Prooxidant and Antioxidant Action of Psoralens

Teresa Michalska

Institute of Physics, Szczecin University of Technology

Al. Piastow 48/49, 70-310 Szczecin, Poland

ABSTRACT: The effect of psoralens (psoralen, 5-methoxypsoralen, 8-methoxypsoralen, khellin and visnagin) on a chemical system involving a superoxide radical was tested using the chemiluminescence method.

High doses of psoralens (1mM) showed prooxidative effects. Incubation of psoralens at lower doses showed khellin (0.8 mM), 8-methoxypsoralen (0.1 mM), visnagin (0.05mM) and psoralen (0.03 mM) have antioxidant effects.

KEYWORDS: psoralens; chemiluminescence; superoxide radicals.

1. Introduction

Psoralens (PSOs) or furanocumarins are well known as photoreactive compounds [1].The compounds are also very often used in dermatology for the photochemotherapy of several diseases such as vitiligo, psoriasis, atopic eczema and others [2,3].The combination of PSOs usage with UV-A irradiation is known as PUVA therapy [4]. It has been shown that UV-A irradiation of PSOs in the air atmosphere generates a large amount of photoproducts, known as reactive oxygen species (ROS) such as: superoxide radical anion (), hydroxyl radical (), hydrogen peroxide () and singlet oxygen () [5-8]. However, it has been observed that PSOs at high doses also generates ROS in PUVA inducing undesirable site effects (e.g. skin cancer, edena, skin erythema) [6,10,11].

This paper deals with experiments concerning the effect of five PSOs: psoralen (PSO), 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), khellin (KHL) and visnagin (VIG) on the light emission from a chemical system generating . These compounds (see structures, Fig. 1) are hypothesized to have anti-oxidative and pro-oxidative properties. The properties may be examined using the chemiluminescent technique, which is the sensitive analytical method for detection, especially for the evaluation of the redox property of a

compound.

Fig.1. The chemical structure of PSOs.

2. Materials and Methods

PSOs, superoxide dismutase (SOD, EC1.15.11, 5600 U/mg) and catalase (19,900 U/mg from bovine liver thymol free) were purchased from Sigma Chemical Co. (st.Louis, MO). Potassium superoxide (KO₂) were from Fluka, Buchs (Switzeralnd). Dimethyl sulfoxide (DMSO) was obtained from Aldrich Chemical Co. (Milwaukee. WI). Crown ether (18-crown-6), Tiron (1,2-dihydroxybenzene-3,5-disulfonic acid), trolox (6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid), 5,5-dimethyl-cyclohexanodion-1,3 (DMCH) and compounds that were used as antioxidant were purchased from Merck (Darmstadt, Germany).

Superoxide anion radical () was prepared according to the following procedure: 60mg of 18-crown-6 was dissolved in 10 mL of dry DMSO and then 7 mg of KO₂ was added quickly to avoid contract with air humidity [12]. The mixture was stirred with a magnetic stirrer for 1 h to give a pole yellow solution of 10 mM which was at least 1 h stable at room temperature. DMSO mixtures of PSOs were prepared at room temperature and kept in the dark.

The influence of PSOs on the system generating was detected by a chemiluminescent technique. The chemiluminescence (CL) intensity was recorded with a set especially designed and constructed in Institute of Physics, Szczecin University of Technology (Fig. 2). The basic instrumentation consists of a specially designed chamber, containing glass cuvette with 50 mm diameter, placed in light-tight box. The cuvette was exhausted and washed using a B-169 vacuum system (Büchi, Flawill Switzerland). The photomultiplier type EMI 9553Q with a S20 cathode, sensitive in the range 200-800 nm, interfaced with a personal computer for date acquisition and handling was used as a detector.

The apparatus used provides the opportunity for simultaneously calculation of the light sum, i.e., area (SI) under the kinetic curve, I = f(t) within any chosen time interval ().

All data are presented as a mean ± SD of at least six different experiments. P-values < 0.05 were considered as statistically significant.

Fig.2. Block diagram of chemiluminescence measurements system.

3. Results and discussion

Reactivity of PSOs with was monitored using the radical at a concentration of 1 mM in DMSO. The mixture elicits a strong CL, (Fig. 3, curves 1). The tested PSOs were added 30s after the start reaction and SI were detected during 60s. An addition of DMSO alone at the same concentration as in the PSOs/DMSO solution resulted in “flash” followed by a increase in CL/Fig. 3, curve 2). An area under curve 2 considered as the control sum(). The quenching ratio was defined as , where represents the light sum without a inhibitor, represents the light sum with a inhibitor.

Fig.3. The effect of DMSO, Tiron and NTB on chemiluminescence intensity from 1 mM generated in DMSO. The arrow indicate the moment of reagent addition

Oostehuizen et al. [13] reported have disproportionation of in the system with KO₂ generating these radicals:

+ + 2H⁺ H₂O₂ + (1)

This reaction is accompanied by generation of small amounts of [14]. An interaction between . and H₂O₂ in the presence of a reducing agent such as DMSO can lead to the formation [15]

+ H₂O₂ + HO^- + (2)

Another possible reaction responsible for the generation under our experimental conditions is on interaction of and radical as follows:

+ HO^- + (3)

as well as disproportionation of H₂O₂

H₂O₂ + H₂O₂ 2H₂O + (4)

The generation of ROS in the examined systems was confirmed by observed quenching effect on the CL of inhibitors specific for , , H₂O₂ and scavengers of ¹O₂ [16] (Table 1).

Among the tested inhibitors Tiron and NTB appeared to be the strongest quenchers of the light emission. In contrast, an addition of SOD (0.1mg/mL) know inhibitor of to the reaction system increased the light of emission (quenching ratio was about -41 %). The observed increase in the CL in the presence of SOD in study may be due to the fact that SOD catalyses the formation of ROS in the presence of H₂O₂ and reducing agent [17,18].

Also the aqueous solution of histidine, DMCH (quenchers of ¹O₂) and catalyse (enzyme responsible for destruction of H₂O₂ ) the CL by 42%, 36% and 48% respectively.

However, water alone at the same amount (5%) as the tested antioxidant / H₂O solution decreased the CL by about 10%

Table 1. Effect of inhibitors ROS on chemiluminescence from 1 mM generated in DMSO.

Compound	Concentration	Quenching %
NBT	1 mM	67 ± 6
Tiron	1 mM	58 ± 5
Trolox	1 mM	36 ± 5
SOD	100 mg/mL	-41 ± 4
SOD	50 mg/mL	-17 ± 3
Catalase*	200 mg/mL	48 ± 6
Catalase*	50 mg/mL	16 ± 2
Histidine*	1 mM	42 ± 4
5,5-dimethylocykloheksanodione-1,3 (DMCH)*	0,5 mM	36 ± 4
Thiourea	1 mM	28 ± 3
Ethanol	1 M	32 ± 4
Mannitol	1 mM	39 ± 5

The compounds were dissolved in DMSO.

* - the compounds dissolved in water.

An addition of thiourea, ethanol, or mannitol (efficient scavenger ) caused a decrease in CL of about 28%, 32% and 39% respectively.

Fig.4. The effect of different concentration of Tiron on CL from 1 mM generated in DMSO (A). Relationship between quenching ratio and concentration Tiron (B).Details are reported under ‘Materials and Method’.

Figure 4A presents quenching of CL in the presence of different concentration of Tiron. Final results were calculated with a help of regression equation and are presented as quenching ratio Q_x = a + b C_x where C_x the logarithmics concentration of Tiron (Fig 4B). Regression coefficients for Tiron are: a = 55,05 ± 0.79 and a value for the slope b = 19.44 ±0.57 (mean ± SD); multiple correlation coefficient r = 0.997. A very similar effect presents KHL in a range 0,8 – 0.002 mM (Fig. 5A). Regression coefficients for KHL are: a = 32,85 ± 0.59 and b = 11,06 ± 0.39; multiple correlation coefficient r = 0.996.

A set experiments was performed to determine the reactivity of PSOs: psoralen, 5-methoxypsoralen, 8-methoxypsoralen, khellin and visnagin towards using the CL technique. The decrease or increase in the light emission from the system generating was dependent on the concentration added PSO and their kind (Fig 5).

Fig.5. The effect of psoralens concentration on chemiluminescence intensity from 1 mM generated in DMSO.

The data demonstrate that all tested PSOs in doses of 1 mM increased the CL (Table 2), thus showing prooxidative effects; however the enhancing effects exerted by KHL were very small in comparison with 5-metoxypsoralen. Incubation of PSOs at lower doses with the generating system showed that KHL (0.8 mM), 8-MOP (0.1 mM), VIG (0.05 mM) and PSO (0.03 mM) exerted quenching effect on the light emission. In contrast 5-MOP at that concentration 0.5 mM did not exert the quenching effects (Fig 5F).

Table 2. Effect of 1 mM Psoralens on chemiluminescence from 1 mM generated in DMSO

Psoralens	Enhancement, R %
	R 60 s	R 120 s
5-metoxypsoralen	2861 ± 10	4522 ± 15
Psoralen	440 ± 5	633 ± 6
Visnagin	104 ± 4	226 ± 5
8-metoxypsoralen	68 ± 3	127 ± 4
Khellin	-37 ± 2	1,2 ± 0,3

The enhancement R was calculated using by , where is the light sum with a PSO and is the light sum without a PSO. R_60s, R_120s – light sum detected during 60s and 120s, respectively.

The chemiluminescence sums increased in the order KHL < 8-MPO < VIG < PSP < 5-MOP, indicating increased production of ROS. We observed a very similar sequence of the PSOs behavior as enhancers of formation via Fenton reaction in dark in experiments using reducion of ferricytochrome c by [9].

4. Conclusion

The chemiluminescence method is a simple and convenient technique to examine the PSOs redox properties at ambient temperature. Incubation of tested PSOs in the system producing light emission showed that khellin, 8-methoxypsoralen, visnagin and psoralen at low concentration show antioxidative effect in the system generating superoxide anion radical. When high doses of these compounds had been used they showed prooxidant property.

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