Time dependency of metabolic changes in rat lens after in vivo UVB irradiation analysed by HR-MAS 1H NMR spectroscopy.
The lens ability to protect against, and repair ultraviolet radiation (UVR) induced damages, is of crucial importance to avoid cataract development. The influence of UVR-induced damage and repair processes on the lens metabolites are not fully understood. Observation of short- and long-term changes in light scattering and the metabolic profile of pigmented rat lenses after threshold UVR exposure might serve to better understand the protective mechanisms in the lens. By using high resolution magic angle spinning (HR-MAS) 1H NMR spectroscopy it was possible to investigate the metabolites of intact rat lenses. Brown-Norway rats were exposed to 15 kJm(-2) UVB irradiation. One eye was exposed and the contralateral served as control. The rats were sacrificed 5, 25, 125, and 625 hr post-exposure and the lenses were removed. The degree of cataract was quantified by measurement of lens forward light scattering. Thereafter, proton NMR spectra from intact lenses were obtained and relative changes in metabolite concentrations were determined. The light scattering in the lens peaked at 25 hr post-exposure and decreased thereafter. The lowest level of light scattering was measured 625 hr after exposure. No significant changes in concentration were observed for the metabolites 5 and 25 hr post-exposure except the total amount of adenosine tri- and diphosphate (ATP/ADP) that showed a significant decrease already 5 hr after exposure. At 125 hr the lens concentrations of lactate, succinate, phospho-choline, taurine, betaine, myo-inositol, and ATP/ADP showed a significant decrease (p<0.05). Phenylalanine was the only metabolite that revealed a significant increase 125 hr post-exposure. At 625 hr most of the metabolic changes seemed to normalise back to control levels. However, the concentration of betaine and phospho-choline were still showing a significant decrease 625 hr after UVB irradiation. The impact of UVB irradiation on the metabolic profile did not follow the same time dependency as the development of cataract. While the light scattering peaked at 25 hr post-exposure, significant changes in the endogenous metabolites were observed after 125 hr. Both the metabolic changes and the light scattering seemed to average back to normal within a month after exposure. Significant decrease in osmolytes like taurine, myo-inositol and betaine indicated osmotic stress and loss of homeostasis. This study also demonstrated that HR-MAS 1H NMR spectroscopy provides high quality spectra of intact lenses. These spectra contain a variety of information that might contribute to a better understanding of the metabolic response to drugs or endogenous stimuli like UVB irradiation.
Risa
Oystein
O
Faculty of Natural Sciences and Technology, The Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway. oystein.risa@ntnu.no
Saether
Oddbjørn
O
Kakar
Manoj
M
Mody
Vino
V
Löfgren
Stefan
S
Söderberg
Per G
PG
Krane
Jostein
J
Midelfart
Anna
A
D000818:Animals; D007908:Lens, Crystalline / Q000378:metabolism* / Q000528:radiation effects*; D009682:Magnetic Resonance Spectroscopy; D011833:Radiation Injuries, Experimental / Q000209:etiology / Q000378:metabolism*; D051381:Rats; D011914:Rats, Inbred BN; D012542:Scattering, Radiation; D013997:Time Factors; D014466:Ultraviolet Rays / Q000009:adverse effects*