Assessment of the genotoxic potential of tetrachlorvinphos insecticide by cytokinesis-block micronucleus and sister chromatid exchange assays

Tetrachlorvinphos is an organophosphate that is classified as a carcinogen in humans by several authorities. Due to very limited data regarding the genotoxic potential, we aimed to comprehensively investigate in vitro genotoxic potential of tetrachlorvinphos. We performed our study by applying the cytokinesis-block micronucleus cytome and sister chromatid exchange (SCE) assays to human peripheral blood lymphocytes. We evaluated micronucleus (MN) and SCE frequencies and cytokinesis-block proliferation index in both exposed and non-exposed lymphocytes. We also calculated the chromosomal instability level in response to exposure by combining the results of MN and SCE. We found that MN frequency did not increase with exposure to tetrachlorvinphos (0–50 µg/ml). In contrast, we observed that SCE frequencies significantly increased with exposure to ≥5 µg/ml tetrachlorvinphos. Furthermore, exposure to tetrachlorvinphos at concentrations of 50 µg/ml induced a significant increase in chromosomal instability level ( p < 0.05). Cytokinesis-block proliferation index level did not significantly decrease in response to tetrachlorvinphos exposure. Our findings reveal that tetrachlorvinphos resulted in different DNA damages that were measured by two assays. Furthermore, our findings suggested that exposure to tetrachlorvinphos increased chromosomal instability that is a hallmark of many malignancies. We conclude that although tetrachlorvinphos does not significantly increase the MN level, the significant increase of both SCE and CIN frequencies indicates the genotoxic potential of tetrachlorvinphos in human peripheral lymphocytes. Additionally, tetrachlorvinphos is not cytotoxic in the range of tested concentrations.

TRIM28 inhibits alternative lengthening of telomere phenotypes by protecting SETDB1 from degradation

Background About 10–15% of tumor cells extend telomeres through the alternative lengthening of telomeres (ALT) mechanism, which is a recombination-dependent replication pathway. It is generally believed that ALT cells are related to the chromatin modification of telomeres. However, the mechanism of ALT needs to be further explored. Results Here we found that TRIM28/KAP1 is preferentially located on the telomeres of ALT cells and interacts with telomeric shelterin/telosome complex. Knocking down TRIM28 in ALT cells delayed cell growth, decreased the level of C-circle which is one kind of extrachromosomal circular telomeric DNA, increased the frequency of ALT-associated promyelocytic leukemia bodies (APBs), led to telomere prolongation and increased the telomere sister chromatid exchange in ALT cells. Mechanistically, TRIM28 protects telomere histone methyltransferase SETDB1 from degradation, thus maintaining the H3K9me3 heterochromatin state of telomere DNA. Conclusions Our work provides a model that TRIM28 inhibits alternative lengthening of telomere phenotypes by protecting SETDB1 from degradation. In general, our results reveal the mechanism of telomere heterochromatin maintenance and its effect on ALT, and TRIM28 may serve as a target for the treatment of ALT tumor cells.

Caenorhabditis elegans RMI2 functional homolog-2 (RMIF-2) and RMI1 (RMH-1) have both overlapping and distinct meiotic functions within the BTR complex

Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange. Here, the BTR complex, consisting of the Bloom helicase (HIM-6 in worms), topoisomerase 3 (TOP-3), and the RMI1 (RMH-1 and RMH-2) and RMI2 scaffolding proteins, is essential for dismantling joint DNA molecules to form non-crossovers (NCOs) via decatenation. In contrast, in meiosis COs are essential for accurate chromosome segregation and the BTR complex plays distinct roles in CO and NCO generation at different steps in meiotic recombination. RMI2 stabilizes the RMI1 scaffolding protein, and lack of RMI2 in mitosis leads to elevated sister chromatid exchange, as observed upon RMI1 knockdown. However, much less is known about the involvement of RMI2 in meiotic recombination. So far, RMI2 homologs have been found in vertebrates and plants, but not in lower organisms such as Drosophila, yeast, or worms. We report the identification of the Caenorhabditis elegans functional homolog of RMI2, which we named RMIF-2. The protein shows a dynamic localization pattern to recombination foci during meiotic prophase I and concentration into recombination foci is mutually dependent on other BTR complex proteins. Comparative analysis of the rmif-2 and rmh-1 phenotypes revealed numerous commonalities, including in regulating CO formation and directing COs toward chromosome arms. Surprisingly, the prevalence of heterologous recombination was several fold lower in the rmif-2 mutant, suggesting that RMIF-2 may be dispensable or less strictly required for some BTR complex-mediated activities during meiosis.

In Vitro Genotoxicity and Molecular Docking Study of Ellagic Acid

Background: Ellagic Acid (EA) is a polyphenolic compound that is classified in the natural antioxidants group. Polyphenolic compounds that exert antioxidant activity possess particular importance for scientists, food producers and consumers due to their positive effects on human health. However, despite considerable evidence that EA shows antigenotoxic activity by binding to DNA, there is no systematic genotoxicity study of this substance, which can covalently bind to DNA. This study aims to reveal the possible genotoxic activity of EA using widely accepted assays for the assessment of DNA clastogenic activity: sister chromatid exchange, chromosome aberration, micronucleus and comet assays as well as to predict the interactions among EA and DNA through molecular docking. Methods: Different assays were carried out to identify the clastogenic activity of EA on human lymphocyte DNA using Sister Chromatid Exchange (SCE), Chromosome Aberration (CA), Micronucleus (MN) and single-cell gel electrophoresis (SCGE/comet) assays. For this aim, human peripheral blood lymphocytes were treated with EA (60, 80 and 100 μg/ml) for 24 and 48 hrs in the SCE, CA and MN assays and for 1 hr in the comet assay. Furthermore, molecular docking experiments were also performed to calculate the binding energy of EA on human B-DNA structure (B-DNA dodecamer) as well as to predict noncovalent interactions among these macromolecules. Results: At the concentrations and treatment times (24- or 48-hr) tested, EA did not induce either SCE or Chromosome Aberrations (CAs) as compared to the negative and solvent controls. Although EA slightly increased the percentage of Micronucleated Binuclear (%MNBN) cells as well as the percentage of Micronucleus (%MN) in 24 or 48-hr treatment periods at all concentrations, this increase was not statistically significant as compared to both controls. The effect of EA on DNA replication (nuclear division) was determined by the Proliferation Index (PI), the Nuclear Division Index (NDI) and the Mitotic Index (MI). No statistically significant differences were observed in the PI or NDI in 24- or 48-hr treatment periods in human lymphocyte cultures treated with EA at various concentrations. EA generally had no significant effect on the MI, as observed with the PI and NDI. Discussion: Although the concentrations of 60 and 80 μg/mL at a 24-hr treatment period and the concentrations of 60 μg/mL and 100 μg/mL at 48-hr treatment period generally decreased the MI, those decreases were not statistically significant when compared to negative and solvent controls. Moreover, none of the concentrations of EA tested in this study were able to increase DNA damage determined by the tail DNA length, %DNA in tail and tail moment parameters in the comet assay. Although the amount of DNA damage in the comet assay decreased with increasing concentrations of EA, this decrease was not statistically significant as compared to both controls. However, molecular docking experiments interestingly showed that the binding free energy of EA with B-DNA was -7.84 kcal/mol-1, indicating a strong interaction between the two molecules. Conclusion : Although the findings of our study show that EA does not have genotoxic potential in human chromosomes, molecular docking experiments revealed strong hydrogen bonding between EA and B-DNA molecules. Therefore, it has been proposed that the prevailing information suggesting that the molecules that bind to DNA cause genotoxic effects should be reconsidered from a wider perspective.

Environmental Monitoring of PAHs Exposure, Biomarkers and Vital Status in Coke Oven Workers

A follow-up study of a cohort of workers from a coke plant compared with a control group from the same industrial area was conducted in 2019. The recruitment and environmental and biomarker measurements were performed during 1993/1994. The environmental concentrations of polycyclic aromatic hydrocarbons (PAH), B(a)P, pyrene and nitro-PAH were measured. Personal data were collected via an individual semi-structured questionnaire by a trained physician. All biomarkers were measured after a specific blood drawing for every test. Significant risks (ORs) were observed for nitro-PAH (≥0.12 µg/m3) [OR = 7.96 (1.01–62.82)], urinary 1-hydroxypyrene (1-OHpy) (≥0.99 µmoles/moles of creatinine) [OR = 11.71 (1.47–92.90)], PAH DNA adducts (P32) (≥2.69 adducts/108 nucleotides) [OR = 5.46 (1.17–25.58)], total nitro-PAH hemoglobin adducts (≥161.68 fg/µg of Hb) [OR = 5.92 (1.26–27.86)], sister chromatid exchange (SCE) with TCR (≥377.84 SCE/cell chromosomes) [OR = 13.06 (3.95–93.10)], sister chromatid exchange with T (≥394.72 total SCE) [OR = 13.06 (3.95–93.10)], and sister chromatid exchange with X (≥8.19 mean SCE) [OR = 13.06 (3.95–93.10)]. Significant risk of death for all causes and chromosomal aberrations (48 h) (OR = 7.19 [1.19–43.44]) or micronuclei in culture at 48 h (OR = 3.86 [1.04–14.38]) were also found.