Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells

Abstract

Violet-blue light is toxic to mammalian cells, and this toxicity has been linked with cellular production of H2O2. In this report, we show that violet-blue light, as well as UVA, stimulated H2O2 production in cultured mouse, monkey, and human cells. We found that H2O2 originated in peroxisomes and mitochondria, and it was enhanced in cells overexpressing flavin-containing oxidases. These results support the hypothesis that photoreduction of flavoproteins underlies light-induced production of H2O2 in cells. Because H2O2 and its metabolite, hydroxyl radicals, can cause cellular damage, these reactive oxygen species may contribute to pathologies associated with exposure to UVA, violet, and blue light. They may also contribute to phototoxicity often encountered during light microscopy. Because multiphoton excitation imaging with 1,047-nm wavelength prevented light-induced H2O2 production in cells, possibly by minimizing photoreduction of flavoproteins, this technique may be useful for decreasing phototoxicity during fluorescence microscopy.

Figure 1

Cytochemical detection of H₂O₂ production in 3T3 cells. Cells were irradiated with blue light (450–490 nm at 6.3 W/cm²) for 20 min in saline containing AEC followed by thorough washing to remove large nonspecific crystals. (A) In normal saline, irradiated cells (circular area) displayed moderately dark reaction product (polyAEC) with a punctate distribution. (B) In cells pretreated with catalase, staining was darker and clearly localized in the cytoplasm. (C) Staining was markedly attenuated in cells fixed for 1.5 hr in buffered 4% paraformaldehyde solution before irradiation. (D) Staining was reduced and punctate in cells pretreated with vitamin C. Arrows denote cells with partial staining. [Bar = 100 μm and 50 μm (Inset).]

Figure 2

Ultrastructural localization of DAB reaction product (polyDAB) in 3T3 cells fixed and stained with osmium after irradiation with blue light (450–490 nm at 6.3 W/cm²) for 40 min. Staining was distributed uniformly within peroxisomes (p in A), whereas it was concentrated in the inner and outer membranes of mitochondria (m in B). Inset in A is ×2 magnification.

Figure 3

Detection of H₂O₂ production in 3T3 cells by using conventional (one-photon) fluorescence microscopy. Cells were pretreated with vitamin C, loaded with DHF-DA, and irradiated with blue light (485–495 nm) at either 1 W/cm² (A) or 0.2 W/cm² (B). (A) After 1 sec of irradiation (Left), fluorescence emanated from mitochondria, worm-like structures (Inset) dispersed throughout the cell. After 6 sec of continuous irradiation (Right), fluorescence was diffuse and brightest around or within the nucleus. (B) Fluorescent responses developed more slowly when irradiated at lower intensity. [Bar = 2 μm (A), 1 μm (A Inset), and 20 μm (B).]

Figure 4

Fluorescent image of CV1 cells loaded with vitamin C and C-DCDHF-DA-AM and irradiated with blue light (485–495 nm at 2.3 W/cm²). Fluorescence was localized in small, spherical organelles, which were most likely peroxisomes (see text). [Bar = 2 μm and 1 μm (Inset).]

Figure 5

Fluorescence was reduced in catalase-treated cells and enhanced in AOX- and XO-transfected cells. Cells were loaded with C-DCDHF-DA-AM and irradiated with violet-blue light (445–455 nm) at 1.6 W/cm² (Upper) or 5.3 W/cm² (Lower) during the time indicated. (Upper) Average response of HK and 3T3 cells (Cont) was significantly reduced after preincubation in catalase (Cat). (Lower) AOX- and XO-transfected cells produced significantly larger responses than nontransfected cells (note difference in scale between Upper and Lower graphs).

Figure 6

Fluorescent responses measured with MPE (two-photon) imaging. Cells were loaded with C-DCDHF-DA-AM and imaged before and after irradiation with violet-blue light (445–455 nm at 1.6 W/cm²). (Top) 3T3 cells irradiated for 1 sec on four consecutive occasions displayed step increases in fluorescence without subsequent photobleaching. (Middle) Average responses of CV1 and 3T3 cells (Cont) were reduced by catalase treatment (Cat). Oxyrase (Oxy) was also effective at suppressing the response. The residual response in catalase-treated cells may reflect radical generation, insufficient catalase, or both. (Bottom) Average responses of AOX- and XO-transfected cells was significantly larger than in nontransfected CV1 cells. The response of XO-transfected cells was reduced to control levels by 200 nM allopurinol (Allo). P values are 0.1 (∗), 0.02 (∗∗), 0.005 (∗∗∗), and 0.001 (^∧∧). Black bars represent basal levels of fluorescence because of autooxidation of the dye.

Figure 7

A model depicting the molecular events underlying light-induced H₂O₂ production and detection in cells. The process is initiated by photoreduction of flavins and/or flavin-containing oxidases within peroxisomes and mitochondria (and probably cytoplasmic sites as well) resulting in H₂O₂ production. Endogenous peroxidase and oxidase enzymes subsequently convert H₂O₂ to water coupled with oxidation of DHF and DAB to fluorescein (F) and polyDAB, respectively. Catalase, which can also oxidize DAB to polyDAB, converts H₂O₂ to water and dioxygen. The latter apparently competes with oxidation of DHF derivatives. When the level of H₂O₂ exceeds enzymatic activities, it diffuses into the cytoplasm where additional dye reduction occurs. H₂O₂ and its metabolite, hydroxyl radicals, may produce toxicity through genetic mutation, inhibition of glycolysis and DNA synthesis, and lipid peroxidation.