Damage In Human Peripheral Lymphocytes Research Articles

New insights into the size-independent bioactive potential of pristine and UV-B aged polyethylene microplastics

The present study investigates the morphological, physicochemical, and structural changes occurred by the UV-B aging process of low-density polyethylene microplastics (LDPE MPs), as well as the bioactive potential of both pristine and UVaged MPs towards healthy peripheral blood lymphocytes. Specifically, LDPE MPs (100–180 μm) prepared by mechanical milling of LDPE pellets, were UV-B irradiated for 120 days (wavelength 280 nm; temperature 25 °C; relative humidity 50 %) and further examined for alterations in their particle size and surface, their functional groups, thermal stability, and crystallinity (by means of SEM, FTIR spectroscopy, XRD patterns, and TGA measurements, respectively). In parallel, isolated human peripheral blood lymphocytes were treated with different concentrations (25–500 μg mL−1) of either pristine or aged MPs (UVfree and UV120d LDPE MPs) for assessing the cytogenotoxic (by means of trypan blue exclusion test and the cytokinesis-block micronucleus assay using cytochalasin-B) and oxidative effects (using the DCFH-DA staining) in both cases. According to the results, UVfree and UV120d-LDPE MPs, with a size ranging from 100 to 180 μm, can differentially promote cytogenotoxic and oxidative alterations in human lymphocytes. In fact, UVfree LDPE MPs not being able to be internalized by cells due to their size, could indirectly promote the onset of mild oxidative and cytogenotoxic damage in human peripheral lymphocytes, via a dose-dependent but size-independent manner. The latter is more profound in case of the irregular-shaped UV120d-LDPE MPs, bearing improved dispersibility and sharp edges (by means of cracks and holes), as well as oxygen-containing and carbonyl groups. To our knowledge, the present findings provide new data regarding the bioactive behavior of pristine and UV-B aged LDPE MPs, at least in the in vitro biological model tested, thus giving new evidence for their size-independent and/or indirect mode of action.

Protective effects of olive oil phenolics oleuropein and hydroxytyrosol against hydrogen peroxide-induced DNA damage in human peripheral lymphocytes.

This study investigates antioxidant capacity and protective effects of phenolic compounds oleuropein (OLP) and hydroxytyrosol (HT), present in olive oil and olive leaves, against H2O2-induced DNA damage in human peripheral lymphocytes. Antioxidant potency was determined using the measurement of radical-scavenging activity (ABTS∙+ assay), ferric reducing power (FRAP assay) and cupric reducing antioxidant capacity (CUPRAC assay). Both substances were found to be potent antioxidant agents due to their free radical-scavenging activities. Antigenotoxic effects of oleuropein and hydroxytyrosol against H2O2-induced damage in human lymphocytes were evaluated in vitro by alkaline comet assay. At tested concentrations (1, 5, 10 µmol L-1), oleuropein and hydroxytyrosol did not induce a significant increase of primary DNA damage in comparison with the negative control. Pretreatment of human lymphocytes with each of the substances for 120 min produced a dose-dependent reduction of primary DNA damage in the tested cell type. Hydroxytyrosol showed a better protective effect against H2O2-induced DNA breaks than oleuropein which could be associated with their free radical-scavenging efficacy.

In vitro cytotoxicity and genotoxicity of Furia®180 SC (zeta-cypermethrin) and Bulldock 125®SC (β-cyfluthrin) pyrethroid insecticides in human peripheral blood lymphocytes

In the present study, human peripheral blood lymphocytes were exposed in vitro to 0, 6, 12, 18, 24, and 30 μg/mL Furia®180 SC (zeta-cypermethrin) and 0, 6.3, 12.5, 18.8, 25, and 31.3 μg/mL Bulldock®125 SC (β-cyfluthrin). Exposure to 32 µg/mL bleomycin for 24 h served as a positive control. The cytotoxic and genotoxic effects of each insecticide were analyzed using alkaline comet and trypan blue dye exclusion assays. DNA damage was evaluated through three genotoxicity parameters: tail length (TL), tail moment (TM) and tail intensity (TI). Furia®180 SC and Bulldock®125 SC pyrethroid insecticides and bleomycin significantly increased DNA damage in a concentration-dependent manner. Bulldock®125 SC induced more DNA damage than Furia. Lymphocyte viability did not change after exposure to different concentrations of the two pyrethroid insecticides and bleomycin. Moreover, genotoxic results demonstrated that Furia®180 SC and Bulldock®125 SC insecticides caused in vitro DNA damage in human peripheral lymphocytes.

In vitro evaluation of the protective effects of 4-thujanol against mitomycin-C and cyclophosphamide-induced genotoxic damage in human peripheral lymphocytes

4-Thujanol (sabinene hydrate), a bicyclic monoterpene alcohol, is found in the essential oils of many aromatic and medicinal plants and is widely used as a fragrance and flavouring agent in many different products. The aim of this study was to evaluate the protective effects of 4-thujanol against the genotoxic effects induced by mitomycin C (MMC) and cyclophosphamide (CP) in human lymphocytes, using the chromosome aberrations, sister chromatid exchanges, and micronucleus tests, in the absence and in the presence of S9 mix, respectively. The cells were treated with 0.25 µg/mL MMC and 28 µg/mL CP as alone and cotreated with 13 + 0.25, 26 + 0.25, and 52 + 0.25 µg/mL 4-thujanol + MMC and with 13 + 28, 26 + 28, and 52 + 28 µg/mL 4-thujanol + CP as a mixture. The present study showed that 4-thujanol was unable to reduce the genetic damage induced by MMC, in the absence of S9 mix. On the other hand, probably the metabolites of 4-thujanol act as an antagonist and markedly antagonize CP-induced genotoxicity, in the presence of S9 mix. In general, 4-thujanol + MMC and 4-thujanol + CP decreased the mitotic index, proliferation index and nuclear division index to the same extent or more than those of individual exposure of MMC or CP. In conclusion, 4-thujanol significantly reduced (p < 0.001) the genotoxic damage induced by CP but not MMC when compared with the respective positive control alone. We can suggest that 4-thujanol may improve the chemopreventive effects and may also reduce the harmful side effects of CP, which is widely used in chemotherapy against cancer, without reducing its antiproliferative activities.

Differences in the activities of resveratrol and ascorbic acid in protection of ethanol-induced oxidative DNA damage in human peripheral lymphocytes

Previous studies have shown that ethanol induces oxidative DNA damage in human peripheral lymphocytes. In the present study, protective effect of resveratrol and ascorbic acid on ethanol-induced oxidative DNA damage in human peripheral lymphocytes in vitro were comparatively investigated. Pretreatments with resveratrol at 5, 25, and 50μM, which were in the concentration range of in vitro research, significantly inhibited ethanol-induced oxidative DNA damage in 24h, whereas ascorbic acid showed such DNA protective activity only in 1h. Further study showed that both compounds could directly scavenge hydroxyl radical produced during ethanol metabolism. Resveratrol significantly inhibited ethanol metabolism by regulating alcohol dehydrogenase 1B (ADH1B) and acetaldehyde dehydrogenase 2 (ALDH2) mRNA expressions. Moreover, resveratrol also activated the base excision repair (BER) system in mRNA and protein levels in DNA auto-repair process. However, ascorbic acid showed no effect on ethanol metabolic pathway and BER system. Thus, the present study provided the first evidence that even though both resveratrol and ascorbic acid are anti-oxidants, they possessed differential mechanisms of action in protection against ethanol-induced oxidative DNA damage in human peripheral lymphocytes.

Evaluation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) adduct levels and DNA strand breaks in human peripheral blood lymphocytes exposed in vitro to polycyclic aromatic hydrocarbons with or without animal metabolic activation

The polycyclic aromatic hydrocarbons (PAHs) dibenzo(a,h)anthracene, benzo(ghi)perylene, benzo(b)fluoranthene and benzo(a)pyrene have been identified in urban air from Mexico City and some of them are classified as human carcinogens. In the present study, human peripheral blood lymphocytes were exposed in vitro to different concentrations of PAHs with (+S9) or without (−S9) metabolic activation. The genotoxic and cytotoxic effects of each PAH were examined with an alkaline comet assay and trypan blue dye exclusion, and oxidative DNA damage was determined via the detection of 8-hydroxy-2′-deoxyguanosine (8-OhdG) adduct levels by enzyme-linked immunosorbent assay (ELISA). The DNA damage was evaluated with two genotoxicity parameters: the frequency of comets and the comet tail length. Concentrations of 20, 40, 80, 160 and 320 µM DB(a,h)A−S9; 20, 40, 80, 160 and 240 µM B(ghi)P−S9; 20, 30, 40, 60 and 80 µM B(b)F−S9; and 80 µM B(a)P−S9 for 24 h induced a small but significant increase in the means of comet frequency, in the tail length and in the 8-oHDg levels in relation to the control (0.5% DMSO−S9). However, all PAHs+S9 produced a more significant increase in DNA strand breaks and the level of 8-OHdG compared with the control (0.5% DMSO+S9), with a concentration-effect relationship. The viability of lymphocytes exposed to all PAHs−S9 and PAHs+S9 was not modified compared with the control. The results of this study demonstrate that the comet and ELISA are rapid, suitable and sensitive methods to detect in vitro PAH-induced DNA damage in human peripheral lymphocytes.

Genotoxicity evaluation of dentine bonding agents by comet assay

To evaluate the genotoxicity of four different adhesives, Clearfil SE Bond, SL Bond, i Bond and Clearfil Protect Bond and the primers of Clearfil SE Bond and Clearfil Protect Bond. Genotoxicity assessment of the adhesives and primers was carried out in vitro in human lymphocytes at different elution concentrations, using the alkaline single-cell gel electrophoresis technique (comet assay). After the incubation of lymphocytes with varying volumes of the test agent, cells were embedded in a low-melting-point agarose suspension and then lysed in alkaline (pH>13) conditions. Electrophoresis was performed on the suspended lysed cells followed by visual analysis with staining of DNA. Fluorescence was than calculated to determine the extent of DNA damage using imaging software. Statistical comparison of the results was carried out by one-way analysis of variance (anova). A significant increase (P<0.001) compared to untreated controls in DNA damage was observed with 'Clearfil Protect Bond' and 'Clearfil SE Bond' primer in human lymphocytes at concentrations of 2.5 and 5.0 mg mL(-1). Clearfil Protect Bond and Clearfil SE Bond adhesives induced significant (P < 0.001) DNA damage only at the higher concentration of 5.0 mg mL(-1) . No significant increase in DNA damage was observed with SL Bond and i Bond. No significant DNA damage was observed with any dentine bonding agents at the lower concentration of 1.25 mg mL(-1) . 'Clearfil Protect Bond' and 'Clearfil SE Bond' primers/adhesives increased DNA damage in human peripheral lymphocytes in high doses.

Protection of resveratrol and its analogues against ethanol-induced oxidative DNA damage in human peripheral lymphocytes

Diseases related to ethanol abuse, especially binge drinking, are becoming one of the most costly health problems in the world. Ethanol-induced DNA damage plays a key role in the etiology of these diseases. New compounds are expected to offer new options against ethanol-induced genotoxicity. It was found, for the first time, that resveratrol and three analogues with 3,5-dimethoxyl groups in the A-ring, such as ( E)-4-(3,5-dimethoxystyryl)phenol (RV32), or with a quinolyl in the B-ring, such as ( E)-5-[2-(quinolin-4-yl)vinyl]benzene-1,3-diol (RV01) and ( E)-4-(3,5-dimethoxystyryl)quinoline (RV02), strongly inhibited ethanol-induced oxidative DNA damage in human peripheral lymphocytes in vitro. Resveratrol and RV32 with more hydroxyl groups in structures showed stronger direct scavenging activity of hydroxyl radicals than RV01 and RV02. Moreover, all compounds reduced hydroxyl radical generation by regulating the mRNA expression of alcohol dehydrogenase 1B and acetaldehyde dehydrogenase 2. Further studies proved resveratrol and three analogues activated the base excision repair system in transcriptional and protein levels in DNA repair process. Both 3,5-dimethoxyl groups and quinolyl modification may enhance such activity. In summary, resveratrol and its three analogues revealed significant protective activity against ethanol-induced oxidative DNA damage in human peripheral lymphocytes, which demonstrates their potential for use in prevention and treatment of the diseases related to ethanol abuse.

The Anti-Genotoxic Effect of Taurine on Aluminum Sulphate-Induced DNA Damage in Human Peripheral Lymphocytes

Aluminum sulphate Al 2 (SO 4 ) 3 is commonly used as a coagulant in the purification of drinking water in many regions of the world. Aluminum (Al) is also involved in several pharmaceutical and cosmetic products. However, Al is suspected to cause serious disorders such as Alzheimer's by oxidative DNA damage. On the other hand, taurine (TA) is an amino acid found in mammalian tissues and it has been suggested to play a role in the defense against cellular damage. The objective of this study was to determine whether the supplemetation of a natural antioxidant, TA conferred the protection against Al exposure in human blood cultures. For this aim, sister chromatid exchange (SCE) and micronucleus (MN) rates were assessed. Present results showed that 10 μg/ml of Al 2 (SO 4 ) 3 has no effect on SCE and MN rates, but 20 μg/ml of this compound increased the rates of SCEs and MN. Besides, the TA at all studied concentrations (25, 50 and 100 μg/ml) did not alter the mean SCE and MN values as compared to controls. Moreover, the negative cytogenetic alterations induced by Al could be significantly ( P<0.05 ) reduced by the application of TA. This study reveals for the first time the ameliorative role of TA against Al-induced DNA damage in vitro .

Possible Metabolic Pathways of Ethanol Responsible for Oxidative DNA Damage in Human Peripheral Lymphocytes

Ethanol abuse, especially binge drinking, can be toxic to human organs. However, there have been few studies on the genotoxicity induced by ethanol in human peripheral lymphocytes under binge drinking conditions. The purpose of this study was to investigate the oxidative DNA damage induced by ethanol in human peripheral lymphocytes in vitro and the possible mechanism associated with ethanol metabolism. The concentrations of ethanol investigated in this study were 50 and 100 mM, which are equal to the ethanol concentrations in blood after binge drinking. The maximum concentration we used was 150 mM although it is not the typical blood ethanol concentration seen during binge drinking, and most people may die at such a high concentration. The purpose of using this maximum concentration was to obtain more detailed evidence about the genotoxicity induced by ethanol. The DNA repair process was also studied. Peripheral lymphocytes were isolated from donors who were nonsmokers and not ethanol drinkers. Oxidative DNA damage, possible metabolic pathways of ethanol in human peripheral lymphocytes, and the repair system involved in the DNA auto-repair process were examined by comet assay, flow cytometry, time-of-flight mass spectrometry (TOF-MS), reverse transcription-polymerase chain reaction (RT-PCR), and western blotting. The results showed that ethanol at the concentrations of 50, 100, and 150 mM significantly induced the oxidative DNA damage in human peripheral lymphocytes in vitro, which was accompanied by a parallel increase in the generation of 8-OHdG, intracellular hydroxyl radical, and reactive oxygen species (iROS). The DNA damage induced by ethanol could be attenuated by alcohol dehydrogenase 1B (ADH1B) or acetaldehyde dehydrogenase 2 (ALDH2) inhibitor, and the mRNA expression levels of ADH1B and ALDH2 were increased markedly by ethanol. The inhibitor of cytochrome P450 2E1 (CYP2E1) had no effect on ethanol-induced DNA damage, and CYP2E1 mRNA expression was not affected by ethanol. Furthermore, ethanol-induced DNA damage could be auto-repaired by lymphocytes. The expression of 8-oxoguanine DNA glycosylase 1 (OGG1) and the X-ray repair cross-complementation group 1 (XRCC1), 2 core enzymes in the base excision repair (BER) system, were increased in both of transcriptional and protein levels after ethanol treatment. This study provides direct evidence that ethanol can induce oxidative DNA damage in human peripheral lymphocytes in vitro, and its mechanism may be associated with the metabolism of ethanol by the ADH1B/ALDH2 pathway. Moreover, ethanol-induced DNA damage can be auto-repaired by human peripheral lymphocytes possibly mediated by the BER system.

Auraptene from Ferula szowitsiana protects human peripheral lymphocytes against oxidative stress

The antigenotoxicity effects of auraptene on DNA damage in human peripheral lymphocytes were studied using alkaline single cell gel electrophoresis. Auraptene at concentrations of 5, 10, 25, 50, 100, 200 and 400 microM was tested under simultaneous treatment with 25 microM H(2)O(2). The data are expressed as % tail DNA and compared with ascorbic acid at concentrations of 25, 50, 100, 200 and 400 microM. Auraptene significantly reduced the genotoxicity of H(2)O(2 )at concentrations higher than 25 microM (p < 0.001). Interestingly, the antigenotoxicity activity of auraptene was higher than ascorbic acid (p < 0.01), however, at some concentrations (25, 50 and 200 microM) there was no significant difference between auraptene and ascorbic acid (p > 0.05). It seems that the significant antigenotoxicity effects of auraptene may be due to the prenyl moiety and also the suppression of superoxide anion (O(2) (-)) generation. This study suggests that the antigenotoxic property of auraptene is of great pharmacological importance and might be beneficial for cancer prevention.

Protective effect of Gingko biloba against radiation-induced cellular damage in human peripheral lymphocytes and murine spleen cells

The present study was designed to determine the possible protective effects of Gingko biloba extract (EGb) against cellular damage induced by irradiation (IR) in human lymphocytes and C56BL/6 murine cells. Murine spleen cells were exposed to IR after pretreatment with either saline or EGb, and treatments were repeated immediately after IR. Then the mice were decapitated at either 6 h or 72 h after whole-body IR, where intraperitoneal injections of EGb or saline were repeated once daily. Human peripheral lymphocytes were observed for the determination of the frequencies of apoptotic cells. All samples were also examined microscopically and assayed for the frequencies of apoptotic cells. The frequencies of apoptotic cells in human peripheral lymphocytes increased significantly in the saline-treated IR groups, but decreased significantly in the EGb-treated IR groups. The sizes of the murine spleen in the saline-treated IR groups decreased significantly compared with those in EGb-treated IR groups. The data demonstrated that EGb, through its free-radical scavenging and antioxidant properties, attenuates IR-induced apoptosis in radiosensitive cells, suggesting that EGb may have a potential benefit in enhancing radioprotective effects.

Genetic damage in human peripheral lymphocytes exposed to antimicrobial endodontic agents

Formocresol, paramonochlorophenol, or calcium hydroxide have been widely used in dental practice to eradicate bacteria and consequently to produce root canal disinfection. Taking into consideration strong evidence for a relationship between DNA damage and carcinogenesis, the purpose of the present study was to evaluate the genotoxic effects of antimicrobial endodontic compounds in human peripheral lymphocytes by single-cell gel (comet) assay. This technique detects DNA strand breaks in individual cells. A total of 10 microL of the tested substance solution (formocreso1, paramonochlorofeno1, and calcium hydroxide at 100-microg/mL concentration) was added to human peripheral lymphocytes from 10 volunteers for 1 hour at 37 degrees C. The negative control group was treated with vehicle control (PBS) for 1 hour at 37 degrees C, as well. For the positive control group, lymphocytes were exposed to hydrogen peroxide at 100 microM during 5 minutes on ice. No DNA breakage was detected after a treatment of peripheral lymphocytes by formocresol, paramonochlorophenol, or calcium hydroxide at 100 microg/mL. In summary, our results indicate that exposure to formocresol, paramonochlorophenol, or calcium hydroxide may not be a factor that increases the level of DNA lesions in human peripheral lymphocytes as detected by single-cell gel (comet) assay.

Evaluation of genetic damage in human peripheral lymphocytes exposed to mineral trioxide aggregate and Portland cements

summary Mineral trioxide aggregate (MTA) and Portland cement are being used in dentistry as root-end-filling material for periapical surgery and for the sealing of communications between the root canal system and the surrounding tissues. However, genotoxicity tests for complete risk assessment of these compounds have not been conducted up to now. In the present study, the genotoxic effects of MTA and Portland cements were evaluated in peripheral lymphocytes from 10 volunteers by the alkaline single cell gel (comet) assay. The results pointed out that the single cell gel (comet) assay failed to detect the presence of DNA damage after a treatment of peripheral lymphocytes by MTA and Portland cements for concentrations up to 1000 mug mL(-1). In summary, our results indicate that exposure to MTA or Portland cements may not be a factor that increases the level of DNA lesions in human peripheral lymphocytes as detected by single cell gel (comet) assay.

Induction of poly(ADP-ribosyl)ation and DNA damage in human peripheral lymphocytes after treatment with (−)-epigallocatechin-gallate

With regard to a future use of tea polyphenols in intervention trials with individuals at high cancer risk, the effects of the tea ingredient (−)-epigallocatechin gallate (EGCG) on poly(ADP-ribose) (PAR) levels and on DNA damage were investigated in human lymphocytes. A dose- and time-dependent elevation of both PAR formation as assessed by quantitative immunofluorescence analysis and DNA damage as assessed by the comet assay were observed after treatment with EGCG at 20, 40 and 80 μM for 10–240 min. Maximum levels of PAR formation and of DNA damage were observed after 10 min at all concentrations tested. Increased PAR levels were still detectable by 240 min in the 40 and 80 μM groups. At the lowest concentration, which is near the physiological peak values found after tea ingestion, PAR formation was not correlated with DNA damage. Here, EGCG led to pronounced PAR levels, whereas the comet assay was almost negative. In contrast, such marked differences in time course and extent of both genotoxicity and PAR formation following EGCG treatment were not detected after γ-irradiation. Our results suggest that the known chemopreventive effects of EGCG, the main constituent of tea, may be partly attributed to an induction of PAR formation.

DNA damage assessment by comet assay of human lymphocytes exposed to jet propulsion fuels.

Exposure to jet fuel damages DNA and results in a number of physiological changes in liver, lung, immune, and neurological tissue. In this study the single-cell gel electrophoresis assay or comet assay was used to compare the DNA damage in human peripheral lymphocytes produced by three jet propulsion fuels: JP-8, JP-5, and JP-8+100. These fuels consist of complex mixtures of aliphatic, aromatic, and substituted naphthalene hydrocarbons. Two exposure times were investigated which correspond to estimated occupational exposure times and concentrations of fuels were used that were based on previous fuel toxicity studies. Analysis of samples for the extent of DNA damage as determined by tail moment and percent tail DNA was performed on exposed cells following a brief recovery time. All fuels produced significant increases in DNA damage; however, only JP-8+100 was genotoxic at the lowest exposure concentration (1:500). At the highest exposure concentration (1:75), the mean tail moments for JP-8 and JP-8+100 (32.041 +/- 2.599 and 45.774 +/- 4.743, respectively) were significantly greater than for JP-5 (1.314 +/- 0.474). These results indicate that JP-8+100 is the most potent inducer of DNA damage in human peripheral lymphocytes and that both JP-8+100 and JP-8 are capable of damaging lymphocyte DNA to a greater extent than JP-5.

The effect of paracetamol on oxidative damage in human peripheral lymphocytes

Unscheduled DNA synthesis (UDS ox) and lipid peroxidation (LPO) induced by non-enzymatic activation of molecular oxygen ( Fe 2+ + H 2 O 2) were measured in human peripheral lymphocytes from healthy volunteers. The effect of paracetamol (PC) in a final concentration range of 0.05–10 mmole/l on these oxidative processes and on DNA repair induced by MNNG (UDS mut) was investigated. The level of induced LPO was measured by the thiobarbituric acid assay, UDS ox and UDS mut were determined by scintillometric measurement of incorporated [methyl- 3H]thymidine into damaged DNA. PC at concentrations lower than 1 mmole/l significantly potentiates the non-enzymatically induced LPO and UDS ox with the maximum of the activation being around 0.1 mmole/l. In contrast, PC at concentrations higher than 1 mmole/l exhibits an inhibitory effect on both LPO and UDS ox. On the other hand, concentrations higher than 1 mmole/l significantly suppressed DNA-repair synthesis induced by MNNG.

Caffeine inhibition of prereplication repair of mitomycin C-induced DNA damage in human peripheral lymphocytes.

Caffeine increases the number of sister-chromatid exchanges (SCE) induced by mitomycin C (MMC) in human peripheral lymphocytes in culture. This enhancement decreases when the treated cells are held in medium before phytohemagglutinin (PHA) stimulation, or when caffeine is added to cultures some time after PHA stimulation but prior to DNA synthesis. There thus appears to be a caffeine-sensitive prereplication repair system, presumably an excision mechanism, capable of repairing a fraction of the MMC-induced DNA lesions.