Sabrina Colafarina

Scavenging system efficiency is crucial for cell resistance to ROS-mediated methylglyoxal injury.

Methylglyoxal is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. Recent research indicates that methylglyoxal is a potent growth inhibitor and genotoxic agent. The antiproliferative activity of methylglyoxal has been investigated for pharmacological application in cancer chemotherapy. However, various cells are not equally sensitive to methylglyoxal toxicity. Therefore, it would be important to establish the cellular factors responsible for the different cell-type specific response to methylglyoxal injury, in order to avoid the risk of failure of a therapy based on increasing the intracellular level of methylglyoxal. To this purpose, we comparatively evaluated the signaling transduction pathway elicited by methylglyoxal in human glioblastoma (ADF) and neuroblastoma (SH-SY 5Y) cells. Results show that methylglyoxal causes early and extensive reactive oxygen species generation in both cell lines. However, SH-SY 5Y cells show higher sensitivity to methylglyoxal challenge due to a defective antioxidant and detoxifying ability that, preventing these cells from an efficient scavenging action, elicits extensive caspase-9 dependent apoptosis. These data emphasize the pivotal role of antioxidant and detoxifying systems in determining the grade of sensitivity of cells to methylglyoxal.

Methylglyoxal induces oxidative stress-dependent cell injury and up-regulation of interleukin-1β and nerve growth factor in cultured hippocampal neuronal cells

Methylglyoxal (MG) is one of the most powerful glycating agents of proteins and other important cellular components and has been shown to be toxic to cultured cells. Under hyperglycaemic conditions, an increase in the concentration of MG has been observed in human body fluids and tissues that seems to be responsible for diabetic complications. Recent data suggest that diabetes may cause impairment of cognitive processes, according to a mechanism involving both oxidative stress and advanced glycation end product (AGE) formation. In this work, we explored the molecular mechanism underlying MG toxicity in neural cells, by investigating the effect of MG on both the interleukin-1beta (IL-1beta), as the major inducer of the acute phase response, and the nervous growth factor (NGF) expression. Experiments were performed on cultured neural cells from rat hippocampus, being this brain region mostly involved in cognitive processes and, therefore, possible target of diabetes-mediated impairment of cognitive abilities. Results show that MG treatment causes in hippocampal neural cells extensive, oxidative stress-mediated cell death, in consequence of a strong catalase enzymatic activity and protein inhibition. MG also causes a very significant increase in both transcript and protein expression of the NGF as well as of the pro-inflammatory cytokine IL-1beta. MG co-treatment with the antioxidant N-acetylcysteine (NAC) completely abrogates the observed effects. Taken together, these data demonstrate that hippocampal neurons are strongly susceptible to MG-mediated oxidative stress.

AGE-DEPENDENT ULTRASTRUCTURAL ALTERATIONS AND BIOCHEMICAL RESPONSE OF RAT SKELETAL MUSCLE AFTER HYPOXIC OR HYPEROXIC TREATMENTS

This work deals with the antioxidant enzymatic response and the ultrastructural aspects of the skeletal muscle of young and aged rats kept under hypoxic or hyperoxic normobaric conditions. It is in fact well known that the supply of oxygen at concentrations higher or lower than those occurring under normal conditions can promote oxidative processes that can cause tissue damage. The enzymes investigated were both those directly involved in reactive oxygen species (ROS) scavenging (superoxide dismutase, catalase and selenium-dependent glutathione peroxidase), and those challenged with the detoxication of cytotoxic compounds produced by the action of ROS on biological molecules (glutathione transferase, glyoxalase I, glutathione reductase), in order to obtain a comparative view of the defence strategies used with respect to aging. Our results support the hypothesis that one of the major contributors to the aging process is the oxidative damage produced at least in part by an impairment of the antioxidant enzymatic system. This makes the aged organism particularly susceptible to oxidative stress injury and to the related degenerative diseases, especially in those tissues with high demand for oxidative metabolism.

PPARγ-dependent effects of conjugated linoleic acid on the human glioblastoma cell line (ADF)

Conjugated linoleic acid (CLA) has been shown to exert beneficial effects against carcinogenesis, atherosclerosis and diabetes. It has been demonstrated that CLA modulates lipid metabolism through the activation of peroxisome proliferator‐activated receptors (PPARs). The PPAR family comprises 3 closely related gene products, PPAR α, β/δ and γ, differing for tissue distribution, developmental expression and ligand specificity. It has also been demonstrated that activated PPARγ results in growth inhibition and differentiation of transformed cells. These observations stimulated a great interest toward PPARγ ligands as potential anticancer drugs to be used in a differentiation therapy. Glioblastomas are the most commonly diagnosed primary tumors of the brain in humans. The prognosis of patients with high‐grade gliomas is poor and only marginally improved by chemotherapy. The aim of this work was to study the effects of CLA and of a specific synthetic PPARγ ligand on cell growth, differentiation and death of a human glioblastoma cell line as well as on parameters responsible for the metastatic behavior of this tumor. We demonstrate here that CLA and PPARγ agonist strongly inhibit cell growth and proliferation rate and induce apoptosis. Moreover, both treatments decrease cell migration and invasiveness. The results obtained show that CLA acts, directly or indirectly, as a PPARγ activator, strongly suggesting that this naturally occurring fatty acid may be used as brain antitumor drug and as a chemopreventive agent. Moreover, the γ‐agonist, once experimented and validated on man, may represent a useful coadjuvant in glioblastoma therapy and in the prevention of recurrences.

Morphofunctional mitochondrial response to methylglyoxal toxicity in Bufo bufo embryos.

Methylglyoxal (2-oxopropanal) is a reactive alpha-oxoaldehyde that can be formed endogenously mainly as a by-product of glycolytic pathway. It is a cytotoxic compound with significant antiproliferative properties as it can bind, under physiological conditions, to nucleic acids and proteins, forming stable adducts. We have recently shown that exogenous methylglyoxal (150-600 microM) is highly toxic for amphibian embryos where it produces, when added to the culture water, inhibition of cell proliferation in the early developmental stages, followed by severe malformations and strongly reduced embryonic viability. In this work we investigate the morphofunctional effect of methylglyoxal on the common toad B. bufo embryo mitochondria in order to verify if its dysmorphogenetic action might be also ascribed to impairment of mitochondrial functions. The mitochondria were isolated from embryos at the developmental stages of morula, neural plate and operculum complete and developing in the presence of 600 microM methylglyoxal. The results show that exogenous methylglyoxal is highly toxic at mitochondrial level, where it produces proliferation, swelling and membrane derangement. As a consequence, mitochondria from treated embryos show decreased oxidative phosphorylation efficiency, as indicated by the significant reduction both of the respiratory control index values and of the embryonic ATP content. On the basis of these data, it is possible that the methylglyoxal-induced embryonic malformations as well as the strongly reduced viability might be also ascribed to energy depletion.

Transgenerational Effects of NMs

Nanomaterials are present in a number of commercially available products but there are uncertainties as to whether the unique properties that support their commercial use may also pose potential health risks. Information is missing concerning the influence of nanomaterials on the overall reproductive outcome and transgenerational effects in animals and plants. To obtain this information, long-term studies would be required using animal models phylogenetically close to humans and exposure conditions that reflect realistic scenarios with regard to dosages and admission. The nanoreprotoxicology literature published to date is largely descriptive in nature regarding the effects of nanoparticles. The mechanisms, which determine particle reproduction compatibility, are mostly elusive at the moment. Thus, it is recommended that future research explore the interactions between nanomaterials and transgenerational matter on a molecular level. It would, for instance, be of major importance to understand the behaviour of nanoparticles inside the cells but also their genotoxic and epigenetic effects. Recent studies have shown that intravenous and/or intra-abdominal administration of nanoparticles to mice results in their accumulation in the cells of many tissues, including the brain and the testis, suggesting that they easily pass through the blood-brain and blood-testis barriers. In parallel embryo development after exposure to nanoparticles should be comparatively investigated. The majority of studies on embryo toxicology have concentrated on piscine embryos, mostly derived from zebrafish. Plants for human food as an important component of the ecosystem need also to be taken into account when evaluating transgenerational effects of engineered nanomaterials in crops.

Antioxidant and GSH-related enzyme response to a single teratogenic exposure to the anticonvulsant phenytoin: Temporospatial evaluation

BACKGROUND It has been proposed that the anticonvulsant drug phenytoin (PHT) requires bioactivation to reactive intermediate(s) to achieve its recognized teratogenic potential and that embryonal detoxification power may play a fundamental role in the teratogenic response. On this basis, we sought to investigate the potential effects of a teratogenic exposure to PHT on the activities of antioxidant and GSH-related detoxifying enzymes in gestational murine tissues. METHODS Pregnant Swiss mice were injected intraperitoneally with 0 (vehicle) or 65 mg/kg of PHT on gestation day (GD) 12 (plug day = GD 1). Biochemical determinations, including activities of glutathione transferase, glutathione peroxidase, glutathione reductase, glyoxalase I, glyoxalase II, catalase, and superoxide dismutase, were carried out on maternal and embryonic/fetal livers and in placentas on GD 14 and 19. RESULTS The major findings of this study show that (1) organogenesis-stage conceptal tissues have detectable levels of all the tested enzymes; (2) most of the embryonic liver and placental enzymes investigated undergo a significant induction within 48 hr (GD 14) after PHT administration; and (3) in the same tissues a down-regulation of enzyme activities is noted near term (GD 19). CONCLUSIONS Overall, these findings show that teratogenic exposure to PHT is associated with a modulation of reactive-intermediates-scavenging enzyme activities, and provide further support for role of generation of reactive intermediates in PHT-induced teratogenesis.

Tyrosinase expression during black truffle development: From free living mycelium to ripe fruit body

The present work studies the expression of tyrosinase (monophenol:diphenol oxygen oxidoreductase, EC 1.14.18.1) during the development of the black truffle Tuber melanosporum Vittad., an ectomycorrhizal fungus of great biological and economic interest. As widely reported in the literature, melanins and the enzymes that synthesize them, are of paramount importance in fungal development and sexual differentiation. Tyrosinase and laccase are the enzymes that produce melanins from monophenols and diphenols. We have detected tyrosinase expression from the stage of free living mycelium, through the mychorrizal stage and the six fruit body developmental stages by measuring the levels of tyrosinase mRNA by quantitative PCR (q-PCR), spectrophotometry, histochemistry, immunohistochemistry and electrophoresis. Tyrosinase is always expressed, from the free living mycelium to the ripe fruit body developmental stages, when it is very low. The switching off of the tyrosinase gene during T. melanosporum development when the fruit body is ripe and no more cell walls are to be built is discussed in relation of thioflavour production. Specific primers, prepared from the cloned T. melanosporum tyrosinase cDNA were used for the q-PCR and the deduced aminoacid sequences of the CuA and CuB binding sites were compared to those of various ascomycetes and basidiomycetes.

Human glioblastoma ADF cells express tyrosinase, L-tyrosine hydroxylase and melanosomes and are sensitive to L-tyrosine and phenylthiourea.

Melanocytes and neuroblasts share the property of transforming L‐tyrosine through two distinct metabolic pathways leading to melanogenesis and catecholamine synthesis, respectively. While tyrosinase (TYR) activity has been shown to be expressed by neuroblastoma it remains to be established as to whether also glioblastomas cells are endowed with this property. We have addressed this issue using the human continuous glioblastoma cell line ADF. We demonstrated that these cells possess tyrosinase as well as L‐tyrosine hydroxylase (TH) activity and synthesize melanosomes. Because the two pathways are potentially cyto‐genotoxic due to production of quinones, semiquinones, and reactive oxygen species (ROS), we have also investigated the expression of the peroxisomal proliferators activated receptor α (PPARα) and nuclear factor‐kB (NFkB) transcription factor as well the effect of L‐tyrosine concentration on cell survival. We report that L‐tyrosine down‐regulates PPARα expression in ADF cells but not neuroblastoma and that this aminoacid and phenylthiourea (PTU) induces apoptosis in glioblastoma and neuroblastoma.

Glutathione dependent enzymes and antioxidant defences in truffles : organisms living in microaerobic environments

Truffles live in an environment poor in oxygen, and are a good model for investigation of enzymatic adaptation to microaerobic conditions. We studied the antioxidant and glutathione dependent enzymatic endowment of Tuber truffles. Superoxide dismutase, catalase, glutathione peroxidase Se dependent, glutathione reductase, glyoxalase 1 and glyoxalase 2 are expressed and correlate with the microaerobic metabolism, growth rate and mycorrhizal symbiosis of truffles. A very low or undetectable glutathione S-transferase activity was found. For comparison the same enzyme activities were investigated in Agaricus bisporus which is epigeous and non-mycorrhizal.