Naturally Occurring Hydroxy Napthoquinones and Their Iron Complexes as Modulators of Radiation Induced Lipid Peroxidation in Synaptosomes

The modulation of radiation induced lipid peroxidation in synaptosomes by iron (II) and iron (III) complexes of two naturally occurring and therapeutically relevant naphthoquinones viz. 5,hydroxy-1,4 naphthoquinone; juglone and 2,hydroxy-1,4 naphthoquinone; lawsone, have been studied. At lower concentrations the complexes enhance lipid peroxidation predominantly through redox cycling as observed for Fe(II)- juglonate while at higher concentrations the complexes tend to limit lipid peroxidation through fast recombinations.


Introduction
Lawsone (I) and Juglone (11) are a pair of naturally occurring isomeric hydroxynaphthoquinones which have been cultivated in Africa and India for medicinal and dyeing purposes [1]. The former is the coloring pigment readily extractable from the leaves of Lawsonia alba [2] and had been used by the desert travelers as a constituent of mud-plaster used on hands and face to protect against the effects of sun-burns during desert safaris. Juglone is the active principle of Juglans regia exuded by its leaves and roots and has been known to exert allelopathic effects [3] on plants growing in the vicinity of the black walnut tree. Both compounds have been employed as radiation modulating agents in Indian Folk medicine but require authentication.

II
Earlier work in our laboratory had _shown that the hydroxyquinones exert most of their biological activities through chelation of trace metals [4]. In this regard complexes of iron and copper were especially four)_d to be biologically active. Since the involvement of iron _as the initiator [5] and propagator [6] of lipid peroxidatio_n is known and well established [7] it was thought to be interesting to examine the effect of the low molecular weight ferrous and ferric complexes of & II on the radiation induced lipid .peroxidation in the model system under physiological-conditions. Our results indicate that the ferrous complex of_ll is especially remarkable in enhancing lipid peroxidation through redox cycling while fer_ric complex of is also active but indePenlent of redox cycling. These results are relevant for formulating 5osmetic materials or therapeutic preparations which include some quantities of I and IJ.

Modulators of Radiation Induced Lipid Peroxidation in Synaptosomes
Preparation of Synaptosomes Synaptosomes prepared, from the_ brains of Swiss Albino mice were irradiated with vari.ous doses of y-radiation at the rate of 0.9 Gy/s and the extent of lipid peroxidation was_ evaluated in terms of malondialdehyde (MDA) formed [10]. To determine the concentration of MDA. in the s_uspension, 1 ml of synaptosomes with or without the drug were transferred into the centrifuge tubes followed by an addition of 1 ml of suspension medium (0.15 M KCI + 10 mM tris_HC]), 0.5 ml of 30% trichloroacetic acid (TCA) and 0.5 ml of. 52 mM thiobarbituric acid. The tubes were covered with an aluminium foil and placed in the water bath for 30 minutes at 80oC after which they were cooled in an ice bath for 10 minutes and centrifuged at room temperature for 10 minutes. The absorbance of the clear supernatant was measured at 531 nm using a UV 260 Schimadzu spectrophotometer.

Irradiation
The irradiation (0.9 Gy/s) of synaptosomes (0.5mg proteins/ml) was carried out in a gamma chamber (5500Ci 60Co) obtained from Bhabha Atomic Research Center, Bombay (India), at room temperature with the required radiation dose. The dose rate was determin.ed using Fricke's dosimetry. The synaptosomes were used immediately after irradiation for measuring lipid peroxidation. Cyclic Voltarnmetry The cyclic voltammetric profiles of the hydroxynaphthoquinones and their iron complexes were recorded as described previously [9].

Results and Discussion
Syna_ptosomes were irradiated with different doses of radiation (0 to 456 Gy) at a dose rate of 0.9 Gy/s. Lipid peroxidation was found to increase with increase in radiation dose in a sigmoidal manner [11]. Various concentrations (0-50 lLtg/ml) of ! and II were added to the synaptosome preparations irradiated at 54 Gy. The synaptosomes without these ligands served as the control samples. It is observed that both the ligands enhance lipid peroxidation in a concentration depen_dent manner ( Figure !). The lowered lipid p_eroxdation in case of II, is probably the result o1' extremely fast recombination processes of free radicals generated by II due to facilitated redox cycling [12]. A comparison of the modification of lipid peroxidation by iron(ll) and iron(Ill) complexes of II is shown in Figure 2. The order of the lipid peroxidation is Fe+(Juglone) > Fe3+KJu.glone) > Juglone which is in accord with their reduction potentials for the qunone to semqunone conversions [9]. One plausible mechanism is the oxidation of Fe+ at the expense of dioxygen leading to the formation of superoxide (02.-), hydrogen peroxide (HO) and eventually hydroxyl (OH.) radicals respectively by reactions commonly referred to as the Haber-Weiss and Fenton reactions [13]. The OH. radicals are extremely reactive and initiate lipid peroxidation. It has been shown that the chelating agents can greatly influence the extent to which the above reactions may proceed. Ligands with low affinities for Fe+ do not affect the rate of autooxidation, while the chelators having oxygen donor atoms tend to enhance Fe+ oxidations perhaps due to their greater affinity for Fe3+. For example, chelation with EDTA shifts the Fe+/Fe/ couple potential from -0.77 V to -0.12 V making autooxidation considerably favourable. Similarly the citrate ions are also capable of shifting the Fe+/Fe3/ couple to -0.33 V which leads to a significant enhancement in the rate of Fe/ oxidation. The shift in the E/2 values for the Fe2//Fe+ couple by some of the chelating agents together with I and I/are shown in Table I Our cYclic voltammetric studies on the Fe(ll) complex ofll show that the Fe+/Fe/ redox couple in this complex is observed at the potential of +0.41V [17] while there is no Fe//Fe/ couple observed in case of Fe/-(juglone) ( Figure 3B). This suggests that the oxidation, of Fe(ll) to Fe(lll) should be very facile, in the former case which !s a pre-requisite for the Fenton type reaction leading to the enhanced lipid peroxidation as observed. Anotherpossible explanation is that the reduced iron compounds react with the lipid hydroxides (lipid-OH) toyield alkoxy (lipid-O.) radicals while the oxidised iro.n compounds y=eld peroxy radical (lipid-O .). Both alkoxy and peroxy radicals can stimulate the chainreactions leading finally to lip=d peroxidation [13].  Synaptosome suspensions were also irradiated at the radiation doses of 114, 228 and 456 Gy in the presence of different concentrations (10,25, 50 I g/ml) of II and its Fe(ll) and Fe(lll} complexes. It is observed that in the lower dose region (0-332 Gy) these compounds enhance radiation induced lipid peroxidation while in the higher dose region (452 Gy) these compounds inhibit radiation-induced lipid peroxidation (Figures 4 to 6).  co trbut=ng factor =n the I=p=d perox=dat=on =nduced by lawsone der=vat=ves. Th=s =s reasonable in view of the quasi-reversible ligand-based redox peaks observed in their electrochemical profiles [9]. Almost all of the observed cyclic voltammetric peaks for a.nd its iron complexes are primarily ligand-based ( Figure 3A). As observed earliethese compounds also inhibit radiation induced lipid perox=dation at the higher dose levels ( Figure   8 to 10). Thus lipid peroxidation induced by lawsone and its iron complexes follow a mechanism independent of redox cycling corroborating our observations on their hepatocytes toxicities [9]. The present work has thus shown that two naturally occurring isomeric hydroxynaphthoquinones, viz. !awsone and juglone are capable of modulating lipid peroxidation in synaptosomes either through redox coupling or enzymatically. The radiat=on pro.tection offered by these compounds at higher doses may be useful.for improving radiation therapy of cancer whle the radiation damage induced by.them at lower concentration needs to be borne in mind when formulating them in the cosmetic or therapeutic preparations.