Many colorectal cancers (CRCs) develop in genetically susceptible individuals most of whom are not carriers of germ line mismatch repair or APC gene mutations and much of the heritable risk of CRC appears to be attributable to the co-inheritance of multiple low-risk variants. The accumulated experience to date in identifying this class of susceptibility allele has highlighted the need to conduct statistically and methodologically rigorous studies and the need for the multi-centre collaboration. This has been the motivation for establishing the COGENT (COlorectal cancer GENeTics) consortium which now includes over 20 research groups in Europe, Australia, the Americas, China and Japan actively working on CRC genetics. Here, we review the rationale for identifying low-penetrance variants for CRC and the current and future challenges for COGENT.
Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Epidemiological risk factors for CRC included dietary fat intake; consequently, the role of genes in the fatty acid biosynthesis and metabolism pathways is of particular interest. Moreover, hyperlipidaemia has been associated with different type of cancer and serum lipid levels could be affected by genetic factors, including polymorphisms in the lipid metabolism pathway. The aim of this study is to assess the association between single-nucleotide polymorphisms (SNPs) in fatty acid metabolism genes, serum lipid levels, body mass index (BMI) and dietary fat intake and CRC risk; 30 SNPs from 8 candidate genes included in fatty acid biosynthesis and metabolism pathways were genotyped in 1780 CRC cases and 1864 matched controls from the Molecular Epidemiology of Colorectal Cancer study. Information on clinicopathological characteristics, lifestyle and dietary habits were also obtained. Logistic regression and association analysis were conducted. Several LIPC (lipase, hepatic) polymorphisms were found to be associated with CRC risk, although no particular haplotype was related to CRC. The SNP rs12299484 showed an association with CRC risk after Bonferroni correction. We replicate the association between the T allele of the LIPC SNP rs1800588 and higher serum high-density lipoprotein levels. Weak associations between selected polymorphism in the LIPC and PPARG genes and BMI were observed. A path analysis based on structural equation modelling showed a direct effect of LIPC gene polymorphisms on colorectal carcinogenesis as well as an indirect effect mediated through serum lipid levels. Genetic polymorphisms in the hepatic lipase gene have a potential role in colorectal carcinogenesis, perhaps though the regulation of serum lipid levels.
Patients with chronic kidney disease (CKD) have signs of genomic instability and, as a consequence, extensive genetic damage, possibly due to accumulation of uraemic toxins, oxidative stress mediators and other endogenous substances with genotoxic properties. We explored factors associated with the presence and background levels of genetic damage in CKD. A cross-sectional study was performed in 91 CKD patients including pre-dialysis (CKD patients; n = 23) and patients undergoing peritoneal dialysis (PD; n = 33) or haemodialysis (HD; n = 35) and with 61 healthy subjects, divided into two subgroups with the older group being in the age range of the patients, serving as controls. Alkaline comet assay and cytokinesis-block micronucleus assay in peripheral blood lymphocytes were used to determine DNA and chromosome damage, respectively, present in CKD. Markers of oxidative stress [malondialdehyde (MDA), advanced glycation end products (AGEs), thiols, advanced oxidation protein products and 8-hydroxy-2′-deoxyguanosine] and markers of inflammation (C-reactive protein, interleukin-6 and tumour necrosis factor alpha) were also measured. Micronucleus (MN) frequency was significantly higher (P < 0.05) in the CKD group (46±4‰) when compared with the older control (oC) group (27.7±14). A significant increase in MN frequency (P < 0.05) was also seen in PD patients (41.9±14‰) versus the oC group. There was no statistically significant difference for the HD group (29.7±15.6‰; P = NS) versus the oC group. Comet assay data showed a significant increase (P < 0.001) of tail DNA intensity in cells of patients with CKD (15.6±7%) with respect to the total control (TC) group (11±1%). PD patients (14.8±7%) also have a significant increase (P < 0.001) versus the TC group. Again, there was no statistically significant difference for the HD group (12.5±3%) compared with the TC group. Patients with MN values in the upper quartile had increased cholesterol, triglycerides, AGEs and MDA levels and lower albumin levels. Multiple logistic regression analysis showed that male gender, diabetes and treatment modality were independently associated with higher levels of DNA damage. Our results suggest that oxidative stress, diabetes, gender and dialysis modality in CKD patients increased DNA and chromosome damage. To confirm these data, prospective clinical trials need to be performed.
Recent evidences have highlighted an influence of micronutrients in the maintenance of telomere length (TL). In order to explore whether diet-related telomere shortening had any physiological relevance and was accompanied by significant damage in the genome, in the present study, TL was assessed by terminal restriction fragment (TRF) analysis in peripheral blood lymphocytes of 56 healthy subjects for which detailed information on dietary habits was available and data were compared \with the incidence of nucleoplasmic bridges (NPBs), a marker of chromosomal instability related to telomere dysfunction visualised with the cytokinesis-blocked micronucleus assay. To increase the capability to detect even slight impairment of telomere function, the incidence of NPBs was also evaluated on cells exposed in vitro to ionising radiation. Care was taken to control for potential confounding factors that might influence TL, viz. age, hTERT genotype and smoking status. Data showed that higher consumption of vegetables was related with significantly higher mean TL (P = 0.013); in particular, the analysis of the association between micronutrients and mean TL highlighted a significant role of antioxidant intake, especially beta-carotene, on telomere maintenance (P = 0.004). However, the diet-related telomere shortening did not result in associated increased spontaneous or radiation-induced NPBs. The distribution of TRFs was also analysed and a slight prevalence of radiation-induced NPBs (P = 0.03) was observed in subjects with higher amount of very short TRFs (<2 kb). The relative incidence of very short TRFs was positively associate with ageing (P = 0.008) but unrelated to vegetables consumption and daily intake of micronutrients, suggesting that the degree of telomere erosion related with low dietary intake of antioxidants observed in this study was not so extensive to lead to chromosome instability.
Chronic inflammatory diseases are characterised by systemically elevated levels of tumour necrosis factor (TNF)-α, a proinflammatory cytokine with pleiotropic downstream effects. We have previously demonstrated increased genotoxicity in peripheral leukocytes and various tissues in models of intestinal inflammation. In the present study, we asked whether TNF-α is sufficient to induce DNA damage systemically, as observed in intestinal inflammation, and whether tumour necrosis factor receptor (TNFR) signalling would be necessary for the resultant genotoxicity. In the wild-type mice, 500 ng per mouse of TNF-α was sufficient to induce DNA damage to multiple cell types and organs 1-h post-administration. Primary splenic T cells manifested TNF-α-induced DNA damage in the absence of other cell types. Furthermore, TNFR1−/−TNFR2−/− mice demonstrated decreased systemic DNA damage in a model of intestinal inflammation and after TNF-α injection versus wild-type mice, indicating the necessity of TNFR signalling. Nuclear factor (NF)-κB inhibitors were also able to decrease damage induced by TNF-α injection in wild-type mice. When TNF-α administration was combined with interleukin (IL)-1β, another proinflammatory cytokine, DNA damage persisted for up to 24 h. When combined with IL-10, an anti-inflammatory cytokine, decreased genotoxicity was observed in vivo and in vitro. TNF-α/TNFR-mediated signalling is therefore sufficient and plays a large role in mediating DNA damage to various cell types, subject to modulation by other cytokines and their mediators.
The primary aim of this study was to quantify genetic and environmental influences on the frequency of spontaneously occurring micronuclei in children and adults. To meet this aim, a total of 63 male and female twin pairs and 19 singletons (145 individuals) were evaluated, ranging in age from 7 to 85 years. Micronuclei frequencies significantly increased with age for both genders (r = 0.49, P < 0.001), with the lowest and highest rates being seen in the 7- to 9 (mean = 0.56%, SD = .28) and 60- to 69-year-olds (mean = 2.12%, SD = 1.0), respectively. This age effect was significantly more pronounced in females than males (P = 0.017). In addition to the main effect of age, the completion of puberty in either gender (P = 0.036) and menopause in females (P = 0.024) was associated with a significant increase in micronuclei frequencies. Genetic model fitting indicated that influences from both additive genetic (65.2% of variance) and unique environmental (34.8% of variance) sources best explained the observed micronuclei frequencies in monozygotic and dizygotic twin pairs. Self-reported health conditions associated with an increased frequency of micronuclei included a history of allergies (P < 0.007) and migraines (P = 0.026). Multivitamin use was also associated with increased micronuclei frequencies (P = 0.004). In contrast, significantly lower micronuclei frequencies were associated with arthritis (P = 0.002), as well as consuming fruit (P = 0.014), green, leafy vegetables (P < 0.001) and/or folate-enriched bread (P = 0.035). A sex-specific effect, resulting in a significantly increased frequency of micronuclei with tobacco usage, was observed for females (but not males). Gender differences also moderated the impact of vitamin D and calcium consumption. In conclusion, the frequency of spontaneously arising micronuclei in humans is a complex trait, being influenced by both heritable genetic and environmental components. Recognition of factors contributing to baseline levels of micronuclei should provide guidance to researchers in designing studies to evaluate agents hypothesised to influence chromosomal instability.
Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments.
Exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs) induces cytochrome P450 (CYP) 1A1 and 1B1 enzymes, which biotransform PAHs resulting in the formation of DNA adducts. We hypothesised that 2,3′,4,5′-tetramethoxystilbene (TMS), an analogue of resveratrol and a potent CYP1B1 inhibitor, may inhibit r7, t8, t9-trihydroxy-c-10-(N2deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[a]pyrene (BPdG) adduct formation in cells exposed to benzo[a]pyrene (BP). To address this, MCF-7 cells were cultured for 96 h in the presence of 1 μM BP, 1 μM BP + 1 μM TMS or 1 μM BP + 4 μM TMS. Cells were assayed at 2–12 h intervals for: BPdG adducts by r7, t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence immunoassay; CYP1A1 and 1B1 gene expression changes by relative real-time polymerase chain reaction; and CYP1A1/1B1 enzyme activity by ethoxyresorufin-O-deethylase (EROD) assay. Whereas maximal BPdG levels were similar for all exposure groups, the times at which the maxima were reached increased by 16 and 24 h with the addition of 1 and 4 μM TMS, respectively. The maximal expression of CYP1A1 and CYP1B1 occurred at 16, 24 and 48 h, but the maximal level for EROD-specific activity was reached at 24, 48 and 60 h, in cells exposed to 1 μM BP, 1 μM BP + 1 μM TMS or 1 μM BP + 4 μM TMS, respectively. The area under the curve from 4 to 96 h of exposure (AUC4–96 h) for BPdG adduct formation was not increased in the presence of TMS, but for CYP1A1 and CYP1B1 expression fold increase AUC4–96 h and EROD-specific activity AUC4–96 h, there were significant (P < 0.05) increases in the presence of 4 μM TMS. Therefore, during 96 h of exposure in MCF-7 cells, the combination of BP plus TMS caused a slowing of BP biotransformation, with an increase in CYP1A1 and CYP1B1 expression and EROD activity, and a slowing, but no change in magnitude of BPdG formation.
Styrene is widely used in the manufacture of synthetic rubber, resins, polyesters and plastics. Styrene and the primary metabolite styrene-7,8-oxide are genotoxic and carcinogenic. Long-term chemical carcinogenesis bioassays showed that styrene caused lung cancers in several strains of mice and mammary cancers in rats and styrene-7,8-oxide caused tumours of the forestomach in rats and mice and of the liver in mice. Subsequent epidemiologic studies found styrene workers had increased mortality or incidences of lymphohematopoietic cancers (leukaemia or lymphoma or all), with suggestive evidence for pancreatic and esophageal tumours. No adequate human studies are available for styrene-7,8-oxide although this is the primary and active epoxide metabolite of styrene. Both are genotoxic and form DNA adducts in humans.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous components of polluted air. The Mexico City Metropolitan Area (MCMA), one of the most densely populated areas in the world, is 2240 m above sea level. At this altitude, less oxygen is available, making combustion less efficient and therefore producing more PAH pollutants. According to the Automatic Monitoring Network in Mexico City (RAMA, for its Spanish initials; http://www.sma.df.gob.mx/simat2/informaciontecnica/index.php?opcion=5&opciondifusion_bd=90), which performs environmental monitoring, the critical air pollutants in Mexico City are ozone and particulate matter (PM). PM emissions increase during the dry season (winter to spring) and decrease during the rainy season (summer to autumn). The bioactivation of some PAHs produces reactive metabolites that bind to DNA, and the presence of elevated levels of PAH–DNA adducts in tissues such as blood lymphocytes represents an elevated risk for the development of cancer. We have compared the levels of PAH–DNA adducts and the percentage of cells with chromosomal aberrations (CWAs) using a matched set of peripheral blood lymphocytes obtained on two separate occasions from young non-smoking inhabitants of the MCMA (n = 92) during the 2006 dry season and the following rainy season. PAH–DNA adducts were analysed using the r7, t8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)–DNA chemiluminescence immunoassay (CIA). The percentages of CWA were determined in cultured lymphocytes from the same individuals. Both DNA adduct levels and chromosomal aberrations were tested for correlation with lifestyle and the polymorphisms of cytochromes P450 CYP1A1 and CYP1B1 as well as glutathione-S-transferases GSTM1 and GSTT1. The levels of PAH–DNA adducts were significantly higher (P < 0.001) in the dry season (10.66 ± 3.05 per 109 nt, n = 92) than during the rainy season (9.50 ± 2.85 per 109 nt, n = 92) and correlated with the seasonal levels of particulate matter with a diameter of ≤10 μm (PM10). The percentage of CWA was not seasonally related; however, significant associations between the number of risk alleles and adduct levels in the dry (R = 0.298, P = 0.048) and in the wet seasons (R = 0.473, P = 0.001) were observed.
There are numerous studies reporting on the effects of inhalation anaesthesia in cells of exposed individuals but not much is known about the ability of isoflurane (ISF) to induce oxidative DNA damage. However, surgery is often associated with a temporary perioperative immunological alteration, and some volatile anaesthetics seem to contribute to a transient lymphocytopenia after surgery. We conducted a study to evaluate a possible genotoxic effect, including oxidative DNA damage, and apoptosis in peripheral lymphocytes of 20 patients American Society of Anaesthesiologists physical status I undergoing minor elective surgery lasting at least 120 min, under anaesthesia with ISF. We also investigated the expression of several genes in blood cells. Blood samples were collected at three time points: before anaesthesia (T1), 2 h after the beginning of anaesthesia (T2) and on the first post-operative day (T3). General DNA damage and oxidised bases (Fpg and endo III-sites) in blood lymphocytes were evaluated using the comet assay. Lymphocytes were phenotyped and apoptosis was evaluated by flow cytometry. In addition, expressions of hOGG1 and XRCC1, genes involved in DNA repair, and BCL2, a gene related to apoptosis, were assessed by quantitative real-time polymerase chain reaction. Results showed no statistically significant difference in the level of DNA damage and oxidised bases among the three sampling times. Anaesthesia with ISF did not increase the percentage of cells in early or late apoptosis in cytotoxic or helper T lymphocytes. Lower hOGG1 and BCL2 expressions were detected at T3 in comparison to the other two previous time points, and there was significantly lower expression of XRCC1 at T3 in relation to T2. In conclusion, the exposure to ISF did not result in genotoxicity and cytotoxicity in lymphocytes and in toxicogenomic effect in leukocytes, although DNA repair and apoptosis-related genes were down-regulated on the first post-operative day.
Ionising radiation (IR) is a known carcinogen and poses a significant risk to the haematopoietic system for the development of leukaemia in part by induction of genomic instability. Induction of chronic oxidative stress has been assumed to play an important role in mediating the effect of IR on the haematopoietic system. However, there was no direct evidence to support this hypothesis prior to our studies. In our recent studies, we showed that exposure of mice to total body irradiation (TBI) induces persistent oxidative stress selectively in haematopoietic stem cells (HSCs) at least in part via up-regulation of nicotinamide adenine dinucleotide phosphate oxidase (NOX) 4. Now, we found that post-TBI treatment with diphenylene iodonium (DPI), a pan NOX inhibitor, not only significantly reduces TBI-induced increases in reactive oxygen species (ROS) production, oxidative DNA damage and DNA double-strand breaks in HSCs but also dramatically decreases the number of cells with unstable chromosomal aberrations in the clonal progeny of irradiated HSCs. The effects of DPI are comparable to Mn (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin, a superoxide dismutase mimetic and a potent antioxidant. These findings demonstrate that increased production of ROS by NOX in HSCs mediates the induction of haematopoietic genomic instability by IR and that NOX may represent a novel molecular target to inhibit TBI-induced genomic instability.
Significant inter-individual variation in G2 chromosomal radiosensitivity, measured as radiation-induced chromatid-type aberrations in the subsequent metaphase, has been reported in peripheral blood lymphocytes of both healthy individuals and a range of cancer patients. One possible explanation for this variation is that it is driven, at least in part, by the efficiency of G2–M checkpoint control. The hypothesis tested in the current analysis is that increased G2 chromosomal radiosensitivity is facilitated by a less efficient G2–M checkpoint. The study groups comprised 23 childhood and adolescent cancer survivors, their 23 partners and 38 of their offspring (Group 1) and 29 childhood and young adult cancer survivors (Group 2). Following exposure to 0.5 Gy of 300 kV X-rays, lymphocyte cultures were assessed for both G2 checkpoint delay and G2 chromosomal radiosensitivity. In Group 1, the extent of G2 checkpoint delay was measured by mitotic inhibition. No statistically significant differences in G2 checkpoint delay were observed between the cancer survivors (P = 0.660) or offspring (P = 0.171) and the partner control group nor was there any significant relationship between G2 checkpoint delay and G2 chromosomal radiosensitivity in the cancer survivors (P = 0.751), the partners (P = 0.634), the offspring (P = 0.824) or Group 1 taken as a whole (P = 0.379). For Group 2, G2 checkpoint delay was assessed with an assay utilising premature chromosome condensation to distinguish cell cycle stage. No significant relationship between G2 checkpoint delay and G2 chromosomal radiosensitivity was found (P = 0.284). Thus, this study does not support a relationship between G2–M checkpoint efficiency and variation in G2 chromosomal radiosensitivity.
Reduced host cell reactivation (HCR) of a reporter gene containing 8-oxoguanine (8-oxoG) lesions in Cockayne syndrome (CS) fibroblasts has previously been attributed to increased 8-oxoG-mediated inhibition of transcription resulting from a deficiency in repair. This interpretation has been challenged by a report suggesting reduced expression from an 8-oxoG containing reporter gene occurs in all cells by a mechanism involving gene inactivation by 8-oxoG DNA glycosylase and this inactivation is strongly enhanced in the absence of the CS group B (CSB) protein. The observation of reduced gene expression in the absence of CSB protein led to speculation that decreased HCR in CS cells results from enhanced gene inactivation rather than reduced gene reactivation. Using an adenovirus-based β-galactosidase (β-gal) reporter gene assay, we have examined the effect of methylene blue plus visible light (MB + VL)-induced 8-oxoG lesions on the time course of gene expression in normal and CSA and CSB mutant human SV40-transformed fibroblasts, repair proficient and CSB mutant Chinese hamster ovary (CHO) cells and normal mouse embryo fibroblasts. We demonstrate that MB + VL treatment of the reporter leads to reduced expression of the damaged β-gal reporter relative to control at early time points following infection in all cells, consistent with in vivo inhibition of RNA polII-mediated transcription. In addition, we have demonstrated HCR of reporter gene expression occurs in all cell types examined. A significant reduction in the rate of gene reactivation in human SV40-transformed cells lacking functional CSA or CSB compared to normal cells was found. Similarly, a significant reduction in the rate of reactivation in CHO cells lacking functional CSB (CHO-UV61) was observed compared to the wild-type parental counterpart (CHO-AA8). The data presented demonstrate that expression of an oxidatively damaged reporter gene is reactivated over time and that CSA and CSB are required for normal reactivation.
Children may be more susceptible to the effects of the environmental exposure and medical treatments than adults; however, limited information is available about the differences in genotoxic effects in children by age, sex and health status. Micronucleus (MN) assay is a well established method of monitoring genotoxicity, and this approach is thoroughly validated for adult lymphocytes by the Human Micronucleus Biomonitoring project (HUMN.org). Similar international undertaking is in progress for exfoliated buccal cells. Most of the MN studies in children are focused on analyses of lymphocytes but in the recent years, more investigators are interested in using exfoliated cells from the oral cavity and other cell types that can be collected non-invasively, which is particularly important in paediatric cohorts. The baseline MN frequency is relatively low in newborns and its assessment requires large cohorts and cell sample counts. Available results are mostly consistent in conclusion that environmental pollutants and radiation exposures lead to the increase in the MN frequency in children. Effects of medical treatments are less clear, and more studies are needed to optimise the doses and minimise genotoxicity without compromising therapy outcomes. Despite the recent progress in MN assay in children, more studies are warranted to establish the relationship between MN in lymphocytes and exfoliated cells, to clarify sex, age and genotype differences in baseline MN levels and the changes in response to genotoxicants. One of the most important types of MN studies in children are prospective cohorts that will help to clarify the predictive value of MN and other cytome end points for cancer and other chronic diseases of childhood and adulthood. Emerging ‘omic’ and other novel molecular technologies may shed light on the molecular mechanisms and biological pathways associated with the MN levels in children.
Laser scanning cytometry (LSC) provides a novel approach for automated scoring of micronuclei (MN) in different types of mammalian cells, serving as a biomarker of genotoxicity and mutagenicity. In this review, we discuss the advances to date in measuring MN in cell lines, buccal cells and erythrocytes, describe the advantages and outline potential challenges of this distinctive approach of analysis of nuclear anomalies. The use of multiple laser wavelengths in LSC and the high dynamic range of fluorescence and absorption detection allow simultaneous measurement of multiple cellular and nuclear features such as cytoplasmic area, nuclear area, DNA content and density of nuclei and MN, protein content and density of cytoplasm as well as other features using molecular probes. This high-content analysis approach allows the cells of interest to be identified (e.g. binucleated cells in cytokinesis-blocked cultures) and MN scored specifically in them. MN assays in cell lines (e.g. the CHO cell MN assay) using LSC are increasingly used in routine toxicology screening. More high-content MN assays and the expansion of MN analysis by LSC to other models (i.e. exfoliated cells, dermal cell models, etc.) hold great promise for robust and exciting developments in MN assay automation as a high-content high-throughput analysis procedure.
Epidemiological studies reveal strong association between micronutrient deficiencies and development of cancer. Since chromosome breaks and abnormal chromosome segregation, identified as micronuclei (MN), are central to malignant transformation, the influence of micronutrient status upon MN frequency has been the subject of intense research. Motivating this effort is the idea that marginal micronutrient deficiencies lead to allocation of scarce cellular resources towards immediate survival at the expense of maintaining genomic integrity, placing the individual at greater risk for degenerative diseases and cancer in old age. The challenge in identifying an association between individual micronutrients and MN frequency stems from the complexity of human diet, simultaneous presence of multiple micronutrient deficiencies, variable genetic susceptibility and methodological difficulties. A unique model for studying MN in humans is provided by a group of haematological diseases, the chronic haemolytic anaemias associated with high reticulocyte count and absence of splenic function. These disorders may prove valuable for assessing the influence of micronutrient status once the effect of abnormal erythropoiesis on MN formation is adequately understood. Eventually, large population-based studies that can account for the baseline variability in MN frequency, lifestyle and genetic factors may be needed to uncover the DNA-damaging effect of poor diet. Understanding the link between micronutrient status and MN frequency will contribute towards determining optimal micronutrient intake to preserve long-term health.
We previously established an Escherichia coli strain capable of re-circularizing linear plasmid DNA by expressing the Mycobacterium tuberculosis Ku (Mt-Ku) and Mycobacterium tuberculosis ligase D (Mt-LigD) proteins from the E.coli chromosome. Repair was predominately mutagenic due to deletions at the termini. We hypothesized that these deletions could be due to a nuclease activity of Mt-LigD that was previously detected in vitro. Mt-LigD has three domains: an N-terminal polymerase domain (PolDom), a central domain with 3′-phosphoesterase and nuclease activity and a C-terminal ligase domain (LigDom). We generated bacterial strains expressing Mt-Ku and mutant versions of Mt-LigD. Plasmid re-circularization experiments in bacteria showed that the PolDom alone had no re-circularization activity. However, an increase in the total and accurate repair was found when the central domain was deleted. This provides further evidence that this central domain does have nuclease activity that can generate deletions during repair. Deletion of only the PolDom of Mt-LigD resulted in a complete loss of accurate repair and a significant reduction in total repair. This is in agreement with published in vitro work indicating that the PolDom is the major Mt-Ku-binding site. Interestingly, the LigDom alone was able to re-circularize plasmid DNA but only in an Mt-Ku-dependent manner, suggesting a potential second site for Ku–LigD interaction. This work has increased our understanding of the mutagenic repair by Mt-Ku and Mt-LigD and has extended the in vitro biochemical experiments by examining the importance of the Mt-LigD domains during repair in bacteria.
Genome stability is essential for normal foetal growth and development. To date, genome stability in human lymphocytes has not been studied in relation to late pregnancy diseases, such as pre-eclampsia (PE) and intrauterine growth restriction (IUGR), which can be life-threatening to mother and baby and together affect >10% of pregnancies. We performed a prospective cohort study investigating the association of maternal chromosomal damage in mid-pregnancy (20 weeks gestation) with pregnancy outcomes. Chromosome damage was measured using the cytokinesis-block micronucleus cytome (CBMNcyt) assay in peripheral blood lymphocytes. The odds ratio for PE and/or IUGR in a mixed cohort of low- and high-risk pregnancies (N = 136) and a cohort of only high-risk pregnancies (N = 91) was 15.97 (P = 0.001) and 17.85 (P = 0.007), respectively, if the frequency of lymphocytes with micronuclei (MN) at 20 weeks gestation was greater than the mean + 2 SDs of the cohort. These results suggest that the presence of lymphocyte MN is significantly increased in women who develop PE and/or IUGR before the clinical signs or symptoms appear relative to women with normal pregnancy outcomes. The CBMNcyt assay may provide a new approach for the early detection of women at risk of developing these late pregnancy diseases and for biomonitoring the efficacy of interventions to reduce DNA damage, which may in turn ameliorate pregnancy outcome.
Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU Cosmetics Directive, have fuelled renewed interest in CTAs. Research is currently ongoing to improve the objectivity of the assays, reveal the underlying molecular changes leading to transformation and explore the use of novel cell types. The UK NC3Rs held an international workshop in November 2010 to review the current state of the art in this field and provide directions for future research. This paper outlines the key points highlighted at this meeting.
Identification of de novo minisatellite mutations in the offspring of parents exposed to mutagenic agents offers a potentially sensitive measure of germ line genetic events induced by ionizing radiation and genotoxic chemicals. Germ line minisatellite mutations (GMM) are usually detected by hybridizing Southern blots of unamplified size-fractionated genomic DNA with minisatellite probes. However, this consumes a relatively large amount of DNA, requires several steps and may lack sensitivity. We have developed a polymerase chain reaction (PCR)-based GMM assay, which we applied to the hypermutable minisatellite, CEB1. Here, we compare the sensitivity and specificity of this assay with the conventional Southern hybridization method using DNA from 10 spouse pairs, one parent of each pair being a survivor of cancer in childhood, and their 20 offspring. We report that both methods have similar specificity but that the PCR method uses 250 times less DNA, has fewer steps and is better at detecting GMM with single repeats provided that specific guidelines for allele sizing are followed. The PCR GMM method is easier to apply to families where the amount of offspring DNA sample is limited.
DNA–protein cross-links (DPCs) are caused by a large number of human carcinogens and anti-cancer drugs. However, cellular processes involved in decreasing a burden of these genotoxic lesions remain poorly understood. Here, we examined the impact of nucleotide excision repair (NER), which is a principal repair pathway for bulky DNA adducts, and the main cellular reducers on removal of chromium(VI)-induced DPC. We found that standard and ascorbate-restored cultures of isogenic XPA-null (NER deficient) and XPA-complemented human fibroblasts had very similar repair of Cr–DPC (60–65% average DPC removal after 24 h). However, XPA absence caused depletion of G1 and accumulation of G2 cells at low Cr(VI) doses, suggesting that Cr–DPC were not a significant cause of cell cycle perturbations. Interestingly, although pro-oxidant metabolism of Cr(VI) in glutathione-depleted cells generated significantly fewer DPC, they were repair resistant irrespective of the NER status of cells. Inhibition of proteasome activity by MG132 abolished DPC repair in both XPA-null and XPA-complemented cells. XPA loss caused two to three times higher initial DPC formation, demonstrating the importance of NER in removal of the precursor lesions. Our results indicate that human NER is not involved in removal of Cr–DPC containing non-histone proteins but it acts as a defence mechanism against these large lesions by preventing their formation. Therefore, individual differences in NER activity are expected to alter sensitivity but not persistence of DPC as a biomarker of hexavalent Cr.
The induction of localized DNA damage within a discrete nuclear volume is an important tool in DNA repair studies. Both charged particle irradiation and laser microirradiation (LMI) systems allow for such a localized damage induction, but the results obtained are difficult to compare, as the delivered laser dose cannot be measured directly. Therefore, we revisited the idea of a biological dosimetry based on the microscopic evaluation of irradiation-induced Replication Protein A (RPA) foci numbers. Considering that local dose deposition is characteristic for both LMI and charged particles, we took advantage of the defined dosimetry of particle irradiation to estimate the locally applied laser dose equivalent. Within the irradiated nuclear sub-volumes, the doses were in the range of several hundreds of Gray. However, local dose estimation is limited by the saturation of the RPA foci numbers with increasing particle doses. Even high-resolution 4Pi microscopy did not abrogate saturation as it was not able to resolve single lesions within individual RPA foci. Nevertheless, 4Pi microscopy revealed multiple and distinct 53BP1- and γH2AX-stained substructures within the lesion flanking chromatin domains. Monitoring the local recruitment of the telomere repeat-binding factors TRF1 and TRF2 showed that both proteins accumulated at damage sites after UVA–LMI but not after densely ionizing charged particle irradiation. Hence, our results indicate that the local dose delivered by UVA–LMI is extremely high and cannot be accurately translated into an equivalent ionizing radiation dose, despite the sophisticated techniques used in this study.
Reactive oxygen species (ROS) generated endogenously or from exogenous sources produce mutagenic DNA lesions. If not repaired, these lesions could lead to genomic instability and, potentially, to cancer development. NEIL2 (EC 220.127.116.11), a mammalian base excision repair (BER) protein and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single-stranded structures, suggesting its involvement in transcription-coupled DNA repair. Perturbation in NEIL2 expression may, therefore, significantly impact BER capacity and promote genomic instability. To characterize the genetic and environmental factors regulating NEIL2 gene expression, we mapped the human NEIL2 transcriptional start site and partially characterized the promoter region of the gene using a luciferase reporter assay. We identified a strong positive regulatory region from nucleotide −206 to +90 and found that expression from this region was contingent on its being isolated from an adjacent strong negative regulatory region located downstream (+49 to +710 bp), suggesting that NEIL2 transcription is influenced by both these regions. We also found that oxidative stress, induced by glucose oxidase treatment, reduced the positive regulatory region expression levels, suggesting that ROS may play a significant role in regulating NEIL2 transcription. In an initial attempt to characterize the underlying mechanisms, we used in silico analysis to identify putative cis-acting binding sites for ROS-responsive transcription factors within this region and then used site-directed mutagenesis to investigate their role. A single-base change in the region encompassing nucleotides −206 to +90 abolished the effect of oxidative stress that was observed in the absence of the mutation. Our study is the first to provide an initial partial characterization of the NEIL2 promoter and opens the door for future research aimed at understanding the role of genetic and environmental factors in regulating NEIL2 expression.
Nucleotide excision repair (NER) is a complex multistage process involving many interacting gene products to repair a wide range of DNA lesions. Genetic defects in NER cause human hereditary diseases including xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy and a combined XP/CS overlapping symptom. One key gene product associated with all these disorders is the excision repair cross-complementing 3/xeroderma pigmentosum B (ERCC3/XPB) DNA helicase, a subunit of the transcription factor IIH complex. ERCC3 is involved in initiation of basal transcription and global genome repair as well as in transcription-coupled repair (TCR). The hamster ERCC3 gene shows high degree of homology with the human ERCC3/XPB gene. We identified new mutations in the Chinese hamster ovary cell ERCC3 gene and characterized the role of hamster ERCC3 protein in DNA repair of ultraviolet (UV)-induced and oxidative DNA damage. All but one newly described mutations are located in the protein C-terminal region around the last intron–exon boundary. Due to protein truncations or frameshifts, they lack amino acid Ser751, phosphorylation of which prevents the 5′ incision of the UV-induced lesion during NER. Thus, despite the various locations of the mutations, their phenotypes are similar. All ercc3 mutants are extremely sensitive to UV-C light and lack recovery of RNA synthesis (RRS), confirming a defect in TCR of UV-induced damage. Their limited global genome NER capacity averages ∼8%. We detected modest sensitivity of ercc3 mutants to the photosensitizer Ro19-8022, which primarily introduces 8-oxoguanine lesions into DNA. Ro19-8022-induced damage interfered with RRS, and some of the ercc3 mutants had delayed kinetics. All ercc3 mutants showed efficient base excision repair (BER). Thus, the positions of the mutations have no effect on the sensitivity to, and repair of, Ro19-8022-induced DNA damage, suggesting that the ERCC3 protein is not involved in BER.