Breaks In Lymphocytes Research Articles

Strawberry Consumption Is Associated with Increased Antioxidant Capacity in Serum

Strawberries are known to contain antioxidants, but the significance of ingesting antioxidant-rich fruits remains to be established. In order to determine whether the consumption of strawberries impacted measures of in vivo antioxidant capacity, frozen strawberries (250 g) were administered daily for 3 weeks to 21 healthy female volunteers. Compliance was confirmed by quantitating pelargonidin-glucuronide, urolithin A-glucuronide, and 2,5-dimethyl-4-hydroxy-3-[(2)H]furanone-glucuronide in plasma and urine by liquid chromatography-mass spectrometry and antioxidant capacity in serum measured by the increase in lag phase of low-density lipoprotein after copper sulfate exposure, DNA strand breaks in lymphocytes, and activity of phase II enzymes. Among these measures lipid peroxidation lag time increased by 20% (P < .01), whereas other measures did not change significantly. The potent antioxidant defenses in humans make determination of changes due to dietary ingestion in healthy individuals difficult. In summary, daily consumption of strawberries resulted in a modest but significant increase in antioxidant capacity in a healthy population.

Cellular DNA breakage by soy isoflavone genistein and its methylated structural analogue biochanin A

Epidemiological studies have indicated that populations with high isoflavone intake through soy consumption have lower rates of breast, prostate, and colon cancer. The isoflavone polyphenol genistein in soybean is considered to be a potent chemopreventive agent against cancer. In order to explore the chemical basis of chemopreventive activity of genistein, in this paper we have examined the structure-activity relationship between genistein and its structural analogue biochanin A. We show that both genistein and its methylated derivative biochanin A are able to mobilize nuclear copper in human lymphocyte, leading to degradation of cellular DNA. However, the relative rate of DNA breakage was greater in the case of genistein. Further, the cellular DNA degradation was inhibited by copper chelator (neocuproine/bathocuproine) but not by compounds that specifically bind iron and zinc (desferrioxamine mesylate and histidine, respectively). We also compared the antioxidant activity of the two isoflavones against tert-butylhydroperoxide-induced oxidative breakage in lymphocytes. Again genistein was found to be more effective than biochanin A in providing protection against oxidative stress induced by tert-butylhydroperoxide. It would therefore appear that the structural features of isoflavones that are important for antioxidant properties are also the ones that contribute to their pro-oxidant action through a mechanism that involves redox cycling of chromatin-bound nuclear copper.

Spontaneous and radiation-induced chromosomal instability and persistence of chromosome aberrations after radiotherapy in lymphocytes from prostate cancer patients

The aim of the study was to compare the spontaneous and ex vivo radiation-induced chromosomal damage in lymphocytes of untreated prostate cancer patients and age-matched healthy donors, and to evaluate the chromosomal damage, induced by radiotherapy, and its persistence. Blood samples from 102 prostate cancer patients were obtained before radiotherapy to investigate the excess acentric fragments and dicentric chromosomes. In addition, in a subgroup of ten patients, simple exchanges in chromosomes 2 and 4 were evaluated by fluorescent in situ hybridization (FISH), before the onset of therapy, in the middle and at the end of therapy, and 1 year later. Data were compared to blood samples from ten age-matched healthy donors. We found that spontaneous yields of acentric chromosome fragments and simple exchanges were significantly increased in lymphocytes of patients before onset of therapy, indicating chromosomal instability in these patients. Ex vivo radiation-induced aberrations were not significantly increased, indicating proficient repair of radiation-induced DNA double-strand breaks in lymphocytes of these patients. As expected, the yields of dicentric and acentric chromosomes, and the partial yields of simple exchanges, were increased after the onset of therapy. Surprisingly, yields after 1 year were comparable to those directly after radiotherapy, indicating persistence of chromosomal instability over this time. Our results indicate that prostate cancer patients are characterized by increased spontaneous chromosomal instability. This instability seems to result from defects other than a deficient repair of radiation-induced DNA double-strand breaks. Radiotherapy-induced chromosomal damage persists 1 year after treatment.

Complex Oncogenic Translocations with Gene Amplification Are Initiated by Specific DNA Breaks in Lymphocytes

Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. Whereas chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double-strand break repair in suppression of oncogenic genome instability, the genomic elements required for chromosome rearrangements, especially complex lesions, have not been elucidated. Using a mouse model of B-lineage lymphoma, characterized by complicon formation involving the immunoglobulin heavy chain (Igh) locus and the c-myc oncogene, we have now investigated the requirement for specific genomic segments as donors for complex rearrangements. We now show that specific DNA double-strand breaks, occurring within a narrow segment of Igh, are necessary to initiate complicon formation. By contrast, neither specific DNA breaks nor the powerful intronic enhancer Emu are required for complicon-independent oncogenesis. This study is the first to delineate mechanisms of complex versus simple instability and the first to identify specific chromosomal elements required for complex chromosomal aberrations. These findings will illuminate genomic cancer susceptibility and risk factors.

Increased capacity of lymphocytes from hereditary hemochromatosis patients homozygous for the C282Y HFE mutation to respond to the genotoxic effect of diepoxybutane

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by systemic iron overload with consequent tissue damage. The vast majority of HH patients are homozygous for the C282Y mutation in HFE, a non-classical MHC class-I gene located in chromosome 6, whose role in the regulation of systemic iron metabolism is still not completely understood. Iron enhances the formation of reactive oxygen species, with increasing risk of DNA damage induced by oxidative stress, and consequently an increased susceptibility to chromosome instability. In the present work we examined spontaneous and diepoxybutane (DEB)-induced chromosome instability in PHA-stimulated lymphocyte cultures from 23 HH patients, all homozygous for the C282Y HFE mutation, in comparison to 29 normal controls. In addition, three patients with secondary forms of iron overload, not related to HFE, were studied as controls to test the role of iron overload on DEB-induced chromosome instability. Our results show a significantly higher frequency of spontaneous chromosome breaks in lymphocytes from the HH patients, when compared with lymphocytes from normal controls ( p < 0.0001). In addition, there is a significant correlation between the percentage of cells with spontaneous chromosomal breaks and the transferrin saturation ( r = 0.53, p = 0.0041) or serum-ferritin levels ( r = 0.66, p = 0.0001) in patients. Surprisingly, the frequency of DEB-induced chromosome breaks was significantly lower in lymphocytes from HH patients than in lymphocytes from both controls and patients with secondary forms of hemochromatosis ( p = 0.0029). No correlation was observed between the percentage of cells with DEB-induced chromosome breaks and the transferrin saturation or serum-ferritin values. These results suggest that lymphocytes from HH patients may have an increased capacity to respond to DEB-induced chromosome breakage, and that this capacity is somehow related to the presence of the C282Y HFE mutation.

MACLEAYA CORDATA EXTRACT AND SANGROVIT GENOTOXICITY. ASSESSMENT IN VIVO

Sanguinarine (SG) has been reported to form DNA adducts in vitro and increase the levels of DNA single strand breaks in the blood and bone marrow of mice treated intraperitoneally with SG. Recently, we showed no genotoxic effects of orally administrated 120 mg/kg feed Macleaya cordata extract (a mixture of sanguinarine and chelerythrine) in pigs or rats in 90-day studies. The goal of this paper was to assess the possible genotoxicity of M. cordata extract when included as a dietary admixture to rodents at concentrations providing 600 mg/kg feed and 100, 7000 or 14000 mg/kg feed Sangrovit (natural feed additive containing M. cordata extract and powdered M. cordata) in a 90-day pilot study. The rats consumed ad libitum either the standard diet or the diets containing 367 ppm of sanguinarine and chelerythrine in M. cordata extract, and 5, 330, or 660 ppm of total alkaloids in Sangrovit for 90 days. The DNA adducts formation in liver was analyzed by (32)P-postlabeling technique and DNA single strand breaks in lymphocytes were evaluated by Comet assay. The results showed that M. cordata extract and/or Sangrovit induced no DNA damage to rat lymphocytes or hepatocytes after 90-days oral administration. Data from the studies described in this paper and the fact that Sangrovit given to the rats in our experiments were higher than the recommended dose (50 to 100 mg/kg feed), argue strongly in favour of the use of Sangrovit in live stock.

Effects of multivitamin supplementation on DNA damage in lymphocytes from elderly volunteers

The aim of this study was to evaluate the effect of a mixture of vitamins and minerals on oxidative DNA damage and the resistance of DNA to H 2O 2-induced DNA strand breaks in lymphocytes from 80 elderly volunteers ex vivo by means of Comet assay. The intervention with vitamin complex decreased significantly the levels of DNA damage. Our results demonstrate that the vitamin complex was able to decrease H 2O 2-induced DNA breakage. Our data suggest that the consumption of some vitamins may reduce the effects of oxidative DNA damage and may be useful for attaining healthy aging.

ATM Prevents the Persistence and Propagation of Chromosome Breaks in Lymphocytes

DNA double-strand breaks (DSBs) induce a signal transmitted by the ataxia-telangiectasia mutated (ATM) kinase, which suppresses illegitimate joining of DSBs and activates cell-cycle checkpoints. Here we show that a significant fraction of mature ATM-deficient lymphocytes contain telomere-deleted ends produced by failed end joining during V(D)J recombination. These RAG-1/2 endonuclease-dependent, terminally deleted chromosomes persist in peripheral lymphocytes for at least 2 weeks in vivo and are stable over several generations in vitro. Restoration of ATM kinase activity in mature lymphocytes that have transiently lost ATM function leads to loss of cells with terminally deleted chromosomes. Thus, maintenance of genomic stability in lymphocytes requires faithful end joining as well a checkpoint that prevents the long-term persistence and transmission of DSBs. Silencing this checkpoint permits DNA ends produced by V(D)J recombination in a lymphoid precursor to serve as substrates for translocations with chromosomes subsequently damaged by other means in mature cells.

Unstabilized DNA Breaks in Lymphocytes of Patients with Different Subsets of Systemic Sclerosis

The clastogenic effects on DNA, proven by the presence of micronuclei (MN) and the protective cellular mechanisms normally used to stabilize DNA breaks were investigated in three subsets of patients with systemic sclerosis (SSc). The frequency of MN found in cultures of peripheral lymphocytes in patients with anticentromere and antitopoisomerase I antibodies was significantly higher than that in the control group. The group with anticentromere antibody showed a significantly higher frequency of MN than did the subjects with antitopoisomerase antibody (4.22% versus 2.34%, P < 0.001). Patients with anti-RNA polymerase III, instead, had a low prevalence of typical micronucleated cells (0.98%), not significantly different from that of the healthy controls (0.82%). Moreover, when MN was characterized for the presence or absence of DNA fragments with free 3'-OH ends by digoxigenin-dUTP (DIG-dUTP) using terminal deoxynucleotidil transferase, its frequency was found to be increased in the groups with anticentromere and antitopoisomerase I antibodies with respect to that in the controls. The increase was significantly higher in the lymphocytes of the patients with anticentromere than in those with antitopoisomerase I antibody (35% versus 20.08%, P < 0.001). Nonetheless, the prevalence of unstable DNA fragments in patients with anti-RNA polymerase III antibody was low (2.05%) and not significantly different from that of the control group (1.18%). Our results indicate that there is a clastogenic effect on DNA and an interference in the protective cellular mechanisms normally stabilizing DNA breaks only in some subsets of SSc patients.

Does metformin prevent short term oxidant-induced DNA damage? In vitro study on lymphocytes from aged subjects

Metformin(1-(diaminomethylidene)-3,3-dimethyl-guanidine), an anti-hyperglycemic agent, also has antioxidant effects. Although the origin is not clearly understood, the antioxidant activity of metformin might result from a direct effect on reactive oxygen species (ROS) or could have an indirect action on the superoxide anions produced by hyperglycemia. The ability of metformin to modulate DNA damage produced by oxidative stress is not known. For this reason, we examined the short term effect of metformin (50 microM, 2 h) on the DNA damage of cumene hydroperoxide (CumOOH)-induced lymphocytes from aged and young control groups (n = 10 each). In this study, DNA damage elicited by CumOOH (1 mM) was detected with the Comet Assay and the ELISA technique. Our results showed a significant increase in apoptotic DNA fragmentation and DNA strand breaks (Comet assay tail factor %) that was detected before and after CumOOH induction in lymphocytes of healthy elderly subjects when compared with healthy young control. Metformin significantly decreased CumOOH-induced apoptotic DNA fragmentation and DNA strand breaks in lymphocytes from aged subjects, although it did not produce a long-term effect. The in vitro results indicate that the short-term effect of metformin can protect against prooxidant stimulus-induced DNA damage in lymphocytes from elderly subjects.

Decreased oxidative DNA damage in Ogg1/Myh‐deficient mice treated with pomegranate extract

Oxidative DNA damage is linked to carcinogenesis. Base excision repair (BER) is critical for the repair of oxidative DNA lesions such as 8-hydroxy-2′-deoxyguanosine (8OHdG). 8-oxoguanine-DNA glycosylase 1 (hOGG1) activity comprises the first step of the BER pathway. The mutY gene encodes an additional DNA glycosylase (Myh), which removes adenosine when paired with 8OhdG to accommodate the removal of 8OHdG. The ratio of 8OhdG to total dG as determined by high performance liquid chromatography (HPLC) was increased in the DNA extracted from tissues of mice deficient in Ogg1 / Myh. After daily treatments with 0.8 mg of PomX for 23 days the ratio 8OhdG/dG was decreased by 30, 55 and 33% in liver, kidney and lung, respectively, compared to vehicle control treated mice. Total strand breaks in lymphocytes from whole blood of the same mice as determined by comet assay were decreased by 49% as well. Pomegranate extract (PomX) was prepared from skins and arils minus the seeds and contains 37% of punicalagin (hydrolysable ellagitannins), which is hydrolysed in the intestine to release ellagic acid (EA). EA in turn can be metabolized to urolithins in the intestine and absorbed into the circulation. Using LC/ESI-MS analysis trace amounts of EA metabolites were found in the tissues of mice treated with PomX. We conclude that pomegranate phytochemicals protect against oxidative DNA damage through their strong antioxidant activity.

Alkaline unwinding flow cytometry assay to measure nucleotide excision repair

Nucleotide excision repair (NER), one of the DNA repair pathways, is the primary mechanism for repair of bulky adducts caused by physical and chemical agents, such as UV radiation, cisplatin and 4-nitroquinolones. Variations in DNA repair may be a significant risk factor for several cancers, but its measurement in epidemiological studies has been hindered by the high variability, complexity and laborious nature of currently available assays. An alkaline unwinding flow cytometric assay using UV-C radiation as a DNA-damaging agent was adapted for measurement of NER-mediated breaks. This assay was based on the principle of alkaline unwinding of strand breaks in double-stranded DNA to yield single-stranded DNA with the number of strand breaks being proportional to the amount of DNA damage. This assay measured 50,000 events per sample with several samples being analyzed per specimen, thereby providing very reliable measurements, which can be performed on a large-scale basis. Using area under the curve (AUC) to quantitate amount of NER-mediated breaks, this assay was able to detect increased NER-mediated breaks with increasing doses of UV-C radiation. The assay detected NER-mediated breaks in lymphocytes from normal donors and not in xeroderma pigmentosum lymphoblastoid cell lines indicating specificity for the detection of NER-mediated breaks. The assay measured NER-mediated breaks within G(1), S and G(2)/M phases of the cell cycle; thereby decreasing variability in measurements of NER-mediated breaks due to differences in cell cycle phases. Intraindividual variability for AUC after 120 min of repair was 15% with interindividual variability being approximately 43% for cells in the G(1) phase, indicating substantial between-subject variation and relatively low within-subject variation. Thus, the alkaline unwinding flow cytometry-based assay provides a high-throughput method for the specific measurement of NER-mediated breaks in lymphocytes.

Regulation of Reactive Oxygen Species by Atm Is Essential for Proper Response to DNA Double-Strand Breaks in Lymphocytes

The ataxia telangiectasia-mutated (ATM) gene plays a pivotal role in the maintenance of genomic stability. Although it has been recently shown that antioxidative agents inhibited lymphomagenesis in Atm(-/-) mice, the mechanisms remain unclear. In this study, we intensively investigated the roles of reactive oxygen species (ROS) in phenotypes of Atm(-/-) mice. Reduction of ROS by the antioxidant N-acetyl-l-cysteine (NAC) prevented the emergence of senescent phenotypes in Atm(-/-) mouse embryonic fibroblasts, hypersensitivity to total body irradiation, and thymic lymphomagenesis in Atm(-/-) mice. To understand the mechanisms for prevention of lymphomagenesis, we analyzed development of pretumor lymphocytes in Atm(-/-) mice. Impairment of Ig class switch recombination seen in Atm(-/-) mice was mitigated by NAC, indicating that ROS elevation leads to abnormal response to programmed double-strand breaks in vivo. Significantly, in vivo administration of NAC to Atm(-/-) mice restored normal T cell development and inhibited aberrant V(D)J recombination. We conclude that Atm-mediated ROS regulation is essential for proper DNA recombination, preventing immunodeficiency, and lymphomagenesis.

Prolonged exercise does not cause lymphocyte DNA damage or increased apoptosis in well-trained endurance athletes

Recent research has demonstrated that lymphocyte apoptosis sensitivity appears to be related to training status and exercise intensity. This work investigated the effect of prolonged, submaximal treadmill running on percentage (%) apoptosis, % necrosis and DNA strand breaks in lymphocytes and related these to changes in total lymphocyte and blood cortisol concentrations in well-trained runners. Venous blood samples (n = 14) were taken immediately before (PRE), immediately after (IPE) and 3 h after (3PE) 2.5 h of treadmill running at 75% of VO2 max from eight well-trained male endurance athletes (age 34.2 +/- 2.44 years) and analysed for cellular content and serum cortisol concentrations. Lymphocytes were isolated from whole blood and % apoptotic and necrotic cell were detected by flow cytometry using Annexin V-FITC and propidium iodide uptake. DNA strand breaks were measured by single-cell gel electrophoresis. Despite a significant (P < 0.001) exercise-induced increase in mean serum cortisol concentrations and reduction in lymphocyte counts, the mean % Annexin-V positive cells (13.3 +/- 6.78 in PRE, 11.3 +/- 5.51 in IPE and 12.8 +/- 6.75 in 3PE samples) were not significantly different at the three time-points (P > 0.05). Mean DNA strand breaks in the lymphocytes also did not change significantly (P > 0.05) rising from 25.7 +/- 2.16 to 26.9 +/- 1.89 and 27.1 +/- 1.38 microm in IPE and 3PE samples, respectively. The exercise-induced changes in total blood lymphocyte counts and cortisol concentrations did not result in a significant change in % apoptotic lymphocytes or DNA strand breaks in the endurance-trained athletes during this prolonged, submaximal exercise.

Microarray Analysis of Bleomycin-Exposed Lymphoblastoid Cells for Identifying Cancer Susceptibility Genes

The uncovering of genes involved in susceptibility to the sporadic cancer types is a great challenge. It is well established that the way in which an individual deals with DNA damage is related to the chance to develop cancer. Mutagen sensitivity is a phenotype that reflects an individual's susceptibility to the major sporadic cancer types, including colon, lung, and head and neck cancer. A standard test for mutagen sensitivity is measuring the number of chromatid breaks in lymphocytes after exposure to bleomycin. The aim of the present study was to search for the pathways involved in mutagen sensitivity. Lymphoblastoid cell lines of seven individuals with low mutagen sensitivity were compared with seven individuals with a high score. RNA was isolated from cells exposed to bleomycin (4 hours) and from unexposed cells. Microarray analysis (19K) was used to compare gene expression of insensitive and sensitive cells. The profile of most altered genes after bleomycin exposure, analyzed in all 14 cell lines, included relatively many genes involved in biological processes, such as cell growth and/or maintenance, proliferation, and regulation of cell cycle, as well as some genes involved in DNA repair. When comparing the insensitive and sensitive individuals, other differentially expressed genes were found that are involved in signal transduction and cell growth and/or maintenance (e.g., BUB1 and DUSP4). This difference in expression profiles between mutagen-sensitive and mutagen-insensitive individuals justifies further studies aimed at elucidating the genes responsible for the development of sporadic cancers.

DNA strand breaks in the lymphocytes of workers exposed to diisocyanates: indications of individual differences in susceptibility after low-dose and short-term exposure

Diisocyanates are chemically reactive and induce asthma, but data on genotoxic effects of diisocyanates in humans are limited. The investigation presented here used short term diisocyanate chamber exposure to study DNA strand breaks in lymphocytes of 10 healthy individuals and of 42 workers, with airway symptoms, who had previously been exposed to diisocyanates. The alkaline version of the Comet assay was used to analyse DNA strand breaks in lymphocytes. In addition, blood samples of 10 further control individuals without any exposure to diisocyanates were studied. Substances studied were 4,4'-methylenediphenyldiisocyanate (MDI, n=25), 2,4-toluenediisocynate and 2,6-toluenediisocyanate (TDI, n=5), and 1,6-hexamethylenediisocyanate (HDI, n=12), at concentrations between 5 and 30 ppb for 2 h. Lymphocytes isolated from the subjects before exposure and 30 min and 19 h after were used to evaluate DNA damage. No significant changes in DNA strand-break frequencies were measured, as Olive tail moment (OTM), either between groups or before and after diisocyanate exposure. OTM was similar in subjects with an asthmatic reaction (MDI, n=5; TDI, n=1; HDI, n=1) and in subjects without such a reaction. However, a small and susceptible group (about 10% of the individuals studied) could be identified with higher frequencies of DNA strand breaks in lymphocytes after chamber exposure. The occurrence of DNA damage in this group may be based on indirect mechanisms such as oxidative stress or apoptosis.

DNA repair capacity in lymphocytes of nasopharyngeal cancer patients

Possible hereditary factors in the tumorigenesis of nasopharyngeal cancer (NPC) have not yet been clearly identified. In the present study, the DNA repair capacity of lymphocytes after exposure to the nitrosamine NDEA was quantified in order to elucidate whether this measure may be a factor in susceptibility to NPC. The alkaline single-cell microgel electrophoresis (Comet) assay was used to quantify chemically induced DNA damage and repair capacity in lymphocytes of 30 NPC patients (NPC) and 29 non-tumor donors (NTD). The induction of DNA single strand breaks, alkali labile and incomplete excision repair sites after exposure of lymphocytes to NDEA was assessed as differences between repair intervals of 0 min, 15 min, 30 min and 60 min, respectively. A RC(total) was assessed using the difference between the OTMs of 0 min of repair time and the 60-min repair interval for both groups. Repair capacities (RC) were calculated for the intervals according to the Olive Tail Moment (OTM), a quantitative measure for DNA migration in the Comet assay for the group of NPC patients and the NTD, accordingly. RCs were compared between the two groups using the Mann-Whitney U-Test. RC(15 min), RC(30 min) RC(60 min) and the RC(total) after a 60-min repair interval demonstrated no significant difference between the two groups. Furthermore, when comparing grades of DNA migration (OTM<2, 2-5, 5-10, 10-20, 20-30 and >30), there were no differences evident. In this investigation, rejoining of DNA single strand breaks in lymphocytes of NPC and NTD appeared to be accomplished to an equal degree and in equal time periods. However, the applied method does not give evidence concerning the quality of the single strand break rejoining processes. In this group of patients, tumorigenesis in NPC could not be associated with a decreased DNA repair capacity.

Effects of age and dietary restriction on oxidative DNA damage, antioxidant protection and DNA repair in rats.

Experimentally imposed dietary restriction is known to extend the lifespan of rodents, perhaps by slowing the accumulation of oxidative damage that is thought to be one of the causes of aging. We examined the effects of restricted total food intake, and protein and calorie restriction, on DNA oxidation and related biomarkers in rats. From 1 to 17 months, rats in group 1 received normal diet ad libitum. Group 2 received 70% of the quantity consumed by the first group. Group 3 had the same quantity as group 2, but with a reduction in protein (from 18% to 10% of the diet by weight), and group 4 were further restricted with a 30% decrease in calories. Lymphocytes were isolated from blood samples taken every two months. DNA breaks, oxidised pyrimidines, resistance to H2O2-induced damage, and strand break repair were measured with the comet assay. Organs were isolated from rats killed at 17 months, with 1 month-old rats for comparison; DNA oxidation and antioxidant enzyme activities were measured. DNA breaks in lymphocytes increased from 1 to 3 months but thereafter declined with age, except in ad libitum fed rats. Oxidised pyrimidines did not change significantly. Resistance to H2O2-induced damage was least at 3 months, and increased with age. Repair of DNA strand breaks was efficient at all ages. Diet had little effect on these endpoints. Diet had no influence on 8-oxo-7.8-dihydroguanine levels in DNA from liver, testis and brain of 17 month old rats. Combining data from all four groups, the levels in brain and liver were significantly higher at 17 months compared with 1 month. Antioxidant enzyme activities tended to increase between 1 and 17 months; effects of diet were not so consistent. While DNA damage shows a modest increase with age in some organs, antioxidant status and DNA strand break repair do not decline with age. Restricted diets (including protein and calorie restriction) have no effect on any of these markers of genetic stability.

Differential susceptibility to chromatid breaks induced by bleomycin in sub-fertile and fertile bovines

The rate of chromatid breaks was studied in cows with a history of sub-fertility by means of a test based on measurement of the average of breaks induced in lymphocytes of peripheral blood cultures. Fourteen female specimens were divided into two groups: fertile and sub-fertile. Peripheral blood lymphocytes were cultured and prepared for cytogenetic analysis. Two types of culture were established for each animal to evaluate the response of peripheral blood lymphocyte cultures to the genotoxic effects of bleomycin. The first culture did not receive bleomycin treatment (spontaneous chromosome aberrations). Our results showed that median breaks per cell (b/c) (± semirange) for spontaneous culture of the fertile and sub-fertile animals and bleomycin sensitivity assay for fertile and sub-fertile animals were 0.00 ± 0.06, 0.02 ± 0.03, 0.08 ± 0.05 and 0.22 ± 0.09, respectively. There was no significant difference ( P > 0.05) in the chromosomal breakage in lymphocytes not exposed to bleomycin; however, in comparing the number of chromatid breaks per cell in cultures treated with bleomycin, the sub-fertile group showed a significantly higher ( P < 0.05) level than the fertile group. These findings have implications both for identifying cattle with less than optimum fertility as well as for providing potential avenues to study the origins of sub-fertility.

Non-random distribution of chromatid breaks in lymphocytes of laryngeal squamous cell carcinoma patients

The frequency and localization of mutagen-induced chromatid breaks in peripheral blood lymphocytes (PBLs) were investigated for association with squamous cell carcinoma (SCC) of the larynx. The case-control study was performed in 52 patients with laryngeal SCC and in 47 cancer-free controls. The analyses were based on the bleomycin sensitivity assay. The differences between cases and controls were estimated using Mann-Whitney U test and unconditional logistic regression. The total number of chromatid breaks in PBLs of patients was significantly higher compared with healthy controls (p<0.0001); the increase was observed in almost all chromosomal arms. In a number of chromosomal regions, the relative frequency of breaks was higher in patients; this increase was statistically significant for 1p22, 5q31, 6q23 and 10q24. The majority of sites with the increased proportion of breaks in patients were identified as regions containing loci involved in DNA repair, cell cycle regulation, suppressor genes and oncogenes. Revealing non-random distribution of chromatid breaks specifically associated with laryngeal SSC may be instrumental for defining regions involved in the etiology of this disease.