Role of Oxidative Stress Mediated by Glutathione-S-transferase in Thiopurines' Toxic Effects

Azathioprine (AZA), 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG) are antimetabolite drugs, widely used as immunosuppressants and anticancer agents. Despite their proven efficacy, a high incidence of toxic effects in patients during standard-dose therapy is recorded. The aim of this study is to explain, from a mechanistic point of view, the clinical evidence showing a significant role of glutathione-S-transferase (GST)-M1 genotype on AZA toxicity in inflammatory bowel disease patients. To this aim, the human nontumor IHH and HCEC cell lines were chosen as predictive models of the hepatic and intestinal tissues, respectively. AZA, but not 6-MP and 6-TG, induced a concentration-dependent superoxide anion production that seemed dependent on GSH depletion. N-Acetylcysteine reduced the AZA antiproliferative effect in both cell lines, and GST-M1 overexpression increased both superoxide anion production and cytotoxicity, especially in transfected HCEC cells. In this study, an in vitro model to study thiopurines' metabolism has been set up and helped us to demonstrate, for the first time, a clear role of GST-M1 in modulating AZA cytotoxicity, with a close dependency on superoxide anion production. These results provide the molecular basis to shed light on the clinical evidence suggesting a role of GST-M1 genotype in influencing the toxic effects of AZA treatment

Long Noncoding RNA GAS5: A Novel Marker Involved in Glucocorticoid Response

Glucocorticoids (GCs) exert their effects through regulation of gene expression after activation in the cytoplasm of the glucocorticoid receptor (GR) encoded by NR3C1 gene. A negative feedback mechanism resulting in GR autoregulation has been demonstrated through the binding of the activated receptor to intragenic sequences called GRE-like elements, contained in GR gene. The long noncoding RNA growth arrest-specific transcript 5 (GAS5) interacts with the activated GR suppressing its transcriptional activity. The aim of this study was to evaluate the possible role of GAS5 and NR3C1 gene expression in the antiproliferative effect of methylprednisolone in peripheral blood mononuclear cells and to correlate the expression with individual sensitivity to GCs. Subjects being poor responders to GCs presented higher levels of GAS5 and NR3C1 in comparison with good responders. We suggest that abnormal levels of GAS5 may alter GC effectiveness, probably interfering with the mechanism of GR autoregulation

Toxicological effects of palytoxin after cutaneous exposure

2010/2011Palytoxin (PLTX) is a marine toxin identified in Palythoa zoanthid corals and Ostreopsis dinoflagellates, representing an increasing hazard for human health. Human poisonings attributed to PLTX exposure are usually associated to ingestion of contaminated seafood and to marine aerosol exposure during Ostreopsis blooms. However, also dermatological problems have been recently associated to PLTX cutaneous exposure during Ostreopsis blooms as well as after handling of Palythoa corals. Despite the increasing human cases of dermotoxicity attributed to PLTX, very few data about its dermal toxicity are presently available. Hence, the aim of this study is to investigate the cutaneous effects of PLTX characterizing its mechanism of action. Thus, this toxicological in vitro study has been carried out on spontaneously immortalized human keratinocytes (HaCaT cells), as a first-round screening of dermotoxicity. The entity of cytotoxicity induced by PLTX has been firstly investigated. A short time exposure (4 h) to PLTX reduces mitochondrial activity (MTT assay), cell mass (SRB assay) and plasma membrane integrity (LDH leakage) with different potencies (EC50 values of 6.1±1.3x10-11, 4.7±0.9x10-10 M and 1.8±0.1x10-8 M, respectively). All these effects are ouabain-sensitive corroborating the dependency of PLTX effects on the interaction with Na+/K+-ATPase. These results indicate that among the chain of intracellular events following the interaction of PLTX with the Na+/K+-ATPase the earliest is mitochondrial damage. This sustained cytotoxic effect can be explained by the high affinity of binding to HaCaT cells. Indeed, saturation experiment reveals a Kd affinity constant of 3.0±0.4x10-10 M after an exposure time as short as 10 minutes. A possible mechanism of mitochondrial dysfunction can be reactive oxygen species (ROS) overproduction. Among all, only superoxide anion (O2-) seems to be produced by the toxin after only 1 h, whereas neither nitric oxide nor peroxynitrite formation are detected. Hence, the mechanism of O2- production has been investigated. Real time PCR analysis together with western blot analysis suggest a possible involvement of NADPH oxidase (NOX) and inducible nitric oxide synthetase (iNOS) since an early increase of their gene and protein expression was observed after short (1 – 4 h) but not longer (24 h) exposure times. On the contrary, other enzymes involved in ROS production (i.e. COX-1, COX-2, XOD) seem to be not involved in PLTX effects. Moreover, using selective inhibitors of these enzymes, we found that only DPI, a nonspecific inhibitor of both NOX and NOS, is able to inhibit by 15%, 26% and 43% O2- production induced by 10-10, 10-9 and 10-8 M PLTX, respectively. However, NMMA, inhibitor of NOS, significantly reduces only O2- produced by high (10-8 M) but no by low (10-9 and 10-10 M) PLTX concentrations, whereas the selective inhibitor of NOX apocynin is totally ineffective. Moreover, since their co-administration does not reproduce DPI effect, a prominent role of these enzymes in causing PLTX-induced oxidative stress seems unlikely. Another feasible source of O2- is mitochondria itself and its production is regulated by H+ fluxes through mitochondrial membranes. Indeed, in presence of nigericin, an ionophore that reduces the H+ imbalance, PLTX-induced O2- is significantly reduced by 23% (10-9 M PLTX) and 24% (10-8 M PLTX). Furthermore, the co-administration with rotenone, a complex I inhibitor, that per se is ineffective, results in a further inhibition of O2- production (-32% and -43% in the presence of 10-9 and 10-8 M PLTX, respectively). Moreover, O2- production turned out to be ouabain-sensitive and Na+-dependent but Ca2+-independent. Thus, on the basis of these results it has been hypothesized that PLTX binding to Na+/K+-ATPase induces intracellular overload of Na+ followed by intracellular increase of H+ with a consequent ΔpH increase across H+-impermeable mitochondrial inner membrane and O2- overproduction by reverse electron transports through mitochondrial chain. Under oxidative stress conditions, mitochondrial dysfunction can be mediated by mitochondrial permeability transition pore (MPTP), which opening, indeed, is induced by PLTX already after only 5 minutes exposure. MPTP opening, which turned out to be cyclosporine A-independent, seems to be mainly induced by the sustained ionic imbalance, since in Na+-free, Ca2+-free medium and in presence of nigericin PLTX effect is strongly inhibited. The very rapid Na+-dependent opening of MPTP suggests that this is the peculiar mechanism of PLTX cytotoxicity and cell death primum movens. Cell death induced by the toxin seems to occur with necrotic-like features. PLTX, indeed, induces a concentration- and time-dependent as well as irreversible uptake of PI after only 1 h exposure and confocal images revealed dramatic morphological alterations such as plasma membrane ruptures and leakage of cytolpasmic content after 4 h. By contrast, caspasis 3/7, 8 and 9 are not activated by PLTX up to 24 h, neither under recovery conditions. Moreover, apoptotic bodies formation is not observed, discarding apoptosis occurrence. Finally, PLTX effects on some pro-inflammatory mediators such as cytokines (IL-1α, IL-6, IL-8 and TNF-α) and arachidonic acid metabolism products (PGE2 and LTB4) have been evaluated. The toxin (10-11 M) induces an early release of PGE2 that is time-dependent after 2 h exposure. On the contrary, even if an early gene expression (1–4 h) is observed, the toxin induces a delayed release of IL-6 and IL-8 (24 h), whereas no effects have been observed evaluating IL-1α and TNF-α. In conclusion, this study highlights the toxic in vitro properties of PLTX on human keratinocytes. The intracellular pathway of the sustained PLTX cytotoxicity leading to cell death has been characterized, as well as the inflammatory mediators involved in skin irritant properties of the toxin. These results can corroborate the use of non steroidal anti-inflammatory drugs in association with anti-inflammatory corticosteroids.La palitossina (PLTX) è una tossina marina identificata in coralli zoantidi appartenenti al genere Palythoa e dinoflagellati del genere Ostreopsis. Intossicazioni umane attribuite alla PLTX sono state solitamente associate all'ingestione di prodotti ittici contaminati, nonché da un'esposizione ad aerosol marino durante le fioriture di Ostreopsis. Tuttavia, anche problemi dermatologici sono stati recentemente associati alla PLTX in seguito ad esposizione cutanea durante fioriture di Ostreopsis o manipolando coralli Palythoa. Nonostante i crescenti casi di dermotossicità attribuiti alla PLTX, pochissimi dati sulla sua tossicità cutanea sono attualmente disponibili. Lo scopo di questo studio è stato, pertanto, indagare gli effetti cutanei della PLTX caratterizzando il suo meccanismo d'azione. E’ stato quindi effettuato uno studio tossicologico in vitro su cheratinociti umani spontaneamente immortalizzati (cellule HaCaT), considerate metodo predittivo per uno screening preliminare di dermotossicità. In primo luogo è stato caratterizzato il grado di citotossicità indotta dalla tossina. Un breve tempo d'esposizione (4 h) alla PLTX riduce l'attività mitocondriale (saggio MTT), la massa cellulare (saggio SRB) e l'integrità della membrana plasmatica (perdita LDH) con diversi valori di EC50 (6.1 ± 1.3x10-11, 4.7 ± 0.9x10-10 M e 1.8 ± 0.1x10-8 M, rispettivamente). Tutti questi effetti sono sensibili alla ouabaina, corroborando la dipendenza degli effetti della PLTX sull'interazione con la Na+/K+-ATPasi. Questi risultati indicano che fra la catena di eventi intracellulari dopo l'interazione con l’ATPasi il più sensibile è un danno mitocondriale. Questo effetto può essere spiegato dall’alta affinità di legame della tossina con le cellule HaCaT. Infatti, esperimenti di saturazione rivelano una costante di affinità (Kd) pari a 3,0 ± 0.4x10-10 M dopo un tempo di esposizione molto breve (10 minuti). Uno dei possibili meccanismi di disfunzione mitocondriale è una sovrapproduzione di specie reattive dell'ossigeno (ROS). Tra tutti, solo l’anione superossido (O2-) sembra essere prodotto dalla tossina dopo 1 h, mentre né ossido nitrico né formazione di perossinitrito sono stati rilevati. Quindi, il meccanismo di produzione di O2- è stato studiato. Analisi real time-PCR ed analisi western blot suggeriscono un possibile coinvolgimento della NADPH ossidasi (NOX) e della forma inducibile dell’ossido nitrico sintetasi (iNOS) poiché un aumento precoce della loro espressione genica e stata osservata dopo brevi (1 - 4 h) ma non lunghi (24 h) tempi di esposizione. Al contrario, altri enzimi coinvolti nella produzione di ROS (COX-1, COX-2, XOD) sembrano non essere coinvolti nel meccanismo di produzione di O2- da parte della tossina. Inoltre, tramite l'utilizzo di inibitori selettivi di questi enzimi, è emerso che solo il DPI, un inibitore non specifico sia di NOX che di NOS, è in grado di inibire del 15%, 26% e 43% la produzione di O2- indotta da 10-10, 10-9 e 10-8 M PLTX, rispettivamente. Tuttavia, l’NMMA, inibitore delle NOS, riduce in modo significativo solo O2- prodotto da alte (10-8 M), ma non basse (10-9 e 10-10 M) concentrazioni di PLTX, mentre l'inibitore selettivo delle NOX apocinina è totalmente inefficace. Inoltre, poiché la loro co-somministrazione non riproduce l’effetto inibitorio del DPI, un ruolo preminente di questi enzimi nel causare stress ossidativo sembra improbabile. Un'altra fonte possibile di O2- è il mitocondrio. La sua produzione è regolata dal flusso di H+ attraverso le membrane mitocondriali. Infatti, in presenza di nigericina, uno ionoforo che riduce lo squilibrio protonico, i livelli di O2- indotti dalla PLTX vengono significativamente ridotti del 23% (10-9 M PLTX) e 24% (10-8 M PLTX). Inoltre, la co-somministrazione con il rotenone, un inibitore del complesso I della catena mitocondriale di trasporto degli elettroni, che è di per sé inefficace, induce un’ulteriore inibizione di produzione di O2- (-32% e -43% in presenza di 10-9 e 10-8 M PLTX, rispettivamente). Inoltre, la produzione di O2- risulta essere ouabaina-sensibile e Na+-dipendente, ma Ca2+-indipendente. Pertanto, sulla base di questi risultati è stato ipotizzato che il legame della PLTX con la Na+/K+-ATPasi induce un aumento intracellulare di Na+ seguito da aumento intracellulare di H+ con un conseguente aumento di ΔpH attraverso la membrana mitocondriale interna con una sovrapproduzione di O2- indotta dal trasporto inverso degli elettroni attraverso la catena mitocondriale. In condizioni di stress ossidativo, la disfunzione mitocondriale può essere mediata dall’apertura dei pori di transizione mitocondriali (MPTP). La loro apertura, infatti, viene indotta dalla PLTX già dopo soli 5 minuti di esposizione. Tale apertura, che si è rivelata ciclosporinaA-indipendente, sembra principalmente indotta dallo squilibrio ionico indotto dalla tossina, poiché in terreni privo di Na+ e privo di Ca2+ e in terreno contenente nigericina, l’attività della tossina è fortemente inibita. La rapidissima apertura di MPTP suggerisce che questo è il peculiare meccanismo di citotossicità della tossina e il primum movens della cellule morte. La morte cellulare sembra verificarsi con un danno necrotico. La PLTX, infatti, induce un uptake di PI (marker di necrosi) in maniera concentrazione e tempo-dipendente. Tale uptake è inoltre irreversibile, dopo solo 1 h di esposizione e immagini ottenute al microscopio confocale rivelano drammatiche alterazioni morfologiche, quali rotture della membrana plasmatica e la perdita di contenuto citoplasmatico dopo 4 h. Al contrario, le caspasi 3/7, 8 e 9 non sono attivate dalla PLTX fino a 24 h, né sotto condizioni di recovery. Inoltre, la formazione di corpi apoptotici non è stata rilevata, scartando l’ipotesi di una morte di tipo apoptotico. Infine, gli effetti della PLTX su alcuni mediatori proinfiammatori quali citochine (IL-1α, IL-6, IL-8 e TNF-α) e metaboliti dell’acido arachidonico (PGE2 e LTB4) sono stati valutati. La tossina (10-11 M) induce una rapida produzione di PGE2 che è tempo-dipendente dopo 2 ore di esposizione. Al contrario, la tossina induce un rilascio ritardato di IL-6 e IL-8 (24 h), anche se alterazioni dell'espressione genica si sono osservate dopo breve tempo di contatto con la tossina (1-4 h). mentre non sono stati osservati effetti valutando IL-1α e TNF -α. In conclusione, questo studio mette in evidenza le proprietà tossiche in vitro della PLTX su cheratinociti umani. L’elevata citotossicità indotta dalla tossina conduce ad una morte cellulare di tipo necrotico mediata dai mitocondri. Infine, i mediatori infiammatori coinvolti nella proprietà irritanti della pelle della tossina sono stati caratterizzati, ponendo delle basi molecolari per spiegare l'utilizzo di farmaci anti-infiammatori non steroidei in associazione con corticosteroidi.XXIV Ciclo198

Glucocorticoid pharmacogenetics in pediatric idiopathic nephrotic syndrome

Idiopathic nephrotic syndrome represents the most common type of primary glomerular disease in children: glucocorticoids (GCs) are the first-line therapy, even if considerable interindividual differences in thepir efficacy and side effects have been reported. Immunosuppressive and anti-inflammatory effects of these drugs are mainly due to the GC-mediated transcription regulation of pro- and anti-inflammatory genes. This mechanism of action is the result of a complex multistep pathway that involves the glucocorticoid receptor and several other proteins, encoded by polymorphic genes. Aim of this review is to highlight the current knowledge on genetic variants that could affect GC response, particularly focusing on children with idiopathic nephrotic syndrome

In vitro assessment of skin irritation and corrosion properties of graphene-related materials on a 3D epidermis

The increasing use of graphene-related materials (GRMs) in many technological applications, ranging from electronics to biomedicine, needs a careful evaluation of their impact on human health. Skin contact can be considered one of the most relevant exposure routes to GRMs. Hence, this study is focused on two main adverse outcomes at the skin level, irritation and corrosion, assessed following two specific Test Guidelines (TGs) defined by the Organization for Economic Co-operation and Development (OECD) (439 and 431, respectively) that use an in vitro 3D reconstructed human epidermis (RhE) model. After the evaluation of their suitability to test a large panel of powdered GRMs, it was found that the latter were not irritants or corrosive. Only GRMs prepared with irritant surfactants, not sufficiently removed, reduced RhE viability at levels lower than those predicting skin irritation (≤50%, after 42 min exposure followed by 42 h recovery), but not at levels lower than those predicting corrosion (<50%, after 3 min exposure or <15% after 1 h exposure). As an additional readout, a hierarchical clustering analysis on a panel of inflammatory mediators (interleukins: IL-1α, IL-1β, IL-6, and IL-18; tumor necrosis factor-α and prostaglandin E2) released by RhE exposed to these materials supported the lack of irritant and pro-inflammatory properties. Overall, these results demonstrate that both TGs are useful in assessing GRMs for their irritant or corrosion potential, and that the tested materials did not cause these adverse effects at the skin level. Only GRMs prepared using toxic surfactants, not adequately removed, turned out to be skin irritants

Assessment of skin sensitization properties of few-layer graphene and graphene oxide through the Local Lymph Node Assay (OECD TG 442B)

: Skin contact is one of the most common exposure routes to graphene-based materials (GBMs) during their small-scale and industrial production or their use in technological applications. Nevertheless, toxic effects in humans by cutaneous exposure to GBMs remain largely unexplored, despite skin contact to other related materials has been associated with adverse effects. Hence, this in vivo study was carried out to evaluate the cutaneous effects of two GBMs, focusing on skin sensitization as a possible adverse outcome. Skin sensitization by few-layer graphene (FLG) and graphene oxide (GO) was evaluated following the Organization for Economic Cooperation and Development (OECD) guideline 442B (Local Lymph Node Assay; LLNA) measuring the proliferation of auricular lymph node cells during the induction phase of skin sensitization. Groups of four female CBA/JN mice (8-12 weeks) were daily exposed to FLG or GO through the dorsal skin of each ear (0.4-40 mg/mL, equal to 0.01-1.00 mg/ear) for 3 consecutive days, and proliferation of auricular lymph node cells was evaluated 3 days after the last treatment. During this period, no clinical signs of toxicity and no alterations in body weight and food or water consumptions were observed. In addition, no ear erythema or edema were recorded as signs of irritation or inflammation. Bromo-deoxyuridine (BrdU) incorporation in proliferating lymphocytes from ear lymph nodes (stimulation indexes <1.6) and the histological analysis of ear tissues excluded sensitizing or irritant properties of these materials, while myeloperoxidase activity in ear biopsies confirmed no inflammatory cells infiltrate. On the whole, this study indicates the absence of sensitization and irritant potential of FLG and GO

Skin biocompatibility of hexagonal boron nitride: An in vitro study on HaCaT keratinocytes and 3D reconstructed human epidermis

Hexagonal boron nitride (hBN) is a promising two-dimensional (2D) material of interest to the scientific community and industry due to its revolutionary physico-chemical features. Skin contact is one of the most feasible exposure routes both for workers, producing hBN, and consumers, using hBN-enabled nanotechnologies. Hence, the toxic potential of hBN at the cutaneous level was evaluated following an in vitro approach with different degree of complexity, using a simplified cell model (HaCaT keratinocytes), and a more predictive and complete skin tissue (a 3D model of human epidermis). Despite its significant uptake by keratinocytes, hBN exerted only weak adverse effects, such as slight alterations of cells parameters indices of cytotoxicity (cell viability, cell mass and plasma membrane integrity) and mitochondrial-related dysfunctions (mitochondrial depolarization, ATP depletion and reactive oxygen species production), detectable only at high concentrations (>25 μg/mL) and mainly after a long exposure (72 h). In addition, adoption of the OECD TG 431 and 439 on the 3D reconstructed human epidermis model demonstrated hBN as a non-corrosive and non-irritant material, with an extremely low pro-inflammatory potential. These results denote a good biocompatibility of hBN at the skin level

Occupational exposure to graphene based nanomaterials: Risk assessment

Graphene-based materials (GBMs) are a family of novel materials including graphene, few layer graphene (FLG), graphene oxide (GO), reduced graphene oxide (rGO) and graphene nanoplatelets (GNP). Currently, the risk posed by them to human health is associated mainly with the occupational exposure during their industrial and small-scale production or waste discharge. The most significant occupational exposure routes are inhalation, oral, cutaneous and ocular, inhalation being the majorly involved and most studied one. This manuscript presents a critical up-to-date review of the available in vivo toxicity data of the most significant GBMs, after using these exposure routes. The few in vivo inhalation toxicity studies (limited to 5-days of repeated exposure and only one to 5 days per week for 4 weeks) indicate inflammatory/fibrotic effects at the pulmonary level, not always reversible after 14/90 days. More limited in vivo data are available for the oral and ocular exposure routes, whereas the studies on cutaneous toxicity are at the initial stage. A long persistence of GBMs in rodents is recorded, while contradictory genotoxic data are reported. Data gap identification is also provided. Based on the available data, the occupational exposure limit cannot be determined. More experimental toxicity studies according to specific guidelines (tentatively validated for nanomaterials) and more information on the actual occupational exposure level to GBMs are needed. Furthermore, ADME (Absorption, Distribution, Metabolism, Excretion), genotoxicity, developmental and reproductive toxicity data related to the occupational exposure to GBMs have to be implemented. In addition, sub-chronic and/or chronic studies are still needed to completely exclude other toxic effects and/or carcinogenicity

Sanitary problems related to the presence of Ostreopsis spp. in the Mediterranean Sea: a multidisciplinary scientific approach

The increased presence of potentially toxic microalgae in the Mediterranean area is a matter of great concern. Since the end of the last century, microalgae of the genus Ostreopsis have been detected more and more frequently in the Italian coastal waters. The presence of Ostreopsis spp. has been accompanied by the presence of previously undetected marine biotoxins (palytoxins) into the ecosystem with the increased possibility of human exposure. In response to the urgent need for toxicity characterization of palytoxin and its congeners, an integrated study encompassing both in vitro and in vivo methods was performed

Graphene-based materials are not skin sensitizers: adoption of the in chemico/in vitro OECD test guidelines

The boost in the market size of graphene-based materials (GBMs) requires a careful evaluation of their impact on human health, acquiring robust and reliable data, also suitable for regulatory purposes. Considering cutaneous contact as one of the most relevant GBM exposure routes, this study is focused on skin sensitization, aimed at assessing the possibility to adopt the three in chemico/in vitro test guidelines (TGs) defined by the Organization for Economic Cooperation and Development (442C, D and E) to predict the first three phases of the skin sensitization adverse outcome pathway. Being originally validated for chemicals, modifications allowing their adoption for GBMs were evaluated. TG 442C was found to be not suitable for testing GBMs due to their reactivity, leading to possible misclassifications. In contrast, TG 442D and E can generally be applied for GBMs. However, protocol adjustments were required to assess cell viability reducing interferences for TG 442D, whereas caution should be exercised regarding dose-finding selection and GBM dispersion stability for TG 442E. When applying these modifications, GBMs were found to be unable to activate keratinocytes and promote dendritic cell differentiation, so they can be considered non-sensitizers. Overall, these results significantly contribute to understanding the safety profiles of GBMs and to improve testing methodologies to obtain reliable toxicological data