Roberto Chiarelli

Heavy metals and metalloids as autophagy inducing agents: focus on cadmium and arsenic.

In recent years, research on the autophagic process has greatly increased, invading the fields of biology and medicine. Several markers of the autophagic process have been discovered and various strategies have been reported studying this molecular process in different biological systems in both physiological and stress conditions. Furthermore, mechanisms of metalloid- or heavy metal-induced toxicity continue to be of interest given the ubiquitous nature and distribution of these contaminants in the environment where they often play the role of pollutants of numerous organisms. The aim of this review is a critical analysis and correlation of knowledge of autophagic mechanisms studied under stress for the most common arsenic (As) and cadmium (Cd) compounds. In this review we report data obtained in different experimental models for each compound, highlighting similarities and/or differences in the activation of autophagic processes. A more detailed discussion will concern the activation of autophagy in Cd-exposed sea urchin embryo since it is a suitable model system that is very sensitive to environmental stress, and Cd is one of the most studied heavy metal inductors of stress and modulator of different factors such as: protein kinase and phosphatase, caspases, mitochondria, heat shock proteins, metallothioneins, transcription factors, reactive oxygen species, apoptosis and autophagy.

Sea urchin embryos as a model system for studying autophagy induced by cadmium stress

It is well known that sea urchin embryos are able to activate different defense strategies against stress. We previously demonstrated that cadmium treatment triggers the accumulation of metal in embryonic cells and the activation of defense systems depending on concentration and exposure time, through the synthesis of heat shock proteins and/or the initiation of apoptosis. Here we show that Paracentrotus lividus embryos exposed to Cd adopt autophagy as an additional stratagem to safeguard the developmental program. At present, there are no data focusing on the role of this process in embryo development of marine organisms.

Marine Invertebrates as Bioindicators of Heavy Metal Pollution

Atmosphere, earth and water compose the environment. The presence of heavy metals in the environment has grown because of their large employment in some industrial and agricultural activities. Although these metals are terrestrial products, they flow into the sea through effluents and sewage or are directly discharged from industries placed on the seawater front. It should be considered that metals concentrations vary widely according to different seawater latitudes and depths and can be strongly influenced by fresh water discharges from heavily polluted rivers. In this review recent studies on heavy metal pollution in marine ecosystems and their organisms will be presented. Metal speciation, bioaccumulation in biota, as well as abiotic and biotic factors affecting their bioavailability will be reviewed. Moreover, the use of bioindicator organisms for the biomonitoring of heavy metal toxicity and their ecological effects will be defined. Many marine invertebrate species fulfill the following criteria: Sensitivity to a wide range of chemicals (especially to heavy metals), cost-effectiveness for repeatable tests, readily interpretable biological consequences of pollution. Among the most important marine invertebrates used as bioindicators, the sea urchin embryo is one of the most suitable, especially to assess metal/heavy metal pollution.

Sea urchin embryos exposed to cadmium as an experimental model for studying the relationship between autophagy and apoptosis

The sea urchin embryo is a suitable model that offers an excellent opportunity to investigate different defence strategies activated in stress conditions. We previously showed that cadmium accumulates in a dose- and time-dependent manner into embryonic cells, activating different stress and defence mechanisms, including the synthesis of HSPs and the onset of apoptosis and/or autophagy. In this paper we investigated the functional relationship between autophagy and apoptosis, evaluating apoptosis signals in cadmium-exposed Paracentrotus lividus embryos with inhibited autophagy. We found that the inhibition of autophagy produced the concurrent reduction of apoptosis, suggesting that the two phenomena are functionally related. Considering the catabolic role of autophagy, an energetic hypothesis to explain the relationship was evaluated. Using a substrate for ATP production, we found that apoptosis, assessed by TUNEL and cleaved caspase-3 immunocytochemistry, was substantially restored in cadmium-treated embryos where autophagy was inhibited by 3-Methyladenine. On the basis of these results, we propose that, autophagy could play a crucial role in stress response of this organism because autophagy could energetically contribute to apoptotic execution through its catabolic role.

Autophagy is required for sea urchin oogenesis and early development

Autophagy is a major intracellular pathway for the degradation and recycling of cytosolic components. Emerging evidence has demonstrated its crucial role during the embryo development of invertebrates and vertebrates. We recently demonstrated a massive activation of autophagy in Paracentrotus lividus embryos under cadmium stress conditions, and the existence of a temporal relationship between induced autophagy and apoptosis. Although there have been numerous studies on the role of autophagy in the development of different organisms, information on the autophagic process during oogenesis or at the start of development in marine invertebrates is very limited. Here we report our recent data on the occurrence of autophagy at these key phases of development. In order to investigate autophagy trends we performed in vivo assays to detect autophagolysomes, as well as in situ analysis with anti-LC3 antibody to detect autophagosomes before the fusion with lysosomes. From data generated through confocal laser scanning microscopy and quantification of autophagic signals we have drawn several unequivocal conclusions. The results showed a copious and rising number of autophagic organelles that had specific localization. Interestingly the increase in autophagy that occurred just after fertilization has been proved to be crucial for correct initiation of the developmental programme: irreversible developmental delays and morphologic anomalies were induced by short autophagic inhibition. This work focused on the sea urchin model system and corroborates evidence on the need for self-digestion during development, enriching the knowledge on autophagy, a biological mechanism belonging to evolutionarily different organisms.

Autophagy as a defense strategy against stress: focus on Paracentrotus lividus sea urchin embryos exposed to cadmium

Autophagy is used by organisms as a defense strategy to face environmental stress. This mechanism has been described as one of the most important intracellular pathways responsible for the degradation and recycling of proteins and organelles. It can act as a cell survival mechanism if the cellular damage is not too extensive or as a cell death mechanism if the damage/stress is irreversible; in the latter case, it can operate as an independent pathway or together with the apoptotic one. In this review, we discuss the autophagic process activated in several aquatic organisms exposed to different types of environmental stressors, focusing on the sea urchin embryo, a suitable system recently included into the guidelines for the use and interpretation of assays to monitor autophagy. After cadmium (Cd) exposure, a heavy metal recognized as an environmental toxicant, the sea urchin embryo is able to adopt different defense mechanisms, in a hierarchical way. Among these, autophagy is one of the main responses activated to preserve the developmental program. Finally, we discuss the interplay between autophagy and apoptosis in the sea urchin embryo, a temporal and functional choice that depends on the intensity of stress conditions.

The histone deacetylase inhibitor JAHA down-regulates pERK and global DNA methylation in MDA-MB231 breast cancer cells

The histone deacetylase inhibitor N1-(ferrocenyl)-N8-hydroxyoctanediamide (JAHA) down-regulates extracellular-signal-regulated kinase (ERK) and its activated form in triple-negative MDA-MB231 breast cancer cells after 18 h and up to 30 h of treatment, and to a lesser extent AKT and phospho-AKT after 30 h and up to 48 h of treatment. Also, DNA methyltransferase 1 (DNMT1), 3b and, to a lesser extent, 3a, downstream ERK targets, were down-regulated already at 18 h with an increase up to 48 h of exposure. Methylation-sensitive restriction arbitrarily-primed (MeSAP) polymerase chain reaction (PCR) analysis confirmed the ability of JAHA to induce genome-wide DNA hypomethylation at 48 h of exposure. Collective data suggest that JAHA, by down-regulating phospho-ERK, impairs DNMT1 and 3b expression and ultimately DNA methylation extent, which may be related to its cytotoxic effect on this cancer cytotype.

Relationship between apoptosis and survival molecules in human cumulus cells as markers of oocyte competence

To select from a single patient the best oocytes able to reach the blastocyst stage, we searched for valuable markers for oocytes competence. We evaluated the DNA fragmentation index (DFI) and the level of some survival molecules, such as AKT, pAKT and pERK1/2, in individual cumulus cell-oocyte complexes (COC). The study included normo-responder women. The average age of the patients was 34.3. DFI in cumulus cells was evaluated using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) assay in situ. AKT, pAKT and pERK1/2 were measured by immunological assay and densitometric analysis of fluorescent signals using NIS-Elements BR 3.10 image software. Statistical analysis was performed using STATA SE/14.1. The study focused on 53 patients involved after informed consent. Out of 255 MII oocytes, 197 were fertilized and the derived embryos had the following evolution: 117 completed the development to blastocyst and were transferred to uterus; 57 were vitrified at the blastocyst stage; and 23 were arrested during in vitro culture at different stages of cleavage. We found a significant statistical difference between the DFI of cumulus cells of the arrested embryos and the transferred blastocysts (P = 0.004), confirming that DFI could be considered as a valuable marker of oocyte competence. In addition, the pAKT/DFI ratio was higher in cumulus cells of oocytes able to produce blastocysts, indicating that DFI is significantly lower when pAKT is higher (P = 0.043). This study demonstrates for the first time that the relationship between apoptosis and survival molecules can be used as a marker to select the best oocytes.

The Role of Autophagy and Apoptosis During Embryo Development

Programmed cell death (PCD) and cell survival are two sides of the same coin. Autopha‐ gy and apoptosis are crucial processes during embryo development of Invertebrates and Vertebrates organisms, as they are necessary for the formation of a new organism, start‐ ing from a fertilized egg. Fertilization triggers cell remodeling from each gamete to a toti‐ potent zygote. During embryogenesis, the cells undergo various processes, thus allowing the transformation of the embryo into an adult organism. In particular, cells require the appropriate tools to suddenly modify their morphology and protein content in order to respond to intrinsic and external stimuli. Autophagy and apoptosis are involved in cell proliferation, differentiation and morphogenesis. Programmed cell death is a key physio‐ logical mechanism that ensures the correct development and the maintenance of tissues and organs homeostasis in multicellular organisms. PCD has been classified into three types, according to the morphology that the dying cells acquire and the molecular machi‐ nery involved: PCD type I or apoptosis; PCD type II or autophagy and PCD type III or necrosis (not involved in physiological development). These different types of cell death have specific features that can be used to be identified and characterized. Apoptosis is a highly conserved, genetically-controlled process through which certain cells destroy themselves. Autophagy is an evolutionarily conserved pathway used by eukaryotes for degrading and recycling various cellular constituents, such as long-lived proteins and en‐ tire organelles, that was mainly detected in those tissues where abundant cell death is re‐ quired. Both autophagy and apoptosis are induced under stress conditions as an adaptive response against stress. Usually, environmental stress cause severe effects on embryonic development. Embryos of different species, exposed to different types of physical or chemical stress, temporarily suspend their development and activate several protective strategies, including PCD II and PCD III. Research has yet to elucidate the interplay be‐ tween these key processes. Not all types of PCD are always detected in association with a developmental process. Unlike the degeneration of tissues of some invertebrates, the tis‐ sues of vertebrates undergo PCD preferentially through an apoptotic mechanisms. In this chapter, we will briefly describe some specific features of apoptotic and autophagic proc‐ esses. We will focus our attention in some useful model systems of invertebrates and ver‐ tebrates organisms, where autophagy and apoptosis occur both in physiological and stress conditions; specifically, we will analyze embryos of: the nematode Caenorhabditis el‐

APOPTOSIS RATE IN CUMULUS CELLS AS POSSIBLE MOLECULAR BIOMARKER FOR OOCYTE COMPETENCE.

Several lines of evidence showed that apoptosis rate of cumulus cells in oocytes derived by assisted reproductive technologies could be used as an indicator of fertilizing gamete quality. Aim of the study was to investigate the effects of three different ovarian stimulation protocols on the biological and clinical outcome in hyporesponder patients. Collected data showed a higher significant rate of DNA fragmentation index (DFI) in U group (patients treated with Highly Purified human Menopausal Gonadotrophin) than in P group (treated with recombinant human Follicle Stimulating Hormone (r-hFSH) combined with recombinant human Luteinizing Hormone (r-hLH)). Both groups R (treated with r-hFSH alone) and P showed a significant increase in collected and fertilized oocytes number, embryo quality number. This study showed that combined r-hFSH/r-hLH therapy could represent the best pharmacological strategy for controlled ovarian stimulation and suggests to use DFI as a biomarker of ovarian function in hyporesponder patients.