Hair follicle response to the variable l.e.t. of a helium ion beam

A homogeneous population of hair follicles in the rat tail has been used to analyse the in vivo response of a biological system to heavy particle irradiation. The conical configuration of the rat tail gives rise to a variable energy degradation of the beam thus yielding information on the damage elicited by the increasing l.e.t. of the helium beam at different sites on the same sample. By means of scanning electron microscopy three different zones were observed as direct evidence of helium ion penetration in the tail. Quantitative evaluation of radiation damage yielded results concerning hair follicle damage after increasing doses for different regions of the ionization curve.     

In vivo response to the different LET regions of a 55 MeV helium ion beam

We evaluated the in vivo response to heavy particle irradiation in rat tail epidermis using silver-stained nucleolar organizer regions (AgNOR) as the end-point. The energy degradation of the beam across the circular section of the tail allowed us to study the damage elicited by two different LET regions of a helium beam, i.e. non-Bragg peak (NBP) and Bragg peak (BP), at different sites on the same sample. The tails were locally irradiated with a helium ion beam at different fluences. AgNOR exhibited marked variations between tissue areas only a few micrometers apart within each tail exposed to a given beam fluence. An analysis of the AgNOR variations in NBP and BP areas of tails exposed to different beam fluences revealed a dose-dependent effect. The AgNOR provide quantitative evidence of differential damage in neighboring tissue areas exposed to different LET regions of a helium-ion beam.     

Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay

A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984 (Biochem. Biophys. Res. Commun. 123, 291-298). Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a "comet" with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate "features" of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor cells were separated from macrophages using a cell-sorting method based on differential binding of FITC-conjugated goat anti-mouse IgG. The "tail moment", the product of the amount of DNA in the tail and the mean distance of migration in the tail, was the most informative feature of the comet image. Tumor and normal cells showed significant heterogeneity in damage produced by ionizing radiation, although the average amount of damage increased linearly with dose (0-15 Gy) and suggested similar net radiosensitivities for the two cell types. Similarly, DNA repair rate was not significantly different for tumor and normal cells, and most of the cells had repaired the damage by 30 min following exposure to 15 Gy. The heterogeneity in response did not appear to be a result of differences in response through the cell cycle.     

Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay

A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984 (Biochem. Biophys. Res. Commun. 123, 291-298). Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a "comet" with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate "features" of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor cells were separated from macrophages using a cell-sorting method based on differential binding of FITC-conjugated goat anti-mouse IgG. The "tail moment", the product of the amount of DNA in the tail and the mean distance of migration in the tail, was the most informative feature of the comet image. Tumor and normal cells showed significant heterogeneity in damage produced by ionizing radiation, although the average amount of damage increased linearly with dose (0-15 Gy) and suggested similar net radiosensitivities for the two cell types. Similarly, DNA repair rate was not significantly different for tumor and normal cells, and most of the cells had repaired the damage by 30 min following exposure to 15 Gy. The heterogeneity in response did not appear to be a result of differences in response through the cell cycle.     

对二甲苯对皱纹盘鲍肝胰腺细胞DNA的损伤

为研究对二甲苯对皱纹盘鲍肝胰腺的毒性作用,设置4个浓度(0.5、1.0、1.5和2.0 mg·L^-1)和对照组,开展为期21 d的对二甲苯对皱纹盘鲍的亚慢性毒性试验,采用彗星试验技术进行皱纹盘鲍肝胰腺细胞DNA损伤分析,采用CASP分析软件对拖尾率、彗星尾长、彗尾DNA相对含量、Olive矩等损伤指标进行统计。结果表明: 与对照组相比,各染毒组皱纹盘鲍肝胰腺细胞DNA均受到损伤,且损伤程度存在显著性差异。随着染毒浓度的增加,肝胰腺细胞DNA受损程度加重,高浓度甚至可以引发细胞凋亡,呈现一定的剂量-损伤效应。中浓度对二甲苯短时间暴露即可对皱纹盘鲍肝胰腺细胞造成DNA损伤,随着暴露时间延长,细胞DNA受损程度加重,呈现一定的时间-损伤效应。但长时间暴露细胞DNA各损伤指标有所减小,这可能与细胞自身的DNA修复机制和生物体解毒系统的代谢机制有关。研究表明,对二甲苯可对皱纹盘鲍肝胰腺细胞产生氧化损伤,导致DNA断裂,高浓度的对二甲苯长时间暴露可导致其细胞凋亡。     

Epithelial cells of different organs exhibit distinct patterns of p53-dependent and p53-independent apoptosis following DNA insult.

The present study shows that DNA damage induces different patterns of p53-dependent and p53-independent apoptosis in epithelial cells of various organs of adult mice. Genotoxic stress induced a biphasic apoptotic response in the small intestine and tongue. While the first immediate apoptotic wave was p53-dependent, the second was slower in rate and was p53-independent. Under the same experimental conditions a single rapid, but a more extended, p53-independent response was evident in the skin of the tail. Indeed, exposure of p53+/+ mice to 400 R induced in epithelium of the small intestine and tongue an immediate rapid response that was followed by a second delayed p53-independent apoptotic wave. p53-/- mice exhibited in these organs the second wave only. However, epithelium of the tail derived from the same mice showed a single rapid apoptotic response that lasted much longer than the p53-dependent response and was similar in the p53-/- and the p53+/+ mice. Variations in apoptotic patterns observed in epithelial cells derived of the different tissues may point to differences in the physiological pathways expressed.     

He–Ne laser (632.8 nm) pre‐irradiation gives protection against DNA damage induced by a near‐infrared trapping beam

We report the results of a study carried out to investigate the effect of He–Ne laser (632.8 nm) pre‐irradiation on DNA damage induced by continuous wave 1064 nm trapping beam exposure in MCF‐7 cells. A significant decrease in % tail DNA (p < 0.05) was observed in MCF‐7 cells pre‐exposed to He–Ne laser beam. The dependence of the induced protection against 1064 nm trapping beam irradiation induced DNA damage on the time interval between the two irradiations as well as the He–Ne laser pre‐irradiation parameters is presented. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Changes in activity and object manipulation before tail damage in finisher pigs as an early detector of tail biting

Tail damage within the production of finisher pigs is an animal welfare problem. Recent research suggests that removal of known risk factors may not be enough to eliminate tail biting, especially in undocked pigs, thus a different strategy is worth investigating. This could be early detection of tail biting, using behavioural changes observed before tail damage. If these early stages of tail biting can be detected before tail damage occurs, then tail damage could be prevented by early interventions. The first step in developing such a strategy is to identify the types of behaviour changes that emerge during early stages of tail biting. Thus, the aim of the current study was to investigate whether pen level activity and object manipulation evolved differently during the last 7 days before the scoring of tail damage (day 0) for pens scored with tail damage (tail damage pens) and pens not scored with tail damage (matched control pens). The study included video recordings for twenty-four tail damage pens and thirty-two matched control pens. Activity level and object manipulation were observed the last 7 days before day 0 during the morning (0600 to 0800 h), afternoon (1600 to 1800 h) and evening (2200 to 2400 h, only activity level). Both activity level and object manipulation were analysed using generalised linear mixed effects models with a binomial distribution for activity level and a negative binomial distribution for object manipulation. The probability of being active was higher in tail damage pens compared to control pens during the afternoon the last 5 days before day 0 (P<0.001). This was seen due to a decrease in activity level in the control pens, which makes it difficult to identify future tail damage pens from this difference. Object manipulation was lower in tail damage pens compared to the control pens on all 7 days before day 0, but only in pens with undocked pigs (P<0.01). Thus, it is still unknown when this difference in object manipulation initiated. It was concluded that both activity level and object manipulation seemed related to ongoing tail biting and should be investigated through more detailed observations and for a longer time to establish the normal behaviour pattern for a particular pen. Thus, it is suggested that future research focusses on developing automatic monitoring methods for pen level activity and object manipulation and applies algorithms that establish and detect deviations from the normal behaviour pattern of the pen before tail damage.     

Induction and repair of DNA damage as measured by the Comet assay and the yield of somatic mutations in gamma-irradiated tobacco seedlings

The advantage of using the tobacco (Nicotiana tabacum var. xanthi) mutagenicity assay is the ability to analyze and compare on the same plants under identical treatment conditions both the induced acute DNA damage in somatic cells as measured by the Comet assay and the yield of induced leaf somatic mutations. Gamma-irradiation of tobacco seedlings induced a dose-dependent increase in somatic mutations from 0.5 (control) to 240 per leaf (10Gy). The increased yield of somatic mutations was highly correlated (r = 0.996) with the increased DNA damage measured by the Comet assay immediately after irradiation. With increased dose of gamma-irradiation, the averaged median tail moment values ( +/- S.E.) significantly increased from 1.08 +/- 0.10 (control) to 20.26 +/- 1.61 microm (10Gy). Nuclei isolated from leaves 24h after irradiation expressed tail moment values that were not significantly different from the control (2.08 +/- 0.11). Thus a complete repair of DNA damage induced by gamma-irradiation and measurable by the Comet assay was observed, whereas the yield of somatic mutations increased in relation to the radiation dose. Data on the kinetics of DNA repair and of DNA damage induced by gamma-radiation on isolated tobacco nuclei, and on nuclei isolated from irradiated leaves and roots are presented.     

Context-dependent effects of tail-ornament damage on mating success in black grouse

Male black grouse (Tetrao tetrix) may receive damage to theirtail ornaments, the lyre, during goshawk predation attemptsand during fights with other males. In this study we confirma previous observation that black grouse males with damagedtail ornaments suffer reduced mating success. In males thatheld territories on the edge of the leks, tail damage was unrelatedto mating success, whereas in central males damage was negativelycorrelated with mating success. We tested experimentally whetherabsence of damage is used by females in mate choice. In maleswith edge territories, intact, control males had higher matingsuccess than males with cut tails, but in males with centralterritories, lyre cutting had no effect on mating success. Theseresults suggest two interpretations. First, female choice alsodepends on factors other than tail damage such as position onthe lek and dominance. Second, the effect of tail damage iscontext dependent; in males that otherwise meet females standards(e.g., dominant males), the effect of tail damage is negligible,but in less dominant males, tail damage could be used by femalesin mate selection. The second interpretation provides an explanationfor why the data on unmanipulated and manipulated birds differ.In experimental central birds, factors other than tail damageprobably determine male mating success, whereas in experimentaledge birds such factors are probably absent and therefore taildamage is relatively more important. In central unmanipulatedbirds, however, males with natural damage are probably not chosenbecause tail damage and absence of other attractive traits arecorrelated. The absence of an effect on peripheral unmanipulatedbirds may be explained by their overall low mating success     

不同程度的尾损伤对镇海林蛙(Rana zhenhaiensis)蝌蚪游泳速度的影响

无尾两栖类蝌蚪的尾巴通过产生强大的游泳速度在反捕食中起到了重要的作用。以镇海林蛙(Rana zhenhaiensis)蝌蚪为实验动物来评估断尾的运动代价。以74尾具有完整尾蝌蚪作为实验组,通过截去不同尾长片段,人为分成轻微尾损伤组(30%)和严重尾损伤组(30%)并测定两组蝌蚪在断尾前后的游泳速度。以16尾完整尾蝌蚪作为对照组在实验组断尾前后同时进行游泳速度的测定。实验结果显示断尾影响蝌蚪的游泳速度,但仅在尾损伤程度达到尾长的30%以上时才产生不利的影响。这表明轻微尾损伤并不对镇海林蛙蝌蚪的游泳速度产生严重影响。在断尾前后实验组蝌蚪的游泳速度均与尾长呈正相关。在相同尾长状态下,尾损伤蝌蚪的相对游泳速度明显快于完整尾蝌蚪。因此,尾损伤的镇海林蛙蝌蚪有可能通过改变尾和身体的摆动频次等方式在断尾后对游泳速度进行了一定的补偿。尾损伤在野外频繁发生于蝌蚪的尾远端,据此推测镇海林蛙蝌蚪在自然条件下的尾损伤并不会产生严重运动代价。     

Increased genotoxic susceptibility of breast epithelial cells to ethylene oxide

This study was carried out with the aim of elucidating the organ-specific effects of ethylene oxide in comparison with the sensitivity of cells from different tissues. An increased incidence of leukemia and lymphoma has been observed in workers exposed to ethylene oxide. However, contradictory findings exist regarding its ability to induce other tumor types, such as breast cancer. We characterized the genotoxicity of ethylene oxide by means of the alkaline version of comet assay in in vitro systems, in order to investigate the hypothesized role of this substance in the development of breast cancer. For this study, we used primary and secondary cultures of lymphoblasts (well-known target cells of the genotoxicity of ethylene oxide), breast epithelial cells (hypothesized target), peripheral blood lymphocytes (cells commonly used in biomonitoring), and of keratinocytes and cervical epithelial cells. DNA damage was measured and expressed as tail DNA, tail length, and tail moment. In the concentration range 0-100 microM, ethylene oxide induced a dose-dependent increase of DNA damage in the investigated cell types without notable cytotoxicity. A statistically significant increase of DNA damage could be observed after treatment with 20 microM ethylene oxide in lymphoblasts (51% increase of tail moment over the background), breast epithelial cells (26% increase) and peripheral lymphocytes (71% increase). In keratinocytes (5% increase) and cervical epithelial cells (5% increase) significant DNA damage could not be detected at this dose, but at higher concentrations (50-100 microM), such an increase was observed. These results are indicative of an increased sensitivity of breast epithelial cells towards genotoxic insults of ethylene oxide. Our observations provide additional data to evaluate the hypothesis that exposure to ethylene oxide may play a role in breast cancer, and the findings may contribute to the development of screening tests for monitoring an early response to genotoxic insults in occupational settings.     

紫外辐射诱导烟草原生质体中DNA损伤的彗星电泳检测

以烟草原生质体为材料,采用彗星电泳检测用0.5W·m^-2紫外线以不同时间（0、5、10、30、60和120s）诱导的烟草原生质体中DNA的损伤。结果表明,在0～10s的时间内代表DNA损伤程度的尾矩、Olive尾矩等参数与紫外线照射时间具有良好的时间依赖关系。本文建立的烟草原生质体体系采用彗星电泳技术,可以快速而灵敏地检测紫外线对植物细胞的损伤程度。     

Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals

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.     

Biophysics and synchrotron radiation where the marriage fails. X-ray damage of lipid membranes and mesophases.

The call for brighter synchrotron X-radiation sources for use in structural biology research is barely audible as we enter the new millennium. Our brightest sources are already creating havoc when used at design specifications because of radiation damage. The time is long overdue to take stock of where we are and where we wish to go with regards to using existing sources and to designing new ones. The problem of radiation damage is particularly severe in studies involving kinetics and mechanism where cryotechniques are not always viable. Accordingly, we need to understand the very nature of radiation damage and to devise means for minimizing it. This is the thrust of the current study as applied to lipid membranes and mesophases. Here, we report on two very different types of radiation damage. One involves a dramatic phase transformation and the other a disordering of lamellar stacking. How beam energy and dose/rate affect damage is also discussed. The work highlights the nature of the damage process and the need for additional studies if we are to make most efficient use of an important resource, synchrotron radiation.     

Protective effect of Crocus sativus stigma extract and crocin (trans-crocin 4) on methyl methanesulfonate-induced DNA damage in mice organs

This study was designed to examine the effect of aqueous extract of Crocus sativus stigmas (CSE) and crocin (trans-crocin 4) on methyl methanesulfonate (MMS)-induced DNA damage in multiple mice organs using the comet assay. Adult male NMRI mice in different groups were treated with either physiological saline (10 mL/Kg, intraperitoneal [ip]), CSE (80 mg/Kg, ip), crocin (400 mg/Kg, ip), MMS (120 mg/Kg, ip), and CSE (5, 20, and 80 mg/Kg, ip) 45 min prior to MMS administration or crocin (50, 200, and 400 mg/Kg, ip) 45 min prior to MMS administration. Mice were sacrificed about 3 h after each different treatment, and the alkaline comet assay was used to evaluate the effect of these compounds on DNA damage in different mice organs. The percent of DNA in the comet tail (% tail DNA) was measured. A significant increase in the % tail DNA was seen in nuclei of different organs of MMS-treated mice. In control groups, no significant difference was found in the % tail DNA between CSE- or crocin-pretreated and saline-pretreated mice. The MMS-induced DNA damage in CSE-pretreated mice (80 mg/Kg) was decreased between 2.67-fold (kidney) and 4.48-fold (lung) compared to those of MMS-treated animals alone (p < 0.001). This suppression of DNA damage by CSE was found to be depended on the dose, which pretreatment with CSE (5 mg/Kg) only reduced DNA damage by 6.97%, 6.57%, 7.27%, and 9.90% in liver, lung, kidney, and spleen, respectively (p > 0.05 as compared with MMS-treated group). Crocin also significantly decreased DNA damage by MMS (between 4.69-fold for liver and 6.55-fold for spleen, 400 mg/Kg), in a dose-dependent manner. These data indicate that there is a genoprotective property in CSE and crocin, as revealed by the comet assay, in vivo.     

Effect of mitochondrial toxins on evoked somatosensory activity in rats

Mitochondrial toxins represent an interesting group of neurotoxicants related both to causation and modelling of central nervous damage. 3-nitropropionic acid (3NP), a neurotoxin of herbal and microfungal origin, inhibits succinate dehydrogenase leading thereby to various biochemical and morphological alterations in the brain. Experimental animals treated by 3NP are used to model Huntington’s disease. Manganese, often present in occupational settings and as environmental pollutant, inhibits complex II and III of the mitochondria and is known to cause Parkinson-like CNS damage. In this work, rats were administered a single acute dose of Mn (50 mg Mn²⁺/kg body weight) or 3-NP (20 mg/kg b.w.) and the alterations of the somatosensory cortical evoked potential elicited by stimulation of the whisker pad and the tail base were observed, together with the changes of the action potential in the tail nerve. Latency and amplitude of the two cortical responses changed in parallel, while those of the tail nerve response remained more or less unaltered. The two mitochondrial toxins studied seem to exert their action centrally, primarily on synaptic transmission, rather than peripherally. Recording of evoked activity could be used to follow-up the nervous system effects of mitochondrial toxins, but it requires further investigation.     

Herbivore damage-induced production and specific anti-digestive function of serine and cysteine protease inhibitors in tall goldenrod, Solidago altissima L. (Asteraceae)

Plant protease inhibitors (PIs) are among the most well-studied and widely distributed resistance traits that plants use against their herbivore attackers. There are different types of plant PIs which putatively function against the different types of proteases expressed in insect guts. Serine protease inhibitors (SPIs) and cysteine protease inhibitors (CPIs) are hypothesized to differentially function against the predominant gut proteases in lepidopteran and coleopteran herbivores, respectively. Here, we test the hypothesis that tall goldenrod, Solidago altissima, can specifically respond to damage by different herbivores and differentially induce SPIs and CPIs in response to damage by lepidopteran and coleopteran herbivores. Moreover, we ask if the concerted induction of different types of PIs accounts for variation in induced resistance to herbivory. We altered and optimized a rapid and effective existing methodology to quantitatively analyze both SPI and CPI activity simultaneously from a single tissue sample and to use the same plant extracts directly for characterization of inhibitory effects on insect gut protease activity. We found that both SPIs and CPIs are induced in S. altissima in response to damage, regardless of the damaging herbivore species. However, only SPIs were effective against Spodoptera exigua gut proteases. Our data suggest that plant PI responses are not necessarily specific to the identity of the attacking organism but that different components of generally induced defense traits can specifically affect different herbivore species. While providing an efficient and broadly applicable methodology to analyze multiple PIs extracted from the same tissue, this study furthers our understanding of specificity in induced plant resistance.     

Indicators of ecological change: comparison of the early response of four organism groups to stress gradients

A central goal in monitoring and assessment programs is to detect change early before costly or irreversible damage occurs. To design robust early-warning monitoring programs requires knowledge of indicator response to stress as well as the uncertainty associated with the indicator(s) selected. Using a dataset consisting of four organism groups (fish, macrophytes, benthic diatoms and macroinvertebrates) and catchment, riparian and in-stream physico-chemical variables from 77 mountain and 85 lowland streams we determined the relationships between indicator response and complex environmental gradients. The upper (>75th percentile) and lower (<25th percentiles) tails of principal component (PC) gradients were used to study the early response of the four organism groups to stress. An organism/metric was considered as an early warning indicator if the response to the short gradients was more robust (higher R² values, steeper slope and lower error) than the null model (organism response to the full PC gradient). For mountain streams, both fish and macrophyte CA scores were shown to exhibit an early warning response to the upper tail of the 1st PC gradient when compared to the null model. Five of the eight metrics showed better response to the upper tail of the 2nd PC gradient compared to the null model, while only one metric (macrophyte CA scores) showed improvement when compared to the lower tail of the 2nd PC gradient. For lowland streams all four organism-groups showed better response (CA scores) to the upper tail of the PC gradient when compared to the null model. Only one metric (fish CA scores) regressed against the lower tail of the 2nd PC gradient was found to be more robust than the PC2 null model. These findings indicate that the nonlinear relationships of organism/metric response to stress can be used to select potentially robust early warning indicators for monitoring and assessment.     

Toxicity of aflatoxin B1 to penaeid shrimp.

Single intramuscular injections of aflatoxin B1 into the tail muscle of Penaeus stylirostris produced 24- and 96-h median lethal doses of 100.5 (78.3 to 129.0) and 49.5 (29.8 to 82.3) mg/kg, respectively. A toxicity curve showed no threshold at the levels tested. The mortality response in a feeding study with P. vannamei was not dose dependent, but tissue and organ damage were similar to that seen in injected animals.