EFFECT OF DIFFERENT VEHICLES ON HEPATO- PROTECTIVE EFFICIENCY OF DESFERRIOXAMNE IN RADIATED CAR= TETRACHLORIDE TREATED MICE

The effect of desferrioxamine (DFO) in different vehicle ( Aqueous and oily) against hepatotoxicity induced by carbon tetrachloride (CC14) in irradiated mice and irradiated carbon tetrachloride (IR-CCI4) in normal mice was investigated. A single dose of CC14 and IR-CCI4 (20 @/kg, i.p.) in irradiated mice (IR-mice) and normal mice induced hepatotoxicrty, manifested biochemically by significant elevation of serum enzyme activities, such as alanine trarsaminase (ALT, EC:2.6.1.2 ) and aspartate transaminase (AST, EC:2.6.1.1). Hepatotoxiaty was further evidenced by significant decrease of total sulfttydryl (-SH) content, and catalase (EC: 1.1 1.1.6) activrty in hepatic tissues and significant increase in hepatic lipid peroxidation measured as malondialdhyde (MDA). Pretreatment of normal mice and IR-mice with DFO (200 mg/kg i.p dissolved either in water or arachis oil vehicle) 1 h before CC14 or IR-CC14 injection ameliorated the hepatotoxicrty as evidenced by a significant reduction in the elevated levels of serum enzymes as well as a significant decrease in the hepatic MDA content and a significant increase in the total sulfhydryl content 24 h after CC14 or IR-CCI4 administration.. These results indicated that both of oily and watery DFO can effectively ameliorated the hepatotoxicity induced by CC14 in IR-mice or IR-CC14 in normal mice. Although, the efficiency of the hepatoprotective effect of DFO in oily vehicle was higher than that DFO in aqueous vehicle. The hepatoprotective effect of DFO possibly through inhibition of the production of oxygen free radicals that cause lipid peroxidation.


Introduction
Irradiation is a complex phenomenon in which molecules are excited, ion pairs are formed, and chain reactions are completed by free radicals [l].Radiation offers several advantages as a sterilization method that makes it attractive in growing number of situations.Articles sterilized by radiation include a large variety of medical products [2,3].
The extent and nature of the damage produced in organic substances by gamma radiation was attributed to radicals produced by the irradiation [4].
CCI4 is a xenobiotic which produces hepatotoxiclty in humans as well as in animals [9,10].The hepatotoxic effect of CCI4 is thought to result from its reductive dehalogenation by cytochrome P-450 enzyme system to the highly reactive free radical, trichloromethyl radical ' CC13 [ I 11.This radical quickly adds molecular oxygen to form trichloromethylperoxy radical [12].Removal of hydrogen atoms from unsaturated fatty acids by such radical created carbon-centered lipid radicals [ I 11.These lipid radicals quickly add molecular oxygen to form lipid peroxyl radicals, thereby, initiating the process of lipid peroxidation.
Unless scavenged by radical scavengers, these lipid peroxyl radicals in turn abstract hydrogen atoms from other lipids molecules, thereby propagating the process of lipid peroxidation [13].Iron can catalyze oxygen free radical reactions that lead to peroxidation of membrane lipids or inactivation of antioxidant enzyme activities [14], and consequently damage of microsomes, mitochondria and nuclei that lead to imparied physiological functions of hepatocytes [15].Also, it has been reported that iron plays a role as a mediator of CCI4-hepatotoxiclty [16].
Administration of some antioxidants such as cystearnine [17], silymarin [12], tetrahydroneopeterin [18], schisandrin B [19] and vitamin E [20] has been shown to significantly suppress CC14-induced hepatotoxictty in mice.Desferrioxamine is an iron chelator that is mainly used to treat iron poisoning [21].The iron chelating properties of DFO has been shown to play a primary role in protecting the liver against CC14-induced hepatotoxicrty [22].It has been demonstrated that administration of DFO prevents lipid peroxidation induced by CC14 [16].Also, it has been reported that DFO could significantly decrease LDH leakage and reactive oxygen species production by cyanobacteria extractinduced hepatotoxiclty in primary cultured rat hepatocyte [23].Previously, we have shown that DFO attenuated hepatotoxictty induced by CCI4 [24].
Therefore, the goal of the present study was to investigate whether DFO can attenuate hepatotoxiclty induced by IR-CC14 and CCl4 in normal and IR-mice respectively.
In addition, whether using different vehicles (water or arachis oil) can affect the hepatoprotective efficiency of DFO.

C h i c a b :
Thiobarbituric acid (TBA) and arachis oil were a product of Fluka (Buchs, Switzerland).DFO (Desferal) and carbon tetrachloride were obtained from Ciba Geigy (Basel, Switzerland) and Aldrich Chemical Company (Milwaukee, WI, USA) respectively.All the remaining chemicals were of the highest analytical grade.

Animals:
Male Swiss albino mice weighing 22-25 g were used in all experiments.They were obtained from the Experimental Animal Care Center of King Saud University, Riyadh, KSA.Animals were maintained under standard conditions of humidity with regular lightldark cycle and free access to food (Purina Chow) and water.

Gammarsdiation:
A .~o f c a r t m n ~~ 50 ml of carbon tetrachloride (CC14) were filled into plastic tubes and were exposed to gamma radiation from Cobalt-60 source in a Gammacell 220 (Nordion International Inc., Kanata, Canada).The selected dose of the radiation in the present study was 25 kGy depending on our own preliminary experiment.The dose rate was 1.15 Graykecond.The exposure time was 6.04 hrs.The zero dose (non-radiated CC14) provided as a control on temperature/ storage/ transportation effects on the samples.

B. Rsdistion of mice
A group of mice are subjected to the same conditions of radiation as in CCh procedure.
The selected dose of radiation in the present study was 50 Gray.The dose rate was 1.15 Gray1 second and the exposure time was 1 min.

Expefimntal protocol:
CC14 (0.2 % vlv) was dissolved in corn oil and a dose of 20 pllkg of CCb was intraperitoneally administered to mice to induce liver damage.The rise in serum ALT was taken as evidence for impaired liver function.In a preliminary experiment, CC14 was administered to IR-mice and IR-CCb in normal mice at different doses ranging from 10 to 25 ullkg and the dose of 20 uWkg was selected in the present model, since it produces moderate hepatotoxicity.Thus the protective effects of DFO could be evaluated.In our own previous study, the peak of an increase in serum ALT activity, an index of liver injury, in mice receiving CC14 alone, was observed at 24 h and started to decrease after 48 and 72 h [24].A portion of DFO was dissolved in aqueous vehicle while the other was suspended in oily vehicle.
The animals were divided into 9 groups, the first group (control) received corn oil (10 mllkg i.p), the second group IR-mice wrth 50 Gray.The third group received CC14 (20 pllkg i.p).
The fourth group was given IR-CCI4, 25 kGy (20 @/kg i.p).The fivth group was IR-mice with 50 Gray treated with 20 pllkg CC14.The last four groups were given DFO in oily and aqueous vehicle 1 h before CC14 and IR-CC14 injection in IR-mice or normal mice.In the present study, the dose of 200 mglkg of DFO was selected, since it showed the optimum hepatoprotective effect against CC14 as evidenced from a significant reduction of the serum enzymes tested.
At 24 h after of CC14 dosing, blood samples were drawn from the orbital plexus, under light ether anaesthesia, into non-heparinized capillary tubes.Serum was separated by centrifugation for 5 min at 4000 rpm and stored at -20°C until analysis.The liver was isolated, washed with saline, weighed and homogenized in ice cold saline, and then 10% homogenate of the liver was made in a Branson sonifiir (250, VWR Scientific, Danbury,Conn.,USA).Serum ALT and AST were determined kinetically as described earlier by 'Reitman and Frankul [25] using commercially available diagnostic kits (Biosystems, Barcelona, Spain).Malondialdhyde (MDA) production as an index of lipid peroxidation, total sulfhydryl content and catalase activlty in the liver tissues were determined according to the methods of Ohkawa et al [26], Ellman [27] and Higgings et al

R e s u b
Effect of DFO on hepatotoxicity induced by CC14 and IR-CC14 in IR-mice and normal mice respectively.
Table 1 and 2 shows that i.p. injection of CCI4 and IR-CC14 (25 kGy) in IR-mice and normal mice induced hepatotoxicity.Serum ALT and AST were significantly increased.
Pretreatment with DFO (200 mglkg i.p) dissolved in arachis oil vehicle prevent the significant increase in serum enzymes activities.Serum ALT and AST were significantly decreased nearly to the normal value in both groups treated (normal mice treated with IR-CC14 or IR-mice treated with CC4).However, administration of DFO in aqueous vehicle ameliorated hepatotoxicrty in both groups (IR-CC14 in normal mice and CC14 in IR-mice).
These were evidenced by the significant reduction in serum enzymes activities, ALT and AST.However,, it was still significant from control

Effects of DFO (aqueous and oily) on lipid peroxide (MDA), reduced glutathione (GSH)
and catalase activity in hepatic tissues.CC14 injection in IR-mice, as the level of the serum enzymes were reduced back almost to the normal.In addition, it prevents the rise in hepatic lipid peroxide.
The biochemical mechanism involved in the development of CC14 hepatotoxicw has been investigated.It is generally believed that this toxictty is due to lipid peroxidation caused by carbon trichloromethyl radical, CC13 [30].In the present study, the development of liver cell injury by a single administration of CCI4 in 18-mice or IR-CCI4 in normal mice (20 pllkg, i.p.) was associated with a significant increase in hepatic lipid peroxide content and a significant decrease in hepatic total sulfhydryl content, a potent factor in the control of lipid peroxidation [31].These findings are consistent with a previous report [32].
The hepatoprotective effect of DFO against CC14 (20 ~l l k g , i.p.) or IR-CC14-induced hepatotoxictty, as demonstrated by the significant reduction of increased serum ALT and AST was associated with a parallel significant inhibition of elevated hepatic lipid peroxide content and a significant increase in total sulfhydryl content in liver tissues.These findings may indicate that inhibition of lipid peroxidation is the initial event in the mechanism by which DFO ameliorates CC4 or IR-CCI4-induced hepatotoxictty and is compatible wrth the study demonstrating that CC14 toxicity could be ameliorated by pretreatment with inhibitors of CC14 metabolism (33) or antioxidants [MI.This does not, however, support the work of Stacy and Klassen [35] who have reported that inhibition of lipid peroxidation can not prevent CC14-induced cellular injury in isolated rat hepatocytes.However, In our previous study, DFO was a potent lipid peroxidation inhibitor in an in vitro study using liver homogenates of 'normal mice as a source of lipids and ~e~'/ascorbate to induce lipid peroxidation [24].
In the present study DFO protected the liver of the mice against CCI4-induced hepatic damage.This finding is in harmony with the previously reported finding showed that DFO inhibits adriamycin-induced hepatotoxicity by suppression of lipid peroxidation [36].Moreover, it has been shown that DFO could significantly decrease LDH leakage and reactive oxygen species production by cyanobacteria extract-induced hepatotoxicrty in primary cultured rat hepatocytes [23].In addition, the antidotal effects of DFO in experimental liver injury were studied by Siegers et al [22].The authors have demonstrated that DFO can protect against hepatotoxicity induced by CCb, but not by bromobenzene, although a marked in vivo lipid peroxidation is observed, that appears to be irondependent, and have indicated that lipid peroxide is not mainly responsible for bromobenzene-induced hepatotoxicdy.
In conclusion: The results of the present study clearly demonstrate that both of oily and watery DFO can effectively ameliorated the hepatotoxicdy induced by CC14 in IR-mice or IR-CCI4 in normal mice.Although, the efficiency of the hepatoprotective effect of DFO in oily vehicle was higher than that DFO in aqueous vehicle.Further studies are needed to explore the possible mechanisms of cytoprotective actions.

[ 28 ]
, respectively.Slatistics1 analysis: Data are expressed as (means + SEM).Statistical comparison between different groups were done using one way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison test.Significance was accepted at PC 0.05.

Table 3 and
4demonstrates that administration of IR-CC14 or CCI, in normal or IR-mice induced hepatotoxiclty as evidenced by significant elevation of lipid peroxide measured as MDA and significant reduction of reduced glutathione.Pretreatment with DFO (oily) significantly decreased its elevated level of lipid peroxide in both groups (CCI, in IR-mice and IR-CCI4 in normal mice).In addition, it prevents the significant reduction of reduced glutathione induced by CC14.Administration of Tab. 1. Effects of DFO on serum ALT and AST enzymes activities of mice injected with IR-CC14 DFO (200 mglkg i.p.) was given either in aqueous and oily vehicle1 h prior to IR-CC14(20 pllkg) injection.In IR-m~ce.Our prevlous study demonstrated that treatment of mice wRh DFO (200 mglkg, i.p.) neither induced any change in the activities of serum ALT and AST measured nor influenced liver lipid peroxide, total sulfhydryl contents and catalase activtty[24].In the present study, pretreatment wrth Tab.3.Effects of DFO on hepatic lipid peroxide and total sulfhydryl contents and catalase activrty in liver homogenates of mice injected with IR-CC14.DFO (200 mglkg i.p.) was given either in aqueous and oily vehicle1 1 h prior to IR-CCI4(20 pllkg) injection.Effects of DFO on hepatic lipid peroxide and total sulfhydryl contents and catalase activtty in liver homogenates of IR-mice injected wrth CC14.DFO (200 mglkg i.p.) was given either in aqueous and oily vehicle1 h prior to CC14(20 pllkg) injection.