Results And Discussion

Carlini melanoma cells were tested for resistance to methylene blue (MB), irradiation, or both (Tables 1 and 2). These cells are resistant to 10 ^M MB for times as long as 60 min (Table 1) and do not suffer when irradiated in the absence of the dye (data not shown). For protection experiments an incubation time of 10 min was chosen to ensure good viability and negligible toxic effects of that vital dye. The presence of MB during irradiation strongly decreases viability (Table 2). This treatment produces in solution singlet oxygen, one of the most reactive ROS. Viability tests after 24, 48, 72 h confirm the high toxicity of singlet oxygen towards cells.

Cells were irradiated in the presence or in absence of hypotaurine or taurine (800 ^M) in combination with methylene blue for 10 min. Table 3 shows the effect on cell viability of taurine and hypotaurine at the same concentrations, when cell cultures were stressed with irradiated methylene blue.

The presence of hypotaurine markedly decreases cell death respect to the not treated cells. After 24 h viability in the presence of hypotaurine was 72% and 58% in the presence of taurine. These results indicate that, in the presence of both taurine and hypotaurine, cells are protected against oxidative stress induced by singlet oxygen and that hypotaurine ensures a more efficient protection in this system. It is known that hypotaurine specifically "traps" singlet oxygen in vitro12 whereas taurine apparently does not react with it [Pecci, unpublished result]. Hypotaurine is also a much more efficient scavenger of hydroxyl radicals than taurine1. We could hypothesize that the less efficient protective effect of taurine involves a different mechanism.

Table 3. Cell viability (evaluated by trypan blue exclusion) in melanoma cells after different treatments: dye irradiation was 10 min in all conditions. Hyp: hypotaurine; Tau: taurine; MB: methylene blue

Sample Control Hyp Tau MB 10 MB 10 nM MB 10 nM +

800 nM 800 nM nM + light+Hyp light + Tau _+ light 800 nM_800 |iM

Viability 100 107 85 42 72 58

Table 4. Ratios of enzymatic specific activities in cytosolic fractions of cultured melanoma cells 24 h after treatment

SAMPLES Ratio of enzymatic activities

Control

MB + light

Control + hypotaurine

MB + light + hypotaurine

Catalase/ SOD

13.2

28.6

18.1

17.1

GSH Px/ SOD

0.030

0.11

< 0.006

< 0.006

GSSG Rx/GSH Px

2.13

2.29

> 13.4

>20

methylene blue was 10 |M, hypotaurine 800 |M and the irradiation time 10 min.

methylene blue was 10 |M, hypotaurine 800 |M and the irradiation time 10 min.

After an irradiation time of 10 min the effect of hypotaurine was high and cell viability allowed measurement of enzymatic activities, recorded 24 h after treatment on the cytosol of cultured cells. Ratios of specific activities of some detoxifying enzymes are reported in Table 4. Absolute activity values are not shown.

The ratios catalase/SOD and GSH Px/SOD are indicative of the efficiency of enzymatic systems to scavenge ROS: an increase indicates a positive response of the cells, a decrease may be indicative of the risk of oxidative damage13. The ratio GSSG Rx/GSH Px is indicative of the capability of the cells to recycle glutathione: a decrease indicates a possible oxidative damage, because the cell cannot produce enough GSH to get rid of hydrogen peroxide, organic hydroperoxides or ROS.

The efficiency of ROS scavenging measured as a ratio between specific activities of catalase and SOD show some important modifications. Catalase specific activity markedly increased in methylene blue + light treated cells. SOD specific activity does not strongly change in any of the considered conditions, even if there is a little decrease when cells are stressed in the presence or in the absence of hypotaurine. The ratio catalase/SOD doubles with respect to the control cells, indicating a positive response of the cells to oxidative stress. The ROS scavenging efficiency is increased even if it is not sufficient to avoid cell death. In the presence of hypotaurine this ratio does not change when cells are stressed and it is a little over the control. These results suggests that the protection by hypotaurine is not imputable to an induction of antioxidant enzymatic response.

The activity of GSH Px parallels that of catalase, as it increases in stressed conditions. In the presence of hypotaurine, however, both in the control and in stressed cells, GSH Px activity is undetectable, and this explains the very low GSH Px/SOD ratio values.

In cells without hypotaurine, the activity of GSSG Rx parallels that of peroxidase, suggesting a relatively enhanced efficiency of glutathione recycling in stressed cells respect to the control. The observed high increase in the GSSG Rx/GSH Px ratio in hypotaurine-treated cells is due mainly to the above reported very low GSH Px activity.

It is well known that GSH Px is responsible for the detoxification of hydrogen peroxide and organic hydroperoxides. Its activity increases in those conditions where peroxide concentration increases in cells. Hypotaurine reacts very slowly6 with H2O2 and is not a valuable candidate for peroxide scavenging.

The unexpected disappearance of GSH Px activity in the presence of hypotaurine may indicate that peroxide production is lowered by the scavenging activity of hypotaurine towards singlet oxygen and/or hydroxyl radicals and that recycling of glutathione as a peroxide-scavenging system is not very important in these conditions.

In conclusion, in cells treated with methylene blue + light the activation of enzymatic defences is not sufficient to counteract ROS attack and we observe cell death. In hypotaurine treated cells viability is higher respect to the control and this would indicate that the enzymatic scavenging activation is not necessary for cell surviving. The protective effect of hypotaurine does not involve detoxifying enzymes; probably hypotaurine decreases the amount of ROS, in particular singlet oxygen, thus avoiding modification in enzyme activities.

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