Comparative toxicities of selected rare earth elements: Sea urchin embryogenesis and fertilization damage with redox and cytogenetic effects

Background: Broad-rangingadverseeffectsareknownforrareearthelements(REE),yetonlyafew studies testedthetoxicityofseveralREE,promptingstudiesfocusingonmulti-parameterREEtoxicity. Methods: TrichloridesaltsofY,La,Ce,Nd,Sm,EuandGdweretestedin Paracentrotuslividus sea urchin embryos andspermfor:(1)developmentaldefectsineitherREE-exposedlarvaeorintheoffspringof REE-exposedsperm;(2)fertilizationsuccess;(3)mitoticanomaliesinREE-exposedembryosandinthe offspring ofREE-exposedsperm,and(4)reactiveoxygenspecies(ROS)formation,andmalondialdehyde (MDA)andnitricoxide(NO)levels. Results: REEs affected P.lividus larvaewithconcentration-relatedincreaseindevelopmentaldefects, 106 to 104 M, rankingas:Gd(III)4Y(III)4La(III)4Nd(III)≅Eu(III)4Ce(III)≅Sm(III). Nominalcon- centrations ofREEsaltswereconfirmed byinductivelycoupledplasmamassspectrometry(ICP-MS). Significant increasesinMDAlevels,ROSformation,andNOlevelswerefoundinREE-exposedembryos. Sperm exposuretoREEs(105 to 104 M) resultedinconcentration-relateddecreaseinfertilization success alongwithincreaseinoffspringdamage.Decreasedmitoticactivityandincreasedaberration ratesweredetectedinREE-exposedembryosandintheoffspringofREE-exposedsperm. Conclusion: REE-associated toxicityaffectingembryogenesis,fertilization,cytogeneticandredoxend- points showeddifferentactivitiesoftestedREEs.Damagetoearlylifestages,alongwithredoxandcy- togeneticanomaliesshouldbethefocusoffutureREEtoxicitystudies

Thrombocytopenia and Mortality Risk in Patients With Atrial Fibrillation: An Analysis From the START Registry

Background: Thrombocytopenia is associated with increased mortality in the general population, but few data exist in patients with atrial fibrillation (AF) taking oral anticoagulants. We investigated factor determinants of thrombocytopenia in a large cohort of patients affected by AF and its association with total mortality. Methods and Results: Multicenter prospective cohort study, including 5215 patients with AF from the START (Survey on Anticoagulated Patients Register) registry, 3877 (74.3%) and 1338 (25.7%) on vitamin K or non\u2013vitamin K antagonist oral anticoagulants, respectively. Thrombocytopenia was defined by a platelet count <150 7109/L. Determinants of thrombocytopenia were investigated, and all-cause mortality was the primary survival end point of the study. Thrombocytopenia was present in 592 patients (11.4%). At multivariable logistic regression analysis, chronic kidney disease (odds ratio [OR], 1.257; P=0.030), active cancer (OR, 2.065; P=0.001), liver cirrhosis (OR, 7.635; P<0.001), and the use of diuretics (OR, 1.234; P=0.046) were positively associated with thrombocytopenia, whereas female sex (OR, 0.387; P<0.001) and the use of calcium channel blockers (OR, 0.787; P=0.032) were negatively associated. During a median follow-up of 19.2 months (9942 patient-years), 391 deaths occurred (rate, 3.93%/year). Mortality rate increased from 3.8%/year to 9.9%/year in patients with normal platelet count and in those with moderate-severe thrombocytopenia, respectively (log-rank test, P=0.009). The association between moderate-severe thrombocytopenia and mortality persisted after adjustment for CHA2DS2 VASc score (hazard ratio, 2.431; 95% CI, 1.254\u20134.713; P=0.009), but not in the fully adjusted multivariable Cox regression analysis model. Conclusions: Thrombocytopenia is common in patients with AF. Despite an increased incidence of mortality, thrombocytopenia was not associated with mortality at multivariable analysis. Thrombocytopenia may reflect the presence of comorbidities associated with poor survival in AF

Shedding light on ovothiol biosynthesis in marine metazoans

Ovothiol, isolated from marine invertebrate eggs, is considered one of the most powerful antioxidant with potential for drug development. However, its biological functions in marine organisms still represent a matter of debate. In sea urchins, the most accepted view is that ovothiol protects the eggs by the high oxidative burst at fertilization. In this work we address the role of ovothiol during sea urchin development to give new insights on ovothiol biosynthesis in metazoans. The gene involved in ovothiol biosynthesis OvoA was identified in Paracentrotus lividus genome (PlOvoA). PlOvoA embryo expression significantly increased at the pluteus stage and was up-regulated by metals at concentrations mimicking polluted sea-water and by cyclic toxic algal blooms, leading to ovothiol biosynthesis. In silico analyses of the PlOvoA upstream region revealed metal and stress responsive elements. Structural protein models highlighted conserved active site residues likely responsible for ovothiol biosynthesis. Phylogenetic analyses indicated that OvoA evolved in most marine metazoans and was lost in bony vertebrates during the transition from the aquatic to terrestrial environment. These results highlight the crucial role of OvoA in protecting embryos released in seawater from environmental cues, thus allowing the survival under different condition

Gene expression analysis in the progeny of <i>P</i>. <i>lividus</i> females exposed to manganese 3.6 x 10⁻⁵ M for 2 days (A,B) and 9 days (C,D).

<p>The embryos were reared in sea water (A,C) and in manganese-containing sea water (B,D). Data are reported as a fold difference in the expression levels of the analyzed genes, compared to controls (mean ± SD), offspring of females kept during the whole experimental period in sea water without addition of metal. Fold differences equal or greater than ± 2 (see horizontal guidelines at values of 2 and—2) were considered significant. Experiments were repeated at least on 3 biological replicates.</p

Synopsis of the patterns of up- and down-regulation of different classes of genes in the offspring of <i>P</i>. <i>lividus</i> females exposed to cadmium (Cd) or manganese (Mn) for 2 and 9 days.

<p>The two arrows indicate the up- or down-regulation of genes in different developmental stages.</p

Morphological analysis of the progeny of <i>P</i>. <i>lividus</i> females exposed to cadmium and manganese.

<p>Females were treated for 2 and 9 days with cadmium (Cd) 10⁻⁶ M and manganese (Mn) 3.6 x 10⁻⁵ M, as described in Materials and Methods. Offspring was reared in sea water (sw) or in the presence of the metal. The development was monitored after 48 hpf. A. Percentage of normal and abnormal plutei; B. Representative pictures of the main abnormalities (bar = 100 μm). Significant differences compared to the control **<i>P</i><0.01, ***<i>P</i><0.001; Two-way ANOVA (<i>P</i><0.05), with Bonferroni’s Post Test. Light grey: normal plutei; dark grey: abnormal plutei. N = 8.</p

Maternal Exposure to Cadmium and Manganese Impairs Reproduction and Progeny Fitness in the Sea Urchin <i>Paracentrotus lividus</i>

<div><p>Metal contamination represents one of the major sources of pollution in marine environments. In this study we investigated the short-term effects of ecologically relevant cadmium and manganese concentrations (10^-6 and 3.6 x 10^-5 M, respectively) on females of the sea urchin <i>Paracentrotus lividus</i> and their progeny, reared in the absence or presence of the metal. Cadmium is a well-known heavy metal, whereas manganese represents a potential emerging contaminant, resulting from an increased production of manganese-containing compounds. The effects of these agents were examined on both <i>P</i>. <i>lividus</i> adults and their offspring following reproductive state, morphology of embryos, nitric oxide (NO) production and differential gene expression. Here, we demonstrated that both metals differentially impaired the fertilization processes of the treated female sea urchins, causing modifications in the reproductive state and also affecting NO production in the ovaries. A detailed analysis of the progeny showed a high percentage of abnormal embryos, associated to an increase in the endogenous NO levels and variations in the transcriptional expression of several genes involved in stress response, skeletogenesis, detoxification, multi drug efflux processes and NO production. Moreover, we found significant differences in the progeny from females exposed to metals and reared in metal-containing sea water compared to embryos reared in non-contaminated sea water. Overall, these results greatly expanded previous studies on the toxic effects of metals on <i>P</i>. <i>lividus</i> and provided new insights into the molecular events induced in the progeny of sea urchins exposed to metals.</p></div

Reproductive state and nitric oxide (NO) production in <i>P</i>. <i>lividus</i> females exposed to cadmium and manganese.

<p>GSI (A), total NO concentration (B), spawning (C) and fertilization success (D) in females exposed to cadmium (Cd) 10⁻⁶ M and manganese (Mn) 3.6 x 10⁻⁵ M for 2 and 9 days. Significant differences compared to the controls (sw 2 and 9 days): *P<0.05, ***P<0.001. Two-way ANOVA, Bonferroni’s post test (P<0.05). N = 8.</p

Gene expression analysis in the progeny of <i>P</i>. <i>lividus</i> females exposed to manganese 3.6 x 10⁻⁵ M for 2 days (A,B) and 9 days (C,D).

<p>The embryos were reared in sea water (A,C) and in manganese-containing sea water (B,D). Data are reported as a fold difference in the expression levels of the analyzed genes, compared to controls (mean ± SD), offspring of females kept during the whole experimental period in sea water without addition of metal. Fold differences equal or greater than ± 2 (see horizontal guidelines at values of 2 and—2) were considered significant. Experiments were repeated at least on 3 biological replicates.</p