1,2-Naphthoquinone Stimulates Lipid Peroxidation and Cholesterol Domain Formation in Model Membranes

Abstract

Purpose.: Naphthalene induces cataract formation through the accumulation of its reactive metabolite, 1,2-naphthoquinone (1,2-NQ), in the ocular lens. 1,2-NQ increases lens protein oxidation and disrupts fiber cell membrane function; however, the association of these effects with changes in membrane structure is not understood. The goal of this study was to determine the direct effects of 1,2-NQ on membrane lipid oxidation and structural organization.

Methods.: Iodometric approaches were used to measure the effects of naphthalene and 1,2-NQ on lipid hydroperoxide (LOOH) formation in model membranes composed of cholesterol and dilinoleoylphosphatidylcholine. Membrane samples were prepared at various cholesterol-to-phospholipid mole ratios and subjected to autoxidation at 37°C for 48 hours in the absence or presence of either agent alone (0.1–5.0 μM) or in combination with vitamin E. Small-angle x-ray diffraction was used to measure the effects of naphthalene and 1,2-NQ on membrane structure before and after exposure to oxidative stress.

Results.: 1,2-NQ increased LOOH formation by 250% (P < 0.001) and 350% (P < 0.001) at 1.0 and 5.0 μM, respectively, whereas naphthalene decreased LOOH levels by 25% (P < 0.01) and 10% (NS). The pro-oxidant effect of 1,2-NQ was inversely affected by membrane cholesterol enrichment and completely blocked by vitamin E. 1,2-NQ also increased cholesterol domain formation by 360% in membranes exposed to oxidative stress; however, no significant changes in membrane lipid organization were observed with naphthalene under the same conditions.

Conclusions.: These data suggest a novel mechanism for naphthalene-induced cataract, facilitated by the direct effects of 1,2-NQ on lipid peroxidation and cholesterol domain formation.

Introduction

Naphthalene is a bicyclic aromatic hydrocarbon that is found as a natural constituent of crude and refined oil products and is derived largely from their combustion as well as that of wood, tobacco, coal, and other organic materials. ¹ It is also used in the manufacture of a wide variety of industrial products, including dyes, pesticides, synthetic resins, and household fumigants. ^2–4 Overexposure to naphthalene, particularly through ingestion or inhalation, is considered hazardous to health and has been reported to cause hemolytic anemia, ^5–10 respiratory inflammation, ^11–14 and renal and hepatic dysfunction ^15,16 in humans and experimental animals. Naphthalene is especially toxic to tissues of the eye, where it induces, among other degenerative effects, the formation of lenticular cataracts. ^17–29 This hallmark feature of naphthalene-induced toxicity is accompanied by increased swelling, discoloration, and vacuole formation in the ocular lens. ^30–34 These morphologic changes are similar to those observed in human, age-related cataracts and have suggested naphthalene to be a valuable experimental tool for studying basic mechanisms associated with cataract development. ^32,34,35  

It is now widely accepted that naphthalene itself does not cause cataracts but, instead, is biotransformed by cytochrome P450 into various metabolites, some of which are known to be directly cataractogenic. ^34,36–38 The most prominent and cytotoxic of these metabolites is 1,2-naphthoquinone (1,2-NQ), which is produced by the intralenticular modification of its hepatic precursor, naphthalene-1,2-dihydrodiol (Fig. 1). ^2,28,34 1,2-NQ has pronounced electrophilic properties that enable it to bind covalently, via Michael addition, to cellular proteins and other macromolecules. ^29,37,39 The highly reactive nature of 1,2-NQ, and its ability to arylate susceptible proteins, is attributed to its unsaturated, α,β-carbonyl system, which also facilitates efficient redox cycling and the production of reactive oxygen species. ^40–42 Numerous studies have indeed confirmed that 1,2-NQ binds to and disrupts the function of glutathione S-transferase, crystallins, and other metabolically active proteins in the ocular lens. ^39,43–45 Such reactions are typically accompanied by an increase in the uptake of oxygen and the formation of hydrogen peroxide and other free radicals. ^39,42,44 Oxidative damage resulting from direct exposure to 1,2-NQ has been shown to initiate other pathologic sequelae in the ocular lens, including mobilization of intracellular calcium reserves, activation of proteolytic enzymes (e.g., calpain), and disruption of fiber cell morphology and organization. ^46,47 All of these changes culminate in the formation of irreversible cataracts. 

Figure 1

 

Metabolism scheme associated with naphthalene and its contribution to cataract formation. Naphthalene is converted by liver enzymes into naphthalene-1,2-dihydrodiol, which is released into the circulation for eventual excretion. This trans-dihydrodiol intermediate is readily taken up by tissues of the eye, including the ocular lens, ³³ where it is further metabolized by aldose reductase to form naphthalenediol (or 1,2-dihydroxynaphthalene). Naphthalenediol undergoes rapid autoxidation to form the toxic end product, 1,2-naphthoquinone, which directly contributes to cataract formation by stimulating various mechanisms of oxidative stress, binding covalently to lens proteins and other macromolecules, and altering fiber cell membrane structure and function. Adapted with permission from Tao RV, Holleschau AM, Rathbun WB. Naphthalene-induced cataract in the rat. II. Contrasting effects of two aldose reductase inhibitors on glutathione and glutathione redox enzymes. Ophthalmic Res. 1991;23:272–283.

Although the pro-oxidant and cataractogenic effects of 1,2-NQ have been well characterized in whole animal and tissue culture studies, very little is known about its direct effects on membrane lipid oxidation and structural organization. Such effects are particularly important in the ocular lens, where lipid peroxidation has been shown to play a significant role in cataract development. ⁴⁸ Lipid peroxidation products, including lipid carbonyls, diene conjugates, and lipid hydroperoxides (LOOH), accumulate in the human lens as a function of age ⁴⁹ and have been found to correlate with the degree of lenticular opacity. ^50,51 These and other lipid peroxidation products have been shown to directly induce cataract formation in various animal models ^52,53 as well as rat and human lenses examined in organ culture. ^54,55 Such oxidative damage is believed to alter lens fiber cell membrane composition and structural organization. ^56–58 One consequence of these changes, as demonstrated by research conducted in our laboratory, is the increased separation of membrane cholesterol into discrete domains. The lens fiber cell plasma membrane, due to its intrinsically high levels of monomeric cholesterol, is typically organized into a biphasic, cholesterol-rich and cholesterol-poor, structural motif ⁵⁹ ; however, cholesterol domains become more prominent and increasingly stable in cataractous lenses despite marked reductions in overall cholesterol levels. ⁶⁰ Similar changes in cholesterol domain formation have been observed in model membranes exposed to oxidative stress, ⁶¹ suggesting a mechanistic link between lipid peroxidation, membrane structural perturbation, and cataract formation. 

In this study, we examined the direct effects of naphthalene and 1,2-NQ on lipid peroxidation and cholesterol domain formation in model membranes prepared as binary mixtures of cholesterol and dilinoleoylphosphatidylcholine. Peroxidation-induced changes in membrane structure were measured using iodometric and small-angle x-ray diffraction approaches. Naphthalene and 1,2-NQ were discovered to have disparate effects on LOOH and cholesterol domain formation in keeping with their distinct biochemical properties. The results of this study suggest a novel mechanism for naphthalene-induced cataract while further implicating oxidative stress and membrane lipid reorganization in the etiology of this disorder. 

Materials and Methods

Materials

1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) and monomeric cholesterol (isolated from ovine wool) were purchased from Avanti Polar Lipids (Alabaster, AL) and solubilized in chloroform at 25 and 10 mg/mL, respectively. Naphthalene and 1,2-NQ were obtained from Sigma-Aldrich (St. Louis, MO) and prepared in ethanol at 40 μM. Vitamin E (α-tocopherol) was also purchased from Sigma-Aldrich and prepared in ethanol at 56.8 μM (ε = 3.06 × 10⁴ M⁻¹ cm⁻¹ at 294 nm) just prior to experimental use. CHOD-iodide color reagent (stock) was prepared, with slight modification, as described by El-Saadani et al. ⁶² and consisted of 0.2 M K₂HPO₄, 0.12 M KI, 0.15 mM NaN₃, 10 μM ammonium molybdate, and 0.1 g/L benzalkonium chloride. Before experimental use, the CHOD reagent was activated by adding 24 μM EDTA, 20 μM butylated hydroxytoluene, and 0.2% Triton X-100. ⁶¹  

Preparation of Membrane Lipid Vesicles

Multilamellar vesicles (MLVs) were prepared as binary mixtures of DLPC (1.0 or 2.5 mg total phospholipid per sample) and cholesterol at cholesterol-to-phospholipid (C/P) mole ratios ranging from 0 to 1.0. Component lipids (in chloroform) were transferred to 13 × 100-mm borosilicate culture tubes and combined with vehicle (ethanol) or an equal volume of naphthalene, 1,2-NQ, or vitamin E stock solutions, each adjusted to achieve desired treatment concentrations (0.1, 1.0, and 5.0 μM for naphthalene and 1,2-NQ; 0.5, 1.0, and 5.0 μM for vitamin E). Samples were shell-dried under nitrogen gas and placed under vacuum for 3 hours to remove residual solvent. After desiccation, each sample was resuspended in saline buffer (0.5 mM HEPES, 154 mM NaCl, pH 7.3) to yield a final phospholipid concentration of 1.0 mg/mL (for lipid peroxidation or x-ray diffraction analysis, respectively). Lipid suspensions were then vortexed for 3 minutes at ambient temperature to form MLVs. ⁶³  

Lipid Peroxidation Analysis

All MLV samples were subjected to time-dependent autoxidation by incubating at 37°C in an uncovered water bath. This method allows lipid peroxidation to occur gradually without requiring the use of exogenous initiators. Small aliquots (10–20 μL) of each sample were removed, immediately following MLV preparation (0 hour) and after exposing samples to oxidative conditions for 48 hours, and combined with 1.0 mL of activated CHOD reagent. Test samples were covered and incubated in darkness at room temperature for at least 4 hours. Sample absorbances were then measured against a CHOD blank at 365 nm using a Beckman DU-640 spectrophotometer (Beckman Coulter, Inc., Fullerton, CA). The CHOD colorimetric assay is based on the oxidation of iodide (I^–) by lipid hydroperoxide (LOOH) to form tri-iodide (I₃ ^–), the quantity of which is directly proportional to the amount of LOOH present in the lipid sample. The molar absorptivity (ε) of I₃ ^– is 2.46 × 10⁴ M⁻¹ cm⁻¹ at 365 nm. ⁶²  

Membrane Structural Analysis

Membrane lipid vesicles were oriented for x-ray diffraction analysis as previously described. ⁶⁴ Briefly, a 100-μL aliquot (containing 250 μg MLV) was aspirated from each sample and transferred to a Lucite sedimentation cell fitted with an aluminum foil substrate on which sample MLVs could be collected by centrifugation. Samples were then loaded into a Sorvall AH-629 swinging bucket rotor (Dupont Corp., Wilmington, DE) and centrifuged at 35,000g, 5°C, for 90 minutes. 

After centrifugal orientation, sample supernatants were aspirated and aluminum foil substrates, each supporting a single membrane pellet, were removed from sedimentation cells. Sample pellets were dried for 5 to 10 minutes at ambient conditions, mounted onto curved glass supports, and placed in hermetically sealed, brass or glass containers (for immediate analysis or temporary storage, respectively). All x-ray diffraction experiments were conducted at 20°C, 74% relative humidity. The latter was established by exposing membrane samples to saturated solutions of L-(+) tartaric acid (K₂C₄H₄O₆ · ½H₂O). Samples were incubated at these conditions for at least 1 hour before experimental analysis. 

Oriented membrane samples were aligned at grazing incidence with respect to a collimated, monochromatic CuK_α x-ray beam (K_α1 and K_α2 unresolved; λ = 1.54 Å) produced by a Rigaku Rotaflex RU-200, high-brilliance microfocus generator (Rigaku-MSC, The Woodlands, TX). ⁶⁵ Diffraction data were collected on a one-dimensional, position-sensitive electron detector (Hecus X-ray Systems, Graz, Austria) at a sample-to-detector distance of 150 mm. Detector calibration was performed by the manufacturer and verified using crystalline cholesterol monohydrate. 

This technique allows for precise measurement of the unit cell periodicity, or d-space, of the membrane lipid bilayer, which is the distance from the center of one lipid bilayer to the next, including surface hydration. The d-space for any given membrane multibilayer is calculated from Bragg's Law, h λ = 2 d sin θ, where h is the diffraction order, λ is the wavelength of the x-ray radiation (1.54 Å), d is the membrane lipid bilayer unit cell periodicity, and θ is the Bragg angle equal to one-half the angle between the incident beam and scattered beam. 

The presence of cholesterol domains in a given membrane sample results in the production of distinct Bragg (diffraction) peaks having singular periodicity values of 34 and 17 Å (typically referred to as first- and second-order cholesterol domain peaks). ⁶⁶ Under the specific temperature and relative humidity conditions established for these experiments, the second-order, 17-Å cholesterol domain peak was well-delineated from other, neighboring cholesterol and phospholipid diffraction peaks and was thus used to quantitate relative cholesterol domain peak intensity. Routines written in Origin 7.5 (OriginLab Corporation, Northampton, MA) were used to determine total peak area (associated with all diffraction peaks in a given pattern) against which the second-order cholesterol domain peak was normalized. 

Statistical Analyses

Data are presented as mean ± SD for (N) separate samples or treatment groups. Lipid peroxidation and cholesterol domain peak intensity measurements were conducted in sextuplicate and octuplicate, respectively. Differences between groups were analyzed using ANOVA followed by Student-Newman-Keuls multiple comparisons post hoc analysis. Alpha error was set to 0.05 in this study. 

Results

We tested the comparative and concentration-dependent effects of naphthalene and 1,2-NQ on LOOH formation in membrane lipid vesicles enriched in polyunsaturated fatty acids and prepared at a C/P mole ratio representative of typical cell membranes. At the lowest dose tested (0.1 μM), neither agent significantly affected membrane lipid peroxidation as compared to vehicle treatment alone (Fig. 2). However, at 1.0 and 5.0 μM, 1,2-NQ increased LOOH formation by 250% (P < 0.001) and 350% (P < 0.001), respectively, as compared with control. Naphthalene, at the same treatment levels, reduced LOOH formation by 25% (P < 0.01) and 10% (NS), respectively. These disparate effects on membrane oxidation, as observed at the higher treatment levels, corresponded to a 5-fold increase in LOOH levels with 1,2-NQ as compared to its parent compound. 

Figure 2

 

Comparative effects of 1,2-NQ and naphthalene on membrane lipid peroxidation. Membrane vesicles were prepared as binary mixtures of DLPC and cholesterol (0.6 C/P mole ratio) in the absence or presence of 1,2-NQ or naphthalene at various concentrations and subjected to autoxidation for 48 hours at 37°C. Lipid hydroperoxide (LOOH) formation was measured using the CHOD-iodide colorimetric assay. *P < 0.01 and **P < 0.001 versus control; †P < 0.001 versus equimolar naphthalene treatment (Student-Newman-Keuls multiple comparisons test; overall ANOVA: P < 0.0001, F = 423.96).

Cholesterol enrichment has been shown to increase the efficiency of free radical propagation through the membrane bilayer by increasing the packing and overall structural order of component lipids. ⁶¹ This effect can be enhanced by certain conditions, including hyperglycemia, as recently reported by our laboratory. ⁶⁷ In this study, we tested the influence of naphthalene and 1,2-NQ (each at 1.0 μM) on membrane lipid peroxidation as a function of increasing cholesterol content (Fig. 3). Consistent with the data shown in Figure 2, naphthalene had a neutral or antioxidant effect at all cholesterol treatment levels; however, as also observed for vehicle-treated controls, the extent of its effect on LOOH formation was largely unaffected by discrete changes in membrane cholesterol content. In contrast, 1,2-NQ was consistently pro-oxidant at each C/P ratio; but the extent of its effect decreased as a function of increasing cholesterol content. Under these treatment conditions, LOOH levels measured at 1.0 C/P, for example, were 42% (P < 0.001) and 27% (P < 0.001) lower than those measured at 0 and 0.6 C/P, respectively. 

Figure 3

 

Membrane cholesterol enrichment attenuated the pro-oxidant effect of 1,2-NQ. DLPC membranes were prepared in the absence or presence of 1,2-NQ or naphthalene (each at 1.0 μM) and examined as a function of increasing cholesterol content. All other experimental conditions were as previously described. *P < 0.01 and **P < 0.001 versus control; †P < 0.001 versus control or naphthalene treatment (Student-Newman-Keuls multiple comparisons test; overall ANOVA: P < 0.0001, F = 92.035).

We also examined the effects of vitamin E on naphthalene- and 1,2-NQ-induced changes in membrane lipid peroxidation. As shown in Figure 4, vitamin E, at all treatment levels, completely blocked the pro-oxidant effect of 1,2-NQ; however, vitamin E had no significant effect on LOOH formation in control or naphthalene-treated samples. 

Figure 4

 

Vitamin E blocked the pro-oxidant effect of 1,2-NQ. The dose-dependent effects of vitamin E were examined in DLPC membranes (0.6 C/P) prepared in the absence or presence of 1,2-NQ or naphthalene (each at 1.0 μM). *P < 0.001 versus all other treatments (Student-Newman-Keuls multiple comparisons test; overall ANOVA: P < 0.0001, F = 25.941).

Lipid peroxidation is highly disruptive to the structural organization of biological membranes and has been shown, in various model systems, to contribute directly to the formation of cholesterol crystalline domains. ^61,66 Given the pronounced pro-oxidant activity of 1,2-NQ, as observed in this study, we hypothesized that this agent would increase membrane cholesterol domain formation in a peroxidation-dependent manner and beyond levels expected for naphthalene or vehicle control treatments. To test this hypothesis, we used small-angle x-ray diffraction to characterize the structural properties of membranes prepared in the absence or presence of naphthalene or 1,2-NQ, before and after exposure to oxidative conditions (Fig. 5). Immediately following sample preparation, and in advance of any substantive exposure to oxidative injury, naphthalene and 1,2-NQ were observed to have no appreciable effect on membrane structure as compared to vehicle treatment alone. Scattering data collected from each membrane preparation yielded four diffraction orders (or peaks), all of which were mathematically related, with an average unit cell periodicity (d-space value) of 51 Å, and consistent with a homogeneously distributed lipid bilayer phase. Following exposure to oxidative conditions for 48 hours, additional peaks, with an average d-space value of 34 Å and consistent with a cholesterol domain phase, were observed in the separate diffraction patterns collected from each treatment group. However, naphthalene and 1,2-NQ had apparent and disparate effects on the extent of cholesterol domain formation as compared to vehicle-treated controls. Quantitative assessment of cholesterol domain peak intensity (expressed as the quotient of cholesterol- to total lipid-peak area) indicated that naphthalene had no significant effect on cholesterol domain formation as compared to control (Fig. 6). In contrast, 1,2-NQ increased relative cholesterol domain peak intensity by 360% (P < 0.001) and 280% (P < 0.001) as compared to vehicle or naphthalene treatments, respectively. 

Figure 5

 

1,2-NQ increased the peroxidation-dependent formation of cholesterol domains in model membranes. X-ray diffraction patterns were obtained from DLPC membranes prepared at 0.6 C/P in the absence or presence of naphthalene or 1,2-NQ (each at 1.0 μM), before (0 hour) and after (48 hours) exposure to oxidative stress. At 0 hour, each sample exhibited a single lipid bilayer phase with an average periodicity (d-space value) of 51 Å (represented by diffraction peaks 1, 2, and 4). At 48 hours, cholesterol crystalline domains, having a characteristic d-space value of 34 Å and represented by a set of distinct diffraction peaks (shown in gray fill), were also observed in all control and drug-treated membrane samples. Cholesterol domain peak intensity was disproportionately greater in samples treated with 1,2-NQ as compared with those treated with naphthalene or vehicle.

Figure 6

 

Quantitative assessment of the effects of 1,2-NQ and naphthalene on peroxidation-induced cholesterol domain formation. Relative cholesterol peak intensity values were derived by integrating the second-order cholesterol domain peak and normalizing to total peak area associated with a given diffraction pattern. *P < 0.001 versus control or naphthalene treatment (Student-Newman-Keuls multiple comparisons test; overall ANOVA: P = 0.0002, F = 12.928).

Discussion

The toxic effects of naphthalene are primarily facilitated through its biotransformation to reactive metabolites (Fig. 1) that directly or indirectly increase oxidative stress in sensitive organ systems such as the ocular lens. ^22,44 Many attempts have been made to identify the proximal metabolite responsible for naphthalene-induced cataract. Early studies implicated 1,2-NQ in this capacity as it was shown to readily react with lens proteins while promoting oxygen uptake and hydrogen peroxide formation in the ocular lens. ^28,39,44 Martynkina et al. ⁴⁶ showed that direct injection of 1,2-NQ into the anterior chamber of the mouse eye resulted in rapid cataract formation, the onset of which was significantly delayed by cotreatment with superoxide dismutase. Using similar approaches, Qian and Shichi ⁴⁷ demonstrated that 1,2-NQ increased intracellular calcium release and calpain activation in lens epithelial cells before inducing the formation of cataracts. These studies, together with well-established chemical reactivity associated with quinone structures, suggest that 1,2-NQ plays a prominent role in naphthalene-induced cataract formation. ⁴⁴  

The importance of naphthalene biotransformation in cataract formation has also been demonstrated by the protective effects provided by certain aldose reductase inhibitors, ⁶⁸ especially in transgenic mice that experience more severe and rapid cataract onset due to the overexpression of aldose reductase. ⁶⁹ Aldose reductase inhibitors appear to exert their anticataractogenic effects by blocking the formation of lens protein adducts in animals treated with naphthalene. ⁷⁰ This would suggest that these agents function by disrupting the enzymatic formation of reactive metabolites (such as 1,2-NQ) instead of simply reducing sugar alcohol (polyol) levels in the ocular lens (the primary pharmacologic mechanism associated with the inhibition of aldose reductase). Naphthalene, in fact, has been shown to have no effect on lens polyol levels in treated versus untreated (control) animals. ¹⁸  

Oxidative stress, in the form of lipid peroxidation, is believed to play a significant role in cataract formation. ⁴⁸ Infrared spectroscopy approaches have been used to demonstrate that lipid peroxidation increases in the human ocular lens as a function of age and in a manner associated with structural perturbation of lens membrane lipids. ⁴⁹ Primary lipid peroxidation products, such as diene conjugates and LOOH, accumulate in the earlier stages of cataract development, whereas secondary products, such as Schiff bases, aldehydes, and ketones, are more prevalent in later stages and correlate with the degree of lenticular opacification. ^50,51 Direct injection of lipid peroxidation products into the vitreous humor has been shown to induce cataracts in rabbits, ⁵³ whereas similar changes have been observed in rat and human lenses incubated with lipid peroxidation products in organ culture. ^54,55 Electron microscopy approaches have confirmed that increased exposure to lipid peroxidation and peroxidation products results in the progressive deterioration of the lens fiber cell plasma membrane, leading to membrane vesiculation and cataract formation. ^50,51  

In this study, 1,2-NQ was shown to have very pronounced pro-oxidant activity as compared with its parent compound, naphthalene, which had neutral or modest antioxidant effects at the various doses tested. The effects of 1,2-NQ on lipid peroxidation were attenuated by cholesterol enrichment, suggesting a role for cholesterol in coordinating the structural and functional interactions of this compound with the lipid bilayer. In previous studies, using similar model membrane preparations, cholesterol has been shown to increase lipid peroxidation, ⁶¹ presumably in keeping with its condensing effect on biological membranes. ⁷¹ Cholesterol has also been shown to increase the rate of lipid peroxidation in liposomes treated with membrane stabilizing agents or exposed to γ-radiation, all of which increase membrane rigidity. ⁷² As the opposite effect was observed with 1,2-NQ treatment in this study, our results suggest that this agent interacts with some component of the membrane bilayer, perhaps cholesterol, effectively reducing phospholipid packing and overall membrane rigidity. 

It appears likely that 1,2-NQ contributes to membrane dysfunction by facilitating the production and propagation of free radicals through the hydrocarbon core region of the membrane bilayer. This hypothesis is supported by our experimental analysis of vitamin E, which was observed to completely block the peroxidative effects of 1,2-NQ. Vitamin E is highly lipophilic and intercalates deep into the membrane bilayer, with its long axis oriented parallel to the acyl chain segments of resident phospholipids. In this position, vitamin E is able to stabilize the membrane bilayer and block peroxyl chain free radical propagation reactions. ⁷³ Our results are also consistent with other studies that have shown vitamin E to have protective effects on naphthalene-induced cataracts. In a 9-week study, Nagata et al. ⁷⁴ showed that daily administration of eye drops containing 1% vitamin E acetate was sufficient to significantly delay the onset of cataracts in naphthalene-treated rats. Lee and Chung ⁶⁹ reported that vitamin E delayed cataract development in transgenic mice engineered to overexpress aldose reductase, an effect they attributed to increased removal of oxidative species and preservation of membrane integrity in the ocular lens. Vitamin E has also been shown to enhance glutathione recycling in the ocular lens ⁷⁵ and to prevent cataract development resulting from diabetes and galactosemia. ⁷⁶  

The key finding in this study is that 1,2-NQ, as a consequence of its pro-oxidant effects, increased the formation of highly ordered, cholesterol crystalline domains in model membranes exposed to oxidative conditions. Lipid peroxidation has been shown, in the absence of any pharmacologic intervention, to induce the aggregation of unesterified, monomeric cholesterol into membrane-restricted domains, even in lipid vesicles containing low or normal amounts of cholesterol. ^61,66 The basis for this effect is attributed to a disruption in normal phospholipid-sterol interactions resulting from oxidative damage to polyunsaturated fatty acids, a common and essential component of most biological membranes. ⁶⁶ Such changes in the physicochemical properties of the cell membrane have been directly implicated in the etiology of age-related cataracts. We previously reported, for example, that cholesterol domains are more pronounced and more highly ordered in cataractous lenses as compared with normal, age-matched controls. ^59,60 Other reports substantiate that lens membrane composition, ^77,78 bilayer structure, ^79–81 and overall function ^82,83 are significantly altered in cataracts. 

Cholesterol monohydrate domains were identified in this study on the basis of characteristic diffraction peaks corresponding to a unit cell periodicity of 34 Å. This repeating structural motif was first observed in multilamellar model membranes prepared as ternary mixtures of phosphatidylcholine, galactocerebroside, and cholesterol ⁸⁴ and is consistent with a bilayer arrangement of monomeric cholesterol. ^85,86 Cholesterol domains have also been characterized in various lens membrane lipid mimetic systems using differential scanning calorimetry, nuclear magnetic resonance (NMR), and electron spin resonance techniques. ^87,88 These highly organized structures may serve as nucleating sites for the formation of extracellular crystals, as characterized in vitro using various microscopy approaches. ⁸⁹ The association of such progressive changes in cholesterol organization with cataract formation in the ocular lens remains an area of ongoing inquiry. 

Other procataractogenic agents may effect lenticular dysfunction by altering membrane structural order and lipid organization in ways similar to those observed for 1,2-NQ. The oxidosqualene cyclase inhibitor, U18666A, was found to intercalate deep into bovine lens lipid model membranes where it produced a broad condensing effect and increased membrane structural order. ⁹⁰ Glucose, which is implicated in the development of diabetic cataracts, ⁹¹ has also been shown to increase LOOH and cholesterol domain formation in model membrane vesicles while reducing overall membrane bilayer width. ⁶⁷ In addition, several experimental lipoxygenase inhibitors, CJ-12,918 and CJ-13,454, known to cause cataracts in rats, have been observed to intercalate into the hydrocarbon core region of lens lipid model membranes (Aleo MD and Jacob RF, unpublished observations, 2003). Other cataractogenic agents, such as acetaminophen and its quinone metabolites, may induce cataract formation through mechanisms similar to that shown for naphthalene and 1,2-NQ in this study. 

The model membrane system used in this study does not recapitulate the diverse and structurally complex nature of the lens fiber cell membrane; however, the very pronounced pro-oxidant and lipid reordering effects observed with 1,2-NQ, coupled with numerous studies demonstrating an etiologic role for lipid oxidation in cataract formation, suggest that peroxidative damage of susceptible lipids could serve as a potential mechanism in naphthalene-induced cataracts. Although the ocular lens contains relatively low amounts of polyunsaturated fatty acids, it is uniquely enriched in sphingomyelin and monoenoic fatty acids, ⁹² both of which have been shown to undergo extensive oxidative modification as a function of age and cataract development. ^49,93 The 1,2-NQ may impact these specific lipids and fatty acid moieties in the ocular lens in a manner similar to that observed in our basic model system. Future studies, using intact or reconstituted lens membranes, will be useful in extending these findings to a more detailed understanding of 1,2-NQ-induced toxicity. 

Acknowledgments

Supported by a Pfizer Drug Safety and Technology and TA Enabler Grant (NRA9010104). 

Disclosure: R.F. Jacob, None; M.D. Aleo, None; Y. Self-Medlin, None; C.M. Doshna, None; R.P. Mason, Pfizer (C)