Radioprotective Role of Thiola and Olive Oil During The Treatment of Some Eye Lesions With b-particles

Sherif S. Mahmoud and Mona M. Gamal

Biophysics department, Research Institute of Ophthalmology

2 El-Ahram Street, Giza, Egypt.

Abstract

This study was conducted to evaluate the radioprotective efficacy of topical application of the radioprotector thiola when combined with olive oil on the biophysical characteristics of lens lipids. Two groups of NewZealand white rabbits were used in this study. The first group (thiola group) was topically administered thiola thirty minutes and one minute before the treatment with two doses of b-particles (24 Gy and 100 Gy). The second group (olive oil and thiola group) was also received thiola as in the first group then olive oil was topically applied after the irradiation and continue daily till decapitation. The lens lipids were studied using UV-spectroscopy, fourier transform infrared spectroscopy and phase transition measurements. In addition, cholesterol and phospholipids concentrations were measured. The resulted typical absorption spectra of the lens lipids indicated that the native lipid peak was shifted towards higher wavelength for both doses (24 Gy and 100 Gy) in both olive oil group and the thiola group. The results also indicated that there was fluctuated changes in lens stability and fluidity concomitant with conformational and compositional changes in both groups. It may be concluded from the present study that application of thiola only or combined with olive oil seems to be promising tools in preventing the peroxidation of lens lipids induced by b-radiation treatments.

Key words: b-radiation, Olive oil, Thiola, Lipids, Lens, Eye.

Introduction

Radiotherapy occupied an important place in the treatment of occular diseases and tumors (Lommatzsch, 1986). The use of b-radiation for superficial treatment of the eye had been recommended for several diseases (Walter, 1994). Complications may arise with all forms of radiotherapy but vary with the different ocular tissues involved and the type of radiation used. The most serious of these include corneal perforations, cataract formation and goblet cell loss with keratinization resulting in dry eye (Hancock, 1986). In these instances, radioprotectors which can protect against or enhance recovery from radiation injury would be beneficial.

Radioprotectors are compounds that are absorbed by normal tissue cells and reduce their response to radiation without significant or comparable reduction of cytotoxic effect of radiation (Perez and Brady, 1992). Certain sulphydryl bearing compounds proved to exert significant role in preventing of radiation toxicity. Among these is aminothiol group which includes thiola. Thiola, as a synthetic compound, proved to be non-toxic and exerting radioprotection characters in man and animals (Sugahara et al., 1970).

Recently, attention has also focused on a variety of non-vitamin antioxidants, such as phenolic compounds, that might also contribute to cellular defense mechanisms (Decker, 2003). These phenolic compounds are found in many plant species and are present at very high concentrations in many components including olive oil (Ho et al., 1992). These components play a major role in controlling oxidative reactions in vivo, thus exhibiting anticarcinogenic and antiatherogenic activities (Decker, 2003 and Staveric 1994). Studies about the possible mechanisms of phenolic compounds indicate that these compounds are able to scavenge free radicals and to break peroxidative chain reactions. Phenolic acid can prevent lipid peroxidation by metal chelation (Staveric, 1994). The scattered litrature on the effect of daily uptake of olive oil revealed that it has: protective effect on the low density lipoprotein susceptible to oxidative modification (Ramirez et al., 1999), protective effect on breast cancer (Lipworth et al.,1997), antioxidant properties (Caterina et al., 1997) and reduce tissue lipid peroxidation (De La Cruz et al., 2000). Olive oil topically applied was found to reduce UV-B induced murine skin tumors (Budiyanto et al., 2001).

The complications arised from therapeutic doses of b-radiation on lens lipids were previously studied (Sherif, 1999), so the present work was designed to overcome these complications through studying the protective role of topically applied extra virgin olive oil when combined with the chemical radioprotector thiola on the molecular changes of lens lipids resulted from b-radiation.

Materials and Methods

The irradiation source used in this study was strontium-90 eye applicator (type SIA-20, Amersham, England). Two doses of beta particles were chosen in order to cover the multiple application of b-radiation in ophthalmology; 24 Gy (used for treatment of ptyerigium) and 100 Gy (used for treatment of melanoma). The dose rate was 10 Gy/week.

The radioprotector thiola [ N-(2-mercaptopropionyl glycin)] was purchased from Aldrich chemical company with purity 99%. Thiola was dissolved in bidistilled water to yield a final concentration of 250 mg/ml as described by Stonceipher et al., (1992). Both eyes of radioprotected animals were topically pretreated with thiola (50 ml/eye) applied thirty minutes and one minute before b-irradiation. Thiola was freshly prepared just before use.

Healthy mature NewZealand white rabbits weighing 2.5-3.0 Kg were used in this study. Animals were randomley classified into two groups, each composed of fifteen rabbits, as follows:

Group I : subdivided into three subgroups; normal , treated with 24 Gy and the third group was treated with 100 Gy b-radiation. The three subgroups were topically received thiola in both eyes thirty minutes and one minute pre-exposure to b-radiation. This group will given the name “thiola group”.

Group II: also, subdivided into three subgroups and received the same manipulation as group I except, after b-irradiation, olive oil was topically applied to the eyes of all animals including the normal ones till the decapitation time (three and ten weeks respectively). This group will given the name “olive oil and group”. All b-radiation dosage values refer to surface dose.

After the treatment with the demonstrated doses of b-particles, animals were sacrificed and the eyes were enucleated. The lenses were removed and weighed then lyophillized. The lyophillized lenses were mixed with cholorofrom:methanol mixture (2:1 v/v) and subjected to lipid extraction and purification according to Folch et al., (1957). Cholesterol and phospholipids were measured in an aliquotes of the extracted lipids spectrophotometrically, after evaporating the solvents , according to the procedure of Zigman et al., (1984) and Broekhyse (1968), respectively.

Phase diagram has been measured using Shimadzu UV-240a spectrophotometer , where the temperature was controlled through the attached thermobath (type Shimadzu TB-85). The accuracy of the temperature control was ± 0.5 oC. The temperature changing rate was 1 oC/minute according to Kremer et al., (1977). After recording the phase diagram of each sample, it was normalized according to the normal diagram of olive oil group or thiola group. The resulted normalized phase diagram was differentiated and plotted against the temperature.

The infrared spectra were aquired with a Shimadzu fourier transform infrared spectrophotometer model 8200 SNO (Japan), equipped with a standard detector. Measurements were made with an IR cell (KBr windows). Interferograms were recorded, coaded and apodized with a Happ-Genzel function prior to fourier transform yielding an effective resolution of 2.0 cm-1 according to William et al., (1996). All the spectra were recorded at physiological temperature.

The resulted data were statistically evaluated by the Student’s “t” test and, correlated by Pearson correlation coefficient.

Results

The typical absorption spectra of lens lipids prepared from thiola group after exposure to a dose of 24 Gy or 100 Gy b-radiation are shown in figure (1). The figure indicates the presence of two peaks; one at about 235 nm, while the other peak (broad) at 274-288 nm region. The normal lens lipid spectra are characterized by its high intensity peaks when compared with thiola group. On the other hand, after topical application of olive oil (figure 2) the absorption spectra of lens lipids showed a completely different pattern; the two peaks were apeared at different wavelengths, the first peak was appeared at 220-225 nm region and the other peak at 272 – 280 nm region.

The estimated lens cholesterol and lens phospholipids are illustrated in figures (3) and (4). In thiola group, both cholesterol and phospholipids were decreased after irradiation with 24 Gy then significantly increased after 100 Gy. After topical application of olive oil post-exposure to b-radiation, both lens cholesterol and phospholipids were non-significantly decreased after 24 Gy and significantly decreased after 100 Gy. There are strong correlation between lens cholesterol of eyes received 24 Gy and 100 Gy (olive oil group), r = 0.961 and also strong correlation was recorded between their phospholipids contents, r = 0.991. Table (1) shows the calculated stability (cholesterol / phospholipids ratio) for both studied groups.

Table (1): The measured values of lens cholesterol, phospholipids (mg/g dry weight) and their calculated ratio in both studied groups.

Thiola group
/ Olive oil and thiola group
Normal
/ 24 Gy / 100 Gy / Normal / 24 Gy / 100 Gy
Cholesterol / 1.89± 0.53 / 0.85 ± 0.2
P<0.01 / 6.48 ± 0.5
P<0.001 / 1.28± 0.23 / 0.96 ± 0.14
N.S. / 0.72 ± 0.1
P<0.01
Phospholipids / 1.99 ± 0.34 / 0.79 ± 0.37
P<0.01 / 6.47 ± 0.17
P<0.001 / 1.07 ±0.29 / 0.74 ± 0.23
N.S. / 0.53 ± 0.14
P<0.01
Choles./Phosphol. / 0.95 / 1.1 / 1.0 / 1.2 / 1.3 / 1.4

Figure (5a) shows the fourier transform infrared (FTIR) spectra of lens lipids prepared from thiola group. The CH2 stretching region (3100 – 2800 cm-1) is used to probe the lipid order. The intensity of the asymmetric stretching band was increased as the exposure dose increased from 24 Gy to 100 Gy relative to the normal band. The fingerprint region (1800 – 500 cm-1) indicates that the intensity of the CH out-of-plane (OOP) band was significantly increased after the exposure to both doses of b-radiation; 24 Gy and 100 Gy. In addition, the CH scissoring band around 1464 cm-1 indicates the presence of more different components than the normal pattern. On the other hand, the CH2 symmetric band frequency of the normal pattern appeared at 2862.2 cm-1 was appeared at different frequencies in the lens irradiated with 24 Gy (2839.0 cm-1) and at 2869.9 cm-1 in that irradiated with 100 Gy.

When olive oil was topically applied after b-radiation treatments (figure 5b), the infrared spectra showed that the intensity of CH – OOP band was increased relative to the normal after the administration of 24 Gy or 100 Gy. The intensity of CH2 asymmetric band was markedly increased than that of the normal pattern but, decreased when compared with that band in the thiola group. In addition, there was a shift in its frequency from 2954.7 cm-1 in the normal pattern to 2939.3 cm-1 after treatment with 24 Gy and to 2947.0 cm-1 after treatment with 100 Gy. The CH scissoring band can be noticed in the spectra of the “olive oil and thiola” group treated with 24 Gy, then this band was completely disappeared after treatment with 100 Gy of b-radiation.

Lipid bilayer systems exhibit two major physical states: the gel phase (hydrocarbon chains relatively immobile and closely packed) and the liquid crystalline phase (hydrocarbon chains mobile and loosely packed) with a characteristic transition temperature or temperature range at which there is a change from one dominant state to the other. Figure 6 (a,b) illustrates the differential phase transition of lens lipids of both groups (thiola and olive oil). The phase diagram of thiola group indicated the presence of two major endothermic transitions at 25 oC and 30 oC for both doses of b-radiation. In addition, one exothermic transition at 20 oC was detected in the case of 100 Gy. The polymorphic changes below or above these two major transitions were obviousley clear. In figure (6b) when olive oil was used, a major phase transition at 30 oC was clearly noticed and associated with less polymorphic changes relatively to thiola group.The phase behaviour in both applied doses (24 Gy and 100 Gy) are similar but differ in its magnitude.

Discussion

The obtained data indicated that the characteristic absorption spectra of lens lipids were completely different among the studied groups. Although two peaks were detected in both studied groups, they had different wavelengths. To understand this behaviour and whether it affected by the topical application of olive oil and/or thiola or it resulted from the exposure to b-radiation, the characteristics of both thiola and olive oil-thiola mixture were studied spectrophotometrically and shown in figure (7). Considering thiola, there was an absorption maximum at 230-240 nm region (plateaue) and a shoulder at 290 nm. On the other hand, when the olive oil and thiola were mixed and studied, the absorption maximum was appeared at 220 nm, in addition to a shoulder at 290 nm.

Accordingly, it is clear that thiola has the ability to penetrate the ocular barriers and incorporated into the lens lipids resulted in the observed shift in the native lipid peak (normally appeared at 210 nm) to 230 - 235 nm region. The broad band at 274 – 288 nm was resulted from the same behaviour of thiola. It is clear that thiola as a radioprotector exerts its effect by protecting the lipid component from oxidation. When both thiola and olive oil were topically applied, the resulted complex also has the ability to pass through the lenticular barriers and prevents the formation of lipid peroxides but, with a different manner than thiola i.e., it seems to produce a mild – but valuable- protecting effect.

Cholesterol and phospholipids are essential membrane constituents. Cholesterol play an important role in the stability (rigidity) of the membranes, while phospholipids due to their unique characteristics are suitable components to serve as major constituent for biological interface within cell membranes; therefore they have an important role in the membrane fluidity. The physicochemical properties of membrane lipids affect the membrane function as protein – lipid interaction, Na+ - K+ ATPase activity and enzyme activities (Zigman et al., 1984). It is clear from table (1) that although “olive oil and thiola” group was characterized by decreased cholesterol and phospholipids contents, its lipids were characterized by high degree of stability relative to the thiola group. The lipid stability is dominant in both doses. On the other hand, the thiola group showed the same behaviour but the stability was decreased as the dose increased from 24 Gy to 100 Gy ( still greater than the normal value). The overview indicates that olive oil-thiola mixture is more effective , compared with thiola only , in enhancing the physical stability of the lens lipids regardless the applied dose of b-radiation.