxetine is more potent in molluscs than in humans. The observation by Sumpter and Margiotta-Casaluci is absolutely correct and deserves answers. We are aware of the ‘fish plasma model’ proposed by Huggett et al. (2004), based on the comparison between human blood concentrations and those of treated fish. We also acknowledge the paper by Rand-Weaver et al. (2013) and the discussion on pharmacodynamics of fluoxetine. Unfortunately, such comparisons may be not that direct with mussel haemolymph, which operates in an open-circulating system and cells are reached almost one by one by the fluid. Nevertheless, these are taken as suggestions for designing future experiments. As a matter of fact, so far only a few laboratories have had the interest to explore interactions of fluoxetine in invertebrates at the molecular/cellular levels. Despite the facts that serotonin is one of the most active neuromodulators in invertebrates and that it is used in farms to induce spawning, knowledge about its receptors, re-uptake transporters (which type, in which tissues, etc.) is extremely poor. We must admit, however, that only small funds are given to these research-based topics, whereas much more interest is given to wide-scale environmental approaches, which often unfortunately neglect to answer emerging and crucial questions such as specificity of action, mechanisms, and potential for disruption of homeostasis by pharmaceuticals. In conclusion, none of the available reports have demonstrated ecological consequences of environmental pharmaceuticals. However, they have clearly pointed out effects on animal homeostasis. Thus, the regulatory issue has to be considered as an important reason to do research to the highest standards possible; however, the regulatory field must seriously consider MOA-related aspects, and possibly promote, guide and support newly oriented research activities.
265
References Ankley, G.T., Bennett, R.S., Erickson, R.J., Hoff, D.J., Hornung, M.W., Johnson, R.D., Mount, D.R., Nichols, J.W., Russom, C.L., Schmieder, P.K., Serrrano, J.A., Tietge, J.E., Villeneuve, D.L., 2010. Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment. Environ. Toxicol. Chem. 29, 730–741. Ankley, G.T., Brooks, B.W., Huggett, D.B., Sumpter, J.P., 2007. Repeating history: pharmaceuticals in the environment. Environ. Sci. Technol. 41, 8211–8217. Contardo-Jara, V., Lorenz, C., Pflugmacher, S., Nützmann, G., Kloas, W., Wiegand, C., 2011. Exposure to human pharmaceuticals carbamazepine ibuprofen and bezafibrate causes molecular effects in Dreissena polymorpha. Aquat. Toxicol. 105, 428–437. Dailianis, S., Domouhtsidou, G.P., Raftopoulou, E., Kaloyianni, M., Dimitriadis, V.K., 2003. Evaluation of neutral red retention assay, micronucleus test, acetylcholinesterase activity and a signal transduction molecule (cAMP) in tissues of Mytilus galloprovincialis (L.), in pollution monitoring. Mar. Environ. Res. 56, 443–470. Fabbri, E., Capuzzo, A., 2010. Cyclic AMP signaling in bivalve molluscs: an overview. J. Exp. Zool. A 313, 179–200. Franzellitti, S., Buratti, S., Valbonesi, P., Fabbri, E., 2013. The mode of action (MOA) approach reveals interactive effects of environmental pharmaceuticals on Mytilus galloprovincialis. Aquat. Toxicol. 140–141, 249–256. Franzellitti, S., Fabbri, E., 2013. Cyclic-AMP mediated regulation of ABCB mRNA expression in mussel haemocytes. PLoS ONE 8, e61634. Gunnarsson, L., Jauhiainen, A., Kristiansson, E., Nerman, O., Larsson, D.G.J., 2008. Evolutionary conservation of human drug targets in organisms used for environmental risk assessments. Environ. Sci. Technol. 42, 5807–5813. Huggett, D.B., Ericson, J.F., Cook, J.C., Williams, R.T., 2004. Plasma concentrations of human pharmaceuticals as predictors of pharmacological responses in fish. In: Kummerer (Ed.), Pharmaceuticals in the Environment: Sources, Fate, effects and Risks. , 2nd ed. Springer-Verlag, Berlin (Chapter 27). Lam, D.D., Heisler, L.K., 2007. Serotonin and energy balance: molecular mechanisms and implications for type 2 diabetes. Expert Rev. Mol. Med. 9, 1–24. Lazzara, R., Blázquez, M., Porte, C., Barata, C., 2012. Low environmental levels of fluoxetine induce spawning and changes in endogenous estradiol levels in the zebra mussel Dreissena polymorpha. Aquat. Toxicol. 106–107, 123–130. Martin-Diaz, M.L., Franzellitti, S., Buratti, S., Valbonesi, P., Capuzzo, A., Fabbri, E., 2009. Effects of environmental concentrations of the antiepileptic drug carbamazepine on biomarkers and cAMP-mediated cell signaling in the mussel Mytilus galloprovincialis. Aquat. Toxicol. 94, 177–185. Mennigen, J.A., Sassine, J., Trudeau, V.L., Moon, T.W., 2010. Waterborne fluoxetine disrupts feeding and energy metabolism in the goldfish Carassius auratus. Aquat. Toxicol. 100, 128–137. Raftopoulou, E.K., Dailianis, S., Dimitriadis, V.K., Kaloyianni, M., 2006. Introduction of cAMP and establishment of neutral lipids alterations as pollution biomarkers using the mussel Mytilus galloprovincialis. Correlation with a battery of biomarkers. Sci. Total Environ. 368, 597–614. Rand-Weaver, M., Margiotta-Casaluci, L., Patel, A., Panter, G.H., Owen, S.F., Sumpter, J.P., 2013. The read-across hypothesis and environmental risk assessment of pharmaceuticals. Environ. Sci. Technol. 47, 11384–11395. Schmitt, H., Boucard, T., Garric, J., Jensen, J., Parrott, J., Péry, A., Römbke, J., Straub, J.-O., Hutchinson, T.H., Sánchez-Argüello, P., Wennmalm, A., Duis, K., 2010. Recommendations on the environmental risk assessment of pharmaceuticals: effect characterization. Integr. Environ. Assess. Manag. 6, S588–S602.
