20 research outputs found
Development and characterization of a gold nanoparticles glassy carbon modified electrode for dithiotreitol (DTT) detection suitable to be applied for determination of atmospheric particulate oxidative potential
A gold nanostructured electrochemical sensor based on modified GC electrode for thiols' detection is described and characterized. This sensor is a suitable device for the measurement of the oxidative potential (OP) of the atmospheric particulate matter (PM), considered a global indicator of adverse health effects of PM, as an alternative to the classic spectrophotometric methods. The operating principle is the determination of the OP, through the measurement of the consumption of DTT content. The DTT-based chemical reactivity is indeed a quantitative acellular probe for assessment of the capacity of the atmospheric PM to catalyze reactive oxygen species generation which contributes to the induction of oxidative stress in living organisms and in turn to the outcome of adverse health effects. To make the sensors, glassy carbon electrodes, traditional (GC) and screen printed (SPE) electrodes, have been electrochemically modified with well-shaped rounded gold nanoparticles (AuNPs) by using a deposition method that allows obtaining a stable and efficient modified surface in a very simple and reproducible modality. The chemical and morphological characterization of the nano-hybrid material has been performed by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy coupled with electron dispersive spectroscopy analysis (SEM/EDS). The electrochemical properties have been evaluated by cyclic voltammetry (CV) and chrono-amperometry (CA) in phosphate buffer at neutral pH as requested in DTT assay for OP measurements. The electroanalytical performances of the sensor in DTT detection are strongly encouraging showing low LODs (0.750 μM and 1.5 μM), high sensitivity (0.0622 μA cm−2 μM−1 and 0.0281 μA cm−2 μM−1), wide linear and dynamic ranges extending over 2-4 orders of magnitude and high selectivity. FIA preliminary results obtained on measuring the DTT rate consumption in six PM aqueous extracts samples showed a good correlation with measurements obtained in parallel on the same set of samples by using the classic spectrophotometric method based on the Ellman's reactive use. These results confirm the high selectivity of the method and its suitability for application to be applied in PM oxidative potential measurements
Oxidative potential, cytotoxicity, and intracellular oxidative stress generating capacity of PM10: a case study in South of Italy
Long and short-term exposure to atmospheric particulate matter (PM) has detrimental effects on human health. The effective mechanisms leading to PM toxicity are still not fully understood, even if it is known that physical-chemical properties, strongly influenced by sources and atmospheric processes, are known to play an important role. In this work, PM10 samples were collected, at an urban background site in southern Italy, to determine cytotoxicity (using MTT test on A549 cells), genotoxicity (using the comet assay), and intracellular oxidative stress on A549 cells exposed for 24h to aqueous extracts of PM10 samples. Organic carbon (OC) and elemental carbon (EC) content of PM10 and acellular determination of oxidative potential with DTT assay was performed with the objective to compare results of acellular and cellular biological assays. Cellular (OSGCV and MTTV) and acellular (OPDTTV) outcomes, normalised in volume, are well correlated (statistical significant results) with carbon content suggesting that combustion sources play an important role in deter-mining cellular oxidative stress and cytotoxicity of PM10. Even if the number of data is limited, genotoxicity results are well correlated (Pearsons > 0.95) with OSGCV and MTTV and a weaker, but statistically significant correlation was observed with OPDTTV. OSGCV is well correlated with the cell mortality observed with MTTV test and a lower, but still statistical significant correlation is observed between MTTV and OPDDTV. A statistically significant correlation was found between OPDTTV and OSGCV results. When the outcomes of cellular and acellular assay are compared normalised in mass (i.e. intrinsic values), the correlations become significantly weaker suggesting that the different sources acting on the site produces particulate matter with different toxicological potential influ-encing differently the biological tests studie
Correlation of Oxidative Potential with Ecotoxicological and Cytotoxicological Potential of PM10 at an Urban Background Site in Italy
: It is known that exposure to atmospheric particulate matter (PM) has detrimental effects on health. However, specific mechanisms of toxicity are still not fully understood depending on several physical and chemical properties of PM. In recent years, there has been a growing evidence that oxidative stress is an important mechanism of PM toxicity leading to the hypothesis to use acellular evaluation of oxidative potential (OP) as a global indicator of potential health effects of PM. However, when OP data are correlated with the outcomes of in vitro (or in vivo) toxicological tests, there are contrasting results. In this work an analysis of PM10 health effect indicators was done, using the acellular DTT assay to retrieve OPDTT, the Microtox® test on Vibrio fischeri bacterium to assess the ecotoxicological potential, and the in vitro MTT assay on the human cell line A549 to estimate the cytotoxicological potential. The objective was to evaluate the correlation among acellular OPDTT and the results from toxicological and ecotoxicological bioassays and how these health-related indicators are correlated with atmospheric PM10 concentrations collected at an urban background site in Southern Italy. Results indicated that both bioassays showed time-dependent and dose-dependent outcomes. Some samples presented significant ecotoxic and cytotoxic response and the correlation with PM10 concentration was limited, suggesting that these health endpoints depend on PM10 chemical composition and not only on exposure concentrations. OPDTT showed a statistically significant correlation with PM10 concentrations. MTT and Microtox outcomes were not correlated suggesting that the two toxicological indicators are sensitive to different physical-chemical properties of PM10. Intrinsic oxidative potential OPDTTM (DTT activity normalised with PM10 mass) was correlated with mortality observed with MTT test (normalized with PM10 mass), however, it was not correlated with Microtox outcome
Sensori elettrochimici (amperometrici/potenziometrici) basati su elettrodi nano/microstrutturati come misura del potenziale ossidativo del particolato atmosferico (PM)
Apoptotic volume decrease (AVD) in A549 cells exposed to water-soluble fraction of particulate matter (PM10)
Exposure to atmospheric particulate matter (PM) is recognized as a human health risk factor of great concern. The present work aimed to study the cellular mechanisms underlying cytotoxic effects of airborne particulate matter <10 mu m in size (PM10), sampled in an urban background site from January to May 2020, on A549 cells. In particular, the study addressed if PM10 exposure can be a main factor in the induction of the Apoptotic Volume Decrease (AVD), which is one of the first events of apoptosis, and if the generation of intracellular oxidative stress can be involved in the PM10 induction of apoptosis in A549 cells. The cytotoxicity of PM10 samples was measured by MTT test on cells exposed for 24 h to the PM10 aqueous extracts, cell volume changes were monitored by morphometric analysis of the cells, apoptosis appearance was detected by annexin V and the induction of intracellular oxidative stress was evaluated by the ROS sensitive CM-H2DCFDA fluorescent probe. The results showed cytotoxic effects ascribable to apoptotic death in A549 cells exposed for 24 h to aqueous extracts of airborne winter PM10 samples characterized by high PM10 value and organic carbon content. The detected reduced cell viability in winter samples ranged from 55% to 100%. Normotonic cell volume reduction (ranging from about 60% to 30% cell volume decrease) after PM10 exposure was already detectable after the first 30 min clearly indicating the ability of PM10, mainly arising from biomass burning, to induce Apoptotic Volume Decrease (AVD) in A549 cells. AVD was prevented by the pre-treatment with 0.5 mM SITS indicating the activation of Clefflux presumably through the activation of VRAC channels. The exposure of A549 cells to PM10 aqueous extracts was able to induce intracellular oxidative stress detected by using the ROS-sensitive probe CM-H2DCFDA. The PM10induced oxidative stress was statistically significantly correlated with cell viability inhibition and with apoptotic cell shrinkage. It was already evident after 15 min exposure representing one of the first cellular effects caused by PM exposure. This result suggests the role of oxidative stress in the PM10 induction of AVD as one of the first steps in cytotoxicity
Correlation of PM10 oxidative potential with ecotoxicological and cytotoxicological potential measured at an urban background site in Italy
Atmospheric particulate matter is a concern in most of
the European towns because it has potential negative
effects on human health (Davidson et al., 2005;
Lelieveld et al., 2015). Although the toxic effects of PM
have been correlated with some of its chemical and
physical properties, the toxicity mechanisms are not yet
fully known. Different in vitro toxicological tests are
often necessary to characterise potential health effects
and, often, it is found significant correlation only among
a few of the possible tests. In addition, contrasting
results could be obtained comparing in vitro tests with
acellular assays like those used to determine oxidative
potential (Steenhof et al., 2011; Van Den Heuvel et al.,
2018). The aim of the work was to study the oxidative
potential (OP) of PM10, determined with the acellular
DTT assay, in relationship with its ecotoxicological and
cytotoxicological potential.
The study was carried out on aqueous extracts of
10 samples of airborne PM10 randomly selected among
the samples collected between 16/09/2017 and
25/12/2017. Samples were collected using a low-volume
(2.3 m3
/h) sampler (SWAM, Fai Instruments srl) on 47
mm quartz fibre filters (Whatman) pre-fired at 700 °C
for 2 hours in order to reduce contamination. Samples
were exposed for 24 hours (starting at midnight) at the
Environmental-Climate Observatory of ISAC-CNR in
Lecce (Southern Italy), regional station of the Global
Atmosphere Watch (GAW) network, characterised as an
urban background site (Cesari et al., 2018). The aqueous
extraction was performed in an ultrasonic bath for 80
min using 10 ml Milli-Q water.
The ecotoxicological potential of PM10 was
assessed by the bioluminescence inhibition assay based
on the Gram-negative non-pathogenic bacterium Vibrio
fischeri (Microtox® test), which physiologically emits
light as a results of its metabolic activity. The natural
bioluminescence of V. fisheri is inhibited by the
exposure to a number of chemical pollutants, including
organic and inorganic compounds (Abbass et al., 2018).
Different exposure times (5, 15, and 30 mins) were used
and inhibition results, obtained with five repetitions, are
reported as a net effect corrected using field blanks.
The cytotoxicological potential of PM10 was
assessed on the same extracts by the MTT assay on the
cell line A549. The MTT assay is based on a
colorimetric reaction dependent on mitochondrial
respiration of the cells and indirectly allows assessing
the cellular energy capacity of a cell (Stockert et al.,
2012). The MTT assay was applied to the A549 cell line,
representative of the alveolar type II pneumocytes of the
human lung (Foster et al., 1998). Cell mortality after 24h
exposition is evaluated, in relative terms, considering
the net effect of PM10 using field blanks for correction.
Six repetitions were done.
The water-soluble fraction of PM10 was also used
for the analysis of the OP, performed with the
dithiothreitol assay (DTT), a surrogate for cellular
antioxidants, which analyses the rate of DTT depletion
catalysed by chemical species present in the PM
(Chirizzi et al., 2017). An aliquot of the extracts was
diluted with deionised water (1:4 factor). Diluted
samples were incubated at 37 °C with DTT (0.1 mM) in
0.1 M potassium phosphate buffer at pH 7.4 for times
varying from 5 to 90 min. At designated times
(specifically at 5, 10, 15, 20, 30, 45, 60, and 90 min) an
aliquot of incubation mixture was picked up and 10%
trichloroacetic acid was added to stop the reaction. Then,
this reaction mixture was mixed with a solution
containing 10 mM DTNB. The concentration of the
formed 5-mercapto-2-nitrobenzoic acid was measured
by its optical density absorption at 412 nm using a Eon
BioTek Microplate Spectrophotometer. The
consumption of DTT over time was determined through
the linear fitting of the absorbance with the time in
which it was made the withdrawal. The DTT depletion
rate was used to determine OP values as DTT-activity
21
normalized in terms of sampled air volume (OPV) or in
terms of mass of collected aerosols (OPM). The OPV and
OPM values were comparable with previous
measurements in this area or in other Italian towns
(Chirizzi et al., 2017).
In all the 10 samples analysed a significant
inhibition of the Vibrio fisheri bioluminescence was
observed as a results of the exposure of bacteria to the
undiluted extracts for 5, 15 and 30 min, suggesting the
presence in the PM10 of components able to induce an
ecotoxic effect. Four samples (samples n. 2,3,4, and 7)
showed a % of inhibition ranging from 30% to 50%,
ascribable to a slight toxic effect, while six samples
(samples 1,5,6, 8,9, and 10) showed a % of inhibition
above 50% after 30 min exposure, suggesting the
presence of a toxic effect. The correlation analysis
between the sampled mass and the Vibrio fisheri
bioluminescence inhibition showed a significant
positive correlation (p<0.0462) for nine of the data pairs,
while one sample (sample n. 6) was out of trend.
As regards cytotoxicity, in all the 10 samples
analysed a significant cell mortality was observed,
ranging from 35% to 65% following exposure of the
cells for 24h to the undiluted samples. 4 samples showed
a slight cytotoxicity (mortality below 50%, samples n.
3,6,8, and 9), while the other showed a mortality higher
than 50% (maximum mortality recorded 65%). The
correlation analysis between the sampled mass and the
A459 showed a highly significant positive correlation
(p<0.0016) for eight of the data pairs, while two sample
(sample n. 2 and 7) were out of trend.
The OPV values were correlated with PM10 mass
concentrations (Pearson 0.85). The specific oxidative
potential (OPM) was well correlated with the results of
the MTT assay (normalised for the mass collected).
Instead, the correlation of OPM with Microtox test
(normalised for the mass collected) was significantly
lower.
The correlation analysis between the Microtox
test results and the MTT assay results on the same
extracts was not significant, suggesting that each test,
based on a specific biological system, can show its
proper specificity and sensibility to different chemical
components of PM. This highlight the need to improve
the use of suites of biological assay in order to detect the
multifaceted aspects associated to the toxicity of PM10
that can be the resulting aspect of multiple contaminants
simultaneously present in the particulate.
The financial support of the project PAPER (Paper
Analyser for Particulate Exposure Risk, funded within
POR Puglia FESR-FSE 2014-2020 – Asse prioritario 1
– Azione 1.6 – Bando Innonetwork – Aiuti a sostegno
delle attività di R&S.).
Abbas, M., M. Adil, S. Ehtisham-ul-Haque, B. Munir,
M. Yameen, A. Ghaffar, G. Abba Sharc, M.A. Tahir,
M. Iqbal. (2018) Sci. Tot. Environ. 626, 1295-1309.
Cesari, D., De Benedetto, G. E., Bonasoni, P., Busetto,
M., Dinoi, A., Merico, E., Chirizzi, D., Cristofanelli,
P., Donateo, A., Grasso, F., Marinoni, A., Pennetta,
A., Contini, D. (2017). Sci. Total Environ. 612, 202-
213.
Chirizzi, D., Cesari D., Guascito, M.R., Dinoi, A.,
Giotta, L., Donateo, A., Contini, D., (2017) Atmos.
Environ. 163, 1-8.
Davidson, C.I., Phalen, R.F., Solomon, P.A., (2005).
Aerosol Science and Technology 39, 737–749.
Foster K.A., Oster C.G., Mayer M.M., Avery M.L.,
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Lelieveld, J., Evans, J.S., Fnais, M., Giannadaki, D.,
Pozzer, A., (2015) Nature Letter 525, 367.
Steenhof, M., Gosens, I., Strak, M., Godri, K.J., Hoek,
G., Cassee, F.R., Mudway, I.S., Kelly, F.J., Harrison,
R.M., Lebret, E., Brunekreef, B., Janssen, N.A.H.,
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Correlation of PM10 oxidative potential with ecotoxicological and cytotoxicological indicators
Exposure to atmospheric particulate matter (PM) has detrimental effects on health. These are due to several physical and chemical properties of PM related to their sources and to atmospheric chemical transformations. However, specific mechanisms of toxicity are still not fully understood. In recent years, there has been a growing evidence that oxidative stress is an important mechanism of toxicity; however, when acellular oxidative potential (OP) data are correlated with the outcomes of in vitro (or in vivo) toxicological tests there are contrasting results (Øvrevik, 2019).
In this work, an analysis of the correlation among different indicators of PM10 health effects was done, using the acellular Dithiotreitol (DTT) assay to retrieve OPDTT, the Microtox® test on Vibrio fischeri bacterium to assess the ecotoxicological potential, and the in vitro MTT assay on the human cell line A549 to estimate the cytotoxicological potential.
The study was carried out on selected PM10 samples collected between 16/09/2017 and 25/12/2017 at the Environmental-Climate Observatory of ISAC-CNR in Lecce (Southern Italy, 40°20’08” N—18°07’028” E, 37 m asl), regional station of the Global Atmosphere Watch (GAW) network, characterized as an urban background site (Lionetto et al., 2019). Water-soluble fraction of PM10 was obtained, using the whole filter, in 10 mL ultrapure water (Milli-Q). The ecotoxicological potential was assessed by the bioluminescence inhibition assay based on the Gram-negative non-pathogenic bacterium Vibrio fischeri (Microtox® test, exposure 30 mins). The cytotoxicological potential was assessed by the MTT assay (based on a colorimetric reaction dependent on mitochondrial respiration of the cells) on the cell line A549 representative of the alveolar type II pneumocytes of the human lung with an exposure of 24h. The OP, was evaluated with the dithiothreitol assay (DTT), a surrogate for cellular antioxidants, which analyses the rate of DTT depletion catalysed by chemical species present in PM10. Results of all assays are presented as net effect (subtracting blank values) and uncertainties are obtained by analysing repeatability.
Results show that PM10 and Microtox outcomes were correlated (Pearson 0.67, p<0.05) for nine of the data pairs (one sample out of trend). PM10 and MTT results were correlated (Pearson 0.91, p<0.05) for eight of the data pairs (two samples out of trend). OPDTTV and PM10 were correlated (R 0.91, p<0.05) with no samples out of trend. MTT and Microtox outcomes were not correlated suggesting that the two toxicological indicators are sensitive to different physical-chemical properties of PM10. Intrinsic oxidative potential OPDTTM (DTT activity normalized with PM10 mass) was correlated with mortality observed with MTT test (normalized with PM10 mass), however, it was not correlated with Microtox outcomes. This suggests that acellular OP DTT could be an indicator of cytotoxicological effects of PM on A549 cells
Size-Resolved Redox Activity and Cytotoxicity of Water-Soluble Urban Atmospheric Particulate Matter: Assessing Contributions from Chemical Components
Throughout the cold and the warm periods of 2020, chemical and toxicological characterization of the water-soluble fraction of size segregated particulate matter (PM) (7.2 μm) was conducted in the urban agglomeration of Thessaloniki, northern Greece. Chemical analysis of the water-soluble PM fraction included water-soluble organic carbon (WSOC), humic-like substances (HULIS), and trace elements (V, Cr, Mn, Fe, Ni, Cu, Zn, As, Cd and Pb). The bulk (sum of all size fractions) concentrations of HULIS were 2.5 ± 0.5 and 1.2 ± 0.3 μg m−3, for the cold and warm sampling periods, respectively with highest values in the mDTT, WSOC, HULIS and the MTT cytotoxicity of PM. Multiple Linear Regression (MLR) showed a good relationship between OPMDTT, HULIS and Cu
Pegaspargase-modified risk-oriented program for adult acute lymphoblastic leukemia: results of the GIMEMA LAL1913 trial
: Pediatric-inspired chemotherapy is the standard of care for younger adults with Philadelphia-negative acute lymphoblastic leukemia/lymphoma (Ph- ALL/LL). The GIMEMA LAL1913 trial tested a modified regimen adding pegaspargase 2000 IU/m2 to courses 1, 2, 5 and 6 of an eight-block protocol for patients 18-65 years, with dose reductions in patients >55 years. Responders were risk-stratified to allogeneic hematopoietic stem cell transplantation (HCT) or maintenance according to clinical characteristics and minimal residual disease (MRD). Out of 203 study patients (median age 39.8 years, 68.5% B-lineage), 185 (91%) achieved a complete remission (CR). The 3-year overall survival (OS), event-free (EFS) and disease-free (DFS) survival rates were 66.7% (95% confidence intervals, 64.4-60.1%), 57.7% (51.0-65.3%) and 63.3% (56.3-71.1%), respectively, fulfilling the primary study endpoint of a 2-year DFS >55%. While by intention-to-treat DFS was 74% and 50% in the chemotherapy (n=94) and HCT (n=91) assigment cohorts, a time-dependent analysis proved the value of HCT in eligible patients (DFS HCT 70% vs. no HCT 26%, P<0.0001). In multivariate analysis, age and MRD (n=151) were independent prognostic factors associated with DFS rates of 86% (age less/equal to 40/MRD-negative, n=66), 65% (age >40/MRD-negative, n=48), 64% (age less/equal to 40/MRD-positive, n=17) and 25% (age >40/MRD-positive, n=20) (P<0.0001). Grade 2/greater pegaspargase toxicity was mainly observed at course 1 (41 episodes in 32 patients), contributing to induction death in 2 patients, but was rare and milder thereafter. This pegarspargase-containing risk-oriented program was feasible and improved outcome of Ph- ALL/LL patients up to 65 years in a multicenter national setting. ClinicalTrials.gov #NCT02067143