Breaks In Lymphocytes Research Articles

Poly(ADP-Ribose) Polymerase Activity and DNA Strand Breaks Are Affected in Tissues of Niacin-Deficient Rats

The niacin cofactor, NAD, is the substrate for poly(ADP-ribose) polymerase, an enzyme associated with DNA repair. We investigated, therefore, whether hepatic poly(ADP-ribose) polymerase activity was altered and DNA strand breaks in lymphocytes and liver were greater in niacin-deficient rats. A niacin deficiency was established in weanling rats with diets containing 1.5 mg/kg of niacin. Based on lower growth rates and NAD concentrations in blood, liver and skeletal muscle, this diet maintained rats in a deficient state for 1 mo, and, when the dietary niacin was reduced to 0.5 mg/kg, rats remained deficient for an additional month. The hepatic poly(ADP-ribose) polymerase activity was decreased in one experiment when mean hepatic NAD concentrations were 0.60 and 0.51 mumol/g at d 34 and d 60, respectively, compared with 0.77 and 0.80 mumol/g in pair-fed controls. Enzyme activity, however, was greater than in controls when hepatic NAD concentrations were < 0.30 mumol/g. Strand breaks in DNA did not accumulate except after tissues were exposed to hypoxanthine-xanthine oxidase, a free radical-generating system. Exposure to this system caused more DNA strand breaks in lymphocytes and hepatic nuclei from niacin-deficient rats compared with the same tissues from controls. The results suggest that, in rats, although hepatic poly(ADP-ribose) polymerase activity can be elevated, a severe niacin deficiency may increase the susceptibility of DNA to oxidative damage, likely due to a lower availability of NAD.

Comparing the efficacy of γ- and electron-irradiation of PBMCs to promote secretion of paracrine, regenerative factors

Cell-free secretomes represent a promising new therapeutic avenue in regenerative medicine, and γ-irradiation of human peripheral blood mononuclear cells (PBMCs) has been shown to promote the release of paracrine factors with high regenerative potential. Recently, the use of alternative irradiation sources, such as artificially generated β- or electron-irradiation, is encouraged by authorities. Since the effect of the less hazardous electron-radiation on the production and functions of paracrine factors has not been tested so far, we compared the effects of γ- and electron-irradiation on PBMCs and determined the efficacy of both radiation sources for producing regenerative secretomes. Exposure to 60 Gy γ-rays from a radioactive nuclide and 60 Gy electron-irradiation provided by a linear accelerator comparably induced cell death and DNA damage. The transcriptional landscapes of PBMCs exposed to either radiation source shared a high degree of similarity. Secretion patterns of proteins, lipids, and extracellular vesicles displayed similar profiles after γ- and electron-irradiation. Lastly, we detected comparable biological activities in functional assays reflecting the regenerative potential of the secretomes. Taken together, we were able to demonstrate that electron-irradiation is an effective, alternative radiation source for producing therapeutic, cell-free secretomes. Our study paves the way for future clinical trials employing secretomes generated with electron-irradiation in tissue-regenerative medicine.

Late Biochemical and Cytogenetic Changes in Plasma and Peripheral Blood Lymphocytes in People Exposed to Low-dose Radiation as a Result of the Accident at the Chernobyl Nuclear Power Plant

A study was conducted to evaluate the biochemical and cytogenetic parameters in peripheral blood lymphocytes in participants in the Chernobyl accident liquidation who were exposed to external and internal radiation in the dose range (0.1–70 cSv) and children living in territories with different levels of radionuclide contamination (1–20 Ci/km2) as a result of the Chernobyl accident. The effects of low-intensity radiation were studied in a comparative analysis of the relationship between the chromatid break index, the rate of mutagenesis in somatic cells (lymphocytes), and the level of reduced glutathione in blood plasma. The increased frequency of chromatid breaks in lymphocytes and an increased level of reduced glutathione in the blood plasma in children and liquidators exposed to low doses of radiation (0.1–20 cSv) were found to be statistically significant compared to the control. As the radiation dose increased (20–70 cSv), the increase in cytogenetic abnormalities in the peripheral blood lymphocytes of liquidators and children from Chechersk (Gomelsk oblast, Belarus) was accompanied by a decrease in the water-soluble antioxidant level in the plasma. The data on the relationship of the reduced glutathione level with the major indices of the cytogenetic status are important for identification of later pathological health-related conditions in people exposed to chronic ionizing radiation.

Induction and rejoining of DNA double-strand breaks in the lymphocytes of prostate cancer patients after radium-223 treatment as assessed by the γH2AX foci assay.

The aim of this study is to assess if the number of radiation-induced double strand breaks (DSB) in lymphocytes of prostate cancer patients is affected after repeated Ra-223 therapies. In addition, we investigated the repair of ex vivo induced DSB to investigate the repair proficiency in patient's lymphocytes over the therapy course. Before each of six therapy cycles, blood samples were obtained from seventeen patients. After separation of lymphocytes, the cells were subjected to immunofluorescence staining for detection of DSB-marking γH2AX foci. The number of foci per cell per patient sample was determined for each cycle (X1-X6, baseline foci per cell). Additionally, appropriate samples were exposed ex vivo to an X-ray dose of 1 Gy. The number of γH2AX foci per cell were analyzed after 0.5 h, 2 h and 24 h of recovery. Patient-specific linear regression of the baseline foci per cell over the therapy cycles revealed no significant slopes in the regression lines. Likewise, the mean baseline foci per cell of all patients for cycles X2-X6 was not significantly elevated in comparison to the pre-therapeutic value (X1). The differences between the percentages of residual DSB and cycles were not significant, both at 2 h and 24 h repair time. Consideration of the X6/X1 ratios of both the number of lymphocytes and the amount of residual damage at 24 h indicated a significant correlation. Our findings indicate that the number of γH2AX foci per cell was not changed in dependence on the Ra-223 therapy cycles. The ability of patient's lymphocytes to repair ex vivo induced DSB remained unaffected throughout the entire therapy course.

Influence of Acute Exercise on DNA Repair and PARP Activity before and after Irradiation in Lymphocytes from Trained and Untrained Individuals.

Several studies indicate that acute exercise induces DNA damage, whereas regular exercise increases DNA repair kinetics. Although the molecular mechanisms are not completely understood, the induction of endogenous reactive oxygen species (ROS) during acute exhaustive exercise due to metabolic processes might be responsible for the observed DNA damage, while an adaptive increase in antioxidant capacity due to regular physical activity seems to play an important protective role. However, the protective effect of physical activity on exogenously induced DNA damage in human immune cells has been poorly investigated. We asked the question whether individuals with a high aerobic capacity would have an enhanced response to radiation-induced DNA damage. Immune cells are highly sensitive to radiation and exercise affects lymphocyte dynamics and immune function. Therefore, we measured endogenous and radiation-induced DNA strand breaks and poly (ADP-ribose) polymerase-1 (PARP1) activity in peripheral blood mononuclear cells (PBMCs) from endurance-trained (maximum rate of oxygen consumption measured during incremental exercise V’O2max > 55 mL/min/kg) and untrained (V’O2max < 45 mL/min/kg) young healthy male volunteers before and after exhaustive exercise. Our results indicate that: (i) acute exercise induces DNA strand breaks in lymphocytes only in untrained individuals, (ii) following acute exercise, trained individuals repaired radiation-induced DNA strand breaks faster than untrained individuals, and (iii) trained subjects retained a higher level of radiation-induced PARP1 activity after acute exercise. The results of the present study indicate that increased aerobic fitness can protect immune cells against radiation-induced DNA strand breaks.

Environmentally realistic exposure to weathered North Sea oil: Sublethal effects in Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus)

ABSTRACTWith increasing oil and gas activities and transport in the Arctic, there is a need to understand how operational or accidental releases of substances affect marine organisms from a pristine environment. The aim of the current study was to describe and compare the responses of two marine fish species, Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus), following exposure to three levels (low, medium, high) of the water-soluble fraction of a North Sea crude oil for 16 days. The exposure system simulated environmental exposure by allowing clean seawater to percolate through gravel covered in weathered oil before being introduced to aquaria. Both polycyclic aromatic hydrocarbon (PAH) metabolite bile concentrations and cytochrome P4501A (CYP1A) levels and activity increased markedly in comparison with controls in both species, but there were no significant differences between the three exposures. Turbot possessed 4–5-fold higher concentrations of two PAH bile metabolites compared to Atlantic cod by day 8. In contrast, hepatic CYP1A activity in cod was consistently 2–6-fold higher than in turbot with increasing differences over the experimental period. Baseline DNA strand breaks in lymphocytes and kidney cells were low in both species, but was elevated for all treatments by day two. There were no marked indications of the treatments affecting immune functions in either species. This investigation demonstrated that there may be significant differences in responses between species receiving identical exposures and that DNA strand breaks in lymphocytes and kidney cells are sensitive to confinement stress. Data also indicate that some species, such as turbot, may adapt to treatments within days and weeks.

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Mobilization of Nuclear Copper by Green Tea Polyphenol Epicatechin-3-Gallate and Subsequent Prooxidant Breakage of Cellular DNA: Implications for Cancer Chemotherapy.

Epidemiological as well as experimental evidence exists in support of chemopreventive and anticancer properties of green tea and its constituents. The gallocatechin, epicatechin-3-gallate is a major polyphenol present in green tea, shown responsible for these effects. Plant-derived polyphenolic compounds are established natural antioxidants which are capable of catalyzing oxidative DNA degradation of cellular DNA, alone as well as in the presence of transition metal ions, such as copper. Here we present evidence to support that, similar to various other polyphenoic compounds, epicatechin-3-gallate also causes oxidative degradation of cellular DNA. Single cell alkaline gel electrophoresis (Comet assay) was used to assess DNA breakage in lymphocytes that were exposed to various concentrations of epicatechin-3-gallate. Inhibition of DNA breakage in the presence of scavengers of reactive oxygen species (ROS) suggested involvement of ROS generation. Addition of neocuproine (a cell membrane permeable Cu(I) chelator) inhibited DNA degradation, dose-dependently, in intact lymphocytes. In contrast, bathocuproine, which does not permeate cell membrane, was observed to be ineffective. We further show that epicatechin-3-gallate degrades DNA in cell nuclei, which can also be inhibited by neocuproine, suggesting mobilization of nuclear copper in this reaction as well. Our results are indicative of ROS generation, possibly through mobilization of endogenous copper ions, and support our long-standing hypothesis of a prooxidant activity of plant-derived polyphenols as a mechanism for their documented anticancer properties.

Cancer Therapy by Catechins Involves Redox Cycling of Copper Ions and Generation of Reactive Oxygen species.

Catechins, the dietary phytochemicals present in green tea and other beverages, are considered to be potent inducers of apoptosis and cytotoxicity to cancer cells. While it is believed that the antioxidant properties of catechins and related dietary agents may contribute to lowering the risk of cancer induction by impeding oxidative injury to DNA, these properties cannot account for apoptosis induction and chemotherapeutic observations. Catechin (C), epicatechin (EC), epigallocatechin (EGC) and epigallocatechin-3-gallate (EGCG) are the four major constituents of green tea. In this article, using human peripheral lymphocytes and comet assay, we show that C, EC, EGC and EGCG cause cellular DNA breakage and can alternatively switch to a prooxidant action in the presence of transition metals such as copper. The cellular DNA breakage was found to be significantly enhanced in the presence of copper ions. Catechins were found to be effective in providing protection against oxidative stress induced by tertbutylhydroperoxide, as measured by oxidative DNA breakage in lymphocytes. The prooxidant action of catechins involved production of hydroxyl radicals through redox recycling of copper ions. We also determined that catechins, particularly EGCG, inhibit proliferation of breast cancer cell line MDA-MB-231 leading to a prooxidant cell death. Since it is well established that tissue, cellular and serum copper levels are considerably elevated in various malignancies, cancer cells would be more subject to redox cycling between copper ions and catechins to generate reactive oxygen species (ROS) responsible for DNA breakage. Such a copper dependent prooxidant cytotoxic mechanism better explains the anticancer activity and preferential cytotoxicity of dietary phytochemicals against cancer cells.

Genetic Variants That Predispose to DNA Double-Strand Breaks in Lymphocytes From a Subset of Patients With Familial Colorectal Carcinomas

DNA structural lesions are prevalent in sporadic colorectal cancer. Therefore, we proposed that gene variants that predispose to DNA double-strand breaks (DSBs) would be found in patients with familial colorectal carcinomas of an undefined genetic basis (UFCRC). We collected primary T cells from 25 patients with UFCRC and matched patients without colorectal cancer (controls) and assayed for DSBs. We performed exome sequence analyses of germline DNA from 20 patients with UFCRC and 5 undiagnosed patients with polyposis. The prevalence of identified variants in genes linked to DNA integrity was compared with that of individuals without a family history of cancer. The effects of representative variants found to be associated with UFCRC was confirmed in functional assays with HCT116 cells. Primary T cells from most patients with UFCRC had increased levels of the DSB marker γ(phosphorylated)histone2AX (γH2AX) after treatment with DNA damaging agents, compared with T cells from controls (P < .001). Exome sequence analysis identified a mean 1.4 rare variants per patient that were predicted to disrupt functions of genes relevant to DSBs. Controls (from public databases) had a much lower frequency of variants in the same genes (P < .001). Knockdown of representative variant genes in HCT116 CRC cells increased γH2AX. A detailed analysis of immortalized patient-derived B cells that contained variants in the Werner syndrome, RecQ helicase-like gene (WRN, encoding T705I), and excision repair cross-complementation group 6 (ERCC6, encoding N180Y) showed reduced levels of these proteins and increased DSBs, compared with B cells from controls. This phenotype was rescued by exogenous expression of WRN or ERCC6. Direct analysis of the recombinant variant proteins confirmed defective enzymatic activities. These results provide evidence that defects in suppression of DSBs underlie some cases of UFCRC; these can be identified by assays of circulating lymphocytes. We specifically associated UFCRC with variants in WRN and ERCC6 that reduce the capacity for repair of DNA DSBs. These observations could lead to a simple screening strategy for UFCRC, and provide insight into the pathogenic mechanisms of colorectal carcinogenesis.

Biological Effects of Cardiac Magnetic Resonance on Human Blood Cells

Cardiac magnetic resonance (CMR) is increasingly used for the diagnosis and management of cardiac diseases. Recent studies have reported immediate post-CMR DNA double-strand breaks in T lymphocytes. We sought to evaluate CMR-induced DNA damage in lymphocytes, alterations of blood cells, and their temporal persistence. In 20 prospectively enrolled healthy men (31.4±7.9 years), blood was drawn before and after (1-2 hours, 2 days, 1 month, and 1 year) unenhanced 1.5T CMR. Blood cell counts, cell death, and activation status of lymphocytes, monocytes, neutrophils, and platelets were evaluated. The first 2-hour post-CMR were characterized by a small increase of lymphocyte B and neutrophil counts and a transient drop of total lymphocytes because of a decrease in natural killer cells. Among blood cells, only neutrophils and monocytes displayed slight and transient activation. DNA double-strand breaks in lymphocytes were quantified through flow cytometric analysis of H2AX phosphorylation (γ-H2AX). γ-H2AX intensity in T lymphocytes did not change early after CMR but increased significantly at day 2 ≤1 month before returning to baseline levels of 1-year post-CMR. Unenhanced CMR is associated with minor but significant immediate blood cell alterations or activations figuring inflammatory response, as well as DNA damage in T lymphocytes observed from day 2 until the first month but disappearing at 1-year follow-up. Although further studies are required to definitely state whether CMR can be used safely, our findings already call for caution when it comes to repeat this examination within a month.

G407(P) Macrocytosis – a rare but serious problem!

Aims A case report to highlight the importance of macrocytosis. Methods A 7½ year old boy was referred with macrocytosis (MCV 108). He was fit and thriving child (weight, height 50th centile). History and examination was unremarkable. Bloods done 5 years ago also showed high MCV (95). Routine tests for evaluation of macrocytosis were normal. Later FBC showed thrombocytopenia, leucopenia and neutropenia. He was finally diagnosed to have Fanconi anaemia. He has been put on register for bone marrow transplant, and whole family is being tested for gene mutations. Fanconi anaemia is an autosomal recessive or X-linked form of inherited aplastic anaemia. Sixteen mutations (FA-A to FA-Q) have been identified. Hematologic findings evolve over months to years, with 70% of children developing bone marrow failure by age 10. They are at high risk for developing myelodysplastic syndrome, acute myeloid leukaemia, and squamous cell carcinoma. Characteristic congenital malformations are present in up to 70% of affected children (short stature, cafe-au-lait spots, hypopigmented spots, thumb abnormality, microcephaly, hydrocephaly, hypogonadism, developmental delay). Diagnosis is made by presence of increased chromosomal breakage in lymphocytes cultured in the presence of DNA cross-linking agents (mitomycin C or diepoxybutane). Bone marrow transplant is the only available curative treatment. Other treatment options include androgens, hematopoietic growth factors, and blood product transfusions with supportive care. Results Hb 122, WCC 8.6, Platelets 164, MCV 102 in 2011. Hb 118, WCC 5.4, Neutrophils 1.34, Platelets 130, MCV 108 in 2014. U&E, LFT, immunoglobulins, B12, Folate, Ferritin, and TFT were normal. Bone marrow biopsy revealed mild aplasia, and no myelodysplastic syndrome or leukaemia. HIV, Hepatitis screen and viral serology were negative. Fanconi Anaemia test came back positive. Conclusions Children presenting with macrocytosis should be carefully evaluated and referred to a haematologist, as it could be the first manifestation of a serious underlying bone marrow problem. Macrocytosis in age range of six months to 12 years age is MCV >90. Causes of macrocytosis include disorders of folate/B12 metabolism, liver disease, congenital heart disease, Down’s syndrome, hypothyroidism, drugs (anticonvulsants, zidovudine, hydroxyurea, immunosuppresants), myelodysplastic syndromes, bone marrow failure, and rare genetic syndromes (eg, Fanconi anaemia, thiamine-responsive megaloblastic anaemia syndrome).

15q12 Variants, Sputum Gene Promoter Hypermethylation, and Lung Cancer Risk: A GWAS in Smokers

Lung cancer is the leading cause of cancer-related mortality worldwide. Detection of promoter hypermethylation of tumor suppressor genes in exfoliated cells from the lung provides an assessment of field cancerization that in turn predicts lung cancer. The identification of genetic determinants for this validated cancer biomarker should provide novel insights into mechanisms underlying epigenetic reprogramming during lung carcinogenesis. A genome-wide association study using generalized estimating equations and logistic regression models was conducted in two geographically independent smoker cohorts to identify loci affecting the propensity for cancer-related gene methylation that was assessed by a 12-gene panel interrogated in sputum. All statistical tests were two-sided. Two single nucleotide polymorphisms (SNPs) at 15q12 (rs73371737 and rs7179575) that drove gene methylation were discovered and replicated with rs73371737 reaching genome-wide significance (P = 3.3×10(-8)). A haplotype carrying risk alleles from the two 15q12 SNPs conferred 57% increased risk for gene methylation (P = 2.5×10(-9)). Rs73371737 reduced GABRB3 expression in lung cells and increased risk for smoking-induced chronic mucous hypersecretion. Furthermore, subjects with variant homozygote of rs73371737 had a two-fold increase in risk for lung cancer (P = .0043). Pathway analysis identified DNA double-strand break repair by homologous recombination (DSBR-HR) as a major pathway affecting susceptibility for gene methylation that was validated by measuring chromatid breaks in lymphocytes challenged by bleomycin. A functional 15q12 variant was identified as a risk factor for gene methylation and lung cancer. The associations could be mediated by GABAergic signaling that drives the smoking-induced mucous cell metaplasia. Our findings also substantiate DSBR-HR as a critical pathway driving epigenetic gene silencing.

Pro-oxidant DNA breakage induced by the interaction of L-DOPA with Cu(II): a putative mechanism of neurotoxicity.

There are reports in scientific literature that the concentration of copper ions in Parkinsonian brain is at a level that could promote oxidative DNA damage. The possibility of copper chelation by antioxidants excited us to explore the generation of reactive oxygen species (ROS) and DNA damage by the interaction of L-DOPA with Cu(II) ions. In the present manuscript, L-DOPA was tested for its ability to bind with Cu(II) and reduce it to Cu(I). The generation of ROS, such as superoxide anion (O(2)(-)) and hydroxyl radical (OH(•)), was also ascertained. As a result of L-DOPA and Cu(II) interaction, the generation of O(2)(-) was found to be increased in a time-dependent manner. Moreover, the formation of OH(•) was also found to be enhanced with increasing concentrations of L-DOPA. Furthermore, Comet assay results clearly showed significantly higher cellular DNA breakage in lymphocytes treated with L-DOPA and Cu(II) as compared to those that were treated with L-DOPA alone. However, such DNA degradation was inhibited to a significant extent by scavengers of ROS and neocuproine, a membrane permeable Cu(I)-specific sequestering agent. These findings demonstrate that L-DOPA exhibits a pro-oxidant activity in the presence of copper ions.

Evaluation of the Comet Assay for Assessing the Dose-Response Relationship of DNA Damage Induced by Ionizing Radiation

Dose- and time-response curves were combined to assess the potential of the comet assay in radiation biodosimetry. The neutral comet assay was used to detect DNA double-strand breaks in lymphocytes caused by γ-ray irradiation. A clear dose-response relationship with DNA double-strand breaks using the comet assay was found at different times after irradiation (p < 0.001). A time-response relationship was also found within 72 h after irradiation (p < 0.001). The curves for DNA double-strand breaks and DNA repair in vitro of human lymphocytes presented a nice model, and a smooth, three-dimensional plane model was obtained when the two curves were combined.

The human bitumen study hits the headlines

Besides the current cutting-edge topics in toxicology, such as oxidative stress research (Baird and Dinkova-Kostova 2011; Barcelos et al. 2011; Cadenas et al. 2010), metal toxicity (Antonini et al. 2011; Bolt et al. 2011; Sinicropi et al. 2010), nanotoxicology (Weiss and Diabate 2011), in vitro systems (Heise et al. 2012; Knobeloch et al. 2012; Zellmer et al. 2010), and modelling (Mielke et al. 2011; Hoehme et al. 2010), bitumen research has led an essentially wallflower existence. Fortunately, this has dramatically changed since the recent publications from the Human Bitumen Study (Schluter 2011; Raulf-Heimsoth et al. 2011a). Bitumen was already regarded as a suspected carcinogen in the 1980s (Schluter 2011; Greim 2001). However, the discussion surrounding bitumen exposure as a potential health hazard was always controversial. On the one side, a long-term carcinogenicity study with dermal application yielded positive results (Sivak et al. 1997). Conversely, a long-term inhalation study––probably the more relevant route of application––did not lead to increased tumour incidence in rats (Fuhst et al. 2007). Furthermore, a small biomonitoring study did not show increased levels of DNA strand breaks in lymphocytes of bitumen workers (Fuchs et al. 1996). However, some evidence of increased DNA damage was found in roofers who process bitumen at higher temperatures (Fuchs et al. 1996). Therefore, key questions are whether bitumen causes genotoxicity in exposed workers and whether inhalation is toxic to the airways. The recently published Human Bitumen Study is the largest worldwide and best-controlled study in this field, encompassing 500 exposed workers from 80 construction sites. The results, in a nutshell, show that DNA adducts and strand breaks in bitumen workers were within the range found in healthy individuals. On the other hand, bitumen workers showed chronic inflammation in the lower airways. The practical consequences for both industry and occupational medicine include the need to reduce the processing temperature of bitumen as much as possible, as temperature represents the most critical factor influencing bitumen vapour exposure. Finally, follow-up studies are needed to assess the relevance of the identified airway inflammation. In particular, the relevance of subtle changes of inflammatory mediators in the lower airways (e.g. interleukins) is of general importance in chemically induced airway inflammation and currently discussed in regulatory toxicology. To give our readers a rapid overview, we summarised the key findings of the Human Bitumen Study (Table 1).

Genotoxicity and reproductive toxicity of bisphenol A and X-ray/bisphenol A combination in male mice

This study was designed to investigate the effects of 2 weeks of exposure of male mice to bisphenol A (BPA) alone or in a combination with X-rays on the sperm count and quality as well as induction of DNA strand breaks in somatic and germ cells. Pzh:SFIS male mice were exposed to X-rays (0.05 and 0.10 Gy) or BPA (5, 10, 20, and 40 mg/kg) or to a combination of both (0.05 Gy + 5 mg/kg body weight of BPA and 0.10 Gy + 10 mg/kg of BPA). Both X-rays and BPA administered alone decreased sperm count and quality. X-rays induced DNA strand breaks in spleen cells, whereas BPA induced DNA strand breaks in lymphocytes and in cells from spleen, kidneys, and lung and in germ cells. After combined exposure to both agents, sperm count and quality were similar as after exposure to each agent alone and significantly reduced, compared to control. Levels of DNA damage in somatic and germ cells after combined exposure to lower, as well as higher, doses were significantly reduced, compared to the effects of BPA alone. Results confirmed the mutagenic ability of BPA. Combined exposure to X-rays and BPA leads to the prevention of DNA damage in somatic and germ cells of mice.

Mechanism of Action of Pentostatin and Cladribine in Hairy Cell Leukemia

Pentostatin (2′-deoxycoformycin; dCF) and cladribine (2-chlorodeoxyadenosine; CdA) are highly effective agents for the treatment of hairy cell leukemia. Although their precise mechanisms of action in this disease are still unknown, a number of mechanisms have been postulated. dCF is a potent inhibitor of adenosine deaminase (ADA), and treatment results in the accumulation of deoxyadenosine (dAdo) and adenosine (Ado) in the plasma. dAdo is phosphorylated by deoxycytidine kinase in lymphocytes to deoxyadenosine monophosphate (dAMP), which is subsequently converted to deoxyadenosine triphosphate (dATP). CdA is the chlorinated derivative of deoxyadenosine, is resistant to degradation by ADA, and accumulates in lymphocytes as CdATP. Both dATP and CdATP cause an initial accumulation of DNA strand breaks in lymphocytes and this results in the activation of p53, the release of cytochrome c from mitochondria, and apoptosis. CdA has several unique mechanisms of action over dAdo and these include the incorportation of CdATP into DNA, the inhibition of DNA polymerase β, and the phosphorylation of CdA to CdATP by deoxyguanosine kinase in mitochondria. These additional modes of action produce further DNA breaks in CdA-treated cells and explain the more potent activity of CdA compared to dCF and the greater myelosuppression with this agent. The cells die by apoptosis, but the DNA strand breaks also cause the activation of poly(ADP-ribose) polymerase (PARP), with resultant cellular depletion of nicotinamide adenine dinucleotide (NAD) and ATP. The induction of necrosis by PARP activation may explain the activity of these analogs in some patients with p53 mutations.

Genome Instability and Bleomycin Sensitivity Test

Estimation of individual susceptibility to mutagens is often a part of epidemiological studies monitoring the appearance of malignant disease in different populations. Genome exposure to mutagens can lead to DNA damage. The rate of damage depends on individual differences in response, which are usually associated with differences in DNA repair capacity. Cytogenetic studies have shown that the genome of tumour cells is less stable than normal cells and therefore accumulates more damage. Tumour patients show a higher frequency of chromatid and chromosomal aberrations and a predisposition to certain types of tumours. One of the common biomarkers used in monitoring tumour appearance and changed response to DNA damage is the bleomycin test. In its active form, bleomycin (glycopeptid) is a radiomimetic cytostatic that can damage the DNA molecule and cause multiple single and double strands. The bleomycin test is simple and inexpensive, and is based on scoring chromatid breaks in lymphocytes in vitro exposed to bleomycin during the late G2 phase of the cell cycle. This review looks into different factors that may affect test results and discusses its wide implementation in studies of genome instability usually caused by a combination of factors.

The antioxidant ascorbic acid mobilizes nuclear copper leading to a prooxidant breakage of cellular DNA: implications for chemotherapeutic action against cancer

Ascorbic acid is an essential micronutrient and is considered to have an antioxidant function in living systems. For the past several decades, ascorbic acid has been the subject of considerable interest as an anticancer agent. Several studies have shown that ascorbic acid is cytotoxic to a variety of cancer cells, whereas normal cells are relatively resistant to such cytotoxic action. In this study, we propose a putative molecular mechanism that accounts for the preferential cytotoxicity of ascorbic acid against cancer cells. Standard and lysed version of alkaline single-cell gel electrophoresis (Comet assay); ferrous oxidation-xylenol orange (FOX) assay. We show that ascorbic acid acts as a prooxidant and leads to oxidative DNA breakage in lymphocytes and lymphocyte nuclei. Scavengers of reactive oxygen species were able to inhibit ascorbic acid-induced DNA breakage, suggesting the involvement of reactive oxygen species in this reaction. We further show that such DNA breakage is inhibited by both iron and copper chelators in cells, whereas in nuclei, similar inhibition was achieved only by copper chelators, indicating an important role of chromatin-bound copper in the prooxidant cellular DNA breakage by ascorbic acid. We propose that the copper-dependent cellular redox status is an important element in the cytotoxic action of ascorbic acid against cancer cells. It is well established that cellular copper levels are considerably elevated in various malignancies. Therefore, cancer cells may be more subject to electron transfer between copper and ascorbate to generate reactive oxygen species. In light of these observations and those in literature, in this paper we explain that the preferential cytotoxicity of ascorbic acid against cancer cells is the result of elevated copper levels in such cells. Further, this study identifies nuclear copper as a novel molecular target for cytotoxic action of ascorbic acid, which has implications for its chemotherapeutic properties against cancer.