Dose-response relationship for α-tocopherol prevention of ultraviolet radiation induced cataract in rat

Abstract

The purpose of this study is to establish the dose response relationship for α-tocopherol protection of ultraviolet radiation (UVR) induced cataract in the rat. Four groups of 20 six-week-old albino Sprague Dawley rats received 5, 25, 50, and 100 IU/day α-tocopherol, whilst another group of 20 rats without any α-tocopherol feeding was the control group. After 4 weeks of feeding, each rat was unilaterally exposed to 8 kJ/m² UVR-300 nm for 15 min. At 1 week after exposure, the rats were sacrificed and lens light scattering was measured quantitatively. Lens total reduced (GSH) and oxidized (GSSG) glutathione; glutathione reductase (GR) and peroxidase (GPx) were determined spectrophotometrically. The UVR-exposed lenses in the α-tocopherol fed groups developed superficial cataract, whereas lenses in the control group developed cortical and equatorial opacities. Light scattering in lenses from the α-tocopherol-supplemented rats was lower than in lenses from the control group. The difference of light scattering between the exposed and contralateral non-exposed lens decreased with increasing doses of α-tocopherol to an asymptote level. UVR-exposure caused a significant depletion of lens GSH in rats without or at low α-tocopherol supplementation. The depletion of GSH became less with higher α-tocopherol supplementation. There was no detectable difference in lens GSSG, GR or GPx at any level of α-tocopherol supplementation. Orally administered α-tocopherol dose dependently protects against UVR-induced cataract. The protection is associated with an α-tocopherol dose-dependent GSH depletion secondary to UVR exposure. UVR-induced light scattering only occurs if the GSH depletion exceeds a threshold.

Introduction

In this project, we aimed to determine the dose response relationship for α-tocopherol protection of in vivo ultraviolet radiation (UVR) induced cataract.

Cataract is the major cause of blindness in the world (West, 2000, Thylefors, 2001). The increasing size and age of the world population is predicted to cause an escalating health economic burden of cataract management, particularly in the developing countries where cataract occurs at an earlier age and cataract surgery is often inaccessible (Brian and Taylor, 2001). In a world perspective, it is therefore important to find a method that prevents or delays the onset of cataract.

Cataract is known to be associated with hereditary factors, metabolic disorders and exposure to ultraviolet radiation (UVR) (McCarty et al., 2000). In recent years, the pollution-related depletion of the atmospheric ozone layer has increased the penetration of UVR to the earth (UNEP, 1998). Clinical and epidemiological studies have demonstrated that solar UVR is the most important avoidable risk factor for human cataract development (Zigman et al., 1979, Taylor et al., 1988, Cruickshanks et al., 1992, West et al., 1998).

UVR causes cataract through oxidative damage. UVR-induced reactive oxygen species (ROS) such as superoxide radicals and hydrogen peroxide lead to DNA destruction and activation of protein kinases (Nishi et al., 1991). Oxidative damage to lens proteins secondary to UVR exposure has been postulated as one of the mechanisms for cataract formation (Spector et al., 1995, Taylor et al., 1995). Cells have developed an antioxidant defense system against ROS and their destruction. Antioxidants act either by preventing ROS production or eliminating them.

Glutathione in the reduced form (GSH) is the most important non-enzymatic antioxidant in the lens (Clark, 1994). GSH plays a vital role in defending against exogenous and endogenous ROS and keeps lens protein in a reduced state. A dynamic balance is maintained between GSH synthesis, its recycling from the oxidized form (GSSG), and its utilization. The amount of GSH in the lens diminishes with aging and oxidative stress. Several previous studies have demonstrated that UVR exposure results in a depletion of the GSH concentration in lenses of rabbits (Hightower and McCready, 1992) and rats (Risa et al., 2004, Risa et al., 2005, Tessem et al., 2006, Wang et al., 2010). Hightower exposed rabbit lenses in vitro to UVR-315 nm, but the dose of UVR was not provided. The other studies were in vivo exposures of rats to 2–15 kJ/m² of UVR-300 nm. Ayala and co-worker did not find any significant depletion of GSH after in vivo UVR exposure of rats (Ayala and Söderberg, 2004), but this may have been due to insufficient resolution in the GSH measurement. Hightower showed a slight increase in lens GSSG, but argued that the increase was too small to account for the loss of GSH observed based on oxidation of GSH (Hightower and McCready, 1992). The in vivo exposures of rats did not demonstrate a detectable change of total lens GSSG concentration (Risa et al., 2004, Risa et al., 2005, Tessem et al., 2006, Wang et al., 2010).

Alpha-tocopherol (type V vitamin E) is an extrinsic antioxidant molecule with reducing action. It is an important dietary constituent. Alpha-tocopherol functions in vivo as a lipid antioxidant and as a free radical scavenger (Bieri et al., 1983, Burton et al., 1983). It exerts its prevention of lipid peroxidation on biological membranes in vivo by functioning as a chain-breaking antioxidant and helping to maintain glutathione level (Shang et al., 2003, Kutlu et al., 2005). Moreover, as an extrinsic antioxidant molecule with reducing power, α-tocopherol may indirectly prevent the consumption of GSH through oxidation of α-tocopherol, preventing the –SH groups on GSH from being oxidized. Additionally, α-tocopherol may directly stimulate GSH synthesis by up-regulating some of GSH-related enzymes in the lens, such as γ-glutamylcysteine synthetase and GSH synthetase (Seth and Kharb, 1999, Masaki et al., 2002).

The recommended daily allowance (RDA) for vitamin E is 12 IU for females and 15 IU for males (Halliwell and Gutteridge, 1985) (1.49 IU of vitamin E are equivalent to 1 mg α-tocopherol). Vitamin E has a low human toxicity and oral administration is not toxic even at high doses. An intake of 1000 mg/day is without risk and 3200 mg/day has been shown to be without any substantial risk (Diplock et al., 1998). The upper tolerance level (UL) for α-tocopherol was set at 1000 mg/day using data from studies in rats (Food and Nutrition Board and Institute of Medicine, 2000). In addition, studies have suggested that supplementation with at least 100 mg/day of vitamin E may decrease the risk of heart disease (Stampfer and Rimm, 1995). This is well above the current RDA and is far greater than what can be received with a well balanced diet. It has been postulated that vitamin E protects against photoperoxidation of lens lipids (Varma et al., 1982, Libondi et al., 1985, Robertson et al., 1989, Ohta et al., 1996, Karslioglu et al., 2004). The pioneering work done by Bhuyan shows that vitamin E is effective in the therapy of certain forms of cataract in the rabbit and the rat (Bhuyan et al., 1981). The outcome of clinical trials aiming for cataract prevention by vitamin E supplementation is inconclusive. Some studies find a beneficial effect (Rouhiainen et al., 1996, Leske et al., 1998, Lyle et al., 1999, Mares-Perlman et al., 2000), whereas others indicate no significant association (Hankinson et al., 1992, Seddon et al., 1994, Chasan-Taber et al., 1999).

Recent studies from our research group have demonstrated that per oral supplementation with α-tocopherol increases lens α-tocopherol and protects against in vivo UVR-induced cataract in rat (Ayala and Söderberg, 2004, Ayala and Söderberg, 2005).

The aim of the present study was to determine the dose dependence for α-tocopherol prevention of in vivo UVR-induced cataract in rat lens. Further, the impact of α-tocopherol supplementation on the lens concentration of GSH, GSSG and the activities of glutathione reductase (GR) and glutathione peroxidase (GPx) was to be determined.