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Knockout of the Ergothioneine Transporter ETT in Zebrafish Results in Increased 8‐Oxoguanine Levels
Author(s) -
Krueger Johanna,
Pfeiffer Carolin,
Gruendemann Dirk
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.lb517
Subject(s) - zebrafish , ergothioneine , carnitine , wild type , transporter , chemistry , danio , malondialdehyde , lipid peroxidation , biology , microbiology and biotechnology , biochemistry , oxidative stress , gene , antioxidant , mutant
Ergothioneine (ET) is expected to provide antioxidative properties due to its double‐bonded sulfur. It is acquired by plants and vertebrates solely through dietary means. However, the precise physiological function of ET remains unclear. Case‐control studies suggest an association of polymorphisms in the human ET transporter (ETT) gene with chronic inflammatory diseases like rheumatoid arthritis [Nat Genet 35, 341‐348], Crohn's disease [Nat Genet 36, 471‐475] and diabetes [Biochem J 25, 171].
Methods Here we have procured, based on retroviral insertion [Genes Dev 13:2713‐24], an ETT knock‐out zebrafish ( Danio rerio) . The expression profile of ETT in wild‐type zebrafish was analyzed by RT‐PCR and real‐time RT‐PCR and validated by measuring ET tissue content. Results Strong signals were obtained with kidney, intestine, skin, brain and eye. In whole‐fish lysates of homozygous knock‐out animals, ET content (0.06 ± 0.01 ng/g wet mass; n=3) was reduced more than 1000‐fold compared to wild‐type (138 ± 61 ng/g wet mass; n = 3). Similarly, all examined organs contained virtually no ET. Carnitine and acetylcarnitine were reduced across all examined tissues of knock‐out fish by a factor of 2.0 ± 0.2, and 1.7 ± 0.1, respectively. Incubation in sublethal concentrations of Pb 2+ and Cu 2+ led to an increase of the lipid peroxidation markers 4‐hydroxy‐2,3‐trans‐nonenal (HNE) and malondialdehyde (MDA) in knock‐out versus wild‐type zebrafish. Comparison of unstressed fish at the level of small molecules by LC‐MS difference shading [Proc. Natl. Acad. Sci. U.S.A. 102:5256‐61] revealed a 3.8‐fold increase of 8‐oxoguanine in the skin of ETT knockout fish. Conclusion Based on this phenotype we propose that the universal and specific purpose of ET is to eliminate singlet oxygen.