The arNOX Enzyme: Implications for Intrinsic Aging

Oct 1, 2009 | Contact Author | By: Helen Knaggs, PhD, Nu Skin Enterprises
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Title: The arNOX Enzyme: Implications for Intrinsic Aging
agingx sourcex arNOXx fine linesx wrinklesx UV radiationx free radicalsx
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Keywords: aging | source | arNOX | fine lines | wrinkles | UV radiation | free radicals

Abstract: This article describes a membrane-bound enzyme found in skin whose activity increases as biological age increases. The enzyme, located on the external surface of fibroblasts and keratinocytes, generates free radicals. The present work identifies the biological mechanism of the enzyme and its relationship to the appearance of aging in skin.

Aging is the result of a complex, natural process. Appearance is a key factor to gauge age, health and even emotional well-being. Studies suggest that an attractive appearance may reflect health and a longer lifespan. Additionally, fine lines, wrinkles and skin tone may affect the perception of age. Although considerable research has been conducted on the role of UV radiation in producing free radicals, there are still many unknown factors that explain why humans age and why they age differently. An area of great interest is the biological or intrinsic processes that affect appearance over time. The present study examines factors known to produce an aged appearance in skin, and provides evidence of a potential source of the aged appearance.

Appearance and Aging
The notion that an attractive and youthful appearance is not just about vanity, but also reflects an individual’s overall health has been strengthened by an increasing number of scientific publications demonstrating that appearance may be linked to health¹ and even life expectancy. In one study investigating more than 350 sets of twins, assessments were made as to which twin appeared older and the twins were followed for two years. By 2003, 49 deaths were reported and of these deaths, the twin who “looked older” had an increased risk of mortality.²

Aging is the result of a complex, natural process. Appearance is a key factor to gauge age, health and even emotional well-being. Studies suggest that an attractive appearance may reflect health and a longer lifespan. Additionally, fine lines, wrinkles and skin tone may affect the perception of age. Although considerable research has been conducted on the role of UV radiation in producing free radicals, there are still many unknown factors that explain why humans age and why they age differently. An area of great interest is the biological or intrinsic processes that affect appearance over time. The present study examines factors known to produce an aged appearance in skin, and provides evidence of a potential source of the aged appearance.

Appearance and Aging

The notion that an attractive and youthful appearance is not just about vanity, but also reflects an individual’s overall health has been strengthened by an increasing number of scientific publications demonstrating that appearance may be linked to health¹ and even life expectancy. In one study investigating more than 350 sets of twins, assessments were made as to which twin appeared older and the twins were followed for two years. By 2003, 49 deaths were reported and of these deaths, the twin who “looked older” had an increased risk of mortality.² 

Other research has shown an association between facial wrinkles and decreased renal function.³ This research suggested that dermatological changes are a manifestation of the total oxidative stress to which an individual is exposed, and that this oxidative stress could reflect the redox state of internal organs. The data provided evidence that severe facial wrinkles were associated with higher lipid peroxidation levels and reduced glomerular filtration in the kidneys, implying that facial wrinkles are a predictive marker and the biological consequence of underlying high levels of oxidative stress. 

From this, one could speculate that a diet high in antioxidants would provide benefits for facial skin due to the role of antioxidants in mitigating potential damage from oxidative stress by combining with and neutralizing free radicals. For example, a diet rich in vitamin C and linoleic acid has resulted in fewer facial wrinkles and better overall health, in agreement with lower internal oxidative stress.⁴ Additionally, dietary carotenoids have correlated with reduced skin roughness, smoother skin and less furrows and wrinkles.⁵

The perception of an aged appearance is influenced by many skin features, and ongoing work is being conducted to determine which features carry the most weight in contributing to age perception. Uneven skin coloration, for instance, is significantly and positively correlated with the perception aged skin and is negatively correlated with the perception of health and beauty.6-8 Changes in facial discoloration occur with age due to sun exposure and/or as a result of hormonal influences. Even skin color or tone has been shown to selectively attract attention toward the female face for longer periods of time, and this increased attention has resulted in more positive statements regarding the appearance of the face.⁹ Further work has elaborated these findings by showing the importance of skin surface topography in cueing skin age, while skin color discoloration may play a greater influence on cueing skin health.¹⁰

There are two primary types of aging that affect an individual’s appearance over time. One type can be attributed to lifestyle choices, such as smoking or outdoor activities; this is often referred to as extrinsic aging.11,12 There are also intrinsic causes of aging, which are less understood but nevertheless the subject of a great deal of research and investigation. These include genetic factors, such as a familial predisposition to diseases such as cancer and heart disease, as well as how and when hormonal changes affect the body. Intrinsic aging is analogous to an individual’s internal clock ticking away. 

Extrinsic Factors of Age

One of the most well-studied extrinsic factors of aging is sun exposure. Certainly, sun damage is a leading cause of aging, even from indoor exposure or suberythemal levels of UV radiation.¹³ Smoking is another factor that contributes toward the production of fine lines and wrinkles, especially around the mouth area.¹⁴ In Asian skin, smoking is also a major contributor to the early onset of fine lines and wrinkles,¹⁵ which typically are not expressed until much later in life, compared with Caucasian skin.¹⁶ Photoprotected skin shows a greater degree of fine lines in smokers than in nonsmokers, which increases with age.¹⁷Additionally, the percentage of subjects with severe wrinkles—i.e., greater than a grade of four in wrinkle severity, based on a photographic scale and visually assessed by a dermatologist—showed a dose-dependent effect of increased number of wrinkles with increased number of cigarette packs smoked and years of smoking.15 

Wrinkles also exhibited the same dose-dependent effect with sun exposure; the more hours an individual was exposed to the sun, the more likely they displayed a wrinkle grade of four or higher.¹⁵ Investigators also looked at individuals who both had smoked and been exposed to sun, and these individuals, unsurprisingly, showed the highest level of facial wrinkling. Thus the effect of sun exposure and smoking on skin is well-noted. Stress, marital status and depression are other factors considered to be environmental or extrinsic causes of aging.¹¹

Free Radicals and Aging

The proposed and favored mechanism by which both smoking and sun exposure are believed to cause detrimental effects in skin is through the generation of free radical species.¹⁸ For example, when UV radiation enters the skin, the absorption of the radiation by photo-unstable molecules produces free radicals when electrons are lost. This generates unstable intermediates called reactive oxygen species (ROS), e.g., superoxide, hydrogen peroxide, hydroxyl radicals and/or singlet oxygen, which can rapidly combine with other cellular compounds and cause damage to proteins, DNA and the cell membrane.¹⁹ In this case, the skin’s own antioxidant network consisting of, for example, vitamins E20 and C helps to absorb these radicals and prevent damage from occurring; however, when an insufficient number of antioxidants are available to cope with the production of free radicals, they can overload and circumvent the cellular defense mechanisms and cause damage to the skin.

While the skin does have repair mechanisms to address this situation, over ttime, these repair mechanisms may falter and become less efficient. Free ROS oxidize unsaturated fats in the cell membrane, forming lipid peroxides; the membrane can no longer adequately protect the cell since nutrients cannot penetrate the cells. In addition, cellular waste builds up and the cell cannot function efficiently. Some oxidized fats may convert to aldehydes, which flow into the cells and initiate an inflammatory process eventually causing apoptosis. Additionally, ROS can increase the level of collagenase and matrix metalloproteinases.21,22 The DNA of cells can also be directly damaged by ROS-forming thymine dimers.

The cumulative effects of these changes over time have been associated with skin aging and even with skin cancer.²³ ROS are not only implicated in facial aging but are also believed to play a major role in many significant diseases such as heart or other vascular diseases. The free radical theory of aging is supported by research; however, it is still not clear whether free radicals are the initiating trigger or are a part of the degenerative process.²⁴ 

A Mechanism for Intrinsic Aging

Even if an individual practices sun protection and makes positive lifestyle choices, he/she will still show signs of an aged appearance with time. These changes are thought to be influenced by genes and hormones. In relation to this, one group of investigators has identified an enzyme belonging to the external NADH oxidase or ENOX (ECTO-NOX) proteins whose activity was strongly correlated with whether an individual looked young or old for their age. This enzyme was named age-related NOX (arNOX), based on the relationship of increasing enzyme activity with increasing age (see Figure 1).²⁵

Interestingly, unlike other members of the ENOX enzyme family that carry out four electron transfers to molecular oxygen to form water, the arNOX enzyme uniquely generates superoxide at the cell surface.²⁵ It can be speculated that these free radicals could cause considerable damage to cells if they are not absorbed by the antioxidant defense system, leading to the accumulation of free radicals on the cell surface and potentially accelerating aging-related changes. 

It was therefore important to investigate whether this mechanism existed in skin cells and if so, whether the generated free radicals were capable of causing skin damage.

Primary keratinocytes and fibroblasts from human donors of various ages were isolated from either breast or facial surgical tissue and cultured in vitro before being assessed for the presence of arNOX activity. Neonatal cells were also obtained and analyzed. The results indicated that arNOX was active in skin cells and that its activity increased with age (see Table 1). This data was further confirmed in samples of dermis and epidermis obtained from skin punch biopsies. The activity of arNOX was detected in skin from donors beginning at about age 30, with maximum activity found in skin from donors ages 55–65 (see Figure 2).²⁶

In a further study, five dermatologists were asked to judge a subject’s age based on his or her facial skin appearance.⁸ The graders did not know the inclusion/exclusion criteria for the study and were not informed of the age range specified by the protocol. Estimates of age were averaged and compared to the subjects’ actual age. Samples were also obtained from the subjects and measured for arNOX activity. The data collected indicated that arNOX enzyme activity correlated with an aged appearance. For example, in subjects perceived by dermatologists to have a higher age than their actual biological age, the arNOX activity levels were higher, and conversely, when arNOX activity was lower, individuals were scored to look younger than their actual biological age. According to these findings, a correlation could be established between arNOX activity levels and errors in estimating participants’ chronological skin ages as estimated by professional graders. In other words, the apparent visible ages of those with higher arNOX activity levels were estimated to look an average of seven years older than their chronological age, while the appearance of those individuals with lower arNOX activity levels was estimated to look an average of seven years younger than their chronological age. This suggests that low activity levels of arNOX may help to contribute to skin that looks visibly younger as humans chronologically age.

In aging skin, there are at least two generally accepted sources of ROS production in cells; the first is UV-induced direct photon generation of ROS while the second is the subsequent inflammatory reactions that liberate free radicals. The end result is the alteration of cells through oxidation. In addition, the presented data indicates the existence of another internal source of free radicals, whose activity increases with age. Taken together, the body’s oxidative defenses may become overwhelmed if adequate antioxidants are not present. 

To date, age has been the most predominant influence of arNOX enzyme activity identified. Extrinsic factors such as smoking and/or sun exposure were also examined to detect whether they influence arNOX enzyme activity. A comparison of arNOX activity in sun-exposed and sun-protected skin sites showed that activity of the enzyme was less influenced by sun exposure and more influenced by age (see Figure 3). Additionally, smoking frequency did not have an apparent effect on the enzyme’s activity.

Conclusion

The present article describes factors that influence an aged appearance. Extrinsic factors include sun exposure and smoking, which have been well-established in the development of fine lines and wrinkles. Intrinsic factors and the interplay between these remain an area of investigation. The newly identified arNOX enzyme appears to be related to age and appearance, and may be implicated in the internal aging process via the production of superoxide free radicals. This work demonstrates that arNOX activity is found in both the epidermis and dermis, and draws a correlation between the enzyme’s activity and an aged appearance. These insights may provide cosmetic formulators with unique and previously unexplored routes to design antiaging products for improved skin appearance.

 

References

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Table 1. Measurement of arNOX activity in skin cells

 Table 1. Measurement of arNOX activity in skin cells 

Figure 1: The increase of arNOX with age

 Figure 1: The increase of arNOX with age

Figure 2: Measurement of arNOX activity

 Figure 2: Measurement of arNOX activity 

Figure 3: Average arNOX activity

 Figure 3: Average arNOX activity8

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