First evidence for toxic defense based on the multixenobiotic resistance (MXR) mechanism in Daphnia magna.
The water flea Daphnia magna is widely used as test species in ecotoxicological bioassays. So far, there is no information available to which extent ATP binding cassette (ABC) transporter based multixenobiotic resistance (MXR) counteracts adverse chemical effects in this species. This, however, would be important for assessing to which extent the bio-active potential of a compound determined with this species depends on this cellular defense. We here present molecular, functional and toxicological studies that provide first evidence for ABC transporter-based MXR in D. magna. We cloned putatively MXR-related partial abcb1, abcc1/3, abcc4 and abcc5 coding sequences; respective transcripts were constitutively expressed in different D. magna life stages. MXR associated efflux activity was monitored in D. magna using the fluorescent substrate dyes rhodamine 123, rhodamine B and calcein-AM combined with inhibitors of human ABCB1 and/or ABCC transporter activities reversin 205, MK571 and cyclosporin A. With inhibitors present, efflux of dye substrates was reduced in D. magna in a concentration-dependent mode, as indicated by elevated accumulation of the dyes in D. magna tissues. In animals pre-exposed to mercury, pentachlorophenol or dacthal applied as inducers of ABC transporter expression, levels of some ABC transporter transcripts were increased in some cases showing that these genes can be chemically induced. Likewise, pre-exposure of animals to these chemicals decreased dye accumulation in tissue, indicating enhanced MXR transporter activity, likely associated with higher transporter protein levels. Toxicity assays with toxic transporter substrates mitoxantrone and chlorambucil that were applied singly and in combination with inhibitors were performed to study the tolerance role of Abcb1 and Abcc efflux transporters in D. magna. Joint toxicities of about half of the binary combinations of test compounds applied (substrate/inhibitor, substrate/substrate, inhibitor/inhibitor) were greater than joint effects predicted with mixture toxicity models, which can be explained by chemosensitization through MXR efflux transporter interference. Our data indicate the presence of an MXR efflux system in D. magna. It needs to be considered when assessing the bioactive potential of test compounds with this species. Further, chemosensitization may explain joint toxicities of compound mixtures to D. magna that are higher than expected.
Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18, 08034 Barcelona, Spain.
Bruno
B
Campos
UFZ-Helmholtz Centre for Environmental Research, Permoserstr. 15, D-04318 Leipzig, Germany.
Rolf
R
Altenburger
Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18, 08034 Barcelona, Spain.
Cristian
C
Gómez
Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18, 08034 Barcelona, Spain.
Silvia
S
Lacorte
Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18, 08034 Barcelona, Spain.
Benjamin
B
Piña
Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18, 08034 Barcelona, Spain. Electronic address: cbmqam@cid.csic.es.
Carlos
C
Barata
UFZ-Helmholtz Centre for Environmental Research, Permoserstr. 15, D-04318 Leipzig, Germany.
Till
T
Luckenbach
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D003621:Daphnia / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D018432:Drug Resistance, Multiple / Q000187:drug effects; D004355:Drug Stability; D005456:Fluorescent Dyes / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D008018:Life Cycle Stages / Q000187:drug effects; D014874:Water Pollutants, Chemical / Q000633:toxicity*
D018528:ATP-Binding Cassette Transporters; D005456:Fluorescent Dyes; D014874:Water Pollutants, Chemical
Chemosensitizer; Daphnia magna; Environment–tissue barrier; MXR; Mixture toxicity; Multixenobiotic resistance