Measurement of DNA damage in rat urinary bladder transitional cells: Improved selective harvest of transitional cells and detailed Comet assay protocols

Urinary bladder transitional epithelium is the main site of bladder cancer, and the use of transitional cells to study carcinogenesis/genotoxicity is recommended over the use of whole bladders. Because the transitional epithelium is only a small fraction of the whole bladder, the alkaline single cell gel electrophoresis assay (Comet assay), which requires only a small number of cells per sample, is especially suitable for measuring DNA damage in transitional cells. However, existed procedures of cell collection did not yield transitional cells with a high purity, and pooling of samples was needed for Comet assay. The goal of this study was to develop an optimized protocol to evaluate DNA damage in the urinary bladder transitional epithelium. This was achieved by an enzymatic stripping method (trypsin–EDTA incubation plus gentle scraping) to selectively harvest transitional cells from rat bladders, and the use of the alkaline Comet assay to detect DNA strand breaks, alkaline labile sites, and DNA–protein crosslinks. Step by step procedures are reported here. Cells collected from a single rat bladder were sufficient for multiple Comet assays. With this new protocol, increases in DNA damage were detected in transitional cells after in vitro exposure to the positive control agents, hydrogen peroxide or formaldehyde. Repair of the induced DNA damage occurred within 4 h. This indicated the capacity for DNA repair was maintained in the harvested cells. The new protocol provides a simple and inexpensive method to detect various types of DNA damage and to measure DNA damage repair in urinary bladder transitional cells.     

A comparison of cell-collecting methods for the Comet assay in urinary bladders of rats

Conducting the single-cell gel electrophoresis (Comet) assay in the urinary bladders of rodents is technically problematic because the bladder is small and thin, which makes it difficult to collect its mucosal cells by scraping. We performed the Comet assay using a simple mincing method in which tissues are minced with scissors. We then compared data obtained with this method with data obtained using the scraping method. Sprague-Dawley rats of both sexes were orally given twice the known carcinogens N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), or o-anisidine (OA). Three hours after the second administration, the bladder of each rat was divided into two parts and each part was processed by either the mincing or the scraping method. Both mincing and scraping methods detected DNA damage in MNU-, EMS-, but not OA-treated rats, and thus the mincing method had a sufficient capability to detect DNA damaging agents. The morphological analysis of the prepared cell suspensions revealed that more than 80% of the cells collected by the mincing method were from the epithelium. Because the mincing method requires only one-half of a bladder, the other half remains intact and can be used for histopathological examination. We conclude that the mincing method is easier and more appropriate for the Comet assay in urinary bladder tissue than the scraping method.     

Modification of the alkaline Comet assay to allow simultaneous evaluation of mitomycin C-induced DNA cross-link damage and repair of specific DNA sequences in RT4 cells

The alkaline Comet assay is a simple, sensitive method for measuring the extent of DNA strand breaks in individual cells. Several modifications to the original assay have been developed to increase its applications. One such modification allows the measurement of DNA cross-links by assessing the relative reduction in DNA migration induced by a strand-breaking agent. Another modification includes the application of fluorescent in situ hybridisation (FISH) to investigate the localisation of specific gene domains within a cell. Although several studies have used these approaches separately, no report to date has combined these two versions of the Comet assay. The current study describes the modification of the Comet assay, to allow both measurement of mitomycin C (MMC)-induced cross-links and the subsequent application of FISH to study repair in the TP53 gene region. RT4 human bladder cancer cells were treated with 0, 5, 50 and 200 microg/ml MMC to study dose response, whilst for cross-link repair studies, they were treated with 50 microg/ml MMC and allowed to repair for up to 24 h. A clear dose response to MMC was displayed, demonstrable by a marked reduction in DNA migration, whilst repair studies showed that MMC-induced cross-links take at least 24 h to repair fully in RT4 cells. For Comet-FISH experiments, the number and location of TP53 hybridisation spots was also recorded for each cell. In dose response experiments, the number of spots per cell, and per Comet tail, decreased as MMC dose increased. In repair experiments, the number of spots, particularly in the Comet tail, increased as repair time increased. Furthermore, our results suggest that repair of the TP53 gene region is most rapid within the first 4 h following MMC treatment. We conclude that the novel experimental protocol presented here has considerable potential in evaluating DNA damage and sequence-related repair responses to cross-linking agents.     

The presence of G1 and G2 populations in normal epithelium of rat urinary bladder

The cell population kinetics of transitional epithelium of the rat urinary bladder was analysed by (3H) thymidine autoradiography and pararosanilin Feulgen DNA cytofluorometry. By flash and 72 h continuous DNA labelling, the generative cells of the transitional epithelium were found to be well localized in the basal layer, and it was postulated that che cells produced by cell proliferation in the basal layer would migrate towards the surface, maintaining direct attachment to the basement membrane by anchorage of a cellular process. Analyses of normal and wounded transitional epithelium revealed that 58.8% of all basal cells are G0 cells in G1 phase (G1-population), and 59.0% of the remaining basal cells reside in prolonged (75.1-108.0 h) G2 phase, preserving the ability to divide (G2-population). The cell cycle time of the generative basal cells including the long G2 phase was calculated as 129.1-162 h. All the cells existing in upper layers were found to be also G0 cells in G1 phase, with the DNA amounts of 2C class. No polyploid cells could be detected except for 2C-2C binucleated cells in the superficial layer. The existence of a G2-population may serve for the urgent need of cell incrementation to repair cell loss as the cells in G2 phase can divide without the time-delay needed for DNA synthesis. The rat transitional epithelium, which is composed exclusively of proliferating and potentially proliferative cells, will have much greater capability to repair damage than stratified squamous epithelia.     

Effects of ochratoxin A on DNA evaluated<Emphasis Type="Italic">in vitro</Emphasis> with the single cell gel electrophoresis (Comet assay)

In cell cultures of Madin Darby canine kidney (MDCK) cells, the mycotoxin ochratoxin A (OTA) induced single strand breaks (ssb) in a concentration dependent manner detected with the single cell gel electrophoresis (Comet assay). When an external metabolizing enzyme system (S9-mix from rat liver) was added, this genotoxic effect was significantly stronger. By addition of methotrexate (MT), a substrate of the hepatic organic anion transporter, the effect of OTA can be completely blocked at concentrations >100 μM methotrexate.When DNA repair was inhibited by addition of cytosine arabinose (araC) and hydroxyurea (HU), the tail length in the Comet assay increased dramatically and all treated cells showed ssb. A further culture of the damaged cells in the absence of any supplement resulted in a complete repair of the damaged DNA within three hours.Compared with MDCK cells, primary cultured porcine urinary bladder epithelial cells (PUBECs) showed weaker effects in the Comet assay if treated with OTA. The presence of S9-mix did not significantly enhance the response. Methotrexate only partially reduced the OTA-induced effects, because in PUBECs methotrexate induced ssb at high concentrations. If DNA repair was inhibited, also in PUBECs clearly more ssb were induced by OTA, an effect which was reversible.These results demonstrate that OTA induces single strand breaks in vitro. The damaged DNA can be repaired more effectively in primary cultured epithelial cells (PUBECs) compared to cells of a cell line (MDCK cells). By competitive inhibition of OTA uptake, DNA damage can be prevented with suitable substrates.     

New Application of the Comet Assay: Chromosome�CComet Assay

The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains.     

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.     

Applicability of the alkaline elution procedure as modified for the measurement of DNA damage and its repair in nonradioactively labeled cells

We have critically evaluated various modifications of the alkaline elution methodology that were required to adapt the method for measuring DNA damage in cells from animal tissues treated in vivo. These modifications involved the use of a fluorometric assay for the eluted DNA using the dye Hoechst 33258, which in turn required the use of a different combination of filter and lysis conditions than those used in conventional assays. This protocol was compared with the conventional protocols by examining the DNA damage produced in cultured Chinese hamster ovary cells after treatment with three agents (gamma-rays, cis-dichlorodiammineplatinum (DDP) and trans-DDP) that differ widely in the type and repairability of the DNA lesions that they induce. For both gamma-rays and trans-DDP, the results obtained by the various protocols were equivalent with respect to the amount, type, and rate of repair of the DNA damage produced. On the other hand, for cis-DDP, where the repair time for DNA crosslinks was significantly long relative to the cell-cycle time, DNA replication appeared to be a potentially complicating factor in the measurement of crosslink repair. However, even after treatment of rapidly dividing cultured cells, where any discrepancy between the radioactivity and Hoechst assays due to DNA replication should be maximal, the resulting difference in the amount of repair measured using the two assays was relatively small. Finally, in experiments using cis-DDP and trans-DDP, the data suggested that when polycarbonate and polyvinyl chloride filters were compared using the same cell lysis conditions, their relative sensitivity to detect DNA-protein versus DNA-interstrand crosslinking were comparable. The modified alkaline elution protocol for the measurement of DNA damage in vivo therefore appears, in most cases, to produce results comparable with those obtained by the conventional protocols.     

Modulation of ochratoxin A induced DNA-damage in urothelial cell cultures

Despite good evidence for a genotoxic potential of ochratoxin A (OTA), the mechanism of OTA-induced genotoxicity (direct or indirect?) is still unclear. This calls for a further characterization of OTA-related DNA damage, and investigations of factors that may modulate dose-effect relationships in cells. Since bladder epithelium is a target tissue for the toxicity of OTA, its effects were studied in cultures of human bladder carcinoma (H5637) cells. Cytotoxicity of OTA, assessed by Neutral red (NR) uptake or Alamar-Blue assay, is concentration- and time-dependent: Upon 24 h treatment of 5637 cells, NR uptake is reduced by 50% with OTA concentrations of ≥0.2 microM, but not with 3 h treatment of the cells. Since cytotoxicity of OTA was not affected by addition of xenobiotic metabolizing enzymes (S-9 mix), it appears to be unrelated to biotransformation of the mycotoxin. Also, addition of S-9 mix did not significantly affect the genotoxicity of OTA as studied by alkaline single cell gel electrophoresis (Comet assay). DNA damage was detectable after 3 h treatment of cells at OTA concentrations between 0.1 and 1 microM, and increased further at higher concentrations. The magnitude of OTA-induced DNA damage did not increase with longer treatment times (18, 24 h), probably due to repair processes in the cells. Repair of OTA-induced lesions is quite efficient in kidney (Arch Toxicol 2002, 75, 734–741) and in porcine bladder cells (Föllmann and Lebrun, 2005, Mycotoxin Research, this volume). Interestingly, the genotoxicity of OTA is modulated by the pH of the culture medium, with higher damage at pH 5 compared to pH 7.5. In line with this, uptake studies with tritiated OTA show a higher cellular accumulation of the mycotoxin at pH 5 than in buffer of pH 7.5. Thus, bladder cells exposed to OTA in slightly acidic urine (which facilitates reabsorption) may be at higher risk.     

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.     

紫外辐射诱导植物叶片DNA损伤敏感性差异

单细胞凝胶电泳(彗星检测, comet assay)技术已广泛应用于动物细胞DNA损伤检测, 但在植物细胞DNA损伤检测中的应用尚不多见。本研究通过对动物细胞彗星检测方法的改进, 利用植物细胞原生质体作为材料, 研究了不同发育期九里香(Murraya panicuata)叶片对UV-B诱导的DNA损伤的敏感性差异。彗星检测结果表明, 九里香叶片DNA的损伤程度与UV-B辐射的剂量呈正相关; 在相同UV-B辐射剂量下, 九里香幼嫩叶片比成熟叶片的DNA损伤量大, 表明其幼嫩叶片对UV-B辐射的敏感性比成熟叶片高。     

Limited repair of 8-hydroxy-7,8-dihydroguanine residues in human testicular cells

Oxidative damage in testicular DNA is associated with poor semen quality, reduced fertility and increased risk of stillbirths and birth defects. These DNA lesions are predominantly removed by base excision repair. Cellular extracts from human and rat testicular cells and three enriched populations of rat male germ cells (primary spermatocytes, round spermatids and elongating/elongated spermatids) all showed proficient excision/incision of 5-hydroxycytosine, thymine glycol and 2,6-diamino-4-hydroxy-5-formamidopyrimidine. DNA containing 8-oxo-7,8-dihydroguanine was excised poorly by human testicular cell extracts, although 8-oxoguanine-DNA glycosylase-1 (hOGG1) was present in human testicular cells, at levels that varied markedly between 13 individuals. This excision was as low as with human mononuclear blood cell extracts. The level of endonuclease III homologue-1 (NTH1), which excises oxidised pyrimidines, was higher in testicular than in somatic cells of both species. Cellular repair studies of lesions recognised by formamidopyrimidine-DNA glycosylase (Fpg) or endonuclease III (Nth) were assayed with alkaline elution and the Comet assay. Consistent with the enzymatic activities, human testicular cells showed poor removal of Fpg-sensitive lesions but efficient repair of Nth-sensitive lesions. Rat testicular cells efficiently repaired both Fpg- and Nth-sensitive lesions. In conclusion, human testicular cells have limited capacity to repair important oxidative DNA lesions, which could lead to impaired reproduction and de novo mutations.     

Effect of PARP-1 deficiency on DNA damage and repair in human bronchial epithelial cells exposed to Benzo(a)pyrene

Benzo[a]pyrene is a ubiquitously distributed environmental pollutant known to cause DNA damage, whereas PARP-1 is a nuclear enzyme that is activated by damaged DNA and plays an important role in base excision repair and genomic stability. Here, 16HBE and its PAPR1-deficient cells were exposed to BaP, and the DNA damage level and repair ability of both cell lines were measured by alkaline comet assay. The results showed that cell viability of both cell lines decreased in a dose-dependent manner when exposed to BaP, but there was no significant difference between two cell lines. Comet assay showed that BaP caused DNA damage in both cell lines at an obvious dose- and time-dependent manner. Compare with 16HBE, the PARP1-deficient cells were more sensitive to the damage caused by BaP. The results of DNA repair experiment showed that both cell lines can recover from the damage in a time-dependent pattern. The relative repair percentage of PARP1-deficient cells were generally lower than that of 16HBE at all exposed concentrations at the early stage of repair, but tended to be closer between two cell lines at the later period. According to results, we came to the conclusion that PARP1-deficient cells were more sensitive to BaP in contrast to normal 16HBE; DNA repair capacity in PARP1-deficient cells decreased significantly at the early stage of repair, but increased to the equivalent level of normal 16HBE in the later period. PARP-1 plays an important role in early repair of DNA damage caused by BaP in 16HBE notwithstanding the main repair work is taken by NER pathway.     

脂质体介导人β防御素2基因转染膀胱上皮的实验研究

目的 探讨脂质体介导人β防御-2（humaβ-defensin2,hBD2）基因体内、外转染膀胱上皮细胞的可行性。方法采用脂质体法体外转染人膀胱上皮细胞株T24及膀胱灌注大鼠体内转染,ELISA检测细胞上清及大鼠尿液中hBD：的表达;RT—PCR及Western印迹检测细胞及大鼠膀胱中hBD：的表达;组织免疫荧光检测hBD,在膀胱黏膜的表达。结果RT—PCR及Western印迹检测显示,脂质体介导pCAGG—hBD2体、内外转染膀胱上皮细胞后均可表达重组hBD2;转染细胞上清及大鼠尿液中hBD2浓度分别为（36．5±3．2）ng／10。细胞和（4．77±1．4）ng／ml;转基因hBD2主要表达于膀胱黏膜上皮层。结论脂质体介导hBD2基因体内、外转染膀胱上皮细胞后均能获得高效表达,为泌尿系感染的防御素基因治疗提供了实验依据。     

紫外辐射诱导植物叶片DNA损伤敏感性差异

单细胞凝胶电泳（彗星检测,cometassay）技术已广泛应用于动物细胞DNA损伤检测,但在植物细胞DNA损伤检测中的应用尚不多见。本研究通过对动物细胞彗星检测方法的改进,利用植物细胞原生质体作为材料,研究了不同发育期九里香（Murraya panicuata）叶片对UV-B诱导的DNA损伤的敏感性差异。彗星检测结果表明,九里香叶片DNA的损伤程度与UV-B辐射的剂量呈正相关：在相同UV—B辐射剂量下,九里香幼嫩叶片比成熟叶片的DNA损伤量大,表明其幼嫩叶片对UV-B辐射的敏感性比成熟叶片高。     

The permeability of rat transitional epithelium. Kertinization and the barrier to water

Permeability barriers must exist in transitional epithelium to prevent the free flow of water from underlying blood capillaries through the epithelium into the hypertonic urine, and such a barrier has now been demonstrated in isolated bladders. This barrier is passive in function and can be destroyed by damaging the luminal surface of the transitional epithelium with sodium hydroxide and 8 M urea solutions, by digesting it with trypsin, lecithinase C, and lecithinase D, or by treating it with lipid solvents such as Triton x 100 and saponin. From this it is concluded that the barrier depends on the integrity of lipoprotein cell membranes. The barrier function is also destroyed by sodium thioglycollate solutions, and electron microscope investigations show that sodium thioglycollate damages the thick asymmetric membrane which limits the luminal face of the superficial squamous cell. Cytochemical staining shows the epithelium to contain disulfide and thiol groups and to have a concentration of these groups at the luminal margin of the superficial cells. It thus appears that the permeability barrier also depends on the presence of disulfide bridges in the epithelium, and it is presumed that these links are located in keratin. Because of the effect of thioglycollates, both on the barrier function and on the morphology of the membrane, it is suggested that keratin may be incorporated in the thick barrier membrane. It is proposed that the cells lining the urinary bladder and ureters should be regarded as a keratinizing epitheluim.     

A modified alkaline Comet assay for in vivo detection of oxidative DNA damage in Drosophila melanogaster

Modifications to the alkaline Comet assay by using lesion-specific endonucleases, such as formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (ENDOIII, also known as Nth), can detect DNA bases with oxidative damage. This modified assay can be used to assess the genotoxic/carcinogenic potential of environmental chemicals. The goal of this study was to validate the ability of this modified assay to detect oxidative stress-induced genotoxicity in Drosophila melanogaster (Oregon R(+)). In this study, we used three well known chemical oxidative stress inducers: hydrogen peroxide (H(2)O(2)), cadmium chloride (CdCl(2)) and copper sulfate (CuSO(4)). Third instar larvae of D. melanogaster were fed various concentrations of the test chemicals (50-200μM) mixed with a standard Drosophila food for 24h. Alkaline Comet assays with and without the FPG and ENDOIII enzymes were performed with midgut cells that were isolated from the control and treated larvae. Our results show a concentration-dependent increase (p<0.05-0.001) in the migration of DNA from the treated larvae. ENDOIII treatment detected more oxidative DNA damage (specifically pyrimidine damage) in the H(2)O(2) exposed larvae compared to FPG or no enzyme treatment (buffer only). In contrast, FPG treatment detected more oxidative DNA damage (specifically purine damage) in CuSO(4) exposed larvae compared to ENDOIII. Although previously reported to be a potent genotoxic agent, CdCl(2) did not induce more oxidative DNA damage than the other test chemicals. Our results show that the modified alkaline Comet assay can be used to detect oxidative stress-induced DNA damage in D. melanogaster and thus may be applicable for in vivo genotoxic assessments of environmental chemicals.     

A modified alkaline comet assay for measuring DNA repair capacity in human populations

Trzeciak, A. R., Barnes, J. and Evans, M. K. A Modified Alkaline Comet Assay for Measuring DNA Repair Capacity in Human Populations. Radiat. Res. 169, 110-121 (2008). Use of the alkaline comet assay to assess DNA repair capacity in human populations has been limited by several factors, including lack of methodology for use of unstimulated cryopreserved peripheral blood mononuclear cells (PBMCs), insufficient control of interexperimental variability, and limited analysis of DNA repair kinetics. We show that unstimulated cryopreserved PBMCs can be used in DNA repair studies performed using the comet assay. We have applied data standardization for the analysis of DNA repair capacity using negative and positive internal standards as controls for interexperimental variability. Our standardization procedure also uses negative controls, which provides a way to minimize the interference of interindividual variation in baseline DNA damage levels on DNA repair capacity measurements in populations. DNA repair capacity was assessed in a small human cohort using the parameters described in the literature including initial DNA damage, half-time of DNA repair, and residual DNA damage after 30 and 60 min. We have also introduced new DNA repair capacity parameter, initial rate of DNA repair. There was no difference in DNA repair capacity between fresh and cryopreserved PBMCs when measured by the Olive tail moment and tail DNA. The use of DNA repair capacity parameters in assessment of fast and slow single-strand break repair components is discussed.     

Comet-assay parameters as rapid biomarkers of exposure to dietary/environmental compounds -- an in vitro feasibility study on spermatozoa and lymphocytes

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.