A B S T R A C T Background: Hydroqu

A B S T R A C T
Background: Hydroquinone (HQ) with or without retinoic acid (RA) is routinely used for the treatment of hyperpigmented conditions. Skin irritation is a major problem with popular depigmenting agents, resulting in postinflammatory hyperpigmentation. Objective: To examine the molecular mechanism associated with skin irritation by RA or HQ. Methods: A genome-wide transcriptional profiling analysis was performed using monolayer cultures of human keratinocytes treated with or without irritant doses of RA, HQ, or sodium lauryl sulfate (SLS), a representative irritant. Differentially expressed genes (DEGs) were mapped on human chromosomes using a Manhattan plot. For the validation of candidate DEGs, the chemicals with different concentrations of varying irritation intensities were applied in vitro and in vivo and analyzed using real time-PCR and Western blotting. Results: DEGs mapped to the 1q21 locus, which is composed of a cluster of genes encoding the cornified envelope precursors, showed an inverse expression pattern in response to HQ and RA. Concentrations of RA and HQ that induced a broad range of irritant responses in cultured cells or mice skin also induced inverse effects on the expression of cornified envelope-associated proteins. Conclusions: Genetic modulation on cornified envelope-associated proteins by RA-induced irritation, which may be involved in physiological skin barrier disturbance, could be inverse to that by HQ- or SLS-induced irritation. [1]
1. Introduction
Topical phenolic agents, including hydroquinone (HQ) and non-phenolic agents, including retinoids are used for hyperpig- mentation disorders. Topical HQ is the most popular treatment modality and is frequently used in combination therapies, such as a triple combination with topical all-trans retinoic acid and corticosteroids [1–4]. Although HQ is used in over-the-counter formulations for lightening skin pigmentation, it can cause skin irritation [5]. The most common adverse event associated with topical retinoic acid (RA), is also an irritant reaction of variable intensity, which presents with dryness, scaling, erythema, burning, and/or stinging [6–8]. Because skin irritation could lead to post-inflammatory hyperpigmentation, efforts have focused on the reduction of the irritant potential without loss of efficacy. Skin irritation can be induced by different mechanisms, such as skin barrier disruption, induction of a cytokine cascade, and involve- ment of the oxidative stress network [9,10]. Different classes of substances could potentially exert skin irritation through differ- ent mechanisms. A dose–response relationship is seen for adverse events, as well as effectiveness [11–13]. Irritant reaction from the same chemical shows variable intensities in vivo, if their concentrations are different. Gene expression may also differ at different intensities of irritation. Although concentrations that inhibit cell viability by 50% have been used as the threshold concentration of irritation on human skin [14], the exact proportion of cell survival or cytotoxicity that is indicative of irritation in vivo is not defined. Therefore, validation using sequentially increasing concentrations [1]
including concentration of 50% cell survival may be helpful in the identification of molecular
mechanism of irritation. Skin irritation is one of the most common adverse reactions to chemicals. Every substance is an irritant to some degree. To reduce irritant reactions, the molecular mechanisms require to be identified. This study was performed to elucidate the molecular mechanisms associated with skin irritation by RA and HQ. Toward this aim, a genome-wide transcriptional profiling analysis and a Manhattan plot were performed after treatment of cultured normal human keratinocytes, a main constituent of skin, with subcytotoxic doses of HQ or RA. The data were validated by in vitro mRNA studies using increasing concentrations of each chemical (subcytotoxic, 50%, and 20–30% cell survival). The expression of corresponding proteins was examined in vivo by using increasing concentrations which induced mild erythema, definite erythema, and severe erythema with focal erosion, to corroborate the in vitro result. [1]
2. Discussion
Global and local graphing of the test results is a necessary step for interpreting genome-wide association studies. A widely used graphical viewing method is the Manhattan plot, which helps to quickly identify genome locations in a chromosome [23]. There-fore, a genome-wide transcriptional profiling analysis and Manhattan plotting were used to examine the mechanism of irritation of RA and HQ, which suggested that chromosome 1q21 was one of the loci associated with DEGs (Fig. 1). The expression pattern of DEG cluster in chromosome 1q21 in response to RA treatment was generally inverse to that by HQ and SLS (Fig. 1). In addition, the validation to evaluate expression of genes encoding structural proteins in chromosome 1q21 using subcytotoxic doses of RA, HQ, and SLS control, which were used for microarray analysis to minimize changes in gene expression due to cell death [24], identified the transcriptional profile results (Fig. 2 and Table S1A and B). Loricrin expression, although reportedly too low to be detectable in monolayered cultures [25], was examined at the mRNA level. RA decreased Loricrin mRNA, while HQ or SLS treatment caused an increase (Fig. 2). In chromosomal region 1q21, there is a cluster of genes encoding the cornified envelope precursors, such as loricrin, involucrin and small proline-rich proteins (SPRRs); intermediate filament-associated proteins, such as profilaggrin and trichohyalin; and several S100A calcium-binding proteins [18–21]. The cornified envelope con- stitutes the principal barrier to percutaneous penetration of exogenous substances. Therefore, these results suggested that effect of RA on the modulation of structural genes of the physiological skin barrier may be different from that of HQ or SLS (Fig. 2). Irritant potential in vitro is correlated with reduced cell viability and increased cytotoxicity. Because concentrations that inhibit cell viability by 50% have been used as threshold concentrations of irritation on human skin [14], subcytotoxic doses may not be sufficient to identify the irritation mechanism. Concentration-dependent effects on cornified envelope-associated proteins expression, including TG-1 showed that RA reduced the expression levels of filaggrin, SPRR1A/1B, and TG-1 at the concentrations producing

النتائج (العربية) 1:

A B S T R A C T Background: Hydroquinone (HQ) with or without retinoic acid (RA) is routinely used for the treatment of hyperpigmented conditions. Skin irritation is a major problem with popular depigmenting agents, resulting in postinflammatory hyperpigmentation. Objective: To examine the molecular mechanism associated with skin irritation by RA or HQ. Methods: A genome-wide transcriptional profiling analysis was performed using monolayer cultures of human keratinocytes treated with or without irritant doses of RA, HQ, or sodium lauryl sulfate (SLS), a representative irritant. Differentially expressed genes (DEGs) were mapped on human chromosomes using a Manhattan plot. For the validation of candidate DEGs, the chemicals with different concentrations of varying irritation intensities were applied in vitro and in vivo and analyzed using real time-PCR and Western blotting. Results: DEGs mapped to the 1q21 locus, which is composed of a cluster of genes encoding the cornified envelope precursors, showed an inverse expression pattern in response to HQ and RA. Concentrations of RA and HQ that induced a broad range of irritant responses in cultured cells or mice skin also induced inverse effects on the expression of cornified envelope-associated proteins. Conclusions: Genetic modulation on cornified envelope-associated proteins by RA-induced irritation, which may be involved in physiological skin barrier disturbance, could be inverse to that by HQ- or SLS-induced irritation. [1]1. Introduction Topical phenolic agents, including hydroquinone (HQ) and non-phenolic agents, including retinoids are used for hyperpig- mentation disorders. Topical HQ is the most popular treatment modality and is frequently used in combination therapies, such as a triple combination with topical all-trans retinoic acid and corticosteroids [1–4]. Although HQ is used in over-the-counter formulations for lightening skin pigmentation, it can cause skin irritation [5]. The most common adverse event associated with topical retinoic acid (RA), is also an irritant reaction of variable intensity, which presents with dryness, scaling, erythema, burning, and/or stinging [6–8]. Because skin irritation could lead to post-inflammatory hyperpigmentation, efforts have focused on the reduction of the irritant potential without loss of efficacy. Skin irritation can be induced by different mechanisms, such as skin barrier disruption, induction of a cytokine cascade, and involve- ment of the oxidative stress network [9,10]. Different classes of substances could potentially exert skin irritation through differ- ent mechanisms. A dose–response relationship is seen for adverse events, as well as effectiveness [11–13]. Irritant reaction from the same chemical shows variable intensities in vivo, if their concentrations are different. Gene expression may also differ at different intensities of irritation. Although concentrations that inhibit cell viability by 50% have been used as the threshold concentration of irritation on human skin [14], the exact proportion of cell survival or cytotoxicity that is indicative of irritation in vivo is not defined. Therefore, validation using sequentially increasing concentrations [1]including concentration of 50% cell survival may be helpful in the identification of molecular mechanism of irritation. Skin irritation is one of the most common adverse reactions to chemicals. Every substance is an irritant to some degree. To reduce irritant reactions, the molecular mechanisms require to be identified. This study was performed to elucidate the molecular mechanisms associated with skin irritation by RA and HQ. Toward this aim, a genome-wide transcriptional profiling analysis and a Manhattan plot were performed after treatment of cultured normal human keratinocytes, a main constituent of skin, with subcytotoxic doses of HQ or RA. The data were validated by in vitro mRNA studies using increasing concentrations of each chemical (subcytotoxic, 50%, and 20–30% cell survival). The expression of corresponding proteins was examined in vivo by using increasing concentrations which induced mild erythema, definite erythema, and severe erythema with focal erosion, to corroborate the in vitro result. [1]2. Discussion Global and local graphing of the test results is a necessary step for interpreting genome-wide association studies. A widely used graphical viewing method is the Manhattan plot, which helps to quickly identify genome locations in a chromosome [23]. There-fore, a genome-wide transcriptional profiling analysis and Manhattan plotting were used to examine the mechanism of irritation of RA and HQ, which suggested that chromosome 1q21 was one of the loci associated with DEGs (Fig. 1). The expression pattern of DEG cluster in chromosome 1q21 in response to RA treatment was generally inverse to that by HQ and SLS (Fig. 1). In addition, the validation to evaluate expression of genes encoding structural proteins in chromosome 1q21 using subcytotoxic doses of RA, HQ, and SLS control, which were used for microarray analysis to minimize changes in gene expression due to cell death [24], identified the transcriptional profile results (Fig. 2 and Table S1A and B). Loricrin expression, although reportedly too low to be detectable in monolayered cultures [25], was examined at the mRNA level. RA decreased Loricrin mRNA, while HQ or SLS treatment caused an increase (Fig. 2). In chromosomal region 1q21, there is a cluster of genes encoding the cornified envelope precursors, such as loricrin, involucrin and small proline-rich proteins (SPRRs); intermediate filament-associated proteins, such as profilaggrin and trichohyalin; and several S100A calcium-binding proteins [18–21]. The cornified envelope con- stitutes the principal barrier to percutaneous penetration of exogenous substances. Therefore, these results suggested that effect of RA on the modulation of structural genes of the physiological skin barrier may be different from that of HQ or SLS (Fig. 2). Irritant potential in vitro is correlated with reduced cell viability and increased cytotoxicity. Because concentrations that inhibit cell viability by 50% have been used as threshold concentrations of irritation on human skin [14], subcytotoxic doses may not be sufficient to identify the irritation mechanism. Concentration-dependent effects on cornified envelope-associated proteins expression, including TG-1 showed that RA reduced the expression levels of filaggrin, SPRR1A/1B, and TG-1 at the concentrations producing <50% cell survival, as well as those producing 80% cell survival (Fig. 3A). On the other hand, HQ and SLS did not reduce expression of these genes at the concentrations producing 50% cell survival. Decrease in expression of these genes was not examined even at the concentration that induced 20% or 30% of cell survival (Fig. 3B and C). The results from in vitro studies with RA and HQ were also similar to the results from in vivo studies. The concentrations that produced mild erythema, definite erythema and severe erythema with focal erosion in vivo corresponded to concentrations producing 70–80%, 50% and 20–30% cell survival in vitro (Figs. 3A and B, 4A and B). Different concentrations of chemicals could induce irritant reaction of varying intensities. Adverse effects, including irritation from chemicals manifest a dose–response relationship [11–13]. Nonetheless, similar changes in cornified envelop-associated proteins expression by same chemical even in different concentrations suggested that mecha-nism of irritation by HQ and SLS could differ from that by RA. The effects of RA are mediated by the nuclear RA receptors (RARs) and retinoid X receptors (RXRs). As observed in our microarray data which showed more than 2-fold increase of RAR-a, expression of RAR-a was increased concentration-dependently by only RA treatment in vitro and in vivo (Fig. S1A and B, respectively), suggesting a potential role of RAR-a in the RA-induced skin irritation. [1]