ω-3 and ω-6 Polyunsaturated fatty acids (PUFAs) play a role in the pathogenesis of colon cancer. Upon oxidation, PUFAs generate α,β-unsaturated aldehydes or enals, such as acrolein (Acr) and (E)-4-hydroxy-2-nonenal (HNE), which can form cyclic adducts of deoxyguanosine (Acr-dG and HNE-dG, respectively) in DNA. Both Acr-dG and HNE-dG adducts have been detected in human and animal tissues and are potentially mutagenic and carcinogenic. In vivo levels of Acr-dG in DNA are at least two orders of magnitude higher than those of HNE-dG. In addition to the facile reaction with Acr, the higher levels of Acr-dG than HNE-dG in vivo may be due to a lower rate of repair. Previous studies have shown that HNE-dG adducts are repaired by the NER pathway (Choudhury et al., Biochemistry 43 (2004) 7514–7521). We hypothesize that Acr-dG adducts are repaired at a slower rate than HNE-dG and that HNE-dG in DNA may influence the repair of Acr-dG. In this study, using a DNA repair synthesis assay and a LC-MS/MS method, we showed that Acr-dG in a plasmid DNA is repaired by NER proteins, but it is repaired at a much slower rate than HNE-dG in human colon cell extracts, and the slow repair of Acr-dG is likely due to poor recognition/excision of the lesions in DNA. Furthermore, using a plasmid DNA containing both adducts we found the repair of Acr-dG is significantly inhibited by HNE-dG, however, the repair of HNE-dG is not much affected by Acr-dG. This study demonstrates that the NER repair efficiencies of the two major structurally-related in vivo cyclic DNA adducts from lipid oxidation vary greatly. More importantly, the repair of Acr-dG can be significantly retarded by the presence of HNE-dG in DNA. Therefore, this study provides a mechanistic explanation for the higher levels of Acr-dG than HNE-dG observed in tissue DNA.
Acrolein; 4-hydroxy-2-nonenal; DNA adduct; nucleotide excision repair (NER); repair kinetics; human colon cells
Single CD34+ cells from adult human peripheral blood show mtDNA sequence heterogeneity. In this study, we compared mtDNA sequence variation in single CD34+ cells from peripheral blood (PB) mononuclear cells (MNCs) from the same donors but under different conditions of storage and transport: group I, MNCs from heparinized PB that inadvertently required six days to be transported to the testing laboratory; group II, MNCs which were isolated from PB within a day of phlebotomy and frozen prior to transportation and storage. We observed more cell death for MNCs of group I than group II. Concordantly, group I CD34+ cells had a very low potential for hematopoietic colony formation in vitro compared with group II cells. CD34+ cells of group II showed an unexpectedly higher level of mtDNA sequence heterogeneity than was present group I cells. These observations suggest that reduced mtDNA sequence heterogeneity in single CD34+ cells of group I was likely due to elimination of cells harboring mutations. CD34+ cells that survive stress ex vivo may be more enriched in quiescent primitive hematopoietic stem cells, with fewer mtDNA mutations than committed progenitors. Technically, attention is required for conditions of preparation of human blood samples for single cell mtDNA analysis.
mtDNA; single cell analysis; mutation; hematopoietic stem cell; committed progenitors
Chromosomal double strand breaks provoke an extensive reaction in neighboring chromatin, characterized by phosphorylation of histone H2AX on serine 139 of its C-terminal tail (to form “γH2AX”). The γH2AX response contributes to the repair of double strand breaks encountered in a variety of different contexts, including those induced by ionizing radiation, physiologically programmed breaks that characterize normal immune cell development and the pathological exposure of DNA ends triggered by telomere dysfunction. γH2AX also participates in the evolutionarily conserved process of sister chromatid recombination, a homologous recombination pathway involved in the suppression of genomic instability during DNA replication and directly implicated in tumor suppression. At a biochemical level, the γH2AX response provides a compelling example of how the “histone code” is adapted to the regulation of double strand break repair. Here, we review progress in research aimed at understanding how γH2AX contributes to double strand break repair in mammalian cells.
DNA double strand breaks (DSBs) constitute one of the most dangerous forms of DNA damage. In actively replicating cells, these breaks are first recognized by specialized proteins that initiate a signal transduction cascade that modulates the cell cycle and results in the repair of the breaks by homologous recombination (HR). Protein signaling in response to double strand breaks involves phosphorylation and ubiquitination of chromatin and a variety of associated proteins. Here we review the emerging structural principles that underlie how post-translational protein modifications control protein signaling that emanates from these DNA lesions.
double-strand break signaling; phosphorylation signaling; ubiquitin; BRCT domains; FHA domains; Ubc13; UIM
Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients’ response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.
nucleotide pool; ITPA gene polymorphism; pharmacogenetics; base analogs; mercaptopurines; protein stability; dominant negative
Repair of double strand breaks (DSBs) is essential for cell survival and genome integrity. While much is known about the molecular mechanisms involved in DSB repair and checkpoint activation, the roles of nuclear dynamics of radiation-induced foci (RIF) in DNA repair are just beginning to emerge. Here, we summarize results from recent studies that point to distinct features of these dynamics in two different chromatin environments: heterochromatin and euchromatin. We also discuss how nuclear architecture and chromatin components might control these dynamics, and the need of novel quantification methods for a better description and interpretation of these phenomena. These studies are expected to provide new biomarkers for radiation risk and new strategies for cancer detection and treatment.
The aminoglycoside streptomycin binds to ribosomes to promote mistranslation and eventual inhibition of translation. Streptomycin kills bacteria, whereas many other non-aminoglycoside inhibitors of translation do not. Because mistranslation is now known to affect DNA replication, we asked if hydroxyurea, a specific inhibitor of DNA synthesis, affects killing, and find that hydroxyurea significantly attenuates killing by streptomycin. We find that the hydroxyl radical scavengers D-mannitol and thiourea have either no effect or only a modest protective effect. The iron chelator 2,2′-dipyridyl eliminated killing by streptomycin, but further investigation revealed that it blocks streptomycin uptake. Prior treatment of cells with low-levels of methyl methanesulfonate to induce the adaptive response to alkylation leads to a significant attenuation of killing, which, together with the hydroxyurea effect, suggests roles for DNA replication and repair functions in cell killing by streptomycin.
mistranslation; DNA replication and repair; dipyridyl; antibiotic uptake; hydroxyl radicals; DNA alkylation damage; alkB
Cigarette smoke causes direct oxidative DNA damage as well as indirect damage through inflammation. Epidemiological studies show a strong relationship between secondhand smoke and cancer; however, the mechanisms of secondhand smoke-induced cancer are not well understood. Animal models with either i) deficient oxidative DNA damage repair, or ii) a decreased capacity to combat oxidative stress may help determine the pathways important in mitigating damage caused by smoke. In this study, we used mice lacking Ogg1 and Myh, both of which are involved in base excision repair by removing oxidatively damaged DNA bases. Gclm-deficient mice, which have decreased levels of glutathione (GSH), were used to look at the role of smoke-induced oxidative damage. Ex vivo experiments show significantly elevated levels of DNA single-strand breaks and chromosomal aberrations in peripheral blood lymphocytes from Ogg1−/−Myh−/− double knockout mice compared to wild type (WT) mice after 24 hours of exposure to cigarette smoke extract (CSE). The average γH2AX foci per cell was significantly elevated 3 hours after exposure to CSE in cells from Ogg1
−/− double knockout mice compared to wildtype mice. In vivo we found that all mice had increased markers of DNA damage after exposure to side-stream tobacco smoke (SSTS). Ogg1−/−
Myh−/− and Gclm−/− mice had altered levels of peripheral blood glutathione after SSTS exposure whereas wild type mice did not. This may be due to differential regulation of glutathione synthesis in the lung. We also found that Ogg1−/−Myh−/− mice had a decreased lifespan after oral gavage with benzo[a]pyrene compared to wildtype mice and sham-exposed Ogg1−/−Myh−/− mice. Our results are important in investigating the roles of oxidative stress and oxidative DNA damage repair in cigarette smoke-induced cancers and characterizing the role of genetic polymorphisms in smoke-related disease susceptibility.
DNA repair; Glutathione; Ogg1; Myh; DNA damage
Existing research has not fully explained how different types of ionizing radiation (IR) modulate the responses of cell populations or tissues. In our previous work, we showed that gap junction intercellular communication (GJIC) mediates the propagation of stressful effects among irradiated cells exposed to high linear energy transfer (LET) radiations, in which almost every cells is traversed by an IR track. In the present study, we conducted an in-depth study of the role of GJIC in modulating the repair of potentially lethal damage (PLDR) and micronuclei formation in cells exposed to low- or high-LET IR. Confluent human fibroblasts were exposed in the presence or absence of a gap junction inhibitor to 200 kV X rays (LET ∼ 1.7 keV/µm), carbon ions (LET ∼ 76 keV/µm), silicon ions (LET ∼ 113 keV/µm) or iron ions (LET ∼ 400 keV/µm) that resulted in isosurvival levels. The fibroblasts were incubated for various times at 37 °C. As expected, high-LET IR were more effective than were low-LET X rays at killing cells and damaging DNA shortly after irradiation. However, when cells were held in a confluent state for several hours, PLDR associated with a reduction in DNA damage, occurred only in cells exposed to X rays. Interestingly, inhibition of GJIC eliminated the enhancement of toxic effects, which resulted in an increase of cell survival and reduction in the level of micronucleus formation in cells exposed to high, but not in those exposed to low-LET IR. The experiment shows that gap-junction communication plays an important role in the propagation of stressful effects among irradiated cells exposed to high-LET IR while GJIC has only a minimal effect on PLDR and DNA damage following low-LET irradiation. Together, our results show that PLDR and induction of DNA damage clearly depend on gap-junction communication and radiation quality.
Gap junction intercellular communication; Potentially lethal damage repair; Linear energy transfer; Ionizing radiation; Heavy-ion beams
Many metals serve as micronutrients which protect against genomic instability. Chromium is most abundant in its trivalent and hexavalent forms. Trivalent chromium has historically been considered an essential element, though recent data indicate that while it can have pharmacological effects and value, it is not essential. There are no data indicating that trivalent chromium promotes genomic stability and, instead may promote genomic instability. Hexavalent chromium is widely accepted as highly toxic and carcinogenic with no nutritional value. Recent data indicate that it causes genomic instability and also has no role in promoting genomic stability.
chromium; chromate; trivalent chromium; hexavalent chromium; genomic instability
Hexavalent chromium (Cr[VI]) is an industrial waste product known to cause nasal and lung cancer in exposed workers. Intracellularly, Cr[VI] undergoes a series of enzymatic reductions resulting in the formation of reactive chromate intermediates and oxygen free radicals. These metabolites react with DNA to cause numerous types of genomic lesions, but the cellular response to these genotoxic insults is poorly understood. Recently, we demonstrated that in response to DNA damage induced by Cr[VI], an ataxia-telangiectasia mutated (ATM) and structural maintenance of chromosomal protein 1 (SMC1)-dependent S-phase checkpoint is activated. Interestingly, this checkpoint response was only ATM-dependent in cells exposed to low doses of Cr[VI], we demonstrate that the ATM and Rad3 related kinase, ATR, is required to activate the S-phase checkpoint. In response to all doses of Cr[VI], ATR is activated and phosphorylates SMC1 to facilitate the checkpoint. Further, chromatin binding ability of Rad17 is required for this process. Taken together, these results indicate that the Rad17-ATR-SMC1 pathway is essential for Cr[VI]-induced S-phase checkpoint activation.
Chromium; ATM; ATR; S-phase arrest
Hexavalent chromium (Cr(VI)) is a widespread environmental contaminant and a known human carcinogen, generally causing bronchial cancer. Recent studies have shown that the particulate forms of Cr(VI) are the potent carcinogens. Particulate Cr(VI) is known to induce a spectrum of DNA damage such as DNA single strand breaks, Cr-DNA adducts, DNA-protein crosslinks and chromosomal aberrations. However, particulate Cr(VI)-induced DNA double strand breaks (DSBs) have not been reported. Thus, the aim of this study was to determine if particulate Cr(VI)-induces DSBs in human bronchial cells. Using the single cell gel electrophoresis assay (comet assay), showed that lead chromate-induced concentration dependent increases in DSBs with 0.1, 0.5, 1 and 5 μg/cm2 lead chromate inducing a 20, 50, 67 and 109% relative increase in the tail integrated intensity ratio, respectively. Sodium chromate at concentrations of 1, 2.5 and 5 μM induced 38, 78 and 107% relative increase in the tail integrated intensity ratio, respectively. We also show that genotoxic concentrations of lead chromate activate the ataxia telangiectasia mutated (ATM) protein, which is thought to play a central role in the early stages of DSB detection and controls cellular responses to this damage. The H2A.X protein becomes rapidly phosphorylated on residue serine 139 in cells when DSBs are introduced into the DNA by ionizing radiation. By using immunofluorescence, we found that lead chromate-induced concentration-dependent increases in phosphorylated H2A.X (r-H2A.X) foci formation with 0.1, 0.5, 1, 5 and 10 μg/cm2 lead chromate inducing a relative increase in the number of cells with r-H2A.X foci formation of 43, 51, 115 and 129%, respectively.
Hexavalent chromium; DNA double strand breaks; ATM; Smc1; Lead chromate
Emerging evidence provide credible support in favor of the potential role of bioactive products derived from ingesting cruciferous vegetables such as broccoli, brussel sprouts, cauliflower and cabbage. Among many compounds, 3,3′-Diindolylmethane (DIM) is generated in the acidic environment of the stomach following dimerization of Indole-3-Carbinol (I3C) monomers present in these classes of vegetables. Both I3C and DIM have been investigated for their use in preventing, inhibiting, and reversing the progression of cancer- as a chemopreventive agent. In this review, we summarize an updated, wide-ranging pleiotropic anti-tumor and biological effects elicited by DIM against tumor cells. It is unfeasible to point one single target as basis of cellular target of action of DIM. We emphasize key cellular and molecular events that are effectively modulated in the direction of inducing apoptosis and suppressing cell proliferation. Collectively, DIM orchestrates signaling through Ah receptor, NF-κB/Wnt/Akt/mTOR pathways impinging on cell cycle arrest, modulation of key cytochrome P450 enzymes, altering angiogenesis, invasion, metastasis and epigenetic behaviors of cancer cells. The ability of DIM to selectively induce tumor cells to undergo apoptosis has been observed in preclinical models, and thus it has been speculated in improving the therapeutic efficacy of other anticancer agents that have diverse molecular targets. Consequently, DIM has moved through preclinical development into phase-I clinical trials, thereby suggesting that DIM could be a promising and novel agent either alone or as an adjunct to conventional therapeutics such as chemo-radio therapy, and targeted therapies. An important development has been the availability of DIM formulation with superior bioavailability for humans. Therefore, DIM appears to be a promising chemopreventive agent or chemo-radio-sensitizer for the prevention of tumor recurrence and/or for the treatment of human malignancies.
3,3′- Diindolylmethane; Brassica sps; Prevention; Therapy
Non-DNA targeted effects of ionizing radiation, which include genomic instability, and a variety of bystander effects including abscopal effects and bystander mediated adaptive response, have raised concerns about the magnitude of low-dose radiation risk. Genomic instability, bystander effects and adaptive responses are powered by fundamental, but not clearly understood systems that maintain tissue homeostasis. Despite excellent research in this field by various groups, there are still gaps in our understanding of the likely mechanisms associated with non-DNA targeted effects, particularly with respect to systemic (human health) consequences at low and intermediate doses of ionizing radiation. Other outstanding questions include links between the different non-targeted responses and the variations in response observed between individuals and cell lines, possibly a function of genetic background. Furthermore, it is still not known what the initial target and early interactions in cells are that give rise to non-targeted responses in neighbouring or descendant cells. This paper provides a commentary on the current state of the field as a result of the Non-targeted effects of ionizing radiation (NOTE) Integrated Project funded by the European Union. Here we critically examine the evidence for non-targeted effects, discuss apparently contradictory results and consider implications for low-dose radiation health effects.
Ionizing radiation; low dose; non-targeted effects; non-cancer disease; health risk
Our previous findings demonstrated that DNA damage by polynuclear aromatic hydrocarbons (PAHs) triggers a cellular protective response of growth inhibition (G1-S cell cycle arrest and inhibition of DNA synthesis) in human fibroblasts associated with accumulation of p53 protein, a growth-inhibitory transcription factor. Here, we report that BPDE (the ultimate carcinogenic metabolite of the PAH benzo[a]pyrene) treatment triggers a variable extent of inhibition of DNA synthesis/cell growth, which does not correspond to the extent of increased p53 accumulation. BPDE treatment of cells significantly attenuates expression of p34cdc2, a cell cycle activating protein. Although the role of cdc2 down-regulation in inhibition of cell cycle progression is well known, cdc2 down-regulation in response to cellular insult by PAHs has not been reported. Unlike p53 accumulation, there is a correspondence between DNA synthesis/cell growth inhibition and cdc2 down-regulation by BPDE. BPDE-induced cdc2 down-regulation is p53 dependent, although there is no correspondence between p53 accumulation and cdc2 down-regulation. BPDE-induced cdc2 down-regulation corresponded with accumulation of the cell cycle inhibitor protein p21 (transactivation product of p53). DNA synthesis/cell growth inhibition in response to DNA-damaging PAHs may involve down-regulation of cdc2 protein mediated by p53 activation (transactivation ability), and the extent of p53 accumulation is not the sole determining factor in this regard.
(±)-anti-Benzo[a]pyrene-7,8-diol-9,10-epoxide; p34cdc2; p53; DNA synthesis; p21; cell growth inhibition
Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental carcinogens. Here we review the evidence that support the role of aristolochic acid (AA) in inducing a mutational fingerprint in the tumor suppressor gene TP53 in urothelial carcinomas of the upper urinary tract (UUT). Exposure to AA, a nitrophenathrene carboxylic acid present in certain herbal remedies and in flour prepared from wheat grain contaminated with seeds of Aristolochia clematitis, has been linked to chronic nephropathy and UUT. TP53 mutations in UUT of individuals exposed to AA reveal a unique pattern of mutations characterised by A to T transversions on the non-transcribed strand, which cluster at hotspots rarely mutated in other cancers. This unusual pattern, originally discovered in UUTs from two different populations, one in Taiwan, and one in the Balkans, has been reproduced experimentally by treating mouse cells that harbour human TP53 sequences with AA. The convergence of molecular epidemiological and experimental data establishes a clear causal association between exposure to the human carcinogen AA and UUT cancer. Despite bans on the sale of herbs containing AA, their use continues, raising global public health concern and an urgent need to identify populations at risk.
Aristolochic acid(s); TP53 mutation; nephropathy; herbal remedies; hotspot mutations; urothelial cancer
In this article we review health effects in offspring of human populations exposed as a result of radiotherapy and some groups exposed to chemotherapy. We also assess risks in offspring of other radiation-exposed groups, in particular those of the Japanese atomic bomb survivors and occupationally and environmentally exposed groups. Experimental findings are also briefly surveyed.
Animal and cellular studies tend to suggest that the irradiation of males, at least at high doses (mostly 1 Gy and above), can lead to observable effects (including both genetic and epigenetic) in the somatic cells of their offspring over several generations that are not attributable to the inheritance of a simple mutation through the parental germ line. However, studies of disease in the offspring of irradiated humans have not identified any effects on health. The available evidence therefore suggests that human health has not been significantly affected by transgenerational effects of radiation. It is possible that transgenerational effects are restricted to relatively short times post-exposure and in humans conception at short times after exposure is likely to be rare. Further research that may help resolve the apparent discrepancies between cellular/animal studies and studies of human health are outlined.
transgenerational effects; radiation; genetic effects; minisatellites; radiotherapy; chemotherapy
Human DNA mismatch repair (MMR) recognizes and binds 5-fluorouracil (5FU) incorporated into DNA and triggers a MMR-dependent cell death. Absence of MMR in a patient's colorectal tumor abrogates 5FU's beneficial effects on survival. Changes in the tumor microenvironment like low extracellular pH (pHe) may diminish DNA repair, increasing genomic instability. Here, we explored if low pHe modifies MMR recognition of 5FU, as 5FU can exist in ionized and non-ionized forms depending on pH. We demonstrate that MMR-proficient cells at low pHe show downregulation of hMLH1, whereas expression of TDG and MBD4, known DNA glycosylases for base excision repair (BER) that can remove 5FU from DNA, were unchanged. We show in vitro that 5FU within DNA pairs with adenine (A) at high and low pH (in absence of MMR and BER). Surprisingly, 5FdU:G was repaired to C:G in hMLH1 -deficient cells cultured at both low and normal pHe, similar to MMR-proficient cells. Moreover, both hMSH6 and hMSH3, components of hMutSα and hMutSβ, respectively, bound 5FU within DNA(hMSH6 > hMSH3) but is influenced by hMLH1. We conclude that an acidic tumor microenvironment triggers downregulation of hMLH1, potentially removing the execution component of MMR for 5FU cytotoxicity, whereas other mechanisms remain stable to implement overall 5FU sensitivity.
Colon cancer; 5-Fluorouracil; DNA mismatch repair; Cell death; Chemotherapy; Acidic tumor microenvironment
The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6–8 h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21Cip1/Waf1 post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70–80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase η, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol η protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.
Caffeine; Checkpoints; DNA repair; ATM; ATR; Ionizing radiation; Ultraviolet radiation
Recent studies have indicated that extranuclear or extracellular targets are important in mediating the bystander genotoxic effects of α-particles. In the present study, human–hamster hybrid (AL) cells were plated on either one or both sides of double-mylar dishes 2–4 days before irradiation, depending on the density requirement of experiments. One side (with or without cells) was irradiated with α-particles (from 0.1 to 100 Gy) using the track segment mode of a 4 MeV Van de Graaff accelerator. After irradiation, cells were kept in the dishes for either 1 or 48 h. The non-irradiated cells were then collected and assayed for both survival and mutation. When one side with cells was irradiated by α-particles (1, 10 and 100 Gy), the surviving fraction among the non-irradiated cells was significantly lower than that of control after 48 h co-culture. However, such a change was not detected after 1 h co-culture or when medium alone was irradiated. Furthermore, co-cultivation with irradiated cells had no significant effect on the spontaneous mutagenic yield of non-irradiated cells collected from the other half of the double-mylar dishes. These results suggested that irradiated cells released certain cytotoxic factor(s) into the culture medium that killed the non-irradiated cells. However, such factor(s) had little effect on mutation induction. Our results suggest that different bystander end points may involve different mechanisms with different cell types.
Bystander effect; α-Particle irradiation; Double-mylar; Mutagenesis
It is well accepted that oxidative DNA repair capacity, oxidative damage to DNA and oxidative stress play central roles in aging and disease development. However, the correlation between oxidative damage to DNA, markers of oxidant stress and DNA repair capacity is unclear. In addition, there is no universally accepted panel of markers to assess oxidative stress in humans. Our interest is oxidative damage to DNA and its correlation with DNA repair capacity and other markers of oxidative stress. We present preliminary data from a small comet study that attempts to correlate single strand break (SSB) level with single strand break repair capacity (SSB-RC) and markers of oxidant stress and inflammation. In this limited study of four very small age-matched 24-individual groups of male and female whites and African-Americans aged 30–64 years, we found that females have higher single strand break (SSB) levels than males (p=0.013). There was a significant negative correlation between SSB-RC and SSB level (p=0.041). There was a positive correlation between SSBs in African American males with both heme degradation products (p=0.008) and high-sensitivity C-reactive protein (hs-CRP) (p=0.022). We found a significant interaction between hs-CRP and sex in their effect on residual DNA damage (p=0.002). Red blood cell reduced glutathione concentration was positively correlated with the levels of oxidized bases detected by endonuclease III (p=0.047), heme degradation products (p=0.015) and hs-CRP (p=0.020). However, plasma carbonyl levels showed no significant correlation with other markers. The data from the literature and from our very limited study suggest a complex relationship between measures of oxidative stress and frequently used clinical parameters believed to reflect inflammation or oxidative stress.
oxidative stress; aging; DNA oxidative lesions; DNA repair; comet assay; red blood cell glutathione (RBC GSH); fluorescent heme degradation products; high-sensitivity C-reactive protein (hs-CRP)
DNA polymerase η (pol η) plays a critical role in suppressing mutations caused by the bypass of cis-syn cyclobutane pyrimidine dimers (CPD) that escape repair. There is evidence this is also the case for the oxidative lesion 7,8-dihydro-8-oxo-guanine (8-oxoG). Both of these lesions cause moderate to severe blockage of synthesis when encountered by replicative polymerases, while pol η displays little no to pausing during translesion synthesis. However, since lesion bypass does not remove damaged DNA from the genome and can possibly be accompanied by errors in synthesis during bypass, the process is often called ‘damage tolerance’ to delineate it from classical DNA repair pathways. The fidelity of lesion bypass is therefore of importance when determining how pol η suppresses mutations after DNA damage. As pol η has been implicated in numerous in vivo pathways other than lesion bypass, we wanted to better understand the molecular mechanisms involved in the relatively low-fidelity synthesis displayed by pol η. To that end, we have created a set of mutant pol η proteins each containing a single amino acid substitution in the active site and closely surrounding regions. We determined overall DNA synthesis ability as well as the efficiency and fidelity of bypass of thymine-thymine CPD (T-T CPD) and 8-oxoG containing DNA templates. Our results show that several amino acids are critical for normal polymerase function, with changes in overall activity and fidelity being observed. Of the mutants that retain polymerase activity, we demonstrate that amino acids Q38, Y52, and R61 play key roles in determining polymerase fidelity, with substation of alanine causing both increases and decreases in fidelity. Remarkably, the Q38A mutant displays increased fidelity during synthesis opposite 8-oxoG but decreased fidelity during synthesis opposite a T-T CPD.
Mutagenesis; DNA damage; translesion synthesis; DNA polymerase eta; 8-oxoG; thymine-thymine CPD
Ku80 forms a heterodimer with Ku70, called Ku, that repairs DNA double-strand breaks (DSBs) via the nonhomologous end joining (NHEJ) pathway. As a consequence of deleting NHEJ, Ku80-mutant cells are hypersensitive to agents that cause DNA DSBs like ionizing radiation. Here we show that Ku80 deletion also decreased resistance to ROS and alkylating agents that typically cause base lesions and single-strand breaks (SSBs). This is unusual since base excision repair (BER), not NHEJ, typically repairs these types of lesions. However, we show that deletion of another NHEJ protein, DNA ligase IV (Lig 4), did not cause hypersensitivity to these agents. In addition, the ROS and alkylating agents did not induce γ-H2AX foci that are diagnostic of DSBs. Furthermore, deletion of Ku80, but not Lig 4 or Ku70, reduced BER capacity. Ku80 deletion also impaired BER at the initial lesion recognition/strand scission step; thus, involvement of a DSB is unlikely. Therefore, our data suggests that Ku80 deletion impairs BER via a mechanism that does not repair DSBs.
nonhomologous end joining; base excision repair; double strand breaks; single strand breaks; base lesions
Exposure to ambient air pollution has been associated with adverse health effects including lung cancer. A recent epidemiology study has established that each 10 μg/m3 elevation in long term exposure to average PM2.5 ambient concentration was associated with approximately 8% of lung cancer mortality. The underlying mechanisms of how PM contributes to lung carcinogenesis, however, remain to be elucidated. We have recently found that transition metals such as nickel and chromium and oxidative stress induced lipid peroxidation metabolites such as aldehydes can greatly inhibit nucleotide excision repair (NER) and enhance carcinogen-induced mutations. Because PM is rich in metal and aldehyde content and can induce oxidative stress, we tested the effect of PM on DNA repair capacity in cultured human lung cells using in vitro DNA repair synthesis and host cell reactivation assays. We found that PM greatly inhibits NER for ultraviolet (UV) light and benzo(a)pyrene diol epoxide (BPDE) induced DNA damage in human lung cells. We further demonstrated that PM exposure can significantly increase both spontaneous and UV-induced mutagenesis. These results together suggest that the carcinogenicity of PM may act through its combined effect on suppression of DNA repair and enhancement of DNA replication errors.
Particulate matter; Nucleotide excision repair (NER); Mutagenesis; Host cell reactivation (HCR)