Daniel Vare

DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cells

DNA interstrand crosslinks (ICLs) are highly toxic lesions that covalently link both strands of DNA and distort the DNA helix. Crosslinking agents have been shown to stall DNA replication and failure to repair ICL lesions before encountered by replication forks may induce severe DNA damage. Most knowledge of the ICL repair process has been revealed from studies in bacteria and cell extracts. However, for mammalian cells the process of ICL repair is still unclear and conflicting data exist. In this study we have explored the fate of psoralen-induced ICLs during replication, by employing intact mammalian cells and novel techniques. By comparative studies distinguishing between effects by monoadducts versus ICLs, we have been able to link the block of replication to the ICLs induction. We found that the replication fork was equally blocked by ICLs in wild-type cells as in cells deficient in ERCC1/XPF and XRCC3. The formation of ICL induced double strand breaks (DSBs), detected by formation of 53PB1 foci, was equally induced in the three cell lines suggesting that these proteins are involved at a later step of the repair process. Furthermore, we found that forks blocked by ICLs were neither bypassed, restarted nor restored for several hours. We propose that this process is different from that taking place following monoadduct induction by UV-light treatment where replication bypass is taking place as an early step. Altogether our findings suggest that restoration of an ICL blocked replication fork, likely initiated by a DSB occurs relatively rapidly at a stalled fork, is followed by restoration, which seems to be a rather slow process in intact mammalian cells.

Neisseria gonorrhoeae infection causes DNA damage and affects the expression of p21, p27 and p53 in non-tumor epithelial cells

Summary The constant shedding and renewal of epithelial cells maintain the protection of epithelial barriers. Interference with the processes of host cell-cycle regulation and barrier integrity permits the bacterial pathogen Neisseria gonorrhoeae to effectively colonize and invade epithelial cells. Here, we show that a gonococcal infection causes DNA damage in human non-tumor vaginal VK2/E6E7 cells with an increase of 700 DNA strand breaks per cell per hour as detected by an alkaline DNA unwinding assay. Infected cells exhibited elevated levels of DNA double-strand breaks, as indicated by a more than 50% increase in cells expressing DNA damage-response protein 53BP1-positive foci that co-localized with phosphorylated histone H2AX (&ggr;H2AX). Furthermore, infected cells abolished their expression of the tumor protein p53 and induced an increase in the expression of cyclin-dependent kinase inhibitors p21 and p27 to 2.6-fold and 4.2-fold of controls, respectively. As shown by live-cell microscopy, flow cytometry assays, and BrdU incorporation assays, gonococcal infection slowed the host cell-cycle progression mainly by impairing progression through the G2 phase. Our findings show new cellular players that are involved in the control of the human cell cycle during gonococcal infection and the potential of bacteria to cause cellular abnormalities.

Genotoxicity of alcohol is linked to DNA replication-associated damage and homologous recombination repair

Although alcohol consumption is related to increased cancer risk, its molecular mechanism remains unclear. Here, we demonstrate that an intake of 10% alcohol for 4 weeks in rats is genotoxic due to induction of micronuclei. Acetaldehyde (AA), the first product of ethanol metabolism, is believed to be responsible for DNA damage induced by alcohol. Here, we observe that AA effectively blocks DNA replication elongation in mammalian cells, resulting in DNA double-strand breaks associated with replication. AA-induced DNA damage sites colocalize with the homologous recombination (HR) repair protein RAD51. HR measured in the hypoxhantineguaninefosforibosyltransferase (HPRT) gene is effectively induced by AA and recombination defective mammalian cells are hypersensitive to AA, clearly demonstrating that HR is essential in the repair of AA-induced DNA damage. Altogether, our data indicate that alcohol genotoxicity related to AA produces replication lesions on DNA triggering HR repair.

Quantification and repair of psoralen-induced interstrand crosslinks in human cells

Bi-functional alkylating agents that cause crosslinks are commonly used in chemotherapy. However, there is no conclusive knowledge for human cells regarding the number of induced interstrand crosslinks (ICLs) and the unhooking rate when the lesion is removed from one of the DNA strand. Using a newly developed method, we quantified the number of induced ICLs for the five furocoumarins; psoralen, 5-methoxypsoralen, 8-methoxypsoralen, tri-methoxypsoralen and angelicin. In quantitative terms, the results were in agreement with the values found by others. In kinetic studies using mammalian cells, we found that half of the psoralen-induced ICLs were unhooked within 2.5h. The rate in normal human diploid fibroblasts was found to be 20,000 ICLs/h/cell. In comparison to survival, 2500 ICLs per cell led to 50% toxicity, indicating that the unhooking of the ICLs is not the crucial step for ICL tolerance. Surprisingly, only 3500 ICLs per cell corresponded to a significant delay in the replication fork elongation. The results indicate involvements of additional pathway(s) for the delay since the effect on replication elongation could be monitored when only 10% of the replication forks encounter an ICL.

Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

Quantification of the mutagenic potency and repair of glycidol-induced DNA lesions

Glycidol (Gly) is an electrophilic low-molecular weight epoxide that is classified by IARC as probably carcinogenic to humans. Humans might be exposed to Gly from food, e.g. refined vegetable oils, where Gly has been found as a food process contaminant. It is therefore important to investigate and quantify the genotoxicity of Gly as a primary step towards cancer risk assessment of the human exposure. Here, quantification of the mutagenic potency expressed per dose (AUC: area under the concentration-time curve) of Gly has been performed in Chinese hamster ovary (CHO) cells, using the HPRT assay. The dose of Gly was estimated in the cell exposure medium by trapping Gly with a strong nucleophile, cob(I)alamin, to form stable cobalamin adducts for analysis by LC-MS/MS. Gly was stable in the exposure medium during the time for cell treatment, and thus the dose in vitro is the initial concentration×cell treatment time. Gly induced mutations in the hprt-gene at a rate of 0.08±0.01 mutations/10(5) cells/mMh. Through comparison with the effect of ionizing radiation in the same system a relative mutagenic potency of 9.5rad-eq./mMh was obtained, which could be used for comparison of genotoxicity of chemicals and between test systems and also in procedures for quantitative cancer risk assessment. Gly was shown to induce strand breaks, that were repaired by base excision repair. Furthermore, Gly-induced lesions, present during replication, were found to delay the replication fork elongation. From experiments with repair deficient cells, homologous recombination repair and the ERCC1-XPF complex were indicated to be recruited to support in the repair of the damage related to the stalled replication elongation. The type of DNA damage responsible for the mutagenic effect of Gly could not be concluded from the present study.

Adductomic Screening of Hemoglobin Adducts and Monitoring of Micronuclei in School-Age Children

Electrophilic compounds/metabolites present in humans, originating from endogenous processes or exogenous exposure, pose a risk to health effects through their reactions with nucleophilic sites in proteins and DNA, forming adducts. Adductomic approaches are developed to screen for adducts to biomacromolecules in vivo by mass spectrometry (MS), with the aim to detect adducts corresponding to unknown exposures from electrophiles. In the present study, adductomic screening was performed using blood samples from healthy children about 12 years old (n = 51). The frequencies of micronuclei (MN) in erythrocytes in peripheral blood were monitored as a measure of genotoxic effect/genotoxic exposure. The applied adductomic approach has been reported earlier by us and is based on analysis of N-terminal valine adducts in hemoglobin (Hb) by liquid chromatography tandem mass spectrometry (LC-MS/MS). High resolution MS was introduced for refined screening of previously unknown N-terminal Hb adducts. Measured adduct levels were compared with MN frequencies using multivariate data analysis. In the 51 individuals, a total of 24 adducts (whereof 12 were previously identified) were observed and their levels quantified. Relatively large interindividual variations in adduct levels were observed. The data analysis (with partial least-squares regression) showed that as much as 60% of the MN variation could be explained by the adduct levels. This study, for the first time, applies the combination of these sensitive methods to measure the internal dose of potentially genotoxic chemicals and genotoxic effects, respectively. The results indicate that this is a valuable approach for the characterization of exposure to chemical risk factors for the genotoxic effects present in individuals of the general population.

A novel micronucleus in vitro assay utilizing human hematopoietic stem cells

The induction of micronucleated reticulocytes in the bone marrow is a sensitive indicator of chromosomal damage. Therefore, the micronucleus assay in rodents is widely used in genotoxicity and carcinogenicity testing. A test system based on cultured human primary cells could potentially provide better prediction compared to animal tests, increasing patient safety while also implementing the 3Rs principle, i.e. replace, reduce and refine. Hereby, we describe the development of an in vitro micronucleus assay based on animal-free ex vivo culture of human red blood cells from hematopoietic stem cells. To validate the method, five clastogens with direct action, three clastogens requiring metabolic activation, four aneugenic and three non-genotoxic compounds have been tested. Also, different metabolic systems have been applied. Flow cytometry was used for detection and enumeration of micronuclei. Altogether, the results were in agreement with the published data and indicated that a sensitive and cost effective in vitro assay to assess genotoxicity with a potential to high-throughput screening has been developed.

In vitro genotoxicity of airborne Ni‐NP in air–liquid interface

Studies using advanced toxicological methods enabling in vitro conditions that are more realistic are currently needed for understanding the risks of pulmonary exposure to airborne nanoparticles. Owing to the carcinogenicity of certain nickel compounds, the increased production of nickel nanoparticles (Ni‐NPs) raises occupational safety concerns. The aim of this study was to investigate the genotoxicity of airborne Ni‐NPs using a recently developed air–liquid interface exposure system. The wild‐type Chinese hamster lung fibroblast cell line (V79) was used and cytotoxicity, DNA damage and mutagenicity were studied by testing colony forming efficiency, alkaline DNA unwinding and HPRT mutation assays, respectively. Additionally, co‐exposure to a PARP‐1 inhibitor was performed to test possible involvement of base excision repair (BER) in repair of Ni‐induced DNA damage. The results showed that cell viability was reduced significantly (to 45% and 46%) after 48 hours Ni‐NP exposure at concentrations of 0.15 and 0.32 μg cm−2. DNA damage was significantly increased after Ni‐NP exposure in the presence of the BER inhibitor indicating that Ni‐NP‐induced DNA damages are subsequently repaired by BER. Furthermore, there was no increased HPRT mutation frequency following Ni‐NP exposure. In conclusion, this study shows that Ni‐NP treatment of lung fibroblasts in an air–liquid interface system that mimics real‐life exposure, results in increased DNA strand breaks and reduced cellular viability. These DNA lesions were repaired with BER in an error‐free manner without resulting in mutations. This study also underlines the importance of appropriate quantification of the actual exposure concentrations during air–liquid interface exposure studies.