Quantification Of Malondialdehyde Research Articles

The Quest for Reliable Drought Stress Screening in Tetraploid Wheat (Triticum turgidum spp.) Seedlings: Why MDA Quantification after Treatment with 10% PEG-6000 Falls Short.

Drought stress poses significant productivity challenges to wheat. Several studies suggest that lower malondialdehyde (MDA) content may be a promising trait to identify drought-tolerant wheat genotypes. However, the optimal polyethylene glycol (PEG-6000) concentration for screening seedlings for drought tolerance based on MDA quantification is not clear. The aim of this study was to verify whether a 10% (w/v) PEG-6000 concentration-induced water stress was reliable for discriminating between twenty-two drought-susceptible and drought-tolerant tetraploid wheat (Triticum turgidum spp. durum, turanicum, and carthlicum) accessions based on MDA quantification. To do so, its correlation with morpho-physiological traits, notoriously related to seedling drought tolerance, i.e., Seedling Vigour Index and Seedling Water Content, was evaluated. Results showed that MDA content was not a reliable biomarker for drought tolerance, as it did not correlate significantly with the aforementioned morpho-physiological traits, which showed, on the contrary, high positive correlation with each other. Combining our study with the cited literature, it clearly emerges that different wheat genotypes have different "water stress thresholds", highlighting that using a 10% PEG-6000 concentration for screening wheat seedlings for drought tolerance based on MDA quantification is not reliable. Given the conflicting results in the literature, this study provides important insights for selecting appropriate methods for evaluating wheat seedling drought tolerance.

Lipid peroxidation and liver damage in double and simple common bile duct ligation models in male Sprague-Dawley rats

AimsBacterial translocation, defined as the presence of living bacteria or bacterial fragments in both mesenteric lymph nodes or systemic circulation, can cause a severe inflammatory reaction in patients with cirrhosis. This study aimed to compare lipid peroxidation associated with liver damage in different experimental models of bile duct ligation: proximal double ligation and transection versus proximal simple ligation versus sham. Materials and methodsSixty-two male rats underwent one of three bile duct surgical interventions: proximal double ligation and transection (n = 22); proximal simple ligation (n = 19); or sham operation (n = 21). We performed microbiological culture of mesenteric lymph nodes; portal and cava blood, spleen and liver cultures; and histological analysis of liver parenchyma. Samples of blood and liver were obtained at laparotomy for malondialdehyde quantification. Key findingsSerum malondialdehyde levels were significantly higher in simple ligature animals (3.7 nmol/mg, standard deviation [SD] 2.1) compared to controls (1.6 nmol/mg SD 0.5; p = 0.001) or double ligature (0.3 nmol/mg SD 0.3; p = 0.001). Liver malondialdehyde levels were significantly higher in animals subjected to double ligation vs controls (9.0 nmol/mg SD 2.8 vs. 1.7 nmol/mg SD 1.0; p = 0.0007) and simple ligature (2.9 nmol/mg SD 2.0; p = 0.0001). Overall incidence of bacterial translocation was similar in simple and double ligatures (22.2 % and 21 % respectively), and significantly higher than in controls. Significancethe type of bile duct ligation influences the type and localization of lipid peroxidation, but does not influence the development of bacterial translocation.

Teratogen Potential Evaluation of the Aqueous and Hydroalcoholic Leaf Extracts of Crataegus oxyacantha in Pregnancy Rats.

Crataegus oxyacantha is used in the treatment of cardiovascular diseases. The aim of this study was to evaluate the transplacental genotoxicity effect of aqueous (AE) and hydroalcoholic extract (HE) of leaves C. oxyacantha in a rat model and the quantification of malondialdehyde (MDA) in the liver. Three different doses of the AE and HE of the C. oxyacantha leaf were administered orally (500, 1000 and 2000 mg/kg) to Wistar rats during 5 days through the pregnancy term (16-21 days), and sampling in rats occurred every 24 h during the last 6 days of gestation, while only one sample was taken in neonates at birth. A sample of the mother's and the neonate's liver was taken for the determination of MDA. The results show that, at the hepatic level, the evaluated doses of extracts C. oxyacantha in pregnant rats and their pups did not show cytotoxicity. However, the AE and HE generated cytotoxic and genotoxic damage in the short term. On the other hand, only the AE showed a teratogenic effect. Based on these results, the AE and HE of the C. oxyacantha leaf should not be administered during pregnancy.

The ferroptosis activity is associated with neurological recovery following chronic compressive spinal cord injury.

Chronic compressive spinal cord injury in compressive cervical myelopathy conditions can lead to rapid neurological deterioration in the early phase, followed by partial self-recovery, and ultimately an equilibrium state of neurological dysfunction. Ferroptosis is a crucial pathological process in many neurodegenerative diseases; however, its role in chronic compressive spinal cord injury remains unclear. In this study, we established a chronic compressive spinal cord injury rat model, which displayed its most severe behavioral and electrophysiological dysfunction at 4 weeks and partial recovery at 8 weeks after compression. Bulk RNA sequencing data identified enriched functional pathways, including ferroptosis, presynapse, and postsynaptic membrane activity at both 4 and 8 weeks following chronic compressive spinal cord injury. Transmission electron microscopy and malondialdehyde quantification assay confirmed that ferroptosis activity peaked at 4 weeks and was attenuated at 8 weeks after chronic compression. Ferroptosis activity was negatively correlated with behavioral score. Immunofluorescence, quantitative polymerase chain reaction, and western blotting showed that expression of the anti-ferroptosis molecules, glutathione peroxidase 4 (GPX4) and MAF BZIP transcription factor G (MafG), in neurons was suppressed at 4 weeks and upregulated at 8 weeks following spinal cord compression. There was a positive correlation between the expression of these two molecules, suggesting that they may work together to contribute to functional recovery following chronic compressive spinal cord injury. In conclusion, our study determined the genome-wide expression profile and ferroptosis activity of a consistently compressed spinal cord at different time points. The results showed that anti-ferroptosis genes, specifically GPX4 and MafG, may be involved in spontaneous neurological recovery at 8 weeks of chronic compressive spinal cord injury. These findings contribute to a better understanding of the mechanisms underlying chronic compressive spinal cord injury and may help identify new therapeutic targets for compressive cervical myelopathy.

Microfluidic paper-based colorimetric quantification of malondialdehyde using silver nanoprism toward on-site biomedical analysis: a new platform for the chemical sensing and biosensing of oxidative stress.

Malondialdehyde (MDA) is a critical product of polyunsaturated adipose acid peroxidation and represents a common biomarker of oxidative stress. The effect of different MDA concentrations on human biofluids reflects pathological changes, which has been seen in diverse types of sickness, such as leukemia, diabetes, cancer, cardiovascular disease, and age-related macular degeneration and liver disease. In this study, different types of silver nanoparticles, including silver nanoprism (AgNPrs), silver nanowires (AgNWs), and silver nanospheres (AgNSs), were synthesized and used for the chemosensing of MDA by colorimetric and spectrophotometric methods. Colorimetric tests were performed to identify malondialdehyde in the solution as well as the one-droplet-based microfluidic paper substrate as a miniaturization device for the monitoring of analytes in human real samples. The analytical quantification of the MDA was done using the UV-Vis method. Also, the utilization of the designed chemosensor for the analysis of MDA in real sample was evaluated in human urine samples. Using the spectrophotometric method, MDA was deformed in the linear range of 0.01192 to 1.192 mM with a low limit of quantification of 0.12 μM. Essential significant features of this study include the first application of AgNPrs with high stability and great optical properties without any reagent as an optical sensing probe of MDA and optimized OD-μPCD toward on-site and on-demand MDA screening in real samples diagnosis and the innovative time/color semi-analytical recognition strategy. Moreover, the prepared OD-μPCD decorated by AgNPrs could be a prized candidate for commercialization due to the benefits of the low-cost materials used, like paper and paraffin, and portability. This innovative process led to uniform hydrophilic micro-channels on the surface of cellulose, without the use of a UV lamp, clean room, and organic solvents. This report could be a pioneering work, inspiring simple and effective on-site semi-analytical recognition devices for harmful substances or illegal drugs, which simply consist of a piece of lightweight paper and one drop of the required reagent.

202 SPIRULINA, GANODERMA LUCIDUM AND MORINGA IMPROVES CARDIAC FUNCTION AND REDUCES CARDIOTOXIC BIOMARKERS IN PRECLINICAL MODELS OF SHORT-TERM DOXORUBICIN MEDIATED CARDIOTOXICITY

Abstract Anthracyclines are essential adjuvant therapies for a variety of cancers, particularly breast, and gastric and esophageal cancers. Whilst prolonging cancer-related survival, these agents can in-duce drug-related cardiotoxicity. Spirulina, Reishi (Ganoderma Lucidum) and Moringa are three nutaceuticals with anti-inflammatory effects that are currently used in cancer patients as Com-plementary and Alternative Medicines to improve quality of life and fatigue. We hypothesize that the nutraceutical combination of Spirulina, Reishi and Moringa (Singo) could reduce in-flammation and cardiotoxicity induced by anthracyclines. Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with short-term doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), Singo at 12 mg/kg (Singo, n=6) or doxorubicin combined to Singo (DOXO-Singo, n=6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100, Fujifilm). Myocardial expression of NLRP3, DAMPs (galectine 3 and calgranu-linS100) and 13 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF) were quantified through selective mouse ELISA methods. Myocadial fi-brosis, necrosis and hypertrophy were analyzed through Immunohistochemistry (IHC). Human cardiomyocytes (HFC cell line) were exposed to subclinical concentration of doxorubicin (200 nM) alone or in combination to Singo (at 10, 25 and 50 µg/ml) for 24 and 48h. Cell viability studies were performed through MTS assay. Quantification of malondialdehyde and 4-hydroxynonenal were performed through spectrophotometric methods. Anti-inflammatory studies (expression of NLRP3 and p65/NF-kB) were made through selective ELISA methods. In-tracellular concentration of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF were also performed. In preclinical models of short-term DOXO cardiotoxicity, Singo improved significantly EF and FS and prevented the reduction of radial and longitudinal strain after 10 days of treatment with DOXO. A reduced expression of myocardial NLRP3 and NF-kB levels in cardiac tissues were seen in DOXO-Singo group compared to DOXO mice (p<0.05). Myocardial expression of pro-inflammatory cytokines were significantly reduced after treatment with Singo indicating anti-inflammatory properties. Myocardial levels of Cal-granulin S100 and galectine-3 were strongly enhanced in DOXO group; on the other hand their expression were reduced in Singo-DOXO group vs DOXO (p<0.05). Immunohistochemistry anal-ysis indicates that Singo reduces fibrosis and hypertrophy in myocardial tissue of mice during exposure to DOXO. In human cardiomyocytes exposed to DOXO, IL1α-β, IL-6, IL-17α, IL-18 and TNF-α levels were strongly enhanced compared to untreated cells. When co-incubated with Singo, cytokine levels were significantly reduced in cells exposed to Singo compared to only DOXO-treated cells. An opposite behavior was seen for IL-10 intracellular levels in cells co-incubated with Singo and DOXO, compared to DOXO. In Conclusion, in models of DOXO-induced cardiotoxicity, Singo is able to improve cardiac function and reduce biomarkers involved in heart failure and fibrosis. Cardioprotective properties of Singo are mediated by the reduction of lipid peroxidation products and expression of NLRP3-NF-kB –cytokine pathways. The overall picture of this study indicates that Singo could be a potential complementary and alternative medicine for primary prevention of cardiomyopathies induced by anthracyclines in cancer patients

Silicon reduces zinc absorption and triggers oxidative tolerance processes without impacting growth in young plants of hemp (Cannabis sativa L.).

Hemp (Cannabis sativa L.) is a promising crop for non-food agricultural production on soils contaminated by moderate doses of heavy metals, while silicon, as a beneficial element, is frequently reported to improve stressed plant behavior. Using a hydroponic system, plants of Cannabis sativa (cv. Santhica 27) were exposed for 1week to 100µM Zn in the presence or absence of 2mM Si. Zinc accumulated in all plant organs but was mainly sequestered in the roots. Additional Si reduced Zn absorption but had no impact on Zn translocation. Zn accumulation had a slight negative impact on leaf number, stem length, and chlorophyll content, and additional Si did not mitigate these symptoms. Exogenous Si reduced the Zn-induced membrane lipid peroxidation (assessed by malondialdehyde quantification) and increased the total antioxidant activities estimated by the FRAP index. In the absence of Si, leaf phytochelatin and total glutathione were the highest in Zn-treated plants and Si significantly decreased their concentrations.

The Effect of Green Tea as an Adjuvant to Enzyme Replacement Therapy on Oxidative Stress in Fabry Disease: A Pilot Study.

Enzymatic replacement therapy (ERT) is not very effective in halting the progression of Fabry disease (FD) toward cardiovascular (CV)-renal remodeling, particularly in case of late diagnosis. FD patients have increased oxidative stress (OS), critical for the induction of CV-renal remodeling. We investigated the effects of an adjuvant antioxidant treatment to ERT on OS and the possible advantages for related complications. OS was evaluated in 10 patients with FD before ERT, after 12 months of ERT, and after 6 months of adjuvant green tea (GT) to ERT by the following experiments: expression of p22phox; phosphorylation state of MYPT-1 and ERK 1/2 (by western blotting); and quantification of malondialdehyde (MDA) and heme oxygenase (HO)-1 levels (by ELISA). p22phox and MYPT-1 phosphorylation decreased after ERT and significantly further decreased after GT. ERK 1/2 phosphorylation and MDA levels remained unchanged after ERT, but significantly decreased after GT. HO-1 significantly increased after ERT and further increased after GT. This study provides preliminary data highlighting the antioxidant effect exerted by ERT itself, further amplified by the adjuvant antioxidant treatment with GT. The results of this study provide evidence of the positive effect of early additive antioxidant treatment to reduce OS and prevent/alleviate cardio and cerebrovascular-renal complications related to OS.

Dapagliflozin associated to sacubitril/valsartan and relationship with cardioprotection in human cardiac cells exposed to doxorubicin and HER2-blocking agents through MyD88, NLRP3 mediated pathways.

587 Background: The cumulative incidence of cardiac events in breast cancer patients treated with anthracycline and trastuzumab at 1 year after the diagnosis of cancer was 16.4%, at 2 years 23.8 %, and at 3 years 28.2%. Sodium glucose co-transporter 2 inhibitors, a new class of antidiabetic drugs, has shown measurable benefits in reduction of HF hospitalization and cardiovascular mortality. LCZ696 (neprilysin inhibitor + valsartan) as able to lower the risk of cardiovascular events in chronic heart failure. We hypothesize that dapagliflozin associated to LCZ696 could exerts cardioprotective effects in cellular models of doxorubicin and trastuzumab-induced cardiotoxicity. Methods: Human cardiomyocytes (HL-1 cells) were exposed to subclinical concentration of doxorubicin and trastuzumab (100 nM) alone or in combination with dapagliflozin (50 nM) or LCZ696 (at 100 mM) or both in combination for 48h. Cell viability, apoptosis and necrosis were performed. Quantification of malondialdehyde, 4-hydroxynonenal and intracellular Ca2+ were performed through spectrophotometric methods. Anti-inflammatory studies were also performed (expression of NLRP3 inflammasome, TLR4/MyD88 pathways, nuclear expression of NF-kB). Intracellular concentration of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF were also performed. Results: Dapagliflozin and LCZ696 increased synergistically the cell viability during exposure to doxorubicin and trastuzumab. Combination of dapagliflozin and LCZ696 reduces intracellular Ca2+ overload (-68,4% vs cells treated only to anticancer drugs; p<0,001), lipid peroxidation (mean reduction of 57-63,4 % compared to cells exposed only to anticancer drugs; p<0,05). The expression of MyD88, NLRP3 and NF-kB were strongly reduced after treatment with dapagliflozin and LCZ-696 (-52,5, -43,7 and -57,3 % vs cells exposed only to anticancer drugs, respectively; p<0.05). Notably, combination of dapagliflozin and LCZ-696 enhanced the expression Of IL-10; contrary, pro-inflammatory cytokines were reduced, such as IL-1α, IL-1β, IL-6, IL-8, IL17-α and IL-18 (p<0.05). Conclusions: During exposure to doxorubicin and trastuzumab, dapagliflozin associated to LCZ-696 exerts additive cardioprotective and anti-inflammatory effects compared to each drug alone. Their properties are mediated by the reduction of iCa2+ content that consequently reduces peroxidation and NLRP3- MyD88 expression. These results indicating the potential use of SGLT-2 inhibitors associated to LCZ696 in preclinical models of cardiotoxicity.

A nutraceutical combination of spirulina, reishi and moringa exerts significant cytoprotective effects againts doxorubicin and trastuzumab cardiotoxicity.

e24056 Background: Anthracycline and trastuzumab are essential adjuvant therapies for a variety of cancers, particularly breast, and gastric and esophageal cancers. Whilst prolonging cancer-related survival, these agents can induce drug-related cardiotoxicity. Cardioprotective agents used to mitigate cardiotoxicity, such as angiotensin antagonists, angiotensin receptor blockers and beta‐blockers, are often poorly tolerated in these patients due to intravascular volume fluctuations, which are further escalated by the hemodynamic side effects of these agents. Spiruline, Reishi and Moringa are nutaceuticals with anti-inflammatory effects that are currently used in cancer patients to improve quality of life and fatigue. We hypothesize that the combination of Spirulina, Reishi and Moringa could reduce inflammation and cardiotoxicity of anthracyclines and trastuzumab. Methods: Human cardiomyocytes were exposed to subclinical concentration of doxorubicin and trastuzumab (200 nM) alone or in combination with a formulation composed by Spiruline, Reishi and Moringa (at 10, 25 and 50 µg/ml) for 48h. Cell viability, apoptosis and necrosis were performed. Quantification of malondialdehyde and intracellular Ca2+ were performed through spectrophotometric methods. Anti-inflammatory studies were also performed (expression of NLRP3, TLR4/MyD88 pathways, nuclear expression of NF-kB). Intracellular concentration of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL17-α were also performed. Results: Spiruline, Reishi and Moringa in combination increased synergistically the cell viability during exposure to doxorubicin and trastuzumab. The nutraceutical formulation reduces intracellular Ca2+ overload (-48,8% vs cells treated only to anticancer drugs; p < 0,001), lipid peroxidation (- 47,2 % compared to cells exposed only to anticancer drugs; p < 0,001). The expression of MyD88 and NLRP3 inflammasome were also reduced (-36,3 and -28,58 % vs cells exposed only to anticancer drugs, respectively; p < 0.001). Notably, Spiruline, Reishi and Moringa increased of 26,3% the production of IL-10 and IL-2. Cytokines involved in cardiotoxicity and chemoresistance were reduced, such as IL-1α and IL-1β (-18,3 and -27,4 % vs DOXO-TRA group), IL-6 (-31,2 % vs DOXO-TRA group) and IL17-α (-27,3 % vs DOXO-TRA group) (p < 0.01 for all). Conclusions: During exposure to doxorubicin and trastuzumab, the combination of Spiruline, Reishi and Moringa exerts cardioprotective and anti-inflammatory effects. Their properties are mediated by the reduction of iCa2+ content that consequently reduces lipid peroxidation and the expression of NLRP3- MyD88. These results indicating the potential use of this nutraceutical formulation in preclinical models of anthracycline and anti HER2 antibodies-mediated cardiotoxicity.

Induction of oxidative stress upon low level exposure of arsenic using lipid peroxidation assay in freshwater fish Channa punctatus

Elevated level of arsenic exposure through drinking water induces noticeable change in the level of oxidative stress parameter such as lipid peroxidation. However, there is paucity of available information at low level exposure of arsenic for inducing oxidative damage. Present work was undertaken to investigate the oxidative stress inducing potential of arsenic at its low level exposure in fresh water fish Channa puntatus using lipid peroxidation, an established biomarker of oxidative stress. Fish specimen were exposed to 10, 50 (low level exposure) and 500μgL-1(high level exposure) of arsenic using sodium arsenite as a test substance. Samples of gill, liver, kidney and brain tissues were collected after 14 days of exposure and the lipid peroxidation assay was performed through the quantification of malondialdehyde (MDA) by TBARS assay for determination of oxidative damage in different tissues of fish. Significant increased levels of LPO were observed in all the organs. The present study showed that gills were the most sensitive organ, therefore, showed maximum peroxidative damage at 500 μgL-1 of arsenic exposure.

“You look sleepy…” The impact of sleep restriction on skin parameters and facial appearance of 24 women

Total sleep deprivation has a visible impact on subjective facial appearance. However, there is a lack of knowledge on how moderate sleep restriction objectively impairs skin quality and facial aspect. Twenty-four healthy good-sleeping women, aged 30-55, volunteered for this study on the impact of sleep restriction (SR) on their facial skin. SR was limited to 3h per night for 2 consecutive nights. We assessed the following parameters at the same time of day, before and after SR: sebumetry (Sebumeter SM 815), hydration (Corneometer CM 825), trans-epidermal water loss (Tewameter TM 210), biomechanical properties (Cutometer MPA 580), pH (PH-meter 900), desquamation quantification (D-Squameter and microscopy), and image analysis (ColorFace - Newtone Technologies). We also obtained skin samples (swab) for malondialdehyde quantification (MDA). We observed that some skin parameters are significantly associated with SR in both the morning and afternoon, including: lower hydration (p<0.001), increased trans-epidermal water loss (PIE) (p<0.001), and decreased extensibility (Uf; p=0.015) and viscosity (Uv; p<0.001) of the skin. The average pH increased from 4.8 (±0.2) to 4.9±0.4; p<0.001. For face photography, brightness and saturation also significantly decreased with SR in mornings and afternoons (p<0.001 for all tests). Finally, we observed a significant decrease in isolated corneocytes after desquamation associated with SR (p<0.001 for all tests). SR was also associated with significantly increased MDA levels (p<0.001 for all tests). Two nights of SR significantly altered the skin and facial appearances in our test group of typically good-sleeping women.

Abstract 9383: Dapagliflozin, an Sglt2 Inhibitor, Reduces Expression of Myd88, Nlrp3 and Nf-kb During Exposure to Doxorubicin and Trastuzumab in Cellular Models

Introduction: The cumulative incidence of cardiac events in women treated with anthracycline and trastuzumab at 1 year after the diagnosis of breast cancer was 16.4%, at 2 years 23.8 %, and at 3 years 28.2%. Sodium glucose cotransporter 2 inhibitors, reduces HF hospitalization and cardiovascular mortality. Hypothesis: We hypothesize that dapagliflozin could exerts cardioprotective effects in cellular models of doxorubicin and trastuzumab-induced cardiotoxicity Methods: Human cardiomyocytes (HL-1 cells) were exposed to subclinical concentration of doxorubicin and trastuzumab (100 nM) alone or in combination with dapagliflozin at 50 nM for 48h. Cell viability, apoptosis and necrosis were performed. Quantification of malondialdehyde, 4-hydroxynonenal and intracellular Ca 2+ were performed through spectrophotometric methods. Anti-inflammatory studies were also performed (expression of NLRP3 inflammasome, TLR4/MyD88 pathways, nuclear expression of NF-kB). Intracellular concentration of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF were also performed. Results: Dapagliflozin increased cell viability during exposure to doxorubicin and trastuzumab. Dapagliflozin reduces intracellular Ca 2+ overload ( -47,6% vs cells treated only to anticancer drugs; p&lt;0,001), lipid peroxidation (mean reduction of 35-43 % compared to cells exposed only to anticancer drugs; p&lt;0,001). The expression of Myd88, NLRP3 and NF-kB was significantly reduced after treatment with dapagliflozin (-37,5, -28 and -37 % vs cells exposed only to anticancer drugs, respectively; p&lt;0.001). A specific set of cytokines, such as IL-1α, IL-1β, IL-6, IL17-α, TNF-α and IL-18 were significantly reduced after treatment with dapagliflozin indicating its anti-inflammatory, anti-fibrotic and cytoprotective properties which could prevent arrhythmic and myocardial events. Conclusions: Dapagliflozin exerts significant cardioprotective properties mediated by iCa 2+ content that consequently reduces peroxidation and NLRP3- Myd88 expression; inhibition of NLRP3 inflammasome by dapagliflozin decreased the expression of cytokines involved in cardiotoxicity.

Method Validation and Characterization of the Associated Uncertainty for Malondialdehyde Quantification in Exhaled Breath Condensate.

There are several methods for quantifying malondialdehyde (MDA), an oxidative stress biomarker, in exhaled breath condensate (EBC). However, due to the very diluted nature of this biological matrix, a high variability is observed at low concentrations. We aimed to optimize a 2,4-dinitrophenylhydrazine-based method using liquid chromatography coupled to tandem mass spectrometry and characterize the uncertainty associated with this method. We investigated the following parameters for the method validation: calibration linearity, limit of detection (LOD), precision, recovery, and matrix effect. The results were used to identify the main sources of uncertainty and calculating the combined uncertainty. The applicability of this method was evaluated in an ongoing epidemiological study by analyzing 164 EBC samples collected from different professional groups in subway environments. The optimized method was sensitive (LOD: 70 pg/mL), precise (inter-day variation < 19%) and accurate (recovery range: 92–106.5%). The calculated analytical uncertainty was the highest at the LOQ level and reached 23%. Although the analytical uncertainty was high at low MDA concentrations, it was significantly lower than that the observed inter-individual variability. Hence, this method performs sufficiently well and can be recommended for future use in epidemiological researches relying on between-subject differences.

Enniatin B and Deoxynivalenol Activity on Bread Wheat and on Fusarium Species Development.

Fusarium head blight (FHB) is a devastating wheat disease, mainly caused by Fusarium graminearum (FG)—a deoxynivalenol (DON)-producing species. However, Fusarium avenaceum (FA), able to biosynthesize enniatins (ENNs), has recently increased its relevance worldwide, often in co-occurrence with FG. While DON is a well-known mycotoxin, ENN activity, also in association with DON, is poorly understood. This study aims to explore enniatin B (ENB) activity, alone or combined with DON, on bread wheat and on Fusarium development. Pure ENB, DON, and ENB+DON (10 mg kg−1) were used to assess the impacts on seed germination, seedling growth, cell death induction (trypan blue staining), chlorophyll content, and oxidative stress induction (malondialdehyde quantification). The effect on FG and FA growth was tested using ENB, DON, and ENB+DON (10, 50, and 100 mg kg−1). Synergistic activity in the reduction of seed germination, growth, and chlorophyll degradation was observed. Conversely, antagonistic interaction in cell death and oxidative stress induction was found, with DON counteracting cellular stress produced by ENB. Fusarium species responded to mycotoxins in opposite directions. ENB inhibited FG development, while DON promoted FA growth. These results highlight the potential role of ENB in cell death control, as well as in fungal competition.

A Simple HPLC/DAD Method Validation for the Quantification of Malondialdehyde in Rodent's Brain.

In the present study, a HPLC/DAD method was set up to allow for the determination and quantification of malondialdehyde (MDA) in the brain of rodents (rats). Chromatographic separation was achieved on Supelcosil LC-18 (3 μm) SUPELCO Column 3.3 cm × 4.6 mm and Supelco Column Saver 0.5 μm filter by using a mobile phase acetonitrile (A) and phosphate buffer (20 mM, pH = 6) (B). Isocratic elution was 14% for (A) and 86% for (B). The injection volume (loop mode) was 100 μL with an analysis time of 1.5 min. Flow rate was set at 1 mL/min. The eluted compound was detected at 532 nm by a DAD detector by keeping the column oven at room temperature. The results indicated that the method has good linearity in the range of 0.2–20 μg/g. Both intra- and inter-day precision, expressed as RSD, were ≤15% and the accuracies ranged between ±15%. The lower limit of quantification (LLOQ), stability, and robustness were evaluated and satisfied the validation criteria. The method was successfully applied in a study of chronic toxicology following different treatment regimens with haloperidol and metformin.

Kinetic spectroscopic quantification using two-step chromogenic and fluorogenic reactions: From theoretical modeling to experimental quantification of biomarkers in practical samples

Kinetic chromogenic (CG) and fluorogenic (FG) quantification deduces analyte concentration based on the reaction rate between the CG/FG probe and its targeted molecule. Little progress has been made in the past half century in either the theory or the applications of the kinetic spectroscopic quantification methods. Current kinetic CG/FG quantification is limited only to a subset of CG/FG reactions that can be approximated as the single-step process, and more problematically, to research samples with no matrix interferences. Reported herein is a kinetic quantification model established for multistep CG/FG reactions and a proof-of-concept demonstration of direct kinetic FG quantification of biomarkers in practical samples. The kinetic spectral intensity of the CG/FG reactions with two rate-limiting steps comprises three temporal regions: an accelerating period where rate of signal change is increasingly rapid, a linear region where the rate of signal change is approximately constant, and a deceleration region where the rate of signal increase becomes progressively small. Kinetic quantification is performed through simple linear-curve-fitting of the kinetic signal in its linear time-course region. The theoretical model is validated with the dual CG/FG 2-thiobarbituric acid (TBA) and malondialdehyde (MDA) reaction. Proof-of-concept kinetic spectroscopic quantification of analytes in practical samples is demonstrated with the FG quantification of MDA in canned chicken. The only sample preparation is bench-top centrifugation followed by two sequential syringe filtrations. The total kinetic FG assay time is less than 10 min, more than 10 times more efficient than the current equilibrium-based MDA assay. The theoretical model and the measurement design strategies offered by this work should help transform the current kinetic spectroscopic quantification from a niche research tool to an indispensable technique for time-sensitive applications.

The protective effect of a nutraceuticals combination in an experimental model of liver ischemia-reperfusion

Presenter: Carlos A Pantanali MD | Hospital das Clinicas of University of Sao Paulo Background: Ischemia-reperfusion (IR) injury is an unresolved problem in liver resection and transplantation. It has a complex pathophysiology and its injury key factors are inflammatory response, oxidative stress and apoptosis. In this context, the preoperatve nutritional state considerably affects postoperative metabolism and liver function. We aimed to evaluate the effect of a nutraceuticals combination on liver injury induced by IR in a rat model. Methods: Thirty-two male Wistar rats were used and the liver IR was performed by clamping the hepatic pedicle of the middle lobe for 60 minutes, followed by reperfusion for 4 hours. The rats were equally divided into 4 groups: group 1 – received the nutraceuticals combination by gavage for 7 days, before being subjected to liver IR; group 2 – submitted only to liver IR; group 3 – received only the nutraceuticals combination for 7 days; group 4 – normal rats (control). Blood was collected, all rats were euthanized and then samples from the injured liver were collected. Hepatocellular injury was evaluated by the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), apoptosis was analyzed using TUNEL method, TNF-alpha and caspase 3 in the liver tissue were analyzed by immunohistochemistry, lipid peroxidation was examined by the quantification of malondialdehyde (MDA) through the HPLC method and the inflammatory response was evaluated by serum interleukins 6, 10 and TNF-alpha. Results: The serum levels of AST, cytokine TNF-alpha and apoptosis were significant decreased in group 1 compared to group 2. The ALT also was reduced in group 1, but it didn’t have statistical difference with group 2. The interleukins 6 and 10, and the MDA were similar in both groups. Looking at apoptosis, the TNF-α and Caspase 3 in the liver tissue were statistically lower in group 1 compared to 2. Comparing group 2 with group 3, the nutraceuticals combination demonstrated lower levels of all studied variables. Group 2 had all variables higher than the control. Group 3 had similar levels of both transaminases, apoptosis, Caspase 3, MDA and inflammatory variables compared to normal rats, but TNF-alpha in the liver was higher this group. Conclusion: The combination of nutraceuticals given before liver IR injury is able to prevent apoptosis and inflammatory response by TNF-α, as well as limit the hepatocelullar injury. Thus, a specific nutrition can protect the hepatocytes and its mechanisms of action can be promising therapeutic targets.

Icariin enhances cell survival in lipopolysaccharide-induced synoviocytes by suppressing ferroptosis via the Xc-/GPX4 axis.

The mechanism of action of synovitis, as the vital pathological process of rheumatoid arthritis and osteoarthritis, remains to be elucidated. The effects and the mechanism of icariin (ICA), which is a promising therapeutic agent in synovitis, was investigated in the present study. In addition, ferroptosis, a vital cell process involved in several diseases, was also studied in synovitis for the first time. Lipopolysaccharide (LPS)-induced synoviocytes served as a synovitis cell model. The cells were divided into control, LPS and experimental groups and were treated with different concentrations of ICA. Cell viability was determined by Cell Counting Kit-8 assay and cell death was determined by flow cytometry. The expression levels of proteins (GPX4, SLC7A11, SLC3A2L, TRF, Nrf2 and NCOA4) were measured by western blotting. Quantification of malondialdehyde (MDA), iron and glutathione peroxidase 4 (GPX4) activity levels were performed via using corresponding assay kits. Cell death was increased, and cell viability was decreased in LPS-induced synoviocytes. Furthermore, MDA levels and iron content were elevated and GPX levels was reduced in LPS-induced synoviocytes. Transferrin receptor protein 1 and nuclear receptor coactivator 4 were upregulated and proteins of the Xc-/GPX4 axis, as well as nuclear factor erythroid 2-related factor 2, were decreased by LPS treatment. All aforementioned LPS affects were alleviated by ICA via a concentration-dependent manner. ICA counteracted the effects of RSL3, a ferroptosis activator, on cell viability, lipid peroxidation, iron content and relative protein expression of ferroptosis in synoviocytes. ICA protects the cells from death in synoviocytes induced by LPS, via the inhibition of ferroptosis by activating the Xc-/GPX4 axis, which can be exploited as a new therapeutic strategy for synovitis.

A colorimetric paper-based smart label soaked with a deep-eutectic solvent for the detection of malondialdehyde

Malondialdehyde (MDA) is a mutagenic secondary product of the peroxidation of polyunsaturated fatty acids, often used as a target for the food oxidation state, whose determination is frequently performed by its slow reaction with thiobarbituric acid (TBA), dissolved in acidified aqueous-ethanol medium, to produce a pink colored MDA-TBA adduct. In this investigation, we have verified that this reaction also occurs in comparable times in a deep eutectic solvent (DES) such as choline chloride-malonic acid (ChCl-MA) displaying intrinsic acid properties. This finding has allowed us to set up a MDA detection procedure suitable for simply assembling colorimetric smart labels of possible use in packaged food. It is based on the use of paper discs soaked with a controlled concentration of TBA dissolved in the mentioned DES, which are exposed to the vapors released from controlled amounts of analyzed samples, whose temperature is of course a constraint for the formation of the pink colored adduct. It profits from the negligible volatility of DESs which makes them ideal candidates to replace volatile organic and aqueous media in colorimetric sensing and, moreover, it allows any preliminary sample extraction to be avoided. In order to avoid only rough MDA determinations by color comparison with standard discs, also a device allowing MDA quantification by smartphone-based reflectance measurements was assembled. The application of this procedure to real samples consisting of fried seed oil gave results in good agreement with values previously reported in literature for similar samples. Finally, it deserves to be underlined that this approach could be easily applied to the colorimetric sensing of other several analytes.