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Singlet oxygen (1O2) is produced by leucocytes during inflammatory reactions, various biochemical reactions and during photoreactions. It deactivates by reacting with a number of targets to produce reactive oxygen species (ROS) and peroxides (that in turn produce ROS). To verify whether serum had the same capability to deactivate secondary oxidants after exposure to 1O2, we provoked a photoreaction using rose bengal added to sera of 53 healthy donors and, after light delivery, reduced 2',7'-dichlorofluorescein (DCFH) was added at the end of irradiation and fluorescence of the oxidized derivative (DCF) was recorded. To avoid optical artifacts, we analyzed the influence of hemolysis. Deactivation capability of secondary oxidants after exposure to (1)O(2) was stable over a long period of time, slightly different between men and women, but standard biochemistry parameters had little influence. Hemolysis, age and platelet number reduced deactivation of 1O2-induced secondary oxidants. Addition of lysed cancer cells had no influence. Blood sampling in clot act tubes gave a better signal than in heparinized tubes. Red blood cells (RBCs) loaded with antioxidants strongly decreased deactivation of secondary oxidants. Assays are in progress to evaluate the clinical implications of these findings.

Photodynamic therapy (PDT) produces singlet oxygen and reactive oxygen species (ROS) that damage tumor cells and the vasculature. The resulting effect is a balance between photo-oxidations through primary or secondary ROS and scavenging activity. Sensitizers are distributed in the extracellular space before and during cell sensitization, suggesting that PDT could act directly on cell structures and on extracellular compartments, including sera. In this study we endeavored to determine whether the application of PDT to culture medium could affect cell survival. Culture medium [RPMI 1640 supplemented with fetal calf serum (FCS)] was incubated with Rose Bengal and irradiated before being added to cells for various contact times as a replacement for untreated medium. Cells were then kept in darkness until the survival assay. Treated medium reduced cell survival by up to 40% after 30 min of contact for 10 microg/ml of Rose Bengal and 20 J/cm(2). Rose Bengal or m-THPC alone or irradiated in water had no effect. This effect was dependent on the doses of Rose Bengal and light and decreased when FCS was replaced by human serum mixed with FCS. The reduction in survival observed with treated medium was more pronounced when the cell doubling time was shorter. Analysis of ROS or peroxide production in treated medium by DCFH added at the end of irradiation of Rose Bengal in serum-containing medium revealed a long-lasting oxidizing activity. Our findings support the hypothesis of an ROS- or peroxide-mediated, PDT-induced, long-lasting cell toxicity.

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Singlet oxygen ((1)O(2)) produced during inflammatory reactions and during photodynamic therapy deactivates by producing in tissues secondary reactive oxygen species and peroxides (SOS) as well as other degradation products. We investigated the influence of animal species on SOS production secondary to standardized (1)O(2) production by performing in vitro experiments with rose bengal as the (1)O(2) producer, human serum (HS) as a control, sera derived from various animal species, and dichloro-dihydro-fluorescein as a nonspecific marker that becomes fluorescent when oxidized. The overall SOS production in HS from a presumed healthy cohort of 53 donors (31 males and 22 females) gave a mean "normal" value of 0.91 compared with a previous pool of 75 male sera samples. SOS production after a photo-reaction was two or four times lower in HS than in fetal calf serum or mice sera, respectively. In mice, the "nude" characteristic increased even more than in the SOS production. In the Aves order, this production appeared to be distributed randomly according to the number of branches after the appearance of Amniotas. For primates, SOS production appeared to decrease linearly with the number of branches (R(2) = 0.98). Adding hemolysates from complete bloods to the corresponding sera induced an increase in SOS production in all species, proportional to the production in sera. These findings should be kept in mind when interpreting results from studies of secondary reactive oxygen species-induced pathways following (1)O(2) production, regardless of its origin.

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