Photodynamic Treatment of Culture Medium Containing Serum Induces Long-Lasting ToxicityIn Vitro

Abstract

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.

Influence of Animal Species on Secondary Oxidant Activation in Serum Exposed to Singlet Oxygen

“…The capacity of SOS production varied from one healthy person to another (Olivier and Douillard et al 2009b) but was constantly lower than in other primates. It had been found to be higher for Microcebus murinus (pre-primates), Platyrrhini, Catarrhini, and among them for humans (Figures 1 and 2), in this order of magnitude, thus following the phylogeny of primates (classified accordingly to the existence of primitive anthropomorphic characters (Thorington and Anderson 1984)).…”

“…In a previous paper we found the capacity of O deactivation to vary widely between FCS and HS (Olivier and Douillard et al 2009b). In the current study, we used similar experimental conditions (RB and serum concentration, temperature, and irradiation) for each serum tested, and we extended the range of species studied to animals that are only rarely assessed for their resistance to ROS.…”

“…When pooled according to the number of branches within primates, the series of ratios (assayed serum AUC/HS pooled sera AUC) correlated with the number of branches (Kruskal-Wallis one-way analysis of variance on ranks, p < 0.001, Dunn's method, p < 0.05). In a previous paper we (Olivier et al 2009b) noted that the capacity for SOS production widely varied between FCS and HS. In the current study, using similar experimental conditions (RB and serum concentration, temperature, and irradiation) for each serum tested, our data indicate that the capacity of mouse sera for SOS production was about twice as high as that of FCS, and four times higher than that of HS.…”

“…The measurement principle (Olivier and Douillard et al 2009b) that we used was based on the analysis of ROS and peroxides production, induced by photodynamically produced, by means of the DCFH/dichloro-dihydro-fluorescent (DCF ) system after adding activated DCFH to each sample assayed immediately after the end of light delivery. Briefly, we prepared a solution extemporaneously in glass cuvettes specialized for fluorescence (1 cm x 1 cm).…”

“…O , an excited form of "normal" molecular O and ROS are also involved as a cause or a consequence of many diseases, inflammatory reactions, and metabolic pathways. We recently demonstrated that secondary ROS and peroxides production was much lower in human serum than in fetal calf serum (FCS ), with production differing from one person to another (Olivier and Douillard et al 2009b). The aim of the present study was to determine whether sera obtained from the blood of several animal species had the same capacity for secondary ROS and peroxides (SOS ) neutralization as sera prepared from human blood.…”