Potential value of the comet assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds

An in situ study of the relationship between marine contamination and genotoxic effects was performed on female dab (Limanda limanda) collected from different sites in the eastern English Channel (France) known to be contaminated by polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs). DNA adducts in liver and DNA strand breaks in blood cells were determined respectively by the nuclease P1-enhanced post-labelling technique and an alkaline version of the comet assay. The extent of DNA base oxidation was also assessed for three of the six sampling sites in the study, using a comet assay in combination with a specific DNA repair enzyme, formamidopyrimidine glycosylase (Fpg).With Comet data, two groups of sites that seem in accordance with the pollution level have been distinguished. The extent of DNA strand breaks was higher in adult than juvenile female dab. From a technical point of view, comet assay sensitivity was affected by high intra-individual variability that accounted for nearly 70% of total variance (the site factor represented no more than 26%). The combined use of the comet assay and Fpg showed the presence of DNA oxidised bases in environmentally exposed dab.Although qualitative differences between the sampling sites were observed in DNA adduct profiles, no significant differences were found for total DNA adduct levels. DNA adducts did not appear to be associated with PAH exposure.Histopathological studies showed hepatic steatosis in most of the animals examined. Only one pre-cancerous lesion (an early stage of hyperplasia) was detected (associated frequency of 0.8%).     

A permanent fish cell line (EPC) for genotoxicity testing of marine sediments with the comet assay.

Genotoxicity and cytotoxicity were evaluated in an in vitro system with a permanent cell line Epithelioma papulosum cyprini (EPC) derived from a skin tumour of carp (Cyprinus carpio L.). EPC cells were exposed to different concentrations of organic sediment extracts from the North Sea for 24h. After incubation the cells were analysed for viability and DNA strand breaks with the comet assay or single cell gel electrophoresis (SCGE). The results confirm the sensitivity of this assay. Out of 10 marine sediment samples from the North Sea, 9 showed a dose-dependent genotoxic effect. The EC(50) of sediment extracts ranged from 7 to 307 mg sediment dry weight/ml assay volume. Hepatic microsomal enzymes from dab (Limanda limanda L.) was proposed for enzymatic activation of benzo[a]pyrene (BAP) or sediment extracts, respectively. The suitability of this in vitro test system for assessing genotoxic and cytotoxic effects of marine sediment extracts on EPC cells could be demonstrated.     

A permanent fish cell line (EPC) for genotoxicity testing of marine sediments with the comet assay

Genotoxicity and cytotoxicity were evaluated in an in vitro system with a permanent cell line Epithelioma papulosum cyprini (EPC) derived from a skin tumour of carp (Cyprinus carpio L.). EPC cells were exposed to different concentrations of organic sediment extracts from the North Sea for 24 h. After incubation the cells were analysed for viability and DNA strand breaks with the comet assay or single cell gel electrophoresis (SCGE). The results confirm the sensitivity of this assay. Out of 10 marine sediment samples from the North Sea, 9 showed a dose-dependent genotoxic effect. The EC₅₀ of sediment extracts ranged from 7 to 307 mg sediment dry weight/ml assay volume. Hepatic microsomal enzymes from dab (Limanda limanda L.) was proposed for enzymatic activation of benzo[a]pyrene (BAP) or sediment extracts, respectively. The suitability of this in vitro test system for assessing genotoxic and cytotoxic effects of marine sediment extracts on EPC cells could be demonstrated.     

A strong genotoxic effect in mouse skin of a single painting of coal tar in hairless mice and in MutaMouse

The dorsal skin of C3H/Tif/hr hairless mice was painted with coal tar, pharmacological grade. Epidermal cells and hepatocytes were isolated after 4, 24, 48 and 96 h and DNA strand breaks were determined as tail moment by the alkaline comet assay. The tail moment of epidermal cells was significantly greater at the time points 4, 24, 48 and 96 h after exposure compared to the controls, with the most DNA strand breaks at 24 h. The DNA strand breaks in epidermal cells increased linearly with the dose of coal tar. In hepatocytes, no difference in DNA strand breaks was found between exposed animals and controls. DNA adducts were determined by the 32P-postlabeling assay. For epidermal cells, the mean DNA adduct level was 12-fold greater in coal tar painted mice after 24 h than in controls. Again, a linear dose/response relationship was seen 24 h after painting. For liver DNA, the mean DNA adduct level was 3-fold greater than for controls. The mutation frequency in epidermal and liver cells was examined in lambdalacZ transgenic mice (MutaMouse). Thirty-two days after painting, the mutation frequency in epidermal cells was 16-fold greater in coal tar treated mice compared to controls. No effect was detected in hepatocytes. We found that a single painting of coal tar resulted in strong genotoxic effects in the murine epidermis, evidenced by induction of DNA strand breaks and DNA adducts in hairless mice and lambdalacZ mutations in the MutaMouse. This demonstrates that it is possible to detect genotoxic effects of mixtures with high sensitivity in mouse skin by these end-points.     

Estragole: a weak direct-acting food-borne genotoxin and potential carcinogen

We evaluated the genotoxicity of the food-flavouring agent estragole in V79 cells using the sister chromatid exchange (SCE) assay and the alkaline comet assay. Unexpectedly, we observed an increase in SCE without an exogenous biotransformation system (S9) and a decrease in its presence. Positive results were also observed in the alkaline comet assay without S9, indicating DNA strand breakage. To ascertain repair of damage, we performed the comet assay in V79 cells after two hours of recovery, and observed a reduction of the genotoxic response. Estragole did not produce strand breaks in plasmid DNA in vitro. We then evaluated the formation of DNA adducts in V79 cells by use of the (32)P-postlabelling assay and detected a dose-dependent formation of DNA adducts, which may be responsible for its genotoxicity. We then assayed estragole in the comet assay with two CHO cell lines, a parental AA8 cell line, and an XRCC1-deficient cell line, EM9. Results confirmed the genotoxicity of estragole without biotransformation in both cell lines, although the genotoxicity in EM9 cells compared with that in AA8 cells was not significantly different, suggesting that the XRCC1 protein is not involved in the repair of estragole-induced lesions. Estragole induces apoptosis, but only with high doses (2000μM), and after long treatment periods (24h). Overall, our results suggest that estragole, besides being metabolized to genotoxic metabolites, is a weak direct-acting genotoxin that forms DNA adducts.     

甲醛对DNA损伤的彗星实验研究

甲醛是一种遗传毒性物质。国内外学者的大量研究证实,甲醛可以引起DNA-DNA、DNA-蛋白质分子交联,但对于甲醛是否能够引起DNA分子的断裂,学界却存在分歧。本实验以颊黏膜细胞作为实验材料,通过彗星实验对甲醛的遗传毒性——尤其是DNA分子断裂作用进行了系统的研究。结果显示甲醛在较低浓度(5μmol／L,7,5μmol／L,10μmol／L)时具有断裂作用,在较高浓度(15μmol／L,30μmol／L,50μmol／L)时则具有交联作用。根据本实验的结果,本文还首次论证了甲醛断裂作用的断裂峰值(7．5μmol／L)。     

Vanadate induces DNA strand breaks in cultured human fibroblasts at doses relevant to occupational exposure

To study possible genotoxic effects of occupational exposure to vanadium pentoxide, we determined DNA strand breaks (with alkaline comet assay), 8-hydroxy-2'deoxyguanosine (8-OHdG) and the frequency of sister chromatid exchange (SCE) in whole blood leukocytes or lymphocytes of 49 male workers employed in a vanadium factory in comparison to 12 non-exposed controls. In addition, vanadate has been tested in vitro to induce DNA strand breaks in whole blood cells, isolated lymphocytes and cultured human fibroblasts of healthy donors at concentrations comparable to the observed levels of vanadium in vivo. To investigate the impact of vanadate on the repair of damaged DNA, co-exposure to UV or bleomycin was used in fibroblasts, and DNA migration in the alkaline and neutral comet assay was determined. Although, exposed workers showed a significant vanadium uptake (serum: median 5.38microg/l, range 2.18-46.35microg/l) no increase in cytogenetic effects or oxidative DNA damage in leukocytes could be demonstrated. This was consistent with the observation that in vitro exposure of whole blood leukocytes and lymphocytes to vanadate caused no significant changes in DNA strand breaks below concentrations of 1microM (50microg/l). In contrast, vanadate clearly induced DNA fragmentation in cultured fibroblasts at relevant concentrations. Combined exposure of fibroblasts to vanadate/UV or vanadate/bleomycin resulted in non-repairable DNA double strand breaks (DSBs) as seen in the neutral comet assay. We conclude that exposure of human fibroblasts to vanadate effectively causes DNA strand breaks, and co-exposure of cells to other genotoxic agents may result in persistent DNA damage.     

The maternal effect in infantile autism: Elevated DNA damage degree in patients and their mothers

Infantile autism is a common disorder of mental development; besides hereditary predisposition, various environmental factors, including the condition of the mother’s body during pregnancy (“maternal effect”), have a significant impact on its appearance. Oxidative stress is suggested to play a key role in the pathogenesis of infantile autism. It causes a prominent genotoxic effect, which is realized through appearance of single and double strand breaks of the nuclear DNA. In patients with infantile autism and their mothers we evaluated the degree of DNA damage by using the DNA comet assay and determining the comet tail moment and DNA percent ratio in the tail. These two parameters demonstrated strong correlation (r = 0.90). Mean and median values of both parameters were significantly higher in samples from autistic children, than in agematching healthy controls. Interestingly, these parameters were also higher in healthy mothers of autistic children and they did not differ from the values in the group of autistic children. In the control group of healthy women of reproductive age, who had no children with autism, the mean value of the DNA comet tail moment significantly differed from the group of mothers of autistic children, but not from the control group of healthy children. The results suggest that mentally healthy mothers of autistic children have some genotoxic factors, which can determine development of the pathological process in the fetus via the environmental “maternal effect” during gestation.     

DNA damage induced by genotoxicants in zebrafish (Danio rerio) embryos after contact exposure to freeze-dried sediment and sediment extracts from Laguna Lake (The Philippines) as measured by the comet assay

Laguna Lake, Philippines, is utilised as a resource for drinking and irrigation water and a major source of animal protein for more than 11 million residents. However, it also serves as a huge sink for anthropogenic pollutants from a variety of direct or indirect discharges. Given their well-known properties to accumulate organic pollutants, Lake Laguna sediments have been investigated by use of a newly developed sediment-contact assay to determine the bioavailable fraction of the total genotoxic hazard potential. Freshly fertilized zebrafish eggs (2 h after fertilization) were allowed to develop while exposed to different concentrations of freeze-dried sediments (exposure to bioavailable compounds) as well as organic extracts (complete genotoxic hazard potential including bioaccumulation) prepared from sediments collected at five sites within the lake. Following a 96 h exposure, single-cell suspensions were prepared from macerated larvae and analyzed for DNA strand-breaks by use of the comet assay. Genotoxicity could be identified in both solid-phase exposure scenarios as well as after exposure to organic sediment extracts. Only the South Bay (SB) site did not show a significant genotoxic burden relative to controls in tests of both freeze-dried sediment and organic extracts. In contrast, the Northwest Bay site displayed pronounced genotoxicity to the zebrafish embryos in both exposure scenarios. Perylene and copper in the sediments could be identified as potential causes of the genotoxic response. To compare the results (maximum induction coefficients) of zebrafish embryos with an established comet protocol, rainbow trout liver cells (RTL-W1) were exposed to the same extracts. The findings correlated well (Spearman correlation r=0.90), proving a good reliability of the results from zebrafish primary cells. In conclusion, the present study demonstrates that the bioavailable fraction of the genotoxic pollutants may pose a threat for both benthic organisms and human health via drinking-water and fish consumption.     

Use of whole blood directly for single-cell gel electrophoresis (comet) assay in vivo and white blood cells for in vitro assay

The present study investigated the use of whole blood from humans and rats directly for single-cell gel electrophoresis (comet) assay. As little as 20 microl of whole blood was sufficient for comet assay, and the comet images obtained from whole blood were not different from those obtained from isolated lymphocytes. The DNA remained intact up to 4 h at 4 degrees C after isolation and had no observable strand breakage, when whole blood was cryopreserved (at -80 degrees C) in 10% pre-cooled DMSO up to 60 days. To demonstrate that the whole-blood technique could be applied to in vivo studies, we injected rats with a known carcinogen Fe/NTA and measured DNA strand breaks in whole blood in comparison with isolated lymphocytes. We showed that Fe/NTA injection resulted in similar extent of DNA strand breakage in both whole blood and lymphocytes, indicating that whole-blood method can be used for in vivo genotoxic studies. One disadvantage of the whole-blood technique is that whole blood cannot be used for in vitro studies because of the interferences from red blood cell (RBC) components. However, this problem can be overcome by prior hemolysis of RBCs and a brief centrifugation to obtain white blood cells (WBCs), which can then be used for in vitro incubation with genotoxic compounds before comet assay. Overall, this whole-blood technique for comet assay is expected to provide a simple, rapid, and cost-effective alternative for the existing comet assay using isolated lymphocytes in situations such as when time and cost are limiting factors.     

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.     

Sudan I induces genotoxic effects and oxidative DNA damage in HepG2 cells

Sudan I, a synthetic lipid soluble azo pigment, is widely used in various industrial fields. However, Sudan I has not been approved at any level of food production, since there are many inconclusive reports relating to its genotoxicity and carcinogenicity in humans. The aim of this study was to assess the genotoxic effects of Sudan I and to identify and clarify the reaction mechanisms by use of human hepatoma HepG2 cells. To study the genotoxic effects of Sudan I, the comet assay and micronucleus test (MNT) were used. In the comet assay and MNT, we found increase of DNA migration and of the micronuclei frequencies at all tested concentrations (25-100 microM) of Sudan I in a dose-dependent manner. The data suggest that Sudan I caused DNA strand breaks and chromosome breaks. To elucidate the underlying mechanism of this difference, we monitored the level of reactive oxygen species (ROS) production with the 2,7-dichlorofluorescein diacetate assay. The level of the oxidative DNA damage and lipid peroxidation was evaluated using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS). Significantly increased levels of ROS, 8-OHdG and TBARS were observed in HepG2 cells at higher concentrations, the doses being 100, 50-100 and 50-100 microM, respectively. We conclude that Sudan I causes genotoxic effects, probably via ROS-induced oxidative DNA damage at the higher doses.     

Methylnitrosourea as challenge mutagen in assessment of the DNA mismatch repair (MMR) activity: Association with some types of cancer

Total repair capability is a widely used phenotypic marker of predisposition to cancer. Evaluation of this parameter implies using a challenge mutagen in an in vitro system to unmask latent genetic instability and repair insufficiency in the target cells. Traditionally, these investigations involve two tests, evaluation of mutagenic susceptibility (chromosomal aberrations) and genotoxic effect (DNA comet assay). The present study was focused on analysis of the effect of methylnitrosourea (MNU) on resting and PHA-stimulated lymphocytes from healthy donors and patients with gynecological cancer. Cytotoxic effect of MNU (apoptotic lymphocyte death) was estimated using two parameters, interaction of the cells with the annexin V-FITC complex, and morphological changes of the nuclei after their staining with the mixture of two DNA-tropic dyes. The genotoxic effect of MNU, namely, secondary double-strand DNA breaks, was scored using the neutral comet assay, modified for the calculation of the comets produced exclusively by BrUdr-labeled proliferating lymphocytes. The proportion of these comets was represented as the proliferative cell index. It was shown that resting lymphocytes were resistant to genotoxic and cytotoxic effects of MNU. The response of proliferating cells to the action of MNU was expressed as the development of secondary DNA breaks (P < 0.01), along with the increased frequency of apoptosis (P < 0.05). The genotoxic effect of MNU on stimulated lymphocytes of gynecological cancer patients was fourfold lower compared to healthy donor lymphocytes. In response to the MNU action, patient lymphocytes did not change their proliferative index, while in healthy donor lymphocytes proliferative index was two times decreased in response to the MNU action. The data obtained pointed to the association between the cytotoxic response of the lymphocytes to the action of MNU and gynecological cancer. Since only proliferating lymphocytes response to the genotoxic effect of MNU, and the effect is revealed a day after the mutagen action, it is suggested that this phenomenon is associated with postreplicative repair, MMR, the substrate of which is O⁶-methylguanin. The MMR deficiency in patient lymphocytes determines their tolerance to the action of MNU. Genotoxic response of lymphocytes to the action of MNU can serve as a marker of MMR, as well as of the MMR deficiency-associated gynecological cancer.     

DNA damage,repair monitoring and epigenetic DNA methylation changes in seedlings of Chernobyl soybeans

This pilot study was carried out to assess the effect of radio-contaminated Chernobyl environment on plant genome integrity 27 years after the accident. For this purpose, nuclei were isolated from root tips of the soybean seedlings harvested from plants grown in the Chernobyl area for seven generations. Neutral, neutral-alkaline, and methylation-sensitive comet assays were performed to evaluate the induction and repair of primary DNA damage and the epigenetic contribution to stress adaptation mechanisms. An increased level of single and double strand breaks in the radio-contaminated Chernobyl seedlings at the stage of primary root development was detected in comparison to the controls. However, the kinetics of the recovery of DNA breaks of radio-contaminated Chernobyl samples revealed that lesions were efficiently repaired at the stage of cotyledon. Methylation-sensitive comet assay revealed comparable levels in the CCGG methylation pattern between control and radio-contaminated samples with a slight increase of approximately 10% in the latter ones. The obtained preliminary data allow us to speculate about the onset of mechanisms providing an adaptation potential to the accumulated internal irradiation after the Chernobyl accident.Despite the limitations of this study, we showed that comet assay is a sensitive and flexible technique which can be efficiently used for genotoxic screening of plant specimens in natural and human-made radio-contaminated areas, as well as for safety monitoring of agricultural products.     

Diepoxybutane induces the formation of DNA-DNA rather than DNA-protein cross-links, and single-strand breaks and alkali-labile sites in human hepatocyte L02 cells

1,3-Butadiene (BD) is an air pollutant and a known carcinogen. 1,2,3,4-Diepoxybutane (DEB), one of the major in vivo metabolites of BD, is considered the ultimate culprit of BD mutagenicity/carcinogenicity. DEB is a bifunctional alkylating agent, being capable of inducing the formation of monoalkylated DNA adducts and DNA cross-links, including DNA-DNA and DNA-protein cross-links (DPC). In the present study, we investigated DEB-caused DNA cross-links and breaks in human hepatocyte L02 cells using comet assay. With alkaline comet assay, it was observed that DNA migration increased with the increase of DEB concentration at lower concentrations (10-200μM); however, at higher concentrations (200-1000μM), DNA migration decreased with the increase of DEB concentration. This result indicated the presence of cross-links at >200μM, which was confirmed by the co-treatment experiments using the second genotoxic agents, tert-butyl hydroperoxide and methyl methanesulfonate. At 200μM, which appeared as a threshold, the DNA migration-retarding effect of cross-links was just observable by the co-treatment experiments. At <200μM, the effect of cross-links was too weak to be detected. The DEB-induced cross-links were determined to be DNA-DNA ones rather than DPC through incubating the liberated DNA with proteinase K prior to unwinding and electrophoresis. However, at the highest DEB concentration tested (1000μM), a small proportion of DPC could be formed. In addition, the experiments using neutral and weakly alkaline comet assays showed that DEB did not cause double-strand breaks, but did induce single-strand breaks (SSB) and alkali-labile sites (ALS). Since SSB and ALS are repaired more rapidly than cross-links, the results suggested that DNA-DNA cross-links, rather than DPC, were probably responsible for mutagenicity/carcinogenicity of DEB.     

Contribution of apoptosis to responses in the comet assay

Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substance and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. The cells were incubated for 24h with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent DNA damage-inducing agent, staurosporine, an inhibitor of protein kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source of reactive oxygen species. Apoptosis was measured with the Annexin V affinity assay which detects the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was detected by single cell microgel electrophoresis, also called "comet assay". The results show that apoptosis does not necessarily need to correlate or coincide with DNA damage observed with genotoxic substances in the comet assay. The representative apoptosis-inducing agent (staurosporine) did not induce strand breaks in the tested concentrations (0.5 and 1.0microM); genotoxic doses of the strand break inducing agent MNNG did not induce apoptosis. Therefore, the comet assay can be used as a specific test for detecting genotoxicity, and the results are not necessarily confounded by concomittant processes leading to apoptosis.     

Enhanced stimulation of chromosomal translocations by radiomimetic DNA damaging agents and camptothecin in Saccharomyces cerevisiae rad9 checkpoint mutants

Several chemical mutagens and carcinogens, including polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs, are adsorbed to the surface of diesel exhaust particles (DEP). DEP can induce formation of reactive oxygen species and cause oxidative DNA damage as well as bulky carcinogen DNA adducts. Lung tissue is a target organ for DEP induced cancer following inhalation. Recent studies have provided evidence that the lung is also a target organ for DNA damage and cancer after oral exposure to other complex mixtures of PAHs. The genotoxic effect of oral administration of DEP was investigated, in terms of markers of DNA damage, mutations and repair, in the lung of Big Blue^® rats fed a diet with 0, 0.2, 0.8, 2, 8, 20 or 80 mg DEP/kg feed for 21 days. There was no significant increase in the mutation frequency in the cII gene. However, an increase of DNA damage measured as DNA strand breaks (comet assay) and bulky DNA adducts (³²P post labeling) was observed. The level of DNA strand breaks increased significantly at all dose levels while the level of DNA adducts increased significantly only at the intermediate dose levels. Similarly, the number of oxidized DNA bases measured as endonuclease III and fapyguanine glycosylase (FPG) sensitive sites increased at the intermediate dose levels. The induction of DNA damage by DEP exposure did not increase the expression of the repair genes OGG1 and ERCC1 at the mRNA level. The present study indicates that the lung is a target organ for primary DNA damage following oral exposure to DEP. DNA damage was induced following exposure to relatively low levels of DEP, but under the conditions used in the present experiment DNA damage did not result in an increased mutation rate.     

DNA damage by carbonyl stress in human skin cells

Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the alpha-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N(epsilon)-(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress.     

Sex and age differences in habitat use by invasive cane toads (Rhinella marina) and a native anuran (Cyclorana australis) in the Australian wet–dry tropics

Although generalized habitat use may contribute to the success of invasive taxa, even species that are typically described as habitat generalists exhibit non‐random patterns of habitat use. We measured abiotic and biotic factors in 42 plots (each 100 × 10 m) along a 4.2‐km long unpaved road in tropical Australia, at a site that had been invaded by cane toads (Rhinella marina Bufonidae) seven years previously. We also counted anurans at night in each of these plots on 103 nights during the tropical wet season, over a five‐year period, beginning soon after the initial toad invasion. Spatial distributions differed significantly among adult male toads (n = 1047), adult female toads (n = 1222), juvenile toads (n = 342) and native frogs (Cyclorana australis Hylidae, n = 234). Adult male toads were closely associated with water bodies used as calling and/or spawning sites, whereas adult female toads and native frogs were most commonly encountered in drier forested areas on sloping ground. Juvenile toads used the margins of the floodplain more than conspecific adults did, but the floodplain itself was rarely used. Understanding which components of the habitat are most important to specific age and sex classes within a population, or how invasive species differ from native species in this respect, can clarify issues such as the spatial and temporal location of ecological impact by an invader, and the most effective places for control of the invader with minimal collateral effects on the native biota.     

Genotoxicity of the volatile anaesthetic desflurane in human lymphocytes in vitro, established by comet assay

The aim of the present study was to estimate the genotoxicity of desflurane, applied as a volatile anaesthetic. The potential genotoxicity was determined by the comet assay as the extent of DNA fragmentation in human peripheral blood lymphocytes in vitro. The comet assay detects DNA strand breaks induced directly by genotoxic agents as well as DNA fragmentation due to cell death. Another anaesthetic, halothane, already proved to be a genotoxic agent, was used as a positive control. Both analysed drugs were capable of increasing DNA migration in a dose-dependent manner under experimental conditions applied. The results of the study demonstrated that the genotoxicity of desflurane was comparable with that of halothane. However, considering the pharmacodynamics of both drugs, the genotoxic activity of desflurane may be connected with a less harmful effect on the exposed patients or medical staff.