Amount Of DNA Damage Research Articles

Efficacy of three different laser wavelengths for in vitro wound healing

Despite contradictory reports on the effect of laser light on cell proliferation, studies have shown that appropriate doses and wavelengths of laser light are therapeutically beneficial in tissue repair and pain control. This study aimed to establish if the dose and/or wavelength influenced the biological responses of irradiated in vitro fibroblasts--1 h after laser irradiation. This study aimed to establish cellular responses of normal and wounded human skin fibroblasts to helium-neon (632.8 nm), diode (830 nm) and Nd:YAG (1064 nm) laser irradiation using one exposure of 5 or 16 J/cm(2) on day 1 and again on day 4. Wounded cells exposed to 5 J/cm(2) using 632.8 nm showed an increase in cell migration and haptotaxis, a stable increase in the release of interleukin-6 (IL-6), a decrease in caspase 3/7 activity, an increase in ATP viability and an increase in cell proliferation--1 h after the final exposure. The results confirm that changes in parameters such as ATP viability, cytokine expression (IL-6), cell proliferation (alkaline phosphatase enzyme activity) and DNA damage can be observed directly after the laser irradiation. The amount of DNA damage and cytotoxicity may be related to duration of the laser irradiation, which is dependent on the power density (mW/cm(2)) of each laser. The results indicate that 5 J/cm(2) using 632.8 nm results in a stimulatory effect that is more effective than 830 and 1064 nm. The results suggest possible mechanisms by which the wavelength may potentially influence the cellular responses of wounded cells.

A Comparative Study of Chemically Induced DNA Damage in Isolated Nasal Mucosa Cells of Humans and Rats Assessed by the Alkaline Comet Assay

Single-cell microgel electrophoresis (comet) assay was used to study genotoxic effects in human nasal mucosa cells and rat nasal and ethmoidal mucosa cells in vitro. Human cells were obtained from tissue samples of 10 patients (3 females/7 males), who underwent surgery (conchotomy) for treatment of nasal airway obstruction. Rat nasal mucosa cells were derived from male Sprague-Dawley rats. Cells were exposed for 1 h to either N-nitrosodiethanolamine (NDELA), epichlorohydrin (EPI), 1,2-epoxybutane (EPB), ethylene dibromide (EDB), or 1,2-dibromo-3-chloropropane (DBCP). Dimethyl sulfoxide (DMSO) was used as negative control. Alkaline comet assay was performed according to a standard protocol and DNA damage was quantified as Olive tail moment using image analysis system. All test substances induced an increase in DNA damage in human and rat cells. The absolute amount of DNA damage in rat nasal mucosa cells was usually higher than in ethmoidal mucosa cells. Human nasal mucosa cells were found to be less sensitive than rat mucosa cells to the genotoxic activities of DBCP (lowest effective concentration in human cells [LEChuman]: 1.5, in rat cells [LECrat]: 0.01 mM) and NDELA (LEChuman: 25, LECrat: 12.5 mM), whereas EPB-treated cells were almost equal (LEChuman and LECrat 0.78 mM). NDELA induced a marked concomitant cytotoxicity. For EPI (LEChuman and LECrat: 0.097 mM) and EDB (LEChuman: 0.195, LECrat: 0.048 mM), pronounced interindividual differences were observed in human samples.

Mutagen sensitivity in peripheral blood lymphocytes among coke-oven workers

To investigate the sensitivity to bleomycin (BLM) in peripheral blood lymphocytes (PBL) among coke-oven workers. Ninty-four coke-oven workers with exposure to a high level of polycyclic aromatic hydrocarbons and 64 non-coke-oven workers (control) were recruited into this study. PBL was challenged by 8 microg/ml BLM, a known carcinogen, to induce certain amount of DNA damage, the difference of olive tail moment (TM) measured by comet assay before and after BLM treatment reflected the sensitivity towards mutagens. The distribution of age, sex, and prevalence of smoking and drinking were not significantly different between these two groups. The geometric mean of urinary 1-hydroxypyrene (1-OHP) was significantly higher in coke-oven workers than in controls (9.0 versus 1.5 microg/L, t = -9.317, P < 0.01). The coke-oven workers showed significantly higher sensitivity to BLM than controls (17.7 versus 14.9, t = -2.583, P = 0.01). A large inter-group difference in sensitivity to BLM was observed in both controls and coke-oven workers. Stratification analysis revealed the significant association between high 1-OHP level (> 9.0 microg/L) and increased sensitivity to BLM (F = 4.001, P = 0.05) among coke-oven workers. Smoking subjects showed a significant higher value of sensitivity than nonsmokers in controls but not in coke-oven workers. No significant difference was observed between age, drinking status, coking history or external exposure class and BLM sensitivity. Exposure to coke oven emission could increase the sensitivity to mutagens, which might be a reason of high incidence of lung cancer among coke-oven workers.

Evaluation of the in vivo mutagenic potential of hydroalcoholic extracts of the northern highbush blueberry (Vaccinium corymbosum L. Ericales, Ericaceae) on peripheral blood cells of Swiss mice (Mus musculus Rodentia, Muridae)

The northern highbush blueberry (Vaccinium corymbosum L. Ericales, Ericaceae) is very rich in anthocyanins, natural pigments which have strong antioxidant properties and potential health benefits, resulting in the worldwide use the blueberry as a medicinal plant. We investigated the mutagenic potential of simple hydroalcoholic extracts of V. corymbosum acutely administrated by gavage to Swiss mice at doses of 1 g kg-1, 1.5 g kg-1 and 2 g kg-1. Peripheral blood cells were collected 4 h and 24 h post-gavage and assessed by the alkaline comet assay, with further blood samples being collected at 48 h and 72 h for assessment using the micronucleus (MN) assay. Our results show that the V. corymbosum extracts did not induce any statistically significant increase in the average amount of DNA damage in peripheral blood leukocytes. However, we did record a significant increase in the frequency of micronucleated polychromatic erythrocytes at the three doses tested.

Application of cytogenetic endpoints and comet assay on human lymphocytes treated with atorvastatin in vitro

This study investigated the genotoxic potential of atorvastatin on human lymphocytes using comet assay, structural chromosome aberrations (CA) and sister-chromatid exchange (SCE) analysis. Lymphocyte cultures were treated with a single drug at a concentration of 30.21 ng/mL. For comet assay, cells exposed to atorvastatin for 24 h, 48 h and 72 h were embedded in agarose slides, lysed with alkaline lysis solution and exposed to an electric field. DNA migrated within the agarose and formed comets whose length depends on the amount of DNA damage. For analysis of structural CA, cells were grown on medium for 48 h and for SCE analysis for 72 h. Structural CA did not induce significant damage to the genome, although a higher CA frequency was observed in cells treated with atorvastatin for 3 h, 20 h and 48 h than in control samples. Results of the SCE analysis did show statistically significant differences in the mean SCE number between atorvastatin-exposed and control human lymphocytes and between different exposure times. Comet assay also showed increased DNA damage caused in atorvastatin-exposed human lymphocytes than in corresponding control cells for exposure times of 24 h, 48 h and 72 h for the tail length and for 72 h for the tail moment. Results obtained in this study point to the significance of biological indicators providing information on the primary genome damage after long-term exposure, which can help to establish drug therapeutic concentrations that do not put patients with high blood cholesterol to a greater treatment-related risk.

Differential DNA damage induced by H 2O 2 and bleomycin in subpopulations of human white blood cells

The purpose of this study was to characterize the differential sensitivities of various subpopulations of human white blood cells after exposure to H 2O 2 (an oxidant agent) and bleomycin (a radiomimetic glycopeptide), in vitro, using single-cell gel electrophoresis (SCGE). Human peripheral blood was fractionated into mononuclear cells, which were further separated into monocytes, CD4+ T-cells, CD8+ T-cells, B-cells and natural killer cells (NK cells). The separated fractions were exposed to different doses of H 2O 2 and bleomycin, and then used to measure levels of induced and basal DNA damage. There was a significant increase in the amount of DNA damage in CD4+ T-cells, CD8+ T-cells, NK cells and B-cells when treated with H 2O 2 and bleomycin, whereas monocytes had the lowest sensitivity to H 2O 2 compared with the other cell fractions, but no lower sensitivity to bleomycin. Furthermore, CD4+ T-cells and CD8+ T-cells had the highest levels of basal DNA damage. When basal DNA damage was taken into account, NK cells tended to show a higher sensitivity to H 2O 2 than CD4+ T-cells, CD8+ T-cells and monocytes. In addition, B-cells, which showed lower sensitivity to H 2O 2 than CD4+ T-cells, CD8+ T-cells and NK cells when exposed to lower doses of H 2O 2 (<10 μM), showed higher sensitivity to H 2O 2 at higher doses (>20 μM). On the other hand, B-cells showed the highest sensitivity to bleomycin.

Induction and repair rate of DNA damage: A unified model for describing effects of external and internal irradiation and contamination with heavy metals

DNA is a key-target for genotoxic stress. Hence, the knowledge of induction and repair rate of DNA damage are crucial to describe and predict the impact of stress situations. Unfortunately, DNA damage induction and repair rates are generally assessed separately whereas they act either concomitantly or transiently in living organisms. Furthermore, the interplay of induction and repair raises the question whether DNA repair adapts to respond to different amounts of DNA damage.In a previous report, we proposed a stochastic interpretation of the repair rate of the major radiation-induced DNA damage. We provided evidence that the repair rate of individual DNA damage is time-independent whereas that of a population of DNA damage is time-dependent (Foray, N., Charvet, A.-M., Duchemin, D., Favaudon, V., Lavalette, D., 2005. The repair rate of radiation-induced DNA damage: a stochastic interpretation based on the gamma function. J. Theor. Biol. 236, 448–458). Here, to better describe situations in which DNA damage induction and repair occur together, our biostatistical model was modified by the introduction of a DNA damage induction parameter. Theoretical and experimental data were compared and discussed by taking concrete experimental situations: X-rays irradiation at different dose-rates, internal irradiation with radioactive compound, contamination with heavy metal and detection of DNA damage by immunofluorescence.By assuming that DNA repair rate is invariant whatever the amount of DNA damage, our model provides good prediction of experimental data suggesting its relevance for the description of complex situations of co-toxicities.

Does age influence loss of heterozygosity?

The striking correlation between advanced age and an increased incidence of cancer has led investigators to examine the influence of aging on genome maintenance. Because loss of heterozygosity (LOH) can lead to the inactivation of tumor suppressor genes, and thus carcinogenesis, understanding the affect of aging on this type of mutation event is particularly important. Several factors may affect the rate of LOH, including an increase in the amount of DNA damage, specifically double-strand breaks (DSBs), and the ability to efficiently repair this damage via pathways that minimize the loss of genetic information. Because of experimental constraints, there is only suggestive evidence for a change in the rate of DNA damage as humans age. However, recent studies in model organisms find that there are increased rates of LOH with age, and that repair of DNA damage occurs via a different pathway in old cells versus young cells. We speculate that the age-dependent change in DNA repair may explain why there is increased LOH, and that the findings from these model organisms may extend to humans.

Effect of cryopreservation on sperm DNA integrity in patients with teratospermia

To test whether sperm with abnormal head morphology are more likely to undergo DNA damage and/or chromatin modification during the process of freeze-thawing. In this prospective study, the semen samples from forty-four men attending the infertility clinic were included. Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. The sperm morphology and the sperm DNA damage were evaluated before and after cryopreservation. The relationship between sperm head abnormalities and freeze-thaw-induced DNA modification was assessed. University hospital fertility center. Men attending infertility clinic for semen analysis. The normospermic and teratospermic semen samples were evaluated for DNA damage before and after cryopreservation by comet assay and acridine orange bindability test. Elucidation of association between sperm morphologic defect and cryodamage. A threefold increase in the amount of DNA damage was observed in teratospermic samples compared with their normospermic counterparts, indicating a higher susceptibility of morphologically abnormal sperm to cryodamage. The susceptibility of morphologically abnormal sperm to DNA damage/chromatin modification during the freeze-thaw process is significantly higher than that of sperm with normal morphology.

Carbonic anhydrase 9 (CA9) expression in tumor cells enhances sensitivity to tirapazamine

Carbonic anhydrase 9 (CA9) is over-expressed in many human solid tumors under conditions of low oxygen concentration and can be associated with a low probability of survival. In this study, stable CA9-expressing cell lines were established using the CA9 gene-defective human C33a cell line and the HeLa cell line to investigate the role of CA9 in response to ionizing radiation and hypoxia-selective cytotoxin, Tirapazamine (TPZ). Human CA9 cDNA or an empty vector was transfected into the C33a and HeLa cell lines and C33a-vector, C33a-CA9, HeLa-vector, and HeLa-CA9 cell lines were produced accordingly. Sensitivity of the C33a-vector/C33a-CA9 cells to ionizing radiation and TPZ was measured using clonogenic assays. The alkaline comet assay was used to measure single strand DNA breaks caused by TPZ in the C33a-vector, C33a-CA9, HeLa-vector, and HeLa-CA9 cell lines. Radiation sensitivity, as determined with clonogenic survival assays, of C33a-vector/C33a-CA9 cells did not differ under either normoxic or hypoxic conditions. However, increased clonogenic sensitivity to TPZ was observed in C33a-CA9 cells under the hypoxic condition by 26% (95% CI 14-39%, P = 0.02 in comparison to the C33a-vector cells). The comet assay showed significantly greater DNA damage in the C33a-CA9 cells compared with that of the C33a-vector cells with the same treatment under hypoxic conditions, supporting the results of the clonogenic survival data. Because this difference in the amount of DNA damage was not observed for the hypoxic HeLa-CA9/HeLa-vector cell lines, both of which have induced CA9 expression by hypoxia, the enhanced sensitivity of C33a-CA9 cells to TPZ is considered to be due to the specific condition of CA9 over-expression. Our results suggest the possibility that CA9 over-expression in tumors might be exploited to increase the treatment effects of TPZ.

The expression of 8-hydroxydeoxyguanosine in oesophageal tissues and tumours

Purpose The most common marker of oxidative DNA damage is 8-hydroxydeoxyguanosine (8-OHdG), which is linked with several malignancies. In the present study we investigated whether DNA damage linked to oxidative stress (as 8-OHdG) is present in Barrett's mucosa with or without associated adenocarcinoma or high-grade dysplasia and in normal controls' squamous mucosa. Experimental design We measured 8-OHdG in 51 patients (13 Barrett's metaplasia, six Barrett's oesophagus with high-grade dysplasia, 18 adenocarcinoma of the distal oesophagus/oesophagogastric junction and 14 normal controls). The amount of DNA damage was determined by high-performance liquid chromatography in oesophagus samples obtained either from endoscopy or as samples from surgery. The median 8-OHdG concentration was expressed as the ratio of 8-OHdG per 10 5 deoxyguanosine. Results Analysis revealed that 8-OHdG was present in both Barrett's metaplasia with and without dysplasia as well as in adenocarcinoma of the oesophagus/oesophagogastric junction. Although the study group was small the amount of 8-OHdG was significantly increased in the distal oesophagus both in Barrett's epithelium 1.26 (0.08–29.47) and in high-grade dysplasia 1.35 (1.04–1.65) as well as in adenocarcinoma of oesophagus/oesophagogastric junction 1.08 (0.59–1.94) compared to controls 0.06 (0–4.08) ( p = 0.002, p = 0.012, p = 0.001, respectively). Barrett's patients had no significant difference in 8-OHdG levels between their distal and proximal oesophageal samples. Conclusions Our results show the presence of oxidative DNA damage in the distal oesophagus of patients with Barrett's oesophagus and adenocarcinoma of the oesophagus/oesophagogastric junction. This may have a connection to carcinogenesis in Barrett's oesophagus.

Paradox Resolved? The Strange Case of the Radiation-Resistant Bacteria

Paradox Resolved? The Strange Case of the Radiation-Resistant Bacteria

Short‐term culture and γH2AX flow cytometry determine differences in individual radiosensitivity in human peripheral T lymphocytes

Histone H2AX, a subfamily of histone H2A, is phosphorylated and forms proteinaceous repair foci at the sites of DNA double-strand breaks in response to genotoxic insults, such as ionizing radiation. This process is believed to play a key role in the repair of DNA damage. In this study, we established a flow cytometry (FCM) system for measuring radiation-induced phosphorylated histone H2AX (gammaH2AX) in cultured human T lymphocytes to evaluate individual radiation sensitivity in vitro. Irradiation of short-term ( approximately 7 days) cultured T lymphocytes exhibited significant interindividual, but not interexperimental, differences in the cellular content of gammaH2AX 6 hr after 4 Gy of X-irradiation in three independent experiments using peripheral blood lymphocytes from six healthy donors. However, these differences were not as marked in uncultured lymphocytes, or lymphocytes that were cultured for a prolonged period ( approximately 13 days). The variation of gammaH2AX focus formation in lymphocytes of individuals was reproducible, with differences reaching about 1.5-fold following 7 days of culture. Therefore, the FCM-based gammaH2AX measurement appeared to reflect both the temporal course and the amount of DNA damage within the irradiated lymphocytes. Further, we confirmed that the differences in residual lymphocyte subsets were not involved in individual radiosensitivity. These results suggest that the FCM-based gammaH2AX assay using cultured T lymphocytes might be useful for the rapid and reliable assessment of individual radiation sensitivity involved in DNA damage repair.

Effects of low- or medium-pressure ultraviolet lamp irradiation on Microcystis aeruginosa and Anabaena variabilis

A pure culture of Microcystis aeruginosa or Anabaena variabilis, the representatives of water blooming algae, was exposed to low-pressure (LP) or medium-pressure (MP) UV lamps. Irradiated pure culture suspension was subsequently incubated for 7 d under white light fluorescent lamps. During incubation, profiles of the number of cells, DNA damage and photosynthetic activity were determined. When UV fluence was 600 mJ/cm 2, M. aeruginosa cell numbers decreased throughout the 7-d incubation period, to produce 1.5 log reduction (LP) or 1.2 log reduction (MP) compared with control. The amount of DNA damage was 2.02×10 −4 ESS/base (LP) and 3.42×10 −4 ESS/base (MP) just after UV irradiation, which became 0.05×10 −4 ESS/base and 0.23×10 −4 ESS/base, respectively, after 3 d incubation. However, cell number kept decreasing, even after DNA repair. Photosynthetic activity decreased by 1.5 log within 1 d (LP) or 3 d (MP). Thus, reduction in photosynthetic activity could contribute to the reduction in M. aeruginosa cell numbers. A. variabilis cell numbers reduced by 2.3 log (LP) or 2.2 log (MP) during the 7-d incubation period; however, after DNA damage repair, cell number began to increase. The amount of DNA damage was 6.07×10 −4 ESS/base (LP) and 4.48×10 −4 ESS/base (MP) just after UV irradiation, which became 0.23×10 −4 ESS/base and 0.40×10 −4 ESS/base, respectively, after 3 d incubation. No reduction was observed in photosynthetic activity/cell. Therefore, DNA damage is the main contributor of the reduction in cell number of A. variabilis.

Three tandem HRDC domains have synergistic effect on the RecQ functions in Deinococcus radiodurans

The RecQ family of DNA helicases performs essential functions in the maintenance of genomic stability in all organisms. In Deinococcus radiodurans, DR1289 is a special member of RecQ family with unique arrangement of three tandem HRDC domains in the C-terminus. A dr1289 mutant is hypersensitive to γ-irradiation, UV, H 2O 2 and mitomycin C. By complementing the dr1289 mutant with various domains of Dr1289 in vivo, we have determined that the helicase and all three HRDC domains are indispensable for complete DNA damage resistance. Using a continuous fluorescent dye-displacement assay, we investigated the optimal conditions for Dr1289 unwinding function at various concentrations of ATP and metal ions to show that the helicase activity is comparable to what observed in Escherichia coli RecQ. We also found that the helicase domain is necessary for the unwinding and ATPase activity and that the three tandem HRDC domains increase the efficiency of these activities. Based on these data, we propose that the C-terminus of Dr1289 has evolved in D. radiodurans to confront the types and amounts of DNA damage.

Acute exposure to UVB has a more profound effect on plant genome stability than chronic exposure

Environmental factors that damage DNA have various lengths of exposure and intensity levels. Although the results of increasing the intensity of a DNA damaging agent is often predictable, it is not clear whether the stage during development when the exposure is received has any influence on the amount of DNA damage. In this paper we analyzed the influence of UVB on the stability of Arabidopsis thaliana and the Nicotiana tabacum genomes. Our experiments showed that the acute exposure to UVB produces a significantly greater increase in homologous recombination frequency (HRF) and recombination rate (RR) compared with that produced by chronic exposure. The increase in HRF showed a positive correlation with UVB dose and a negative correlation with plant age. In other words, as the UVB dose was increased, there was a concomitant increase in HRF. Conversely, older plants had a lower HRF increase as compared to younger plants. Our experiments suggest that exposure to UVB makes the most significant impact on genome stability during the early stages of plant development.

Mammalian SWI/SNF complexes facilitate DNA double-strand break repair by promoting γ-H2AX induction

Although mammalian SWI/SNF chromatin remodeling complexes have been well established to play important role in transcription, their role in DNA repair has remained largely unexplored. Here we show that inactivation of the SWI/SNF complexes and downregulation of the catalytic core subunits of the complexes both result in inefficient DNA double-strand break (DSB) repair and increased DNA damage sensitivity as well as a large defect in H2AX phosphorylation (gamma-H2AX) and nuclear focus formation after DNA damage. The expression of most DSB repair genes remains unaffected and DNA damage checkpoints are grossly intact in the cells inactivated for the SWI/SNF complexes. Although the SWI/SNF complexes do not affect the expression of ATM, DNA-PK and ATR, or their activation and/or recruitment to DSBs, they rapidly bind to DSB-surrounding chromatin via interaction with gamma-H2AX in the manner that is dependent on the amount of DNA damage. Given the crucial role for gamma-H2AX in efficient DSB repair, these results suggest that the SWI/SNF complexes facilitate DSB repair, at least in part, by promoting H2AX phosphorylation by directly acting on chromatin.

A model for low dose effects of low-LET radiation delivered at high dose rates

In vitro studies show that protective tumour-reducing effects occur for low dose rates (mGy per minute). To account for these phenomena, we have previously developed stochastic and deterministic multi-stage cancer models that include radiation-induced adaptations in DNA repair processes and radical scavenging. Here, these models are extended to account for the induction of radioprotective mechanisms for low doses of low LET radiation delivered at high dose rates. Cellular adaptations in DNA repair are related to temporal changes in the amount of DNA damage in a cell. The combined effects of endogenous DNA damage, background radiation and artificial irradiation are considered.

DNA damage and repair in peripheral blood lymphocytes from healthy individuals and cancer patients: A pilot study on the implications in the clinical response to chemotherapy

Drug resistance is considered the main impediment to successful cancer chemotherapy. The quest for a method useful to predict individual responses to chemotherapy prior to treatment is highly desired. This study was designed to determine the individual influences of doxorubicin and cisplatin on the degree of DNA damage, DNA repair and hMSH2 and the hMLH1 protein expression in peripheral blood lymphocytes (PBL) and their correlations with the clinical response. PBL were obtained from 25 cancer patients (pre- and post-chemotherapy) and from 10 healthy persons, cultured and exposed to doxorubicin or cisplatin. Cells were collected at T0 (immediately after drug treatment) and 24 h after damage (T24). The alkaline comet assay was employed to assess the DNA damage and repair function, and immunocytochemistry to study hMLH1 and hMSH2 expression. Clinical response was evaluated after three cycles of chemotherapy. Pre-chemotherapy PBL from cancer patients showed significantly higher levels of basal DNA damage than healthy persons, with appreciable interindividual variations between them. The in vivo administration of antineoplasic drugs was accompanied by significant DNA damage, and an increased in the number of apoptotic cells. Cancer patients with complete response showed a high number of apoptotic cells. The DNA migration increased at T0 and at T24 in cisplatin-treated patients, reflecting a decreased rate of cisplatin adducts repair than that observed in healthy individuals. The ability to repair DNA lesions in doxorubicin-damaged cells was very similar between healthy individuals and cancer patients. Cisplatin-treated patients that died by the disease showed lower DNA migration than the mean value. The expression of hMLH1 and hMSH2 was practically identical between healthy individuals and cancer patients. Nevertheless, chemotherapy induced a depletion mostly of hMLH1. In 83% of cisplatin-treated patients with CR the hMLH1 and hMSH2 expression at T24 was higher than the mean. In this pilot study the alkaline comet assay offered information about the amount of DNA damage and the DNA repair status in PBL from individual patients and this seems useful in predicting the response to chemotherapy.

DNA Damage in Sperm: Should Semen or the 90% Density Gradient Layer be Used for Prospective Screening?

Sperm chromatin damage may be related to pregnancy outcome in patients undergoing IVF. Therefore, many laboratories have initiated prospective screening of patients using assays to quantitate DNA breaks in sperm. Fresh semen is usually evaluated. The purpose of this study was to evaluate the clinical relevance of evaluating the fresh semen compared to the post-gradient 90% layer used for insemination. A prospective evaluation of chromatin damage in the fresh semen sample and 90% layer following density gradient centrifugation was followed by a retrospective chart review to evaluate semen quality factors which may influence or be related to changes observed in the amount of DNA damage, and to evaluate the results in regard to IVF outcome. Semen samples from 66 patients were evaluated for DNA damage using the TUNEL assay, as previously described. An aliquot of the fresh semen, and an aliquot of the sperm obtained from the 90% layer of the density gradient preparation were analyzed. The samples were classified as “normal” when <15% of the sperm stained positive in the TUNEL assay. Data were evaluated using ANOVA and Chi square analysis. Twenty-six fresh semen samples were classified as “abnormal” (39.4%). Of those patients, 16 samples (61.5%) demonstrated an improvement to the normal range following density gradient preparation. The mean TUNEL score was 24.6 ± 2.8 in the fresh sample and 8.3 ± 1.0 in the 90% layer (p < 0.001). Conversely, 8 patients classified as normal in the fresh semen aliquot (8/40, 20%), had abnormal TUNEL values for sperm in the 90% layer (4.9 ± 1.5 versus 27.8 ± 4.2, p < 0.01). When comparing patients with normal fresh TUNEL values who had abnormal values following density gradient isolation to those who remained in the normal category, significant differences were observed (p < 0.05) in the initial sperm count (38.4 ± 11.3 versus 118.4 ± 23.2), viability (30.9 ± 5.6 versus 45.8 ± 2.6), and HOS reactivity (35.7 ± 5.9 versus 48.8 ± 3.19), but not in white blood cells, morphology, or motility. No significant differences were observed in semen quality between patients who improved following density gradient separation and those who did not. IVF outcomes were more closely aligned to data observed from the 90% layer (p < 0.05). These data demonstrate that the results obtained using the TUNEL assay to evaluate DNA damage may vary in the same patient between the fresh semen and 90% layer used for actual insemination. Negative changes in the TUNEL results may be due to oxidative damage, or other inherent abnormalities in the sperm which result in damage during processing. Conversely, many samples with a high percentage of abnormal sperm seem to benefit from sperm selection during the gradient centrifugation. These results indicate a possible advantage of analyzing sperm actually used for insemination rather than the raw semen. Additionally, the results highlight the need for evaluation of other preparation techniques for sperm with an inherent susceptibility to damage during gradient centrifugation.