In the Mesolithic, birch-bark tar was commonly utilized across Europe and much of Asia as an adhesive to bind, seal and coat surfaces, but also quite frequently it was found to be chewed. The tar is known to contain biomarker triterpenoid compounds like betulin, crucial in preserving food and for medical applications. Aqueous, ethanolic and DMSO extracts were prepared from solid birch-bark tar and evaluated in vitro for the induction of DNA damage using Comet, micronucleus and sister-chromatid-exchange assays. Additionally, apoptosis induction was assessed. For the ethanolic extract, only the Comet assay showed a significant increase of DNA damage. All three extracts were able to significantly induce apoptosis. Thus, birch-bark tar seems capable of inducing genotoxic damage as well as apoptotic effects possibly originating from the triterpenoids’ antimicrobial properties. We examine why prehistoric tar is found with tooth marks, the beneficial effects of birch-bark tar, and evidence for increased genotoxic risk upon exposure.

Twelve chemical compounds have been selected for the European NewGeneris study on the basis of their potential to damage DNA, in order to establish adequate and reliable biomarkers of exposure. These genotoxic chemicals include heterocyclic amines, organochlorines, polycyclic aromatic hydrocarbons, mycotoxins, lipid peroxidation products and alcohol. Damage in somatic cells such as lymphocytes could give rise to cancer, while damage in germ cells could not only give rise to cancer but also to heritable defects. The alkaline Comet assay, with and without metabolic activation, as well as the neutral Comet assay were used to assess DNA integrity in spermatozoa and lymphocytes after in vitro treatment with low, middle and high doses of each chemical. DNA-reactive aldehydes generated by lipid peroxidation, food mutagens such as heterocyclic amines, nitrosamine and benzo[a]pyrene produced the highest amounts of DNA damage, even without metabolic activation. Damage seen with the neutral Comet assay - detecting primarily double-strand breaks - was lower than with the alkaline assay. In general, there was increased damage in the spermatozoa by comparison with the lymphocytes, with altered slopes in the dose-response curves. The Comet assay with sperm was generally very sensitive in assessing genotoxic damage, with the Comet parameters being good biomarkers of induced DNA damage. Establishing reliable biomarkers of exposure for the evaluation of dietary/environmental carcinogens is of utmost importance to protect our health and the health of our offspring.

Oxidative stress has been implicated in the development of smoking-related diseases such as lung cancer, cardiovascular disease and chronic obstructive pulmonary disease. Damage to biological tissues from interactions with free radicals found within the smoke and induced within cells by smoke exposure, is thought to contribute to smoking-related disease development. Many of these radical components are very short-lived in vivo due to their highly reactive nature and the highly efficient detoxification mechanisms possessed by the body to counteract their effects. Hence, biomarkers are needed to assess the extent of radical exposure and subsequent oxidative damage in humans. Oxidised lipids, proteins and DNA bases persist longer in vivo, and have been extensively investigated as surrogate measures of radical damage. Furthermore, assessment of the body's antioxidant defence mechanisms such as antioxidant enzyme activity and antioxidant compounds can help to understand the extent of radical exposure. This mini review critically evaluates various biomarkers falling into these categories and the consistency of their relationship with smoking status, as a preliminary evaluation of their usefulness in dissecting disease pathways in smokers. The future use of such biomarkers is also briefly discussed.

Exposure to genotoxins may compromise DNA integrity in male reproductive cells, putting future progeny at risk for developmental defects and diseases. To study the usefulness of sperm DNA damage as a biomarker for genotoxic exposure, we have investigated cellular and molecular changes induced by benzo[a]pyrene (B[a]P) in human sperm in vitro, and results have been compared for smokers and non-smokers. Sperm DNA obtained from five smokers was indeed more fragmented than sperm of six non-smokers (mean % Tail DNA 26.5 and 48.8, respectively), as assessed by the alkaline comet assay (P < 0.05). B[a]P-related DNA adducts were detected at increased levels in smokers as determined by immunostaining. Direct exposure of mature sperm cells to B[a]P (10 or 25 μM) caused moderate increases in DNA fragmentation which was independent of addition of human liver S9 mix for enzymatic activation of B[a]P, suggesting some unknown metabolism of B[a]P in ejaculates. In vitro exposure of samples to various doses of B[a]P (with or without S9) did not reveal any significant differences in sensitivity to DNA fragmentation between smokers and non-smokers. Incubations with the proximate metabolite benzo[a]pyrene-r-7,t-8-dihydrodiol-t9,10-epoxide (BPDE) produced DNA fragmentation in a dose-dependent manner (20 or 50 μM), but only when formamidopyrimidine DNA glycosylase treatment was included in the comet assay. These levels of DNA fragmentation were, however, low in relation to very high amounts of BPDE–DNA adducts as measured with 32P postlabelling. We conclude that sperm DNA damage may be useful as a biomarker of direct exposure of sperm using the comet assay adapted to sperm, and as such the method may be applicable to cohort studies. Although the sensitivity is relatively low, DNA damage induced in earlier stages of spermatogenesis may be detected with higher efficiencies.

Due to our lifestyle and the environment we live in, we are constantly confronted with genotoxic or potentially genotoxic compounds. These toxins can cause DNA damage to our cells, leading to an increase in mutations. Sometimes such mutations could give rise to cancer in somatic cells. However, when germ cells are affected, then the damage could also have an effect on the next and successive generations. A rapid, sensitive and reliable method to detect DNA damage and assess the integrity of the genome within single cells is that of the comet or single-cell gel electrophoresis assay. The present communication gives an overview of the use of the comet assay utilising sperm or testicular cells in reproductive toxicology. This includes consideration of damage assessed by protocol modification, cryopreservation vs the use of fresh sperm, viability and statistics. It further focuses on in vivo and in vitro comet assay studies with sperm and a comparison of this assay with other assays measuring germ cell genotoxicity. As most of the de novo structural aberrations occur in sperm and spermatogenesis is functional from puberty to old age, whereas female germ cells are more complicated to obtain, the examination of male germ cells seems to be an easier and logical choice for research and testing in reproductive toxicology. In addition, the importance of such an assay for the paternal impact of genetic damage in offspring is undisputed. As there is a growing interest in the evaluation of genotoxins in male germ cells, the comet assay allows in vitro and in vivo assessments of various environmental and lifestyle genotoxins to be reliably determined.

We are constantly confronted with an increasing number of potentially harmful toxic/genotoxic chemicals. Confounding factors such as our lifestyle, the environment in which we live, medical treatments and our innate susceptibility due to our genetic make-up has also to be taken into account. Some of...

Drinking water disinfection byproducts (DBPs) are generated by the chemical disinfection of water and may pose a hazard to the public health. Previously we demonstrated that iodoacetic acid was the most cytotoxic and genotoxic DBP analyzed in a mammalian cell system. Little is known of the mechanisms of its genotoxicity. The involvement of oxidative stress in the toxicity of iodoacetic acid was analyzed with the antioxidants catalase and butylated hydroxyanisole (BHA). lodoacetic acid toxicity was quantitatively measured with and without antioxidants in Salmonella typhimurium strain TA100 and with Chinese hamster ovary (CHO) cells. The endpoints included cytotoxicity in S. typhimurium or in CHO cells, mutagenicity in S. typhimurium, and genotoxicity in CHO cells. Neither catalase nor BHA reduced the level of iodoacetic acid induced cytotoxicity in S. typhimurium. In CHO cells neither antioxidant caused a significant reduction in iodoacetic acid induced cytotoxicity. However, in S. typhimurium, BHA or catalase reduced the mutagenicity of iodoacetic acid by 33.5 and 26.8%, respectively. Likewise, BHA or catalase reduced iodoacetic acid induced genomic DNA damage by 86.5 and 42%, respectively. These results support the hypothesis that oxidative stress is involved in the induction of genotoxicity and mutagenicity by iodoacetic acid.

Cadmium is a toxic metal and no uniform mechanism of toxicity has so far been proposed. The aim of this study was to investigate the biochemical effects of cadmium chloride in a rat hepatoma cell line (HTC cells) and the cellular events mediating DNA damage.HTC cells were exposed to various concentrations of cadmium chloride for 5 and 8 h and lysosomal damage was assessed with the neutral red assay (NR) and fluorescence microscopy. Mitochondrial integrity was assessed from ATP levels and DNA damage determined with the single cell gel electrophoresis/comet assay. The formation of reactive oxygen species (ROS) was also determined under the same experimental conditions with the dichlorofluorescein assay. Cytotoxicity was assessed with the LDH leakage assay the levels of glutathione were measured and correlated with the other effects.The results indicate that lysosomal damage occurs at a lower concentration of cadmium chloride (20 mu M) than DNA damage (500 mu M) in HTC cells. The latter effect was accompanied by an increase of reactive oxygen species without any significant LDH leakage whereas lysosomal damage was significant as determined by the neutral red assay and confirmed with fluorescence microscopy. The effect of CdCl2 on mitochondria and glutathione levels were observed at concentrations or incubation times higher than the ones required to induce lysosomal damage. The data suggest that DNA damage may be due to the formation of reactive oxygen species. It is possible that cadmium induced lysosomal damage is an earlier event than DNA damage and can mediate other cellular events that lead to cell death. (c) 2005 Elsevier Ltd. All rights reserved.

Reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) are produced in the skin under the influence of UV radiation. These compounds are highly reactive and can induce DNA lesions in epidermal cells. Melanin is considered to protect human skin against DNA damage by absorbing UV radiation. We have investigated whether melanin can, in addition, offer protection against the effects of H(2)O(2) in human melanocytes and HaCaT keratinocytes. In the present study, it was shown that 40 and 100 microM H(2)O(2) increased the number of DNA strand breaks as measured using the comet assay, in melanocytes of Caucasian origin. In melanocytes of the same origin in which melanin levels were increased by culturing in presence of 10 mM NH(4)Cl and elevated l-tyrosine, H(2)O(2)-induced DNA damage was reduced compared to that in control melanocytes. Similarly, HaCaT cells that were loaded with melanin were better protected against H(2)O(2)-induced DNA strand breaks than control HaCaT cells. These protective effects of melanin were mimicked by the intracellular Ca(2+)-chelator BAPTA. Thus, BAPTA reduced the level of H(2)O(2)-induced DNA strand breaks in melanocytes. Like BAPTA, melanin is known to be a potent chelator of Ca(2+) and this was confirmed in the present study. It was shown that melanin levels in melanocytic cells correlated directly with intracellular Ca(2+) binding capacity and, in addition, correlated inversely with H(2)O(2)-induced increases in intracellular Ca(2+). Our results show that melanin may have an important role in regulating intracellular Ca(2+) homeostasis and it is suggested that melanin protects against H(2)O(2)-induced DNA strand breaks in both melanocytes and keratinocytes and through its ability to bind Ca(2+).

Reactive oxygen species (ROS) are produced by a wide variety of exogenous chemicals and metabolic processes and cause a broad spectrum of damage to biological systems. As a consequence, ROS react with DNA, among many other biological targets, disrupting its structure and functionality. Estrogen‐like compounds mediate DNA damage by ROS generation, implying that their effects can be modulated by antioxidants such as catalase, superoxide dismutase, and vitamin C. We examined DNA damage in human lymphocytes and sperm after treatment with four estrogen‐like compounds (β‐estradiol, diethylstilbestrol, daidzein, and genistein) and its modulation by flavonoids (quercetin and kaempferol) using the Comet assay. The results indicated that quercetin and kaempferol reduced the DNA damage produced in sperm and lymphocytes by the four estrogenic compounds. The flavonoids also reduced the DNA damage induced by hydrogen peroxide, which was used as a positive control. Our results demonstrate that the antioxidant properties of flavonoids can protect the integrity of human sperm and lymphocyte DNA from ROS induced by estrogenic compounds. Environ. Mol. Mutagen., 2004. © 2004 Wiley‐Liss, Inc.

In recent years, two techniques for detecting genetic damage in the whole genome have gained importance: the alkaline comet assay, to detect DNA damage such as strand breaks and alkali-labile sites, and a multicolour FISH method, spectral karyotyping (SKY), to identify chromosomal aberrations simultaneously in all metaphase chromosomes. In the present study, the induction of DNA damage in human sperm and lymphocytes in vitro has been studied employing an anticancer drug, doxorubicin (DX). An increase in DNA damage was observed with the comet assay as the median per cent head DNA of sperm significantly decreased from 82.07 and 85.14% in the untreated control groups to 63.48 and 72.52% at doses of 0.8 micro M DX. At 1.6 micro M the percentage declined to 60.96% (the corresponding tail moment increased from 4.42 to 12.19). In stimulated lymphocytes, a significant increase was observed in tail moment, from 0.72 and 0.53 in controls to 15.17 and 12.10 at 0.2 micro M DX, continuing at the same level to a final concentration of 1.6 micro M. Structural aberrations found in the parallel SKY study in stimulated lymphocytes at 0.2 micro M DX consisted of 14% chromatid-type and 2% chromosome-type aberrations; none were found in controls. The SKY results correlate very well with the findings of the comet assay in lymphocytes where DNA damage was observed at similar doses. This study is the first reporting use of the comet assay and SKY analysis in parallel after chemical treatment. The potential of the two techniques together is evident, as they represent a set of assays feasible for evaluating damage in human somatic and germ cells after chemical treatment (i) by direct observation of two different end-points, detecting general DNA damage and chromosomal aberrations and (ii) by extrapolation from lymphocytes to sperm, which provides a 'parallelogram' approach in human cells.