In vitro effects of Sunset Yellow on Chromosomal Damage and Sister Chromatid Exchanges in Human Peripheral Lymphocytes

Sunset Yellow (SY) is an organic azo dye that is used extensively as a coloring agent in many industries, such as cosmetics, pharmaceuticals ,and foodstuffs. Many studies have conflicting results about the genotoxicity effect of SY. Thus, the purpose of this study was to provide additional data concerning SY genotoxicity in human lymphocytes by using chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) assay. Four concentrations of Sunset Yellow (1, 5, 20 ,and 50 mg/ml) were used on human lymphocyte cultures. Positive and negative controls were mitomycin C and distilled water, respectively. Compared to the control, SY caused a significant increase in CAs and SCEs frequencies at all concentrations. A total of five types of CAs were observed, such as gaps, fragments, RCF, stickiness,and polyploidy. According to the present results, high concentrations of SY are genotoxic in vitro to cultured human lymphocytes. To determine its full genotoxicity potential, SY should be tested in other test systems.

A Systematic Review of Studies on Genotoxicity and Related Biomarkers in Populations Exposed to Pesticides in Mexico

In agricultural activities, pest control is essential, and the most effective method is the use of chemical agents that also represent an important source of exposure to potentially toxic compounds. Pesticides constitute a heterogeneous group of compounds designed specifically to control different pests. Besides measuring their levels or that of their metabolites in air, plasma, serum, blood, urine, etc., some studies reported increased DNA damage levels after occupational or environmental pesticides exposure, evidenced by several cytogenetic biomarkers such as chromosomal aberrations (CA), sister chromatid exchanges (SCE), micronuclei frequency (MN) together with other nuclear abnormalities (NA), alkaline comet assay, but also changes in oxidative stress parameters and miRNA levels. Single or combined, these techniques have also been used in genotoxic biomonitoring studies of workers occupationally exposed to pesticides in Mexico. Despite being a country with great agricultural activity and reported excessive pesticide use, genotoxic studies have been relatively few and, in some cases, contradictory. A review was made of the studies available (published until the end of 2020 on PubMed, Web of Science, Redalyc and Scielo, both in English and Spanish) in the scientific literature that evaluated occupational exposure of human samples to pesticides assessed with DNA damage and related biomarkers in Mexico.

Sister chromatid exchanges induced by perturbed replication are formed independently of homologous recombination factors

SummarySister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, upon generation of irradiation-induced DNA breaks, SCE induction was compromised in cells deficient for canonical HR factors BRCA1, BRCA2 and RAD51. Contrarily, replication-blocking agents, including PARP inhibitors, induced SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs were enriched at common fragile sites (CFSs), and were accompanied by post-replicative single-stranded DNA (ssDNA) gaps. Moreover, PARP inhibitor-induced replication lesions were transmitted into mitosis, suggesting that SCEs originate from mitotic processing of under-replicated DNA. We found that DNA polymerase theta (POLQ) was recruited to mitotic DNA lesions, and loss of POLQ resulted in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Combined, our data show that PARP inhibition generates under-replicated DNA, which is transferred into mitosis and processed into SCEs, independently of canonical HR factors.

Μελέτη του υδατοδιαλυτού κλάσματος των βαρέων μετάλλων και της συσχέτισής του με τη γονιδιοτοξικότητα των αναπνεύσιμων αιωρούμενων σωματιδίων του αέρα εσωτερικών χώρων

Δείγματα αιωρούμενων σωματιδίων ΡΜ2.5 (σωματίδια με αεροδυναμική διάμετρο ≤2.5 μm) που συλλέχθηκαν από τον εσωτερικό αέρα 20 επαγγελματικών χώρων στην περιοχή της Αλεξανδρούπολης, υποβλήθηκαν σε υδατική εκχύλιση από την οποία προέκυψαν τρία διαδοχικά υδατοδιαλυτά κλάσματα: το αρχικό υδατοδιαλυτό κλάσμα διηθημένο από μεμβράνη πόρων <0,45μm (WSA), το υδατοδιαλυτό κλάσμα διηθημένο από μεμβράνη πόρων <0,22 μm (WSΒ) και το υδατοδιαλυτό κλάσμα μετά από συμπλοκοποίηση με ρητίνη Chelex (WSC). Στα τρία υδατοδιαλυτά κλάσματα προσδιορίστηκαν τα βαρέα μέταλλα Cu, Pb, Mn, Ni, Co, Zn, Cr και Cd με φασματοφωτομετρία ατομικής απορρόφησης και τεχνική φούρνου γραφίτη. Παράλληλα, έγινε προσδιορισμός της γονοτοξικότητας των υδατικών εκχυλισμάτων, εφαρμόζοντας τη μέθοδο των χρωματιδιακών ανταλλαγών (Sister Chromatid Exchanges, SCEs), η οποία εφαρμόστηκε σε ανθρώπινα λεμφοκύτταρα υγιών εθελοντών δοτών. Οι τρεις δείκτες που υπολογίστηκαν για κάθε εκχύλισμα είναι ο δείκτης SCEs/μετάφαση, ο Δείκτης Ρυθμού Πολλαπλασιασμού (ΔΡΠ) και ο Μιτωτικός Δείκτης (ΜΔ). Οι συγκεντρώσεις των αιωρούμενων σωματιδίων PM2.5 και οι συγκεντρώσεις των υδατοδιαλυτών βαρέων μετάλλων παρουσίασαν σημαντική διακύμανση μεταξύ των 20 εργασιακών χώρων, ακόμα και μεταξύ χώρων του ίδιου τύπου. Οι συγκεντρώσεις των PM2.5 κυμάνθηκαν από 11,5 μg m-3 μέχρι 276 μg m-3, ενώ οι συγκεντρώσεις των βαρέων μετάλλων στο αρχικό υδατοδιαλυτό κλάσμα κυμάνθηκαν από 0,67± 2,52 ng m-3 για το Co μέχρι 27,8±19,1 ng m-3 για το Ni.Η επεξεργασία με τη ρητίνη Chelex οδήγησε στη συμπλοκοποίηση και κατακράτηση σημαντικού μέρους του μεταλλικού περιεχομένου του αρχικού υδατοδιαλυτού κλάσματος. Τα μεγαλύτερα ποσοστά (53-67% των αντίστοιχων συγκεντρώσεων των μετάλλων στο αρχικό υδατοδιαλυτό κλάσμα) παρατηρήθηκαν για τα μέταλλα Cd, Mn, Cu και Ni, ενώ για τα μέταλλα Pb, Cr, Co και Zn τα ποσοστά αυτά βρέθηκαν χαμηλότερα (34-42% των αντίστοιχων συγκεντρώσεων των μετάλλων στο αρχικό υδατοδιαλυτό κλάσμα). Με βάση τις συγκεντρώσεις των υδατοδιαλυτών βαρέων μετάλλων στο αρχικό υδατοδιαλυτό κλάσμα WSA και στο συμπλοκοποιημένο κλάσμα WSB-WSC, το οποίο μπορεί να θεωρηθεί ότι εκφράζει το βιοπροσβάσιμο κλάσμα των βαρέων μετάλλων, έγινε εκτίμηση του κινδύνου για την υγεία λόγω εισπνοής ΡΜ2.5 με τον υπολογισμό δύο δεικτών κινδύνου: του κινδύνου μη-καρκινογένεσης και του κινδύνου καρκινογένεσης. Ο συνολικός δείκτης κινδύνου μη-καρκινογένεσης (ΣΤHQ), υπολογιζόμενος για τα μέταλλα Mn, Cd, Ni και Cr (VI), βρέθηκε να υπερβαίνει το αποδεκτό όριο του 1,0 σε όλους τους εργασιακούς χώρους για το αρχικό υδατοδιαλυτό κλάσμα και στους περισσότερους χώρους για το βιοπροσβάσιμο κλάσμα. Ο συνολικός δείκτης κινδύνου καρκινογένεσης (ΣΤR), υπολογιζόμενος για τα μέταλλα Ni, Cd, Pb και Cr (VI), βρέθηκε μικρότερος του επιπέδου αναφοράς 1x10-6 και για τα δύο κλάσματα σε όλους τους χώρους. Σε ό,τι αφορά στην γονοτοξικότητα των υδατικών εκχυλισμάτων των σωματιδίων ΡΜ2.5, τα αποτελέσματα έδειξαν στατιστικά σημαντική γονοτοξικότητα σε όλα τα υδατοδιαλυτά κλάσματα σε σχέση με το control, που ακολουθεί τη σειρά: WSA>WSΒ>WSC. Η γονοτοξικότητα των υδατοδιαλυτών PM2.5 υπολογίσθηκε (α) ως γονοτοξικότητα κανονικοποιημένη ως προς τη μάζα των σωματιδίων (SCEs/mg PM2.5) και (β) γονοτοξικότητα κανονικοποιημένη ως προς τον όγκο του αέρα (SCEs/m3 air). Και στις δύο περιπτώσεις, η γονοτοξικότητα βρέθηκε να εξαρτάται σημαντικά από τις συγκεντρώσεις των μετάλλων Zn και Pb.

Double Strand Breaks (DSBs) as Indicators of Genomic Instability in PATRR-mediated Translocations

Genomic instability contributes to a variety of potentially damaging conditions, including DNA-based rearrangements. Breakage in the form of double strand breaks (DSBs) increases the likelihood of DNA damage, mutations, and translocations. Certain human DNA regions are known to be involved in recurrent translocations, such as the palindrome-mediated rearrangements that have been identified at the breakpoints of several recurrent constitutional translocations: t(11;22)(q23;q11), t(17;22)(q11;q11), and t(8;22) (q24;q11). These breakpoints occur at the center of palindromic AT-rich repeats (PATRRs), which suggests that the structure of the DNA may play a contributory role, potentially through the formation of secondary cruciform structures. The current study analyzed the DSB propensity of these PATRR regions in both lymphoblastoid (mitotic) and spermatogenic cells (meiotic). Initial results found an increased association of sister chromatid exchanges (SCEs) at PATRR regions in experiments that used SCEs to assay DSBs, combining SCE staining with fluorescence in situ hybridization (FISH). Additional experiments used chromatin immunoprecipitation (ChIP) with antibodies for either markers of DSBs or proteins involved in DSB repair along with quantitative polymerase chain reaction (qPCR) to quantify the frequency of DSBs occurring at PATRR regions. The results indicate an increased rate of DSBs at PATRR regions. Additional ChIP experiments with the cruciform binding 2D3 antibody indicate an increased rate of cruciform structures at PATRR regions in both mitotic and meiotic samples. Overall, these experiments demonstrate an elevated rate of DSBs at PATRR regions, an indication that the structure of PATRR containing DNA may lead to increased breakage in multiple cellular environments.

Meiotic sister chromatid exchanges are rare in C. elegans

Sexual reproduction shuffles the parental genomes to generate new genetic combinations. To achieve that, the genome is subjected to numerous double-strand breaks, the repair of which involves two crucial decisions: repair pathway and repair template. Use of crossover pathways with the homologous chromosome as template exchanges genetic information and directs chromosome segregation. Crossover repair, however, can compromise the integrity of the repair template and is therefore tightly regulated. The extent to which crossover pathways are used during sister-directed repair is unclear, because the identical sister chromatids are difficult to distinguish. Nonetheless, indirect assays have led to the suggestion that inter-sister crossovers, or sister chromatid exchanges (SCEs), are quite common. Here we devised a technique to directly score physiological SCEs in the C. elegans germline using selective sister chromatid labeling with the thymidine analog 5-ethynyl-2’-deoxyuridine (EdU). Surprisingly, we find SCEs to be rare in meiosis, accounting for <2% of repair events. SCEs remain rare even when the homologous chromosome is unavailable, indicating that almost all sister-directed repair is channeled into noncrossover pathways. We identify two mechanisms that limit SCEs. First, sister-directed repair intermediates are efficiently inhibited by the RecQ helicase BLMHIM-6. Second, the Synaptonemal Complex–a conserved interface that promotes crossover repair– localizes between the homologous chromosomes and not the sister chromatids. Our data suggest that in C. elegans crossover pathways are only used to generate the single necessary link between the homologous chromosomes. Almost all other breaks, regardless of which repair template they use, are repaired by noncrossover pathways.

Genotoxicity test methods - a tool for DNA and chromosome damage biomonitoring

Nowadays, life is highly influenced by intense growth of various industries, high levels of pollution, and other environmental factors with harmful effects on human health. Therefore, cytogenetic monitoring is essential for detection of changes in the structure of chromosomes, which occur because of the effects of various genotoxic agents. In this review, we shall apprize the theoretical and experimental aspects of several tests for assessment of genotoxicity in humans such as Micronucleus assay, Comet assay, Chromosomal aberrations assessment and Sister chromatid exchanges analysis. These methods are accepted by the World Health Organization as standard tests for genotoxicological screening in humans. The methods are sensitive and confirm the cellular genotoxic effects of various genotoxicants.

The human Shu complex functions with PDS5B and SPIDR to promote homologous recombination

RAD51 plays a central role in homologous recombination during double-strand break repair and in replication fork dynamics. Misregulation of RAD51 is associated with genetic instability and cancer. RAD51 is regulated by many accessory proteins including the highly conserved Shu complex. Here, we report the function of the human Shu complex during replication to regulate RAD51 recruitment to DNA repair foci and, secondly, during replication fork restart following replication fork stalling. Deletion of the Shu complex members, SWS1 and SWSAP1, using CRISPR/Cas9, renders cells specifically sensitive to the replication fork stalling and collapse caused by methyl methanesulfonate and mitomycin C exposure, a delayed and reduced RAD51 response, and fewer sister chromatid exchanges. Our additional analysis identified SPIDR and PDS5B as novel Shu complex interacting partners and genetically function in the same pathway upon DNA damage. Collectively, our study uncovers a protein complex, which consists of SWS1, SWSAP1, SPIDR and PDS5B, involved in DNA repair and provides insight into Shu complex function and composition.