The Novel SSTR3 Agonist ITF2984 Exerts Antimitotic and Proapoptotic Effects in Human Non-Functioning Pituitary Neuroendocrine Tumor (NF-PitNET) Cells.

Somatostatin receptor ligands (SRLs) with high affinity for somatostatin receptors 2 and 5 (SSTR2 and SSTR5) are poorly efficacious in NF-PitNETs, expressing high levels of SSTR3. ITF2984 is a pan-SSTR ligand with high affinity for SSTR3, able to induce SSTR3 activation and to exert antitumoral activity in the MENX rat model. The aim of this study was to test ITF2984's antiproliferative and proapoptotic effects in NF-PitNET primary cultured cells derived from surgically removed human tumors and to characterize their SSTR expression profile. We treated cells derived from 23 NF-PitNETs with ITF2984, and a subset of them with octreotide, pasireotide (SRLs with high affinity for SSTR2 or 5, respectively), or cabergoline (DRD2 agonist) and we measured cell proliferation and apoptosis. SSTR3, SSTR2, and SSTR5 expression in tumor tissues was analyzed by qRT-PCR and Western blot. We demonstrated that ITF2984 reduced cell proliferation (-40.8 (17.08)%, p < 0.001 vs. basal, n = 19 NF-PitNETs) and increased cell apoptosis (+41.4 (22.1)%, p < 0.001 vs. basal, n = 17 NF-PitNETs) in all tumors tested, whereas the other drugs were only effective in some tumors. In our model, SSTR3 expression levels did not correlate with ITF2984 antiproliferative nor proapoptotic effects. In conclusion, our data support a possible use of ITF2984 in the pharmacological treatment of NF-PitNET.

Alterations of Perineuronal Net Expression and Abnormal Social Behavior and Whisker-dependent Texture Discrimination in Mice Lacking the Autism Candidate Gene Engrailed 2.

GABAergic interneurons and perineuronal nets (PNNs) are important regulators of plasticity throughout life and their dysfunction has been implicated in the pathogenesis of several neuropsychiatric conditions, including autism spectrum disorders (ASD). PNNs are condensed portions of the extracellular matrix (ECM) that are crucial for neural development and proper formation of synaptic connections. We previously showed a reduced expression of GABAergic interneuron markers in the hippocampus and somatosensory cortex of adult mice lacking the Engrailed2 gene (En2-/- mice), a mouse model of ASD. Since alterations in PNNs have been proposed as a possible pathogenic mechanism in ASD, we hypothesized that the PNN dysfunction may contribute to the neural and behavioral abnormalities of En2-/- mice. Here, we show an increase in the PNN fluorescence intensity, evaluated by Wisteria floribunda agglutinin, in brain regions involved in social behavior and somatosensory processing. In addition, we found that En2-/- mice exhibit altered texture discrimination through whiskers and display a marked decrease in the preference for social novelty. Our results raise the possibility that altered expression of PNNs, together with defects of GABAergic interneurons, might contribute to the pathogenesis of social and sensory behavioral abnormalities.

A ß-Arrestin 2-Biased Dopamine Receptor Type 2 (DRD2) Agonist Is More Efficacious Than Cabergoline in Reducing Cell Proliferation in PRL-Secreting but Not in Non-Functioning Pituitary Tumor Cells.

The molecular events underlying the variable effectiveness of dopamine receptor type 2 (DRD2) agonists in pituitary neuroendocrine tumors (PitNETs) are not known. Besides the canonical pathway induced by DRD2 coupling with Gi proteins, the ß-arrestin 2 pathway contributes to DRD2's antimitotic effects in PRL- and NF-PitNETs. A promising pharmacological strategy is the use of DRD2-biased agonists that selectively activate only one of these two pathways. The aim of the present study was to compare the effects of two biased DRD2 ligands, selectively activating the G protein (MLS1547) or ß-arrestin 2 (UNC9994) pathway, with unbiased DRD2 agonist cabergoline in PRL- and NF-PitNET cells. In rat tumoral pituitary PRL-secreting MMQ cells, UNC9994 reduced cell proliferation with a greater efficacy compared to cabergoline (-40.2 ± 20.4% vs. -21 ± 10.9%, p < 0.05), whereas the G-protein-biased agonist induced only a slight reduction. ß-arrestin 2 silencing, but not pertussis toxin treatment, reverted UNC9994 and cabergoline's antiproliferative effects. In a cabergoline-resistant PRL-PitNET primary culture, UNC9994 inhibited cell proliferation and PRL release. In contrast, in NF-PitNET primary cultures (n = 23), biased agonists did not show better antiproliferative effects than cabergoline. In conclusion, the preferential activation of the ß-arrestin 2 pathway by UNC9994 improves DRD2-mediated antiproliferative effects in PRL-PitNETs, suggesting a new pharmacological approach for resistant or poorly responsive tumors.

TMPRSS2 Expression and Activity Modulation by Sex-Related Hormones in Lung Calu-3 Cells: Impact on Gender-Specific SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although males and females are at equivalent risk of infection, males are more prone to develop a higher severity disease, regardless of age. The factors that mediate susceptibility to SARS-CoV-2 and transmission are still under investigation. A potential role has been attributed to differences in the immune systems response to viral antigens between males and females as well as to different regulatory actions played by sex-related hormones on the two crucial molecular effectors for SARS-CoV-2 infection, TMPRSS2 and ACE2. While few and controversial data about TMPRSS2 transcript regulation in lung cells are emerging, no data on protein expression and activity of TMPRSS2 have been reported. Aim of the present study was to search for possible modulatory actions played by sex-related hormones on TMPRSS2 and ACE2 expression in Calu-3 cells, to test the effects of sex-steroids on the expression of the 32kDa C-term fragment derived from autocatalitic cleavage of TMPRSS2 and its impact on priming of transiently transfected spike protein. Cells were stimulated with different concentrations of methyltrienolone (R1881) or estradiol for 30 h. No difference in mRNA and protein expression levels of full length TMPRSS2 was observed. However, the 32 kDa cleaved serine protease domain was increased after 100 nM R1881 (+2.36 ± 1.13 fold-increase vs control untreated cells, p < 0.05) and 10 nM estradiol (+1.90 ± 0.64, fold-increase vs control untreated cells, p < 0.05) treatment. Both R1881 and estradiol significantly increased the activating proteolytic cleavage of SARS-CoV-2 Spike (S) transfected in Calu-3 cells (+1.76 ± 0.18 and +1.99±,0.76 increase in S cleavage products at R1881 100nM and 10 nM estradiol treatment, respectively, p < 0.001 and p < 0.05 vs control untreated cells, respectively). Finally, no significant differences in ACE2 expression were observed between hormones-stimulated cells and untreated control cells. Altogether, these data suggest that both male and female sex-related hormones are able to induce a proteolityc activation of TMPRSS2, thus promoting viral infection, in agreement with the observation that males and females are equally infected by SARS-CoV-2.

DRD2 Agonist Cabergoline Abolished the Escape Mechanism Induced by mTOR Inhibitor Everolimus in Tumoral Pituitary Cells.

The mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to display antiproliferative effects on a wide spectrum of tumors. In vitro studies demonstrated that everolimus inhibited pituitary neuroendocrine tumor (PitNET) cell growth in a subset of patients. Sensitivity to everolimus is reduced by an escape mechanism that increases AKT phosphorylation (p-AKT), leading to pro-survival pathway activation. Dopamine receptor type 2 (DRD2) mediates a reduction of p-AKT in a subgroup of non-functioning PitNETs (NF-PitNETs) and in prolactin-secreting tumor cells (MMQ cells) through a ß-arrestin 2-dependent mechanism. The aim of this study was to investigate the efficacy of everolimus combined with DRD2 agonist cabergoline in reducing NF-PitNET primary cells and MMQ cell proliferation and to evaluate AKT phosphorylation and a possible role of ß-arrestin 2. We found that 9 out of 14 NF-PitNETs were resistant to everolimus, but the combined treatment with cabergoline inhibited cell proliferation in 7 out of 9 tumors (-31.4 ± 9.9%, p < 0.001 vs. basal) and reduced cyclin D3 expression. In the everolimus-unresponsive NF-PitNET group, everolimus determined a significant increase of p-AKT/total-AKT ratio (2.1-fold, p < 0.01, vs. basal) that was reverted by cabergoline cotreatment. To investigate the molecular mechanism involved, we used MMQ cells as a model of everolimus escape mechanism. Indeed everolimus did not affect MMQ cell proliferation and increased the p-AKT/total-AKT ratio (+1.53 ± 0.24-fold, p < 0.001 vs. basal), whereas cabergoline significantly reduced cell proliferation (-22.8 ± 6.8%, p < 0.001 vs. basal) and p-AKT. The combined treatment of everolimus and cabergoline induced a reduction of both cell proliferation (-34.8 ± 18%, p < 0.001 vs. basal and p < 0.05 vs. cabergoline alone) and p-AKT/total-AKT ratio (-34.5 ± 14%, p < 0.001 vs. basal and p < 0.05 vs. cabergoline alone). To test ß-arrestin 2 involvement, silencing experiments were performed in MMQ cells. Our data showed that the lack of ß-arrestin 2 prevented the everolimus and cabergoline cotreatment inhibitory effects on both p-AKT and cell proliferation. In conclusion, this study revealed that cabergoline might overcome the everolimus escape mechanism in NF-PitNETs and tumoral lactotrophs by inhibiting upstream AKT activation. The co-administration of cabergoline might improve mTOR inhibitor antitumoral activity, paving the way for a potential combined therapy in ß-arrestin 2-expressing NF-PitNETs or other PitNETs resistant to conventional treatments.

Adrenalectomy Improves Blood Pressure and Metabolic Control in Patients With Possible Autonomous Cortisol Secretion: Results of a RCT.

Objective: The best approach to patients with adrenal incidentaloma (AI) and possible autonomous cortisol secretion (PACS) is debated. The aim of this study was to assess the metabolic effect of adrenalectomy in AI patients with PACS in relation to cortisol secretion parameters, peripheral activation, and glucocorticoid sensitivity. Design: This is a multicenter randomized study (NCT number: NCT04860180). Methods: Sixty-two AI outpatients (40-75 years) with AI >1 cm and cortisol after overnight dexamethasone suppression test (F-1mgDST) between 50 and 138 nmol/L were randomized to adrenalectomy (Arm A) or a conservative approach (Arm B). Fifty-five patients completed the 6-month follow-up, 25 patients in Arm A (17 female patients, aged 62.5 ± 10.4 years) and 30 patients in Arm B (24 female patients, 66.1 ± 9.1 years). Plasma adrenocorticotroph hormone (ACTH), 24-h urinary free cortisol, 24-h urinary free cortisone, F-1mgDST, glucose, lipids, glycated hemoglobin (HbA1c) levels, blood pressure (BP), body weight, and treatment variations were assessed. The 24-h urinary free cortisol/cortisone ratio (an 11-beta hydroxysteroid dehydrogenase type 2 activity marker), BclI, and the N363S variants of glucocorticoid receptor (GR) polymorphisms were also evaluated. Results: BP control improved in 68% and 13% of the subjects in Arm A and Arm B, respectively (p = 0.001), and the glycometabolic control improved in 28% and 3.3% of the subjects in Arm A and Arm B patients, respectively (p = 0.02). Arm A subjects more rarely showed the BP and/or glycometabolic control worsening than Arm B patients (12% and 40%, respectively, p = 0.03). The surgical approach was independently associated with BP amelioration (OR 3.0, 95% CI 3.8-108.3, p < 0.001) but not with age, F-1mgDST levels, BMI, and hypertension and diabetes mellitus presence at baseline. The 24-h urinary free cortisol/cortisone ratio and the presence of sensitizing GR polymorphisms were not associated with the surgical outcome. The receiver operating characteristic (ROC) curve analysis showed that the BP control amelioration was associated with F-1mgDST [area under the curve (AUC), 0.82 ± 0.09 p = 0.012]. The F-1mgDST cutoff with the best compromise in predicting the BP amelioration was set at 75 nmol/L (sensitivity 77%, specificity 75%). Conclusions: AI patients with PACS benefit from surgery in terms of BP and glycometabolic control.

P720R USP8 Mutation Is Associated with a Better Responsiveness to Pasireotide in ACTH-Secreting PitNETs.

Somatic mutations in the ubiquitin specific peptidase 8 (USP8) gene have been associated with higher levels of somatostatin (SS) receptor subtype 5 (SSTR5) in adrenocorticotroph hormone (ACTH)-secreting pituitary neuroendocrine tumors (PitNETs). However, a correlation between the USP8 mutational status and favourable responses to pasireotide, the somatostatin multi-receptor ligand acting especially on SSTR5, has not been investigated yet. Here, we studied the impact of USP8 mutations on pasireotide responsiveness in human and murine corticotroph tumor cells. SSTR5 upregulation was observed in USP8 wild-type primary tumor cells transfected with S718del USP8 mutant. However, cell transfection with S718del USP8 and C40-USP8 mutants in in vitro sensitive cultures from USP8 wild-type tumors abolished their ability to respond to pasireotide and did not confer pasireotide responsiveness to the in vitro resistant culture. Pasireotide failed to reduce ACTH secretion in primary cells from one S718P USP8-mutated tumor but exerted a strong antisecretory effect in primary cells from one P720R USP8-mutated tumor. In agreement, AtT-20 cells transfection with USP8 mutants led to SSTR5 expression increase but pasireotide could reduce ACTH production and cyclin E expression in P720R USP8 overexpressing cells, only. In situ Proximity Ligation Assay and immunoflurescence experiments revealed that P720R USP8 mutant is still able to bind 14-3-3 proteins in AtT-20 cells, without affecting SSTR5 localization. In conclusion, P720R USP8 mutation might be considered as a molecular predictor of favourable response to pasireotide in corticotroph tumor cells.

Natural SINEUP RNAs in Autism Spectrum Disorders: RAB11B-AS1 Dysregulation in a Neuronal CHD8 Suppression Model Leads to RAB11B Protein Increase.

CHD8 represents one of the highest confidence genetic risk factors implied in Autism Spectrum Disorders, with most mutations leading to CHD8 haploinsufficiency and the insurgence of specific phenotypes, such as macrocephaly, facial dysmorphisms, intellectual disability, and gastrointestinal complaints. While extensive studies have been conducted on the possible consequences of CHD8 suppression and protein coding RNAs dysregulation during neuronal development, the effects of transcriptional changes of long non-coding RNAs (lncRNAs) remain unclear. In this study, we focused on a peculiar class of natural antisense lncRNAs, SINEUPs, that enhance translation of a target mRNA through the activity of two RNA domains, an embedded transposable element sequence and an antisense region. By looking at dysregulated transcripts following CHD8 knock down (KD), we first identified RAB11B-AS1 as a potential SINEUP RNA for its domain configuration. Then we demonstrated that such lncRNA is able to increase endogenous RAB11B protein amounts without affecting its transcriptional levels. RAB11B has a pivotal role in vesicular trafficking, and mutations on this gene correlate with intellectual disability and microcephaly. Thus, our study discloses an additional layer of molecular regulation which is altered by CHD8 suppression. This represents the first experimental confirmation that naturally occurring SINEUP could be involved in ASD pathogenesis and underscores the importance of dysregulation of functional lncRNAs in neurodevelopment.

Complementary and Integrative Approaches in Obstetrics and Gynecology: Clinical Updates In Women's Health Care Primary and Preventive Care Review.

ABSTRACT: Integrative medicine is a holistic approach to health care that acknowledges multiple dimensions of health, including its physical, emotional, and spiritual aspects. It approaches health not just as absence of disease but as a state of optimal vitality, and utilizes all appropriate evidence-based modalities, including lifestyle interventions, complementary treatments, and conventional allopathic methods, in one paradigm. Utilization of non-conventional therapies by women and interest in integrative care is very high. Much disinformation exists and many women engage in non-conventional therapies without medical advice, sometimes benefiting from them, but also possibly delaying needed care or placing themselves at risk. Research and clinical interest in non-conventional methods also is rising within the medical community, with increased recognition of the value of an integrative health model for individuals and communities. Obstetrician-gynecologists are in a unique position to support their patients in holistic health optimization by choosing beneficial integrative strategies while identifying potentially harmful practices.

Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel USP8 Gene Variant.

Cushing's Disease (CD) is a rare condition characterized by an overproduction of ACTH by an ACTH-secreting pituitary tumor, resulting in an excess of cortisol release by the adrenal glands. Somatic mutations in the deubiquitinases USP8 and USP48, and in BRAF genes, have been reported in a subset of patients affected by CD. The aim of this study was to characterize the genetic profile of a cohort of 60 patients with ACTH-secreting tumors, searching for somatic mutations in USP8, USP48, and BRAF hotspot regions. Seven patients were found to carry USP8 somatic mutations in the well-characterized 14-3-3 protein binding motif (n = 5 P720R, n = 1 P720Q, n = 1 S718del); 2 patients were mutated in USP48 (M415I); no mutation was identified in BRAF. In addition, a novel USP8 variant, G664R, located in exon 14, upstream of the 14-3-3 protein binding motif, was identified in 1 patient. Functional characterization of USP8 G664R variant was performed in murine corticotroph tumor AtT-20 cells. Transient transfection with the USP8 G664R variant resulted in a significant increase of ACTH release and cell proliferation (+114.5 ± 53.6% and +28.3 ± 2.6% vs. empty vector transfected cells, p < 0.05, respectively). Notably, USP8 proteolytic cleavage was enhanced in AtT-20 cells transfected with G664R USP8 (1.86 ± 0.58-fold increase of N-terminal USP8 fragment, vs. WT USP8, p < 0.05). Surprisingly, in situ Proximity Ligation Assay (PLA) experiments showed a significant reduction of PLA positive spots, indicating USP8/14-3-3 proteins colocalization, in G664R USP8 transfected cells with respect to WT USP8 transfected cells (-47.9 ± 6.6%, vs. WT USP8, p < 0.001). No significant difference in terms of ACTH secretion, cell proliferation and USP8 proteolytic cleavage, and 14-3-3 proteins interaction was observed between G664R USP8 and S718del USP8 transfected cells. Immunofluorescence experiments showed that, contrary to S718del USP8 but similarly to WT USP8 and other USP8 mutants, G664R USP8 displays an exclusive cytoplasmic localization. In conclusion, somatic mutations were found in USP8 (13.3% vs. 36.5% incidence of all published mutations) and USP48 (3.3% vs. 13.3% incidence) hotspot regions. A novel USP8 variant was identified in a CD patient, and in vitro functional studies in AtT-20 cells suggested that this somatic variant might be clinically relevant in ACTH-secreting tumor pathogenesis, expanding the characterization of USP8 functional domains.

USP8 inhibitor RA-9 reduces ACTH release and cell growth in tumor corticotrophs.

Cushing's disease (CD) is a rare endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor. Pasireotide is the only pituitary-targeted drug approved for adult patients. Nevertheless, many side effects are encountered and curative therapy is still challenging. Ubiquitin-specific peptidase 8 (USP8) plays a crucial role in the modulation of corticotroph cells growth and ACTH secretion. Here, we explored the anticancer potential of the USP8 inhibitor RA-9 in USP8-WT human tumor corticotroph cells and murine AtT-20 cells. Our results showed that RA-9 causes cell proliferation decrease (-24.3 ± 5.2%, P < 0.01) and cell apoptosis increase (207.4 ± 75.3%, P < 0.05) in AtT-20 cells, as observed with pasireotide. Moreover, RA-9 reduced ACTH secretion in AtT-20 cells (-34.1 ± 19.5%, P < 0.01), as well as in AtT-20 cells transfected with USP8 mutants, and in one out of two primary cultures in vitro responsive to pasireotide (-40.3 ± 6%). An RA-9 mediated decrease of pERK1/2 levels was observed in AtT-20 cells (-52.3 ± 13.4%, P < 0.001), comparable to pasireotide, and in primary cultures, regardless of their in vitro responsiveness to pasireotide. Upregulation of p27 was detected upon RA-9 treatment only, both in AtT-20 cells (167.1 ± 36.7%, P < 0.05) and in one primary culture tested (168.4%), whilst pCREB level was similarly halved in AtT-20 cells by both RA-9 and pasireotide. Altogether, our data demonstrate that RA-9 is efficient in exerting cytotoxic effects and inhibitory actions on cell proliferation and hormone secretion by modulating the expression of pERK1/2, pCREB and p27. Inhibition of USP8 might represent a novel strategy to target both USP8-WT and USP8-mutated tumors in CD patients.

Filamin A is required for somatostatin receptor type 5 expression and pasireotide-mediated signaling in pituitary corticotroph tumor cells.

Somatostatin receptor type 5 (SST5) represents the main pharmacological target in the treatment of adrenocorticotroph hormone (ACTH)-secreting tumors. However, molecular predictors of responsiveness to pasireotide require further investigation. The cytoskeleton protein filamin A (FLNA) modulates the responsiveness to somatostatin analogs (SSA) treatment in other types of pituitary tumors by regulating somatostatin receptor type 2 (SST2)/dopamine receptor type 2 (DRD2) expression and activity. Here, we aimed to test the involvement of FLNA in the modulation of SST5 response to SSA in human and murine tumor corticotrophs. Western blot analysis of human corticotropinomas showed that FLNA and SST5 correlate. Both in human primary cultures and AtT-20 cells, FLNA genetic silencing caused a decrease of receptor expression level. Moreover, pasireotide-mediated SST5 downregulation observed in AtT-20 control cells was no further detected in FLNA silenced cells. In AtT-20 cells, in situ PLA experiments revealed an increased number of SST5-FLNA complexes following pasireotide incubation. Finally, FLNA knock down abolished pasireotide-induced SST5 actions on hormone secretion, cell proliferation and apoptosis. In conclusion, FLNA is implicated in SST5 expression modulation and signaling.

Beta-Arrestin 2 Is Required for Dopamine Receptor Type 2 Inhibitory Effects on AKT Phosphorylation and Cell Proliferation in Pituitary Tumors.

Dopamine receptor type 2 (DRD2) agonists are the first-choice treatment for prolactin-secreting pituitary tumors but are poorly effective in nonfunctioning pituitary neuroendocrine tumors (NF-PitNETs). DRD2 reduces AKT phosphorylation in lactotrophs, but no data are available in NF-PitNETs. DRD2 effects on AKT are mediated by a ß-arrestin 2-dependent mechanism in mouse striatum. The aim of this study was to investigate DRD2 effects on AKT phosphorylation and cell proliferation in human primary cultured NF-PitNET cells and in rat tumoral lactotroph cells MMQ, and to test ß-arrestin 2 involvement. We found that the DRD2 agonist BIM53097 induced a reduction of the p-AKT/total-AKT ratio in MMQ (-32.8 ± 17.6%, p < 0.001 vs. basal) and in a subset (n = 15/41, 36.6%) of NF-PitNETs (subgroup 1). In the remaining NF-PitNETs (subgroup 2), BIM53097 induced an increase in p-AKT. The ability of BIM53097 to reduce p-AKT correlated with its antimitotic effect, since the majority of subgroup 1 NF-PitNETs was responsive to BIM53097, and nearly all subgroup 2 NF-PitNETs were resistant. ß-Arrestin 2 was expressed in MMQ and in 80% of subgroup 1 NF-PitNETs, whereas it was undetectable in 77% of subgroup 2 NF-PitNETs. In MMQ, ß-arrestin 2 silencing prevented DRD2 inhibitory effects on p-AKT and cell proliferation. Accordingly, ß-arrestin 2 transfection in subgroup 2 NF-PitNETs conferred to BIM53097 the ability to inhibit both p-AKT and cell growth. In conclusion, we demonstrated that ß-arrestin 2 is required for DRD2 inhibitory effects on AKT phosphorylation and cell proliferation in MMQ and NF-PitNETs, paving the way for a potential role of ß-arrestin 2 as a biomarker predicting NF-PitNETs' responsiveness to treatment with dopamine agonists.

Urinary biomarkers of nucleic acid oxidation and methylation in workers exposed to low concentrations of benzene.

The study aims to investigate the influence of exposure to low concentrations of benzene on urinary biomarkers of nucleic acid oxidative damage and methylation. Benzene exposure was characterized for 93 coke production workers by measuring both airborne benzene and S-phenylmercapturic acid (SPMA) and unmodified benzene (U-B) in urine samples, collected at the end of the shift (ES) and at the next morning before shift (next BS). In the same urinary samples, biomarkers of nucleic acid oxidative damage and methylation were determined. Urinary concentrations of cotinine and creatinine were also determined to evaluate the smoking effect and to normalize urinary concentrations of analytes, respectively. The biomarkers of benzene internal dose, of oxidative damage (8-hydroxyy-7,8-dihydroguanine, 8-hydroxy-7,8-dihydroguanosine and 8-hydroxy-7,8-2'deoxyguanosine) and some of the biomarkers of nucleic acid methylation (5-Methyl-Cytosine, 1-Methyl-Guanine and 7-Methyl-Guanine) were higher in the ES than the next BS samples. Positive associations between ES 5-Methyl-Cytosine and both SPMA and U-B were found. In conclusion, occupational exposure to low levels of benzene seems to be related to urinary ES 5-Methyl-Cytosine that could be a possible biomarker to evaluate the changes of the nucleic acid methylation status.

Prediction of hypertension, diabetes and fractures in eucortisolemic women by measuring parameters of cortisol milieu.

PURPOSE: Cortisol secretion, peripheral activation, and sensitivity seem to be associated with hypertension (HY), type 2 diabetes (T2D), and fragility fractures (FX) even in eucortisolemic subjects. The aim of the present study was to determine the cutoff(s) of the parameters of cortisol secretion and peripheral activation for predicting the presence of HY, T2D, and FX (comorbidities). METHODS: In 206 postmenopausal females (157 with ≥1 comorbidities and 49 without any), we assessed the ratio between 24-h urinary free cortisol and cortisone (R-UFF/UFE, cortisol activation index), cortisol after 1 mg-overnight-dexamethasone (F-1mgDST, cortisol secretion index), and the GC receptor N363S single-nucleotide polymorphism (N363S-SNP, cortisol sensitivity index). RESULTS: The cutoffs for F-1mgDST and R-UFF/UFE were set at 0.9 µg/dL (area under the curve, AUC 0.634 ± 0.43, p = 0.005) and 0.17 (AUC 0.624 ± 0.5, p = 0.017), respectively, predicted the presence of ≥1 comorbidities. The presence of F-1mgDST > 0.9 µg/dL plus R-UFF/UFE > 0.17 showed 82.1% specificity for predicting the presence of ≥1 comorbidities, while the simultaneous presence of F-1mgDST ≤ 0.9 µg/dL and R-UFF/UFE ≤ 0.17 showed 88% sensitivity for predicting the absence of comorbidities. The F-1mgDST > 0.9 µg/dL or R-UFF/UFE > 0.17 was associated with 2.8 and 2.1-fold increased risk of having ≥1 comorbidities, respectively. The F-1mgDST ≤ 0.9 µg/dL plus R-UFF/UFE ≤ 0.17 or F-1mgDST > 0.9 µg/dL plus R-UFF/UFE > 0.17 was associated with 2.8-fold reduced or 4.9-fold increased risk of having ≥1 comorbidities regardless of age, BMI, and N363S-SNP. CONCLUSIONS: F-1mgDST > 0.9 µg/dL and R-UFF/UFE > 0.17 may be used for predicting the presence of ≥1 among HY, T2D, and fragility FX.

Cortisol Secretion, Sensitivity, and Activity Are Associated With Hypertension in Postmenopausal Eucortisolemic Women.

CONTEXT: Previous data suggest a possible association between type 2 diabetes (T2D) and fragility fractures (FX) with the degree of glucocorticoid suppressibility (GCS) and peripheral activation or sensitivity even in persons without hypercortisolemia. OBJECTIVE: To investigate whether the degree of GCS, GC sensitivity, and peripheral activation in persons without overt or mild hypercortisolism are associated with hypertension and with the number of the possible consequences of cortisol excess among patients with T2D, fragility FX, and hypertension. DESIGN: Case-control study. SETTING: Outpatient clinic. PATIENTS: A total of 216 postmenopausal women without hypercortisolemia (age, 50 to 80 years; 108 with hypertension); 68 and 99 patients had fragility FX and T2D, respectively. MAIN OUTCOME MEASURES: We assessed 24-hour urinary free cortisol (UFF), cortisone (UFE), their ratio (R-UFF/UFE), (F-1mgDST), and the GC receptor N363S single-nucleotide polymorphism (N363S-SNP). RESULTS: Hypertension was associated with F-1 mgDST [odds ratio (OR), 3.3; 95% CI, 1.5 to 7.5; P = 0.004) and R-UFF/UFE (OR, 101.7; 95% CI, 2.6 to 4004.1; P = 0.014), regardless of age, body mass index, and presence of the N363S single nucleotide polymorphism and of T2D. The progressive increase in the number of possible consequences of cortisol excess was significantly associated with F-1mgDST levels (R2 = 0.125; P = 0.04), R-UFF/UFE (R2 = 0.46; P = 0.02), and the prevalence of N363S heterozygous variant (T = 0.46; P = 0.015), after adjustment for age. CONCLUSIONS: In postmenopausal women without hypercortisolemia, hypertension is associated with GCS and GC peripheral activation. The number of possible consequences of cortisol excess (among patients with hypertension, T2D, and fragility FX) is associated with GCS, GC peripheral activation, and the prevalence of the N363S heterozygous variant.

[Occupational safety and health risks in seaport: an update of narrative review]. / I rischi per la salute e la sicurezza nel lavoro portuale: aggiornamento della revisione narrativa della letteratura.

SUMMARY: Seaports are complex systems in which workers can be exposed to a large variety of safety and health risks. Nevertheless, a little literature is available concerning this topic, if we exclude the specific area of shipbuilding industry. Objectives. The aim of this paper is to update the review of the scientific literature previously published as result of a project concerning the occupational risks in seaports. Methods. Literature on this theme, obtained consulting the main databases (PubMed, Scholar and CCOHS) from 2012 and up to April 2019, was reviewed. Results. 5 of 8 articles published after 2012 were related to risk of release or formation of volatile compounds in restricted and poorly ventilated areas or inhalation of particles from specific goods. Three papers specifically debated musculoskeletal disorders related to loading/unloading procedures, occupational diseases and injuries. Conclusions. The update of the literature highlighted intrinsically dangerous goods, toxic volatile compounds and emissions as critical aspects of seaport activities related to goods handling. Recently, the literature shows a growing interest in occupational health, especially work-related musculoskeletal diseases. Prevention measures and implementation of worker's training and information are identified by all authors as the more effective action to increase health and safety..

Suppressive effects of tenofovir disoproxil fumarate, an antiretroviral prodrug, on mineralization and type II and type III sodium-dependent phosphate transporters expression in primary human osteoblasts.

Tenofovir disoproxil fumarate (TDF) is an antiretroviral drug commonly used for the management of Human Immunodeficiency Virus (HIV) in highly active antiretroviral therapy (HAART) and of chronic Hepatitis B Virus (HBV) infections. Long-term TDF-treated subjects present decrease of bone mineral density and rarely severe osteomalacia. Although these adverse effects have been attributed to the impaired proximal tubule function, a possible direct involvement of TDF on osteoblasts should be taken into account. The aim of this study was to evaluate whether sodium phosphate transporters NPT2A (sodium-dependent phosphate transport protein 2A), NPT2C (sodium-dependent phosphate transport protein 2C), PIT1 (sodium-dependent phosphate transporter 1), and PIT2 (sodium-dependent phosphate transporter 2) were expressed in primary human osteoblasts (HOBs), whether their expression was related to HOBs differentiation and whether TDF could affect mineralization and gene expression. PIT1 and PIT2 were expressed under proliferating conditions and increased after induction of mineralization, while NPT2A and NPT2C were almost undetectable. In HOBs TDF exposure induced a significant dose-dependent decrease in mineralization. Moreover, TDF caused a reduction of COL1A1 and of ATF4 expression in differentiated HOBs. In summary, HOBs do not express NPT2A and NPT2C and do express PIT1 and PIT2, suggesting a role of these two latter in human osteoblast mineralization. TDF impairs osteoblast mineralization, confirming a direct negative effect on bone. Therefore, in clinical practice, bone damage must be suspected and evaluated also in patients receiving TDF without kidney function alterations.

Biological monitoring of exposure to low concentrations of benzene in workers at a metallurgical coke production plant: new insights into S-phenylmercapturic acid and urinary benzene.

CONTEXT: Urinary S-phenylmercapturic acid (SPMA) and benzene (U-Ben) are usually measured at the end of the work shift (ES), although their kinetic of elimination is not clearly known. OBJECTIVE: To investigate SPMA and U-Ben elimination 16 h after the ES, in 93 coke production workers exposed to low benzene concentrations. MATERIALS AND METHODS: Airborne benzene (A-Ben) was measured by passive samplings, while SPMA, U-Ben, methyl-tert-butyl ether (U-MTBE), cotinine (U-Cot) and creatinine were determined on urine samples collected at ES and before the beginning of the next work shift (next BS). RESULTS: Median A-Ben concentrations were 17.2 µg/m3 in the personal and 34.7 µg/m3 in the stationary samplings. SPMA was always detectable, whereas U-Ben was below the limit of quantification in 26.7% of the ES and 35.6% of the next BS samples, and U-MTBE in more than the 80.0% of the samples. At both the sampling times, SPMA and U-Ben showed a positive dependence on personal A-Ben, as well as on creatinine and U-Cot values. DISCUSSION AND CONCLUSION: SPMA and U-Ben at the next BS were dependent on the exposure to low benzene concentrations suffered in the previous work shift, prompting a reconsideration of the urine sampling time recommended by the American Conference Governmental Industrial Hygienists (ACGIH).

DNA damage and repair capacity in workers exposed to low concentrations of benzene.

DNA damage and cellular repair capacity were studied in 18 male fuel tanker drivers and 13 male filling-station attendants exposed to low and very low concentrations of benzene, respectively, and compared to 20 males with no occupational exposure (controls). Exposure to airborne benzene was measured using passive personal samplers, and internal doses were assayed through the biomarkers t,t-muconic acid, S-phenylmercapturic acid and urinary benzene. DNA damage was evaluated using tail intensity (TI) determined by the comet assay in peripheral lymphocytes. Urinary 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) was measured as a biomarker of oxidative damage. DNA repair kinetics were assessed using the comet assay in lymphocytes sampled 20 and 60 min post H2O2 exposure. Benzene exposure differed significantly between the drivers (median 246.3 µg/m(3)), attendants (median 13.8 µg/m(3)), and controls (median 4.1 µg/m(3)). There were no differences in TI and 8-oxodG among the three groups, or between smokers and non-smokers. DNA repair kinetics were similar among the drivers, attendants and controls, although the comet assay on H2 O2 -damaged lymphocytes after 60 min revealed significantly lower levels of TI only in drivers. The DNA repair process in smokers was similar to that observed in drivers. In conclusion, this study found no relationship between low levels of benzene exposure and DNA damage, although there was evidence that exposure interferes with DNA repair kinetics. The biological impact of this finding on the onset of genotoxic effects in exposed workers has still to be ascertained.