Interactions of Phenylalanine Derivatives with Human Tyrosinase: Lessons from Experimental and Theoretical tudies.

The pigmentation of the skin, modulated by different actors in melanogenesis, is mainly due to the melanins (protective pigments). In humans, these pigments' precursors are synthetized by an enzyme known as tyrosinase (TyH). The regulation of the enzyme activity by specific modulators (inhibitors or activators) can offer a means to fight hypo- and hyper-pigmentations responsible for medical, psychological and societal handicaps. Herein, we report the investigation of phenylalanine derivatives as TyH modulators. Interacting with the binuclear copper active site of the enzyme, phenylalanine derivatives combine effects induced by combination with known resorcinol inhibitors and natural substrate/intermediate (amino acid part). Computational studies including docking, molecular dynamics and free energy calculations combined with biological activity assays on isolated TyH and in human melanoma MNT-1 cells, and X-ray crystallography analyses with the TyH analogue Tyrp1, provide conclusive evidence of the interactions of phenylalanine derivatives with human tyrosinase. In particular, our findings indicate that an analogue of L-DOPA, namely (S)-3-amino-tyrosine, stands out as an amino phenol derivative with inhibitory properties against TyH.

Effect of Ultraviolet Radiation and Benzo[a]pyrene Co-Exposure on Skin Biology: Autophagy as a Potential Target.

The skin is the outermost protective barrier of the human body. Its role is to protect against different physical, chemical, biological and environmental stressors. The vast majority of studies have focused on investigating the effects of single environmental stressors on skin homeostasis and the induction of several skin disorders, such as cancer or ageing. On the other hand, much fewer studies have explored the consequences of the co-exposure of skin cells to two or more stressors simultaneously, which is much more realistic. In the present study, we investigated, using mass-spectrometry-based proteomic analysis, the dysregulated biological functions in skin explants after their co-exposure to ultraviolet radiation (UV) and benzo[a]pyrene (BaP). We observed that several biological processes were dysregulated, among which autophagy appeared to be significantly downregulated. Furthermore, immunohistochemistry analysis was carried out to validate the downregulation of the autophagy process further. Altogether, the output of this study provides an insight into the biological responses of skin to combined exposure to UV + BaP and highlights autophagy as a potential target that might be considered in the future as a novel candidate for pharmacological intervention under such stress conditions.

Isoconazole and Clemizole Hydrochloride Partially Reverse the Xeroderma Pigmentosum C Phenotype.

Xeroderma Pigmentosum protein C (XPC) is involved in recognition and repair of bulky DNA damage such as lesions induced by Ultra Violet (UV) radiation. XPC-mutated cells are, therefore, photosensitive and accumulate UVB-induced pyrimidine dimers leading to increased cancer incidence. Here, we performed a high-throughput screen to identify chemicals capable of normalizing the XP-C phenotype (hyper-photosensitivity and accumulation of photoproducts). Fibroblasts from XP-C patients were treated with a library of approved chemical drugs. Out of 1280 tested chemicals, 16 showed ≥25% photo-resistance with RZscore above 2.6 and two drugs were able to favor repair of 6-4 pyrimidine pyrimidone photoproducts (6-4PP). Among these two compounds, Isoconazole could partially inhibit apoptosis of the irradiated cells especially when cells were post-treated directly after UV irradiation while Clemizole Hydrochloride-mediated increase in viability was dependent on both pre and post treatment. No synergistic effect was recorded following combined drug treatment and the compounds exerted no effect on the proliferative capacity of the cells post UV exposure. Amelioration of XP-C phenotype is a pave way towards understanding the accelerated skin cancer initiation in XP-C patients. Further examination is required to decipher the molecular mechanisms targeted by these two chemicals.

Quantitative Proteomic Approach Reveals Altered Metabolic Pathways in Response to the Inhibition of Lysine Deacetylases in A549 Cells under Normoxia and Hypoxia.

Growing evidence is showing that acetylation plays an essential role in cancer, but studies on the impact of KDAC inhibition (KDACi) on the metabolic profile are still in their infancy. Here, we analyzed, by using an iTRAQ-based quantitative proteomics approach, the changes in the proteome of KRAS-mutated non-small cell lung cancer (NSCLC) A549 cells in response to trichostatin-A (TSA) and nicotinamide (NAM) under normoxia and hypoxia. Part of this response was further validated by molecular and biochemical analyses and correlated with the proliferation rates, apoptotic cell death, and activation of ROS scavenging mechanisms in opposition to the ROS production. Despite the differences among the KDAC inhibitors, up-regulation of glycolysis, TCA cycle, oxidative phosphorylation and fatty acid synthesis emerged as a common metabolic response underlying KDACi. We also observed that some of the KDACi effects at metabolic levels are enhanced under hypoxia. Furthermore, we used a drug repositioning machine learning approach to list candidate metabolic therapeutic agents for KRAS mutated NSCLC. Together, these results allow us to better understand the metabolic regulations underlying KDACi in NSCLC, taking into account the microenvironment of tumors related to hypoxia, and bring new insights for the future rational design of new therapies.

Xeroderma Pigmentosum C (XPC) Mutations in Primary Fibroblasts Impair Base Excision Repair Pathway and Increase Oxidative DNA Damage.

Xeroderma Pigmentosum C (XPC) is a multi-functional protein that is involved not only in the repair of bulky lesions, post-irradiation, via nucleotide excision repair (NER) per se but also in oxidative DNA damage mending. Since base excision repair (BER) is the primary regulator of oxidative DNA damage, we characterized, post-Ultraviolet B-rays (UVB)-irradiation, the detailed effect of three different XPC mutations in primary fibroblasts derived from XP-C patients on mRNA, protein expression and activity of different BER factors. We found that XP-C fibroblasts are characterized by downregulated expression of different BER factors including OGG1, MYH, APE1, LIG3, XRCC1, and Polß. Such a downregulation was also observed at OGG1, MYH, and APE1 protein levels. This was accompanied with an increase in DNA oxidative lesions, as evidenced by 8-oxoguanine levels, immediately post-UVB-irradiation. Unlike in normal control cells, these oxidative lesions persisted over time in XP-C cells having lower excision repair capacities. Taken together, our results indicated that an impaired BER pathway in XP-C fibroblasts leads to longer persistence and delayed repair of oxidative DNA damage. This might explain the diverse clinical phenotypes in XP-C patients suffering from cancer in both photo-protected and photo-exposed areas. Therapeutic strategies based on reinforcement of BER pathway might therefore represent an innovative path for limiting the drawbacks of NER-based diseases, as in XP-C case.

Impairment of Base Excision Repair in Dermal Fibroblasts Isolated From Nevoid Basal Cell Carcinoma Patients.

The nevoid basal cell carcinoma syndrome (NBCCS), also called Gorlin syndrome is an autosomal dominant disorder whose incidence is estimated at about 1 per 55,600-256,000 individuals. It is characterized by several developmental abnormalities and an increased predisposition to the development of basal cell carcinomas (BCCs). Cutaneous fibroblasts from Gorlin patients have been shown to exhibit an increased sensitivity to ionizing radiations. Mutations in the tumor suppressor gene PTCH1, which is part of the Sonic Hedgehog (SHH) signaling pathway, are responsible for these clinical manifestations. As several genetic mutations in the DNA repair genes are responsible of photo or radiosensitivity and high predisposition to cancers, we hypothesized that these effects in Gorlin syndrome might be due to a defect in the DNA damage response (DDR) and/or the DNA repair capacities. Therefore, the objective of this work was to investigate the sensitivity of skin fibroblasts from NBCCS patients to different DNA damaging agents and to determine the ability of these agents to modulate the DNA repair capacities. Gorlin fibroblasts showed high radiosensitivity and also less resistance to oxidative stress-inducing agents when compared to control fibroblasts obtained from healthy individuals. Gorlin fibroblasts harboring PTCH1 mutations were more sensitive to the exposure to ionizing radiation and to UVA. However, no difference in cell viability was shown after exposure to UVB or bleomycin. As BER is responsible for the repair of oxidative DNA damage, we decided to assess the BER pathway efficacy in Gorlin fibroblasts. Interestingly, a concomitant decrease of both BER gene expression and BER protein activity was observed in Gorlin fibroblasts when compared to control. Our results suggest that low levels of DNA repair within Gorlin cells may lead to an accumulation of oxidative DNA damage that could participate and partly explain the radiosensitivity and the BCC-prone phenotype in Gorlin syndrome.

High-throughput synthetic rescue for exhaustive characterization of suppressor mutations in human genes.

Inherited or acquired mutations can lead to pathological outcomes. However, in a process defined as synthetic rescue, phenotypic outcome created by primary mutation is alleviated by suppressor mutations. An exhaustive characterization of these mutations in humans is extremely valuable to better comprehend why patients carrying the same detrimental mutation exhibit different pathological outcomes or different responses to treatment. Here, we first review all known suppressor mutations' mechanisms characterized by genetic screens on model species like yeast or flies. However, human suppressor mutations are scarce, despite some being discovered based on orthologue genes. Because of recent advances in high-throughput screening, developing an inventory of human suppressor mutations for pathological processes seems achievable. In addition, we review several screening methods for suppressor mutations in cultured human cells through knock-out, knock-down or random mutagenesis screens on large scale. We provide examples of studies published over the past years that opened new therapeutic avenues, particularly in oncology.

The effects of different bisphenol derivatives on oxidative stress, DNA damage and DNA repair in RWPE-1 cells: A comparative study.

Bisphenol A (BPA) is a well-known endocrine disruptor and it is widely used mainly in the plastics industry. Due to recent reports on its possible impact on health (particularly on the male reproductive system), bisphenol F (BPF) and bisphenol S (BPS) are now being used as alternatives. In this study, RWPE-1 cells were used as a model to compare cytotoxicity, oxidative stress-causing potential and genotoxicity of these chemicals. In addition, the effects of the bisphenol derivatives were assessed on DNA repair proteins. RWPE-1 cells were incubated with BPA, BPF, and BPS at concentrations of 0-600 µM for 24 h. The inhibitory concentration 20 (IC20 , concentration that causes 20% of cell viability loss) values for BPA, BPF, and BPS were 45, 65, and 108 µM, respectively. These results indicated that cytotoxicity potentials were ranked as BPA > BPF > BPS. We also found alterations in superoxide dismutase, glutathione peroxidase and glutathione reductase activities, and glutathione and total antioxidant capacity in all bisphenol-exposed groups. In the standard and modified Comet assay, BPS produced significantly higher levels of DNA damage vs the control. DNA repair proteins (OGG1, Ape-1, and MyH) involved in the base excision repair pathway, as well as p53 protein levels were down-regulated in all of the bisphenol-exposed groups. We found that the BPA alternatives were also cytotoxic and genotoxic, and changed the expressions of DNA repair enzymes. Therefore, further studies are needed to assess whether they can be used safely as alternatives to BPA or not.

Effects of Iron Oxide Nanoparticles (γ-Fe2O3) on Liver, Lung and Brain Proteomes following Sub-Acute Intranasal Exposure: A New Toxicological Assessment in Rat Model Using iTRAQ-Based Quantitative Proteomics.

Iron Oxide Nanoparticles (IONPs) present unique properties making them one of the most used NPs in the biomedical field. Nevertheless, for many years, growing production and use of IONPs are associated with risks that can affect human and the environment. Thus, it is essential to study the effects of these nanoparticles to better understand their mechanism of action and the molecular perturbations induced in the organism. In the present study, we investigated the toxicological effects of IONPs (γ-Fe2O3) on liver, lung and brain proteomes in Wistar rats. Exposed rats received IONP solution during 7 consecutive days by intranasal instillation at a dose of 10 mg/kg body weight. An iTRAQ-based quantitative proteomics was used to study proteomic variations at the level of the three organs. Using this proteomic approach, we identified 1565; 1135 and 1161 proteins respectively in the brain, liver and lung. Amon them, we quantified 1541; 1125 and 1128 proteins respectively in the brain, liver and lung. Several proteins were dysregulated comparing treated samples to controls, particularly, proteins involved in cytoskeleton remodeling, cellular metabolism, immune system stimulation, inflammation process, response to oxidative stress, angiogenesis, and neurodegenerative diseases.

Investigating the toxic effects induced by iron oxide nanoparticles on neuroblastoma cell line: an integrative study combining cytotoxic, genotoxic and proteomic tools.

Nanomaterials have gained much attention for their use and benefit in several fields. Iron Oxide Nanoparticles (IONPs) have been used in Biomedicine as contrast agents for imaging cancer cells. However, several studies reported the potential toxicity of those nanoparticles in different models, especially in cells. Therefore, in our present study, we investigated the effects of IONPs on the SH-SY5Y neuroblastoma cell line. We carried out cytotoxic and genotoxic studies to evaluate the phenotypic effects, and proteomic investigation to evaluate the molecular effects and the mechanisms by which this kind of NPs could induce toxicity. Our results showed that the use of three different sizes of IONPs (14, 22 and 30 nm) induced cell detachment, cell morphological changes, size, and concentration-dependent IONP internalization and cell mortality. IONPs induced slight genotoxic damage assayed by modified comet assay without affecting cell cycle, mitochondrial function, membrane integrity, intracellular calcium level, and without inducing ROS generation. All the studies were performed to compare also the effects of IONPs to the ferric iron by incubating cells with equivalent concentration of FeCl3. In all tests, the NPs exhibited more toxicity than the ferric iron. The proteomic analysis followed by gene ontology and pathway analysis evidenced the effects of IONPs on cytoskeleton, cell apoptosis, and cancer development. Our findings provided more information about IONP effects on human cells and especially on cancer cell line.

The Polyphenol-Rich Extract from Psiloxylon mauritianum, an Endemic Medicinal Plant from Reunion Island, Inhibits the Early Stages of Dengue and Zika Virus Infection.

The recent emergence and re-emergence of viral infections transmitted by vectors, such as the Zika virus (ZIKV) and Dengue virus (DENV), is a cause for international concern. These highly pathogenic arboviruses represent a serious health burden in tropical and subtropical areas of the world. Despite the high morbidity and mortality associated with these viral infections, antiviral therapies are missing. Medicinal plants have been widely used to treat various infectious diseases since millenaries. Several compounds extracted from plants exhibit potent effects against viruses in vitro, calling for further investigations regarding their efficacy as antiviral drugs. Here, we demonstrate that an extract from Psiloxylon mauritianum, an endemic medicinal plant from Reunion Island, inhibits the infection of ZIKV in vitro without exhibiting cytotoxic effects. The extract was active against different ZIKV African and Asian strains, including an epidemic one. Time-of-drug-addition assays revealed that the P. mauritianum extract interfered with the attachment of the viral particles to the host cells. Importantly, the P. mauritianum extract was also able to prevent the infection of human cells by four dengue virus serotypes. Due to its potency and ability to target ZIKV and DENV particles, P. mauritianum may be of value for identifying and characterizing antiviral compounds to fight medically-important flaviviruses.

Loss of Epidermal HIF-1α Blocks UVB-Induced Tumorigenesis by Affecting DNA Repair Capacity and Oxidative Stress.

HIF-1α is constitutively expressed in mouse and human epidermis. It plays a crucial role in skin physiology, including the response of keratinocytes to UVR. However, little information is available about its role in photocarcinogenesis. Using a multistage model of UVB radiation-induced skin cancer, we show that the knockout of Hif-1α in the epidermis prevents tumorigenesis but at the same time triggers the formation of hyperkeratotic plaques. Our results indicate that the absence of oncogenic transformation in Hif-1α-ablated mice is related to increased DNA repair in keratinocytes, whereas the formation of hyperkeratotic plaques is caused by an increase in the levels of reactive oxygen species. Indeed, impairing the DNA repair machinery by ablating xeroderma pigmentosum C restored the UVB-induced neoplastic transformation of Hif-1α-ablated keratinocytes, whereas the development of hyperkeratotic plaques was blocked by chronic antioxidant treatment. We conclude that HIF-1α plays a procarcinogenic role in UVB-induced tumorigenesis.

Human CD8+ CD25 + CD127 low regulatory T cells: microRNA signature and impact on TGF-ß and IL-10 expression.

Regulatory T cells (Tregs) are central for maintaining immune balance and their dysfunction drives the expansion of critical immunologic disorders. During the past decade, microRNAs (miRNAs) have emerged as potent regulators of gene expression among which immune-related genes and their immunomodulatory properties have been associated with different immune-based diseases. The miRNA signature of human peripheral blood (PB) CD8+ CD25 + CD127 low Tregs has not been described yet. We thus identified, using TaqMan low-density array (TLDA) technique followed by individual quantitative real-time polymerase chain reaction (qRT-PCR) confirmation, 14 miRNAs, among which 12 were downregulated whereas two were upregulated in CD8 + CD25 + CD127 low Tregs in comparison to CD8 + CD25 - T cells. In the next step, microRNA Data Integration Portal (mirDIP) was used to identify potential miRNA target sites in the 3'-untranslated region (3'-UTR) of key Treg cell-immunomodulatory genes with a special focus on interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß). Having identified potential miR target sites in the 3'-UTR of IL-10 (miR-27b-3p and miR-340-5p) and TGF-ß (miR-330-3p), we showed through transfection and transduction assays that the overexpression of two underexpressed miRNAs, miR-27b-3p and miR-340-5p, downregulated IL-10 expression upon targeting its 3'-UTR. Similarly, overexpression of miR-330-3p negatively regulated TGF-ß expression. These results highlighted an important impact of the CD8 + Treg mirnome on the expression of genes with significant implication on immunosuppression. These observations could help in better understanding the mechanism(s) orchestrating Treg immunosuppressive function toward unraveling new targets for treating autoimmune pathologies and cancer.

Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny.

The epidermis undergoes constant renewal during its lifetime. This is possible due to a special population of keratinocyte stem cells (KSCs) located at the basal layer. These cells are surrounded by their direct progeny, keratinocyte progenitors or transient amplifying cells (TAs), which arise from cell division. Skin is exposed every day to sun radiation; in particular, UVA radiation penetrates through the epidermis and induces damage to KSCs and TAs. Although keratinocytes in the basal layer are the most likely skin carcinomas and/or photoaging cells of origin, surprisingly few studies have addressed the specific responses of these cells to UV radiation. In this study, we showed for the first time that keratinocyte stem cells were more resistant to UVA irradiation than their direct progeny, transient amplifying cells. Using both the MTT assay and clonogenic assay, we found that KSCs were more photo-resistant compared to TAs after exposure to different doses of UVA (from 0 to 50 J/cm2). Moreover, KSCs had a greater ability to reconstruct human epidermis (RHE) after UVA exposure compared with TAs. Finally, investigations of DNA repair using the comet assay showed that DNA single-strand breaks and thymine dimers were repaired quicker and more efficiently in KSCs compared with TAs. In a previous work, we showed that the same stem cell population was more resistant to ionizing radiation, another carcinogenic agent. Collectively, our results combined with other observations demonstrate that keratinocyte stem cells, which are responsible for epidermal renewal throughout life, are equipped with an efficient arsenal against several genotoxic agents. Our future work will try to identify the factors or signaling pathways that are responsible for this differential photo-sensitivity and DNA repair capacity between KSCs and TAs.

Energy Metabolism Rewiring Precedes UVB-Induced Primary Skin Tumor Formation.

Although growing evidence indicates that bioenergetic metabolism plays an important role in the progression of tumorigenesis, little information is available on the contribution of reprogramming of energy metabolism in cancer initiation. By applying a quantitative proteomic approach and targeted metabolomics, we find that specific metabolic modifications precede primary skin tumor formation. Using a multistage model of ultraviolet B (UVB) radiation-induced skin cancer, we show that glycolysis, tricarboxylic acid (TCA) cycle, and fatty acid ß-oxidation are decreased at a very early stage of photocarcinogenesis, while the distal part of the electron transport chain (ETC) is upregulated. Reductive glutamine metabolism and the activity of dihydroorotate dehydrogenase (DHODH) are both necessary for maintaining high ETC. Mice with decreased DHODH activity or impaired ETC failed to develop pre-malignant and malignant lesions. DHODH activity represents a major link between DNA repair efficiency and bioenergetic patterning during skin carcinogenesis.

Long-term Genoprotection Effect of Sechium edule Fruit Extract Against UVA Irradiation in Keratinocytes.

Photoprotection is essential to prevent the long-term deleterious effects of ultraviolet (UV), including skin cancer and photoaging. So far, there has been an increase in the use of natural bioactive phytochemicals for the development of more effective skin photoprotective agents. However, the molecular mechanisms underlying the photochemoprotection activity of such compounds remain largely unknown. The objective of this study was to investigate the effects of a Sechium edule fruit extract (SEE) in terms of photoprotection against UVA in primary human keratinocytes. We found that SEE protected keratinocytes against UVA-induced cytotoxicity, decreased the intracellular amounts of reactive oxygen species, and reduced oxidatively induced DNA lesions after UVA exposure. Furthermore, SEE decreased the induction of CPD lesions in UVA-irradiated keratinocytes and exhibited increased DNA repair of such photoproducts at 24 h postexposure. Finally, using DNA repair biochips, we demonstrated that SEE-treated keratinocytes had DNA enzymatic repair activities more efficient for abasic sites, CPD and thymine glycols. Therefore, the benefits of SEE against UVA could be explained by a combination of antioxidant activity, the reduction in DNA damage, and the enhancement of DNA repair capacities.

Selenium preserves keratinocyte stemness and delays senescence by maintaining epidermal adhesion.

Skin is constantly exposed to environmental factors such as pollutants, chemicals and ultra violet radiation (UV), which can induce premature skin aging and increase the risk of skin cancer. One strategy to reduce the effect of oxidative stress produced by environmental exposure is the application of antioxidant molecules. Among the endogenous antioxidants, selenoproteins play a key role in antioxidant defense and in maintaining a reduced cellular environment. Selenium, essential for the activity of selenoproteins, is a trace element that is not synthesized by organisms and must be supplied by diet or supplementation. The aim of this study is to evaluate the effect of Selenium supplementation on skin aging, especially on keratinocytes, the main cells of the epidermis. Our results demonstrate for the first time to our knowledge, the major role of Selenium on the replicative life span of keratinocytes and on aging skin. Selenium protects keratinocyte stem cells (KSCs) against senescence via preservation of their stemness phenotype through adhesion to the basement membrane. Additionally, Selenium supplementation maintains the homeostasis of skin during chronological aging in our senescent skin equivalent model. Controlled supplementation with Selenium could be a new strategy to protect skin against aging.

The effects of the cellular and infectious prion protein on the neuronal adaptor protein X11α.

BACKGROUND: The neuronal adaptor protein X11α is a multidomain protein with a phosphotyrosine binding (PTB) domain, two PDZ (PSD_95, Drosophila disks-large, ZO-1) domains, a Munc Interacting (MI) domain and a CASK interacting region. Amongst its functions is a role in the regulation of the abnormal processing of the amyloid precursor protein (APP). It also regulates the activity of Cu/Zn Superoxide dismutase (SOD1) through binding with its chaperone the copper chaperone for SOD1. How X11α production is controlled has remained unclear. METHODS: Using the neuroblastoma cell line, N2a, and knockdown studies, the effect of the cellular and infectious prion protein, PrP(C) and PrP(Sc), on X11α is examined. RESULTS: We show that X11α expression is directly proportional to the expression of PrP(C), whereas its levels are reduced by PrP(Sc). We also show PrP(Sc) to affect X11α at a functional level. One of the effects of prion infection is lowered cellular SOD1 levels, here by knockdown of X11α we identify that the effect of PrP(Sc) on SOD1 can be reversed indicating that X11α is involved in prion disease pathogenesis. CONCLUSIONS: A role for the cellular and infectious prion protein, PrP(C) and PrP(Sc), respectively, in regulating X11α is identified in this work. GENERAL SIGNIFICANCE: Due to the multiple interacting partners of X11α, dysfunction or alteration in X11α will have a significant cellular effect. This work highlights the role of PrP(C) and PrP(Sc) in the regulation of X11α, and provides a new target pathway to control X11α and its related functions.

The effects of di(2-ethylhexyl) phthalate and/or selenium on trace element levels in different organs of rats.

Di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer for synthetic polymers, is known to have endocrine disruptive potential, reproductive toxicity, and induces hepatic carcinogenesis in rodents. Selenium (Se) is a component of several selenoenzymes which are essential for cellular antioxidant defense and for the functions of mammalian reproductive system. The present study was designed to investigate the effects of DEHP exposure on trace element distribution in liver, testis, and kidney tissues and plasma of Se-deficient and Se-supplemented rats. Se deficiency was produced by feeding 3-week old Sprague-Dawley rats with ≤0.05mg Se/kg diet for 5 weeks, and supplementation group were on 1mg Se/kg diet. DEHP treated groups received 1000mg/kg dose by gavage during the last 10 days of feeding period. Se, zinc (Zn), copper (Cu), iron (Fe) and manganese (Mn) levels were measured by inductively coupled plasma mass spectrometry (ICP-MS). Se supplementation caused significant increases in hepatic, renal, and testicular Se levels. With DEHP exposure, plasma Se and Zn, kidney Se, Cu and Mn levels were significantly decreased. Besides, liver Fe decreased markedly in all the DEHP-treated groups. Liver and kidney Mn levels decreased significantly in DEHP/SeD group compared to both DEHP and SeD groups. These results showed the potential of DEHP exposure and/or different Se status to modify the distribution pattern of essential trace elements in various tissues, the importance of which needs to be further evaluated.

Effects of di(2-ethylhexyl)phthalate on testicular oxidant/antioxidant status in selenium-deficient and selenium-supplemented rats.

Di(ethylhexyl)phthalate (DEHP), the most widely used plasticizer, was investigated to determine whether an oxidative stress process was one of the underlying mechanisms for its testicular toxicity potential. To evaluate the effects of selenium (Se), status on the toxicity of DEHP was further objective of this study, as Se is known to play a critical role in testis and in the modulation of intracellular redox equilibrium. Se deficiency was produced in 3-weeks-old Sprague-Dawley rats feeding them ≤0.05 mg Se /kg diet for 5 weeks, and Se-supplementation group was on 1 mg Se/kg diet. DEHP-treated groups received 1000 mg/kg dose by gavage during the last 10 days of the feeding period. Activities of antioxidant selenoenzymes [glutathione peroxidase 1 (GPx1), glutathione peroxidase 4 (GPx4), thioredoxin reductase (TrxR)], catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST); concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), and thus the GSH/GSSG redox ratio; and thiobarbituric acid reactive substance (TBARS) levels were measured. DEHP was found to induce oxidative stress in rat testis, as evidenced by significant decrease in GSH/GSSG redox ratio (>10-fold) and marked increase in TBARS levels, and its effects were more pronounced in Se-deficient rats with ∼18.5-fold decrease in GSH/GSSG redox ratio and a significant decrease in GPx4 activity, whereas Se supplementation was protective by providing substantial elevation of redox ratio and reducing the lipid peroxidation. These findings emphasized the critical role of Se as an effective redox regulator and the importance of Se status in protecting testicular tissue from the oxidant stressor activity of DEHP.