Dulaglutide rescues the elevated testicular dysfunction in a mouse model of high-fat diet-induced obesity.

Obesity is a well-known risk factor for testicular function; however, dulaglutide's effect on the testis in obesity has received little attention. Currently, clinicians prescribe the antidiabetic drug dulaglutide only off-label for weight management in non-diabetics. Investigating the impact of this novel compound on obesity is critical for determining whether it has any disruptive effects on testicular cells. We used a well-known animal model of high-fat diet-induced obesity in this investigation, and testicular dysfunction was determined by sperm DNA damage, spermatocyte chromosomal abnormalities, and spermiogram analysis. Following a 12-week high-fat diet challenge, mice were randomly assigned to dulaglutide (0.6 mg/kg/day) or saline treatments for five weeks. Testes and sperm cells were collected 24 h after the last dulaglutide injection. Untreated obese mice had a lower testes/body weight ratio, more sperm DNA damage, diakinesis-metaphase I chromosomal abnormalities, a lower sperm count/motility, more cell morphological defects, and an altered testicular redox balance. In obese mice, dulaglutide injection efficiently restored all disturbed parameters to their control levels. Dulaglutide injection into healthy mice exhibited no significant harmful effects at the applied regimen. As a result, we infer that dulaglutide therapy might bring obese men additional benefits by recovering testicular dysfunction induced by obesity.

Dapagliflozin suppresses diabetes-induced oxidative DNA damage and hypermethylation in mouse somatic cells.

Diabetes mellitus is a complex metabolic disorder resulting from the interplay of environmental, genetic, and epigenetic factors that increase the risk of cancer development. However, it is unclear whether the increased cancer risk is due to poor glycemic control or the use of some antidiabetic medications. Therefore, we investigated the genetic and epigenetic changes in somatic cells in a mouse model of diabetes and studied whether multiple exposures to the antidiabetic medication dapagliflozin influence these changes. We also elucidated the mechanism(s) of these ameliorations. The micronucleus test and modified comet assay were used to investigate bone marrow DNA damage and methylation changes. These assays revealed that dapagliflozin is non-genotoxic in the tested regimen, and oxidative DNA damage and hypermethylation were significantly higher in diabetic mice. Spectrophotometry also evaluated oxidative DNA damage and global DNA methylation, revealing similar significant alterations induced by diabetes. Conversely, the dapagliflozin-treated diabetic animals significantly reduced these changes. The expression of some genes involved in DNA repair and DNA methylation was disrupted considerably in the somatic cells of diabetic animals. In contrast, dapagliflozin treatment significantly restored these disruptions and enhanced DNA repair. The simultaneous effects of decreased oxidative DNA damage and hypermethylation levels suggest that dapagliflozin can be used as a safe antidiabetic drug to reduce DNA damage and hypermethylation in diabetes, demonstrating its usefulness in patients with diabetes to control hyperglycemia and decrease the development of its subsequent complications.

Alkaline Comet Assay to Assess Genotoxicity in Zebrafish Larvae.

The zebrafish (Danio rerio) is a model organism widely used in several research fields due to its characteristics and numerous advantages, such as optical embryo transparency, fully sequenced genome, orthologous genes to humans, small size, high reproductive rate, easy gene editing and relatively low costs. Thus, a number of protocols have been developed that allow the use of this vertebrate model for toxic effect evaluation at various biological levels, including genotoxicity, using the comet assay technique.The comet assay or single-cell gel electrophoresis is a popular and sensitive method to study DNA damage in cells, which is described in this chapter. Briefly, cells suspended in agarose on a microscope slide are lysed, denatured, electrophoresed, neutralized, and stained to study the migration of DNA strand breaks. As a result, cells with increased DNA damage present a high fluorescence intensity and an increase of comet tail length. For the visual score, comets are classified according to the head integrity, tail intensity, and tail length into five classes, namely, class 0 until class 4 (comets with high damage and with almost all the DNA in the tail). These data are used to calculate the Genetic Damage Index (GDI) expressed as Arbitrary Units (AU).

Short-term Effects of Non-invasive Physical Plasma Treatment on Genomic Stability.

BACKGROUND/AIM: The application of non-invasive physical plasma (NIPP) generates reactive oxygen species. These can lead to chemical oxidation of cellular molecules including DNA. On the other hand, NIPP can induce therapeutically intended apoptosis, which also leads to DNA fragmentation in the late phase. Therefore, to assess unwanted genotoxic effects, the formation of DNA damage was investigated in this study in discrimination from apoptotic processes. MATERIALS AND METHODS: Mutation events after NIPP application were analyzed in CCL-93 fibroblast cells using the hypoxanthine phosphoribosyl transferase assay. Additionally, DNA single-strand breaks (SSB) and double-strand breaks (DSB) were quantified by performing the alkaline comet assay, and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. DSBs were quantified by phospho-histone 2AX-p53-binding protein 1 co-localization DSB focus assay. The data were compared with cell death quantification by the caspase-3/7 apoptosis assay. RESULTS: Treatment with NIPP led to exceedingly rapid damage to genomic DNA and the appearance of DNA SSBs and DSBs in the initial 4 h. However, damage decreased again within the first 4-8 h, then the late phase began, characterized by DNA DSB and increasing caspase-3/7 activation. CONCLUSION: Although NIPP treatment leads to extremely rapid damage to genomic DNA, this damage is reversed very quickly by efficient DNA-repair processes. As a consequence, only those cells whose genome damage can be repaired actually survive and proliferate. Persistent genotoxic effects were not observed in the cell system used.

Application of the Fpg-modified comet assay on three-spined stickleback in freshwater biomonitoring: toward a multi-biomarker approach of genotoxicity.

Aquatic species are exposed to a wide spectrum of substances, which can compromise their genomic integrity by inducing DNA damage or oxidative stress. Genotoxicity biomarkers as DNA strand breaks and chromosomal damages developed on sentinel species have already proved to be relevant in aquatic biomonitoring. However, these biomarkers do not reflect DNA oxidative lesions, i.e., the 8-oxodG, recognized as pre-mutagenic lesion if not or mis-repaired in human biomonitoring. The relevance to include the measure of these lesions by using the Fpg-modified comet assay on erythrocytes of the three-spined stickleback was investigated. An optimization step of the Fpg-modified comet assay considering enzyme buffer impact, Fpg concentration, and incubation time has been performed. Then, this measure was integrated in a battery of genotoxicity and cytotoxicity biomarkers (considering DNA strand breaks, DNA content variation, and cell apoptosis/necrosis and density) and applied in a freshwater monitoring program on six stations of the Artois Picardie watershed (3-week caging of control fish). These biomarkers allowed to discriminate the stations regarding the genotoxic potential of water bodies and specifically by the measure of oxidative DNA lesions, which seem to be a promising tool in environmental genotoxicity risk assessment.

ADP-ribose contributions to genome stability and PARP enzyme trapping on sites of DNA damage; paradigm shifts for a coming-of-age modification.

ADP-ribose is a versatile modification that plays a critical role in diverse cellular processes. The addition of this modification is catalyzed by ADP-ribosyltransferases, among which notable poly(ADP-ribose) polymerase (PARP) enzymes are intimately involved in the maintenance of genome integrity. The role of ADP-ribose modifications during DNA damage repair is of significant interest for the proper development of PARP inhibitors targeted toward the treatment of diseases caused by genomic instability. More specifically, inhibitors promoting PARP persistence on DNA lesions, termed PARP "trapping," is considered a desirable characteristic. In this review, we discuss key classes of proteins involved in ADP-ribose signaling (writers, readers, and erasers) with a focus on those involved in the maintenance of genome integrity. An overview of factors that modulate PARP1 and PARP2 persistence at sites of DNA lesions is also discussed. Finally, we clarify aspects of the PARP trapping model in light of recent studies that characterize the kinetics of PARP1 and PARP2 recruitment at sites of lesions. These findings suggest that PARP trapping could be considered as the continuous recruitment of PARP molecules to sites of lesions, rather than the physical stalling of molecules. Recent studies and novel research tools have elevated the level of understanding of ADP-ribosylation, marking a coming-of-age for this interesting modification.

Acrylamide-induced meiotic arrest of spermatocytes in adolescent mice by triggering excessive DNA strand breaks: Potential therapeutic effects of resveratrol.

Background: Baked carbohydrate-rich foods are the main source of acrylamide (AA) in the general population and are widely consumed by teenagers. Considering the crucial development of the reproductive system during puberty, the health risks posed by AA in adolescent males have raised public concern.Methods: In this study, we exposed 3-week-old male pubertal mice to AA for 4 weeks to evaluate its effect on spermatogenesis using computer-assisted sperm analysis (CASA) and historical analysis. Flow cytometric analysis and meiocyte spreading assay were conducted to assess meiosis in mice. The expression of meiosis-related proteins and double-strand break (DSB) proteins were evaluated by immunoblot analyses. Additionally, isolated spermatocytes were used to explore the role of resveratrol in AA-induced damages of meiosis.Results: Our results showed that AA decreased the testicular and epididymal indexes, reduced sperm count and motility, and induced morphological disruption of the testes in pubertal mice. Subsequent meiotic analysis revealed that AA increased the proportion of 4C spermatocytes and decreased the proportion of 1C spermatids. The expression levels of meiosis-related proteins (SYCP3, Cyclin A1 and CDK2) were downregulated, and signaling proteins (γH2AX, p-CHK2 and p-ATM) expression levels were upregulated in AA-treated mice testes. Similar expression patterns were observed in primary spermatocytes treated with AA and these effects were reversed significantly by resveratrol.Conclusions: Our results indicate that AA induces meiotic arrest via persistent activation of DSBs, which may contribute to AA-compromised spermatogenesis. Resveratrol could serve as a potential therapeutic agent against AA-induced meiotic toxicity. These data highlight the importance of natural product supplementation for treating AA-related reproductive toxicity.

The Effect of Zinc Biofortified Wheat Produced via Foliar Application on Zinc Status: A Randomized, Controlled Trial in Indian Children.

BACKGROUND: Agronomic zinc biofortification of wheat by foliar application increases wheat zinc content and total zinc absorption in humans. OBJECTIVES: To assess the effect of agronomically biofortified whole wheat flour (BFW) on plasma zinc (PZC) compared with a postharvest fortified wheat (PHFW) and unfortified control wheat (CW) when integrated in a midday school meal scheme. METHODS: We conducted a 20-wk double-blind intervention trial in children (4-12 y, n = 273) individually randomly assigned to 3 groups to receive a daily school lunch consisting of 3 chapattis prepared with the 3 different wheat flour types. Measurements of anthropometry, blood biochemistry, and leukocyte DNA strand breaks were conducted. We applied sparse serial sampling to monitor PZC over time, and analysis was performed using linear mixed-effects models. RESULTS: Mean zinc content in BFW, PHFW, and CW were 48.0, 45.1, and 21.2 ppm, respectively (P < 0.001). Mean (standard deviation) daily zinc intakes in the study intervention in BFW, PHFW, and CW groups were 4.4 (1.6), 5.9 (1.9) and 2.6 (0.6) mg Zn/d, respectively, with intake in groups PHFW and BFW differing from CW (P < 0.001) but no difference between BFW and PHFW. There were no time effect, group difference, or group × time interaction in PZC. Prevalence of zinc deficiency decreased in the BFW (from 14.1%-11.2%), PHFW (from 8.9%-2.3%), and CW (9.8%-8.8%) groups, but there was no time × treatment interaction in the prevalence of zinc deficiency (P = 0.191). Compliance with consuming the study school meals was associated with PZC (P = 0.006). DNA strand breaks were not significantly associated with PZC (n = 51; r = 0.004, P = 0.945). CONCLUSIONS: Consumption of either PHFW or BFW provided an additional ∼1.8 to 3.3 mg Zn/d, but it did not affect PZC or zinc deficiency, growth, or DNA strand breaks. This trial was registered on clinicaltrials.gov as NCT02241330 and ctri.nic.in as CTRI/2015/06/005913.

Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity.

DNA single-strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair-defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5-chloro-2'-deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA-defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.

DNA strand break levels in cryopreserved mononuclear blood cell lines measured by the alkaline comet assay: results from the hCOMET ring trial.

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern.

Dissection of DNA damage and repair pathways in live cells by femtosecond laser microirradiation and free-electron modeling.

Understanding and predicting the outcome of the interaction of light with DNA has a significant impact on the study of DNA repair and radiotherapy. We report on a combination of femtosecond pulsed laser microirradiation at different wavelengths, quantitative imaging, and numerical modeling that yields a comprehensive picture of photon-mediated and free-electron-mediated DNA damage pathways in live cells. Laser irradiation was performed under highly standardized conditions at four wavelengths between 515 nm and 1,030 nm, enabling to study two-photon photochemical and free-electron-mediated DNA damage in situ. We quantitatively assessed cyclobutane pyrimidine dimer (CPD) and γH2AX-specific immunofluorescence signals to calibrate the damage threshold dose at these wavelengths and performed a comparative analysis of the recruitment of DNA repair factors xeroderma pigmentosum complementation group C (XPC) and Nijmegen breakage syndrome 1 (Nbs1). Our results show that two-photon-induced photochemical CPD generation dominates at 515 nm, while electron-mediated damage dominates at wavelengths ≥620 nm. The recruitment analysis revealed a cross talk between nucleotide excision and homologous recombination DNA repair pathways at 515 nm. Numerical simulations predicted electron densities and electron energy spectra, which govern the yield functions of a variety of direct electron-mediated DNA damage pathways and of indirect damage by •OH radicals resulting from laser and electron interactions with water. Combining these data with information on free electron-DNA interactions gained in artificial systems, we provide a conceptual framework for the interpretation of the wavelength dependence of laser-induced DNA damage that may guide the selection of irradiation parameters in studies and applications that require the selective induction of DNA lesions.

Temperature Effects on DNA Damage during Hibernation.

AbstractDuring multiday torpor, deep-hibernating mammals maintain a hypometabolic state where heart rate and ventilation are reduced to 2%-4% of euthermic rates. It is hypothesized that this ischemia-like condition may cause DNA damage through reactive oxygen species production. The reason for intermittent rewarming (arousal) during hibernation might be to repair the accumulated DNA damage. Because increasing ambient temperatures (Ta's) shortens torpor bout duration, we hypothesize that hibernating at higher Ta's will result in a faster accumulation of genomic DNA damage. To test this, we kept 39 male and female garden dormice at a Ta of either 5°C or 10°C and obtained tissue at 1, 4, and 8 d in torpor to assess DNA damage and recruitment of DNA repair markers in splenocytes. DNA damage in splenocytes measured by comet assay was significantly higher in almost all torpor groups than in summer euthermic groups. Damage accumulates in the first days of torpor at Ta=5°C (between days 1 and 4) but not at Ta=10°C. At the higher Ta, DNA damage is high at 24 h in torpor, indicating either a faster buildup of DNA damage at higher Ta's or an incomplete repair during arousals in dormice. At 5°C, recruitment of the DNA repair protein 53BP1 paralleled the increase in DNA damage over time during torpor. In contrast, after 1 d in torpor at 10°C, DNA damage levels were high, but 53BP1 was not recruited to the nuclear DNA yet. The data suggest a potential mismatch in the DNA damage/repair dynamics during torpor at higher Ta's.

Induction of DNA strand breaks and oxidative base damages in plasmid DNA by ultra-high dose rate proton irradiation.

PURPOSE: Radiation cancer therapy with ultra-high dose rate (UHDR) exposure, so-called FLASH radiotherapy, appears to reduce normal tissue damage without compromising tumor response to therapy. The aim of this study was to clarify whether a 59.5 MeV proton beam at an UHDR of 48.6 Gy/s could effectively reduce the DNA damage of pBR322 plasmid DNA in solution compared to the conventional dose rate (CONV) of 0.057 Gy/s. MATERIALS AND METHODS: A simple system, consisting of pBR322 plasmid DNA in 1× Tris-EDTA buffer, was initially employed for proton beam exposure. We then used formamidopyrimidine-DNA glycosylase (Fpg) enzymes. which convert oxidative base damages of oxidized purines to DNA strand breaks, to quantify DNA single strand breaks (SSBs) and double strand breaks (DSBs) by agarose gel electrophoresis. RESULTS: Our findings showed that the SSB induction rate (SSB per plasmid DNA/Gy) at UHDR and the induction of Fpg enzyme sensitive sites (ESS) were significantly reduced in UHDR compared to CONV. However, there was no significant difference in DSB induction and non-DSB cluster damages. CONCLUSIONS: UHDR of a 59.5 MeV proton beam could reduce non-clustered, non-DSB damages, such as SSB and sparsely distributed ESS. However, this effect may not be significant in reducing lethal DNA damage that becomes apparent only in acute radiation effects of mammalian cells and in vivo studies.

Quantum Yield of DNA Strand Breaks under Photoexcitation of a Molecular Ruby.

Photodynamic therapy (PDT) used for treating cancer relies on the generation of highly reactive oxygen species, for example, singlet oxygen 1 O2 , by light-induced excitation of a photosensitizer (PS) in the presence of molecular oxygen, inducing DNA damage in close proximity of the PS. Although many precious metal complexes have been explored as PS for PDT and received clinical approval, only recently, the potential of photoactive complexes of non-noble metals as PS has been discovered. Using the DNA origami technology that can absolutely quantify DNA strand break cross sections, we assessed the potential of the luminescent transition metal complex [Cr(ddpd)2 ]3+ (ddpd=N,N'-dimethyl-N,N'-dipyridine-2-ylpyridine-2,6-diamine) to damage DNA in an air-saturated aqueous environment upon UV/Vis illumination. The quantum yield for strand breakage, that is, the ratio of DNA strand breaks to the number of absorbed photons, was determined to 1-4 %, indicating efficient transformation of photons into DNA strand breaks by [Cr(ddpd)2 ]3+ .

Adverse (geno)toxic effects of bisphenol A and its analogues in hepatic 3D cell model.

Bisphenol A (BPA) is one of the most widely used and versatile chemical compounds in polymer additives and epoxy resins for manufacturing a range of products for human applications. It is known as endocrine disruptor, however, there is growing evidence that it is genotoxic. Because of its adverse effects, the European Union has restricted its use to protect human health and the environment. As a result, the industry has begun developing BPA analogues, but there are not yet sufficient toxicity data to claim that they are safe. We investigated the adverse toxic effects of BPA and its analogues (BPS, BPAP, BPAF, BPFL, and BPC) with emphasis on their cytotoxic and genotoxic activities after short (24-h) and prolonged (96-h) exposure in in vitro hepatic three-dimensional cell model developed from HepG2 cells. The results showed that BPFL and BPC (formed by an additional ring system) were the most cytotoxic analogues that affected cell viability, spheroid surface area and morphology, cell proliferation, and apoptotic cell death. BPA, BPAP, and BPAF induced DNA double-strand break formation (γH2AX assay), whereas BPAF and BPC increased the percentage of p-H3-positive cells, indicating their aneugenic activity. All BPs induced DNA single-strand break formation (comet assay), with BPAP (≥0.1 µM) being the most effective and BPA and BPC the least effective (≥1 µM) under conditions applied. The results indicate that not all of the analogues studied are safer alternatives to BPA and thus more in-depth research is urgently needed to adequately evaluate the risks of BPA analogues and assess their safety for humans.

Interspecific differences in oxidative DNA damage after hydrogen peroxide exposure of sea urchin coelomocytes.

Interspecific comparison of DNA damage can provide information on the relative vulnerability of marine organisms to toxicants that induce oxidative genotoxicity. Hydrogen peroxide (H2O2) is an oxidative toxicant that causes DNA strand breaks and nucleotide oxidation and is used in multiple industries including Atlantic salmon aquaculture to treat infestations of ectoparasitic sea lice. H2O2 (up to 100 mM) can be released into the water after sea lice treatment, with potential consequences of exposure in nontarget marine organisms. The objective of the current study was to measure and compare differences in levels of H2O2-induced oxidative DNA damage in coelomocytes from Scottish sea urchins Echinus esculentus, Paracentrotus lividus, and Psammechinus miliaris. Coelomocytes were exposed to H2O2 (0-50 mM) for 10 min, cell concentration and viability were quantified, and DNA damage was measured by the fast micromethod, an alkaline unwinding DNA method, and the modified fast micromethod with nucleotide-specific enzymes. Cell viability was >92% in all exposures and did not differ from controls. Psammechinus miliaris coelomocytes had the highest oxidative DNA damage with 0.07 ± 0.01, 0.08 ± 0.01, and 0.07 ± 0.01 strand scission factors (mean ± SD) after incubation with phosphate-buffered saline, formamidopyrimidine-DNA glycosylase, and endonuclease-III, respectively, at 50 mM H2O2. Exposures to 0.5 mM H2O2 (100-fold dilution from recommended lice treatment concentration) induced oxidative DNA damage in all three species of sea urchins, suggesting interspecific differences in vulnerabilities to DNA damage and/or DNA repair mechanisms. Understanding impacts of environmental genotoxicants requires understanding species-specific susceptibilities to DNA damage, which can impact long-term stability in sea urchin populations in proximity to aquaculture farms.

DNA damage by radiation as a function of electron energy and interaction at the atomic level with Monte Carlo simulation.

In radiotherapy, X-ray or heavy ion beams target tumors to cause damage to their cell DNA. This damage is mainly induced by secondary low energy electrons. In this paper, we report the DNA molecular breaks at the atomic level as a function of electron energy and types of electron interactions using of Monte Carlo simulation. The number of DNA single and double strand breaks are compared to those from experimental results based on electron energies. In recent years, DNA atomistic models were introduced but still the simulations consider energy deposition in volumes of DNA or water equivalent material. We simulated a model of atomistic B-DNA in vacuum, forming 1122 base pairs of 30 nm in length. Each atom has been represented by a sphere whose radius equals the radius of van der Waals. We repeatedly simulated 10 million electrons for each energy from 4 eV to 500 eV and counted each interaction type with its position x, y, z in the volume of DNA. Based on the number and types of interactions at the atomic level, the number of DNA single and double strand breaks were calculated. We found that the dissociative electron attachment has the dominant effect on DNA strand breaks at energies below 10 eV compared to excitation and ionization. In addition, it is straightforward with our simulation to discriminate the strand and base breaks as a function of radiation interaction type and energy. In conclusion, the knowledge of DNA damage at the atomic level helps design direct internal therapeutic agents of cancer treatment.

Polystyrene nanoparticles: the mechanism of their genotoxicity in human peripheral blood mononuclear cells.

Plastic nanoparticles are widely spread in the biosphere, but health risk associated with their effect on the human organism has not yet been assessed. The purpose of this study was to determine the genotoxic potential of non-functionalized polystyrene nanoparticles (PS-NPs) of different diameters of 29, 44, and 72 nm in human peripheral blood mononuclear cells (PBMCs) (in vitro). To select non-cytotoxic concentrations of tested PS-NPs, we analyzed metabolic activity of PBMCs incubated with these particles in concentrations ranging from 0.001 to 1000 µg/mL. Then, PS-NPs were used in concentrations from 0.0001 to 100 µg/mL and incubated with tested cells for 24 h. Physico-chemical properties of PS-NPs in media and suspension were analyzed using dynamic light scattering (DLS), atomic force microscopy (AFM), scanning electron microscopy (SEM) and zeta potential. For the first time, we investigated the mechanism of genotoxic action of PS-NPs based on detection of single/double DNA strand-breaks and 8-oxo-2'-deoxyguanosine (8-oxodG) formation, as well as determination of oxidative modification of purines and pyrimidines and repair efficiency of DNA damage. Obtained results have shown that PS-NPs caused a decrease in PBMCs metabolic activity, increased single/double-strand break formation, oxidized purines and pyrimidines and increased 8oxodG levels. The resulting damage was completely repaired in the case of the largest PS-NPs. It was also found that extent of genotoxic changes in PBMCs depended on the size of tested particles and their ζ-potential value.

Core level ionization or excitation and Auger relaxation induce clustered DNA damage.

Ionizing radiation causes various types of DNA damage, such as single- (SSBs) and double-strand breaks (DSBs), nucleobase lesions, abasic sites (AP sites), and cross-linking between complementary strands of DNA or DNA and proteins. DSBs are among the most harmful type of DNA damage, inducing serious genetic effects such as cell lethality and mutation. Nucleobase lesions and AP sites, on the other hand, may be less deleterious and are promptly repaired by base excision repair (BER) pathways. Recently, biochemical approaches to quantify nucleobase lesions and AP sites have revealed certain types of non-strand break lesions as harmful DNA damage, called clustered DNA damage. Such clusters can retard nucleobase excision repair enzymes, and can sometimes be converted to DSBs by BER catalysis. This unique character of clustered DNA damage strongly depends on the spatial density of ionization or excitation events occurring at the track end of initial radiation or low energy secondary electrons. In particular, the photoelectric effect of elements comprising biological molecules, followed by emission of Auger electrons, are key factors in determining the future fate of each clustered damage site. This chapter describes biological studies of clustered nucleobase lesions with SSBs or AP sites, and mechanistical studies on core level excitation and Auger relaxation giving rise to clustered DNA damage.

Exploring the direct effects of microcystin-LR on DNA via using cross-technical means.

Microcystin-leucine arginine (MC-LR) is the most toxic and abundant microcystin produced by cyanobacteria. Previous studies have demonstrated that MC-LR can lead to DNA damage by increasing intracellular reactive oxygen species content to induce oxidative stress. However, the direct effect of MC-LR on DNA has not been fully described. In this study, the direct effect of MC-LR on DNA was explored by using spectral analysis and molecular biology technology. First, the fluorescent probe Bptp-R2 was developed to monitor different types of DNA and explore the direct interaction between DNA and MC-LR. The significant differences in the fluorescence of probe-plasmid DNA and probe-ds DNA at various MC-LR concentrations (0, 5, 10, 20, and 30 µmol/L) and MC-LR exposure times (0, 6, 12, and 24 h) showed that the direct interaction between DNA and MC-LR was significant (P ≤ 0.01). Gel electrophoresis demonstrated that the direct interaction between DNA and MC-LR cannot cause DNA strand breaks or change DNA configuration. Then, PCR experiments revealed that the direct interaction between DNA and MC-LR cannot affect DNA replication in a PCR system (P ≤ 0.01). This study discovered that the effects of MC-LR on DNA originate mainly from the secondary effects of MC-LR rather than from the direct interaction between DNA and MC-LR.