Genotoxic Exposure Research Articles

Genetic Susceptibility to Therapy-Related Leukemia (t-MDS/AML) After Hodgkin Lymphoma (HL) or Non-Hodgkin Lymphoma (NHL).

Abstract 199Therapy-induced t-MDS/AML is the leading cause of non-relapse mortality after HL or NHL. However, there exists a wide variation in t-MDS/AML susceptibility – potentially explained by individual variability in drug metabolism, DNA repair and apoptosis, or in genetic profiles shared with de novo AML. Using a matched case-control study design, we examined the association between t-MDS/AML and candidate genes (n=29) in relevant biological pathways, including hematopoietic regulation (RUNX1, HLX1); apoptosis (TP53, MDM2); drug metabolism (CYP3A4, CYP1A1, GSTM1, GSTP1, GSTT1, NQO1); DNA repair (MGMT, MUTYH, MLH1, MSH2, MTHFR, RAD51, RAD51C, RAD52, XRCC1, XRCC2, XRCC3, XRCC4, XPD); and genes associated withde novoAML (LAMC2/NMNAT2, SGCE/PEG10, FRAP1, and PTPRT). The study cohort consisted of 46 cases with t-MDS/AML after HL/NHL and 46 controls with HL/NHL, but without t-MDS/AML (matching criteria: primary diagnosis, age and year of primary diagnosis, length of follow-up and genetic ancestry [see Table]). Sequenom MassArray and PCR were used to analyze 55 SNPs and 2 deletions in these genes. After correction for multiple testing, we could not identify significant association between any single SNP and t-MDS/AML. However, we did confirm enrichment of risk genotypes in t-MDS/AML for 2 of 4 loci previously implicated in de novo AML by a genome wide association study in Caucasians: LAMC2/NMNAT2 (minor allele carrier [MAC] odds ratio [OR]=4.0, p=0.1) and PTPRT (MAC OR=4.5, p=0.06). Next, we tested the hypothesis that TP53 plays a role in mediating apoptotic response to DNA damage following genotoxic exposures. We modeled interactions between a common coding SNP of TP53 causing a Pro 72 to Arg variant (P72R: Pro allele is associated with ∼15-fold decreased apoptotic capacity compared to Arg) and other candidate SNPs. We identified significant interaction between TP53 and several SNPs in MTHFR (Min. likelihood ratio pinteraction=0.0003 and 0.04 adjusted after 10000 permutations). Although the homozygous T allele of rs7538516 (associated with lower MTHFR expression) was not associated with t-MDS/AML by itself, it increased the risk 11-fold (OR=11.4, p=0.005) when combined with a Pro/Arg or Pro/Pro genotype of P72R compared to its combination with Arg/Arg. This observation suggests that reduced MTHFR activity (associated with increased risk of chromosomal aberrations during DNA repair), in combination with reduced apoptotic capacity (Pro/Arg or Pro/Pro variant of TP53), increases the risk of t-MDS/AML. Next, we examined expression levels of these 29 genes (79 probe sets in Affymetrix U133 Plus 2.0 microarrays) in a subset of 13 cases matched with 28 controls. Using a general linear model adjusted for age, gender, race, and exposure to alkylating agents, topoisomerase inhibitors and radiation, we detected 11 of the 29 (38%) genes to be differentially expressed between cases and controls (p<0.05). Strong signals (p<0.05 after Holm-Bonferroni adjustment) were observed for TP53 (apoptosis), genes involved in drug metabolism (CYP3A4 [activation], GSTM1, GSTP1, and GSTT1 [detoxification]), in DNA repair (MSH2), and genes associated with de novo MDS/AML (NMNAT2, PEG10). Taken together, these observations provide evidence supporting the following: i) a significant association between individual capacity of apoptosis, drug metabolism, DNA repair, and t-MDS/AML; and ii) shared genetic susceptibility between t-MDS/AML and de novo AML. These observations not only further our understanding of the pathogenesis of t-MDS/AML, but also help identify those at the highest risk, setting the stage for targeted surveillance or pharmacological interventions.TableCharacteristics of Study CohortGenotyping StudyExpression AnalysisCases (46)Controls (46)P-valueCases (13)Controls (28)P-valueHL13 (28.3%)11 (23.9%)0.51 (7.7%)3 (10.7%)1.0NHL33 (71.7%)35 (76.1%)12 (92.3%)25 (89.3%)Age at Diagnosis46.2 (12.9–73.4)46.8 (26.1–72.5)0.648.4 (26.1–66.6)47.9 (25.5–64.2)0.8Time to t-MDS/AML6.9 (0.7–28.9)NANA3.9 (1.2–9.2)NANAMale34 (73.9%)28 (60.9%)0.27 (53.8%)20 (71.4%)0.3Female12 (26.1%)18 (39.1%)6 (46.2%)8 (28.6%)Caucasian37 (80.4%)37 (80.4%)1.07 (53.8%)17 (60.7%)0.9Hispanic6 (13.0%)6 (13.0%)5 (38.5%)9 (32.1%)Other3 (6.5%)3 (6.5%)1 (7.7%)2 (7.1%)HCT33 (71.7%)35 (76.1%)0.513 (100%)28 (100%)1.0Without HCT13 (28.3%)11 (23.9%)00 Disclosures:No relevant conflicts of interest to declare.

Effect of Afobazole on DNA Damage in Patients with Systemic Lupus Erythematosus

Using comet assay we studied the effect of anxiolytic afobazole, exhibiting also antioxidant and antimutagenic properties, on spontaneous and ex-vivo hydrogen peroxide-induced DNA damage in blood cells from patients with systemic lupus erythematosus. Afobazole treatment (30-60 mg/day for 1 month) in addition to standard therapy decreased spontaneous level of DNA damage in blood cells. The level of ex vivo hydrogen peroxide-induced DNA damage decreased by 49% in this group of patients. The number of cells hypersensitive to hydrogen peroxide yielding DNA comets with highly damaged DNA also decreased by 51%. No significant changes in the analyzed parameters were found in the placebo group. Addition of afobazole to complex therapy of patients with systemic lupus erythematosus reduced the level of DNA damage in blood cells and improved cell resistance to oxidative genotoxic exposure.

GST Polymorphisms: Bonassi et al. Respond

We thank Vodicka et al. for drawing attention to a critical issue—the role of genetic polymorphisms in the induction of chromosomal damage. First, we would like to clarify that our study (Rossi et al. 2009) was not designed to evaluate the influence of glutathione S-transferase (GST) polymorphisms on the frequency of chromosomal aberrations (CAs). Instead, we aimed to identify any possible susceptibility alleles that would increase the strength of the relationship between CAs and risk of cancer in subgroups of genetically variant individuals. The specific purpose of this case–control study explains the relative small size: We planned to obtain data on both CAs and enetic polymorphisms in the same individuals, with all subjects at ages appropriate to allow for an informative follow-up. The interesting data reported by Vodicka et al. in their letter confirm the extreme heterogeneity of findings when the frequency of chromosome damage is associated with polymorphisms in genes that influence several pathways. In fact, the lack of association in the whole database described by Vodicka et al. contrasts with the significant results reported in exposure-specific subsets, such as the highest frequency of CAs in GSTT1-null smokers working in the tire industry (Musak et al. 2008). This interpretation is also supported by the study of Iarmarcovai et al. (2008a), who reviewed 72 population studies reporting a significant effect of GSTM1, EPHX (microsomal epoxide hydrolase), and GSTT1 on micronucleus frequency. These authors also reported that the effect observed in specific pathways—such as ALDH2 in connection to chromosome damage due to alcohol consumption, BRCA1 and BRCA2 mutations in breast cancer patients, and MTHFR depending on folate levels—was much more pronounced. A great deal of information about the role of genetic polymorphisms in the induction of chromosomal damage will stem from genome-wide association studies, when enough resources will be available in the field to explore the genetic determinants of chromosome damage using a whole genome scan. Nevertheless, classical association studies are still valid assuming that specific pathways are identified, possibly a priori, linking genotoxic exposure to polymorphisms of genes involved in the correspondent pathway We would also like to address future strategies for exploring the link between the frequency of chromosome damage and the risk of cancer (and other diseases). We fully agree with Vodicka et al.’s recommendation that possible modifying effects of lifestyle, occupational factors, and genetic poly morphisms be taken into account. However, after finding negative results concerning the role of occupational exposure to genotoxic agents and smoking habit (Bonassi et al. 2000), and polymorphisms of metabolic genes (Rossi et al. 2009), we believe that the remaining factor to be investigated is diet. Many studies have reported on the role of various food items and nutrients as modulators of chromosome stability (Fenech 2007); therefore, this parameter now has top priority, although reliable data are difficult to collect retrospectively. The other recommendation from Vodicka et al. seems less promising, because measuring the frequency of chromosome damage in cancer patients (besides the classical discussion about whether the damage is a cause or a consequence of the disease) is possibly more a marker of progression than a predictive marker (Iarmarcovai et al. 2008b).

Evaluation of indoor air quality by odor units (odor threshold ratio)

Buccal micronucleus cytome (BMCyt) assay monitors genetic damage, cell proliferation and cell death in humans exposed to occupational and environmental agents. BMCyt is used as an indicator of genotoxic exposure, since it is associated with chromosomal instability. There is little research on the occupational exposure among road construction workers for genotoxicity testing. In the present study, we evaluated MN frequencies and other nuclear changes, karyorrhexis (KR), karyolysis (KL), broken egg (BE), binucleate (BN), condensed chromatin cell (CCC), and picnotic cell (PC) in buccal mucosa cells of 40 road construction workers (twenty smokers and twenty non-smokers) and 40 control groups consisting of healthy persons (twenty smokers and twenty non-smokers). Microscopic observation was performed of 2000 cells per individual in both road construction workers and control group. In control and worker groups, for each person repair index (RI) was calculated via formula KR+L/BE+MN. The results showed a statistically significant increase in the frequency of MN in buccal epithelial cells of exposed group compared with control group (p<0.001). There is no significant difference between smokers and non-smokers for incidence of MN or nuclear changes and value of RI in exposed group. In road construction workers, RI is lower than the control group. There is a significant difference between workers and control group (p<0.001) for RI. Our data reveal that asphalt fumes during road paving operations are absorbed by workers and that asphalt fume exposure is able to significantly induce cytogenetic damage in buccal mucosa cells of workers after controlling some possible confounding factors, such as age, sex and smoking habits.In addition to determination of nuclear changes and the micronucleus, the determination of RI value presents a new approach to genotoxic bio-monitoring assessment studies of occupationally exposed population.

Etoposide induces MLL rearrangements and other chromosomal abnormalities in human embryonic stem cells

MLL rearrangements are hallmark genetic abnormalities in infant leukemia known to arise in utero. They can be induced during human prenatal development upon exposure to etoposide. We also hypothesize that chronic exposure to etoposide might render cells more susceptible to other genomic insults. Here, for the first time, human embryonic stem cells (hESCs) were used as a model to test the effects of etoposide on human early embryonic development. We addressed whether: (i) low doses of etoposide promote MLL rearrangements in hESCs and hESCs-derived hematopoietic cells; (ii) MLL rearrangements are sufficient to confer hESCs with a selective growth advantage and (iii) continuous exposure to low doses of etoposide induces hESCs to acquire other chromosomal abnormalities. In contrast to cord blood-derived CD34(+) and hESC-derived hematopoietic cells, exposure of undifferentiated hESCs to a single low dose of etoposide induced a pronounced cell death. Etoposide induced MLL rearrangements in hESCs and their hematopoietic derivatives. After long-term culture, the proportion of hESCs harboring MLL rearrangements diminished and neither cell cycle variations nor genomic abnormalities were observed in the etoposide-treated hESCs, suggesting that MLL rearrangements are insufficient to confer hESCs with a selective proliferation/survival advantage. However, continuous exposure to etoposide induced MLL breaks and primed hESCs to acquire other major karyotypic abnormalities. These data show that chronic exposure of developmentally early stem cells to etoposide induces MLL rearrangements and make hESCs more prone to acquire other chromosomal abnormalities than postnatal CD34(+) cells, linking embryonic genotoxic exposure to genomic instability.

Role of NKX2-1 in N-bis(2-hydroxypropyl)-nitrosamine-induced thyroid adenoma in mice

NKX2-1 is a homeodomain transcription factor that is critical for genesis of the thyroid and transcription of the thyroid-specific genes. Nkx2-1-thyroid-conditional hypomorphic mice were previously developed in which Nkx2-1 gene expression is lost in 50% of the thyroid cells. Using this mouse line as compared with wild-type and Nkx2-1 heterozygous mice, a thyroid carcinogenesis study was carried out using the genotoxic carcinogen N-bis(2-hydroxypropyl)-nitrosamine (DHPN), followed by sulfadimethoxine (SDM) or the non-genotoxic carcinogen amitrole (3-amino-1,2,4-triazole). A significantly higher incidence of adenomas was obtained in Nkx2-1-thyroid-conditional hypomorphic mice as compared with the other two groups of mice only when they were treated with DHPN + SDM, but not amitrole. A bromodeoxyuridine incorporation study revealed that thyroids of the Nkx2-1-thyroid-conditional hypomorphic mice had >2-fold higher constitutive cell proliferation rate than the other two groups of mice, suggesting that this may be at least partially responsible for the increased incidence of adenoma in this mouse line after genotoxic carcinogen exposure. Thus, NKX2-1 may function to control the proliferation of thyroid follicular cells following damage by a genotoxic carcinogen.

Cancer Risk and GSTM1 and GSTT1 Polymorphisms: Hansteen et al. Respond

We thank Cetta et al. for the interesting comments regarding our article (Rossi et al. 2009). In their letter they address two main issues. The first refers to the role of genetic polymorphisms in the causal relationships between exposure to carcinogens and cancer occurrence. The second is more conceptual and criticizes the evolution of association studies, claiming a decreased attention to pathogenetic mechanisms in favor of an indiscriminate increase of the study size, with a consequent lack of biological plausibility. We agree that these are important issues. We have addressed the problem of inherited predisposition for DNA damage from a different angle, namely using the frequency of chromosomal aberration (CA) as a response indicator for occupational and environmental exposure to genotoxic agents. An increase in CA level in exposed individuals compared with controls has been documented since the 1990s (Nordic Study Group 1990). The conceptual basis for using this assay has been the hypothesis that the extent of genetic damage in peripheral lymphocytes reflects critical events for the carcinogenic process in target tissues. The key issue—whether the association with cancer risk is attributable to exposure to carcinogenic agents or reflects inherited susceptibility and accumulated damages—was addressed with a nested case–control study on incident and deceased cancer cases in the Nordic and Italian cohorts (Bonassi et al. 2000). The main findings of that study indicated an increase in cancer risk for subjects with high CA levels compared with those with low levels. This increase was independent of exposure history, as further verified in follow-up studies (Bonassi et al. 2008; Hagmar et al. 2004). In all these studies, cancer has been studied as one entity. This summarization was mostly due to statistical needs, although the very early occurrence of chromosome damage in the carcinogenic pathway of most solid cancers provided a valuable rationale (Mitelman et al. 2004). A further reason for summarizing data by cancer type was that damages were measured in surrogate tissues and not in the target, providing only an indirect measure of cancer-related events. However, studying the cancer site in relation to CA frequency was a major interest of our group, because different types of cancers have different pathogenetic models. In our recent article (Rossi et al. 2009), we grouped cancer types into three groups, and we showed for all of these groups that subjects with high levels of CAs are more susceptible to developing cancer than are subjects with low or medium levels of CAs; this indicates that CA is an inherited susceptibility marker for cancer regardless of cancer type. The beginning of Cetta et al.’s letter is misleading. The statement from our article (Rossi et al. 2009) that “GSTM1 [gluta-thione S-transferase M1] and GSTT1 [gluta-thione S-transferase theta 1]polymorphisms [as all individual polymorphisms] . . . are not expected to have a dramatic influence on baseline CA [chromosomal aberration] or overall cancer risk” is not a conclusion of the study, but describes the conclusions of the extensive literature supporting this evidence (Hirschhorn 2009). We agree that it is important to examine the cause of different types of cancer and the role(s) of the different modifying enzymes, including GSTM1 and GSTT1. However, the present study was designed to evaluate a possible modifying effect of GSTM1 and GSTT1 on the cancer predictivity of CA (indicating individual susceptibility to developing cancer). Our main concern was identify individuals more susceptible to damage from known genotoxic exposure. Because only GSTM1 and GSTT1 polymorphisms have been extensively evaluated in human surveillance studies, we tested only these genotypes. Within the consortium of studies included in this project (Bonassi et al. 2008), further follow-up studies to differentiate cancer types or include other genotypes are possible, providing adequate financial support. The issue raised by Cetta et al. of decreased attention to pathogenetic mechanisms in favor of larger studies, with a consequent lack of biological plausibility, is only partially correct. Actually, in association studies that link a genetic polymorphism to the effect of exposure or to the risk of cancer, the lack of specificity is the main reason for failure. Another reason for their failure is small study size, which generates meaningless and often contrasting results. The conflict noted by Cetta et al. is apparent because, as demonstrated by the success of genome-wide association studies, the need of reaching a proper statistical power is as important as studying a genetic polymorphism in a specific pathway.

Urinary levels of oxidative DNA and RNA damage among workers exposed to polycyclic aromatic hydrocarbons in silicon production: Comparison with 1‐hydroxypyrene

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous occupational and environmental pollutants and the urinary excretion of 1-hydroxypyrene (1-OHP) is classically measured for the determination of PAH exposure internal dose. Some of PAH are tumorigenic due to their metabolites ability to generate DNA adducts and oxidative DNA damage through the production of reactive oxygen species during metabolism. 8-hydroxy-7,8-dihydro-2'-deoxyguanosine (8-OHdGuo) is one of the major oxidative DNA lesions and its use as a potential biomarker of genotoxic PAH occupational exposure should be evaluated. Indeed conflicting results are frequently reported in occupational studies in terms of correlation between 8-OHdGuo urinary levels and PAH exposure. The aim of our study was therefore to determine the potential for PAH occupational exposure to increase urinary oxidative DNA damage. The population consisted of 68 male workers employed in silicon production. The urinary concentrations of 8-OHdGuo and its homologue in RNA, 8-hydroxy-7,8-dihydroguanosine (8-OHGuo) were determined using high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry, whereas those of 1-OHP were measured using HPLC with fluorescence detection. Individual variation rates were calculated on a working day and a working week. The results indicated that, while 1-OHP levels strongly increased on a working day and even more on a working week, 8-OHdGuo and 8-OHGuo urinary levels did not show similar significant increases. Moreover, no correlation between 1-OHP and oxidative DNA and RNA lesions was found. Consequently, urinary 8-OHdGuo and 8-OHGuo did not seem to be relevant biomarkers of genotoxic PAH exposure in the case of the silicon plant studied.

Persistence of DNA damage in the freshwater mussel Unio pictorum upon exposure to ethyl methanesulphonate and hydrogen peroxide

An important endpoint in assessing pollution-related toxicity is genotoxicity. To obtain insight into the time-course of oxidative- and alkylation-induced DNA damage in the freshwater mussel, Unio pictorum, mussels were exposed for 24 hr to concentration gradients of pro-oxidant hydrogen peroxide (H(2)O(2)) and a mono-functional alkylating agent, ethyl methanesulfonate (EMS). DNA damage was assessed in haemocytes immediately upon exposure and over the recovery period of up to 72 days by means of comet and micronucleus assays. Following exposure to H(2)O(2), DNA damage as detected by the comet assay returned to control values after one day, except for the mussels exposed to the highest dose when damage was detectable for the next 3 days. In contrast, alkylation-induced DNA damage was detectable even after 72 days of recovery in de-chlorinated water, with a dose-response relationship observable throughout the whole recovery period. Micronucleus frequency was the highest on Day 3 after exposure to EMS; it decreased considerably by Day 7 and returned almost to the control levels 19 days after exposure, while no significant induction of micronuclei was observed in mussels exposed to H(2)O(2). Although the comet assay is considered a biomarker of recent genotoxic exposure, detecting DNA damage of shorter longevity than with the micronucleus assay, results presented here show that in the case of alkylation damage the comet assay reveals genotoxic exposure of U. pictorum in a dose-dependent manner even after 2 months.

The comet assay in human biomonitoring: gene-environment interactions

The comet assay is the method of choice for measuring DNA damage, of various sorts, in human cells such as lymphocytes obtained in the course of population-based studies of environmental and occupational exposure to different genotoxic agents, including radiation, chemicals and oxidative stress. It is noted for its versatility and the breadth of its possible applications. In terms of simplicity, cost, small amount of material required, sensitivity and reliability, the comet assay in its various modifications has few serious competitors. When standardized and validated, the comet assay can provide invaluable information in the areas of hazard identification and risk assessment of environmental and occupational exposure, diseases linked with oxidative stress (e.g. diabetes and cardiovascular disease), nutrition, monitoring the effectiveness of medical treatment and investigating individual variation in response to DNA damage that may reflect genetic or environmental influences. The information obtained could lead to individual advice on lifestyle changes to promote health and especially on relative risks of genotoxic exposure to environmental pollution.

Transcription-coupled nucleotide excision repair in mammalian cells: molecular mechanisms and biological effects

The encounter of elongating RNA polymerase II (RNAPIIo) with DNA lesions has severe consequences for the cell as this event provides a strong signal for P53-dependent apoptosis and cell cycle arrest. To counteract prolonged blockage of transcription, the cell removes the RNAPIIo-blocking DNA lesions by transcription-coupled repair (TC-NER), a specialized subpathway of nucleotide excision repair (NER). Exposure of mice to UVB light or chemicals has elucidated that TC-NER is a critical survival pathway protecting against acute toxic and long-term effects (cancer) of genotoxic exposure. Deficiency in TC-NER is associated with mutations in the CSA and CSB genes giving rise to the rare human disorder Cockayne syndrome (CS). Recent data suggest that CSA and CSB play differential roles in mammalian TC-NER: CSB as a repair coupling factor to attract NER proteins, chromatin remodellers and the CSA- E3-ubiquitin ligase complex to the stalled RNAPIIo. CSA is dispensable for attraction of NER proteins, yet in cooperation with CSB is required to recruit XAB2, the nucleosomal binding protein HMGN1 and TFIIS. The emerging picture of TC-NER is complex: repair of transcription-blocking lesions occurs without displacement of the DNA damage-stalled RNAPIIo, and requires at least two essential assembly factors (CSA and CSB), the core NER factors (except for XPC-RAD23B), and TC-NER specific factors. These and yet unidentified proteins will accomplish not only efficient repair of transcription-blocking lesions, but are also likely to contribute to DNA damage signalling events.

Radiosensitivity of Prostate Cancer and BPH Patients Studied by DNA Repair Capacity, CA and FISH.

An individual’s genetic constitution and lifestyle, e.g., diet and levels of physical activity, can affect the body’s response to various exogenous agents including therapeutic treatment. There is a strong need to combine studies on variability in a cellular response to genotoxic exposure with predisposition of the patient to diseases development and healing. In this study a variation in responses to challenging dose of X rays in lymphocytes from healthy donors and prostate cancer patients were compared on the molecular and mitotic levels. Blood was collected from healthy donors BPH (Benign Prostate Hyperplasia), and prostate cancer patients. Among cancer patients 33% never smoke 46.7% were former smokers. Immediately after collecting the blood a challenging irradiation was performed followed by culturing procedures for classic cytogenetics and FISH techniques with a probe for the whole chromosome 1. In DNA damage investigations, isolated and cryopreserved lymphocytes were thawed and their viability examined before molecular studies. To evaluate individual susceptibility, defrosted lymphocytes were exposed to 4 Gy dose of X-rays and the extent of DNA damage was studied right after irradiation with the alkaline version of the single cell gel electrophoresis (SCGE) assay. To asses variability in the DNA repair competency the residual (unrepaired) DNA damage was detected again after one hour of incubation, during which irradiated cells had the condition allowing to complete the fast DNA repair process. Visible difference between DNA susceptibilities to the challenging dose was observed between investigated groups. However, variation between individuals in repair efficiency of the DNA damage induced by the challenging treatment was significantly higher in lymphocytes of prostate cancer patients than that of BPH and healthy donors (p<.05). That findings correlate with results from cytogenetic studies. Significantly higher amount of chromosomal damage was detected in irradiated lymphocytes of prostate cancer than that in BPH patients by classic cytogenetics. Results obtained by FISH technique have also shown a statistically significantly higher level of translocations frequency (4.3 ± 0.33 vs 2.9 ± 0.31, p<.005) in chromosome 1 of prostate cancer lymphocytes than that of BPH. Our results, clearly suggest that vulnerability of chromosome 1 and DNA repair competence assays have shown a possible potency as predictive assays in preclinical studies. *the MSC foundation’ fellow from Kazakh National University, Almaty, Kazakhstan

The Role of Apoptosis in the Development of Radiation-Induced Genome Instability under Chronic Radiation Expos

Apoptosis resulting from a high frequency of damage to the cell’s genetic material and manifested at late time after exposure is regarded as an evidence of radiation-induced genomic instability. On the other hand, apoptosis disruption may lie behind preservation of cells with genetic anomalies, and become thereby one of the mechanisms of genomic instability. Techa riverside residents, have been chronically exposed to radiation since 1949 as a result of the Mayak PA activities. Maximum dose rates were 0.4-0.5 Gy/year during the early years. Two groups (168 exposed individuals and 118 unexposed controls) were studied with the aim to assess the intensity of peripheral blood lymphocyte apoptosis 55-58 years after the onset of radiation exposure. Apoptosis was assessed based on morphological criteria, using the TUNEL assay, by typing CD95 positive lymphocytes. To assess the apoptotic reserve of the peripheral blood lymphocytes, in-vitro additional irradiation at a dose of 1 Gy, and 24-hour incubation was used. Increase of apoptosis in exposed individual was revealed based on morphological criteria (‰) (12.3±0.6 in exposed vs 9.1±0.8 control subjects) and the findings of the TUNEL assay (‰) (2.4±0,3 in exposed vs 0.9±0.2 control subjects). No dependence of CD95 positive lymphocytes on radiation factor was registered (8.0±0.4 in exposed vs 7.4±0.6 control subjects). A modeling approach was used to estimate the fraction of cells with broken apoptosis from these two groups. In this approach dynamics of cell states after in vitro irradiation is modeled by the 4-compartment model with compartments of i) cell with deficient apoptosis, ii) normal cells, iii) damaged cells, and iv) cells identified as apoptotic. The estimation of model parameters resulted in identifying the fraction of cells with broken apoptosis from the initial blood sample. It can be concluded based on the study results that: (1) chronically exposed individuals manifest an increased rate of peripheral blood lymphocyte apoptosis decades after dose rates returned to the background level, (2) using the method of genotoxic exposure (standard in-vitro irradiation), it was possible to detect a higher frequency of apoptotic abnormalities in chronically exposed individuals vs that in the controls. Thus, the study has provided a convincing evidence of the important role played by apoptosis in the development of radiation-induced genomic instability in man.

Trends in the study of genetic susceptibility

Genotoxicology investigates the molecular basis of cellular responses to DNA damage. During more than three decades of genetic toxicology testing, a large number of tests with varying sensitivity and specificity have been developed. These genotoxicity tests can predict: the likelihood of a substance to be a (rodent) carcinogen, the mechanism of carcinogenic activity of different substances, if the results of genotoxicity studies only predict carcinogenicity or they are a distinct hazard endpoint (Nohynek, 2005). The current genetic toxicity testing batteries represent: Ames test which is a component of all genetic testing batteries. A mammalian cell mutagenicity assay which should confirm or complete the Ames test. Chromosomal aberrations tests which are based on a different endpoint than gene mutations. Positive in vitro results need to be confirmed by in vivo tests; results from test batteries have higher predictive value than results of a single test (Nohynek, 2005). The human molecular epidemiology represents a new trend in the study of genetic susceptibility and interactions between genetic and environmental factors of risk. Doses and biochemical effects biomonitoring have nowadays a tremendous utility providing an efficient means of measuring human exposure to chemical substances. Human biomonitoring considers all routes of uptake and all sources which are relevant making it an ideal instrument for risk assessment and risk management; blood is by far the most approved matrice. Human biomonitoring can be done for most chemical substances which are in the focus of the worldwide discussion of environmental medicine (Angerer et al., 2007). Molecular epidemiology studies of human populations exposed to potential mutagens have the aim to assess the risk of genetic disease or cancer by analysing the relationship between internal exposure and biological effects in target cells under consideration of confounding factors (Faust et al., 2004). Human biomonitoring can identify new chemical exposures, trends and changes in exposure, establish distribution of exposure among the general population, identify vulnerable groups and populations with higher exposures and identify environmental risks at specific contaminated sites. The sensitivity of methods moreover enables the elucidation of human metabolism and toxic mechanisms of the pollutants (Angerer et al., 2007). Biomarkers of effect that indicate exposure to a causative agent and that reflect the individual risk of disease are numerous. The most commonly used biomarkers in cancer epidemiology include: measurements of DNA damage, such as DNA breaks, altered bases, bulky adducts; chromosomal aberrations (CA), micronuclei (MN) and sister chromatid exchange (SCE), which are also the results of DNA damage (Kassie et al., 2000; Collins, 1998). Chromosomal aberrations and micronuclei are biomarkers of damage due to genetic instability or exposure to environmental mutagens or carcinogens. A recent approach is to associate the biomarkers of genetic susceptibility which take into account cancer susceptibility and interindividual differences in the response to a genotoxic exposure, and the analysis of CA and/or MN, which serves as a biomarker of interactions between the environment and the genetic material of the cell. Information is being gathered on how DNA damage and more particularly the frequency of chromosomal aberrations and/or micronuclei depend on the polymorphisms in genes implicated in xenobiotic metabolism (activation and/or detoxification) and DNA lesion repair. For biomonitoring purposes, numerous confounding factors (age, sex, tobacco consumption, etc.) influence the CA and MN biomarker, and thus associating genetic polymorphisms to CA and MN would be useful to better define the prevention and prediction of risk (Iarmarcovai et al., 2007a; Iarmarcovai et al., 2007b). The in vitro genetic toxicology tests used for regulatory purposes measure formation of gene mutations and chromosomal changes following DNA damage induced by the compounds under test, and are used to predict the carcinogenic potential of pharmaceuticals, industrial chemicals, food additives and cosmetic ingredients (Kirkland et al., 2007). In vitro test systems will aid in the identification of the most sensitive species and strains. Also, in vitro systems are good models for studying qualitative and quantitative species differences in toxicity. Further, in vitro systems are excellent models for characterisation of the mode of action/mechanism for critical effects, but findings need to be validated in vivo. In vitro systems will aid in the extrapolation from high to low dose and from experimental animals to humans (Holme et al., 2002).

Gene Expression Profiles of CD34+ Cells from PBSC Grafts Predict for Development of t-MDS Following Autologous Transplantation for HL/NHL.

Therapy-related myelodysplasia (t-MDS) is a lethal complication of autologous hematopoietic cell transplant (HCT) for Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). The development of t-MDS after HCT appears related to pre-HCT genotoxic exposures. Here we investigated whether alterations in gene expression patterns in hematopoietic stem cells (HSC) from peripheral blood stem cell (PBSC) autografts was associated with subsequent development of t-MDS after HCT. We analyzed pre-HCT PBSC samples from 18 patients who developed t-MDS and 38 controls (matched for primary diagnosis, age at HCT, race/ ethnicity, and length of follow-up) that did not develop t-MDS after HCT for HL/NHL. CD34+ cells were selected using flow cytometry. RNA was extracted from 1000 cells, processed using the Affymetrix 2-Cycle Target labeling kit and hybridized on Affymetrix U133 Plus 2.0 microarrays. Following QC assessment, 16 t-MDS and 29 matched controls were selected for further analysis. Raw data were normalized using a RMA algorithm. 35042 transcripts were selected for analysis based on ≥4 arrays having intensity >16. Limma package was used to identify genes differentially expressed between t-MDS and control samples (FDR ≤ 0.01; and > 1.5-fold up or down-regulation) while controlling for matched groups. 877 differentially expressed transcripts representing 781 unique genes were identified. Of several classification algorithms tested, the K nearest neighbor (KNN) and Naïve Bayes (NB) were found to best predict t-MDS. To select the best genes for classification, a step forward method was used for prediction analysis. First, redundant transcripts were removed with only the most significant one of the redundant transcripts being kept and genes were ranked based on p-value. Prediction analyses were performed by starting with top 2 genes and incrementing one more gene at a time. Using this approach the top 5 genes best predicted the class labels using leave-one-out (LOO) cross validation with a prediction error of 22%. To determine the statistical significance of the prediction error rate, a permutation based approach was implemented to establish the null distribution of the error rate. This determines the probability of obtaining a cross-validated classification error as small as would be achieved if there were no difference between t-MDS and control. Sample labels were randomly permuted 10,000 times and cross-validated error rates were recalculated using LOO cross validation. Based on the null distribution of the permutated error rates, the original error rate (ER0=0.22) had a P value of 0.005, indicating that the likelihood of obtaining such a small prediction error for a cross-validated classifier by chance is very low. We are currently analyzing the differentially expressed genes to investigate for altered response to genotoxic stress and stem cell regulation in HSC from patients who subsequently develop t-MDS. In conclusion, we have shown that gene expression profiles of HSC from PBSC autografts from HL/NHL patients can differentiate patients who develop t-MDS after autologous HCT from those who do not develop t-MDS. The prediction power of these gene sets will be further verified using different sets of t-MDS case and control samples.

Influence of GSTM1 null and low repair XPC PAT+ on anti-B[a]PDE-DNA adduct in mononuclear white blood cells of subjects low exposed to PAHs through smoking and diet

The influence of low-activity NER genotypes ( XPC PAT−/ +, XPA- A23G, XPD Asp312Asn, XPD Lys751Gln) and GSTM1 ( active or null) was evaluated on anti-benzo[ a]pyrene diol epoxide-(B[ a]PDE)-DNA adduct formed in the lymphocyte plus monocyte fraction (LMF). The sample population consisted of 291 healthy subjects with low exposure to polycyclic aromatic hydrocarbons (PAHs) (B[ a]P) through their smoking ( n = 126 smokers) or dietary habits ( n = 165 non-smokers with high (≥52 times/year) consumption of charcoaled meat or pizza). The bulky anti-B[ a]PDE-DNA adduct levels were detected by HPLC/fluorescence analysis and genotypes by PCR. Anti-B[ a]PDE-DNA was present (≥0.5 adducts/10 8 nucleotides) in 163 (56%) subjects (median (range) 0.77 (0.125–32.0) adducts/10 8 nucleotides), with smokers showing a significantly higher adduct level than non-smokers with high consumption of PAH-rich meals ( P < 0.01). Our exposed-sample population with unfavourable XPC PAT+/− or +/+ and GSTM1 null genotypes has the significantly highest adduct level ( P < 0.01). Taking into account tobacco smoke and diet as sources of exposure to B[ a]P, low-activity XPC PAT+ shows a major role in smokers ( P < 0.05) and GSTM1 null in non-smokers with frequent consumption of PAH-rich meals ( P < 0.01). The modulation of anti-B[ a]PDE-DNA adduct in the LMF by GSTM1 null and low-activity XPC PAT+ polymorphisms may be considered as potential genetic susceptibility factors that can modify individual responses to low PAH (B[ a]P) genotoxic exposure, with the consequent risk of cancer in the general population.

Aquatic ecotoxicology approaches in Western Mexico

A series of bioindicator organisms for aquatic ecosystems are being maintained under laboratory conditions in order to analyze effects of pollution on aquatic wildlife and potential effects on human health. Growth kinetics of algae Pseudokirchneriella subcapitata was used to evaluate effects of the surfactant nonilphenol (NP). Brachionus calyciflorus was used to set up a model of endocrine disruption using the fungicide vinclozolin (Vc). We exposed salamanders from the genus Ambystoma sp., to different concentrations of both the aneugen colchicine (COL) and the clastogen cyclophosphamide (CP) and we determined the frequency of micronucleated cells (MNC) in their shed skin. The presence of spontaneous micronuclei in peripheral blood erythrocytes from 10 fish species in Lake “La Alberca,” Michoacan (Mexico), was evaluated as a possible biological indicator of genotoxic agents. Results confirm the sensivity of Pseudokirchneriella subcapitata to growth kinetics: the range of concentration of NP (20, 200 and 2000 μ g L− 1) shows an inverted U shape in its maximum growth rate; Dimethyl sulfoxide (DMSO) used as a positive control and to solvate NP induced an inverse stimulatory effect on growth rate in the range of concentrations analyzed (0.0023, 0.023 and 0.23% v v− 1). In the use of the rotifer Brachionus calyciflorus, the range of Vc from 0.185 mg L− 1 to 3 mg L− 1 clearly showed an inverted U shape characteristic of endocrine disruptions. We were able to use shed skin from Ambystoma sp., to measure MNC frequencies induced either by an aneugenic or a clastogenic compound. The MNC frequency was increased significantly by all doses of COL and CP, administered either as single or repeated exposures. The presence of MNC in the shed skin and the speed of sloughing lead us to propose that the sheds of Ambystoma sp., or other amphibians that slough their skin, as suitable alternative models for detecting genotoxic exposures relevant to aquatic environments. In the survey to determine potential biological fish indicators for genotoxic agents, the frequency of spontaneous micronucleated erythrocytes (MNE) found in the goodeid Xenotoca melanosoma (3.7 ± 1.6 MNC) and the cichlid Oreochromis aureus (2.0 ± 1.0 MNC) suggests that these species can be considered as potential biological indicators.

Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure

High stabilities of reporter proteins and their messenger RNAs (mRNAs) interfere with the detection of rapid transient changes in gene expression, such as transcriptional blocks posed by genotoxic DNA lesions. We have modified a green fluorescent protein (GFP) gene within the episomal pMARS vector by addition of a fragment encoding for mouse ornithine decarboxylase (ODC) proline-glutamate-serine-threonine-rich (PEST) sequence in order to target the protein to the proteasomes and achieved an unprecedentedly fast GFP turnover in permanently transfected human cells. As early as 1 h after inhibition of protein synthesis by cycloheximide, the number of fluorescent cells decreased more than 5-fold. Concordantly, treatments with transcription inhibitors a-amanitin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) resulted in progressive depletion of the destabilized GFP, detected as fluorescence decline, while the stable protein levels were not affected under the same conditions. Moreover, fluorescence of the destabilized but not of normal GFP decreased strongly and in a dose-dependent manner following an instant transcription block induced by ultraviolet-C (UVC) irradiation. In agreement with the transient nature of the transcriptional block due to transcription -coupled DNA repair the GFP fluorescence fully recovered after several hours.

Micronuclei and genetic polymorphisms: from exposure to susceptibility

The concept of genetic susceptibility and interactions between genetic and environmental factors of risk is a new trend in molecular epidemiology studies of cancers. Micronuclei are biomarkers of chromosome damage due to genetic instability or exposure to environmental mutagens or carcinogens. The micronucleus assay in combination with fluorescent in situ hybridization discriminates between micronuclei containing acentric chromosome fragments (chromosome breakage) and micronuclei containing whole chromosomes (chromosome loss). A recent approach is to associate the biomarkers of genetic susceptibility, which take into account cancer susceptibility and interindividual differences in the response to a genotoxic exposure, and the micronucleus assay, which serves as a biomarker of interactions between the environment and the genetic material of the cell. Information is being gathered on how DNA damage and more particularly the frequency and centromeric content of micronuclei depend on the polymorphisms of genes implicated in xenobiotic metabolism (activation or detoxication), DNA lesion repair, or folate metabolism. For biomonitoring purposes, numerous confounding factors (age, sex, tobacco consumption) influence the micronucleus biomarker, and thus associating genetic polymorphisms to micronuclei would be useful to better define the prevention and prediction of cancer risk.

Repair competence assay in studies of the influence of environmental exposure to c-PAHs on individual susceptibility to induction of DNA damage

Previous results from studies performed in three European cities suggested a decrease in DNA repair efficiency observed in lymphocytes of subjects occupationally exposed to environmental carcinogenic polycyclic aromatic hydrocarbons (c-PAHs). The aim of this study was to investigate whether a relationship between exposure to environmental c-PAHs and cellular vulnerability to the induction of DNA damage and its repair is confirmed in a pooled group of subjects from Prague, Košice and Sofia. The investigated pool consisted of 144 subjects occupationally exposed to environmental c-PAHs, who were municipal policemen or bus drivers. A control group of 115 matched individuals consisted of males unexposed at work to c-PAHs. The repair efficacy was evaluated by a comparison of the DNA damage detected by the single cell gel electrophoresis (SCGE) immediately after challenging the cells with X-ray irradiation, with residual damage (RD) being measured after an incubation period of 60 min. A stochastic concept for a mechanism of the interaction between DNA and various genotoxic exposures, was applied to analyze a relationship between exposure and biological effect in the studied sample. The outcome of the study confirms that the exposure to environmental c-PAHs or smoking cigarettes, significantly decreases DNA repair efficiency (repair efficiency in the pooled group of exposed individuals was 61.8 ± 11.8% versus 67.9 ± 9.9 in control, p < 0.001, and repair efficiency in group of smoking individuals was 63.0 ± 11.5% versus 65.9 ± 11.1 in nonsmokers, p < 0.005). The repair efficiency can be affected by a genetic polymorphism, such as subjects with a homozygous mutation in polymorphic CYP1A1 (Val/Val) enzyme, or slow NAT2 acetylators, who showed a considerably lower DNA repair efficiency (i.e. average repair efficiency in subgroups of fast acetylators was for the control subgroup 68.1% versus 66.5% in exposed subjects, while in the case of subgroups of slow acetylators, for the control group was 68.0% versus significantly less in the exposed subjects, 60.6%, p < 0.05). Smoking habits, or the diet's vitamin content, significantly affected the process. The results obtained confirm a potential value of the method as a biomarker of susceptibility in molecular epidemiology or preclinical studies, aimed at predicting susceptibility to various genotoxic exposures (environmental, occupational, therapeutic). To conclude, the research proved the influence of environmental c-PAHs, genotypes, and life styles on DNA damage and on its repair efficiency. Even low exposure to environmental c-PAHs altered DNA repair abilities of the subjects, which may result in an increased cancer risk. The findings confirm that c-PAHs should become pollutants that are subject to regulation.