Inhibition of rat microsomal lipid peroxidation by the oral administration of D 002

The effect of D002, a defined mixture of higher primary alcohols purified from bee wax, on in vivo and in vitro lipid peroxidation was studied. The extent of lipid peroxidation was measured on the basis of the levels of thiobarbituric acid reactive substances (TBARS). When D002 (5-100 mg/kg body weight) was administered orally to rats for two weeks, a partial inhibition of the in vitro enzymatic and nonenzymatic lipid peroxidation was observed in liver and brain microsomes. Maximal protection (46%) occurred at a dose of 25 mg/kg. D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes, which suggests that under conditions where microsomal metabolism was favored the protective effect of D002 was increased. D002 (25 mg/kg) also completely inhibited carbon tetrachlorideand toluene-induced in vivo lipid peroxidation in liver and brain. Also, D002 significantly lowered in a dose-dependent manner the basal level of TBARS in liver (19-40%) and brain (28-44%) microsomes. We conclude that the oral administration of D002 (5, 25 and 100 mg/kg) for two weeks protected rat liver and brain microsomes against microsomal lipid peroxidation in vitro and in vivo. Thus, D002 could be useful as a dietary natural antioxidant supplement. More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans. Correspondence R. Menéndez Soto del Valle Laboratory of Biochemistry Center of Natural Products National Center for Scientific Research


Introduction
D002 is a well-defined mixture of higher primary alcohols isolated and purified from bee wax.Triacontanol is its main component, followed by octacosanol, dotriacontanol, hexacosanol and tetracosanol.Tetracontanol is also present as a minor component (1).Previous work has demonstrated a moderate antiinflammatory effect of D002, since oral administration of D002 signifi-cantly inhibited the volume of carrageenaninduced exudate and the pellet dry weight of the granuloma (2).Evidence supports that reactive oxygen species appear to be involved in the inflammatory process, including the generation of chemotactic factors and tissue damage (3)(4)(5)(6).Thus, some antiinflammatory drugs are thought to exert part of their action by decreasing the reactive oxygen species (7)(8)(9).
We report here initial studies directed at the investigation of the effect of oral treatment with D002 on lipid peroxidation.We evaluated the effect of D002 on enzymatic and non-enzymatic rat microsomal peroxidation in liver and brain in vitro.We also studied the action of D002 against in vivo lipid peroxidation by determining its effects on carbon tetrachloride (CCl 4 )-and tolueneinduced lipid peroxidation in liver and brain.

In vitro experiments
Animals and treatment.Wistar rats (180-200 g) were used throughout the study.D002 was suspended in a 2% Tween 20/water vehicle and administered orally (5, 25 and 100 mg/kg) for two weeks by gastric gavage (1 ml/200 g).A control group received an equivalent volume of the vehicle.
Preparation of microsomes.After treatment, rats were anesthetized and the livers were perfused with ice cold KCl (1.15% w/ v).Livers were homogenized in 250 mM sucrose and microsomes were prepared by differential centrifugation as described previously (10).Brains were removed and homogenized in 40 mM Tris buffer, pH 7.4.Microsomes were obtained in the same manner as for liver microsomes.The microsomal protein concentration was determined by a modification of the Lowry procedure (11).
Assay for lipid peroxidation.Lipid peroxidation of liver microsomes was initiated by the addition of either a) 25 µM FeSO 4 , 100 µM ascorbate and 10 mM KH 2 PO 4 , or b) 2 µM FeCl 3 /200 µM ADP (previously complexed) and 200 µM NADPH according to Ruiz-Lerrea et al. (12).When ascorbate system was used, liver microsomes were heat inactivated before being added to the incubation mixture.Microsomal lipid peroxidation of brain was performed according to Cini et al. (13) using microsomes instead of crude homogenates.Microsomes were incubated at 37 o C for 60 min with no addition (spontaneous) or after the addition of a free radical-generating system (100 µM FeCl 2 ).Lipid peroxidation was monitored by the formation of thiobarbituric acid reactive substances (TBARS) according to Ohkawa et al. (14).After extraction with n-butanol and pyridine (15:1 v/v), the amount of TBARS formed was determined by measuring the absorbance of the organic layer at 532 nm.In parallel TMP was used as an external standard.Results are reported as nmol malondialdehyde (MDA)/mg microsomal protein.

In vivo experiments
Animals and treatment.Male Wistar rats were randomly divided into five groups (10 animals/group) and were given free access to standard laboratory diet and water.Two control groups received vehicle alone and three treated groups received 5, 25 and 100 mg D002/kg.For CCl 4 -induced liver peroxidation, food was withdrawn 12-14 h before CCl 4 administration but after dosing rats had free access to food and water.All groups received 1 ml/kg CCl 4 in 10% corn oil (v/v) by intraperitoneal (ip) injection, except the negative control group which received corn oil ip (1 ml/kg).Toluene-induced brain damage was performed according to Hanna et al. (15) and Mattia et al. (16).Rats were injected with a single dose of toluene (1.5 g/ kg) ip.The negative control group was injected ip with comparable volumes of normal saline.All animals were sacrificed by decapitation 24 h after CCl 4 injection and 4 h after toluene injection.Liver and brain microsomes were prepared as described for the in vitro experiments.

Statistical analysis
Data are reported as means ± SD.Statistical analysis was performed by the Mann-Whitney U-test and P<0.05 was considered to be statistically significant.

Results
Treatment of rats with D002 resulted in partial inhibition of liver microsome lipid peroxidation.As shown in Table 1, oral administration of D002 partially prevented the accumulation of TBARS in vitro in liver microsomes in a dose-dependent manner.When the reduced nicotinamide coenzyme was present (enzymatic system) D002 caused a partial inhibition of lipid peroxidation (apparent maximal 32% inhibition after administration of 25 mg/kg).Additional experiments were performed with heat-treated microsomes.Analysis of TBARS accumulation after the administration of similar doses of D002 revealed that lipid peroxidation was partially depressed but to a lesser extent when compared with native microsomes (apparent maximal 16% inhibition at 25 mg D002/kg) (Table 1).We also tested the ability of D002 to inhibit TBARS accumulation by brain microsomes.As shown in Table 2, TBARS are formed in appreciable amounts by brain microsomes after 30 min in the presence of 100 µM FeCl 2 .D002 was able to inhibit iron- induced lipid peroxidation in brain microsomes since TBARS accumulation was significantly decreased after D002 treatment at doses of 5, 25 and 100 mg/kg.D002 provided only partial protection, since a maximal 46% inhibition was observed after administration of 25 mg D002/kg.The antioxidant action of D002 was further demonstrated by in vivo experiments, which showed that oral administration of D002 for 2 weeks significantly lowered in a dose-dependent manner the basal level of lipid peroxidation in liver and brain microsomes (Table 3).Table 4 shows the effect of various doses of D002 administered for 2 weeks prior to the injection of CCl 4 .D002 treatment significantly inhibited the effect of CCl 4 , which occurred at doses of 5, 25 and 100 mg/kg.In addition, D002 prevented the toluene-induced lipid peroxidation in brain (Table 5).Lipid peroxidation by brain microsomes (TBARS) isolated from rats injected ip with toluene was significantly higher than that observed in rats injected with saline.The stimulation of lipid peroxidation produced by toluene was significantly inhibited by D002 administration, since doses of 25 and 100 mg/kg fully inhibited TBARS accumulation.

Discussion
The present study demonstrated that the oral administration of D002 partially inhibited the in vitro and in vivo lipid peroxidation in liver and brain microsomes in a dosedependent manner.Microsome fractions isolated from liver and brain of rats treated with several doses of D002 were more resistant to lipid peroxidation than those isolated from control rats.The maximal effect occurred at a dose of 25 mg/kg, with no additional protection observed with 100 mg/kg.D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes.In studies with denatured microsomes, D002 produced lower protection when compared with native microsomes incubated with NADPH.At present we cannot explain the mechanism underlying these differences, but it seems clear that under conditions where microsome metabolism was favored, the protective effect of D002 was also increased.
We also examined the effect of D002 on in vivo lipid peroxidation.CCl 4 -induced liver microsome lipid peroxidation was used, since the hepatotoxicity of this solvent has been reported to be related to lipid peroxidation (17,18).Also, we studied the effect of D002 on toluene-induced lipid peroxidation in brain microsomes, since toluene is a neurotoxic chemical that acts by increasing the generation of reactive oxygen species in brain (16).When the ability of D002 to inhibit TBARS generation in vivo after both chemicals was tested, an inhibition of TBARS generation was observed.This indicated that D002 not only protected microsomes against in vitro lipid peroxidation but also against in vivo peroxidation.As observed in the in vitro models, the maximal effect was attained at doses of 25 mg/kg.The high degree of protection against oxidative stress in brain exerted by D002 might be biologically relevant.In this regard, it should be considered that brain is highly susceptible to peroxidative damage because of its high degree of unsaturated fatty acids in relation to its antioxidant levels and its high oxygen consumption (19)(20)(21)(22).
It is known that in mammals superoxide dismutase and peroxide-metabolizing enzymes, catalase and glutathione peroxidase represent an efficient defense system against hazards of lipid peroxidation.The data presented here also indicate that D002 decreased the basal level of TBARS in liver and brain.This result, together with differences in the degree of protection against lipid peroxidation in vitro when comparing the enzymatic model and the denaturalized microsomes, suggests that an effect on the defense enzyme system should not be ruled out.Therefore, this possibility should be considered in future investigations.
In conclusion, the above results show that oral administration of D002 (5, 25 and 100 mg/kg) for two weeks protects rat liver and brain microsomes against in vitro and in vivo microsomal lipid peroxidation as assessed by measured TBARS levels.More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans.

Table 1 -
Effects of oral administration of D002 on TBARS accumulation initiated by Fe 3+ /ADP/NADPH in liver native microsomes (enzymatic system) and by Fe 2+ /ascorbate in liver denatured microsomes (non-enzymatic system).TBARS are reported in terms of malondialdehyde (MDA).Data are reported as means ± SD for 10 rats in each group.*P<0.05 compared to control (Mann-Whitney U-test).

Table 2 -
Effect of oral administration of D002 on TBARS accumulation in brain microsomes after incubation with FeCl 2 (100 µM).

Table 4 -
Effect of oral administration of D002 on in vivo CCl 4 -induced lipid peroxidation by liver microsomes.Lipid oxidation was measured in terms of malondialdehyde (MDA).Data are reported as means ± SD for 10 rats in each group.*P<0.05 compared to control group.**P<0.05compared to control + CCl 4 group (Mann-Whitney U-test).

Table 3 -
Effect of oral administration of D002 on basal TBARS levels in liver and brain microsomes.TBARS are reported in terms of malondialdehyde (MDA).Data are reported as means ± SD for 10 rats in each group.*P<0.05 compared to control (Mann-Whitney U-test).

Table 5 -
Effect of oral administration of D002 on in vivo toluene-induced lipid peroxidation by brain microsomes.