Imogolite: an aluminosilicate nanotube endowed with low cytotoxicity and genotoxicity.

High-aspect-ratio nanomaterials (HARN) (typically, single-walled carbon nanotubes (SWCNT) or multiwalled carbon nanotubes (MWCNT)) impair airway barrier function and are toxic to macrophages. Here, we assess the biological effects of nanotubes of imogolite (INT), a hydrated alumino-silicate [(OH)3Al2O3SiOH] occurring as single-walled NT, on murine macrophages and human airway epithelial cells. Cell viability was assessed with resazurin. RT-PCR was used to study the expression of Nos2 and Arg1, markers of classical or alternative macrophage activation, respectively, and nitrite concentration in the medium was determined to assess NO production. Epithelial barrier integrity was evaluated from the trans-epithelial electrical resistance (TEER). Potential genotoxicity of INT was assessed with comet and cytokinesis-block micronucleus cytome assays. Compared to MWCNT and SWCNT, INT caused much smaller effects on RAW264.7 and MH-S macrophage viability. The incubation of macrophages with INT at doses as high as 120 µg/cm(2) for 72 h did not alter either Nos2 or Arg1 expression nor did it increase NO production, whereas IL6 was induced in RAW264.7 cells but not in MH-S cells. INT did not show any genotoxic effect in RAW264.7 and A549 cells except for a decrease in DNA integrity observed in epithelial A549 cells after treatment with the highest dose (80 µg/cm(2)). No significant change in permeability was recorded in Calu-3 epithelial cell monolayers exposed to INT, whereas comparable doses of both SWCNT and MWCNT lowered TEER. Thus, in spite of their fibrous nature, INT appear not to be markedly toxic for in vitro models of lung-blood barrier cells.

Region-specific DNA alterations in focally induced seizures.

We induced brief secondarily generalized seizures of limbic origin in Sprague-Dawley rats by bicuculline microinfusion into the anterior piriform cortex. After 1 h or 5 days we performed comet assay, a sensitive marker for DNA damage, within entorhinal cortex, hippocampus (limbic areas recruited by seizure spreading) and striatum (which is not recruited). DNA damage occurred selectively in the ipsilateral entorhinal cortex and hippocampus at 1 h, but not at 5 days. These data shed new light on molecular genetics as a marker during limbic seizures, the most common in epileptic patients.

A novel fungal metabolite with beneficial properties for agricultural applications.

Trichoderma are ubiquitous soil fungi that include species widely used as biocontrol agents in agriculture. Many isolates are known to secrete several secondary metabolites with different biological activities towards plants and other microbes. Harzianic acid (HA) is a T. harzianum metabolite able to promote plant growth and strongly bind iron. In this work, we isolated from the culture filtrate of a T. harzianum strain a new metabolite, named isoharzianic acid (iso-HA), a stereoisomer of HA. The structure and absolute configuration of this compound has been determined by spectroscopic methods, including UV-Vis, MS, 1D and 2D NMR analyses. In vitro applications of iso-HA inhibited the mycelium radial growth of Sclerotinia sclerotiorum and Rhizoctonia solani. Moreover, iso HA improved the germination of tomato seeds and induced disease resistance. HPLC-DAD experiments showed that the production of HA and iso HA was affected by the presence of plant tissue in the liquid medium. In particular, tomato tissue elicited the production of HA but negatively modulated the biosynthesis of its analogue iso-HA, suggesting that different forms of the same Trichoderma secondary metabolite have specific roles in the molecular mechanism regulating the Trichoderma plant interaction.

Genotoxicity of amorphous silica particles with different structure and dimension in human and murine cell lines.

Although amorphous silica is used in food products, cosmetics and paints and as vector for drug delivery, data on its potential health hazard are limited. The aim of this study was to investigate the cytotoxic and genotoxic potential of silica particles of different sizes (250 and 500nm) and structures (dense and mesoporous). Dense silica (DS) spheres were prepared by sol-gel synthesis, mesoporous silica particles (MCM-41) were prepared using hexadecyltrimethyl ammonium bromide as a structure-directing agent and tetraethylorthosilicate as silica source. Particles were accurately characterised by dynamic light scattering, nitrogen adsorption, X-ray diffraction and field emission scanning electron microscopy. Murine macrophages (RAW264.7) and human epithelial lung (A549) cell lines were selected for investigation. Genotoxicity was evaluated by Comet assay and micronucleus test. Cytotoxicity was tested by the trypan blue method. Cells were treated with 0, 5, 10, 20, 40 and 80 µg/cm(2) of different silica powders for 4 and 24 h. The intracellular localisation of silica was investigated by transmission electron microscopy. Amorphous particles penetrated into the cells, being compartmentalised within endocytic vacuoles. DS and MCM-41 particles induced cytotoxic and genotoxic effects in A549 and RAW264.7 although to different extent in the two cell lines. A549 were resistant in terms of cell viability, but showed a generalised induction of DNA strand breaks. RAW264.7 were susceptible to amorphous silica exposure, exhibiting both cytotoxic and genotoxic responses as DNA strand breaks and chromosomal alterations. The cytotoxic response of RAW264.7 was particularly relevant after MCM-41 exposure. The genotoxicity of amorphous silica highlights the need for a proper assessment of its potential hazard for human health.

Prefrontal dynamics and encoding of flexible rule switching.

Behavioral flexibility, the ability to adjust behavioral strategies in response to changing environmental contingencies and internal demands, is fundamental to cognitive functions. Despite a large body of pharmacology and lesion studies, the underlying neurophysiological correlates and mechanisms that support flexible rule switching remain elusive. To address this question, we trained mice to distinguish complex sensory cues comprising different perceptual dimensions (set shifting). Endoscopic calcium imaging revealed that medial prefrontal cortex (mPFC) neurons represented multiple task-related events and exhibited pronounced dynamic changes during rule switching. Notably, prominent encoding capacity in the mPFC was associated with switching across, but not within perceptual dimensions. We then showed the involvement of the ascending modulatory input from the locus coeruleus (LC), as inhibiting the LC impaired rule switching behavior and impeded mPFC dynamic processes and encoding. Our results highlight the pivotal role of the mPFC in set shifting processes and demonstrate the profound impact of ascending neuromodulation on shaping prefrontal neural dynamics and behavioral flexibility.

Cerinolactone, a hydroxy-lactone derivative from Trichoderma cerinum.

A novel metabolite, 3-hydroxy-5-(6-isopropyl-3-methylene-3,4,4a,5,6,7,8,8a-octahydronaphthalen-2-yl)dihydrofuran-2-one, trivially named cerinolactone (1), has been isolated from culture filtrates of Trichoderma cerinum together with three known butenolides containing the 3,4-dialkylfuran-2(5H)-one nucleus, harzianolide (2), T39butenolide (3), and dehydroharzianolide (4). The structure of 1 was determined by spectroscopic methods, including UV, MS, and 1D and 2D NMR analyses. In vitro tests with the purified compound exhibited activity against Pythium ultimum, Rhizoctonia solani, and Botrytis cinerea.

Genotoxic potential of TiO2 on bottlenose dolphin leukocytes.

Titanium dioxide is extensively used in a variety of products, including industrial materials and cosmetics. Studies mainly performed on human cell lines and in vivo exposure on experimental animals have raised concern about the toxic effects of ultrafine titanium dioxide; however, scarce information is available about its impact on aquatic life. The aim of this article was to assess the genotoxic potential of TiO(2) (anatase and rutile) on bottlenose dolphin leukocytes. Blood samples were obtained from four male and one female specimens reared at the Adriatic SeaWorld "Oltremare" (Riccione, Italy). Leukocytes were isolated by the lyses procedure and in vitro exposed to TiO(2) in RPMI. Experimental solutions were sonicated immediately before dosing the cells. Three exposure times (4, 24 and 48 h) and three doses (20, 50 and 100 microg/ml) were tested. Genotoxicity was detected by the single-cell gel electrophoresis (or comet assay) at pH > or = 13, assessing single/double-strand breaks and alkali-labile sites. Cytotoxicity was also detected by the Trypan blue exclusion method. Results showed that both the crystalline forms of TiO(2) were genotoxic for bottlenose dolphin leukocytes, with a statistically significant increase of DNA fragmentation after exposure to 50 and 100 microg/ml for 24 and 48 h. Although preliminary, these are the first data regarding the genetic susceptibility of toothed cetaceans toward an "emerging" pollutant, such as TiO(2) particles.

Human pharmaceuticals in marine mussels: Evidence of sneaky environmental hazard along Italian coasts.

Despite the increasing interest for pharmaceuticals in the marine environment, their accumulation in wild organisms and consequent environmental hazards are still poorly known. The Mediterranean Sea is highly challenged by the density of coastal populations, large consumption of pharmaceuticals and their often limited removal by Wastewater Treatment Plants (WWTPs). In this respect, the present study aims to provide the first large-scale survey on the distribution of such contaminants of emerging concern in native mussels, Mytilus galloprovincialis from Italian coasts. Organisms were collected from 14 sites representative of relatively unpolluted marine waters along the Adriatic and Tyrrhenian Sea and analysed for 9 common pharmaceuticals including Non-Steroidal Anti-Inflammatory Drugs (NSAIDs: Diclofenac DIC, Ibuprofen IBU, Ketoprofen KET and Nimesulide NIM), the analgesic Acetaminophen AMP, the antiepileptic Carbamazepine CBZ, the antihypertensive Valsartan VAL, the anxiolytic Lormetazepam LOR and the antidepressant Paroxetine PAR. Results indicated the widespread occurrence of the majority of pharmaceuticals in mussel tissues: CBZ was measured in >90% of analysed samples, followed by VAL (>50%), PAR (>40%), and DIC (>30%), while only AMP and KET were never detected. Heterogeneous tissue concentrations ranged from a few units up to hundreds of ng/g (d.w.), while seasonal and interannual variability, investigated over 4 years, did not highlight any clear temporal trend. Limited differences obtained between the Adriatic and Tyrrhenian Sea, as well as coastal versus off-shore sampling sites, suggest that analysed levels of pharmaceuticals in mussels tissues should be considered as baseline concentrations for organisms collected in unpolluted areas of the Mediterranean. This study provided the first unambiguous evidence of the widespread occurrence of pharmaceuticals in marine mussels from Italian coasts, giving novel insights on the potential ecotoxicological hazard from such compounds in marine species.

Dynamics of interaction and effects of microplastics on planarian tissue regeneration and cellular homeostasis.

Increasing microplastics pollution of marine and terrestrial water is a concerning issue for ecosystems and human health. Nevertheless, the interaction of microplastics with freshwater biota is still a poorly explored field. In order to achieve information concerning the uptake, distribution and effect of microplastics in planarians, Dugesia japonica specimens have been fed with mixtures of food and differently shaped and sized plastic particles. Feeding activity and food intake were non-altered by the presence of high concentrations of different types of plastic particles. However, the persistence of microplastic within the planarian body was a function of size/shape, being small spheres (<10 µm in diameter) and short fibers (14 µm large and 5/6 µm length) more persisting than larger spheres and longer fibers which were eliminated almost entirely by ejection in a few hours. Transmission electron microscopy analysis demonstrated that at least part of microplastics was phagocytized by the enterocytes. Chronic exposure to small plastic did not alter the regenerative ability but caused a significant reduction of the gut epithelium thickness and lipid content of enterocytes, together with the induction of apoptotic cell death, modulation of Djgata 4/5/6 expression and reduced growth rate. The ability of microplastic to perturb planarian homeostasis is concerning being them extremely resilient against mechanical and chemical insults and suggests possible harmful effects upon other more susceptible species in freshwater ecosystems.

Somatostatin interneurons in the prefrontal cortex control affective state discrimination in mice.

The prefrontal cortex (PFC) is implicated in processing of the affective state of others through non-verbal communication. This social cognitive function is thought to rely on an intact cortical neuronal excitatory and inhibitory balance. Here combining in vivo electrophysiology with a behavioral task for affective state discrimination in mice, we show a differential activation of medial PFC (mPFC) neurons during social exploration that depends on the affective state of the conspecific. Optogenetic manipulations revealed a double dissociation between the role of interneurons in social cognition. Specifically, inhibition of mPFC somatostatin (SOM+), but not of parvalbumin (PV+) interneurons, abolishes affective state discrimination. Accordingly, synchronized activation of mPFC SOM+ interneurons selectively induces social discrimination. As visualized by in vivo single-cell microendoscopic Ca2+ imaging, an increased synchronous activity of mPFC SOM+ interneurons, guiding inhibition of pyramidal neurons, is associated with affective state discrimination. Our findings provide new insights into the neurobiological mechanisms of affective state discrimination.

Interactions between trace metals (Cu, Hg, Ni, Pb) and 2,3,7,8-tetrachlorodibenzo-p-dioxin in the Antarctic fish Trematomus bernacchii: oxidative effects on biotransformation pathway.

Biological effects of chemical mixtures are an emerging issue when using biomarkers in field conditions, because synergistic or inhibitory interactions, cascade, and indirect mechanisms can both enhance or suppress responses to specific classes of pollutants. The Antarctic rock cod (Trematomus bernacchii) was exposed to various trace metals (Cu, Hg, Ni, and Pb) and to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dosed alone or in combination, to generate hypotheses regarding the mechanisms of action at the cellular level. Concentrations of metals significantly increased in liver of T. bernacchii without significant differences between organisms exposed only to the elements or cotreated with TCDD. The marked induction of cytochrome P450 (CYP) by TCDD was greatly suppressed by cotreatment with Cu at both catalytic and protein expression levels, whereas no significant effects were caused by coexposures with Hg, Ni, and Pb. The oxidative status was measured by integrating individual antioxidants with the total oxyradical scavenging capacity toward peroxyl radicals (ROO*) and hydroxyl radicals (*OH). Synergistic oxidative effects appeared in fish coexposed to Cu and TCDD, suggesting that biotransformation efficiency is down-regulated by higher levels of hydrogen peroxide, hydroxyl radicals, and/or mechanisms limiting availability of heme groups. Major oxidative perturbations also were observed in organisms coexposed to TCDD and Hg; in these organisms, however, the marked increase of glutathione content could explain the absence of oxidative inhibition of the CYP system. More limited oxidative effects were caused by Ni and Pb, and the contemporary absence of inhibitory effects on CYP would further confirm the importance of pro-oxidant mechanisms in modulating the biotransformation pathway. The overall results indicate that a wide battery of biomarkers is necessary to assess the impact of chemical mixtures in field conditions.

Oxytocin Signaling in the Central Amygdala Modulates Emotion Discrimination in Mice.

Recognition of other's emotions influences the way social animals interact and adapt to the environment. The neuropeptide oxytocin (OXT) has been implicated in different aspects of emotion processing. However, the role of endogenous OXT brain pathways in the social response to different emotional states in conspecifics remains elusive. Here, using a combination of anatomical, genetic, and chemogenetic approaches, we investigated the contribution of endogenous OXT signaling in the ability of mice to discriminate unfamiliar conspecifics based on their emotional states. We found that OXTergic projections from the paraventricular nucleus of the hypothalamus (PVN) to the central amygdala (CeA) are crucial for the discrimination of both positively and negatively valenced emotional states. In contrast, blocking PVN OXT release into the nucleus accumbens, prefrontal cortex, and hippocampal CA2 did not alter this emotion discrimination. Furthermore, silencing each of these PVN OXT pathways did not influence basic social interaction. These findings were further supported by the demonstration that virally mediated enhancement of OXT signaling within the CeA was sufficient to rescue emotion discrimination deficits in a genetic mouse model of cognitive liability. Our results indicate that CeA OXT signaling plays a key role in emotion discrimination both in physiological and pathological conditions.

Dioxin-like compounds bioavailability and genotoxicity assessment in the Gulf of Follonica, Tuscany (Northern Tyrrhenian Sea).

The Gulf of Follonica (Italy) is impacted by the chemical pollution from ancient mining activity and present industrial processes. This study was aimed to determine the bioavailability of dioxin-like compounds (DLCs) in coastal marine environment and to assess the genotoxic potential of waste waters entering the sea from an industrial canal. Moderately high levels of DCLs compounds (∑ PCDDs + PCDFs 2.18­29.00 pg/g dry wt) were detected in Mytilus galloprovincialis transplanted near the waste waters canal and their corresponding Toxic Equivalents (TEQs) calculated. In situ exposed mussels did not show any genotoxic effect (by Comet and Micronucleus assay). Otherwise, laboratory exposure to canal waters exhibited a reduced genomic template stability (by RAPD-PCR assay) but not DNA or chromosomal damage. Our data reveal the need to focus on the levels and distribution of DLCs in edible species from the study area considering their potential transfer to humans through the consumption of sea food.

Oxidative and modulatory effects of trace metals on metabolism of polycyclic aromatic hydrocarbons in the Antarctic fish Trematomus bernacchii.

Biological interactions between various classes of pollutants are of great relevance for the Antarctic marine environment, where the naturally elevated bioavailability of metals like cadmium might indirectly influence sensitivity of endemic organisms toward other environmental pollutants, e.g. polycyclic aromatic hydrocarbons (PAHs). To further investigate reciprocal effects of different chemicals, the fish Trematomus bernacchii was exposed to trace metals (Cd, Cu, Hg, Ni, Pb) and benzo[a]pyrene (BaP, as a model PAH), dosed alone and in combinations. Co-exposures revealed that BaP did not influence the accumulation of metals, while these elements caused significant changes on tissue levels of the PAH. The marked EROD induction caused by BaP was completely suppressed by co-exposure with Cd and Cu, but no effects were observed with Ni, Hg and Pb. Similar results were confirmed at the protein level by Western blot analyses while CYP1A1 mRNA levels were reduced only during Cd co-exposures. Clear evidence of oxidative perturbations was observed in fish co-treated with Cd and BaP and the reduced capability to absorb peroxyl and hydroxyl radicals suggested some oxidative pathways by which this element might indirectly modulate the biotransformation efficiency of Cytochrome P450. Partly different and post-transcriptional mechanisms of action could be hypothesized for Cu, while moderate oxidative effects of Hg, Ni and Pb during co-exposures would confirm their limited influence on metabolism of PAHs. In general, the overall results revealed a complex pathway of interactions between different chemicals during co-exposures and the importance of oxidative status in modulating induction and expression of CYP1A1.

Presence and inducibility by beta-naphthoflavone of CYP1A1, CYP1B1 and phase II enzymes in Trematomus bernacchii, an Antarctic fish.

This study investigated some aspects of xenobiotic metabolism in the Nototheniidae Trematomus bernacchii, a key sentinel species for monitoring Antarctic ecosystems. After laboratory exposure to beta-naphthoflavone (betaNF), basal levels and time-course induction of CYP1A, CYP1B and CYP3A were measured as enzymatic activities, immunoreactive protein content and mRNA expression in liver, gills, intestine and heart. Additional analyses in the liver included enzymatic activities of testosterone hydroxylase, (omega)- and (omega-1)-lauric acid hydroxylase and some phase II enzymes related to the AhR battery genes, DT-diaphorase, glutathione S-transferases and UDP-glucuronyl transferases. Responsiveness of hepatic CYP1A1 after exposure to betaNF demonstrated an higher sensitivity of MEROD than EROD activity and long lasting expression of mRNA still induced after 20 days from the treatment. Testosterone metabolism, oxidation of lauric acid and activities of phase II enzymes were not affected by betaNF indicating that their modulation is not mediated by Ah receptor. Induction of CYP1A was more limited in gills and absent in intestine and heart. The first nucleotide sequence for CYP1B1 in an Antarctic fish has been obtained, revealing a homology of 89% and 72% respectively to CYP1B1 of plaice and CYP1B2 of carp. Constitutive expression of CYP1B1 was restricted to gills where it was also induced by betaNF. Obtained results represent an additional contribution to the ecotoxicological characterization of T. bernacchii and further support the use of biomarkers for early detection of chemical pollution in Antarctica.

Vitellogenin gene expression in males of the Antarctic fish Trematomus bernacchii from Terra Nova Bay (Ross Sea): a role for environmental cadmium?

Synthesis of vitellogenin (VTG) in male fish is a widely recognized effect for estrogenic pollutants in temperate environments, while similar investigations are still lacking for Antarctic organisms. In this study, a preliminary characterization of vitellogenin gene expression was performed by RT-PCR in the key species Trematomus bernacchii sampled in different phases of reproductive cycle and food availability. Females exhibited the highest gene expression during the spawning period, but VTG mRNA was always detected also in males; a significant increase of gene expression was observed both in males and females at the end of the feeding season. These results were not fully supported by a differential exposure to phyto- or anthropogenic estrogens during the planctonic cycle; on the other side, the endocrine properties of cadmium, naturally elevated in Terra Nova Bay and increasing during algal bloom, would explain both the presence of VTG mRNA in males and the seasonal changes of gene induction. Laboratory exposures did not reveal short-term estrogenic effects of cadmium while an elevated responsiveness of T. bernacchii was observed toward a classical estrogenic receptor agonist (17beta-estradiol). Different hypotheses were considered to suggest delayed endocrine effects of cadmium, including the early interaction with other cellular detoxification systems or alterations at multiple levels of the hypothalamus-pituitary-gonad-liver axis. Although molecular mechanisms of VTG gene expression in males of T. bernacchii remain unclear, obtained results provide interesting insights on this species which should stimulate future research activities.

Nrf2 and regulation of the antioxidant system in the Antarctic silverfish, Pleuragramma antarctica: Adaptation to environmental changes of pro-oxidant pressure.

Despite the key importance of Nrf2-Keap1 in regulating antioxidant system in vertebrates, this system is still poorly investigated in marine species. The present study focused on the Antarctic silverfish Pleuragramma antarctica which, during the final phases of embryo development in platelet ice, is challenged by a sudden enhancement of environmental oxidative conditions associated to ice melting. Partial coding sequences were identified for Nrf2, its repressor Keap1 and for typical Nrf2-target antioxidant genes, like catalase, glutathione peroxidase isoform 1 and Cu/Zn-dependent superoxide dismutase. Compared to temperate homologues, the protein sequences showed an elevated conservation of amino acids essential for catalytic functions, while a few specific substitutions in non-essential regions may represent a molecular adaptation to improve flexibility and accessibility to active site at cold temperatures. The role of the Nrf2-Keap1 pathway in modulating the activation of antioxidant defences was demonstrated at both transcriptional and functional levels with a clear temporal increase of antioxidant protection in embryos before the hatching. Such findings confirm the importance of Nrf2 and highlight regulation of antioxidants as an adaptive strategy in P. antarctica to protect the early life stages toward the environmental changes of pro-oxidant pressure.

The comet assay as a method of assessment of neurotoxicity: usefulness for drugs of abuse.

Comet assay is a quick and versatile method for assessing DNA damage in individual cells. It allows the detection of single and double DNA strand breaks, as well as the presence of alkali labile sites. DNA breaks may represent the direct effect of some damaging agent, or they may be intermediates in cellular repair. DNA strand breaks may also come from the action of free radicals generated by oxidative stress processes. The present article summarizes some data from our and other groups underlining the suitability of the Comet assay in assessing neurotoxicity and its potential in evaluating drugs of abuse-related genotoxicity.

MDMA induces caspase-3 activation in the limbic system but not in striatum.

Several studies, carried out in chronic (+/-) 3,4-methylenedioxymethamphetamine (MDMA) abusers, have shown memory loss and cognitive impairment, as well as persistent electroencephalographic changes. This suggests that, at least in humans, forebrain areas, including the limbic system, might be altered by MDMA. Consistently, recent experimental evidences suggest that, in rodents, MDMA, besides effects on the basal ganglia, produces alterations in the hippocampus. Therefore, the aim of the present article was to investigate whether treatment with MDMA produces activation of the caspase-3 enzyme, which is part of an enzymatic pathway involved in cell death, within limbic areas (i.e., hippocampus, amygdala, and piriform cortex) and striatum. A marked induction of caspase-3 activity was demonstrated in the amygdala and hippocampus, although MDMA did not affect caspase-3 activity neither in the striatum nor in the frontal cortex. These data indicate that limbic structures possess a high sensitivity to MDMA with respect to the activation of at least one step in the apoptotic pathway. Potential implications and pitfalls of such an experimental observation are reported.

Oxidative responsiveness to multiple stressors in the key Antarctic species, Adamussium colbecki: Interactions between temperature, acidification and cadmium exposure.

High-latitude marine ecosystems are ranked to be among the most sensitive regions to climate change since highly stenothermal and specially adapted organisms might be seriously affected by global warming and ocean acidification. The present investigation was aimed to provide new insights on the sensitivity to such environmental stressors in the key Antarctic species, Adamussium colbecki, focussing also on their synergistic effects with cadmium exposure, naturally abundant in this area for upwelling phenomena. Scallops were exposed for 2 weeks to various combinations of Cd (0 and 40 µgL-1), pH (8.05 and 7.60) and temperature (-1 and +1 °C). Beside Cd bioaccumulation, a wide panel of early warning biomarkers were analysed in digestive glands and gills including levels of metallothioneins, individual antioxidants and total oxyradical scavenging capacity, onset of oxidative cell damage like lipid peroxidation, lysosomal stability, DNA integrity and peroxisomal proliferation. Results indicated reciprocal interactions between multiple stressors and their elaboration by a quantitative hazard model based on the relevance and magnitude of effects, highlighted a different sensitivity of analysed tissues. Due to cellular adaptations to high basal Cd content, digestive gland appeared more tolerant toward other prooxidant stressors, but sensitive to variations of the metal. On the other hand, gills were more affected by various combinations of stressors occurring at higher temperature.