Polyunsaturated fatty acids (PUFA)⁶ are susceptible to oxidation, and the resulting products may be toxic to the cell (Halliwel and Chirico 1993). Fish oil, especially that of cold water fish, is a rich source of long-chain (n-3) PUFA, docosahexaenoic acid [22:6 (n-3)] and eicosapentaenoic acid [20:5 (n-3)]. Fish oil is highly susceptible to oxidation; in rats, increased intake of fish oil is associated with an increased need for vitamin E (Cho and Choi 1994, Saito and Nakatsugawa 1994). Vitamin E is the most important lipid-soluble chain-breaking antioxidant in tissues, red cells and plasma (Burton and Traber 1990). The vitamin may protect cellular components against peroxidative damage via the free radical scavenging mechanism or as a constituent of the membrane (Chow 1991).

Iron is essential for maintaining proper cell functions; it is normally tightly controlled by transport and storage proteins. Iron overload, however, may result in deleterious reactions such as degradation of proteins and nucleic acids, and peroxidation of PUFA (Aust et al. 1986, Halliwell and Gutteridge 1986, Minotti 1993). Although the mechanism by which iron is involved in initiating or promoting oxidative damage is not entirely clear, iron is capable of catalyzing the transformation of hydrogen peroxide to the highly reactive hydroxyl radical via the Haber-Weiss reaction (Van der Zee et al. 1993). In addition, iron can catalyze the decomposition of lipid hydroperoxides to form alkoxyl, peroxyl and other radicals (Halliwell and Gutteridge 1990).

A number of studies have been conducted to study the effect of dietary lipid (Cho and Choi 1994, Javouhey-Donzel et al. 1993, L'Abbé et al. 1991, Leibovitz et al. 1990, Meydani et al. 1987, Saito and Nakatsugawa 1994, Witting 1970), vitamin E (Cho et al.1995, Javouhey-Donzel et al. 1993, Meydani et al. 1987, Witting 1970) or iron (Bacon et al. 1983, Dabbagh et al. 1994, Dillard et al. 1983, Wu et al. 1990) on the extent of oxidative damage. However, relatively little is known about interactions among these dietary components. Also, the experimental evidence for oxidative damage resulting from iron overload, high PUFA intake and vitamin E deprivation remains inconclusive. Therefore, the present study was designed to investigate the effects of dietary lipid, vitamin E and iron on generation of lipid and protein oxidation products in mouse liver. Additionally, the effect of dietary lipids, vitamin E and iron on hepatic antioxidant status was determined.

MATERIALS AND METHODS

Table 1. Sources of lipids, vitamin E and iron in the diets1 [View Table]

Table 2. Fatty acid composition of lipid sources1 [View Table]

RESULTS

Vitamin E supplementation resulted in higher hepatic levels of -tocopherol (Fig. 2A). The levels of -tocopherol, however, were significantly lower in the livers of fish oil- and vitamin E-fed mice compared with those receiving lard and vitamin E. Dietary iron had no significant effect on the levels of -tocopherol in mice receiving either fish oil or lard without vitamin E supplementation. The levels of GSH (Fig. 2B) in the livers of fish oil-fed mice were significantly higher than in those fed lard. Dietary vitamin E/iron had no significant effect on GSH levels in the two lipid groups. The levels of ascorbic acid (Fig. 2C) were not affected by dietary fat or vitamin E/iron. Significant interactions were found between dietary lipid and vitamin E/iron for -tocopherol and glutathione.

The effects of dietary lipid and vitamin E/iron on hepatic activities of antioxidant enzymes are shown in Figures 3 and 4. The activities of Se-GSH-PX (Fig. 3A), non-Se-GSH peroxidase (Fig. 3B), catalase (Fig. 3C) and glutathione reductase (Fig. 4B) were not significantly affected by any of the treatments. The activities of SOD (Fig. 4A) and G-6-PDH (Fig. 4C) in the livers of fish oil-fed mice were significantly lower than in those fed lard. Dietary vitamin E/iron had no significant effect on the activity of SOD or G-6-PDH.

At the time of killing, the body weight of mice fed fish oil without vitamin E was significantly less than those receiving vitamin E and all those receiving lard (Fig. 5A). Similarly, mice fed fish oil without vitamin E had significantly smaller livers than any other group (Fig. 5B). The concentrations of hepatic total lipids were significantly lower in the fish oil-fed groups compared with those fed lard (Fig. 5C). Vitamin E/iron supplementation had no effect on the concentrations of total lipids in either lipid group.

DISCUSSION

Increased susceptibility of PUFA to lipid peroxidation can be overcome by vitamin E supplementation (Javouhey-Donzel et al. 1993, Meydani et al. 1987, Witting 1970). As expected, fish oil-fed mice were more susceptible to the effect of vitamin E depletion than those receiving lard. The hepatic levels of TBARS, conjugated dienes and protein-bound carbonyls were lower in the fish oil-fed group receiving vitamin E than in those receiving no supplementation. The relatively short feeding period (4 wk) may be responsible for the lack of significant differences in the lard groups.

Whether the conditions that lead to lipid peroxidation also increase protein oxidation is a continuing point of interest. In this study, significantly higher levels of protein-bound carbonyls were found in the liver of fish oil-fed mice receiving no vitamin E, with or without supplemental iron, than in the other treatment groups. Although the mechanism by which high fish oil intake leads to increased formation of protein carbonyls is not clear, protein carbonyls may be formed from the reaction between protein and oxidation products of PUFA such as aldehydes and peroxy radicals (Tappel 1972). Because the effect was observed only in the fish oil-fed mice receiving no vitamin E and not in the vitamin E-supplemented group, it appears that the formation of protein carbonyls is secondary to lipid peroxidation. Similarly, higher levels of 8-hydroxydeoxyguanosine have been reported in the liver of fish oil-fed rats receiving 3 mg vitamin E/kg diet compared with those receiving 45 or 209 mg vitamin E/kg (Cho et al. 1995), suggesting that lipid peroxidation can lead to oxidative DNA damage.

Many studies have reported that transition metals play a key role in the initiation and propagation of free radical-induced peroxidative damage (Dillard et al. 1979, Halliwell and Gutteridge 1986, Minotti 1983). Iron overload in rats has been associated with increased generation of lipid peroxidation products, including breath ethane and pentane and hepatic levels of TBARS (Dillard et al. 1979 and 1983). Chronic iron overload has also been shown to increase generation of hepatic lipid peroxidation products in rats (Bacon et al. 1983, Wu et al. 1990). Iron may enhance lipid peroxidation by catalyzing both the formation of hydroxyl radicals via the Harber-Weiss reaction and the decomposition of lipid hydroperoxides and formation of free radicals (Aust et al. 1986, Halliwell and Gutteridge 1990). Because liver is the major recipient of excessive iron, hepatotoxicity is the most common finding in patients with iron overload. In the present study, a significant interaction was found between dietary lipids and vitamin E/iron relative to TBARS and conjugated dienes, but not protein-bound carbonyls. Increased formation of lipid peroxidation products may be due to the action of iron in catalyzing the initiation of free radical reactions and/or the decomposition of lipid hydroperoxides. This event can lead to the production of a wide range of oxidation products, including epoxides, hydrocarbon gases and aldehydes (Halliwell and Gutteridge 1990).

As expected, dietary vitamin E has a profound effect on the hepatic levels of the vitamin. Interestingly, the hepatic level of -tocopherol in mice receiving fish oil was lower than that of mice fed lard. This effect of fish oil, however, was observed only in mice supplemented with vitamin E, and not in the deficient groups. An effect of fish oil on vitamin E has been reported in the liver of both rats (Javouhey-Donzel 1993) and mice (Meydani et al. 1987). The lower levels of hepatic -tocopherol in mice fed fish oil may be due to either an interaction of -tocopherol and fish oil at the gut level (Mouri et al. 1984) or to enhanced postabsorptive utilization of -tocopherol (Drevon 1991, McCay and King 1980, Meydani et al. 1987). Because dietary vitamin E in the present study was provided as -tocopheryl acetate, it is unlikely that oxidation of the compound occurs before absorption. Therefore, increased postabsorptive utilization and/or decreased retention is most likely the major factor responsible for the observed lower hepatic vitamin E concentration.

The status of other antioxidant systems was variably affected by dietary fat and vitamin E. In the current study, the levels of hepatic GSH were significantly higher in the fish oil groups than in the lard-fed mice. Because GSH levels are maintained by the activities of glutathione reductase and GSH synthetases, higher levels of GSH in mice receiving fish oil may be due to an adaptive response to increased oxidative stress. The activities of glutathione reductase and Se-GSH PX, however, were not significantly altered by dietary fat or vitamin E/iron. The hepatic activity of SOD was significantly lower in the fish oil groups than in the lard groups. Similar results have been reported by L'Abbé et al. (1991), who observed lower hepatic total SOD activity in fish oil-fed rats compared with a lard-corn oil-fed group. L'Abbé et al. (1991) also observed lower activities of both Se-GSH PX and non-Se-GSH PX in fish oil-fed rats, whereas we found no differences in the activities of these two enzymes as a result of dietary lipid. The reason for this discrepancy is not clear, although differences in the species of experimental animals used (rats vs. mice), dietary composition and duration of the experiment may be responsible.

The NADPH needed for GSH regeneration and lipogenesis is provided by various NADPH-generating enzymes (Chance et al. 1979). In this study, the activity of the pentose shunt enzyme, G-6-PDH, was significantly higher in the lard-fed groups compared with mice fed fish oil. Higher NADPH generated as a result of higher G-6-PDH activity in mice fed lard may have led to an increase in lipogenesis. This is supported by the higher hepatic levels of total lipids in the lard-fed groups compared with the fish oil-fed groups.

The results obtained from this study provide experimental evidence of the prooxidative effect of vitamin E deprivation and/or high intake of fish oil and iron in vivo. The findings also demonstrate that the type of fat and levels of vitamin E and iron in the diet are important factors in determining cellular susceptibility to oxidative damage. Additionally, the results suggest that vitamin E is capable of protecting not only lipid-soluble compounds but also water-soluble constituents against oxidative damage, and that dietary lipid plays a key role in determining cellular susceptibility to oxidative stress.

ACKNOWLEDGMENT