title: Endogenous hydrogen peroxide production in the epithelium of the developing embryonic lens.
abstract: OBJECTIVE
  Hydrogen peroxide (H2O2) is an endogenously produced reactive oxygen species (ROS) present in a variety of mammalian systems. This particular ROS can play dichotomous roles, being beneficial in some cases and deleterious in others, which reflects the level and location of H2O2 production. While much is known about the redox regulation of ROS by antioxidant and repair systems in the lens, little is known about the endogenous production of H2O2 in embryonic lens tissue or the physiologic relevance of endogenous H2O2 to lens development. This gap in knowledge exists primarily from a lack of reagents that can specifically detect endogenous H2O2 in the intact lens. Here, using a recently developed chemoselective fluorescent boronate probe, peroxyfluor-6 acetoxymethyl ester (PF6-AM), which selectively detects H2O2 over related ROS, we examined the endogenous H2O2 signals in the embryonic lens.
  
  
  METHODS
  Embryonic day 10 chick whole lenses in ex vivo organ culture and lens epithelial cells in primary culture were loaded with the H2O2 probe PF6-AM. To determine the relationship between localization of mitochondria with active membrane potential and the region of H2O2 production in the lens, cells were exposed to the mitochondrial probe MitoTracker Red CMXRos together with PF6-AM. Diphenyleneiodonium (DPI), a flavin inhibitor that blocks generation of intracellular ROS production, was used to confirm that the signal from PF6-AM was due to endogenous ROS production. All imaging was performed by live confocal microscopy.
  
  
  RESULTS
  PF6-AM detected endogenous H2O2 in lens epithelial cells in whole lenses in ex vivo culture and in lens epithelial cells grown in primary culture. No endogenous H2O2 signal could be detected in differentiating lens fiber cells with this probe. Treatment with DPI markedly attenuated the fluorescence signal from the peroxide-specific probe PF6-AM in the lens epithelium, suggesting that basal generation of ROS occurs in this region. The lens epithelial cells producing an endogenous H2O2 signal were also rich in actively respiring mitochondria.
  
  
  CONCLUSIONS
  PF6-AM can be used as an effective reagent to detect the presence and localization of endogenous H2O2 in live lens cells.
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA.
authors.forename: Subhasree
authors.initials: S
authors.lastname: Basu
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA.
authors.forename: Suren
authors.initials: S
authors.lastname: Rajakaruna
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA ; Department of Chemistry, University of California, Berkeley, CA.
authors.forename: Bryan C
authors.initials: BC
authors.lastname: Dickinson
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA ; Department of Molecular and Cell Biology, University of California, Berkeley, CA ; Howard Hughes Medical Institute, University of California, Berkeley, CA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Chang
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA.
authors.forename: A Sue
authors.initials: AS
authors.lastname: Menko
mesh_terms: D000818:Animals; D019069:Cell Respiration; D002642:Chick Embryo; D004847:Epithelial Cells / Q000378:metabolism; D004848:Epithelium / Q000196:embryology* / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007908:Lens, Crystalline / Q000166:cytology / Q000196:embryology* / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D009861:Onium Compounds / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D009861:Onium Compounds; C007517:diphenyleneiodonium; D006861:Hydrogen Peroxide
references.citation: Curr Opin Chem Biol. 2010 Feb;14(1):50-6
references.pmid: 19910238
references.citation: Sci STKE. 2006 Aug 22;2006(349):re8
references.pmid: 16926363
references.citation: Chem Biol Interact. 2013 Feb 25;202(1-3):2-10
references.pmid: 23159885
references.citation: J Cell Sci. 1994 Apr;107 ( Pt 4):799-811
references.pmid: 8056837
references.citation: Science. 2006 Jun 30;312(5782):1882-3
references.pmid: 16809515
references.citation: Biol Reprod. 2013 Jul 25;89(1):17
references.pmid: 23740947
references.citation: Antioxid Redox Signal. 2009 Oct;11(10):2467-80
references.pmid: 19309256
references.citation: Methods Mol Biol. 2009;554:165-81
references.pmid: 19513674
references.citation: Dev Biol. 1984 May;103(1):129-41
references.pmid: 6370757
references.citation: Antioxid Redox Signal. 2011 Jul 1;15(1):147-51
references.pmid: 21375475
references.citation: Proc Natl Acad Sci U S A. 2010 Sep 7;107(36):15681-6
references.pmid: 20724658
references.citation: Mol Cell. 2007 Apr 13;26(1):1-14
references.pmid: 17434122
references.citation: Pathophysiology. 2006 Aug;13(3):151-62
references.pmid: 16765571
references.citation: Invest Ophthalmol Vis Sci. 2006 Oct;47(10):4490-9
references.pmid: 17003444
references.citation: Cell Res. 2011 May;21(5):817-34
references.pmid: 21445095
references.citation: Ophthalmic Res. 1989;21(6):414-9
references.pmid: 2626238
references.citation: Immunity. 2012 Dec 14;37(6):1037-49
references.pmid: 23159440
references.citation: Biomed Eng Online. 2012 Sep 19;11:69
references.pmid: 22992294
references.citation: Exp Eye Res. 2005 May;80(5):709-25
references.pmid: 15862178
references.citation: J Cell Physiol. 1998 Sep;176(3):574-87
references.pmid: 9699510
references.citation: Nat Chem Biol. 2011 Jul 18;7(8):504-11
references.pmid: 21769097
references.citation: Am J Physiol Lung Cell Mol Physiol. 2005 Aug;289(2):L159-73
references.pmid: 16002998
references.citation: Biochim Biophys Acta. 2007 May;1774(5):545-55
references.pmid: 17428749
references.citation: ACS Chem Biol. 2010 Jan 15;5(1):47-62
references.pmid: 19957967
references.citation: Invest Ophthalmol Vis Sci. 2011 Mar 25;52(3):1723-34
references.pmid: 21087961
references.citation: J Biol Chem. 2012 Mar 9;287(11):8384-97
references.pmid: 22275359
references.citation: Biochim Biophys Acta. 2010 Oct;1797(10):1705-15
references.pmid: 20547138
references.citation: Dev Biol. 2002 Nov 15;251(2):195-205
references.pmid: 12435352
references.citation: J Membr Biol. 2007 Mar;216(1):1-16
references.pmid: 17568975
references.citation: Cell Metab. 2011 Apr 6;13(4):361-366
references.pmid: 21459321
references.citation: Antioxid Redox Signal. 2006 Mar-Apr;8(3-4):243-70
references.pmid: 16677071
references.citation: Mol Vis. 2004 Feb 19;10:112-21
references.pmid: 14978478
references.citation: J Gen Physiol. 2011 Jun;137(6):507-20
references.pmid: 21624945
references.citation: Am J Physiol Renal Physiol. 2012 Jan 1;302(1):F95-F102
references.pmid: 21975873
references.citation: Invest Ophthalmol Vis Sci. 2010 May;51(5):2303-6
references.pmid: 20435604
references.citation: Philos Trans R Soc Lond B Biol Sci. 2011 Apr 27;366(1568):1265-77
references.pmid: 21402585
references.citation: J Biol Chem. 2011 Sep 23;286(38):33203-12
references.pmid: 21768092
references.citation: J Biol Chem. 2004 Dec 10;279(50):51715-8
references.pmid: 15364933
references.citation: Exp Eye Res. 2004 Jun;78(6):1057-67
references.pmid: 15109912
references.citation: Biochim Biophys Acta. 2006 Aug;1758(8):994-1003
references.pmid: 16566894
references.citation: FASEB J. 1995 Sep;9(12):1173-82
references.pmid: 7672510
references.citation: Biochemistry. 1998 Apr 21;37(16):5633-42
references.pmid: 9548949
references.citation: J Am Chem Soc. 2008 Jul 30;130(30):9638-9
references.pmid: 18605728
references.citation: Cell Biochem Funct. 2007 May-Jun;25(3):317-21
references.pmid: 17191273
references.citation: Mol Vis. 2007 Mar 14;13:374-87
references.pmid: 17392688
references.citation: Front Biosci (Elite Ed). 2012 Jan 01;4:141-55
references.pmid: 22201860
references.citation: Invest Ophthalmol Vis Sci. 2011 Oct 17;52(11):8231-40
references.pmid: 21896865
references.citation: Antioxid Redox Signal. 2013 Mar 20;18(9):1042-52
references.pmid: 22867279
references.citation: Acc Chem Res. 2011 Sep 20;44(9):793-804
references.pmid: 21834525
references.citation: Methods Enzymol. 2013;526:19-43
references.pmid: 23791092
references.citation: Invest Ophthalmol Vis Sci. 2012 Oct 11;53(11):7087-96
references.pmid: 22969071
references.citation: Prog Retin Eye Res. 2003 Sep;22(5):657-82
references.pmid: 12892645
references.citation: Br J Ophthalmol. 1999 Sep;83(9):1064-8
references.pmid: 10460777
references.citation: Mol Cell Biol. 2004 May;24(10):4384-94
references.pmid: 15121857
references.citation: J Biol Chem. 2005 Jun 10;280(23):22135-45
references.pmid: 15826955
references.citation: Invest Ophthalmol Vis Sci. 2009 May;50(5):2291-300
references.pmid: 19136702
references.citation: PLoS One. 2011 Jan 21;6(1):e14564
references.pmid: 21283738
references.citation: Nat Chem Biol. 2011 Feb;7(2):106-12
references.pmid: 21186346