Miroslav Chovanec

Genotoxic effects of ethylene oxide, propylene oxide and epichlorohydrin in humans: update review (1990-2001)

Ethylene oxide (EtO), propylene oxide (PO) and epichlorohydrin (ECH) are important industrial chemicals widely used as intermediates for various synthetic products. EtO and PO are also environmental pollutants. In this review we summarize data published during the period 1990-2001 concerning both the genotoxic and carcinogenic effects of these epoxides in humans. The use of DNA and hemoglobin adducts as biomarkers of exposure and the role of polymorphism, as well as confounding factors, are discussed. We have also included recent in vitro data comprising genotoxic effects induced by EtO, PO and ECH in mammalian cells. The uncertainties regarding cancer risk estimation still persist, in spite of the large database collected.

Components of a Fanconi-like pathway control Pso2-independent DNA interstrand crosslink repair in yeast.

Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5′-3′ exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA–related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.

Rad52 has a role in the repair of sodium selenite-induced DNA damage in Saccharomyces cerevisiae

Selenium (Se) is a chemo-preventive agent that has been shown to have a protective role against cancer. The inorganic form of Se, sodium selenite (Na2SeO3), has frequently been included in various chemo-prevention studies, and this commercially available form of Se is used as dietary supplement by the public. Because high doses of this Se compound can be toxic, the underlying molecular mechanisms of sodium selenite toxicity need to be elucidated. Recently, we have reported that sodium selenite is acting as an oxidizing agent in the budding yeast Saccharomyces cerevisiae, producing oxidative damage to DNA. This pro-oxidative activity of sodium selenite likely accounted for the observed DNA double-strand breaks (DSB) and yeast cell death. In this study we determine the genetic factors that are responsible for repair of sodium selenite-induced DSB. We report that the Rad52 protein is indispensable for repairing sodium selenite-induced DSB, suggesting a fundamental role of homologous recombination (HR) in this repair process. These results provide the first evidence that HR may have a fundamental role in the repair of sodium selenite-induced toxic DNA lesions.

Intracellular Diagnostics: Hunting for the Mode of Action of Redox-Modulating Selenium Compounds in Selected Model Systems

Redox-modulating compounds derived from natural sources, such as redox active secondary metabolites, are currently of considerable interest in the field of chemoprevention, drug and phytoprotectant development. Unfortunately, the exact and occasionally even selective activity of such products, and the underlying (bio-)chemical causes thereof, are often only poorly understood. A combination of the nematode- and yeast-based assays provides a powerful platform to investigate a possible biological activity of a new compound and also to explore the “redox link” which may exist between its activity on the one side and its chemistry on the other. Here, we will demonstrate the usefulness of this platform for screening several selenium and tellurium compounds for their activity and action. We will also show how the nematode-based assay can be used to obtain information on compound uptake and distribution inside a multicellular organism, whilst the yeast-based system can be employed to explore possible intracellular mechanisms via chemogenetic screening and intracellular diagnostics. Whilst none of these simple and easy-to-use assays can ultimately substitute for in-depth studies in human cells and animals, these methods nonetheless provide a first glimpse on the possible biological activities of new compounds and offer direction for more complicated future investigations. They may also uncover some rather unpleasant biochemical actions of certain compounds, such as the ability of the trace element supplement selenite to induce DNA strand breaks.

A prototypical Fanconi anemia pathway in lower eukaryotes

DNA interstrand cross-links (ICLs) present a major challenge to cells, preventing separation of the two strands of duplex DNA and blocking major chromosome transactions, including transcription and replication. Due to the complexity of removing this form of DNA damage, no single DNA repair pathway has been shown to be capable of eradicating ICLs. In eukaryotes, ICL repair is a complex process, principally because several repair pathways compete for ICL repair intermediates in a strictly cell cycle-dependent manner. Yeast cells require a combination of nucleotide excision repair, homologous recombination repair and postreplication repair/translesion DNA synthesis to remove ICLs. There are also a number of additional ICL repair factors originally identified in the budding yeast Saccharomyces cerevisiae, called Pso1 though 10, of which Pso2 has an apparently dedicated role in ICL repair. Mammalian cells respond to ICLs by a complex network guided by factors mutated in the inherited cancer-prone disorder Fanconi anemia (FA). Although enormous progress has been made over recent years in identifying and characterizing FA factors as well as in elucidating certain aspects of the biology of FA, the mechanistic details of the ICL repair defects in FA patients remain unknown. Dissection of the FA DNA damage response pathway has, in part, been limited by the absence of FA-like pathways in highly tractable model organisms, such as yeast. Although S. cerevisiae possesses putative homologs of the FA factors FANCM, FANCJ and FANCP (Mph1, Chl1 and Slx4, respectively) as well as of the FANCM-associated proteins MHF1 and MHF2 (Mhf1 and Mhf2), the corresponding mutants display no significant increase in sensitivity to ICLs. Nevertheless, we and others have recently shown that these FA homologs, along with several other factors, control an ICL repair pathway, which has an overlapping or redundant role with a Pso2-controlled pathway. This pathway acts in S-phase and serves to prevent ICL-stalled replication forks from collapsing into DNA double-strand breaks.

Rejoining of DNA strand breaks induced by propylene oxide and epichlorohydrin in human diploid fibroblasts

The repair kinetics of DNA single‐ and double‐strand breaks (SSBs, DSBs) induced with two carcinogenic epoxides, propylene oxide (PO) and epichlorohydrin (ECH), was studied in human diploid fibroblasts. The methods used were: alkaline DNA unwinding (ADU), the comet assay, and pulsed field gel electrophoresis (PFGE). About 70% of SSBs, measured by ADU, were rejoined after the treatment with 5 mMh and 10 mMh of PO within 20 hr, and the half‐life was estimated to be ∼15 hr. On the other hand, effective rejoining of SSBs after ECH treatment was observed only at a dose of 1 mMh (a half‐life of ∼15 hr), whereas after 2 mMh treatment, only 26% of SSBs could be rejoined within 20 hr. Furthermore, the use of the comet assay demonstrated that DNA strand breaks were effectively rejoined after PO and ECH treatment at doses of 5–10 mMh and 0.5–1 mMh, respectively. About 76% and 83% of DSBs induced by 5 and 10 mMh of PO, respectively, were rejoined within 4 hr after the treatment (a half‐life of ∼2.5 hr), with little further repair thereafter. At lower dose of ECH (1 mMh) a half‐life for DSBs rejoining was estimated to be ∼2 hr; however, only 29% of DSBs were rejoined within 2 hr at the higher dose of 2 mMh. After 18 hr, the rejoining following treatment with a lower dose was negligible. At a higher dose, a rapid accumulation of DSBs was observed, probably as the result of cell death and DNA degradation. The results demonstrate the capability of human diploid fibroblasts to repair DNA SSBs and DSBs at low‐to‐moderate doses of the epoxides. A weak capacity to rejoin DNA strand breaks induced by higher doses of ECH may be a consequence of its higher DNA alkylation activity and approximately 10 times higher toxicity compared to PO. Environ. Mol. Mutagen. 32:223–228, 1998

Increased DNA double strand breakage is responsible for sensitivity of the pso3-1 mutant of Saccharomyces cerevisiae to hydrogen peroxide.

Escherichia coli endonuclease III (endo III) is the key repair enzyme essential for removal of oxidized pyrimidines and abasic sites. Although two homologues of endo III, Ntgl and Ntg2, were found in Saccharomyces cerevisiae, they do not significantly contribute to repair of oxidative DNA damage in vivo. This suggests that an additional activity(ies) or a regulatory pathway(s) involved in cellular response to oxidative DNA damage may exist in yeast. The pso3-1 mutant of S. cerevisiae was previously shown to be specifically sensitive to toxic effects of hydrogen peroxide (H2O2) and paraquat. Here, we show that increased DNA double strand breakage is very likely the basis of sensitivity of the pso3-1 mutant cells to H2O2. Our results, thus, indicate an involvement of the Pso3 protein in protection of yeast cells from oxidative stress presumably through its ability to prevent DNA double strand breakage. Furthermore, complementation of the repair defects of the pso3-1 mutant cells by E. coli endo III has been examined. It has been found that expression of the nth gene in the pso3-1 mutant cells recovers survival, decreases mutability and protects yeast genomic DNA from breakage following H2O2 treatment. This might suggest some degree of functional similarity between Pso3 and Nth.

Regulation of non-homologous end joining via post-translational modifications of components of the ligation step

DNA double-strand breaks are the most serious type of DNA damage and non-homologous end joining (NHEJ) is an important pathway for their repair. In Saccharomyces cerevisiae, three complexes mediate the canonical NHEJ pathway, Ku (Ku70/Ku80), MRX (Mre11/Rad50/Xrs2) and DNA ligase IV (Dnl4/Lif1). Mammalian NHEJ is more complex, primarily as a consequence of the fact that more factors are involved in the process, and also because higher chromatin organization and more complex regulatory networks exist in mammals. In addition, a stronger interconnection between the NHEJ and DNA damage response (DDR) pathways seems to occur in mammals compared to yeast. DDR employs multiple post-translational modifications (PTMs) of the target proteins and mutual crosstalk among them to ensure highly efficient down-stream effects. Checkpoint-mediated phosphorylation is the best understood PTM that regulates DDR, although recently SUMOylation has also been shown to be involved. Both phosphorylation and SUMOylation affect components of NHEJ. In this review, we discuss a role of these two PTMs in regulation of NHEJ via targeting the components of the ligation step.

The Escherichia coli RecA protein complements recombination defective phenotype of the Saccharomyces cerevisiae rad52 mutant cells.

The Saccharomyces cerevisiae rad52 mutants are sensitive to many DNA damaging agents, mainly to those that induce DNA double‐strand breaks (DSBs). In the yeast, DSBs are repaired primarily by homologous recombination (HR). Since almost all HR events are significantly reduced in the rad52 mutant cells, the Rad52 protein is believed to be a key component of HR in S. cerevisiae. Similarly to the S. cerevisiae Rad52 protein, RecA is the main HR protein in Escherichia coli. To address the question of whether the E. coli RecA protein can rescue HR defective phenotype of the rad52 mutants of S. cerevisiae, the recA gene was introduced into the wild‐type and rad52 mutant cells. Cell survival and DSBs induction and repair were studied in the RecA‐expressing wild‐type and rad52 mutant cells after exposure to ionizing radiation (IR) and methyl methanesulphonate (MMS). Here, we show that expression of the E. coli RecA protein partially complemented sensitivity and fully complemented DSB repair defect of the rad52 mutant cells after exposure to IR and MMS. We suggest that in the absence of Rad52, when all endogenous HR mechanisms are knocked out in S. cerevisiae, the heterologous E. coli RecA protein itself presumably takes over the broken DNA. Copyright

Down-regulation of traditional oncomiRs in plasma of breast cancer patients

Deregulated expression of microRNAs has the oncogenic or tumor suppressor function in cancer. Since miRNAs in plasma are highly stable, their quantification could contribute to more precise cancer diagnosis, prognosis and therapy prediction. We have quantified expression of seven oncomiRs, namely miR-17/92 cluster (miR-17, miR-18a, miR-19a and miR-20a), miR-21, miR-27a and miR-155, in plasma of 137 breast cancer (BC) patients. We detected down-regulation of six miRNAs in patients with invasive BC compared to controls; however, only miR-20a and miR-27a down-regulations were statistically significant. Comparing miRNA expression between early and advanced stages of BC, we observed statistically significant decrease of miR-17 and miR-19a. We identified down-regulation of miR-17 and miR-20a in patients with clinical parameters of advanced BC (lymph node metastasis, tumor grade 3, circulating tumor cells, higher Ki-67-related proliferation, hormone receptor negativity and HER2 amplification), when compared to controls. Moreover, decreased level of miR-17 was found from low to high grade. Therefore, miR-17 could represent an indicator of advanced BC. Down-regulated miR-27a expression levels were observed in all clinical categories regardless of tumor progression. Hence, miR-27a could be used as a potential diagnostic marker for BC. Our data indicates that any changes in miRNA expression levels in BC patients in comparison to controls could be highly useful for cancer-associated pathology discrimination. Moreover, dynamics of miRNA expression changes could be used for BC progression monitoring.