AOP report: Development of an adverse outcome pathway for oxidative DNA damage leading to mutations and chromosomal aberrations.

The Genetic Toxicology Technical Committee (GTTC) of the Health and Environmental Sciences Institute (HESI) is developing adverse outcome pathways (AOPs) that describe modes of action leading to potentially heritable genomic damage. The goal was to enhance the use of mechanistic information in genotoxicity assessment by building empirical support for the relationships between relevant molecular initiating events (MIEs) and regulatory endpoints in genetic toxicology. Herein, we present an AOP network that links oxidative DNA damage to two adverse outcomes (AOs): mutations and chromosomal aberrations. We collected empirical evidence from the literature to evaluate the key event relationships between the MIE and the AOs, and assessed the weight of evidence using the modified Bradford-Hill criteria for causality. Oxidative DNA damage is constantly induced and repaired in cells given the ubiquitous presence of reactive oxygen species and free radicals. However, xenobiotic exposures may increase damage above baseline levels through a variety of mechanisms and overwhelm DNA repair and endogenous antioxidant capacity. Unrepaired oxidative DNA base damage can lead to base substitutions during replication and, along with repair intermediates, can also cause DNA strand breaks that can lead to mutations and chromosomal aberrations if not repaired adequately. This AOP network identifies knowledge gaps that could be filled by targeted studies designed to better define the quantitative relationships between key events, which could be leveraged for quantitative chemical safety assessment. We anticipate that this AOP network will provide the building blocks for additional genotoxicity-associated AOPs and aid in designing novel integrated testing approaches for genotoxicity.

Gene expression changes induced by the human carcinogen aristolochic acid I in renal and hepatic tissue of mice.

Aristolochic acid (AA) is the causative agent of urothelial tumors associated with AA nephropathy and is also implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. These tumors contain AA-characteristic TP53 mutations. We examined gene expression changes in Hupki (human TP53 knock-in) mice after treatment with aristolochic acid I (AAI) by gavage (5 mg/kg body weight). After 3, 12 and 21 days of treatment gene expression profiles were investigated using Agilent Whole Mouse 44K Genome Oligo Array. Expression profiles were significantly altered by AAI treatment in both target (kidney) and nontarget (liver) tissue. Renal pathology and DNA adduct analysis confirmed kidney as the target tissue of AAI-induced toxicity. Gene ontology for functional analysis revealed that processes related to apoptosis, cell cycle, stress response, immune system, inflammatory response and kidney development were altered in kidney. Canonical pathway analysis indicated Nfκb, aryl hydrocarbon receptor, Tp53 and cell cycle signaling as the most important pathways modulated in kidney. Expression of Nfκb1 and other Nfκb-target genes was confirmed by quantitative real-time PCR (qRT-PCR) and was consistent with the induction of Nfκb1 protein. Myc oncogene, frequently overexpressed in urothelial tumors, was upregulated by AAI on the microarrays and confirmed by qRT-PCR and protein induction. Collectively we found that microarray gene expression analysis is a useful tool to define tissue-specific responses in AAI-induced toxicity. Several genes identified such as TP53, Rb1, Mdm2, Cdkn2a and Myc are frequently affected in human urothelial cancer, and may be valuable prognostic markers in future clinical studies.

Polo-like kinase 1 reaches beyond mitosis--cytokinesis, DNA damage response, and development.

Polo-like kinase 1 (Plk1) is a key regulator of cell division in eukaryotic cells. In this review we focus on recent leaps in our understanding of how Plk1 controls cytokinesis, the final stage of cell division. Furthermore, we will go beyond mitosis to highlight unexpected roles of Plk1 during interphase and during animal development. In vertebrate cells, Plk1 has emerged as a novel player in maintaining genomic stability during DNA replication and as an important modulator of the DNA damage checkpoint. Plk1 functions extend past the 'core' cell cycle. Plk1 acts as a link between developmental processes and the cell cycle machinery during asymmetric cell divisions in flies and worms. The term 'mitotic kinase' might not do justice to Plk1 in the light of these recent results.

Plx1 is required for chromosomal DNA replication under stressful conditions.

Polo-like kinase (Plk)1 is required for mitosis progression. However, although Plk1 is expressed throughout the cell cycle, its function during S-phase is unknown. Using Xenopus laevis egg extracts, we demonstrate that Plx1, the Xenopus orthologue of Plk1, is required for DNA replication in the presence of stalled replication forks induced by aphidicolin, etoposide or reduced levels of DNA-bound Mcm complexes. Plx1 binds to chromatin and suppresses the ATM/ATR-dependent intra-S-phase checkpoint that inhibits origin firing. This allows Cdc45 loading and derepression of DNA replication initiation. Checkpoint activation increases Plx1 binding to the Mcm complex through its Polo box domain. Plx1 recruitment to chromatin is independent of checkpoint mediators Tipin and Claspin. Instead, ATR-dependent phosphorylation of serine 92 of Mcm2 is required for the recruitment of Plx1 to chromatin and for the recovery of DNA replication under stress. Depletion of Plx1 leads to accumulation of chromosomal breakage that is prevented by the addition of recombinant Plx1. These data suggest that Plx1 promotes genome stability by regulating DNA replication under stressful conditions.

ATM and ATR promote Mre11 dependent restart of collapsed replication forks and prevent accumulation of DNA breaks.

Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia Rad3-related (ATR) and the Mre11/Rad50/Nbs1 complex ensure genome stability in response to DNA damage. However, their essential role in DNA metabolism remains unknown. Here we show that ATM and ATR prevent accumulation of DNA double-strand breaks (DSBs) during chromosomal replication. Replicating chromosomes accumulate DSBs in Xenopus laevis egg extracts depleted of ATM and ATR. Addition of ATM and ATR proteins to depleted extracts prevents DSB accumulation by promoting restart of collapsed replication forks that arise during DNA replication. We show that collapsed forks maintain MCM complex but lose Pol epsilon, and that Pol epsilon reloading requires ATM and ATR. Replication fork restart is abolished in Mre11 depleted extracts and is restored by supplementation with recombinant human Mre11/Rad50/Nbs1 complex. Using a novel fluorescence resonance energy transfer-based technique, we demonstrate that ATM and ATR induce Mre11/Rad50/Nbs1 complex redistribution to restarting forks. This study provides direct biochemical evidence that ATM and ATR prevent accumulation of chromosomal abnormalities by promoting Mre11/Rad50/Nbs1 dependent recovery of collapsed replication forks.

Radiosensitivity of lymphoblastoid cell lines with a heterozygous BRCA1 mutation is not detected by the comet assay and pulsed field gel electrophoresis.

Lymphoblastoid cell lines (LCL) with a heterozygous mutation in the breast cancer susceptibility gene BRCA1 have been repeatedly used to elucidate the biological consequences of such a mutation with respect to radiation sensitivity and DNA repair deficiency. Our previous results indicated that LCL with a BRCA1 mutation do not generally show the same chromosomal mutagen sensitivity in the micronucleus test as lymphocytes with the same BRCA1 mutation. To further study the radiosensitivity of LCL with a BRCA1 mutation, we now performed comparative investigations with the alkaline (pH 13) and the neutral (pH 8.3) comet assay and pulsed field gel electrophoresis (PFGE). These tests are commonly used to determine the repair capacity for DNA double strand breaks (DNA-DSB). Six LCL (three established from women with a heterozygous BRCA1 mutation and three from healthy controls) were investigated. Induction (2 and 5 Gy) of gamma-ray-induced DNA damage and its repair (during 60 min after irradiation) was measured with the alkaline and neutral comet assay. Comparative experiments were performed with PFGE determining the induction of DNA-DSB by 10-50 Gy gamma-irradiation and their repair during 6 h. There was no significant difference between LCL with and without BRCA1 mutation in any of these experiments. Therefore, using these methods, no indication for a delayed repair of DNA-DSB in LCL with a BRCA1 mutation was found. However, these results do not generally exclude DNA-DSB repair deficiency in these cell lines because the methods applied have limited sensitivity and only measure the speed but not the fidelity of the repair process.

Sensitivity of the FPG protein towards alkylation damage in the comet assay.

The comet assay (single cell gel electrophoresis) is widely used for the evaluation of DNA-damaging effects in genotoxicity testing and population monitoring. In its standard version at pH >13, DNA double strand breaks (DSB), DNA single strand breaks (SSB) and alkali-labile sites (ALS) lead to increased DNA migration. At reduced pH (12.5-12.1) the expression of ALS as SSB can be eliminated and the effect of SSB only can be identified. Specific endonucleases have been used to characterize specific classes of DNA damage. The formamido pyrimidine glycosylase (FPG) protein has been used to assess oxidative DNA base damage because it detects 8-OH guanine and other oxidatively damaged purines. Here, we show that the FPG protein also detects alkylation damage with high sensitivity in the comet assay. Human whole blood, isolated lymphocytes and V79 cells were treated with alkylating agents and post-incubated with FPG. FPG strongly enhanced MMS- and EMS-induced DNA damage but had no significant effect on ENU-induced DNA damage, indicating that the amount of N-7 guanine alkylation is responsible for the observed effect. Reducing the pH during alkali unwinding and electrophoresis to 12.5 to avoid the contribution of ALS to the comet assay effects, strongly decreased the sensitivity of the comet assay with and without FPG treatment and prevented DNA migration. We conclude that enhanced DNA effects in the comet assay by FPG after exposure to genotoxins with unknown mode of action should not directly be regarded as evidence for the presence of oxidative damage. Furthermore, reducing the pH leads to a considerable loss in sensitivity and should not be used in biomonitoring and other applications which require a sensitive protocol.

Enhanced sensitivity of peripheral blood lymphocytes from women carrying a BRCA1 mutation towards the mutagenic effects of various cytostatics.

We are studying the induction and repair of DNA damage in lymphocytes of women from families with familial breast cancer and mutations in the breast cancer susceptibility genes BRCA1 and BRCA2. Our previous results indicated a close relationship between the presence of a BRCA1 mutation and sensitivity for the induction of micronuclei by gamma irradiation and hydrogen peroxide (H2O2). To further characterize the mutagen sensitivity and to better understand the underlying mechanisms, we now tested the effect of various cytostatics on the micronucleus frequencies in lymphocytes of women with various BRCA1 mutations in comparison to controls. The results presented here indicate enhanced sensitivity towards bleomycin, cisplatin, cyclophosphamide and bischloroethylnitosurea (BCNU). However, mutagen sensitivity towards cisplatin and BCNU was not accompanied by enhanced induction of sister chromatid exchanges (SCE), suggesting that intrachromosomal recombination is not affected. In contrast to the various DNA-damaging agents, there was no clear difference in the response to vincristine and taxol. FISH analysis revealed that the two aneugens mainly induced centromere-positive micronuclei to a similar extent in lymphocytes with and without a BRCA1 mutation. We conclude that cells containing a heterozygous mutation in BRCA1 are more sensitive towards different kinds of DNA damage in accordance with the proposed central role of BRCA1 in maintaining genomic integrity. Although BRCA1 has been shown to interact with the mitotic spindle, spindle poisons do not cause enhanced induction of micronuclei. Since some of the DNA-damaging mutagens tested here are used as cytostatics in breast cancer chemotherapy, it might be that women with a BRCA1 mutation are at higher risk for the induction of mutations and secondary cancers by standard therapies.

Investigations on the effect of cigarette smoking in the comet assay.

The comet assay (single-cell gel electrophoresis, SCG) is widely accepted as an in vitro and in vivo genotoxicity test. Because of its demonstrated ability to detect various kinds of DNA damage and its ease of application, the technique is being increasingly used in human biomonitoring. However, the assessment of small genotoxic effects as typically obtained in biomonitoring may be limited by the different sources of assay variability and the lack of an optimal protocol with high sensitivity. To better characterize the suitability of the comet assay for biomonitoring, we are performing a comprehensive investigation on blood samples from smokers and non-smokers. Because tobacco smoke is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds, smokers should be a suitable study group with relevant mutagen exposure. Here, we report our results for the first sample of 20 healthy male smokers and 20 healthy male non-smokers. Baseline and benzo[a]pyrene diolepoxide (BPDE)-induced effects were analysed by two investigators using two image analysis systems. The study was repeated within 4 months. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline and BPDE-induced DNA damage was comparatively analysed. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. None of these approaches revealed significant differences between smokers and non-smokers. Although more data is needed for a final conclusion, this study indicates some limitations of the comet assay with regard to the detection of DNA damage induced by environmental mutagens in peripheral blood cells.

Mutagen sensitivity of human lymphoblastoid cells with a BRCA1 mutation.

Previous results indicated that lymphoblastoid cell lines (LCL) with a BRCA1 mutation are hypersensitive to the chromosome-damaging effects of gamma irradiation or hydrogen peroxide as revealed by the micronucleus test. We now investigated six LCL (three with and three without a BRCA1 mutation) in more detail, to see whether LCL represent a useful model for the investigation of mechanisms responsible for the known mutagen sensitivity of lymphocytes from women carrying a BRCA1 mutation. Our results show that there is no systematic difference in radiation sensitivity between LCL with and without a BRCA1 mutation. Spontaneous and gamma radiation-induced micronucleus frequencies were in same range. Furthermore, cytotoxic effects (reduced cell proliferation, reduced viability) induced by gamma radiation were not different. The only difference found was an induction of micronuclei by 10 microM hydrogen peroxide in BRCA1 cell lines while a concentration of 20 microM hydrogen peroxide was necessary to induce micronuclei in control cells. Comet assay experiments did not reveal differences with regard to the induction and removal of primary DNA damage. Furthermore, expression of BRCA1 mRNA after gamma irradiation showed considerable variability and there was no clear difference between cell lines with and without BRCA mutation. These results indicate that LCL with a BRCA1 mutation do not generally show the same mutagen sensitivity as lymphocytes with the same BRCA1 mutation. Therefore, the use of LCL to study the mechanisms underlying mutagen sensitivity due to a heterozygous BRCA1 mutation seems to be limited.

Oxygenated water does not induce genotoxic effects in the comet assay.

Drinking of oxygenated water (i.e. water with increased concentration of physically dissolved oxygen) is said to improve oxygen availability of the body and will do the consumer good. However, increased oxygen concentrations can also lead to an increased production of reactive oxygen species (ROS). If antioxidant defences are not completely efficient, ROS can cause cell injury including DNA damage. We therefore investigated whether drinking of oxygenated water can lead to increased DNA damage in peripheral blood cells of test subjects. We also tested whether direct exposure of V79 Chinese hamster cells to oxygenated medium or oxygenated Hank's solution for various time periods induces DNA damage. Induction of DNA damage was measured with the alkaline comet assay (single cell gel electrophoresis). The comet assay, in particular the modification with FPG post-treatment for the determination of oxidative DNA base damage, has been proven to be extremely sensitive for the detection of oxygen-induced DNA damage. However, both the in vivo and the in vitro studies with the comet assay in the absence and presence of FPG post-treatment did not provide evidence for a genotoxic effect of oxygenated water.

Mutagen sensitivity of peripheral blood from women carrying a BRCA1 or BRCA2 mutation.

We are studying the induction and repair of DNA damage in lymphocytes of women from families with familial breast cancer and a heterozygous mutation in the breast cancer susceptibility genes BRCA1 or BRCA2. Besides various other functions, BRCA proteins seem to be involved in DNA repair processes like transcription-coupled and double-strand break (dsb) repair. Our previous results indicated a close relationship between the presence of a BRCA1 mutation and sensitivity for the induction of micronuclei (MN) by gamma irradiation and hydrogen peroxide (H2O2). In contrast to the results with the micronucleus assay, we found no significant individual difference between women with and without a BRCA1 mutation with respect to the induction and repair of DNA damage in the alkaline comet assay. We now investigated further cases heterozygous for a BRCA1 mutation and cases heterozygous for a BRCA2 mutation and show that enhanced micronucleus formation after gamma irradiation and H2O2-treatment is also a feature of lymphocytes carrying a BRCA2 mutation. Investigations with the comet assay did not reveal clear differences with regard to the induction of DNA damage on the individual level. There were also no significant differences between blood samples carrying a BRCA1 or BRCA2 mutation and blood samples from normal controls when the repair capacities (i.e. the kinetics of the removal of radiation-induced DNA effects in the comet assay) were compared. Our results indicate that mutagen sensitivity of lymphocytes heterozygous for a BRCA2 mutation is similar to that of cells with a BRCA1 mutation and BRCA1 and BRCA2 cannot be differentiated at present with the micronucleus test (MNT) or the comet assay.