title: Differential modulation of ammonia excretion, Rhesus glycoproteins and ion-regulation in common carp (Cyprinus carpio) following individual and combined exposure to waterborne copper and ammonia.
abstract: The main objective of this study was to understand the mode of interaction between waterborne copper (Cu) and high environmental ammonia (HEA) exposure on freshwater fish, and how they influence the toxicity of each other when present together. For this purpose, individual and combined effects of Cu and HEA were examined on selected physiological and ion-regulatory processes and changes at transcript level in the common carp (Cyprinus carpio). Juvenile carp were exposed to 2.6μM Cu (25% of the 96h LC50value) and to 0.65mM ammonia (25% of the 96h LC50value) singly and as a mixture for 12h, 24h, 48h, 84h and 180h. Responses such as ammonia (Jamm) and urea (Jurea) excretion rate, plasma ammonia and urea, plasma ions (Na(+), Cl(-) and K(+)), muscle water content (MWC) as well as branchial Na(+)/K(+)-ATPase (NKA) and H(+)-ATPase activity, and branchial mRNA expression of NKA, H(+)-ATPase, Na(+)/H(+) exchanger (NHE-3) and Rhesus (Rh) glycoproteins were investigated under experimental conditions. Results show that Jamm was inhibited during Cu exposure, while HEA exposed fish were able to increase excretion efficiently. In the combined exposure, Jamm remained at the control levels indicating that Cu and HEA abolished each other's effect. Expression of Rhcg (Rhcg-a and Rhcg-b) mRNA was upregulated during HEA, thereby facilitated ammonia efflux out of gills. On the contrary, Rhcg-a transcript level declined following Cu exposure which might account for Cu induced Jamm inhibition. Likewise, Rhcg-a was also down-regulated in Cu-HEA co-exposed fish whilst a temporary increment was noted for Rhch-b. Fish exposed to HEA displayed pronounced up-regulation in NKA expression and activity and stable plasma ion levels. In both the Cu exposure alone and combined Cu-HEA exposure, ion-osmo homeostasis was adversely affected, exemplified by the significant reduction in plasma [Na(+)] and [Cl(-)], and elevated plasma [K(+)], along with an elevation in MWC. These changes were accompanied by a decline in NKA activity. Gill H(+)-ATPase mRNA levels and activities were not affected by either Cu or HEA or both. Likewise, NHE-3 expression remained unaltered but tended to be numerically higher during HEA exposure. Overall, these data suggest that at equitoxic concentrations (25% of 96h LC50), the individual effect of Cu is more harmful while HEA induces quicker adaptive responses. Our findings also denote a competitive mode of interaction, exemplified by the inhibition of HEA -mediated adaptive responses in the presence of Cu.
authors.affiliation: Systemic Physiological and Ecotoxicological Research, Department of Biology, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp, Belgium. Electronic address: sinha_cife@rediffmail.com.
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Sinha
authors.affiliation: Systemic Physiological and Ecotoxicological Research, Department of Biology, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp, Belgium.
authors.forename: Mumba
authors.initials: M
authors.lastname: Kapotwe
authors.affiliation: Systemic Physiological and Ecotoxicological Research, Department of Biology, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp, Belgium.
authors.forename: Shambel Boki
authors.initials: SB
authors.lastname: Dabi
authors.affiliation: Systemic Physiological and Ecotoxicological Research, Department of Biology, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp, Belgium; Laboratory of Immunohistochemical and Cellular Ultrastructure, Federal University of Para, Campus Guamá, Rua Augusto Corrêa 1, 66075-900, Belém, Pará, Brazil.
authors.forename: Caroline da Silva
authors.initials: CDS
authors.lastname: Montes
authors.affiliation: Systemic Physiological and Ecotoxicological Research, Department of Biology, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp, Belgium.
authors.forename: Jyotsna
authors.initials: J
authors.lastname: Shrivastava
authors.affiliation: Systemic Physiological and Ecotoxicological Research, Department of Biology, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp, Belgium.
authors.forename: Ronny
authors.initials: R
authors.lastname: Blust
authors.affiliation: Systemic Physiological and Ecotoxicological Research, Department of Biology, University of Antwerp, Groenenborgerlaan 171, BE-2020 Antwerp, Belgium.
authors.forename: Gudrun De
authors.initials: G
authors.lastname: Boeck
mesh_terms: D000641:Ammonia / Q000097:blood / Q000633:toxicity*; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D002347:Carps / Q000254:growth & development / Q000378:metabolism*; D003300:Copper / Q000633:toxicity*; D004781:Environmental Exposure; D005880:Gills / Q000187:drug effects* / Q000378:metabolism; D007477:Ions / Q000097:blood / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012964:Sodium / Q000097:blood; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D014508:Urea / Q000097:blood; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D001798:Blood Proteins; D007477:Ions; D012333:RNA, Messenger; D014874:Water Pollutants, Chemical; D000641:Ammonia; D003300:Copper; D014508:Urea; D012964:Sodium; D025262:Vacuolar Proton-Translocating ATPases; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Ammonia excretion rate; Ion-regulation; Na(+)/NH(4)(+) exchange metabolon; Plasma ions; Rhesus (Rh) glycoprotein