Protein modifications by 4-hydroxynonenal and 4-hydroxyhexenal in light-exposed rat retina

Invest Ophthalmol Vis Sci. 2005 Oct;46(10):3859-68. doi: 10.1167/iovs.05-0672.


Purpose: 4-Hydroxynonenal (4-HNE) and 4-hydroxyhexenal (4-HHE) are reactive aldehydes derived from the nonenzymatic oxidation of n-6 and n-3 polyunsaturated fatty acids, respectively. Increasing evidence suggests that protein modifications by reactive aldehydes are involved in various diseases. The present study was undertaken to test whether protein modifications by 4-HNE and 4-HHE increase in retinal tissues after exposure of rats to damaging levels of light.

Methods: Albino rats were exposed to 1 or 5 klux white fluorescent light for 3 hours and, at various times thereafter, the levels and localizations of aldehyde-modified proteins in retinas were assessed by densitometric analysis of semiquantitative Western dot blots and by immunohistochemistry, using 4-HNE- and 4-HHE-specific antibodies. In some rats, the protective antioxidant phenyl-N-tert-butylnitrone (PBN) was injected (50 mg/kg) before exposure to light. To assess retinal damage, outer nuclear layer (ONL) thickness was measured on hematoxylin-eosin (H&E)-stained sections, and apoptosis was semiquantitatively analyzed by TUNEL staining.

Results: By dot blot analysis, 4-HNE- and 4-HHE-modified proteins were significantly increased in retina (both by 1.7-fold) and RPE fraction (1.5- and 1.8-fold, respectively) after 5-klux exposure. In retina, increases in 4-HNE- and 4-HHE-modified proteins were more prominent at 3 hours than at 24 hours or 48 hours after exposure to light. In rod outer segments, only 4-HHE-modified proteins increased significantly (1.4-fold). Retinal thinning, TUNEL staining in ONL, 4-HNE-, and 4-HHE protein modifications were all found in the same retinal regions. PBN treatment inhibited the light-induced increase of 4-HNE and 4-HHE modified proteins in retina and RPE fractions.

Conclusions: Exposure to intense light increases 4-HNE and 4-HHE protein modifications in the retina, suggesting that free radical initiated, nonenzymatic reactions are involved in this process. These modifications may be early events that precede photoreceptor cell apoptosis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aldehydes / metabolism*
  • Animals
  • Apoptosis
  • Blotting, Western
  • Cyclic N-Oxides
  • Eye Proteins / metabolism*
  • Female
  • Free Radical Scavengers / pharmacology
  • Immunoenzyme Techniques
  • In Situ Nick-End Labeling
  • Light
  • Nitrogen Oxides / pharmacology
  • Protein Processing, Post-Translational*
  • Radiation Injuries, Experimental / metabolism*
  • Radiation Injuries, Experimental / pathology
  • Radiation Injuries, Experimental / prevention & control
  • Rats
  • Rats, Sprague-Dawley
  • Retina / drug effects
  • Retina / pathology
  • Retina / radiation effects*
  • Retinal Degeneration / metabolism*
  • Retinal Degeneration / pathology
  • Retinal Degeneration / prevention & control


  • Aldehydes
  • Cyclic N-Oxides
  • Eye Proteins
  • Free Radical Scavengers
  • Nitrogen Oxides
  • 4-hydroxy-2-hexenal
  • phenyl-N-tert-butylnitrone
  • 4-hydroxy-2-nonenal