Michał Biernacki

The cross-talk between electrophiles, antioxidant defence and the endocannabinoid system in fibroblasts and keratinocytes after UVA and UVB irradiation.

BACKGROUND UV, including UVA and UVB radiation, is one of the most ubiquitous environmental stress factors to human skin and leads to redox imbalance and, consequently, photoaging and cancer development. The aim of the study was to verify which skin cells, keratinocytes or fibroblasts, were more susceptible to UVA or UVB irradiation. OBJECTIVE Keratinocytes and fibroblasts were subjected to UVA and UVB irradiation. METHODS The redox potential (superoxide anion generation and antioxidant level/activity), electrophile level and endocannabinoid system were estimated. RESULTS The results presented in this paper demonstrate a strong relationship between UV-induced oxidative stress and changes in the endocannabinoid system. Simultaneously, in irradiated cells, the transcription factors Nrf1, Nrf2 and NFκB are activated to varying degrees. Fibroblasts have a greater susceptibility to ROS generation and transcription factor activation after both UVA and UVB irradiation than keratinocytes. Keratinocytes are more sensitive to changes in the electrophile levels connected with oxidative stress compared to fibroblasts. CONCLUSION The differences demonstrated in the response of the tested cells to UV irradiation allow for a better understanding of the mechanisms occurring in the human skin, which may be exploited for future therapies in dermatology.

Crosstalk between liver antioxidant and the endocannabinoid systems after chronic administration of the FAAH inhibitor, URB597, to hypertensive rats.

Hypertension is accompanied by perturbations to the endocannabinoid and antioxidant systems. Thus, potential pharmacological treatments for hypertension should be examined as modulators of these two metabolic systems. The aim of this study was to evaluate the effects of chronic administration of the fatty acid amide hydrolase (FAAH) inhibitor [3-(3-carbamoylphenyl)phenyl]N-cyclohexylcarbamate (URB597) on the endocannabinoid system and on the redox balance in the livers of DOCA-salt hypertensive rats. Hypertension caused an increase in the levels of endocannabinoids [anandamide (AEA), 2-arachidonoyl-glycerol (2-AG) and N-arachidonoyl-dopamine (NADA)] and CB1 receptor and the activities of FAAH and monoacylglycerol lipase (MAGL). These effects were accompanied by an increase in the level of reactive oxygen species (ROS), a decrease in antioxidant activity/level, enhanced expression of transcription factor Nrf2 and changes to Nrf2 activators and inhibitors. Moreover, significant increases in lipid, DNA and protein oxidative modifications, which led to enhanced levels of proapoptotic caspases, were also observed. URB597 administration to the hypertensive rats resulted in additional increases in the levels of AEA, NADA and the CB1 receptor, as well as decreases in vitamin E and C levels, glutathione peroxidase and glutathione reductase activities and Nrf2 expression. Thus, after URB597 administration, oxidative modifications of cellular components were increased, while the inflammatory response was reduced. This study revealed that chronic treatment of hypertensive rats with URB597 disrupts the endocannabinoid system, which causes an imbalance in redox status. This imbalance increases the levels of electrophilic lipid peroxidation products, which later participate in metabolic disturbances in liver homeostasis.

Time-dependent effect of rutin on skin fibroblasts membrane disruption following UV radiation

Chronic exposure of the skin to solar UV radiation induces a number of biological alterations, including a redox imbalance; therefore, there is an urgent need for skin cells protective compounds. The aim of this study was to determine the effects of natural, previously extensively examined, polyphenol with antioxidant properties – rutin, on UV-induced skin fibroblasts membrane disruption. Accordingly, fibroblasts exposed to UVA and UVB irradiation were incubated with rutin (12 h before and/or up to 24 h after irradiation), and the structural and metabolic changes were examined. Rutin penetration through the fibroblast phospholipid bilayer was aided by UVA-induced bilitranslocase activity 2–4 h after irradiation, while UVB irradiation led to enhanced phospholipid peroxidation and higher membrane permeability to facilitate the interaction of rutin with phospholipids. Lipidomic analysis revealed that 4 h of rutin treatment also partially prevented UVA/B-induced increase in phosphatidylethanolamine and phosphatidylcholine level, as well as their membrane localization, which resulted in an enhanced zeta potential in the cells and liposomes. Moreover, rutin 2 h following irradiation, in a various degree, prevented the increased in phospholipase A2 activity and ROS generation, and partially protected against the reduction of arachidonic and linoleic acids level and the lipid peroxidation product 4-hydroxynonenal level increase. Rutin effectively prevented against decrease in glutathione peroxidase, glutathione and vitamins E and C activities/levels, particularly 2 h following UVA irradiation. In conclusion, highest skin fibroblasts membrane level of rutin occurred in 2–4 h following UVA/B-radiation results in its strongest effect on biomembrane structure and functions and cellular antioxidant system irrespective of the radiation type.

Redox system and phospholipid metabolism in the kidney of hypertensive rats after FAAH inhibitor URB597 administration

Primary and secondary hypertension is associated with kidney redox imbalance resulting in enhanced reactive oxygen species (ROS) and enzymes dependent phospholipid metabolism. The fatty acid amide hydrolase inhibitor, URB597, modulates the levels of endocannabinoids, particularly of anandamide, which is responsible for controlling blood pressure and regulating redox balance. Therefore, this study aimed to compare the effects of chronic URB597 administration to spontaneously hypertensive rats (SHR) and rats with secondary hypertension (DOCA-salt rats) on the kidney metabolism associated with the redox and endocannabinoid systems. It was shown fatty acid amide hydrolase (FAAH) inhibitor decreased the activity of ROS-generated enzymes what resulted in a reduction of ROS level. Moreover varied changes in antioxidant parameters were observed with tendency to improve antioxidant defense in SHR kidney. Moreover, URB597 administration to hypertensive rats decreased pro-inflammatory response, particularly in the kidneys of DOCA-salt hypertensive rats. URB597 had tendency to enhance ROS-dependent phospholipid oxidation, estimated by changes in neuroprostanes in the kidney of SHR and reactive aldehydes (4-hydroxynonenal and malondialdehyde) in DOCA-salt rats, in particular. The administration of FAAH inhibitor resulted in increased level of endocannabinoids in kidney of both groups of hypertensive rats led to enhanced expression of the cannabinoid receptors type 1 and 2 in SHR as well as vanilloid receptor 1 receptors in DOCA-salt rats. URB597 given to normotensive rats also affected kidney oxidative metabolism, resulting in enhanced level of neuroprostanes in Wistar Kyoto rats and reactive aldehydes in Wistar rats. Moreover, the level of endocannabinoids and cannabinoid receptors were significantly higher in both control groups of rats after URB597 administration. In conclusion, because URB597 disturbed the kidney redox system and phospholipid ROS-dependent and enzymatic-dependent metabolism, the administration of this inhibitor may enhance kidney disorders depending on model of hypertension, but may also cause kidney disturbances in control rats. Therefore, further studies are warranted.

Comparison of protective effect of ascorbic acid on redox and endocannabinoid systems interactions in in vitro cultured human skin fibroblasts exposed to UV radiation and hydrogen peroxide

The mechanisms of biological activity of commonly used natural compounds are constantly examined. Therefore, the aim of this study was to compare ascorbic acid efficacy in counteracting the consequences of UV and hydrogen peroxide treatment on lipid mediators and their regulative action on antioxidant abilities. Skin fibroblasts exposed to UVA and UVB irradiation, treated with hydrogen peroxide and ascorbic acid. The redox system was estimated through reactive oxygen species (ROS) generation (electron spin resonance spectrometer) and antioxidants level/activity (HPLC/spectrometry) which activity was evaluated by the level of phospholipid metabolites: 4-hydroxynonenal, malondialdehyde, 8-isoprostanes and endocannabinoids (GC/LC-MS) in the human skin fibroblasts. Protein and DNA oxidative modifications were also determined (LC). The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), its activators and inhibitors as well as pro/anti-apoptotic proteins and endocannabinoid receptors was examined (Western blot) and collagen metabolism was evaluated by collagen biosynthesis and prolidase activity (spectrometry). UVA and UVB irradiation and hydrogen peroxide treatment enhanced activity of xanthine and NADPH oxidases resulting in ROS generation as well as diminution of antioxidant phospholipid protection (glutathione peroxidase-glutathione-vitamin E), what led to increased lipid peroxidation and decreased endocannabinoids level. Dysregulation of cannabinoid receptors expression and environment of transcription factor Nrf2 caused apoptosis induction. Ascorbic acid partially prevented ROS generation, antioxidant capacity diminution and endocannabinoid systems disturbances but only slightly protected macromolecules such as phospholipid, protein and DNA against oxidative modifications. However, ascorbic acid significantly prevented decrease in collagen type I biosynthesis. Ascorbic acid in similar degree prevents UV (UVA and UVB) and hydrogen peroxide-dependent redox imbalance. However, this antioxidant cannot efficiently protect cellular macromolecules and avert metabolic dysregulation leading to apoptosis.

Repetitive injection field-amplified sample stacking for cationic compounds determination.

The development of a field-amplified sample stacking technique is presented. Sensitivity enhancement in this technique was obtained by repetitive injections of a sample followed by steps of sample matrix removal through the application of counter-pressure. Under optimized conditions the background electrolyte (BGE) was composed of 80 mM H3PO4 while the sample matrix contained 0.5mM H3PO4 and 30% (v/v) methanol. The elaborated method enabled a 4-fold effective injection of the sample (53 s, 0.5 psi). Each injection was followed by a focusing step during which the application of a voltage (2 kV) and counter-pressure (-1 psi) was performed for 0.65 min. The method was developed for the determination of six psychiatric drugs (opipramol, hydroxyzine, promazine, amitriptyline, fluoxetine, and thioridazine). The elaborated method was applied for analysis of human urine samples after a simple liquid-liquid extraction procedure. The detection limits obtained were in the range of 2.23-6.21 ng/mL.

Metabolism of endocannabinoids.

Endocannabinoids belong to a group of ester, ether and amide derivatives of fatty acids, which are endogenous ligands of receptors CB1, CB2, TRPV1 and GPR55 that are included in the endocannabinoid system of the animal organism. The best known endocannabinoids are: N-arachidonylethanolamide called anandamide (AEA) and 2-arachidonoylglycerol (2-AG). They occur in all organisms, and their highest level is observed in the brain. In this review the mechanisms of synthesis and degradation of both AEA and 2-AG are shown. Endocannabinoids are synthesized from phospholipids (mainly phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol) located in the cell membrane. As a result of arachidonic acid transfer from phosphatidylcholine to phosphatidylethanolamine, N-arachidonoyl phosphatidylethanolamine is formed, which is hydrolyzed to AEA by phospholipase D, C and A2. However, 2-AG is formed during the hydrolysis of phosphatidylinositol catalyzed mainly by DAGL. The primary role of endocannabinoids is the activation of cannabinoid receptors. Both AEA and 2-AG are primarily agonists of the CB1 receptor and to a lower degree CB2 and TRPV1r eceptors, but 2-AG has stronger affinity for these receptors. Through activation of receptors, endocannabinoids affect cellular metabolism and participate in the metabolic processes by receptor-independent pathways. Endocannabinoids which are not bound to the receptors are degraded. The main enzymes responsible for the hydrolysis of AEA and 2-AG are FAAH and MAGL, respectively. Apart from hydrolytic degradation, endocannabinoids may also be oxidized by cyclooxygenase-2, lipoxygenases, and cytochrome P450. It has been shown that the metabolites of both endocannabinoids also have biological significance.

Long-term administration of fatty acid amide hydrolase inhibitor (URB597) to rats with spontaneous hypertension disturbs liver redox balance and phospholipid metabolism

PURPOSE The effect of chronic administration of [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate (URB597), inhibitor of fatty acid amide hydrolase (FAAH) that hydrolyzes anandamide, on cross-talk between endocannabinoid system, oxidative status and pro-inflammatory factors in the liver of spontaneously hypertensive rats (SHRs) was investigated. MATERIALS/METHODS Experiments were conducted using SHRs and normotensive control Wistar-Kyoto rats treated by intraperitoneal injection with URB597 for 14 days. The biochemical parameters were assayed in the rats livers. RESULTS In the liver of SHRs an increase in endocannabinoids level, the activity of enzymes degrading them and expression of the cannabinoid receptor type 2 (CB2) receptor as well as a decrease in the expression of the CB1 and vanilloid 1 receptor (TRPV1) were shown. These changes were related to inflammatory conditions as well as oxidative stress resulting from increased reactive oxygen species (ROS) generation due to enhanced activity of enzymes generating ROS accompanied by decrease in the effectiveness of transcription activity of nuclear factor erythroid 2 and the activity of antioxidant enzymes, as well as level of glutathione and vitamins. Chronic administration of URB597 to SHRs caused a decrease in FAAH activity and an increase in anandamide and N-arachidonoyl-dopamine level as well as a decrease in CB2 and an increase in TRPV1 receptor expression. The levels/activities of pro- and antioxidant and inflammatory factors tended to normalize, but phospholipid peroxidation and DNA modifications were increased. CONCLUSION In conclusion, long-term chronic administration of URB597 to SHRs by altering interactions between endocannabinoid and redox systems enhances some liver metabolic disturbances observed in hypertension.

Hypertension and chronic inhibition of endocannabinoid degradation modify the endocannabinoid system and redox balance in rat heart and plasma

The interaction between the endocannabinoid and ROS signaling systems has been demonstrated in different organs. Inhibitors of fatty acid amide hydrolase (FAAH), the key enzyme responsible for degradation of the endocannabinoid anandamide, are postulated to possess anti-hypertensive potential. Here, we compared the effects of hypertension and chronic FAAH inhibition by URB597 on the endocannabinoid system and redox balance in spontaneously hypertensive rats (SHR) and hypertensive deoxycorticosterone acetate (DOCA)-salt rats. Enhanced oxidative stress and lipid peroxidation were found in both hypertension models. Hypertension affected cardiac and plasma endocannabinoid systems in a model-dependent manner: anandamide and 2-arachidonoylglycerol levels decreased in SHR and increased in DOCA-salt. Cardiac CB1 receptor expression increased in both models while higher CB2 receptor expression was only in DOCA-salt. URB597 increased endocannabinoid levels in both models but produced the partial reduction of oxidative stress in DOCA-salt but not in SHR. Notably, URB597 decreased antioxidant defense and increased lipid peroxidation products in normotension. Therefore, the therapeutic potential of FAAH inhibitors should be interpreted cautiously.

The Effect of Long-Term Administration of Fatty Acid Amide Hydrolase Inhibitor URB597 on Oxidative Metabolism in the Heart of Rats with Primary and Secondary Hypertension

Fatty acid amide hydrolase (FAAH) inhibitor [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate (URB597) may influence redox balance and blood pressure through the modulation of endocannabinoids levels. Therefore, this study aimed to compare changes in oxidative metabolism and apoptosis in the hearts of rats with spontaneous hypertension (SHR) and secondary hypertension (11-deoxycorticosterone acetate; DOCA-salt rats) treated by URB597 via intraperitoneal injection for 14 days. The results showed that URB597 decreased the activity of NADPH and xanthine oxidases in both groups of rats. Moreover, in the heart of SHR rats, URB597 led to an increase of enzymatic and nonenzymatic antioxidant activity and levels (catalase, vitamin C, glutathione/glutathione disulfide [GSH/GSSG]) and upregulation of the thioredoxin system; however, NRf2 expression was downregulated. The opposite effect in relation to Nrf2 activity and the thioredoxin system was observed in DOCA-salt rats after URB597 administration. Despite improvement in antioxidant parameters, URB597 enhanced oxidative modifications of phospholipids (4-hydroxynonenal and isoprostanes) and proteins (carbonyl groups) in SHR heart, whereas 4-hydroxynonenal and carbonyl groups levels decreased in the heart of DOCA-salt rats. Obtained results suggest that examined lipid mediators are involved in peroxisome proliferator-activated receptors (PPAR)-independent and PPAR-dependent modulation of cardiac inflammatory reactions. Furthermore, decreased expression of pro-apoptotic proteins (Bax and caspase 3 and 9) was observed after URB597 administration in the heart of both groups of hypertensive rats, whereas expression of the antiapoptotic protein (Bcl-2) increased in SHR rats. Long-term administration of URB597 altered cardiac redox status depending on the type of hypertension. URB597 enhanced oxidative metabolism and reduced pro-apoptotic factors in the heart of SHR rats, increasing the probability of heart metabolic disorders occurrence or progression.