Knockout of the ergothioneine transporter ETT in zebrafish results in increased 8-oxoguanine levels.
Ergothioneine (ET) is a natural compound that humans and other vertebrates must absorb from dietary sources. In general, ET is considered an intracellular antioxidant. However, the precise physiological purpose of ET and the consequences of ET deficiency are still unclear. The ergothioneine transporter ETT (human gene symbol SLC22A4) is a highly specific transporter for the uptake of ET. Here, we sought to identify and knock out ETT from zebrafish (Danio rerio) to determine the function of ET. We cloned and assayed three related proteins from zebrafish, only one of which catalyzed the uptake of ET. RT-PCR analysis revealed that the protein is strongly expressed in the skin, brain, kidney, intestine, and eye. In ETT-knockout animals generated by retroviral insertion into exon 1, ET content was reduced by more than 1000-fold compared to the wild type. Thus, ETT is the sole transporter responsible for uptake of ET into zebrafish. ETT-knockout fish did not exhibit obvious differences in morphology or behavior. In whole-fish homogenates, an increase in 4-hydroxy-2,3-trans-nonenal and malondialdehyde was observed, but only after stress caused by incubation with Pb(2+) or Cu(2+). Comparison of unstressed fish at the level of small molecules by LC-MS difference shading revealed a 3.8-fold increase in 8-oxoguanine (8-oxo-7,8-dihydroguanine) in the skin of ETT-knockout animals. Our knockout represents a new model for examining the consequences of complete absence of ET. Based on the phenotype observed here, we hypothesize that the specific purpose of ET could be to eliminate singlet oxygen.
Department of Pharmacology, University of Cologne, 50931 Cologne, Germany.
Carolin
C
Pfeiffer
Department of Pharmacology, University of Cologne, 50931 Cologne, Germany.
Markus
M
Bach
Department of Pharmacology, University of Cologne, 50931 Cologne, Germany.
Tim
T
Bauer
Department of Pharmacology, University of Cologne, 50931 Cologne, Germany.
Julia
J
Campos da Ponte
Department of Pharmacology, University of Cologne, 50931 Cologne, Germany.
Edgar
E
Schömig
Department of Pharmacology, University of Cologne, 50931 Cologne, Germany. Electronic address: dirk.gruendemann@uni-koeln.de.
Dirk
D
Gründemann
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D000975:Antioxidants / Q000378:metabolism*; D002853:Chromatography, Liquid; D004880:Ergothioneine / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000172:deficiency* / Q000235:genetics; D018384:Oxidative Stress / Q000187:drug effects; D054852:Small Molecule Libraries; D015027:Zebrafish / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000172:deficiency* / Q000235:genetics
D000975:Antioxidants; D026901:Membrane Transport Proteins; D054852:Small Molecule Libraries; D029961:Zebrafish Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D004880:Ergothioneine
8-Oxoguanine; Ergothioneine; Ergothioneine transporter; Singlet oxygen; Zebrafish knockout