A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen.

Aims: The well-documented relationship between sperm oxidation and male infertility strongly encourages the development of assays for reactive oxygen species detection in semen samples. The present study aims to apply the microplate-based 2',7'-dichlorofluorescein diacetate assay to the evaluation of oxidative stress in unprocessed whole semen, thus avoiding sample centrifugations and other manipulations that may cause significant reactive oxygen species increments. Main methods: The fluorescence assay consisted in the quantification of both intracellular and extracellular reactive oxygen species levels in unwashed semen specimens by using the probe 2',7'-dichlorofluorescein diacetate into a 96-well plate. The method was useful for the preliminary assessment of the oxidation levels of whole semen samples from men undergoing standard sperm analysis as well as to evaluate the effect of some pro-glutathione molecules on semen oxidative status. Key findings: The 2',7'-dichlorofluorescein diacetate assay was successfully adapted to the evaluation of oxidative stress in whole semen, effectively revealing the perturbation of the redox homeostasis of the sample. Accordingly, specimens with abnormal sperm parameters (n = 10) presented oxidation indexes significantly higher than those with normospermia (n = 10) [7729 (range 3407-12769) vs. 1356 (range 470-2711), p < 0.001]; in addition, semen oxidation indexes negatively correlated to sperm motility and morphology. Noteworthy, whole semen exposure to pro-glutathione compounds led to reduced semen oxidation levels and sperm protection against oxidative damage. Significance: Based on our pilot experimental data, the microplate-based 2',7'-dichlorofluorescein diacetate assay appears to be a convenient method for the detection of reactive oxygen species levels in whole semen samples, avoiding artifacts due to semen centrifugation steps. At the same time, the test could be a helpful tool for the basic and quick screening of antioxidant molecules able to preserve semen quality.

The effects of Acyclovir administration to NCI-H1975 non-small cell lung cancer cells.

The biochemical mechanisms by which the antiviral drug Acyclovir (ACV) may induce anticancer effects even without detecting human herpesviruses (HHVs) are still poorly understood. Herein, we investigated for the first time how NCI-H1975 non-small cell lung cancer cells responded in vitro to ACV administration by exploring mitochondrial damage and apoptosis induction. We confirmed ACV ability to cause the inhibition of cancer cell growth even without detecting intracellular HHVs; the drug also significantly inhibited the colony formation capacity of NCI-H1975 cells. Cell cycle analysis revealed an increase of the sub-G1 hypodiploid peak after ACV treatment; the activation of caspase-3 and the presence of DNA laddering sustained the capacity of the drug to induce apoptotic cell death. Regarding mitochondrial toxicity, a reduction of mitochondrial membrane potential, altered mitochondrial size and shape, and mtDNA damage were found after ACV administration. Furthermore, an increment of intracellular reactive oxygen species levels as well as the upregulation of NudT3 involved in DNA repair mechanisms were observed. Altogether, these findings suggest that mitochondria may be possible initial targets and/or sites of ACV cytotoxicity within cancer cells in the absence of intracellular HHVs.

Systematic review and meta-analysis of epidemiological studies on the association of occupational exposure to free crystalline silica and systemic lupus erythematosus.

OBJECTIVES: Some evidence suggests that exposure to free crystalline silica may contribute to the risk of developing SLE. A systematic search was carried out for all published epidemiological studies concerning this association. A meta-analysis was conducted on relevant studies. METHODS: We searched PubMed and EMBASE databases for original articles published from 1960 to November 2019 in any language. In addition, we also searched the reference lists of included studies manually for additional relevant articles. Finally, seven studies were included in the systematic review and six studies in the meta-analysis (four case-control and two cohort studies). The odds ratio and 95% CI were calculated using a random effect meta-analysis. RESULTS: The meta-analysis of the studies, applying a random effect model, yielded an overall odds ratio of 3.49 (95% CI, 1.24, 9.83), with I2 = 92.36% (pronounced heterogeneity). We also stratified the meta-analysis by study design; case-control studies: odds ratio 1.85 (95% CI, 0.96, 3.59) with I2 = 75.92%; and cohort studies (cases with silicosis): odds ratio 9.71 (95% CI, 1.13, 83.58) with I2 = 72.65%. CONCLUSIONS: The obtained results support the hypothesis of a possible association between occupational exposure to free crystalline silica and SLE, in particular at higher exposure levels, known to induce silicosis. The studies that have investigated this association are still scarce and the heterogeneity between the studies remains high. New studies are deemed necessary to confirm the association.

Health effects of living near an incinerator: A systematic review of epidemiological studies, with focus on last generation plants.

Huge reductions in incinerators' emissions occurred over time, and results of older studies cannot be directly generalized to modern plants. We conducted a systematic review of the epidemiologic evidence of the health effects of incinerators, classifying plants in three generations, according to emission limits. A systematic search identified 63 epidemiologic studies, published in English, investigating health effects of incinerators on humans. We focused on cancer, cardio-cerebrovascular diseases (CVD) and respiratory diseases, pregnancy outcomes and congenital anomalies. Only six studies in the general population were on third generation incinerators providing data on pregnancy outcomes and congenital anomalies. Given the heterogeneity of methods, the abundance of ecological/semi-ecological studies and the lack of reliable quantitative measures of exposure in several studies we did not perform any meta-analysis. No excesses emerged concerning all cancers and lung cancer. An excess of non-Hodgkin lymphoma was reported in some earlier studies, but not for second generation plants. Possible excesses of soft tissue sarcomas were confined to earlier incinerators and the areas closer to the plants. No clear association emerged for CVD and diseases of the respiratory system. Several different pregnancy outcomes were considered, and no consistent association emerged, in spite of a few positive results. Studies were negative for congenital anomalies as a whole. Sporadic excesses were reported in a few studies for specific types of anomalies, but no consistent pattern emerged. Evaluation of the evidence was hindered by heterogeneity in reporting and classification of outcomes across studies. Direct evidence from third generation plants is scarce. Methodological issues in study design (mainly related to exposure assessment, confounding and ecological design) and analysis make interpretation of results complex. In spite of this, the overall evidence suggests that, if there were any excesses at all for older incinerators, they were modest at most. Additional monitoring of third generation plants needs to overcome methodological weaknesses.

Occupational exposure to formaldehyde and risk of non hodgkin lymphoma: a meta-analysis.

BACKGROUND: Formaldehyde, a widely used chemical, is considered a human carcinogen. We report the results of a meta-analyses of studies on the relationship between occupational exposure to formaldehyde and risk of non-Hodgkin lymphoma (NHL). METHODS: We performed a systematic review and meta-analysis according to international guidelines and we identified 12 reports of occupational populations exposed to formaldehyde. We evaluated inter-study heterogeneity and we applied a random effects model. We conducted a cumulative meta-analysis and a meta-analysis according to estimated average exposure of each study population. RESULTS: The meta-analysis resulted in a summary relative risk (RR) for NHL of 0.93 (95% confidence interval 0.83-1.04). The cumulative meta-analysis suggests that higher RRs were detected in studies published before 1986, while studies available after 1986 did not show an association. No differences were found between different levels of occupational exposure. Conclusions Notwithstanding some limitations, the results of this meta-analysis do not support the hypothesis of an association between occupational exposure to formaldehyde and risk of NHL.

Ischemia-Modified Albumin Expression: Is there a Difference between Male and Female Subjects?

BACKGROUND: Ischemia-modified albumin (IMA) derives from naive albumin, modified in the binding region of bivalent ions, as cobalt and iron. The cobalt, released from some types of hip prosthesis seems to be metabolized differently in males and females but the iron ion is more prevalent than cobalt and is detectable in the healthy population. Our aim was to verify if there are any gender- and age-related differences in IMA concentrations and if IMA correlates with cobalt and iron-related proteins. METHODS: IMA, albumin, iron, ferritin, transferrin, and cobalt were measured in 50 men and 50 women divided into two age/fertility-homogeneous groups. RESULTS: Men < 45-years-old showed a statistically significant lower IMA concentration than men ≥ 45 and fertile and menopausal women. Considering all the population studied, IMA does not seem to be correlated with age and is distributed differently by gender; also, Co distribution was different between males and females. CONCLUSIONS: IMA did not correlate with cobalt, iron, ferritin, and transferrin in any group, except for fertile women where IMA presented a statistically significant correlation with serum iron values. Minor expression of IMA in young males together with the results obtained on serum iron in fertile females, could explain the higher accumulation of circulating Co in women compared to men and their different cobalt metabolism.

Erratum to "Development of a New Sequential Extraction Procedure of Nickel Species on Workplace Airborne Particulate Matter: Assessing the Occupational Exposure to Carcinogenic Metal Species".

[This corrects the article DOI: 10.1155/2018/3812795.].

Occupational and environmental exposure to polychlorinated biphenyls and risk of non-Hodgkin lymphoma: a systematic review and meta-analysis of epidemiology studies.

We carryied out a meta-analysis of studies on exposure to polychlorinated biphenyls (PCBs) and risk of non-Hodgkin lymphoma (NHL). Through a systematic search of the literature, we identified relative risks (RRs) for PCB exposure and NHL risk in 30 populations (10 occupational exposure, seven high environmental exposure, 13 without special exposure). We performed random effects meta-analyses for exposure to all PCBs, specific PCB congeners and risk of all NHL and NHL subtypes. The meta-RR for studies of occupational exposure, high environmental exposure, and no special exposure were 0.94 [95% confidence interval (CI): 0.84-1.03], 1.05 (95% CI: 0.94-1.16), and 1.03 (95% CI: 0.72-1.34), respectively, and the cumulative meta-RR was 0.96 (95% CI: 0.85-1.07). No positive associations were found for exposure to specific congeners, nor for NHL subtypes. The meta-RR for an increase of 100 ppb serum or fat PCB level was 1.02 (95% CI: 1.00-1.04). There was weak indication of publication bias. Our meta-analysis found no association between PCB exposure and NHL risk, in particular in studies of occupational exposures. We detected a weak dose-response relation; the possibility of residual confounding and other sources of bias cannot be ruled out. PCBs are not likely to cause NHL in humans.

Acyclovir induces cell cycle perturbation and apoptosis in Jurkat leukemia cells, and enhances chemotherapeutic drug cytotoxicity.

AIMS AND METHODS: Many antiviral agents have been reported to present direct cytotoxic activity in cancer, showing antiproliferative and proapoptotic effects through different mechanisms. In the present study, we took into account the cytotoxic action of the antiviral drug acyclovir (ACV) on leukemia cells, by investigating cell cycle perturbations and apoptosis induction upon drug administration to three still unexplored cell lines, namely Jurkat, U937, and K562. At the same time, the cytotoxicity of cisplatin (CDDP) and 5­fluorouracil (5­FU) in combination with ACV was assessed, thus to evaluate if the antiviral agent could enhance cancer cell sensitivity to these chemotherapeutic drugs. FINDINGS AND SIGNIFICANCE: Our results showed that ACV cytotoxic action was maximum in Jurkat cells (acute T cell leukemia), which showed a dose- and time-dependent reduction of cell viability after drug exposure. The flow cytometric analysis of cell cycle revealed a delay/block in S phase and an increase of the sub-G1 peak upon ACV administration, thereby indicating apoptotic cell death. The activation of caspase-3 and the presence of nuclear DNA fragmentation confirmed the induction of apoptosis in ACV-treated cells. Interestingly, the pre-treatment of Jurkat cells with ACV for 72 h or 7 days increased CDDP and 5-FU cytotoxicity, suggesting enhanced leukemia cell sensitivity to these anticancer drugs.

Synthesis and biological evaluation of novel heteroring-annulated pyrrolino-tetrahydroberberine analogues as antioxidant agents.

Tetrahydroberberine (THB), otherwise known as canadine, is a natural alkaloid showing significant pharmacological properties and antioxidant protection against oxidative damage. Herein, we synthetized structurally complex THB analogues, namely pyrrolino-tetrahydroberberines (PTHBs) 4a-g, containing the pyrrolino[2,3-b]pyridine system, by means of the reactions of 1,2-diaza-1,3-dienes and 7,8-dihydroberberine. Aim of the study was to explore the in vitro antioxidant properties of PTHBs in comparison to THB thus to identify the most effective against free radical-induced oxidative injury, by using three different antioxidant tests: the ORAC method, the DNA nicking assay, and the DCFH-DA cellular assay. As a result, PTHB 4d emerged among the other THB analogues by exhibiting the best antioxidant properties. First, it was the only compound having an ORAC value completely comparable to that of THB, indicating the same ability to neutralize peroxyl radicals. Secondly, 4d showed an even better antioxidant capacity than THB in protecting DNA against ferrous ion-induced strand breaks. These observations were also confirmed in NCTC-2544 human keratinocytes exposed to hydrogen peroxide. Indeed, 4d protected cells against oxidation more efficiently than THB both in the short (1 and 3 h) and long (24 h) period of incubation, possibly suggesting increased cell membrane permeability and/or intracellular stability of 4d as compared to THB.

Composition of Metallic Elements and Size Distribution of Fine and Ultrafine Particles in a Steelmaking Factory.

BACKGROUND: The characteristics of aerosol, in particular particle size and chemical composition, can have an impact on human health. Particle size distribution and chemical composition is a necessary parameter in occupational exposure assessment conducted in order to understand possible health effects. The aim of this study was to characterize workplace airborne particulate matter in a metallurgical setting by synergistically using two different approaches; Methodology: Analysis of inhalable fraction concentrations through traditional sampling equipment and ultrafine particles (UFP) concentrations and size distribution was conducted by an Electric Low-Pressure Impactor (ELPI+™). The determination of metallic elements (ME) in particles was carried out by inductively coupled plasma mass spectrometry; Results: Inhalable fraction and ME concentrations were below the limits set by Italian legislation and the American Conference of Governmental Industrial Hygienists (ACGIH, 2017). The median of UFP was between 4.00 × 104 and 2.92 × 105 particles/cm³. ME concentrations determined in the particles collected by ELPI show differences in size range distribution; Conclusions: The adopted synergistic approach enabled a qualitative and quantitative assessment of the particles in steelmaking factories. The results could lead to a better knowledge of occupational exposure characterization, in turn affording a better understanding of occupational health issues due to metal fumes exposure.

Impact of the 24-h ultramarathon race on homocysteine, oxidized low-density lipoprotein, and paraoxonase 1 levels in professional runners.

The impact of the 24-h ultramarathon race on homocysteine (Hcy) and oxidized low-density lipoprotein (oxLDL) levels, two well-recognized cardiovascular risk factors, has not been deeply investigated. Similarly, no information exists on paraoxonase 1 (PON1), an antioxidant enzyme associated with high-density lipoproteins, which may detoxify oxLDL and Hcy-thiolactone, hence preventing their proatherogenic action. Taking this into account, a competitive 24-h ultramarathon race was organized in Reggio-Emilia (Italy) recruiting professional runners (n = 14) from the Italian Ultramarathon and Trail Association. Blood samples were collected from each participant before, during (14 h), and immediately after (24 h) the competition, thus to monitor the serum changes in Hcy, oxLDL, and PON1 levels, as well as other oxidative stress-related parameters, namely reactive oxygen metabolites (ROM) and total antioxidant capacity (PAT). As a result, a significant PON1 increase was recorded after 14 h of racing that persisted until the end of the performance. The same trend was observed for PAT values, which positively correlated to PON1 levels (R = 0.643, P<0.001). Hcy, oxLDL, and ROM remained almost unchanged throughout the competition. In conclusion, the present study suggested a protective role of PON1 in sustaining the antioxidant defense system and contrasting lipoprotein oxidative modifications over the 24-h race, and did not specifically evidence either Hcy or oxLDL accumulation in such challenging sporting events.

Serum Non-Ceruloplasmin Non-Albumin Copper Elevation in Mild Cognitive Impairment and Dementia due to Alzheimer's Disease: A Case Control Study.

Several studies showed high serum copper levels in Alzheimer's disease (AD). The present study applied a newly developed method to detect serum copper free from proteins (free-Cu). Forty-four patients affected by dementia due to AD, thirty-six patients affected by mild cognitive impairment (MCI) due to AD, and twenty-eight healthy controls underwent clinical, cognitive, and MRI assessment. The new method showed higher free-Cu concentrations in MCI and dementia due to AD compared to controls (p < 0.0001). No correlation between copper levels, cognitive or MRI measures were found.

Occupational exposure to polychlorinated biphenyls and risk of cutaneous melanoma: a meta-analysis.

The aim of this study was to carry out a meta-analysis of studies on exposure to polychlorinated biphenyls (PCBs) and the risk of malignant melanoma (MM). We searched Scopus, PubMed, and reference lists; among 807 potentially relevant articles, we selected those based on 12 populations. Data were extracted according to a standardized form; the Newcastle-Ottawa Scale was used to assess study quality. Meta-analyses were carried out according to fixed-effect and random-effects models. The fixed-effect summary relative risk (RR) for MM was 0.91 [95% confidence interval (CI): 0.82-1.00]; the random-effects summary RR was 1.05 (95% CI: 0.78-1.32). The random-effects summary RR from eight occupational cohorts was 1.13 (95% CI: 0.91-1.35) and that from four community-based studies was 0.84 (95% CI: 0.36-1.31). The quality of the studies and the methods for PCB exposure assessment did not influence the RR. These results do not support the hypothesis of an association between PCB exposure and the risk of MM.

Free copper in serum: An analytical challenge and its possible applications.

Copper (Cu), as an essential metal, plays a crucial role in biochemical reactions and in physiological regulations. Cu in plasma is mostly bound to proteins; about 65-90% of Cu is tightly binds with caeruloplasmin and the rest of Cu is loosely binds with albumin and transcuprein. A small but significant relatively "free" fraction, probably complexed with amino acids, is present at around 5% of the total concentration. We developed and validated a new method for direct measurement of free Cu in serum by ultrafiltration with AMICON®Ultra 100K device and determination with AAS. Also, we checked that there is no trace of albumin in the ultrafiltrates and we demonstrated the ultrafiltration of a known concentration of Cu added in artificial serum without albumin and, on the contrary, the retention of the Cu in artificial serum with albumin. The ultrafiltration procedure and the instrumental determination showed a good repeatability and a very low limit of detection (1µg/L). The method was applied to 30 healthy subjects, the mean value of the total Cu (994.8µg/L) is included in the normal range for healthy people and the values of free Cu (23.6µg/L) corresponding to 2.37% of the Cu total. The determination of free Cu by this simple and cheap method may be useful to measure the most bioavailable Cu fraction possibly implicated in neuro-degenerative and oxidative-stress related diseases.

Assessment of residual exposure to PCBs in metallurgy.

OBJECTIVE: To evaluate the occupational exposure to polychlorinated biphenyls (PCBs) in 56 workers employed in 6 electric arc furnace steelmaking plants and 2 secondary aluminum smelting plants located in the highly industrialized area of Brescia, Northern Italy. METHODS: Thirty-four PCB congeners were found in both environmental and biological samples from workers engaged in scrap yards, electric arc furnaces, casting and maintenance departments. RESULTS: The highest airborne PCB levels were found in the aluminum plant, even 100 times those detected in the steelwork plants. Dioxin-like PCB congeners (DL-PCBs) were poorly represented in all biological samples, whereas non Dioxin-Like PCB congeners (noDL-PCBs), in particular environmentally widespread congeners (PCB 153, 138, 180), could be detected in almost all samples. The mean total PCB serum level was 3.9 ng/ml, with a range of 1.3-10.3 ng/ml, while the geometric mean for airborne PCBs levels was 9305 pg/m3, with a range of 1138-217806 pg/m3. CONCLUSIONS: Despite the higher PCB values recorded in some metallurgical plant workplaces, we failed to find any significant difference between serum concentrations in workers from steel or aluminum production, even in consideration of different tasks or different job seniority, while positive association was found only according to the age of the workers. A possible explanation may be identified in the effectiveness of the individual and collective preventive measures adopted in the workplace. Assessment of the occupational exposure to such compounds, in consideration of the recent classifications as carcinogenic to humans, should be encouraged.

Reduced cell viability and apoptosis induction in human thyroid carcinoma and mesothelioma cells exposed to cidofovir.

Besides its well-recognized antiviral activity, Cidofovir (CDV) has been shown to exert anticancer properties both within in vitro and in vivo models. The aim of this study was to evaluate the effects of CDV on still unexplored cultured cancer cells from human mesothelioma as well as breast, colon, liver, lung, prostate, and thyroid carcinomas. Overall, a dose- and time-dependent inhibition of cell viability was observed after CDV exposure. To clarify the mechanisms underlying CDV action, apoptotic cell death was investigated in two infected cell lines [Ist-Mes1 and Ist-Mes2 mesothelioma cells (SV40+)] and in two uninfected cell lines (NCI-H2425 mesothelioma cells and FTC-133 thyroid cancer cells), which resulted the most sensitive to CDV treatment. Reduced expression of procaspase-3 and increased expression of PARP p85 fragment were observed in both infected and uninfected mesothelioma cells, indicating apoptosis induction by CDV in a virus-independent manner. Similarly, the increase of the pro-apoptotic proteins p53, cytochrome c and caspase-3, the decrease of the survival protein Bcl-x, and the increment of Bax/Bcl-2 ratio revealed the occurrence of apoptosis in CDV-treated FTC-133. The presence of nuclear DNA fragmentation confirmed apoptotic cell death by CDV. Overall, our findings warrant further investigations to explore the therapeutic potential of CDV for human mesothelioma and follicular thyroid carcinoma.

Oxidative stress and apoptosis induction in human thyroid carcinoma cells exposed to the essential oil from Pistacia lentiscus aerial parts.

BACKGROUND: Essential oils from the aerial parts (leaves, twigs and berries) of Pistacia lentiscus (PLEO) have been well characterized for their antibacterial and anti-inflammatory properties; however, poor information exists on their potential anticancer activity. METHODS: Increasing concentrations of PLEO (0.01-0.1% v/v, 80-800 µg/ml) were administered to a wide variety of cultured cancer cells from breast, cervix, colon, liver, lung, prostate, and thyroid carcinomas. Fibroblasts were also included as healthy control cells. Cell viability was monitored by WST-8 assay up to 72 hours after PLEO administration. The intracellular formation of reactive oxygen species (ROS), the induction of apoptosis, and the enhancement of chemotherapeutic drug cytotoxicity by PLEO were further investigated in the most responsive cancer cell line. RESULTS: A dose-dependent reduction of tumor cell viability was observed upon PLEO exposure; while no cytotoxic effect was revealed in healthy fibroblasts. FTC-133 thyroid cancer cells were found to be the most sensitive cells to PLEO treatment; accordingly, an intracellular accumulation of ROS and an activation of both the extrinsic and intrinsic apoptotic pathways were evidenced in FTC-133 cells after PLEO administration. Furthermore, the cytotoxic effect of the antineoplastic drugs cisplatin, 5-fluorouracil and etoposide was enhanced in PLEO-exposed FTC-133 cells. CONCLUSION: Taking into account its mode of action, PLEO might be considered as a promising source of natural antitumor agents which might have therapeutic potential in integrated oncology.

In vivo response of heme-oxygenase-1 to metal ions released from metal-on-metal hip prostheses.

Metal ion release and accumulation is considered to be a factor responsible for the high failure rates of metal-on-metal (MoM) hip implants. Numerous studies have associated the presence of these ions, besides other factors, including a hypoxia­like response and changes in pH due to metal corrosion leading to the induction of the oxidative stress response. The aim of the present study was to verify whether, in patients with a MoM hip prosthesis, mRNA and protein expression of HMOX­1 was modulated by the presence of metal ions and whether patients without prostheses exhibit a different expression pattern of this enzyme. The study was conducted on 22 matched pairs of patients with and without prostheses, for a total of 44 samples. Ion dosage was determined using inductively coupled plasma mass spectrometry equipped with dynamic cell reaction. HMOX­1 gene expression was quantified by reverse transcription-quantitative polymerase chain reaction and HMOX­1 protein expression was analyzed using an enzyme-linked immunosorbent assay. The results demonstrated that although there were significant differences in the metallic ion concentrations amongst the two groups of patients, there was no correlation between circulating levels of cobalt (Co) and chromium (Cr), and HMOX­1 gene and protein expression. Additionally, there was no significant difference in the protein expression levels of HMOX­1 between the two groups. In conclusion, it was demonstrated that circulating Co and Cr ions released by articular prosthetics do not induce an increase in HMOX­1 mRNA and protein expression at least 3.5 years after the implant insertion. The present study suggests that involvement of HMOX­1 may be excluded from future studies and suggests that other antioxidant enzymes, including superoxide dismutase, glutathione peroxidase and reductase should be investigated.