arNOX: Generator of Reactive Oxygen Species in the Skin and Sera of Aging Individuals Subject to External Modulation

Introduction

O ur work has described an aging-related form of cell-surface nicotinamide adenine dinucleotide (NADH) oxidase associated with sera, saliva, skin, perspiration, and lymphocytes of subjects of age 30 years or older capable of directly reducing ferricytochrome c through generation of superoxide as a key component of the aging cascade. ¹ This activity provides a rational basis for the development of antiaging formulations and supplements as skin-care products and for the prevention of atherogenesis and other oxidative changes in cell membranes and circulating lipoproteins. ²

ECTO-NOX proteins, in general, have been postulated to link the accumulation of lesions in mitochondrial DNA to cell-surface accumulations of reactive oxygen species (ROS) as one consequence of their roles as terminal oxidases in a plasma membrane electron transport chain. ³ Cells with functionally deficient mitochondria become characterized by anaerobic metabolism. ⁴ NADH accumulates from glycolytic production of adenosine triphosphate (ATP) and elevated plasma membrane electron transport activity become necessary to maintain the NAD⁺/NADH homeostasis essential for survival. ⁵ Previous findings have demonstrated that hyperactivity of the plasma membrane electron transport system results in NADH oxidase activity capable of cell-surface generation of ROS. ⁶ This hyperactive activity would serve to propagate the aging cascade both to adjacent cells and to oxidize serum lipoproteins. ³

The production of ROS have long been postulated to lead to random deleterious modifications of macromolecules with an associated progressive development of age-associated systemic disease. ⁷ However, the source of these ROS and the manner in which they are directed to specific targets, some of which appear to be quite distant from the usual sources considered as ROS generators, has been inadequately addressed, if at all.

Methods

Aging-related NADH oxidase (arNOX) was assayed spectrophotometrically as described from the superoxide dismutase (SOD)-inhibited reduction of ferricytochrome c. ⁸ Proteins were determined by the bicinchoninic acid (BCA) procedure. ⁹

Advanced glycation end products (AGEs) were determined using a DiagnOptics (San Diego, CA) fluorescence AGE reader in which a light source excites fluorescent moieties in tissues such as skin, which then emits light of a different wavelength. In the wavelength band used, the major contribution in fluorescence comes from fluorescent AGEs linked mostly to collagen, but also to other proteins and lipids and includes fluorescent adducts of oxidized proteins and lipids.

Results

A number of observations point to arNOX as a primary generator of ROS in the body, especially with regard to oxidation of circulating lipoprotein particles. Mitochondria appear not to be a major source of cellular superoxide anion or hydrogen peroxide (H₂O₂). Uninhibited respiring mitochondria produce very little of either. ^{10 –12} The actual amount of H₂O₂ produced by mitochondria appears to be in the order of 0.1 nmoles formed per min/mg of mitochondrial protein. In contrast, the bulk of the superoxide anion and H₂O₂ appears to be produced by plasma membrane or exterior to the cells themselves.

Aging-related constitutive generation of superoxide anion for subsequent conversion to H₂O₂ is the purview of a special class of ENOX proteins (Fig. 1) measurable in humans after about age 30 ^2,13 as well as in late-passage tissue culture cells and aging plant parts referred to as age related. arNOX proteins generate superoxide anion exclusively as measured by SOD-inhibited reduction of ferricytochrome c, a standard measure of superoxide anion production. At peak production, which occurs on average at about age 65, approximately 0.06 nmol of superoxide is produced/10⁷ cells (human buffy coat or epidermal epithelia). The value in sera is approximately 0.3 nmol/mL per min, resulting in several millimoles of superoxide produced on a daily basis in the proximity of circulating lipoproteins just from the circulating form of arNOX.

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FIG. 1.

Diagram showing the spatial relationships among the intracellular NAD(P)H:quinone reductase, the membrane pool of coenzyme Q (Q₁₀) and the external nicotinamide adenine dinucleotide (NADH) oxidase (NOX) protein across the plasma membrane. In this manner, the ECTO-NOX protein could function as a terminal oxidase of plasma membrane electron transport, donating electrons from cytosolic NADH either to molecular oxygen or to protein disulfides as electron acceptors. Modified from ref. 19.

Well advanced is use of an inhibitor cocktail of natural product sources of arNOX inhibitors consisting of a blend of botanical ingredients to reduce arNOX levels to slow production of free radicals in skin in collaboration with NuSkin Enterprises (Provo, Utah). The product, under the trade name age-LOC, is currently marketed to prevent skin aging at its source. The inhibitors target the arNOX enzyme per se rather than the superoxide anion product, as occurs with SOD inhibition, and combine with arNOX to prevent binding of the substrate. Structure–activity analysis and related arNOX-blocking molecules have been used to identify other arNOX substrate-blocking molecules present in food supplements appropriate to formulation of dietary antiaging interventions. Some identified supplement candidates are efficiently absorbed and extremely effective at dilutions corresponding to <100 ng/mL or approximately 0.1 mg/kg body weight.

arNOX is unique among the ENOX proteins in that it results in the generation of superoxide at the cell surface and, as a shed protein, in the circulation. ^{1,14 –16} More than one arNOX activity appears to be present, depending on the individual examined, which is suggestive of at least five different homologs. The superoxide generated affords an opportunity to form H₂O₂ and ROS for propagation to adjacent cells and tissues and direct oxidation of serum lipoprotein particles. The shed forms of arNOX appear in body fluids (serum, saliva, urine, perspiration, and interstitial fluids) where protein thiols serve as the electron donors. ¹⁷ The ROS generated from superoxide can become accessible to lipoproteins in the circulation resulting in their oxidation and increased atherogenic risk as well as damaging to adjacent cells and extracellular supporting matrices that are important to skin health.

Superoxide can be further converted to H₂O₂ through the action of the enzyme SOD. ¹⁶ arNOX proteins are absent or present at levels below the limit of detection for cells and sera of young individuals. They then increase with increasing age (>30 years) to about ages 60–70. Both cell-bound and shed arNOX are inhibited by coenzyme Q₁₀ (CoQ₁₀). ^2,13,14 This inhibition appears to be at a site unique to arNOX and unrelated to the quinone site involved in binding and oxidation of reduced CoQ₁₀ at the plasma membrane. Estimates of subjects with high arNOX compared to actual age had skin characteristics that made them appear on average 7 years older than their chronological age, whereas subjects with low arNOX activity at the same age had on average 7 years younger-appearing skin than their actual age. ¹⁷ AGEs normally accumulate with aging. AGEs of skin correlate with arNOX activity, suggestive of a causal relationship. ¹⁷

Discussion

Our findings provide a new and novel strategy to direct development of antiaging products and regimens using inhibition or suppression of an arNOX as an analytical end point. We anticipate that the arNOX protein will provide the first ever defined gene product to serve as a facile aging biomarker. Through the use of this biomarker, we can rapidly seek antiaging ingredients and develop nutritional supplements with antiaging properties targeted to the arNOX protein, a major source of ROS in aging populations. Also, it will be possible to evaluate different dietary ingredients and combinations for antiaging properties.

arNOX, a member of the ECTO-NOX family of growth-related and time-keeping proteins, is associated specifically with aging cells in culture and in skin and aging cells in general. ¹⁶ The circulating form of arNOX increases markedly in human sera and in lymphocytes of individuals after age 30. ^2,13 The arNOX is uniquely characterized by an ability to generate superoxide (Fig. 1) and, therefore, can contribute significantly to the imbalance between production of ROS and antioxidant defense by producing reactive oxygen at the cell surface to greatly accelerate aging-related changes, including atherogenesis, skin damage, and other action-at-a-distance aging phenomena. ³

The basic premise of our antiaging research is that the arNOX protein provides a molecular target to explain how CoQ₁₀ and other antiaging substances could offer protection to maintain skin vitality and possibly ablate aging related cardiovascular changes. ^1,13,16 arNOX is inhibited by CoQ₁₀, a substance long implicated as having antiaging properties but for which there was no clear biochemical rationale to completely explain its effects.

In addition to sera and perspiration, human saliva contains arNOX activity. Saliva offers an opportunity to readily monitor arNOX in patients and volunteers ¹³ and to monitor responses to supplements.

One priority objective for future work is to develop noninvasive arNOX assays based on arNOX levels in serum and saliva and to apply these assays to identification of at risk populations and individuals and to monitor intervention efficacy. In ongoing work, an inhibitor cocktail consisting of a blend of botanical ingredients has been developed at NOX Technologies, Inc., which appears to slow production of free radicals in the skin associated with arNOX. ¹⁷ The cocktail is currently under evaluation for this purpose. Also under development at NOX Technologies, Inc., is a nutritional supplement based on use of the arNOX protein as the basis for an intervention strategy directed toward reducing aging-related cardiovascular risk.