Absorption measurements of cell monolayers relevant to mechanisms of laser phototherapy: reduction or oxidation of cytochrome c oxidase under laser radiation at 632.8 nm.

OBJECTIVE: The objective of this work was a further investigation of redox mechanisms of laser phototherapy on the cellular level. BACKGROUND DATA: Cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is believed to work as the photoacceptor to modulate cellular metabolism in laser phototherapy. MATERIALS AND METHODS: The changes in the absorption spectra of HeLa-cell monolayers before and after irradiation at 632.8 nm using fast multi-channel recording were evaluated by the intensity ratio between the peaks at 770 and 670 nm (intensity ratio criterion). RESULTS: By the intensity ratio criterion, the irradiation effects (reduction or oxidation of the photoacceptor) depended on the initial redox status of cytochrome c oxidase. The irradiation (three times at 632.8 nm, dose = 6.3 x 103 J/m(2), tau(irrad.) = 10 sec, tau(record.) = 600 msec) of cells initially characterized by relatively oxidized cytochrome c oxidase caused first a reduction of the photoacceptor, and then its oxidation (a bell-shaped curve). The irradiation by the same scheme of the cells with initially relatively reduced cytochrome c oxidase caused first oxidation and then a slight reduction of the enzyme (a curve opposite to the bell-shaped curve). CONCLUSION: The experimental results of our work demonstrate that irradiation at 632.8 nm causes either a (transient) relative reduction of the photoacceptor, putatively cytochrome c oxidase, or its (transient) relative oxidation, depending on the initial redox status of the photoacceptor. The maximum in the bell-shaped dose-dependence curve or the minimum of the reverse curve is the turning point between the prevailing of oxidation or reduction processes. Our results are evidence that the bell-shaped dose dependences recorded for various cellular responses are characteristic also for redox changes in the photoacceptor, cytochrome c oxidase.

Absorption measurements of a cell monolayer relevant to phototherapy: reduction of cytochrome c oxidase under near IR radiation.

Phototherapy uses monochromatic light in the optical region of 600-1000 nm to treat in a non-destructive and non-thermal fashion various soft-tissue and neurological conditions. This kind of treatment is based on the ability of light red-to-near IR to alter cellular metabolism as a result of its being absorbed by cytochrome c oxidase. To further investigate the involvement of cytochrome c oxidase as a photoacceptor in the alteration of the cellular metabolism, we have aimed our study at, first, recording the absorption spectra of HeLa-cell monolayers in various oxygenation conditions (using fast multichannel recording), secondly, investigating the changes caused in these absorption spectra by radiation at 830 nm (the radiation wavelength often used in phototherapy), and thirdly, comparing between the absorption and action spectra recorded. The absorption measurements have revealed that the 710- to 790-nm spectral region is characteristic of a relatively reduced photoacceptor, while the 650- to 680-nm one characterizes a relatively oxidized photoacceptor. The ratio between the peak intensities at 760 and 665 nm is used to characterize the redox status of cytochrome c oxidase. By this criterion, the irradiation of the cellular monolayers with light at lambda=830 nm (D=6.3 x 10(3)J/m(2)) causes the reduction of the photoacceptor. A similarity is established between the peak positions at 616, 665, 760, 813, and 830 nm in the absorption spectra of the cellular monolayers and the action spectra of the long-term cellular responses (increase in the DNA synthesis rate and cell adhesion to a matrix).