3,5-diiodo-L-thyronine modifies the lipid droplet composition in a model of hepatosteatosis.

BACKGROUND/AIMS: Fatty acids are the main energy stores and the major membrane components of the cells. In the hepatocyte, fatty acids are esterified to triacylglycerols (TAGs) and stored in lipid droplets (LDs). The lipid lowering action of 3,5-diiodo-L-thyronine (T2) on an in vitro model of hepatosteatosis was investigated in terms of fatty acid and protein content of LDs, lipid oxidation and secretion. METHODS: FaO cells were exposed to oleate/palmitate, then treated with T2. RESULTS: T2 reduced number and size of LDs, and modified their acyl composition by decreasing the content of saturated (SFA) vs monounsaturated (MUFA) fatty acids thus reversing the SFA/MUFA ratio. The expression of the LD-associated proteins adipose differentiation-related protein (ADRP), oxidative tissue-enriched PAT protein (OXPAT), and adipose triglyceride lipase (ATGL) was increased in 'steatotic' cells and further up-regulated by T2. Moreover, T2 stimulated the mitochondrial oxidation by up-regulating carnitine-palmitoyl-transferase (CPT1), uncoupling protein 2 (UCP2) and very long-chain acyl-coenzyme A dehydrogenase (VLCAD). CONCLUSIONS: T2 leads to mobilization of TAGs from LDs and stimulates mitochondrial oxidative metabolism of fatty acids, in particular of SFAs, and thus enriches of MUFAs the LDs. This action may protect the hepatocyte from excess of SFAs that are more toxic than MUFAs.

Agreement and discrepancies in patient-clinician reports of DSM-5-TR section III maladaptive personality traits: A study on a mixed outpatient sample.

The assessment of personality pathology based on dimensional models may improve self-other agreement, but previous research mainly adopted a categorical approach and overlooked the role of the person of the therapist. Our study examined patient-clinician agreement in a mixed sample of Italian outpatients using the Personality Inventory for DSM-5 (PID-5) and the PID-5-Informant Form (PID-5-IRF). Moreover, the role of clinician personality traits on agreement was preliminary explored. Sixty-eight outpatients (51.4% male, M = 30.30, SD = 12.05 years) and their treating clinicians (N = 22; 77.3% female, M = 43.77 ± 8.45 years) entered the study. Patients completed the PID-5, whereas clinicians filled-in the PID-5-Brief Form (PID-5-BF) and the PID-5-IRF for each patient they involved. A multilevel Bayesian analysis showed that rank-order agreement was large for domains (mean r = .60) and moderate for facets (mean r = .44). As regards mean-level agreement, patient ratings on cognitive/perceptual dysregulation, distractibility, eccentricity, and emotional lability were higher than clinician ratings, whereas patients' scores on depressivity were lower than clinicians' ones. Scores on the PID-5-BF detachment positively predicted agreement on anhedonia, anxiousness, depressivity, distractibility, separation insecurity, and suspiciousness, while scores on the PID-5-BF negative affectivity, antagonism, and disinhibition negatively predicted agreement on few specific facets. Current findings suggest that clinician personality traits may contribute to agreement on maladaptive personality traits, but areas of discrepancies remain in case of low observable internal ones. Since patient-clinician agreement is crucially involved in therapeutic alliance, further research on this issue is highly encouraged. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Effects of the COVID-19 pandemic on dentists' workforce confidence and workflow.

BACKGROUND: The COVID-19 pandemic has affected the US economy and workforce, including marked effects on small businesses. Researchers have evaluated workers' views of financial confidence and advancement, but there has been limited focus on the dental industry. METHODS: To extend investigations to dentistry, the authors used published scales and pretested questions to determine workforce confidence and workflow changes among dentists. Data were evaluated using descriptive and bivariate statistics. In the wake of the pandemic, surveys were distributed to the memberships of the American Dental Association and American Association of Orthodontists (n = 656). RESULTS: Dentists' top concern was increased cost of providing treatment (57.4%; 95% CI, 53.5% to 61.3%), associated with widely adopted workflow changes including reduced patient volumes (66.0%; 95% CI, 62.4% to 69.6%) and increased safety protocols and equipment (health screening: 75.5%; 95% CI, 72.2% to 78.8%; KN/N95 respirators: 76.7%; 95% CI, 73.5% to 80.0%). However, most respondents did not expect their personal or practice finances to be negatively affected after the pandemic, as only 18.5% (95% CI, 15.4% to 21.7%) predicted their practice's gross revenue would decrease. CONCLUSIONS: Dentists were optimistic in the wake of vaccinations and lifting restrictions. Most expected their finances and practice performance to remain the same or grow in the short term and expected long-term improvements postpandemic. PRACTICAL IMPLICATIONS: Results suggest that despite shutdowns and workflow changes, dentists have rebounded financially and anticipate future growth.

Direct effects of iodothyronines on excess fat storage in rat hepatocytes.

BACKGROUND & AIMS: Previous studies have demonstrated that 3,5-L-diiodothyronine (T(2)) is able to prevent lipid accumulation in the liver of rats fed a high-fat diet. Whether this effect is due to a direct action of T(2) on the liver has not been elucidated. In this study, we investigated the ability of T(2) to reduce the excess lipids in isolated hepatocytes treated with fatty acids (FFAs). The effects of T(2) were compared with those elicited by 3,3',5-L-triiodothyronine (T(3)). METHODS: To mimic the fatty liver condition, primary cultures of rat hepatocytes were overloaded with lipids, by exposure to FFAs ("fatty hepatocytes"), and then treated with T(2) or T(3). Lipid content, morphometry of lipid droplets (LDs), and expression of the adipocyte differentiation-related protein (ADRP) and the peroxisome proliferator-activated receptors (PPAR-α, -γ, -δ) were evaluated. Activities of the lipolytic enzyme acyl CoA oxidase-AOX and the antioxidant enzymes superoxide dismutase-SOD and catalase-CAT were also determined. RESULTS: FFA-induced lipid accumulation was associated with an increase in both number/size of LDs and expression of ADRP, PPAR-γ, and PPAR-δ/ß mRNAs, as well as in the activities of AOX, SOD, and CAT. The addition of T(2) or T(3) to "fatty hepatocytes" resulted in a reduction in: (i) lipid content and LD diameter; (ii) PPAR-γ and PPAR-δ expression; (iii) activities of AOX and antioxidant enzymes. CONCLUSIONS: These data demonstrate, for the first time, a direct action of both T(2) and T(3) in reducing the excess fat in cultured hepatocytes.

Bisphenol-A alters gene expression and functional parameters in molluscan hepatopancreas.

Bisphenol-A (BPA) is a well-known xenoestrogen in mammalian systems that can affect reproduction also in aquatic organisms. In this work the possible effects of BPA were investigated in the hepatopancreas of the bivalve mollusc Mytilus galloprovincialis: mussels were injected with different amounts of BPA (3-60ng/g dw tissue) and tissues sampled at 24h post-injection. Expression of different Mytilus genes was evaluated by RT-Q-PCR: BPA exposure increased the expression of MeER2 and induced downregulation of antioxidant genes, catalase and metallothioneins. Moreover, BPA induced changes in activity of catalase, GSH transferase (GST) and GSSG reductase (GSR), and in total glutathione content. A decrease in lysosomal membrane stability and increased neutral lipid accumulation were also observed. The results were compared with those obtained with similar concentrations of 17beta-estradiol. These data demonstrate that BPA can alter gene expression, activities of enzymes involved in redox balance, and lysosomal function in molluscan hepatopancreas, a tissue involved in the control of metabolism and gamete maturation. Overall, these data indicate that BPA, at environmentally relevant concentrations, can have both estrogen-like and distinct effects in invertebrates like in vertebrates.

Efects of growth hormone and cadmium on the transcription regulation of two metallothionein isoforms.

The effect of growth hormone (GH) and cadmium (Cd) on metallothionein (MT) expression was investigated in hepatoma cells. In fish the constitutive isoform MT-B and the metal-responsive MT-A are expressed. Real-time RT-PCR revealed that: Cd up-regulates mostly MT-A, GH slightly induces MT-B and the GH/Cd combination induces synergistically both MTs. Perturbations in Ca2+ levels suppressed or reduced the Cd-induction of MTs and abolished the GH/Cd synergy. Similar results were obtained by inhibition of tyrosine kinases. Also the signaling molecules recruited by the GH receptor responded differently to GH and Cd, with ERKs showing a synergistic activation upon GH/Cd. The following conclusions can be drawn: (1) cytosolic Ca2+ is mainly involved in MT-A regulation; (2) both Ca2+ and tyrosine phosphorylation are essential for Cd-induction and GH/Cd synergy on MTs. The synergy could depend on interactions in different signaling pathways, leading to a differential recruitment of MTF-1 and AP-1 transcription factors.

Ethanol and fatty acids impair lipid homeostasis in an in vitro model of hepatic steatosis.

Excess ethanol consumption and fatty acid intake lead to a cumulative effect on liver steatosis through still unclear mechanisms. This study aimed to characterize the lipid homoeostasis alterations under the exposure of hepatocytes to ethanol alone or combined with fatty acids. FaO hepatoma cells were incubated in the absence (C) or in the presence of 100 mM ethanol (EtOH) or 0.35 mM oleate/palmitate (FFA) alone or in the combination (FFA/EtOH). Content of intra- and extra-cellular triglycerides (TAGs) and of lipid droplets (LDs), expression of lipogenic and lipolytic genes, and oxidative stress-related parameters were evaluated. Exposure to either FFAs or EtOH given separately led to steatosis which was augmented when they were combined. Our results show that FFA/EtOH: (i) increased the LD number, but reduced their size compared to separate treatments; (ii) up-regulated PPARγ and SREBP-1c and down-regulated sirtuin-1 (SIRT1); (iii) impaired FFA oxidation; (iv) did not change lipid secretion and oxidative stress. Our findings indicate that one of the major mechanisms of the metabolic interference between ethanol and fat excess is the impairment of FFA oxidation, in addition to lipogenic pathway stimulation. Interestingly, ethanol combined with FFAs led to a shift from macrovesicular to microvesicular steatosis that represents a more dangerous condition.

Effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) on cell signaling and function of Mytilus hemocytes: involvement of MAP kinases and protein kinase C.

Brominated flame retardants (BFRs) are a large group of compounds added to or applied as a treatment to polymeric materials to prevent fires. Tetrabisphenol A (TBBPA) is the most important individual BFR used in industry. Although TBBPA and its derivatives can be found in environmental samples, data are very limited on the presence of this compound in biota. Research on mammals indicates that TBBPA has low toxicity in vivo; however, in vitro TBBPA can act as a cytotoxicant, neurotoxicant, immunotoxicant, thyroid hormone agonist and has a weak estrogenic activity; in particular, the effects of TBBPA have been recently ascribed to its interactions with cellular signaling pathways, in particular with mitogen activated protein kinases (MAPKs). TBBPA has high acute toxicity to aquatic organisms, such as algae, molluscs, crustaceans and fish; however, little is known on the mechanisms of action of this compound in the cells of aquatic species. In this work, we investigated the possible effects and mechanisms of action of TBBPA on the immune cells, the hemocytes, of the marine mussel Mytilus galloprovincialis. The results demonstrate that TBBPA in the low micromolar range induces hemocyte lysosomal membrane destabilization. The effect was reduced or prevented by hemocyte pre-treatment by specific inhibitors of MAPKs and of protein kinase C (PKC). TBBPA stimulated phosphorylation of MAPK members and PKC, as evaluated by electrophoresis and Western blotting with anti-phospho-antibodies, although to a different extent and with distinct time-courses. A rapid (from 5 min) and transient increase in phosphoryation of the stress-activated JNK MAPKs and of PKC was observed, followed by a later increase (at 30-60 min) in phosphorylation of extracellularly regulated MAPKs (ERK2 MAPK) and of the stress-activated p38 MAPK. TBBPA significantly stimulated the hemocyte microbicidal activity towards E. coli, lysosomal enzyme release, phagocytic activity and extracellular superoxide (O2-) production. The results demonstrate that TBBPA in vitro activates the immune function of mussel hemocytes through kinase-mediated cell signaling and that common transduction pathways are involved in mediating the effects of this BFR in mammalian and aquatic invertebrate cells.

'In vivo' effects of Bisphenol A in Mytilus hemocytes: modulation of kinase-mediated signalling pathways.

Endocrine disrupting chemicals (EDCs) include a variety of natural and synthetic estrogens, as well as estrogen-mimicking chemicals. We have previously shown that in the hemocytes of the mussel Mytilus galloprovincialis Lam. both natural and environmental estrogens in vitro can rapidly affect the phosphorylation state of components of tyrosine kinase-mediated cell signalling, in particular of mitogen activated protein kinases (MAPKs) and signal transducers and activators of transcription (STAT), that are involved in mediating the hemocyte immune response. These effects were consistent with the hypothesis that 'alternative' modes of estrogen action involving kinase-mediated pathways similar to those described in mammalian systems are also present in invertebrate cells. This possibility was investigated in vivo with Bisphenol A (BPA): mussels were injected with BPA and hemocytes sampled at 6, 12, and 24 h post-injection. The results show that BPA (25 nM nominal concentration in the hemolymph) lead to a significant lysosomal membrane destabilisation at all times post-injection, indicating BPA-induced stress conditions in the hemocytes, whereas lower concentrations were ineffective. BPA induced significant changes in the phosphorylation state of MAPK and STAT members, as evaluated by SDS-PAGE and WB of hemocyte protein extracts with specific antibodies, although to a different degree at different exposure times. In particular, BPA induced a dramatic decrease in phosphorylation of the stress-activated p38 MAPK, whose activation is crucial in mediating the bactericidal activity. Moreover, BPA decreased the phosphorylation of a CREB-like transcription factor (cAMP-responsive element binding protein). The results demonstrate that BPA can affect kinase-mediated cell signalling in mussel hemocytes also in vivo, and suggest that EDCs may affect gene expression in mussel cells through modulation of the activity of transcription factors secondary to cytosolic kinase cascades. Overall, these data address the importance of investigating full range responses to EDCs in ecologically relevant marine invertebrate species.

Triglyceride Mobilization from Lipid Droplets Sustains the Anti-Steatotic Action of Iodothyronines in Cultured Rat Hepatocytes.

Adipose tissue, dietary lipids and de novo lipogenesis are sources of hepatic free fatty acids (FFAs) that are stored in lipid droplets (LDs) as triacylglycerols (TAGs). Destiny of TAGs stored in LDs is determined by LD proteomic equipment. When adipose triglyceride lipase (ATGL) localizes at LD surface the lipid mobilization is stimulated. In this work, an in vitro model of cultured rat hepatocytes mimicking a mild steatosis condition was used to investigate the direct lipid-lowering action of iodothyronines, by focusing, in particular, on LD-associated proteins, FFA oxidation and lipid secretion. Our results demonstrate that in "steatotic" hepatocytes iodothyronines reduced the lipid excess through the recruitment of ATGL on LD surface, and the modulation of the LD-associated proteins Rab18 and TIP47. As an effect of ATGL recruitment, iodothyronines stimulated the lipid mobilization from LDs then followed by the up-regulation of carnitine-palmitoyl-transferase (CPT1) expression and the stimulation of cytochrome-c oxidase (COX) activity that seems to indicate a stimulation of mitochondrial function. The lipid lowering action of iodothyronines did not depend on increased TAG secretion. On the basis of our data, ATGL could be indicated as an early mediator of the lipid-lowering action of iodothyronines able to channel hydrolyzed FFAs toward mitochondrial beta-oxidation rather than secretion.

Signaling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 MAP kinases.

In this work the mechanisms of transduction triggered in Mytilus galloprovincialis hemocytes by bacterial challenge were investigated in an in vitro model of infection of hemocyte monolayers with Escherichia coli. Western blot analyses of hemocyte extracts with phospho-specific anti-MAPK (Mitogen Activated Protein Kinase) antibodies indicate that E. coli induced a time dependent activation of different classes of MAPKs, mainly of the stress-activated p38 MAPK. P38 activation was confirmed by the use of the selective kinase inhibitor SB203580. Moreover, hemocyte pretreatment with SB203580 significantly reduced bacterial killing, whereas PD98059, an inhibitor of extracellularly regulated kinase (ERK) activation, was ineffective. Interestingly, the PI3-kinase (phosphatidylinositol-3-OH-kinase) inhibitor, Wortmannin, reduced both p38 activation and bacterial killing, indicating a critical role also for this lipid kinase in the hemocyte immune response.

Bacteria-hemocyte interactions and phagocytosis in marine bivalves.

Marine bivalves (such as mussels, oysters, and clams) are widespread mollusks in coastal waters at different latitudes; due to their filter-feeding habits, they accumulate large numbers of bacteria from the harvesting waters and may act as passive carriers of human pathogens. To cope with this challenge, bivalves possess both humoral and cellular defense mechanisms with remarkably effective capabilities. The circulating cells, or hemocytes, are primarily responsible for defense against parasites and pathogens; microbial killing results from the combined action of the phagocytic process with humoral defense factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), toxic oxygen intermediates, and various antimicrobial peptides. In this work, current knowledge of the mechanisms underlying the interactions between bacteria and the hemolymph components of marine bivalves is summarized. Bacterial susceptibility to hemolymph killing in different bivalve species may be a consequence of the different ability of bacterial products to attract phagocytes, the presence or absence of specific opsonizing molecules, the hemocyte capability to bind and engulf different bacteria, and the different bacterial sensitivity to intracellular killing. The role of soluble (e.g., agglutinins and opsonins) and surface-bound factors in bacterial phagocytosis by hemocytes of the most common marine bivalve species is described and the possibility that environmental temperatures and other seasonal factors may influence this process is considered. Moreover, the potential strategies used by bacteria to evade phagocytic killing by hemocytes are discussed. From the available data it is clear that several questions need further investigation; the elucidation of the factors influencing phagocytosis in bivalves and the fundamental strategies used by bacteria to escape hemolymph killing are important not only to understand bivalve immune defenses but also to explain the persistence of pathogenic bacteria in bivalve tissues and to predict the consequent impact on human health.

Characterization of metalloproteinase-like activities in barnacle (Balanus amphitrite) nauplii.

The presence of extracellular matrix (ECM) degrading enzymes was investigated in naupliar stages of the barnacle Balanus amphitrite Darwin. The results of substrate gel-zymography and quantitative assays demonstrated that naupliar extracts contain several protease activities that are specific towards gelatin substrates; some caseinolytic activity was also detected. Substrate specificity was observed in all naupliar stages (II-VI). The gelatinolytic activities showed dependence on both Ca(2+) and Zn(2+) and inhibition by EDTA, EGTA, and 1,10-phenanthroline. Also Mg(2+) partially activated the enzymes, whereas Cd(2+), Cu(2+), Hg(2+) and Pb(2+) were inhibitory. The thermal denaturation profile was significantly different in the presence and absence of Ca(2+) and Zn(2+). Overall, the results indicate that the Ca(2+)/Zn(2+)-dependent gelatinase activities in barnacle nauplii belong to the subfamily of matrix metalloproteases. Barnacle larvae MMPs showed biochemical characteristics different from those of vertebrate MMPs but common to other gelatinases from marine invertebrates: they were unaffected by several protease inhibitors and insensitive to specific activators/inhibitors of vertebrate MMPs. The presence of MMP-like activities in different naupliar stages suggests a constitutive role for these enzymes in ECM remodeling during barnacle larvae growth and development.

Effects of PCB congeners on the immune function of Mytilus hemocytes: alterations of tyrosine kinase-mediated cell signaling.

Polychlorinated biphenyls (PCBs) are industrial chemicals which have been released into the environment resulting in widespread and persistent contamination. PCBs exist as 209 different congeners depending on the chlorine substitution on the biphenyl rings; the physical properties and the toxic effects of a PCB congener are structure-dependent. In this work, individual ortho-substituted non coplanar PCB congeners were tested for their effects on the function of mussel (Mytilus galloprovincialis Lam.) hemocytes. Moreover, the possibility that in mussel hemocytes different PCBs may affect the signal transduction pathways involved in the immune response was investigated, with particular regards to relevant components of tyrosine-kinase mediated cell signaling. The results were compared with those obtained with a model of non-ortho-substituted coplanar congener. The results demonstrate that the di-ortho-substituted, non coplanar PCB congeners P47 (2,2',4,4'-tetrachlorobiphenyl) and P153 (2,2',4,4',5,5'-hexachlorobiphenyl) can alter immune parameters of mussel hemocytes, such as microbicidal activity and lysosomal enzyme release, respectively. Both congeners, as well as the non-ortho, coplanar congener P77 (3,3',4,4'-tetrachlorobiphenyl) significantly reduced hemocyte lysosomal membrane stability; however, P77 had no effect on either bacterial killing or lysozyme release. P47, P153 and P77 affected different components of tyrosine kinase-mediated cell signalling; in particular, they lead to a time-dependent increase in the phosphorylation level of the stress activated p38 and JNK Mitogen Activated Protein Kinases (MAPKs), as evaluated by Western blotting of hemocyte protein extracts with specific anti-phospho-MAPK antibodies. P153 also increased the level of phosphorylated ERK (extracellularly regulated) MAPKs. Moreover, non coplanar P47 and P153 caused increased tyrosine phosphorylation of the transcription factor STAT5, thus possibly affecting gene expression, whereas coplanar P77 was ineffective. The results demonstrate that MAPKs, and in particular the stress-activated p38 and JNK MAPKs, that represents a key step in the response of mussel hemocytes to bacterial infection, are a target for different non coplanar and coplanar PCB congeners. The results also show functional differences between different PCB congeners with respect to the hemocyte functions. However, chlorine substitution at the ortho positions is not necessarily related to immunotoxicity: the hexachlorinated P128 (2,2',3,3',4,4'-hexachlorobiphenyl) had no significant effect on mussel hemocytes, whereas its isomer P153, that represents a major component of environmental PCBs, and that is accumulated in mussel tissues, significantly affected both aspects of the immune response and relevant signal transduction pathways. These are the first data on the effects and possible mechanisms of immunotoxicity of non coplanar PCBs in mussel hemocytes. The results support the hypothesis that the innate immune system is a sensitive target for these contaminants in both vertebrates and invertebrates. Moreover, when considering that non coplanar congeners are present both in commercial mixtures and, in higher proportions, in environmental samples, the results suggest that bivalve hemocytes represent a useful model for evaluating the potential immunotoxicity of PCB contamination.

Effects of binge ethanol on lipid homeostasis and oxidative stress in a rat model of nonalcoholic fatty liver disease.

Excess fat accumulation renders the liver more vulnerable to ethanol, but it is still unclear how alcohol enhances lipid dysmetabolism and oxidative stress in a pre-existing steatosis condition. The effects produced by binge ethanol consumption in the liver of male Wistar rats fed a standard (Ctrl) or a high-fat diet HFD were compared. The liver status was checked through tissue histology and standard serum parameters. Alteration of hepatic lipid homeostasis and consequent oxidative unbalance were assessed by quantifying the mRNA expression of the lipid-regulated peroxisome proliferator-activated receptors (PPARs), of the cytochromes CYP2E1 and CYP4A1, and of some antioxidant molecules such as the metallothionein isoforms MT1 and MT2 and the enzymes catalase and superoxide dismutase. The number of adipose differentiation-related protein (ADRP)-positive lipid droplets (LDs) was evaluated by immunohistochemical staining. As a response to the double insult of diet and ethanol the rat liver showed: (1) a larger increase in fat accumulation within ADRP-positive LDs; (2) stimulation of lipid oxidation in the attempt to limit excess fat accumulation; (3) induction of antioxidant proteins (MT2, in particular) to protect the liver from the ethanol-induced overproduction of oxygen radicals. The data indicate an increased susceptibility of fatty liver to ethanol and suggest that the synergistic effect of diet and ethanol on lipid dysmetabolism might be mediated, at least in part, by PPARs and cytochromes CYP4A1 and CYP2E1.

Altered oxidative stress/antioxidant status in blood of alcoholic subjects is associated with alcoholic liver disease.

BACKGROUND: Oxidative stress is implicated in pathogenesis of alcoholic liver disease (ALD). This study investigated the possible correlation among the erythrocyte indices of oxidative stress, the leukocyte panels of antioxidant proteins (metallothioneins), the serum biochemical parameters and the liver steatosis grade. METHODS: A total of 118 cases including 60 alcoholic subjects and 58 controls were enrolled. All the alcoholic subjects were screened for body mass index (BMI), liver steatosis, and blood chemistry and serology. The level of oxidative stress and oxidative stress-related parameters were measured in the blood and correlated with clinical findings. RESULTS: Alcoholic subjects showed higher BMI, moderate/severe hepatic steatosis, increase in the levels of triglycerides, cholesterol, glucose, γ-glutamyl-transpeptidase (GGT), alanine aminotransferase (ALT), bilirubin, alpha 1 and beta 2 globulins, iron and a decrease in the levels of aspartate aminotransferase (AST) and beta 1 globulin with respect to the reference values. Moreover, alcoholic subjects showed: (i) an increase in Thiobarbituric Acid Reactive Substance (TBARS) content representing a good estimation of global oxidative stress; (ii) a stimulation of the activities of the antioxidant enzymes catalase and SOD; (iii) a modulation of expression of metallothioneins, with a down-regulation of MT-1A and an up-regulation of MT-1E isoforms. CONCLUSIONS: Our data suggest that alcoholism is strongly associated with altered pattern of blood metallothioneins; this parameter combined with the score calculated by an ad hoc implemented algorithm (HePaTest) could offer a non-invasive alternative approach for evaluating alcohol-related damages and could be used in follow-up of alcoholic patients.

Co-exposure to n-TiO2 and Cd2+ results in interactive effects on biomarker responses but not in increased toxicity in the marine bivalve M. galloprovincialis.

The increasing production of nanoparticles (NPs) will lead to their release into the aquatic environment, where they could modify the bioavailability/bioconcentration and consequent biological impact of other contaminants. Interactive effects of n-TiO2, one of the most widespread NP type, and Cd(2+), a common heavy metal pollutant, have been described in freshwater species, whereas no information is available in marine organisms. In this work, the effects of co-exposure to n-TiO2 and Cd(2+) were investigated in the marine bivalve Mytilus galloprovincialis. Experimental conditions (100 µg/L, 96 h), were chosen in order to induce early but measurable stress responses (biomarkers) without toxicity. Several biomarkers, from molecular to tissue level, were measured in hemolymph and digestive gland; the effects on embryo development were also evaluated. In hemolymph, Cd(2+) abolished the increase in immune parameters induced by n-TiO2 (NO production and lysozyme activity). In the digestive gland, distinct interactive effects of n-TiO2 and Cd(2+) were observed on different lysosomal biomarkers (lysosomal membrane stability, lipid accumulation and lysosome/cytoplasm volume ratio) and transcription of the immune genes lysozyme and toll-like receptor (TLR). However, n-TiO2 did not affect specific metal-induced responses (metallothionein induction) and tissue metal accumulation. Cd(2+) alone, but not in combination with n-TiO2, affected embryo development. The interactive effects observed on different biomarkers were not apparently due to differences in bioavailability/bioaccumulation of Cd(2+) in the presence of n-TiO2 agglomerates; these effects may result from interactions of either contaminant with both common and distinct targets/mechanisms of action at different levels of biological organization. Overall, the results indicate that co-exposure to n-TiO2 and Cd(2+) did not result in increased adverse effects in M. galloprovincialis. These data underline the need for further knowledge on the potential interactions of NPs with existing contaminants in marine organisms.

Direct effects of Bisphenol A on lipid homeostasis in rat hepatoma cells.

Bisphenol A (BPA), used in the manufacture of polycarbonate plastic and epoxy resin, is one of the most abundant endocrine disruptors in the environment, considered as a xenoestrogen. BPA has recently become of additional public health concern because of increasing evidence of deleterious effects on metabolism. Dietary intake seems the most important route for BPA exposure, followed by rapid biotransformation in the gut and liver and elimination in the urine. Although hepatocytes can represent a significant target for this compound, little is known on the direct effects and mechanisms of action of BPA on lipid homeostasis at the cellular level. In this work, the effects of BPA (0.3-3-30-300 ng mL(-1), 24 h) were investigated in rat FaO hepatoma, a well differentiated liver cell line. At both 30 and 300 ng mL(-1), BPA significantly increased intracellular triglyceride (TAG) content and lipid accumulation in lipid droplets (LDs), without affecting cell viability. The effects of BPA were associated with decreased mRNA levels of the transcription factors Peroxisome Proliferator-Activated Receptor (PPAR) isoforms α and ßδ, as well as of their downstream genes acyl-CoA oxidase (AOX) and carnitine palmitoyl transferase (CPT1) involved in lipid oxidation. No increase in transcription of lipogenic genes was observed. BPA also decreased mRNA levels of ApolipoproteinB (apoB) and the extracellular TAG content, indicating alterations in lipid secretion. FaO cells did not express Estrogen Receptor α (ERα and showed a very low expression of ERß compared to rat liver. All the effects of BPA were prevented by cell pretreatment with Wortmannin, indicating the involvement of phosphatidyl inositol-3 kinase activation. The results demonstrate a direct action of BPA on lipid homeostasis in FaO cells through interference with lipid oxidation and secretion, and add further information on the cellular pathways that can be perturbed by this compound.

In vivo effects of n-TiO2 on digestive gland and immune function of the marine bivalve Mytilus galloprovincialis.

Due to the increasing production of nanoparticles (NPs) and their potential release in the aquatic environment, evaluation of their biological impact on aquatic organisms represents a major concern. Suspension feeding invertebrates, in particular bivalve mollusks, may play a role in NP biotransformation and transfer through food webs and may represent a significant target for NP toxicity. In this work, the in vivo effects of titanium dioxide (n-TiO2), one of the most widespread NPs in use, were investigated in the bivalve Mytilus galloprovincialis, largely utilised as a sentinel for marine contamination. Mussels were exposed for 96h to different concentrations of n-TiO2 suspensions (1, 10 and 100µgL(-1)) and multiple responses were evaluated in the digestive gland and immune cells, the haemocytes. In the digestive gland, n-TiO2 affected lysosomal and oxidative stress biomarkers and decreased transcription of antioxidant and immune-related genes. In the haemocytes, n-TiO2 decreased lysosomal membrane stability-LMS and phagocytosis, increased oxyradical production and transcription of antimicrobial peptides; moreover, pre-apoptotic processes were observed. The effects of n-TiO2 on digestive gland and haemocytes were distinct, also depending on the endpoint and on nominal NP concentrations, with many significant responses elicited by the lowest concentrations tested. The results show that n-TiO2, at concentrations close to predicted environmental levels, significantly affected different functional and molecular parameters of mussel digestive gland and immune cells. In particular, the observed changes in immune parameters that represent significant biomarkers of exposure at the organism level suggest that exposure to n-TiO2 may pose a serious risk to mussel health.

Bivalve molluscs as a unique target group for nanoparticle toxicity.

Due to the continuous development and production of manufactured nanomaterials or nanoparticles (NPs), their uptake and effects in the aquatic biota represent a major concern. Estuarine and coastal environments are expected to represent the ultimate sink for NPs, where their chemical behavior (aggregation/agglomeration) and consequent fate may be critical in determining the biological impact. Bivalve mollusks are abundant from freshwater to marine ecosystems, where they are widely utilized in biomonitoring of environmental perturbations. As suspension-feeders, they have highly developed processes for cellular internalization of nano- and micro-scale particles (endo- and phagocytosis), integral to key physiological functions such as intra-cellular digestion and cellular immunity. Here we will summarise available information on the effects of different types of NPs in different bivalve species, in particular Mytilus spp. Data on the effects and modes of action of different NPs on mussel hemocytes in vitro demonstrate that cell-mediated immunity represents a significant target for NPs. Moreover, in vivo exposure to NPs indicates that, due to the physiological mechanisms involved in the feeding process, NP agglomerates/aggregates taken up by the gills are directed to the digestive gland, where intra-cellular uptake of nanosized materials induces lysosomal perturbations and oxidative stress. Overall, bivalves represent a particularly suitable model for investigating the effects and mechanisms of action underlying the potential toxicity of NPs in marine invertebrates.