DNA Damage In Lymphocytes Research Articles

Sodium benzoate in locally available soft drinks and its effect on DNA damage and liver function in rats

Sodium benzoate (E211) is used as a preservative in several kinds of food. One of the essential uses of E211 is to preserve non-alcoholic carbonated beverages. The amount of sodium benzoate in 17 local soft drink samples was estimated in the present study. The in vivo effects on biochemical aspects of the liver, kidney function, and DNA damage in lymphocytes were also investigated after oral administration of sodium benzoate in rats over 12 days. The control and experimental groups were fed standard pellet diet and distilled water ad libitum; and administered distilled water (control) and different concentrations (30, 60, and 120 mg/kg body weight) of sodium benzoate once daily through a stomach tube (0.5 ml), respectively. The alkaline comet assay was performed to investigate the possible DNA damage induced by E211 in lymphocytes. At the end of the experiment, after overnight fasting the rats were sacrificed and blood samples were collected. Animals showed no significant weight loss or gain. The investigation revealed that twelve samples contained a higher level, and the rest had trace or below the recommended maximum permitted concentration (150 mg/l) of E211 as a preservative in the soft drinks. The percentage of tail DNA (p < 0.01) and olive tail movement (p < 0.05) was significantly increased in lymphocytes that were treated with high concentration (120 mg/kg body weight) of sodium benzoate, indicated substantially higher DNA damage (3.5 times) in lymphocytes compared to control. Moreover, serum AST activity level was increased significantly (p<0.05) in the group treated with a higher dose (120 mg/kg body weight) of E211, indicating malfunction of the liver. The preservative did not significantly affect serum ALT and ALP activity, total bilirubin, creatinine, and urea level. Nonetheless, our findings suggest that caution should be adopted for using sodium benzoate as a preservative in various food products. Dhaka Univ. J. Biol. Sci. 30(3 CSI): 371-383, 2022 (June)

DNA damage in peripheral blood lymphocytes of severely ill COVID-19 patients in relation to inflammatory markers and parameters of hemostasis.

Bearing in the mind that a variety of agents can contribute to genome instability, including viral infections, the aim of this study was to analyze DNA damage in hospitalized COVID-19 patients and its relationship with certain laboratory parameters. The potential impact of applied therapy and chest X-rays on DNA damage was also estimated. The study population included 24 severely COVID-19 patients and 15 healthy control subjects. The level of DNA damage was measured as genetic damage index (GDI) by comet assay. The standard laboratory methods and certified enzymatic reagents for the appropriate autoanalyzers were performed for the determination of the biochemical and hematological parameters. COVID-19 patients had significantly higher level of DNA damage compared with control subjects. The absolute number of neutrophil leukocytes was statistically higher, while the absolute number of lymphocytes was statistically lower in COVID-19 patients than in healthy controls. The analysis of the relationship between DNA damage and laboratory parameters indicated that GDI was positively correlated with interleukin 6 (IL-6) concentration and negatively with platelet count in COVID-19 patients. The level of DNA damage was slightly higher in female patients, in whom it was demonstrated a positive correlation of GDI with C-reactive protein (CRP) and procalcitonin. Likewise, there was a negative relationship of GDI and platelet count, and positive relationship of GDI and activated partial thromboplastin time (aPTT) in female population. The applied therapy (antibiotics, corticosteroid, anticoagulant, and antiviral therapy) as well as chest X rays has been shown to have genotoxic potential. The level of DNA damage significantly corresponds to the inflammatory markers and parameters of hemostasis in COVID-19 patients. In conclusion, inflammation, smoking habit, applied therapy, and chest X rays contribute to a higher level of DNA damage in COVID-19 patients.

The relationship of double-stranded DNA breaks in blood lymphocytes and obesity without carbohydrate metabolism disorder.

The purpose of this study was to investigate the effect of body mass index on DNA damage of lymphocytes in obese patients without carbohydrate metabolism disorder in the Kazakh population. Research design is based on a single-stage descriptive study. The sample included 239 patients aged 18-60years. Among the participants, people with chronic decompensated diseases, with bad habits (smokers, drug users, drinkers) were excluded. Special attention was paid to the exclusion of diabetes mellitus to exclude the effect of hyperglycemia on DNA damage when forming the sample according to WHO criteria. The following were estimated: the diameter of the breaks (Foci dia, µm), the average number of γ-H2AX (n) foci detected per cell. The study of DNA damage of blood lymphocytes in individuals of the Kazakh population showed high rates of DSB with a BMI over 40kg/m2. The number of breaks per cell in women is significantly higher than in men (p = 0.004). The median test revealed a significant difference in the number of DSBs between different age groups (χ2 = 10.39, p = 0.0155). Obesity is now gaining momentum, so the study of the effect of body mass index on lymphocyte DNA damage in obese patients without impaired carbohydrate metabolism gives valuable results in the treatment of this disease.

Assessment of DNA Damage in Blood of Iraqi Museum Workers

: This study was done to evaluate the risk of natural alpha emitter's concentrations in blood of museum employees and any potential DNA damage that may be linked to it. The current research was conducted on employees in State board of antiquities and heritage, Abd alkarim qasim museum and Iraqi museum. Blood samples were collected from 30 participants in employees group and 30 participants in controls group that was collected from general population. Alkaline comet assay has been used to assess DNA damage in lymphocytes of participants. The results showed higher DNA damage and in employees compared with control group high comet L/W value (1.56) (1.1).Thesmokers have increased average L/W value compared to participants who never smoked in employee group. From this study concluded based on these results, exposure of museum employees and archaeologists to natural ionizing radiation may have increased risk to DNA damage and cancer compared to non-occupational employees.

Evaluation of DNA Damage in Peripheral Blood Lymphocytes Exposed to Gamma Radiation

Evaluation of DNA Damage in Peripheral Blood Lymphocytes Exposed to Gamma Radiation

FLASH irradiation induces lower levels of DNA damage ex vivo, an effect modulated by oxygen tension, dose, and dose rate.

FLASH irradiation reportedly produces less normal tissue toxicity, while maintaining tumour response. To investigate oxygen's role in the 'FLASH effect', we assessed DNA damage levels following irradiation at different oxygen tensions, doses and dose rates. Samples of whole blood were irradiated (20 Gy) at various oxygen tensions (0.25-21%) with 6 MeV electrons at dose rates of either 2 kGy/s (FLASH) or 0.1 Gy/s (CONV), and subsequently with various doses (0-40 Gy) and intermediate dose rates (0.3-1000 Gy/s). DNA damage of peripheral blood lymphocytes (PBL) were assessed by the alkaline comet assay. Following 20 Gy irradiation, lower levels of DNA damage were induced for FLASH, the difference being significant at 0.25% (p < 0.05) and 0.5% O2 (p < 0.01). The differential in DNA damage at 0.5% O2 was found to increase with total dose and dose rate, becoming significant for doses ≥20 Gy and dose rates ≥30 Gy/s. This study shows, using the alkaline comet assay, that lower levels of DNA damage are induced following FLASH irradiation, an effect that is modulated by the oxygen tension, and increases with the total dose and dose rate of irradiation, indicating that an oxygen related mechanism, e.g. transient radiation-induced oxygen depletion, may contribute to the tissue sparing effect of FLASH irradiation. This paper is first to directly show that FLASH-induced DNA damage is modulated by oxygen tension, total dose and dose rate, with FLASH inducing significantly lower levels of DNA damage for doses ≥20 Gy and dose rates ≥30 Gy/s, at 0.5% O2.

Analysis of Differences in DNA Damage and Repair Efficacy in Lymphocytes of Patients with Bipolar Disorder.

The aim of this study was to determine DNA damage during euthymic and attack periods, and the oxidative metabolism states that may cause this damage in the pathophysiology of bipolar disorder. The role of DNA repair mechanisms in this process was also investigated. The study included a total of 90 patients aged between 18-65 years who were diagnosed with bipolar disorder according to DSM- 5 diagnostic criteria, with 30 patients in euthymic, 30 in manic and 30 in depressive periods. A control group was formed of 30 healthy subjects matched to the patients by age, gender, body mass index and smoking status and/or alcohol consumption. Oxidative metabolism was investigated using the Comet Assay technique to assess DNA damage, according to the oxidant/antioxidant status in the technique developed by Erel with the Rel ASSAY Diagnostics kit (Turkey). The control and patient groups were compared in respect of gene expression levels of OGG1 and NEIL1 repair genes at mRNA level with Real-Time PCR. Increased DNA damage was found in the euthymic and manic groups and decreased NEIL1 gene expression in the depressive group. The oxidative stress index was found to be decreased in the patient groups compared to the healthy control group. Oxidative imbalance and DNA damage and repair disorders may be effective in the pathophysiology of bipolar disorder. Further studies on this subject are required to clarify the etiology and new treatment goals.

Analysis of Differences in DNA Damage and Repair Efficacy in Lymphocytes of Patients with Bipolar Disorder

The aim of this study was to determine DNA damage during euthymic and attack periods, and the oxidative metabolism states that may cause this damage in the pathophysiology of bipolar disorder. The role of DNA repair mechanisms in this process was also investigated.The study included a total of 90 patients aged between 18-65 years who were diagnosed with bipolar disorder according to DSM- 5 diagnostic criteria, with 30 patients in euthymic, 30 in manic and 30 in depressive periods. A control group was formed of 30 healthy subjects matched to the patients by age, gender, body mass index and smoking status and/or alcohol consumption. Oxidative metabolism was investigated using the Comet Assay technique to assess DNA damage, according to the oxidant/antioxidant status in the technique developed by Erel with the Rel ASSAY Diagnostics kit (Turkey). The control and patient groups were compared in respect of gene expression levels of OGG1 and NEIL1 repair genes at mRNA level with Real-Time PCR.Increased DNA damage was found in the euthymic and manic groups and decreased NEIL1 gene expression in the depressive group. The oxidative stress index was found to be decreased in the patient groups compared to the healthy control group.Oxidative imbalance and DNA damage and repair disorders may be effective in the pathophysiology of bipolar disorder. Further studies on this subject are required to clarify the etiology and new treatment goals.

Flavonoids of the Heartwood of Cotinus coggygria Scop. Showing Protective Effect on Human Lymphocyte DNA

In continuation of our study on Cotinus coggygria from Serbia, 10 known flavonoids (1-10) were isolated from the methylene chloride/methanol extract of the heartwood. They were tested for in vitro protective effect against chromosome aberrations in peripheral human lymphocytes, using the cytokinesis-block micronucleus assay. All tested compounds (in minimal doses of 1 μg/mL) exerted a beneficial effect by decreasing DNA damage of human lymphocytes in the range of 24.2% to 54.5%, better than the radio protectant control, amifostine. Functional groups, such as 3′,4′-dihydroxyphenyl (catechol), 5-OH, 3-OH, and 4-keto in flavonoids (3-keto in aurones), which play a key role in antioxidant activity, are proposed to be responsible for the DNA protective activity of the tested compounds.

Long-Term Persistence of Increased Number of γH2AX+ Peripheral Blood Lymphocytes in Monkeys Exposed to Negative Factors of Space Flights: Ionizing Radiation and Simulated Hypogravity.

We studied the influence of ionizing radiation and hypogravity as negative factors of space flights on DNA damage in peripheral blood lymphocytes of rhesus monkeys at different times after exposure (from 1 to 446 days). The proportion of cells with high numbers of DNA double-strand breaks (DSB), positive for the surrogate DSB marker-protein γH2AX, was monitored using flow cytometry. Some animals were exposed to 7-day antiorthostatic hypokinesia simulating hypogravity, the others to a combined effect of antiorthostatic hypokinesia, whole-body γ-irradiation (2.34 cGy/h, dose 1 Gy), and irradiation of the head with 12C ions (450 MeV, dose 1 Gy). Exposure to antiorthostatic hypokinesia led to a significant increase in the proportion of γH2AX+ lymphocytes only on the first day after exposure, whereas after combined exposure, increased numbers of damaged lymphocytes were recorded up to 42 days after exposure.

Preparation of Aspirin Nanosuspension by Antisolvent Precipitation Method

Preparation of Aspirin Nanosuspension by Antisolvent Precipitation Method

Cytogenetic Damage of Human Lymphocytes in Humanized Mice Exposed to Neutrons and X Rays 24 h After Exposure

Detonation of an improvised nuclear device highlights the need to understand the risk of mixed radiation exposure as prompt radiation exposure could produce significant neutron and gamma exposures. Although the neutron component may be a relatively small percentage of the total absorbed dose, the large relative biological effectiveness (RBE) can induce larger biological DNA damage and cell killing. The objective of this study was to use a hematopoietically humanized mouse model to measure chromosomal DNA damage in human lymphocytes 24 h after in vivo exposure to neutrons (0.3 Gy) and X rays (1 Gy). The human dicentric and cytokinesis-block micronucleus assays were performed to measure chromosomal aberrations in human lymphocytes in vivo from the blood and spleen, respectively. The mBAND assay based on fluorescent in situ hybridization labeling was used to detect neutron-induced chromosome 1 inversions in the blood lymphocytes of the neutron-irradiated mice. Cytogenetics endpoints, dicentrics and micronuclei showed that there was no significant difference in yields between the 2 irradiation types at the doses tested, indicating that neutron-induced chromosomal DNA damage in vivo was more biologically effective (RBE ∼3.3) compared to X rays. The mBAND assay, which is considered a specific biomarker of high-LET neutron exposure, confirmed the presence of clustered DNA damage in the neutron-irradiated mice but not in the X-irradiated mice, 24 h after exposure.

Abstract 808: Functional evaluation of novel germline DNA repair variants identified in patients with early-onset renal cancer

Abstract Background: Early-onset renal cancer (eoRC) is typically associated with significant family history of cancer, and often associated with germline pathogenic variants (PVs) in ‘RC-specific' genes including VHL, MET, FLCN, TSC1, TSC2, FH, SDH, PTEN and BAP1. However, most eoRC patients lack PVs in RC-specific genes; rather, we recently identified an enrichment of likely PVs in DNA repair genes in eoRC patients. Here, using eoRC patient resources (primary and transformed lymphocytes, lymphocyte DNA, and tumor tissue), we functionally evaluated the DNA repair variants identified in eoRC patients. Methods: We performed whole-exome sequencing (WES) on lymphocyte DNA from 22 patients with genetically undefined eoRC, with extensive family history of RC and/or other cancers, provided by Risk Assessment Program and Genitourinary Group at Fox Chase Cancer Center. Prioritized variants were assessed by structural, biochemical and cellular studies. Results: 70% of patients had rare, heterozygous, missense variants in genes that suppress DNA damage. ~18% of the variants were in DNA polymerase genes (POLD1, POLE, POLH, POLK), which mediate DNA replication-repair. Increased tumor mutation burden (&amp;gt;10 mutations/Mb) was observed in tumors carrying predicted damaging polymerase variants. In functional testing, induction of DNA damage in primary lymphocytes from eoRC patients and matched cancer-free controls (n=20 each) selectively elevated γH2AX foci (indicative of DNA damage) in cells from eoRC patients (P&amp;lt;0.001, pairwise T-test). DNA fiber analysis of eoRC patient-derived lymphocyte lines carrying the polymerase variants, showed significantly slower rate of DNA replication, and replication fork restoration after DNA damage versus the matched control-derived lymphocyte lines (P&amp;lt;0.01, Mann-Whitney test). Biochemical analysis of purified polymerase proteins found that the variants differ from wild type polymerase in their ability to extend DNA substrates and bypass DNA lesion(s), and exhibit dominant negative effects. Conclusion and future work: Our results indicate that germline variants in DNA repair genes impact suppression of DNA damage in cells from patients with eoRC. Biological studies testing the impact of the identified variants on renal carcinogenesis are warranted. Overall, genetic testing for variation in DNA repair genes may provide a more comprehensive risk assessment in eoRC patients, and provide novel opportunities for targeting DNA repair vulnerabilities in RC. Citation Format: Elena V. Demidova, Waleed Iqbal, Ilya G. Serebriiskii, Andrea F. Forman, Tatiana Kent, Richard T. Pomerantz, Erica A. Golemis, Michael J. Hall, David Y. Chen, Mary B. Daly, Sanjeevani Arora. Functional evaluation of novel germline DNA repair variants identified in patients with early-onset renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 808.

Influence of dietary fish oil supplementation on DNA damage in peripheral blood lymphocytes of nine healthy dogs.

BackgroundFish oil (FO) supplementation as a source of omega 3 fatty acids is associated with beneficial effects on health. However, high unsaturated fatty acid content in the diet could result in increased lipid peroxidation and damage to proteins, lipids and DNA. We evaluated the effect of dietary FO supplementation on DNA damage in peripheral blood lymphocytes of dogs. Additionally, we determined the effect of FO supplementation on lipid peroxidation and lipid profile of these dogs.MethodsHealthy male dogs (n = 9) were randomly assigned to one of two diets during 90 days: control (CG, n = 4), based on a commercial food, and FO (FOG, n = 5), the same food supplemented with 1000 mg FO. Blood samples were collected on days −1, 30, 60 and 90. DNA damage was assessed with the comet assay, and the damage index was obtained. Malondialdehyde (MDA) levels were determined as an indicator of lipid peroxidation. Lipid profile determination included serum triglyceride, cholesterol, low‐density lipoprotein and high‐density lipoprotein levels (HDL).ResultsDamage index values (arbitrary units) were lower in FOG on day 30 (CG, 13.7 ± 2.5; FOG, 6.5 ± 2.5), 60 (CG, 14.7 ± 2.5; FOG, 3.5 ± 2.5) and 90 (CG, 15.5 ± 2.5; FOG, 3.0 ± 2.5) compared with CG (treatment × time interaction, p < 0.01). Serum MDA and HDL concentrations were lower in FOG compared with CG on day 60 and 90 (treatment × time interaction, p < 0.05).ConclusionThese findings suggest that dietary FO supplementation did not induce DNA damage in peripheral blood lymphocytes of healthy dogs, but rather reduced it.

Evaluating the genetic basiss of anti-cancer property of Taxol in Saccharomyces cerevisiae model.

Taxol has been regarded as one of the most successful anti-cancer drugs identified from natural sources to date. Although Taxol is known to sensitize cells by stabilizing microtubules, its ability to cause DNA damage in peripheral blood lymphocytes and to induce oxidative stress and apoptosis indicates that Taxol may have other modes of cytotoxic action. This study focuses on identifying the additional targets of Taxol that may contribute to its multifaceted cell killing property, using Saccharomyces cerevisiae. We show that yeast oxidative stress response mutants (sod1Δ, tsa1Δ and cta1Δ) and DNA damage response mutants (mre11∆, sgs1∆ and sub1∆) are highly sensitive to Taxol. Our results also show that Taxol increases the level of reactive oxygen species (ROS) in yeast oxidative stress response mutant strains. Further, 4',6-Diamidino-2'-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining show that Taxol induces apoptotic features such as nuclear fragmentation and chromatin condensation in DNA repair mutants. On the whole, our results suggest that Taxol's cytotoxic property is attributed to its multifaceted mechanism of action. Yeast S. cerevisiae anti-oxidant and DNA repair gene mutants are sensitive to Taxol compared to wild-type, suggesting yeast model can be used to identify the genetic targets of anti-cancer drugs.

Intercomparison of Conventional and QuickScan Dicentric Scoring for the Validation of Individual Biodosimetry Analysis in Taiwan

Purpose The dicentric chromosome assay (DCA), the gold standard for radiation biodosimetry, evaluates an individual absorbed radiation dose by the analysis of DNA damage in human lymphocytes. The conventional (C-DCA) and QuickScan (QS-DCA) scoring methods are sensitive for estimating radiation dose. The Biodosimetry Laboratory at Institute of Nuclear Energy Research (INER), Taiwan, participated in intercomparison exercises conducted by Health Canada (HC) in 2014, 2015 and 2018 to validate the laboratory’s accuracy and performance. Material and Methods Blood samples for the conventional dose response curve for Taiwan were irradiated with 0, 0.25, 0.5, 1, 2, 3, 4 and 5 Gy. Ten blind blood samples were provided by HC. Either or both of two methods of conventional (C) or QuickScan (QS) scoring could be chosen for the HC’s intercomparison. For C-DCA triage scoring, only cells with 46 centromeres were counted and each scorer recorded the number of dicentrics in the first 50 metaphases or stopped scoring when 30 dicentrics were reached. Scorers also recorded how much time it took to analyze 10, 20, and 50 cells. Subsequently, the data were entered into the Dose Estimate software (DoseEstimate_v5.1) and dose estimates were calculated. With QS-DCA scoring, a minimum of 50 metaphase cells (or 30 dicentrics) were scored in apparently complete metaphases without verification of exactly 46 centromeres. Results For the blinded blood samples irradiated at HC and shipped to INER, the mean absolute deviation (MAD) derived after scoring 50 cells for C-DCA and QS-DCA was <0.5 Gy for all three intercomparisons, meeting the criteria for acceptance. Conclusion The results indicated that the Biodosimetry Laboratory at INER can provide reliable dose estimates in the case of a large-scale radiation accident.

PERIPHERAL BLOOD MONONUCLEAR DNA DAMAGE IDENTIFIED BY THE «СOMET» METHOD, AS A POSSIBLE INDICATOR OF SENSITIVITY OF MELANOMA TO IMMUNOTHERAPY WITH NIVOLUMAB

P urpose of the study : to assess whether the determination of the grade of dNA damage in the blood lymphocytes of patients with metastatic melanoma can be a criterion for assessing the effectiveness of therapy. Material and Methods. The grade of dNA damage in blood mononuclear cells was studied using the comet assay in 10 patients with progressive metastatic melanoma before therapy with Nivolumab and 3–4 months after Nivolumab therapy. Results. It was revealed that prior to treatment the level of dNA damage in blood mononuclears was higher in patients with melanoma than in controls (healthy subjects). This level significantly decreased when stable disease or complete regression were reached, while it changed less significantly or increased in cases with disease progression. Сonclusion. The initial level and changes in lymphocyte dNA damage in patients with melanoma can serve as a criterion for assessing tumor response to immunotherapy with Nivolumab.

Repeated dose treatment of Tinospora cordifolia polysaccharide rich extract activates splenic antigen presenting cells with no associated organ toxicity

Tinospora cordifolia is a medically valuable plant widely used in Ayurveda and has been attributed with immunomodulatory properties. We compared the immunomodulation of repeated doses of the purified polysaccharide G1-4A and polysaccharide-rich extract (PRE) of T. cordifolia in BALB/c mice. Since both had comparable effects, we further compared the repeated dose effects of 50 and 500 mg/kg b.w PRE per dose with particular emphasis on histological changes in different organs, splenic cell death and DNA damage and immunomodulation. In mice treated with both concentrations of PRE, no gross changes were observed in any organ by histopathological analyses along with normal biochemical parameters similar to control. There was no increase in splenic lymphocyte cell death or DNA damage. A significant increase in the number of splenic macrophages with no increase in phagocytosis was observed. Increased expression of phenotypic maturation markers was observed in splenic dendritic cells and not in bone marrow-derived dendritic cells of PRE-treated mice. Increased activation of B cells in response to mitogen with no change in such a response by T cells was also observed. These results from repeated dose experiments indicate that PRE treatment has no associated organ toxicity and activates antigen-presenting cells and thus can be used instead of G1-4A as a promising immunotherapeutic adjuvant.

Coenzyme Q10 protects isolated human blood cells from TiO2 nanoparticles induced oxidative/antioxidative imbalance, hemolysis, cytotoxicity, DNA damage and mitochondrial impairment.

TiO2 NPs have been investigated for their toxic potential and studies have reported their toxicity is due to generation of oxidative stress. In the present study, we investigated the toxicity of TiO2 NPs and explored the potential of well-known antioxidant coenzyme Q10 (CoQ10) in counteracting the NP-induced toxicity in isolated human blood cells. When the isolated blood cells were treated with varying concentrations of TiO2 NPs (25-100μg/ml), only 50μg/ml dose induced statistically significant hemolysis in erythrocytes and cytotoxicity in lymphocytes (p < 0.05). None of the concentrations induced any significant increase in platelet aggregation. To investigate the protective effect of CoQ10, we incubated the isolated blood cells with 50μg/ml of TiO2 NPs in the presence and absence of 25μM of CoQ10 for 3h. Hemolysis, oxidative stress, LDH leakage and ATPase enzyme activity were studied in erythrocytes; cytotoxic and DNA damaging potential of NPs were determined in lymphocytes, along with mitochondrial membrane potential (MMP) and ADP/ATP ratio. Hemolysis, generation of oxidative stress, LDH leakage and reduced ATPase activity were observed in the erythrocytes treated with NPs alone (50μg/ml), the results were statistically significant at p < 0.05. Oxidative stress was evident by increased levels of malonaldehyde, indicating lipid peroxidation and generation of reactive oxygen species including hydrogen peroxide, together with statistically significant decrease in the activities of catalase and superoxide dismutase and reduced glutathione levels. In the lymphocytes treated with NPs alone (50μg/ml), cytotoxicity in MTT assay and DNA damage in comet assay were observed; in addition, mitochondrial membrane potential collapsed and ADP/ATP ratio increased indicating mitochondrial function impairment. However, in the presence of CoQ10, hemolysis, oxidative stress and LDH leakage in the erythrocytes and lymphocyte cytotoxicity and DNA damage were drastically reduced, enzyme activities, MMP and ADP/ATP ratio were restored towards normal levels. TiO2 NPs induce cytotoxicity, damage DNA in lymphocytes, and induce oxidative/anti-oxidative imbalance in erythrocytes. Antioxidant CoQ10 protects erythrocytes and lymphocytes from toxicity induced by TiO2 NPs.

Impact of Different JAK Inhibitors and Methotrexate on Lymphocyte Proliferation and DNA Damage.

Janus kinase inhibitors (JAKis) represent a new strategy in rheumatoid arthritis (RA) therapy. Still, data directly comparing different JAKis are rare. In the present in vitro study, we investigated the immunomodulatory potential of four JAKis (tofacitinib, baricitinib, upadacitinib, and filgotinib) currently approved for RA treatment by the European Medicines Agency. Increasing concentrations of JAKi or methotrexate, conventionally used in RA therapy, were either added to freshly mitogen-stimulated or preactivated peripheral blood mononuclear cells (PBMC), isolated from healthy volunteers. A comparable, dose-dependent inhibition of lymphocyte proliferation was observed in samples treated with tofacitinib, baricitinib, and upadacitinib, while dosage of filgotinib had to be two orders of magnitude higher. In contrast, antiproliferative effects were strongly attenuated when JAKi were added to preactivated PBMCs. High dosage of upadacitinib and filgotinib also affected cell viability. Further, analyses of DNA double-strand break markers γH2AX and 53BP1 indicated an enhanced level of DNA damage in cells incubated with high concentrations of filgotinib and a dose-dependent reduction in clearance of radiation-induced γH2AX foci in the presence of tofacitinib or baricitinib. Thereby, our study demonstrated a broad comparability of immunomodulatory effects induced by different JAKi and provided first indications, that (pan)JAKi may impair DNA damage repair in irradiated PBMCs.