Emanuela Camera

Modulation of Antioxidant Defense in Aspergillus parasiticus Is Involved in Aflatoxin Biosynthesis: a Role for the ApyapA Gene

ABSTRACT Oxidative stress is recognized as a trigger of different metabolic events in all organisms. Various factors correlated with oxidation, such as the β-oxidation of fatty acids and their enzymatic or nonenzymatic by-products (e.g., precocious sexual inducer factors and lipoperoxides) have been shown to be involved in aflatoxin formation. In the present study, we found that increased levels of reactive oxygen species (ROS) were correlated with increased levels of aflatoxin biosynthesis in Aspergillus parasiticus. To better understand the role of ROS formation in toxin production, we generated a mutant (ΔApyapA) having the ApyapA gene deleted, given that ApyapA orthologs have been shown to be part of the antioxidant response in other fungi. Compared to the wild type, the mutant showed an increased susceptibility to extracellular oxidants, as well as precocious ROS formation and aflatoxin biosynthesis. Genetic complementation of the ΔApyapA mutant restored the timing and quantity of toxin biosynthesis to the levels found in the wild type. The presence of putative AP1 (ApYapA orthologue) binding sites in the promoter region of the regulatory gene aflR further supports the finding that ApYapA plays a role in the regulation of aflatoxin biosynthesis. Overall, our results show that the lack of ApyapA leads to an increase in oxidative stress, premature conidiogenesis, and aflatoxin biosynthesis.

Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry

Sebum is a complex lipid mixture that is synthesized in sebaceous glands and excreted on the skin surface. The purpose of this study was the comprehensive detection of the intact lipids that compose sebum. These lipids exist as a broad range of chemical structures and concentrations. Sebum was collected with SebuTapeTM from the foreheads of healthy donors, and then separated by HPLC on a C8 stationary phase with sub 2 µm particle size. This HPLC method provided high resolution and excellent reproducibility of retention times (RT). Compound mining was performed with time of flight (TOF) and triple quadrupole (QqQ) mass spectrometers (MS), which allowed for the classification of lipids according to their elemental composition, degree of unsaturation, and MS/MS fragmentation. The combination of the two MS systems detected 95 and 29 families of triacylglycerols (TAG) and diacylglycerols (DAG), respectively. Assignment was carried out regardless of positional isomerism. Among the wax esters (WE), 28 species were found to contain the 16:1 fatty acyl moiety. This method was suitable for the simultaneous detection of squalene and its oxygenated derivative. A total of 9 cholesterol esters (CE) were identified and more than 48 free fatty acids (FFA) were detected in normal sebum. The relative abundance of each individual lipid within its own chemical class was determined for 12 healthy donors. In summary, this method provided the first characterization of the features and distribution of intact components of the sebum lipidome.

Sebaceous gland lipids

Principal activity of mature sebaceous glands is producing and secreting sebum, which is a complex mixture of lipids. Sebum composition is different among species and probably this difference can be due to the function that sebum has to absolve. In human sebum there are unique lipids, such as squalene and wax esters, not found anywhere else in the body nor among the epidermal surface lipids. Moreover, they correspond to major components supplying the skin with protection. However, the ultimate role of human sebum as well the metabolic pathways regulating its composition and secretion rate are far from a complete comprehension. Increased sebum secretion is considered, among all features, the major one involved in the pathophysiology of acne. Along with increased sebum secretion rate, quali- and quantitative modifications of sebum are likely to occur in this pathology. Understanding the factors and mechanisms that regulate sebum production is needed in order to identify new targets that could be addressed to achieve a selective modulation of lipid biosynthesis as a novel therapeutic strategy to correct lipid disregulations in acne and other disorders of the pilosebaceous unit.

Astaxanthin, canthaxanthin and β-carotene differently affect UVA-induced oxidative damage and expression of oxidative stress-responsive enzymes

Abstract:  Carotenoids are used for systemic photoprotection in humans. Regarding mechanisms underlying photoprotective effects of carotenoids, here we compared the modulation of UVA‐related injury by carotenoids. Human dermal fibroblasts (HDF) were exposed to moderate doses of UVA, which stimulated apoptosis, increased levels of reactive oxygen species and thiobarbituric acid reactive substances, decreased antioxidant enzymes activities, promoted membrane perturbation, and induced the expression of heme oxygenase‐1 (HO‐1). The carotenoids astaxanthin (AX), canthaxanthin (CX) and β‐carotene (βC) were delivered to HDF 24 h before exposure to UVA. Astaxanthin exhibited a pronounced photoprotective effect and counteracted all of the above‐mentioned UVA‐induced alterations to a significant extent. β‐Carotene only partially prevented the UVA‐induced decline of catalase and superoxide dismutase activities, but it increased membrane damage and stimulated HO‐1 expression. Moreover, βC dose‐dependently induced caspase‐3 activity following UVA exposure. In contrast, CX had no effect on oxidative damage, except for HO‐1 expression, which was augmented. Uptake of AX by fibroblasts was higher than that of the other two carotenoids. The photostability of the three compounds in fibroblasts was AX > CX >> βC. The data indicate that the oxo‐carotenoid AX has a superior preventive effect towards photo‐oxidative changes in cell culture.

Levels of Enzymatic Antioxidants Activities in Mononuclear Cells and Skin Reactivity to Sodium Dodecyl Sulphate

Chemical irritants are able to produce several biological modifications of the skin, including the direct or indirect production of cytokines and reactive oxygen species leading to an inflammatory reaction. This report examines the existence of a possible correlation between the skin sensitivity to the irritant sodium dodecyl sulphate (SDS) and the activity of the enzymatic antioxidants. In twenty-three healthy subjects the evaluation of the epidermal and peripheral blood mononuclear cells (PBMCs) activities of superoxide Dismutase (SOD) and Catalase (Cat) demonstrate a significant correlation (r= 0,85 and p< 0,005 for SOD, and r= 0,87 and p< 0,0001 for Cat). Based on this result, on a further group of normal subjects (n=13) we studied the link between the threshold dose of skin reactivity to SDS and the activities of the enzymatic antioxidants in PBMCs. The degree of skin modification induced by SDS, applied at different concentrations for 24 hrs, was determined by means of Trans Epidermal Water Loss (TEWL), Erythemal Index or by Visual Score (VS). The minimal dose of the irritant capable of inducing skin modifications, was significantly correlated with SOD (r=0,77) and Cat (r=0,81) activities in PBMCs, and the modification of TEWL or EI were inversely correlated with levels of antioxidants in PBMCs (r=−0,62 for SOD and r=−0,66 for Cat). Our results indicate that the skin reactivity to irritants can be modulated by the levels of antioxidants, and suggest a possible therapeutical approach in preventing irritant contact dermatitis.

Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography-electrospray mass spectrometry.

We developed a sensitive and specific liquid chromatography-electrospray mass spectrometric (HPLC-ESI-MS) assay for the simultaneous determination of reduced and oxidized glutathione (GSH and GSSG) in peripheral blood mononuclear cells (PBMC). Following derivatization with N-ethylmaleimide to prevent GSH auto-oxidation, addition of thiosalicylic acid as internal standard, and protein precipitation with cold acetonitrile, the samples were injected into a diol column, eluted with acetonitrile-1% aqueous acetic acid (25:75) and detected by the ESI-MS system. The optimized method exhibited a good detection limit for both analytes (0.01 and 0.05 microM for GSH and GSSG, respectively). Good linearity was reached in the 0.01-20 microM range for GSH and 0.05-20 microM for GSSG. The mean recoveries of GSH and GSSG were 98.5-100.6% and 105.8-111.5%, respectively. The run-to-run repeatability for retention time and peak area was RSD% 0.06 and 1.75 for GSH and 0.18 and 2.50 for GSSG. The optimized method was applied to GSH and GSSG assay in PBMC analyzing 20 healthy individuals.

Lipid mediators in acne.

Multiple factors are involved in acne pathogenesis, and sebum secretion is one of the main ones. The role sebum plays in acne development has not been completely elucidated yet; however, increasing amounts of data seem to confirm the presence of alterations in sebum from acne patients. Altered ratio between saturated and unsaturated fatty acids has been indicated as an important feature to be considered in addition to the altered amount of specific fatty acids such as linoleic acid. Furthermore, particular attention has been focused on squalene peroxide that seems to be able to induce an inflammatory response beyond cytotoxicity and comedones formation. Moreover, recent data suggest that lipid mediators are able to interfere with sebocytes differentiation and sebogenesis through the activation of pathways related to peroxisome proliferators-activated receptors. Understanding the factors and mechanisms that regulate sebum production is needed in order to identify novel therapeutic strategies for acne treatment.

Azelaic acid modulates the inflammatory response in normal human keratinocytes through PPARγ activation

Please cite this paper as: Azelaic acid modulates the inflammatory response in normal human keratinocytes through PPARγ activation. Experimental Dermatology 2010; 19: 813–820.

Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes

The endocannabinoid system (ECS) regulates multiple physiological processes, including cutaneous cell growth and differentiation. Here, we explored the effects of the major nonpsychotropic phytocannabinoid of Cannabis sativa, (-)-cannabidiol (CBD), on human sebaceous gland function and determined that CBD behaves as a highly effective sebostatic agent. Administration of CBD to cultured human sebocytes and human skin organ culture inhibited the lipogenic actions of various compounds, including arachidonic acid and a combination of linoleic acid and testosterone, and suppressed sebocyte proliferation via the activation of transient receptor potential vanilloid-4 (TRPV4) ion channels. Activation of TRPV4 interfered with the prolipogenic ERK1/2 MAPK pathway and resulted in the downregulation of nuclear receptor interacting protein-1 (NRIP1), which influences glucose and lipid metabolism, thereby inhibiting sebocyte lipogenesis. CBD also exerted complex antiinflammatory actions that were coupled to A2a adenosine receptor-dependent upregulation of tribbles homolog 3 (TRIB3) and inhibition of the NF-κB signaling. Collectively, our findings suggest that, due to the combined lipostatic, antiproliferative, and antiinflammatory effects, CBD has potential as a promising therapeutic agent for the treatment of acne vulgaris.

Separation of tocopherols by nano-liquid chromatography

Nanoliquid chromatography (nano-LC) was used for the separation of tocopherols (delta-, gamma-, alpha-TOH), alpha-tocopherol acetate (alpha-TOH-Ac) and an antioxidant compound, namely butylated hydroxytoluene (BHT) used to prevent TOHs autoxidation. The separation was carried out in a fused silica capillary of 100 microm I.D. and 375 microm O.D. packed in our laboratory with RP18 silica stationary phase of either 5- or 3-microm diameter (23-cm long). The mobile phase was composed by mixtures of methanol (MeOH), acetonitrile (MeCN) and water. Typical analyses time for the separation of all the five components of the mixture were 6-9 min depending on the composition of the mobile phase. Efficiency and resolution were strongly influenced by the particle diameter and the highest Rs and N/m values were observed using 3-microm RP18 particles. Experiments performed with capillaries packed with 3-microm RP18 particles provided good limit of detection (LOD) and limit of quantification (LOQ) (for delta-, gamma-TOH, alpha-TOH-Ac were 4 and 8 microg/ml, while for alpha-TOH were 6 and 10 microg/ml, respectively). The optimized method was applied to extracts of serum and pharmaceutical preparation containing alpha-TOH and alpha-TOH-Ac.