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In criminal investigations, postmortem interval (PMI) is important information to be inferred in homicide investigations, as well as the focus and the difficulty in forensic pathology research. Because the DNA content in different tissues is relatively constant and shows changes regularly with the extension of PMI, it has become a research hotspot of PMI estimation. This paper reviews the recent progress of PMI estimation technologies including DNA-based single cell gel electrophoresis, image analysis, flow cytometry, real-time fluorescence quantitative PCR and high-throughput sequencing, hoping to provide references for forensic medicine practice and scientific research.

Accurate verification of the time passed since death is a challenging task in daily forensic investigations, chiefly in homicidal or unwitnessed death. The study evaluated the applicability of the time-dependent changes in histological structure, DNA content,and oxidant/antioxidant parameters in rat's skeletal muscles in an attempt to reach a reliable method for post-mortem interval (PMI) estimation. We used 56 adult male albino rats. These animals were sacrificed then and allocated randomly to seven groups (eight rats each). First group: rats were dissected to obtain samples from quadriceps femoris muscles promptly after death. While the rats in the second, third, fourth, fifth, sixth and seventh groups were dissected to get muscle samples from quadriceps femoris at 6, 24, 48, 72, 96 and 144 hours post-mortem (hpm). The results showed that a significant gradual histopathological alteration of muscle structure started at 48 hpm and progressed afterwards. Also, the DNA degradation percentage increased significantly parallel to the increased oxidant parameter levels from 6 to 144 hpm. Our results concluded that changes in oxidant/antioxidant parameters and DNA degradation, as well as the histological examination of muscle tissues, showed a strong correlation with post-mortem intervals. Consequently, the integration of all these parameters can provide a valuable method of post-mortem intervals estimation. [ABSTRACT FROM AUTHOR]

Introduction: Drug delivery systems could induce cellular toxicity as side effect of nanomaterials. The mechanism of toxicity usually involves DNA damage. The comet assay or single cell gel electrophoresis (SCGE) is a sensitive method for detecting strand damages in the DNA of a cell with applications in genotoxicity testing and molecular epidemiology as well as fundamental research in DNA damage and repair. Methods: In the current study, we reviewed recent drug delivery researches related to SCGE. Results: We found that one preference for choosing the assay is that comet images may result from apoptosis-mediated nuclear fragmentation. This method has been widely used over the last decade in several different areas. Overall cells, such as cultured cells are embedded in agarose on a microscope slide, lysed with detergent, and treated with high salt. Nucleoids are supercoiled DNA form. When the slide is faced to alkaline electrophoresis any breakages present in the DNA cause the supercoiling to relax locally and loops of DNA extend toward the anode as a ‘‘comet tail’’. Conclusion: This article provides a relatively comprehensive review upon potentiality of the comet assay for assessment of DNA damage and accordingly it can be used as an informative platform in genotoxicity studies of drug delivery systems.

Abstract: The haloacetic acids (HAAs) are the second-most prevalent class of drinking water disinfection by-products formed by chemical disinfectants. Previous studies have determined DNA damage and repair of HAA-induced lesions in mammalian and human cell lines; however, little is known of the genomic DNA and chromosome damage induced by these compounds in primary human cells. The aim of this study was to evaluate the genotoxic and clastogenic effects of the monoHAA disinfection by-products in primary human lymphocytes. All monoHAAs were genotoxic in primary human lymphocytes, the rank order of genotoxicity and cytotoxicity was IAA > BAA >> CAA. After 6 h of repair time, only 50% of the DNA damage (maximum decrease in DNA damage) was repaired compared to the control. This demonstrates that primary human lymphocytes are less efficient in repairing the induced damage by monoHAAs than previous studies with mammalian cell lines. In addition, the monoHAAs induced an increase in the chromosome aberration frequency as a measurement of the clastogenic effect of these compounds. These results coupled with genomic technologies in primary human cells and other mammalian non-cancerous cell lines may lead to the identification of biomarkers that may be employed in feedback loops to aid water chemists and engineers in the overall goal of producing safer drinking water. [Copyright &y& Elsevier]

Abstract: This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated as CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays. [Copyright &y& Elsevier]

The article discusses a study which examined the process of DNA degradation in brain and dental pulp cells of mice. The study performed single cell gel electrophoresis assay and professional comet image analysis and processing techniques to elucidate the process. Results showed a decreasing trend in the percentage of head DNA and head area and a high correlation between the parameters and the postmortem interval.

Context: Single cell gel electrophoresis (SCGE) or "comet assay" is a rapid and very sensitive fluorescent microscopic method for detecting various forms of DNA damage at individual cell level. Aims: The aim of the present study was to detect the extent of DNA damage in oral cancer, oral submucous fibrosis (OSMF) and leukoplakia in comparison to normal individual. Settings and Design: A total of 44 consecutive patients with oral cancer (n=26), leukoplakia (n=12) and OSMF (n=6) and 10 healthy normal volunteers with normal oral epithelia (controls) were recruited from Dr. R. Ahmed Dental College and Hospital and were assessed for the extent of DNA damage using SCGE following clinical diagnosis. Materials and Methods: Peripheral blood was collected by venepuncture and comet assay was performed using SCGE. Mean tail length was compared between diagnostic groups and between different oral habit groups using t-tests and analysis of variance (ANOVA). Pearson's product moment correlation was used to examine the linear association between the extent of DNA damage and oral habit pack-years. Scheffe's pair-wise test was employed to adjust for multiple comparisons. Results: None of the controls were associated with any oral habits. Mean (±SD) tail lengths (in µm) for cancer (24.95 ± 5.09) and leukoplakia (12.96 ± 2.68) were significantly greater than in controls (8.54 ± 2.55, P<0.05). After adjustment, well-, moderately, and poorly differentiated carcinomas had significantly greater tail length than controls. Whereas the extent of DNA damage in cancer cases was significantly greater in leukoplakia than in compared to OSMF (11.03 ± 5.92), the DNA damage in latter was not different from controls. DNA damage for people with any oral habit (19.78 ± 7.77) was significantly greater than those with no habits (8.54 ± 2.55; P<0.0001). Conclusions: DNA damage measured by SCGE is greater in leukoplakia and squamous cell carcinoma, but not in OSMF. Deleterious oral habits are also associated with greater DNA damage. [ABSTRACT FROM AUTHOR]

Abstract: Many previous studies have described differential responses to mutagens among human lymphocyte subpopulations. However, there are some confusing data about the sensitivity to mutagens among lymphocyte subpopulations. In this paper we review the studies published to date reporting differential responses among CD4+ T-cells, CD8+ T-cells, B-cells and NK-cells exposed to different mutagens, using different assay methods, such as micronuclei (MN), sister chromatid exchanges (SCEs), single cell gel electrophoresis (SCGE), and fluorescence-activated cell sorting (FACS). These methods are mostly used for genetic biomonitoring of human populations exposed to potential mutagens. Thus, this review may provide a reference of target cells sensitive to mutagens that will be useful for genetic biomonitoring of human populations. [Copyright &y& Elsevier]

Abstract: The aim of the present study was to investigate in vitro toxicological effects of PM2.5 suspensions, their water-soluble fraction and solvent-extractable organics from dust storm on the viability and DNA of rat alveolar macrophages and in vivo toxicological effects of PM2.5 suspensions on DNA of lung cells of rats. PM2.5 samples from dust storm and normal weather were collected in Baotou city, Inner Mongolia Autonomous Region, and Wuwei city, Gansu Province, China, in March, 2004. DNA damage was detected with single cell gel electrophoresis technique and cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The results showed that: (1) In vitro, PM2.5 suspensions, their water-soluble fraction and solvent-extractable organics from both dust storm and normal weather caused a decrease of the cell viability and an increase of DNA damage of rat alveolar macrophages in a dose–response manner; for both Baotou city and Wuwei city, the samples of normal weather showed higher DNA damage than those of dust storm at the highest treated dosage; for both normal weather PM2.5 and dust storm PM2.5, their solvent-extractable organics showed higher DNA damage than the water-soluble fraction. (2) In vivo, PM2.5 from both dust storm and normal weather caused an increase of DNA damage of rat lung cells in a dose–response manner. (3) Baotou city is one of the heavy industrial cities, while Wuwei is one of agricultural cities in Northwest region of China. The effects induced by normal weather samples in Baotou city slightly higher than those in Wuwei city on DNA damage, though there was no significant difference was found between two cities. These results lead to conclusions that dust storm PM2.5 as well as normal weather PM2.5 could lead to DNA damage and the organic compounds and the insoluble particle-core might be the main contributors to DNA damage. Our results suggest that the risk of health effects may be greater during dust storms because dust storm PM2.5 whose airborne mass were much higher. Further studies are needed to determine the components of dust storm particles that may contribute to the particle toxicity. [Copyright &y& Elsevier]

Abstract: Over the years, several lines of evidence have emerged supporting the role of stress in the development and progression of cancer. Stress can cause an increase in the production of reactive oxygen species (ROS) and decrease in the in vivo antioxidant defense systems. A ROS-induced DNA damage in peripheral lymphocytes, liver and skin cells may be revealed by Comet assay. To test whether DNA is damaged by stress/DMBA/stress and DMBA, rats were exposed to multiple doses of DMBA in the presence and absence of restraint stress, and DNA damage was evaluated. Insignificant differences were detected in all the three cells tested (peripheral lymphocytes, liver and skin cells) between control and stress treatment in terms of frequencies of damaged DNA. The extent of DNA migration was enhanced in DMBA treated rats in a dose dependent manner. Pre-stress DMBA treatment showed still higher frequencies of damage in comparison with control, stress alone or DMBA alone groups. Thus, prior exposure to stress clearly enhanced the DMBA induced DNA damage, especially so in the skin cells (target organ of the carcinogen application) than liver and peripheral lymphocytes as observed on the basis of the extent of DNA migration (tail DNA) during single cell gel electrophoresis. [Copyright &y& Elsevier]

Abstract: During the production of sex-sorted spermatozoa from bull semen, the cells are exposed to a number of potential hazards including: dilution, centrifugation, incubation, exposure to DNA stains and laser light. These factors may affect the survival capacity and fertilization potential of the sperm. The objective of this study was to determine whether sex-sorted bull spermatozoa have more DNA damage than sperm from conventional processed bull semen. Two methods were used to determine DNA integrity: the neutral Comet assay (NCA) and the sperm chromatin structure assay (SCSA). The NCA showed that the conventional samples had a higher tail moment (TM) (P &#60;0.017) than the sorted samples and that there was no difference between the samples in tail length (TL) (P =0.36). The SCSA showed that the DNA fragmentation index (DFI) was higher for conventional than the sorted samples (P =0.011), but the standard deviation of DFI (SD-DFI) was higher for the sorted samples (P &#60;0.001). We conclude that the NCA and SCSA can be used in assessing DNA integrity in bovine sperm and that cell sorting by flow cytometry improves the integrity of the sperm cell population. Additionally the results from the SCSA indicated that the sex-sorted sperm had less homogenous sperm chromatin. In the future assessment of sperm DNA integrity may be used to select bulls for sperm sex sorting and optimizing sperm sex sorting procedures. [Copyright &y& Elsevier]

The comet assay is a rapid, sensitive and inexpensive method for measuring DNA strand breaks. The comet assay has advantages over other DNA damage methods, such as sister chromatid exchange, alkali elution and micronucleus assay, because of its high sensitivity and that DNA strand breaks are determined in individual cells. This review describes a number of studies that used the comet assay to determine DNA strand breaks in aquatic animals exposed to genotoxicants both in vitro and in vivo, including assessment of DNA damage in aquatic animals collected from contaminated sites. One difficulty of using the comet assay in environmental work is that of comparing results from studies that used different methods, such as empirical scoring or comet tail lengths. There seems to be a consensus in more recent studies to use both the intensity of the tail and the length of the tail, i.e. DNA tail moment, percentage of DNA in the tail. The comet assay has been used to assess DNA repair and apoptosis in aquatic animals and modifications of the comet assay have allowed the detection of specific DNA lesions. There have been some recent studies to link DNA strand breaks in aquatic animals to effects on the immune system, reproduction, growth, and population dynamics. Further work is required before the comet assay can be used as a standard bio-indicator in aquatic environments, including standardization of methods (such as ASTM method E2186-02a) and measurements. [Copyright &y& Elsevier]

Context: Single cell gel electrophoresis (SCGE) or "comet assay" is a rapid and very sensitive fluorescent microscopic method for detecting various forms of DNA damage at individual cell level. Aims: The aim of the present study was to detect the extent of DNA damage in oral cancer, oral submucous fibrosis (OSMF) and leukoplakia in comparison to normal individual. Settings and Design: A total of 44 consecutive patients with oral cancer (n=26), leukoplakia (n=12) and OSMF (n=6) and 10 healthy normal volunteers with normal oral epithelia (controls) were recruited from Dr. R. Ahmed Dental College and Hospital and were assessed for the extent of DNA damage using SCGE following clinical diagnosis. Materials and Methods: Peripheral blood was collected by venepuncture and comet assay was performed using SCGE. Mean tail length was compared between diagnostic groups and between different oral habit groups using t-tests and analysis of variance (ANOVA). Pearson′s product moment correlation was used to examine the linear association between the extent of DNA damage and oral habit pack-years. Scheffe′s pair-wise test was employed to adjust for multiple comparisons. Results: None of the controls were associated with any oral habits. Mean (±SD) tail lengths (in mm) for cancer (24.95 ± 5.09) and leukoplakia (12.96 ± 2.68) were significantly greater than in controls (8.54 ± 2.55, P