Edward Pelle

An in vitro Model to Test Relative Antioxidant Potential: Ultraviolet-Induced Lipid Peroxidation in Liposomes'

Since antioxidants have been shown to play a major role in preventing some of the effects of aging and photoaging in skin, it is important to study this phenomenon in a controlled manner. This was accomplished by developing a simple and reliable in vitro technique to assay antioxidant efficacy. Inhibition of peroxidation by antioxidants was used as a measure of relative antioxidant potential. Liposomes, high in polyunsaturated fatty acids (PUFA), were dispersed in buffer and irradiated with ultraviolet (UV) light. Irradiated liposomes exhibited a significantly higher amount of hydroperoxides than liposomes containing antioxidants in a dose- and concentration-dependent manner. Lipid peroxidation was determined spectrophotometrically by an increase in thiobarbituric acid reacting substances. To further substantiate the production of lipid peroxides, gas chromatography was used to measure a decrease in PUFA substrate. In order of decreasing antioxidant effectiveness, the following results were found among lipophilic antioxidants: BHA greater than catechin greater than BHT greater than alpha-tocopherol greater than chlorogenic acid. Among hydrophilic antioxidants, ascorbic acid and dithiothreitol were effective while glutathione was ineffective. In addition, ascorbic acid was observed to act synergistically with alpha-tocopherol, which is in agreement with other published reports on the interaction of these two antioxidants. Although peroxyl radical scavengers seem to be at a selective advantage in this liposomal/UV system, these results demonstrate the validity of this technique as an assay for measuring an antioxidants potential to inhibit UV-induced peroxidation.

Aryl Hydrocarbon Receptor Is an Ozone Sensor in Human Skin

Ozone, one of the main components of photochemical smog, represents an important source of environmental oxidative stress to which the skin is exposed, especially during smoggy and ozone-alert days. However, very little is known about the effects of ozone exposure on human skin. Here, we used normal human epidermal keratinocytes (NHEKs) to determine the effects of attainable levels of ozone exposure on the family of cytochrome P450 (CYP) isoforms, which plays a determinant role in the biotransformation of many environmental pollutants. NHEK exposure to ozone (0.3 ppm) resulted in an increase in protein and messenger RNA (mRNA) expression of CYP1A1, CYP1A2, and CYP1B1. NHEK exposure to ozone also resulted in nuclear translocation of the aryl hydrocarbon receptor (AhR) and in phosphorylation of epidermal growth factor receptor (EGFR). The effect of ozone on events downstream of EGFR was an increased activation of phosphoinositide 3-kinase and phosphorylation of protein kinase B and mitogen-activated protein kinases. We found that AhR silencing by small interfering RNA abolished the capacity of these cells to increase the protein and mRNA expression of CYPs on ozone exposure. Thus, AhR signaling is an integral part of the induction of CYPs by ozone. These studies strongly suggest that there are toxicological consequences of ozone to human skin.

Protection against endogenous and UVB‐induced oxidative damage in stratum corneum lipids by an antioxidant‐containing cosmetic formulation

A 16‐week human clinical study was carried out to determine the ability of antioxidants in a cosmetic vehicle to inhibit the induction of lipid peroxidation in stratum corneum lipids. The study consisted of a twice daily application of material for 12 weeks followed by a 4‐week regression phase. Stratum corneum lipids were collected and then exposed to 500 mJ/cm2 of ultraviolet B (UVB) radiation in order to avoid excessive erythemal damage to the subjects. Lipid peroxides were assayed by a methylene blue derivative assay and expressed per unit area of skin. During the treatment period, decreases in the level of lipid peroxides were observed on the sites treated with the compositions containing antioxidants, as compared to the untreated sites, and expressed as percent differences. Decreases were observed in endogenous as well as UV‐induced lipid peroxides followed by a return to baseline levels. These results demonstrate that antioxidants in a topical cosmetic formulation were effective in protecting human stratum corneum lipids against endogenous oxidation or if challenged by 500 mJ/cm2 UVB.

Identification and quantitation of ascorbic acid in extracts of human lymphocytes by high-performance liquid chromatography☆

Abstract Sixty percent methanol is widely used for the extraction of nucleotides from lymphocytes for quantitation by high-performance liquid chromatography. In the course of such studies, we noted that these extracts analyzed on an anion-exchange column showed a major “unknown” uv-absorbing peak which eluted after the nucleosides and before the nucleotides. The material cochromatographed with and had the spectral properties of ascorbic acid. This compound was identified as ascorbic acid by chemical and enzymatic assays. The ascorbate content of human lymphocytes determined by high-performance liquid chromatography, 42.2 ± 3.3 nmol 10 8 cells (mean ± SEM), agreed closely with the levels obtained by standard less sensitive methodology. Evidence is presented that this technique can be used to determine the ascorbate content of lymphocytes where only scanty material or very low levels are found.

Deferoxamine synergistically enhances iron-mediated AP-1 activation: a showcase of the interplay between extracellular-signal-regulated kinase and tyrosine phosphatase.

Deferoxamine (DFO) is a drug widely used for iron overload treatment to reduce body iron burden. In the present study, it was shown in mouse epidermal JB6 cells that all iron compounds transiently induced extracellular signal-regulated kinases (ERK) phosphorylation, whereas DFO further enhanced ERK phosphorylation over long periods. The ERK phosphorylation by DFO treatment appears to be due to the inhibition of MAPK phosphatases (MKP) by DFO. The combined effects of iron-initiated MAPK activation and DFO-mediated MKP inhibition resulted in a synergistic enhancement on AP-1 activities. The results indicate that the interplay between MAPK and MKP is important in regulating the extent of AP-1 activation. It is known that administration of DFO in iron overload patients often results in allergic responses at the injection sites. The results suggest that this synergistic AP-1 activation might play a role in DFO-induced skin immune responses of iron overload patients.

Biological Rhythms in the Skin

Circadian rhythms, ≈24 h oscillations in behavior and physiology, are reflected in all cells of the body and function to optimize cellular functions and meet environmental challenges associated with the solar day. This multi-oscillatory network is entrained by the master pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which directs an organism’s rhythmic expression of physiological functions and behavior via a hierarchical system. This system has been highly conserved throughout evolution and uses transcriptional–translational autoregulatory loops. This master clock, following environmental cues, regulates an organism’s sleep pattern, body temperature, cardiac activity and blood pressure, hormone secretion, oxygen consumption and metabolic rate. Mammalian peripheral clocks and clock gene expression have recently been discovered and are present in all nucleated cells in our body. Like other essential organ of the body, the skin also has cycles that are informed by this master regulator. In addition, skin cells have peripheral clocks that can function autonomously. First described in 2000 for skin, this review summarizes some important aspects of a rapidly growing body of research in circadian and ultradian (an oscillation that repeats multiple times during a 24 h period) cutaneous rhythms, including clock mechanisms, functional manifestations, and stimuli that entrain or disrupt normal cycling. Some specific relationships between disrupted clock signaling and consequences to skin health are discussed in more depth in the other invited articles in this IJMS issue on Sleep, Circadian Rhythm and Skin.

Effects of ozone in normal human epidermal keratinocytes

Ozone is a tropospheric pollutant that can form at ground level as a result of an interaction between sunlight and hydrocarbon engine emissions. As ozone is an extremely oxidative reaction product, epidermal cells are in the outer layer of defense against ozone. We exposed normal human epidermal keratinocytes (NHEK) to concentrations of ozone that have been measured in cities and assayed for its effects. Hydrogen peroxide and IL‐1α levels both increased while ATP levels decreased. We found a decrease in the NAD‐dependent histone deacetylase, sirtuin 3. Lastly, we found that ozone increased DNA damage as evaluated by Comet assay. Taken together, our results show increased damage to NHEK that will ultimately impair normal cellular function as a result of an environmentally relevant ozone exposure.

Sirtuin 4 identification in normal human epidermal keratinocytes and its relation to sirtuin 3 and energy metabolism under normal conditions and UVB‐induced stress

Abstract:  Sirtuins (SIRT) are NAD+‐dependent deacetylases and ADP‐ribosyltransferases that play a critical role in metabolism and epigenetics. SIRT3 and SIRT4 are of particular interest because they are localized in the mitochondria where energy is generated and their expression is inversely proportional to each other. Here, we report data, for the first time, demonstrating the presence of SIRT4 in normal human epidermal keratinocytes (NHEK) and confirm that its expression is inversely related to SIRT3 in these cells and that they follow a temporal cycle. Further, UVB radiation modified their expression, as well as ATP and H2O2 levels. These deviations from the normal sirtuin cycles after UVB exposure can be an epigenetic indicator of lower metabolism levels.

Iron sensitizes keratinocytes and fibroblasts to UVA-mediated matrix metalloproteinase-1 through TNF-α and ERK activation

Abstract:  Oestrogen deficiency is regarded as the main causative factor in postmenopausal skin ageing and photoageing. While women after menopause experience low levels of oestrogen because of cease of ovarian function, they are also exposed to high levels of iron as a result of cessation of menstruation. In this study, we investigated whether this increase in iron presents a risk to the postmenopausal skin. Because of the lack of appropriate animal models to closely mimic the low oestrogen and high iron conditions, we tested the hypothesis in a high iron and low oestrogen culture model. Here, we showed that primary human dermal fibroblasts exposed to iron did not affect the baseline levels of matrix metalloproteinase‐1 (MMP‐1) activity. However, the iron‐exposed fibroblasts were sensitized to UVA exposure, which resulted in a synergistic increase in MMP‐1. UVA activated the three members of MAPK family: ERKs, p38, and JNKs. Additional activation of ERKs by iron contributed to the synergistic increases. Primary normal human epidermal keratinocytes (NHEK) did not respond to iron or UVA exposure as measured by MMP‐1, but produced tumor necrosis factor‐alpha (TNF‐α) in the media, which then stimulated MMP‐1 in fibroblasts. Our results indicate that iron and UVA increase MMP‐1 activity in dermal fibroblasts not only directly through ERK activation but also by an indirect paracrine loop through TNF‐α released by NHEK. We conclude that in addition to oestrogen deficiency, increased iron as a result of menopause could be a novel risk factor by sensitizing postmenopausal skin to solar irradiation.

Protection against ultraviolet A-induced oxidative damage in normal human epidermal keratinocytes under post-menopausal conditions by an ultraviolet A-activated caged-iron chelator: a pilot study

Background/purpose: Human skin is constantly exposed to ultraviolet A (UVA), which can generate reactive oxygen species and cause iron release from ferritin, leading to oxidative damage in biomolecules. This is particularly true in post‐menopausal skin due to an increase in iron as a result of menopause. As iron is generally released through desquamation, the skin becomes a main portal for the release of excess iron in this age group. In the present study, we examined a strategy for controlling UVA‐ and iron‐induced oxidative stress in skin using a keratinocyte post‐menopausal cellular model system.