Novel Insights into Mercury Effects on Hemoglobin and Membrane Proteins in Human Erythrocytes.

Mercury (Hg) is a global environmental pollutant that affects human and ecosystem health. With the aim of exploring the Hg-induced protein modifications, intact human erythrocytes were exposed to HgCl2 (1-60 µM) and cytosolic and membrane proteins were analyzed by SDS-PAGE and AU-PAGE. A spectrofluorimetric assay for quantification of Reactive Oxygen Species (ROS) generation was also performed. Hg2+ exposure induces alterations in the electrophoretic profile of cytosolic proteins with a significant decrease in the intensity of the hemoglobin monomer, associated with the appearance of a 64 kDa band, identified as a mercurized tetrameric form. This protein decreases with increasing HgCl2 concentrations and Hg-induced ROS formation. Moreover, it appears resistant to urea denaturation and it is only partially dissociated by exposure to dithiothreitol, likely due to additional protein-Hg interactions involved in aggregate formation. In addition, specific membrane proteins, including band 3 and cytoskeletal proteins 4.1 and 4.2, are affected by Hg2+-treatment. The findings reported provide new insights into the Hg-induced possible detrimental effects on erythrocyte physiology, mainly related to alterations in the oxygen binding capacity of hemoglobin as well as decreases in band 3-mediated anion exchange. Finally, modifications of cytoskeletal proteins 4.1 and 4.2 could contribute to the previously reported alteration in cell morphology.

Melphalan hypoxic perfusion with hemofiltration for melanoma locoregional metastases in the pelvis.

BACKGROUND: For patients with melanoma metastases in the pelvic and groin regions, the median survival time (MST) was 8 mo with old treatments, whereas today is approximately 20 mo with new target therapy and novel immunotherapy. Unfortunately, approximately 30% of patients are nonresponsive to these new drugs. MATERIALS AND METHODS: Thirty-six patients, previously progressing after standard treatments, collectively received 146 melphalan (30 mg/m2) hypoxic pelvic perfusions with hemofiltration, in association with palliative excision in most cases. RESULTS: The median follow-up time was 15 mo. Among 36 patients, three patients were alive without evidence of disease after 62, 95, and 118 mo, respectively. Thirty-three patients died of melanoma. The overall MST was 15 mo. The 5-y survival rate was 8%. The MST was 37 mo for stage IIIB; 19 mo for stage IIIC; and 6 mo for stage IV. The MST was 11 mo for patients with ≥1 mitosis per mm2 and 20 mo for patients with <1 mitosis per mm2; 17 mo for patients who received excision and 7 mo for patients who did not receive excision; and 19.5 mo for patients who received >2 treatments and 7.5 mo for patients who received ≤2 treatments. CONCLUSIONS: Pelvic/inguinal perfusion is a safe and feasible treatment for patients with advanced melanoma. Further studies are necessary to establish if it may play a role in patients who fail current systemic therapies.

Does Locoregional Chemotherapy Still Matter in the Treatment of Advanced Pelvic Melanoma?

Pelvic Melanoma relapse occurs in 15% of patients with loco regional metastases, and 25% of cases do not respond to new target-therapy and/or immunotherapy. Melphalan hypoxic pelvic perfusion may, therefore, be an option for these non-responsive patients. Overall median survival time (MST), stratified for variables, including BRAF V600E mutation and eligibility for treatments with new immunotherapy drugs, was retrospectively assessed in 41 patients with pelvic melanoma loco regional metastases. They had received a total of 175 treatments with Melphalan hypoxic perfusion and cytoreductive excision. Among the 41 patients, 22 (53.7%) patients exhibited a wild-type BRAF genotype, 11 of which were not eligible for immunotherapy. The first treatment resulted in a 97.5% response-rate in the full cohort and a 100% response-rate in the 22 wild-type BRAF patients. MST was 18 months in the full sample, 20 months for the 22 wild-type BRAF patients and 21 months for the 11 wild-type BRAF patients not eligible for immunotherapy. Melphalan hypoxic perfusion is a potentially effective treatment for patients with pelvic melanoma loco regional metastases that requires confirmation in a larger multicenter study.

Structural segregation in GSR from mercuric primers. A preliminary study.

Since the second half of the XX century, primer mixtures based on mercury fulminate have become a rare occurrence on small ammunition markets in Western Europe and North America. As a consequence, Hg-containing gunshot residue (GSR) particles have not been as deeply investigated as residues from lead-based primer mixtures. As a matter of fact, no mention of GSR particles from mercuric primers is made by the current ASTM standard procedure for gunshot residue analysis. However, those laboratories dealing with ammunition and firearms produced in Eastern Europe or Asia still have a forensic interest in Hg-containing GSR. In this paper, a brief description of chemical composition and inner morphology of GSR particles from three different mercuric primers is reported. Regarding composition, arguments are given to promote SbSnHg residues to Characteristic of GSR particles when mercuric primers are discharged. From a morphological point of view, presence of inner nodules and other inhomogeneities were shown in GSR particles milled in a FIB/SEM. Moreover, mercury vaporization under the electron beam was observed for a particle reduced to a lamella. Mercury evanescence in GSR was interpreted in terms of mercury segregation during particle formation and higher mobility of Hg atoms in presence of defects (vacancies) in a strained lattice.

Molecular and toxicological mechanisms behind the effects of chromium (VI) on the male reproductive system of Mytilus galloprovincialis: First evidence for poly-ADP-ribosylation of protamine-like II.

Studies on the molecular mechanisms of heavy metal toxicity in invertebrate reproduction are limited. Given that PARP-catalysed ADP-ribosylation is also involved in counteracting heavy metal toxicity and maintaining genomic integrity, and that PARylation is implicated in chromatin remodelling but its role in sperm chromatin remains to be elucidated, we investigated the effects of chromium(VI) at 1, 10 and 100 nM on the reproductive health of Mytilus galloprovincialis. The damage to the gonads was assessed by morphological analyses and the damage indices PARP and É£H2A.X were measured. Changes in the binding of protamine-like (PL) to DNA and the possibility of poly(ADP-ribosyl)ation of PL proteins were also investigated. Gonadal chromium accumulation and morphological damage were found, especially when the mussels were exposed to the highest dose of chromium(VI). In addition, the maximum expression of gonadal É£H2A.X and PARP were obtained at 100 and 10 nM Cr(VI), respectively. Interestingly, for the first time in all exposed conditions, poly(ADP)-ribosylation was detected on PL-II, which, together with PL-III and PL-IV, are the major nuclear basic proteins of Mytilus galloprovincialis sperm chromatin. Since PL-II is involved in the final high level of sperm chromatin compaction, this post-translational modification altered the binding of the PL protein to DNA, favouring the action of micrococcal nuclease on sperm chromatin. This study provides new insights into the effects of chromium(VI) on Mytilus galloprovincialis reproductive system and proposes a molecular mechanism hypothesis describing the toxic effects of this metal on PL-DNA binding, sperm chromatin and gonads.

Antioxidant Characterization of Six Tomato Cultivars and Derived Products Destined for Human Consumption.

The consumption of fresh tomatoes and processed tomato products is widespread in the Mediterranean diet. This fruit is a valuable source of antioxidants and plays an important role in preventing oxidative stress. This study aimed to investigate the content of antioxidants and measure the total antioxidant capacity (ABTS and DPPH assays) in the peel, pulp, and seed fractions of six tomato cultivars. Finally, some bioactive compounds and total antioxidant activity were also determined in homemade tomato purees, since such homemade production is commonplace in Southern Italy. The level of antioxidants and total antioxidant capacity in each fraction were also calculated based on their actual fresh weight in the whole tomato. The overall results indicated that the peel and seeds of all analysed tomato cultivars contribute significantly to the antioxidant charge of the fruits. Consequently, consuming tomatoes without peel and seeds results in a substantial loss of compounds beneficial for human health. Our results also showed that phenolic and lycopene content, as well as antioxidant activities in all purees are higher than in fresh tomatoes. Based on this evidence, producing homemade tomato puree is a good practice, and its consumption helps prevent oxidative stress damage.

Evaluation of prednisolone and prednisolone sodium succinate concentrations in human plasma and inner ear perilymph during cochlear implantation 24 h after intratympanic injection.

BACKGROUND: The use of intratympanic (IT) steroids has drastically increased over the past 10-15 years to manage many otological pathologies. OBJECTIVES: This study aimed to compare the concentrations of prednisolone and prednisolone sodium succinate (SS) in the plasma and inner ear perilymph of participants who underwent cochlear implantation 24 h after IT injection. MATERIALS AND METHODS: It was a prospective comparative randomized study. Twenty participants received an IT injection of prednisolone SS ∼24 h before the cochlear implantation. The other five participants received an IT saline injection and represented the control group. Perilymph and blood were sampled during the cochlear implantation surgery. RESULTS: Both prednisolone and prednisolone SS were still present in perilymph ∼24 h after the IT administration. Only prednisolone was present in the blood plasma of seven participants (35%). CONCLUSION: IT injection of prednisolone SS resulted in high perilymph concentrations of prednisolone and prednisolone SS, which could stay in the perilymph for at least 24 h. Using a mini-endoscope during the IT injection may effectively detect barriers infront of the round window membrane, increasing the drug concentration in the inner ear. SIGNIFICANCE: IT injection is an effective method for delivering prednisolone to the inner ear.

Altered Expression of Protamine-like and Their DNA Binding Induced by Cr(VI): A Possible Risk to Spermatogenesis?

Chromium (VI) is the most dangerous oxidation state among the stable forms of chromium. In this work, we evaluated the effect of exposing Mytilus galloprovincialis for 24 h to 1, 10, and 100 nM chromium (VI) on the properties of Protamine-like (PLs) and their gene levels in the gonads. Specifically, we analyzed, by AU-PAGE and SDS-PAGE, PLs extracted from unexposed and exposed mussels. In addition, via EMSA, we evaluated the ability of PLs to bind DNA and also verified their potential to protect DNA from oxidative damage. Finally, we assessed possible alterations in gonadal expression of mt10, hsp70, and genes encoding for PLs-II/PL-IV and PL-III. We found that for all experimental approaches the most relevant alterations occurred after exposure to 1 nM Cr(VI). In particular, a comigration of PL-II with PL-III was observed by SDS-PAGE; and a reduced ability of PLs to bind and protect DNA from oxidative damage was recorded. This dose of chromium (VI) exposure was also the one that produced the greatest alterations in the expression of both mt10 and PL-II/PL-IV encoding genes. All of these changes suggest that this dose of chromium (VI) exposure could affect the reproductive health of Mytilus galloprovincialis.

Thoracic stop-flow perfusion for refractory lymphoma: a phase I-II evaluation trial.

BACKGROUND: Management of patients with heavily pretreated malignant lymphoma failing front-line treatment and salvage high-dose chemotherapy and autologous peripheral stem cell rescue is problematic. A phase I-II evaluation trial was conducted to evaluate thoracic stop-flow perfusion. PATIENTS AND METHODS: nine refractory patients were enrolled in the study. The schedule of thoracic stop-flow perfusion was based on cisplatin (100 mg/m(2)) and melphalan (25 mg/m(2)). Melphalan pharmacokinetic analyses were performed. All patients received hemofiltration during each procedure. RESULTS: Overall 18 cycles of perfusional chemotherapy were administered. During the procedures there were no technical, hemodynamic, or vascular complications, and no deaths occurred during surgery. Hematological and non-hematological toxicity was mild in relation to the use of hemofiltration during the procedures. All 9 patients responded favourably to stop-flow therapy. We observed 5 CR and 4 PR. Four out of five patients in CR relapsed. Three out of these four died of progressive disease after a response duration of 9, 7 and 7 months, respectively. One patient had a duration of CR of 10 months before relapse. He attained a new PR (+ 3 months). The remaining complete responder is still in remission after 37+ months. The 4 patients in PR progressed and died after a response duration of 4, 6, 2 and 1 month, respectively. To date, 8 out of 9 patients have died and one is still alive. CONCLUSION: Thoracic stop-flow perfusion seems very active in this kind of patient.

Development of a method for the determination of vardenafil in human plasma by high performance liquid chromatography with UV detection.

A simple high-performance liquid chromatographic (HPLC) method with photometric detection is described for the determination of vardenafil hydrochloride, a phosphodiesterase V inhibitor, in human plasma. Chromatographic separation of the analyte and internal standard was achieved on an analytical 250 x 4.6 mm i.d. reversed-phase Kromasil KR 100 C(18) (5 microm particle size) column using a mobile phase of acetonitrile-potassium dihydrogen phosphate (30:70 v/v). The run time was less than 15 min. Column eluate was monitored at 230 nm. The linearity over the concentration range of 10-1500 ng/mL for vardenafil was obtained and the limit of quantification (LOQ) was 10 ng/mL. The method has been applied to analysis of the vardenafil concentrations for application in pharmacokinetic studies.

Frog (Pelophylax bergeri, Günther 1986) endocrine disruption assessment: characterization and role of skin poly(ADP-ribose) polymerases.

Model of the our research was the adult male amphibian anura, Pelophylax bergeri, poikilotherm species not considered threatened by the IUCN, sampled in representative sites at different degree. In the first phase, a biochemical characterization of the ADP-ribosylation on the skin of barcoded amphibian anura collected from Matese Lake (clean reference site in CE, Italy) was carried out. Two PARP isoforms were evidence: the first of 66 kDa is localized into nucleus and activated by DNA damage; the second of 150 kDa is in cytoplasm, as demonstrated by biochemical and immunohistochemical analysis. Subsequently, the PARP activity, the quantitative expression of androgen receptor gene, and the levels of arsenic and chromium in skin and testis of frog and soil, water, and sediment collected from sites at different degrees of pollution were measured. A significant variation of PARP activity and androgen receptor expression levels was detected in both tissues of barcoded frogs from Sarno and Scafati, along Sarno River (SA, Italy), suggesting that a PARP activation is correlated to pollution and to steroid-regulated physiology disruption.

Surgical versus percutaneous isolated pelvic perfusion (IPP) for advanced melanoma: comparison in terms of melphalan pharmacokinetic pelvic bio-availability.

BACKGROUND: Isolated pelvic perfusion (IPP) can be used to treat unresectable melanoma metastases of the pelvis. IPP can be performed either by surgical or percutaneous approaches, using different balloon catheters. The aim of this study was to examine whether the surgical and percutaneous approaches were comparable with respect to tumor drug exposure in the pelvis. METHODS: A pharmacokinetic study was performed in 5 melanoma patients treated with surgical IPP and five with percutaneous IPP. Both groups received melphalan at the dose of 30 mg/m2. Melphalan pharmacokinetic analyses were performed and the main parameter used to evaluate pelvic tumor drug-exposure was the ratio of areas under the melphalan plasma concentration curves in the pelvis and the systemic compartment, during the perfusion time (AUC0 to 20). Non-parametric Mann-Whitney tests were employed for statistical comparisons. RESULTS: The median and interquartile range (IQR) values of the ratios between melphalan AUC0 to 20 in pelvic and systemic compartments were 7.9 (IQR 7.2 to 9.9) and 5 (IQR 4 to 7.9) for surgical and percutaneous IPPs, respectively (p = 0.209). CONCLUSIONS: Tumor exposure to drug using these two methods did not statistically differ and both methods, therefore, can be adopted interchangeably, utilizing a perfusion blood flow rate of approximately 120 ml/min. The small sample size is a limitation of this study but our preliminary results can be used to calculate the effect size of a larger trial. Trial Registration Clinical Trials.gov Identifier NCT01920516; date of trial registration: August 6, 2013.

MGMT methylation correlates with melphalan pelvic perfusion survival in stage III melanoma patients: a pilot study.

Approximately 25% of melanoma patients with locoregional metastases are nonresponsive to new molecular target therapy and immunotherapy. When metastases are located in the pelvis, melphalan hypoxic perfusion can be an optional treatment. Because methylation of MGMT promoter increases the efficacy of alkylating agents, studies on melanoma outcome of patients treated with melphalan regional chemotherapy should consider this epigenetic change. This study aims to evaluate whether the survival of stage III melanoma patients treated with melphalan regional chemotherapy may be correlated with MGMT methylation status. The metastatic tissues of 27 stage III melanoma patients with locoregional metastases located in the pelvis subjected to melphalan hypoxic pelvic perfusion were examined. The methylation status of the MGMT promoter was investigated by MS-MLPA probes analysis and the presence of the BRAF V600E mutation was analyzed by CAST-PCR. The median survival times were estimated using the Kaplan-Meier curves and were stratified according to the clinicopathological characteristics of patients and lesions. The overall median survival time was 17 months. The 1-year, 3-year, and 5-year survival rates were 66.7, 18.5, and 7.4%, respectively. Disease stage, burden, and percentage of MGMT methylation significantly affected survival. We estimated an MGMT promoter methylation cut-off of at least 14%, which was significantly associated with a longer survival after melphalan regional chemotherapy. Our data suggest that MGMT promoter methylation could be an important factor in determining which melanoma patients should receive melphalan regional chemotherapy, but its prognostic significance in the routine clinical setting needs to be clarified in a larger study.

Deliberate hypoxic pelvic and limb chemoperfusion in the treatment of recurrent melanoma.

BACKGROUND: The treatment of patients with advanced or recurrent pelvic melanoma, which are often associated with lesions in the lower limbs, is still unsatisfactory and controversial. A simplified hypoxic pelvic and limb perfusion has been recently recommended to provide therapeutic options for palliation and possibly cure. METHODS: A nonrandomized and noncontrolled phase II experimental study was performed in 11 patients with symptomatic unresectable recurrent melanoma of the pelvis and limb. Patients were submitted to hypoxic pelvic and limb perfusion with 25 mg/m(2) of melphalan, 50 mg/m(2) of cisplatin, 300 mg/m(2) of dacarbazine, and 75 mg/m(2) of epirubicin by means of a simplified balloon occlusion technique. Response rate and time to disease progression were the primary endpoints; overall survival was the secondary endpoint. RESULTS: During the procedures there were no technical, hemodynamic, or vascular complications, and no deaths occurred during surgery or in the postoperative period. Response rate was 82% (95% confidence interval, 58% to 100%). Median time to disease progression was 12 months (range 9 to 30 months). Three-year overall survival was 34%. CONCLUSIONS: Hypoxic pelvic and limb perfusion is a safe and good palliative treatment for patients with unresectable recurrent melanoma. Further studies are necessary to to confirm these data and to establish if refinements can be made with acceptable toxicity.

A validated HPLC method with electrochemical detection for simultaneous assay of 5-aminosalicylic acid and its metabolite in human plasma.

A high-performance liquid chromatographic method was developed, validated and applied to the simultaneous determination of 5-aminosalicylic acid (5-ASA) and its acetylated metabolite (acetyl-5-ASA) in human plasma. The method involves liquid-liquid extraction with methanol followed by isocratic reversed-phase chromatography on a Kromasil KR100 C(18) column with electrochemical detection. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of 5-ASA, acetyl 5-ASA and internal standard were investigated. Limits' of detection were 5 ng/mL for 5-ASA and 10 ng/mL for acetyl-5-ASA, respectively. The method can be used for supporting therapeutical drug monitoring and pharmacokinetic studies.