Advancements in non-invasive skin sampling: Clinical conditions characterization via the assessment of skin surface cytokine biomarkers.

The skin is increasingly recognized as a biological active organ interacting with the immune system. Given that the epidermal skin layer actively releases various cytokines, non-invasive skin sampling methods could detect these cytokines, offering insights into clinical conditions. This study aims non-invasively measuring cytokine levels directly from the skin surface to characterize different inflammatory chronic disorders in the adult and elderly population: psoriasis, diabetes type 2, rosacea, chronic kidney disease (CKD) and aging. Cytokines IL-1ß, IL-8 and IL-10 were sampled from healthy subjects and patients aged 18-80 using skin surface wash technique. A well with sterile phosphate-buffered saline solution was placed on the skin for 30 min, and the extracted solution was collected from the well for further cytokine levels analysis using ELISA assay. Results show distinct cytokine profiles in different pathological processes, healthy controls, affected and unaffected areas. Aging was associated with increased IL-1ß, IL-8, and IL-10 levels in skin. In diabetes, IL-1ß and IL-8 levels were elevated in lesional areas, while IL-10 levels were decreased in non-lesional skin. Psoriatic lesions showed elevated levels of IL-1ß and IL-8. Rosacea patients had lower IL-10 levels in both lesional and non-lesional areas. CKD patients exhibited significantly lower IL-10 levels compared to healthy individuals. In conclusion, skin surface wash-derived cytokine profiles could serve as "alert biomarkers" for disease prediction, enabling early detection. Additionally, this method's cost-effectiveness allows pre-screening of molecules in clinical studies and holds potential as a tool for biomarkers and omics analysis, enhancing disorder characterization and disease management.

Saturated and aromatic aldehydes originating from skin and cutaneous bacteria activate the Nrf2-keap1 pathway in human keratinocytes.

Skin homeostasis is constantly challenged by environmental factors, affecting its delicate redox balance. The skin is also home to a wide variety of bacterial species, including Staphylococci. The cutaneous redox state is governed by the Nrf2-keap1 pathway, which is responsible for the induction of phase II cytoprotective enzymes, thus sustaining a healthy oxidative state. As part of normal metabolism, both bacteria and cutaneous tissue emit copious amounts of volatile organic compounds (VOCs), one subgroup of which are aldehydes. α,ß-unsaturated aldehydes are known activators of Nrf2-keap1 pathway by direct oxidation of the keap1 protein. However, we did not encounter reports of Nrf2 activation by saturated or aromatic aldehydes, neither bacteria nor skin-derived. We hypothesized that non-α,ß-unsaturated aldehydes derived from skin or cutaneous bacteria may act as Nrf2-keap1 pathway activators and therefore afford protection against environmental insults. The saturated aldehydes nonanal and decanal (known skin metabolites) and the aromatic aldehyde benzaldehyde (known skin and Staphylococcus epidermidis metabolite) were shown to induce the Nrf2-keap1 pathway in human keratinocytes. We also identified a newly described aromatic aldehyde, 3-furaldehyde (3-FA), emitted from S. aureus and S. epidermidis cultures, which also activated the pathway. Moreover, Nrf2-keap1 induction led to a significant protection against UVB-induced apoptosis. The mechanism involved in this activation has been partially elucidated. This work emphasizes the importance of cutaneous bacteria, as well as healthy skin lipid peroxidation processes in the maintenance and regulation of the cellular antioxidant response, namely with regard to coping with environmental stressors.

Do low molecular weight antioxidants contribute to the Protection against oxidative damage? The interrelation between oxidative stress and low molecular weight antioxidants based on data from the MARK-AGE study.

A redox steady state is important in maintaining vital cellular functions and is therefore homeostatically controlled by a number of antioxidative agents, the most important of which are enzymes. Oxidative Stress (OS) is associated with (or/and caused by) excessive production of damaging reactive oxygen and/or nitrogen species (ROS, RNS), which play a role in many pathologies. Because OS is a risk factor for many diseases, much effort (and money) is devoted to early diagnosis and treatment of OS. The desired benefit of the "identify (OS) and treat (by low molecular weight antioxidants, LMWA)" approach is to enable selective treatment of patients under OS. The present work aims at gaining understanding of the benefit of the antioxidants based on interrelationship between the concentration of different OS biomarkers and LMWA. Both the concentrations of a variety of biomarkers and of LMWA were previously determined and some analyses have been published by the MARK-AGE team. For the sake of simplicity, we assume that the concentration of an OS biomarker is a linear function of the concentration of a LMWA (if the association is due to causal relationship). A negative slope of this dependence (and sign of the correlation coefficient) can be intuitively expected for an antioxidant, a positive slope indicates that the LMWA is pro-oxidative, whereas extrapolation of the OS biomarker to [LMWA] = 0 is an approximation of the concentration of the OS biomarker in the absence of the LMWA. Using this strategy, we studied the effects of 12 LMWA (including tocopherols, carotenoids and ascorbic acid) on the OS status, as observed with 8 biomarkers of oxidative damage (including malondialdehyde, protein carbonyls, 3-nitrotyrosine). The results of this communication show that in a cross-sectional study the LMWA contribute little to the redox state and that different "antioxidants" are very different, so that single LMWA treatment of OS is not scientifically justified assuming our simple model. In view of the difficulty of quantitating the OS and the very different effects of various LMWA, the use of the "identify and treat" approach is questionable.

Nuclear histones: major virulence factors or just additional early sepsis markers? A comment.

In 2009, Xu et al. and Chaput et al. in Nature Medicine had argued that the main cause of death in sepsis is the release from neutrophil nets of nuclear histone, highly toxic to endothelial cells and that these polycations are major and unique virulence factors. Since 2009, numerous researchers have also suggested the involvement of histones in the pathophysiology of many clinical disorders. If histones are indeed major unique virulence toxic agents, then heparin, activated protein C and antibodies to histone should prove excellent antisepsis agents. However, this is provided that these agents are administered to patients early enough before the activation of the cytokine storms, immune responses and the coagulation cascades are irreversibly unleashed. This may not be practical, since a diagnosis of sepsis is usually made much later. Future identifications of novel early markers are therefore needed and a compilation of cocktails of antagonists may replace the faulty single antagonists tried for many years, but in vain, to prevent death in sepsis.

Non-invasive evaluation of skin cytokines secretion: an innovative complementary method for monitoring skin disorders.

In this review, a novel non-invasive approach based on skin surface wash sampling is described. Since the epidermis possesses a high metabolic activity, the secretion of various biomarkers can be exploited to develop non-invasive procedures for skin measurement to monitor disorders and to define a therapeutic strategy. Thus, we developed a method for the quantification of skin surface compounds. In this procedure, a well is placed on skin surface and is attached using an adhesive pad. Extraction buffer is introduced into the well for 30 min incubation period and the secretion of different biomarkers on skin surface can be measured: cytokines, antioxidants, peptides, RNA, DNA volatile organic compounds etc. Here, the focus is on cytokine measurement. After collecting skin samples cytokines can be quantified using ELISA assay. Since so far cytokine levels in skin have been evaluated mostly by invasive and prolonged procedures (punch biopsy, blister fluid and scrapping), employing this method has important implications, because it allows assessing cytokine amount with minimal invasion and high accuracy. We have already applied skin surface wash sampling for cytokine quantification in different clinical conditions: psoriasis, atopic dermatitis and chronic renal failure. A distinct pattern of cytokine secretion has been demonstrated for each disorder. Differences were also observed between lesional and non-lesional areas. The obtained results shed a new light on cutaneous cytokine expression in different clinical conditions. Moreover, the interplay between cytokines and other soluble compounds can give an added value in understanding the mechanism of skin pathologies.

Cutaneous Nrf2-Keap1 pathway modulation by environmental factors: The Dead Sea area as a test case.

The skin is constantly exposed to exogenous environmental stressors and has to cope with excessive oxidative stress and tissue damage. However, exposure to moderate environmental stressors may be beneficial for the cutaneous tissue and assist in protecting against oxidative damage via the enhanced activation of the nuclear factor erythroid 2-related factor 2-Kelch-like ECH-associated protein 1 (Nrf2-Keap1) pathway. Such moderate stressors can be found in various locations around the globe. In this manuscript, we chose to focus on the Dead Sea (DS) area as a test case to study the effect of moderate stressors on the cutaneous tissue because of the unique combinations of moderate stressors in this area. The exceptional location of the DS at an altitude of -438 meters below sea level (the lowest place on earth) is responsible for its rare accumulation of moderate stressors such as high-water salinity, high atmospheric pressure, and unique solar radiation. In this manuscript, we hypothesized that the unique solar radiation in the DS area generates moderate oxidative stress in the skin leading to the induction of intracellular electrophiles, which in turn can activate the protecting Nrf2-Keap1 pathway. We showed that exposure of human skin organ culture from the same donor to solar radiation at the DS resulted in significant activation of the Nrf2-Keap1 pathway, induction of phase II enzymes, and lower apoptotic activity compared to a nearby location at a higher altitude (Jerusalem +700 m). This remarkable effect of activating the Nrf2 protecting pathway and the importance and characteristics of the solar irradiation at the DS is discussed.

Skin organ culture as a model to study oxidative stress, inflammation and structural alterations associated with UVB-induced photodamage.

BACKGROUND: Ultraviolet (UV) irradiation is a major cause of skin damage, of long-term alteration of skin metabolism, homoeostasis and physical structure. The analysis of UV-induced pathogenic processes requires in vitro models allowing biochemical studies, and appropriate for the development of novel, accurate diagnosis methods based on non-invasive procedures. OBJECTIVES: This work was aimed to reproduce the effects of UVB on whole-skin explants ex vivo and to study underlying biochemical mechanisms, especially in correlation with skin autofluorescence. METHODS: Human skin organ cultures were irradiated with UVB and subjected to enzyme assays, Western blots, solid-phase ELISA, HPLC and fluorescence measurements. RESULTS: UVB irradiation was found to enhance ROS production, to deplete the pool of low-molecular-weight antioxidants and to decrease the overall antioxidant capacity in the epidermis, in a manner dependent on xanthine-oxidase activity. Epidermal cell proliferation and mitochondrial activity were transiently stimulated. IκB-α was degraded, and the secretion of inflammatory cytokines was drastically increased. Inducible nitric oxide synthase activity was increased in non-irradiated controls, probably due to the mechanical stress of skin excision, and this phenomenon was suppressed by UVB. Autofluorescence measurements revealed alterations of dermal protein crosslinks following UVB irradiation. CONCLUSIONS: Skin organ culture proved to be an integrated model appropriate for in vitro analysis of UVB biologic effects and their correlations, and for the study of non-invasive diagnostic methods in cellular and molecular terms.

Microbial and host cells acquire enhanced oxidant-scavenging abilities by binding polyphenols.

The dilemma whether supplementations of dietary antioxidants might prevent the adverse consequences of oxidative stress, the inadequacy of the analytical methods employed to quantify oxidant scavenging ability (OSA) levels in whole blood and the distribution and fate of polyphenols and their metabolites in various body compartments following oral consumption are discussed. While none-metabolized polyphenols might exert their antioxidant effects mainly in the oral cavity, metabolized polyphenols might be beneficial in the gastrointestinal tract to counteract the toxicity of oxidants and also of the sequelae of inflammatory processes. Although only micromolar amounts of polyphenols and their metabolites eventually reach the blood circulation, these may nevertheless still be highly effective as scavengers of reactive oxygen and nitrogen species because of their ability to synergize with plasma low molecular-weight antioxidants and with albumin. Polyphenols can avidly bind to surfaces of microorganisms and of blood cells to markedly enhance their OSA, therefore the routine quantifications of antioxidant levels conducted in clinical settings should always use catalase-rich whole blood but not as customary, plasma alone. In addition to their antioxidant and metal chelating properties, polyphenols may also act as signaling agents capable of affecting metabolic, inflammatory, autoimmune, carcinogenic and aging processes.

eDNA-Mediated Cutaneous Protection Against UVB Damage Conferred by Staphylococcal Epidermal Colonization.

The human skin is a lush microbial habitat which is occupied by a wide array of microorganisms. Among the most common inhabitants are Staphylococcus spp., namely Staphylococcus epidermidis and, in ≈20% of healthy individuals, Staphylococcus aureus. Both bacteria have been associated with cutaneous maladies, where they mostly arrange in a biofilm, thus achieving improved surface adhesion and stability. Moreover, our skin is constantly exposed to numerous oxidative environmental stressors, such as UV-irradiation. Thus, skin cells are equipped with an important antioxidant defense mechanism, the Nrf2-Keap1 pathway. In this work, we aimed to explore the morphology of S. aureus and S. epidermidis as they adhered to healthy human skin and characterize their matrix composition. Furthermore, we hypothesized that the localization of both types of bacteria on a healthy skin surface may provide protective effects against oxidative stressors, such as UV-irradiation. Our results indicate for the first time that S. aureus and S. epidermidis assume a biofilm-like morphology as they adhere to ex vivo healthy human skin and that the cultures' extracellular matrix (ECM) is composed of extracellular polysaccharides (EPS) and extracellular DNA (eDNA). Both bacterial cultures, as well as isolated S. aureus biofilm eDNA, conferred cutaneous protection against UVB-induced apoptosis. This work emphasized the importance of skin microbiota representatives in the maintenance of a healthy cutaneous redox balance by activating the skin's natural defense mechanism.

The Cutaneous Physiological Redox: Essential to Maintain but Difficult to Define.

Skin is a unique tissue, possessing extremely efficient protective and regulative mechanisms, similar only to the gut and lungs. These tissues serve as an interface with the environment and are exposed to stressors from both endogenous and exogenous sources. Interestingly, all these stressors lead downstream to a cellular production of reactive oxygen species (ROS) and other electrophiles, which, in turn could have deleterious outcomes for the living organism. Hence, such tissues should always maintain a "high-alert" condition in order to cope with these various insults. Nevertheless, a moderate production of ROS induced by stressors could actually be beneficial, although it is impossible to predict if and which exposure would lead to which outcome. Consequently, a parameter which would indicate the skin's readiness to cope with continuously fluctuating conditions is required. It has been proposed that the redox status may serve as a suitable indicator. In this opinion manuscript, we argue that the redox status is a vague parameter that is difficult to characterized and quantify due to its extremely dynamic nature. The common convention that the redox status is composed solely of the balance between oxidants and reductants (ROS and antioxidants) is also thought-provoking. Since this parameter in vivo behaves in a dynamic and complex manner, it better fits the description of a process, rather than an individual parameter. We suggest that the homeostatic modulation of the physiological redox (PR) should be in focus, rather than the redox status parameter itself. It is further suggested that low molecular weight antioxidants (LMWA) are, in fact, rather insignificant concerning the PR maintenance, and that the major contributors to this delicate modulation are regulative, protein-based systems such as the protective phase II antioxidant enzymes. Moreover, we show that skin microbiome and cutaneous advanced lipid peroxidation end-products (ALEs) take part in sustaining the cutaneous PR homoeostasis via activation of the Nrf2-Keap1 protective pathway.

Protective effects of a cream containing Dead Sea minerals against UVB-induced stress in human skin.

BACKGROUND: Dead Sea (DS) mud and water are known for their unique composition of minerals, and for their therapeutic properties on psoriasis and other inflammatory skin diseases. Their mode of action, however, remains poorly known. OBJECTIVES: To analyse the ability of Dermud, a leave-on skin preparation containing DS mud and other ingredients like DS water, zinc oxide, aloe-vera extract, pro-vitamin B5 and vitamin E, to antagonize biological effects induced by UVB irradiation in skin when topically applied in organ cultures. METHODS: We have used human skin organ cultures as a model to assess the biological effects of UVB irradiation and of Dermud cream topical application. Skin pieces were analysed for mitochondrial activity by MTT assay, for apoptosis by caspase 3 assay, for cytokine secretion by solid phase ELISA, for overall antioxidant capacity by ferric reducing antioxidant power and Oxygen radical absorbance capacity assays (epidermis) or by cyclic voltammetry (external medium), and for uric acid (UA) content by HPLC. RESULTS: We report that UVB irradiation decreases cell viability, total antioxidant capacity and UA contents in the epidermis of skin organ cultures, while increasing the levels of apoptosis in cells and their cytokine secretion. Topical application of Dermud decreased all these effects significantly. CONCLUSIONS: Our results clearly show that Dermud has protective, anti-oxidant and anti-inflammatory properties that can antagonize biological effects of UVB irradiation in skin. It may therefore be able to reduce skin photodamage and photoaging, and more generally to reduce oxidative stress and inflammation in skin pathologies.

A novel function of red wine polyphenols in humans: prevention of absorption of cytotoxic lipid peroxidation products.

Current evidence supports a contribution of polyphenols to the prevention of cardiovascular disease, but their mechanisms of action are not understood. We investigated the impact of red wine polyphenols on postprandial cytotoxic lipid peroxidation products (MDA) levels in humans. In a randomized, crossover study, the effect of red wine polyphenols on postprandial levels of plasma and urine MDA was investigated. Three meals of 250 g turkey cutlets supplemented by water (A); soaked in red wine after heating plus 200 ml of red wine (B); or soaked in red wine prior to heating plus 200 ml of red wine (C) were administered to 10 healthy volunteers. Subject baseline plasma levels of MDA were 50 +/- 20 nM. After a meal of turkey meat cutlets, plasma MDA levels increased by 160 nM (P<0.0001); after (B) there was a 75% reduction in the absorption of MDA (P<0.0001). However, after (C), the elevation of plasma MDA was completely prevented (P<0.0001). Similar results were obtained for MDA accumulation in urine. Our study suggests that red wine polyphenols exert a beneficial effect by the novel new function, absorption inhibition of the lipotoxin MDA. These findings explain the potentially harmful effects of oxidized fats found in foods and the important benefit of dietary polyphenols in the meal.

Bacteria coated by polyphenols acquire potent oxidant-scavenging capacities.

Several microbial species, including probiotic lactic acid bacteria, have the ability to irreversibly bind a large variety of polyphenols (flavonoids) and anthocyanidins found in many colored fruits and vegetables and to enhance their total oxidant-scavenging capacities (TOSC). The binding of flavonoids to microbial surfaces was further increased by the cationic polyelectrolytes ligands poly-L-histidine, chlorhexidine and Copaxone. This phenomenon was confirmed visually, by the FRAP, DPPH, cyclic voltammetry, Folin-Ciocalteu as well as by luminol-dependent chemiluminescence techniques employed to assay TOSC. The possibility is considered that clinically, microbial cells in the oral cavity and in the gastro intestinal tract, complexed with antioxidant polyphenols from nutrients and with cationic ligands, might increase the protection of mammalian cells against damage induced by excessive generation of reactive oxygen species during infections and inflammation.

A randomized controlled clinical trial comparing the efficacy of dead sea mineral-enriched body lotion versus two types of placebo in the treatment of cutaneous dryness, itching, peeling and tightness in hemodialysis patients (EDIT).

BACKGROUND/AIMS: The present study was designed to investigate the short-term safety and efficacy of topical application with body lotion enriched with minerals from the Dead Sea versus 2 different placebo treatments in reducing symptoms of uremic pruritus. METHODS: In this single-center, randomized, double placebo-controlled clinical trial, 78 hemodialysis patients with self-reported uremic pruritus were randomized to twice-daily topical treatment with body lotion enriched with minerals from the Dead Sea (DS) or to each of 2 types of placebo: (1) lotion with no Dead Sea minerals but otherwise identical to DS (P1) or (2) lotion with no active ingredients (P2). Symptoms of uremic pruritus (itching, dryness, peeling, tightness) were evaluated at baseline and 2 weeks (14 days) after treatment intervention using a 5-point Likert scale. RESULTS: Following treatment, significant differences in symptom severity scores between DS and P1 and, separately, between group DS and P2, were not detected. Additionally, when DS was compared to the combined placebo groups (P1 and P2 together), significant post-treatment differences in symptom severity scores were not observed. Symptoms were less severe post-treatment regardless of treatment assignment. CONCLUSIONS: DS was not superior to either of the placebo treatments in the symptomatic relief of uremic pruritus.

Supplementation with antioxidants fails to increase the total antioxidant capacity of several cell lines in culture.

Low molecular weight antioxidants (LMWA) supplements are a popular and routine approach to assist the cell and the whole organism to cope with increasing oxidative stress. Numerous experiments have been conducted in which exogenous antioxidants were supplemented to cells, animals and humans to prevent and delay pathological disorders associated with reactive oxygen species. Recently, many meta-analysis publications have demonstrated the failure of this approach and in some cases even showed an increase in the severity of the disease and all-cause mortality. The reasons for the lack of success are not fully understood and the concept of antioxidant therapy is questionable. We suggest a new explanation concerning the way antioxidants function in the living cells that can elucidate some of the conflicting data published. The aim of this study was to examine the hypothesis that the overall antioxidant capacities of cells in culture remains constant and since the cells tightly regulate this antioxidant network, supplementation with exogenous antioxidants cannot enhance the total antioxidant capacity of the cells. This assumption was examined in HaCaT, Hep3B, PC3 and Caco-2 cells using several types of antioxidant supplements. It has been shown that while the levels of the specific administrated antioxidant increased significantly intracellularly, the overall antioxidant capacity of the cells as evaluated by various methods did not increase, and in some cases, even decreased. These results support the hypothesis and demonstrate that the total antioxidant capacity of these cells in culture is kept under tight regulation and cannot be enhanced by exogenous LMWA.

Multiple adaptive mechanisms to chronic liver disease revealed at early stages of liver carcinogenesis in the Mdr2-knockout mice.

Molecular events preceding the development of hepatocellular carcinoma were studied in the Mdr2-knockout (Mdr2-KO) mice. These mice lack the liver-specific P-glycoprotein responsible for phosphatidylcholine transport across the canalicular membrane. Portal inflammation ensues at an early age followed by hepatocellular carcinoma development after the age of 1 year. Liver tissue samples of Mdr2-KO mice in the early and late precancerous stages of liver disease were subjected to histologic, biochemical, and gene expression profiling analysis. In an early stage, multiple protective mechanisms were found, including induction of many anti-inflammatory and antioxidant genes and increase of total antioxidant capacity of liver tissue. Despite stimulation of hepatocyte DNA replication, their mitotic activity was blocked at this stage. In the late stage of the disease, although the total antioxidant capacity of liver tissue of Mdr2-KO mice was normal, and inflammation was less prominent, many protective genes remained overexpressed. Increased mitotic activity of hepatocytes resulted in multiple dysplastic nodules, some of them being steatotic. Expression of many genes regulating lipid and phospholipid metabolism was distorted, including up-regulation of choline kinase A, a known oncogene. Many other oncogenes, including cyclin D1, Jun, and some Ras homologues, were up-regulated in Mdr2-KO mice at both stages of liver disease. However, we found no increase of Ras activation. Our data suggest that some of the adaptive mechanisms induced in the early stages of hepatic disease, which protect the liver from injury, could have an effect in hepatocarcinogenesis at later stages of the disease in this hepatocellular carcinoma model.

Interplay among oxidants, antioxidants, and cytokines in skin disorders: present status and future considerations.

The pathogenicity of skin disorders involves a complexity of physiological, immunological, environmental, and genetic phenomena. This review focuses on cross-talks between two main agents, the oxidants and cytokines network, which have recently been found to play important roles in the pathophysiology of a large variety of skin disorders, including carcinogenesis, UVB irradiation damages, inflammatory processes, and a series of diseases such as, psoriasis, pyoderma gangrenosum, atopic dermatitis, irritant contact dermatitis, and bacterial skin infections. In particular the review discusses the question how an interplay between oxidants and cytokines might be beneficial in wound-healing and in therapeutic strategies in clinical settings. These involve topical applications and oral administration of antioxidant and inflammatory-cytokines-neutralizing antibodies. Monitoring cytokine expression in skin disorders (inflammatory versus anti-inflammatory, or Th1 versus Th2 types of cytokines) will definitely help to evaluate the severity of injury, its type, and its role in therapy. Furthermore, it is expected that future studies should explore the possible roles of the synergistic interactions between antioxidants and cytokines and their impact on the Th1/Th2 cytokine networks balances.

Chronic treatment with Tempol during acquisition or withdrawal from CPP abolishes the expression of cocaine reward and diminishes oxidative damage.

In previous studies, we reported that pretreatment with the antioxidant Tempol attenuated the development and expression of cocaine-induced psychomotor sensitization in rats and diminished cocaine-induced oxidative stress (OS) in the prefrontal cortex (PFC) and nucleus accumbens (NAc), suggesting a potential role for Tempol in interfering with cocaine-related psychomotor sensitization. The aim of the current study was to examine the role of Tempol in reward and reinforcement using the conditioned place preference (CPP) paradigm. We found that administration of Tempol during the conditioning session abolished the expression of cocaine-induced CPP. We also found that OS was significantly elevated following the establishment of CPP, and that cocaine-induced OS was significantly diminished by pretreatment with Tempol during conditioning. Furthermore, we found that repeated, but not single, administration of Tempol for seven days during withdrawal from CPP resulted in significant attenuation in the expression of CPP. Moreover, Tempol did not affect the expression of food reward. Taken together, these findings provide evidence for the involvement of Tempol in regulating cocaine rewarding properties without affecting natural rewards. Since Tempol was found to be effective in reducing OS and expression of CPP following withdrawal, it may be a potential treatment for cocaine addiction.