Detection of DNA strand breakage in a marine amphipod by agarose gel electrophoresis: exposure to X-rays and copper

This article describes the leading steps to develop an assay of DNA damage for the marine amphipod Gammarus locusta, using agarose gel electrophoresis (AGE). To test the sensitivity and feasibility of the AGE technique, X-ray assays were performed with naked DNA and with live amphipods. These positive controls demonstrated the effectiveness of the AGE technique to not only discriminate distinct levels of DNA strand breakage in a dose-dependent manner, but also to identify and quantify the type of strand breakage induced. It was also shown that it is possible to detect DNA damage using whole-body DNA extracts from amphipods. To explore the potential of this technique for use in ecotoxicological studies with amphipods, a 96-h waterborne-copper toxicity test was performed. Copper-induced DNA strand breakage was first observed after 24 h of exposure, and was recorded again at 96 h, at a copper concentration of 20 μg l -1 . The absence of strand breakage after 48 h of exposure is discussed in the light of the underlying mechanisms of copper toxicity and DNA repair. These studies demonstrated the feasibility of including DNA damage as a biomarker in ecotoxicological studies with amphipods. Information gained from the use of this biomarker would help with the interpretation of chronic toxicity tests and would contribute to our understanding of the impact of genotoxic insult in marine invertebrates, particularly crustaceans.     

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.     

Estimates of DNA strand breakage in bottlenose dolphin (Tursiops truncatus) leukocytes measured with the Comet and DNA diffusion assays

The analysis of DNA damage by mean of Comet or single cell gel electrophoresis (SCGE) assay has been commonly used to assess genotoxic impact in aquatic animals being able to detect exposure to low concentrations of contaminants in a wide range of species. The aims of this work were 1) to evaluate the usefulness of the Comet to detect DNA strand breakage in dolphin leukocytes, 2) to use the DNA diffusion assay to determine the amount of DNA strand breakage associated with apoptosis or necrosis, and 3) to determine the proportion of DNA strand breakage that was unrelated to apoptosis and necrosis. Significant intra-individual variation was observed in all of the estimates of DNA damage. DNA strand breakage was overestimated because a considerable amount (~29%) of the DNA damage was derived from apoptosis and necrosis. The remaining DNA damage in dolphin leukocytes was caused by factors unrelated to apoptosis and necrosis. These results indicate that the DNA diffusion assay is a complementary tool that can be used together with the Comet assay to assess DNA damage in bottlenose dolphins.     

Protecting or promoting effects of spermine on DNA strand breakage induced by iron or copper ions as a function of metal concentration

Polyamines are ubiquitous polycations that participate in cellular processes such as growth, differentiation and cell death. Among the different functions ascribed to these organic cations, the polyamine spermine is known to protect DNA from the damage produced by reactive oxygen species (ROS) generated by different agents including copper ions. We have found that spermine exerts opposite effects on DNA strand breakage induced by Fenton reaction depending on metal concentration. Whereas at low concentration of the transition metals, 10 microM copper or 50 microM Fe(II), 1 mM spermine exerted a protective role, at metal concentrations higher than 25 microM copper or 100 microM Fe(II), spermine stimulated DNA strand breakage. The promotion of the damage induced by spermine was independent of DNA sequence but decreased by increasing the ionic concentration of the media or by the presence of metal-chelating agents. Moreover, spermine did not increase the oxidation of 2-deoxyribose by metal/H2O2 when DNA was substituted by 2-deoxyribose as a target for damage. Our results corroborate that spermine may protect DNA and 2-deoxyribose from the damage induced by ROS but also demonstrate that under certain conditions spermine may promote DNA strand breakage. The fact that this promoting effect of spermine on ROS-induced damage was observed only in the presence of DNA suggests that this polyamine under certain conditions may facilitate the interaction of copper and iron ions with DNA leading to the formation of ROS in close proximity to DNA.     

Repair of cytokine-induced DNA damage in cultured rat islets of Langerhans

Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1beta (IL-1beta), interferon gamma (IFN gamma) and tumour necrosis factor alpha (TNF alpha) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4 h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during,the cytokine treatment and islets are incubated for 4 h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4 h in the presence of hydroxyurea (HU) and 1-beta-D-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.     

Evaluation of the genotoxicity induced by the fungicide fenarimol in mammalian and plant cells by use of the single-cell gel electrophoresis assay

Fenarimol, a systemic pyrimidine carbinol fungicide, is considered to be not genotoxic or weakly genotoxic, although the available toxicological data are controversial and incomplete. Our results obtained in vitro with leukocytes of two different rodent species (rat and mouse) show that fenarimol affects DNA, as detected by the single-cell gel electrophoresis (SCGE, Comet) assay. This fungicide is able to induce DNA damage in a dose-related manner, with significant effectiveness at 36 nM, but without significant interspecies differences. Simultaneous exposure of rat leukocytes to fenarimol (36-290 nM) and a model genotoxic compound (50 microg/ml bleomycin) produced a supra-additive cytotoxic and genotoxic effect. This supports previous findings suggesting possible co-toxic, co-mutagenic, cancer-promoting and co-carcinogenic potential of fenarimol, and modification of the effects of other xenobiotics found to be influenced by this agrotoxic chemical, with consequent different toxicological events. The potential for DNA strand breaks to act as a biomarker of genetic toxicity in plants in vivo was also considered, in view of the fact that higher plants represent reliable sensors in an ecosystem. Significant DNA breakage was observed in the nuclei of Impatiens balsamina leaves after in vivo treatment with fenarimol (145 nM, 1h). More than 50% of the cells showed such DNA damage.     

Bio-techniques for the detection of genetic toxicity in the aquatic environment.

This article reviews the application of sensitive biotechniques in the detection of DNA damage within the aquatic environment as "early warning" indicators of pollution-related genetic toxicity. Bacterial mutagenicity assays have played an important role both in the discovery of mutagens and in the analysis of the ability of aquatic organisms to convert inert substances to reactive genotoxic products. Genetic damage in aquatic species treated with carcinogens has been detected by analysis of DNA adducts, induction of DNA repair, DNA strand breakage and formation of chromosomal aberrations and sister chromatid exchanges. Little information is available on pollution-related DNA damage resulting from field exposure. A 32P-postlabelling technique for the sensitive detection of DNA adducts is a promising development towards this aim and the analysis of changes in oncogene nucleotide sequence in tumour tissue is considered an important future direction.     

Repair of Cytokine-induced DNA Damage in Cultured Rat Islets of Langerhans

Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1β (IL-1β), interferon γ (IFN γ) and tumour necrosis factor α (TNF α) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4?h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor N^G-monomethyl-l-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during, the cytokine treatment and islets are incubated for 4?h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4?h in the presence of hydroxyurea (HU) and 1-β-d-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18?h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.     

Bleomycin-induced DNA-strand damage in isolated male germ cells

DNA strand damage in isolated male germ cells (MGC) was evaluated after in vitro exposure to bleomycin (BLM), a known genotoxin. The alkaline elution technique was used to determine DNA-strand breaks. Concentration-dependent strand damage was established following exposure to bleomycin for 1 h at 37 degrees C. Exposure at 0 degrees C resulted in an increase in the frequency of strand breaks as compared to those observed at 37 degrees C. Pretreatment of cells with deferoxamine (DM), an iron-selective chelating agent, abolished the DNA damage induced by bleomycin. Isolated male germ cells responded in a predictable and reproducible manner thus supporting their use in mechanistic studies of genotoxicity.     

Characterization of DNA lesions produced by HgCl2 in cell culture systems

HgCl2 is extremely cytotoxic to Chinese hamster ovary (CHO) cells in culture since a 1-h exposure to a 75- microM concentration of this compound reduced cell plating efficiency to 0 and cell growth was completely inhibited at 7.5 microM . The level of HgCl2 toxicity depended upon the culture incubation medium and has previously been shown to be inversely proportional to the extracellular concentration of metal chelating amino acids such as cysteine. Thus, HgCl2 toxicity in a minimal salts/glucose maintenance medium was about 10-fold greater than the toxicity in McCoy's culture medium. The HgCl2 toxicity in the latter medium was 3-fold greater than that in alpha-MEM which contains more of the metal chelating amino acids. When cells were exposed to HgCl2 there was a rapid and pronounced induction of single strand breaks in the DNA at time intervals and concentrations that paralleled the cellular toxicity. The DNA damage was shown to be true single strand breaks and not alkaline sensitive sites or double strand breaks by a variety of techniques. Consistent with the toxicity of HgCl2, the DNA damage under an equivalent exposure situation was more pronounced in the salts/glucose than in the McCoy's medium and more striking in the latter medium than in alpha-MEM. Most of the single strand breaks occurred within 1 h of exposure to the metal. We believe that the DNA damage caused by HgCl2 leads to cell death because the DNA single strand breaks are not readily repaired. DNA repair activity measured by CsCl density gradient techniques was elevated above the untreated levels at HgCl2 concentrations that produced little measurable binding of the metal to DNA or few single strand breaks assessed by the alkaline elution procedure. DNA repair activity decreased at HgCl2 concentrations that produced measurable DNA binding and single strand breaks. These irreversible interactions of HgCl2 with DNA may be responsible for its cytotoxic action in cells.     

DNA-protein cross-links induced by nickel compounds in intact cultured mammalian cells

The carcinogenic activity of crystalline NiS has been attributed to phagocytosis and intracellular dissolution of the particles to yield Ni2+ which is thought to enter the nucleus and damage DNA. In this study the extent and type of DNA damage in Chinese hamster ovary CHO cells treated with various nickel compounds was assessed by alkaline elution. Both insoluble (crystalline NiS) and soluble (NiCl2) nickel compounds induced single strand breaks and DNA protein cross-links. The single strand breaks were repaired relatively quickly but the DNA-protein cross-links were present and still accumulating 24 h after exposure to nickel. Single strand breakage occurred at both non-cytotoxic and cytotoxic concentrations of nickel, however, DNA-protein cross-linking was absent when cells were exposed to toxic nickel levels. The concentration of nickel that induced DNA-protein cross-linking correlated with those metal concentrations that reversibly inhibited cellular replication.     

The rate of strand separation in alkali of DNA of irradiated Mammalian cells

When mammalian cells are treated with alkali of pH at about 12, the cells are lysed and the released DNA starts to uncoil. This process of DNA strand separation is accelerated if the cells have been exposed to ionizing radiation, and the effect is clearly detectable in the dose range 10-100 rads. The rate of strand separation is also influenced by temperature and ionic strength of the alkaline solution. The kinetics of DNA strand separation in alkali is studied for three conditions in terms of ionic strength and temperature, chosen in such a way that the effect of irradiation may conveniently be studied in the dose range 10 rads to 20 krads. The accelerating effect of ionizing radiation on DNA strand separation is probably due to DNA strand breakage and the technique described is thus a sensitive method of studying such damage to DNA. A model for the strand-separation process, based on the assumption that strand breakage causes the accelerating effect, is proposed and found to describe the experimental data adequately.     

Alkaline step elution analysis of gamma-ray induced DNA strand breaks and repair in diploid yeast

Gamma-ray induction of DNA strand breaks and their repair was analysed in the diploid yeast strain D7 (Saccharomyces cerevisiae) by means of the alkaline step elution technique. A dose-dependent increase of DNA strand breakage was observed in the dose range 25-2000 Gy corresponding to 100 and 0.01 per cent survival. When, after exposure to gamma-irradiation, the cells were incubated for 2 h in liquid growth medium, the elution profiles reached the pattern of unirradiated controls, thus indicating the restoration of cellular DNA due to repair. The alkaline step elution analysis is found to be a useful and reproducible technique for studying the induction of DNA strand breaks and repair in yeast. In comparison with other current methods, such as alkaline sucrose gradients and DNA unwinding, this method appears to be more rapid, versatile and easier to handle.     

The long amplicon quantitative PCR for DNA damage assay as a sensitive method of assessing DNA damage in the environmental model, Atlantic killifish (Fundulus heteroclitus)

DNA damage is an important mechanism of toxicity for a variety of pollutants, and therefore, is often used as an indicator of pollutant effects in ecotoxicological studies. Here, we adapted a PCR-based assay for nuclear and mitochondrial DNA damage for use in an important environmental model, the Atlantic killifish (Fundulus heteroclitus). We refer to this assay as the long amplicon quantitative PCR (LA-QPCR) assay. To validate this method in killifish, DNA damage was measured in liver, brain, and muscle of fish dosed with 10 mg/kg benzo[a]pyrene. This exposure caused 0.4-0.8 lesions/10 kb. We also measured DNA damage in liver and muscle tissues from killifish inhabiting a Superfund site, confirming the utility of this method for biomonitoring. In both cases, damage levels were comparable in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Since extensive nDNA sequence data are not readily available for many environmentally relevant species, but mitochondrial genomes are frequently fully sequenced, this assay can be adapted to examine mtDNA damage in virtually any species with little development. Therefore, we argue that this assay will be a valuable tool in assessing DNA damage in ecotoxicological studies.     

Oxidative damage to DNA by 1,10-phenanthroline/<Emphasis Type="SmallCaps">l</Emphasis>-threonine copper (II) complexes with chlorogenic acid

The oxidative DNA damage by copper (II) complexes in the presence of chlorogenic acid was explored using agarose gel electrophoresis. The extent of pBR322 DNA damage was enhanced significantly with increasing concentration of [Cu-phen-Thr] complex and incubation time. A fluorescence quenching activity of calf thymus DNA–EB was observed more remarkably with chlorogenic acid than without chlorogenic acid. The fluorescence measurements suggested that [Cu-phen-Thr] complex not only can bind to DNA by intercalation but also can damage the double strand DNA in the presence of chlorogenic acid. Further, 8-hydroxy-2′-deoxyguanosine, a biomarker of DNA oxidative damage was determined by electrochemical method. The control experiments revealed that the structure of copper (II) complexes affected capability of complex to DNA damage. The planar structure copper (II) complex showed high efficiency to DNA damage. The chlorogenic acid as biological reductant could improve copper (II) complex to DNA damage. A mechanism on [Cu-phen-Thr] complex to DNA damage in the presence of chlorogenic acid was proposed.     

Oxidation-reduction reactions in Ehrlich cells treated with copper-neocuproine.

The interaction of 2,9-dimethyl-1,10-phenanthroline (neocuproine or NC) and its copper complex with Ehrlich ascites tumor cells was studied. NC is frequently used as a negative control in studies of in vitro DNA degradation by copper phenanthroline and has also found use as a potential inhibitor of damage from oxidative stress in biological systems. NC inhibited Ehrlich cell growth in monolayer culture over 48 h treatment by 50% at 0.05 nmol/10(5) cells. Addition of 5- to 100-fold ratios of CuCl2 to NC (at 0.035 nmol NC/10(5) cells) produced progressively more growth inhibition. Addition of 1:0.5 ratios of NC to CuCl2 over the range of NC concentrations 0.08-0.2 nmol/10(5) cells/mL resulted in DNA single-strand breakage during 1-h treatments as measured by DNA alkaline elution. Concomitant addition of catalase or dimethyl sulfoxide (DMSO) inhibited DNA strand scission, while superoxide dismutase enhanced breakage. Catalase and DMSO also inhibited induction of membrane permeability by the copper complex of NC. These cellular effects apparently result from the intracellular generation of hydroxyl radical from H2O2. NC facilitated the uptake of copper into cells, though it was initially bound as a copper-histidine-like complex. The internalized copper was reduced to Cu(I), bound mostly as (NC)2Cu(I). To explain the (NC)2Cu-dependent generation of hydroxyl radical, it is hypothesized that glutathione successfully competes for Cu(I), converting it to a redox-active form that can catalyze the reduction of molecular oxygen to .OH. Model studies support this view. Radical scavengers did not reverse growth inhibition produced by NC or NC + CuCl2.     

DNA damage induced by catechol derivatives

We investigated the effect of catechol derivatives, including dopa, dopamine, adrenaline and noradrenaline, on DNA damage and the mechanisms of DNA strand breakage and formation of 8-hydroxyguanine (8HOG). The catechol derivatives caused strand breakage of plasmid DNA in the presence of ADP-Fe(3+). The DNA damage was prevented by catalase, mannitol and dimethylsulfoxide, suggesting hydroxyl radical (HO..)-like species are involved in the strand breakage of DNA. Iron chelators, such as desferrioxamine and bathophenanthroline, and reduced glutathione also inhibited the DNA damage. Deoxyribose, a molecule that is used to detect HO,, was not degraded by dopa in the presence of ADP-Fe(3+). By adding EDTA, however, dopa induced the marked deoxyribose degradation in the presence of ADP-Fe(3+), indicating that EDTA may extract iron from ADP-Fe(3+) to catalyze HO. formation by dopa. Thus, EDTA was a good catalyst for HO.-generation, whereas it did not promote the strand breakage of DNA. However, calf thymus DNA base damage, which was detected as 8-HOG formation, was caused by dopa in the presence of EDTA-Fe(3+), but not in the presence of ADP-Fe(3+). The 8HOG formation was also inhibited by catalase and HO. scavengers, indicating that HO&z.rad; was involved in the base damage. These results suggest that DNA strand breakage is due to ferryl species rather than HO., and that 8HOG formation is due to HO. rather than ferryl species.     

Potentiation of DNA damage by inhibition of poly(ADP-ribosyl)ation: a test of the hypothesis for random nuclease action.

Poly(ADP-ribosyl)ation is a cellular response to DNA strand breaks by which a large array of proteins becomes covalently modified for a brief period during the lifetime of the DNA breaks. Inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide after many types of DNA damage leads to a marked increase in DNA strand breakage, repair replication, cytogenetic damage, mutagenesis, and cell killing. It has been hypothesized that poly(ADP-ribose) polymerase may modify potentially degradative endogenous nucleases that can reduce cellular viability. Thus, in the presence of DNA strand breakage, the polymer would bind these enzymes to inhibit their activity. When synthesis of the polymerase is inhibited, the enzymes would act randomly to produce nonspecific damage in the DNA. We tested this hypothesis by electroporating restriction enzymes into human cells containing the shuttle vector pHAZE. Restriction enzymes cleave at specific recognition sequences in the lacZ target gene of pHAZE, and mutations result from rejoining errors at the cleavage sites. If the hypothesis were correct, enzyme-treated cells cultured with 3-aminobenzamide to inhibit synthesis of poly(ADP-ribose) polymers would result in a significant increase in mutations outside the restriction enzyme sites. The spectrum of mutations observed after electroporation of PvuII (which produces blunt-end double-strand breaks) or PvuI (which produces cohesive-end double-strand breaks) was similar in untreated and 3-aminobenzamide-treated cells. Thus, our results do not support the hypothesis that the increase in damage observed when poly(ADP-ribosyl)ation is inhibited is due to a chaotic, nonspecific attack on DNA by endogenous cellular nucleases.     

Asp-Ala-His-Lys (DAHK) inhibits copper-induced oxidative DNA double strand breaks and telomere shortening

Both DNA and the telomeric sequence are susceptible to copper-mediated reactive oxygen species (ROS) damage, particularly damage attributed to hydroxyl radicals. In this study, ROS-induced DNA double strand breaks and telomere shortening were produced by exposure to copper and ascorbic acid. Asp-Ala-His-Lys (DAHK), a specific copper chelating tetrapeptide d-analog of the N-terminus of human albumin, attenuated DNA strand breaks in a dose dependent manner. d-DAHK, at a ratio of 4:1 (d-DAHKCu), provided complete protection of isolated DNA from double strand breaks and, at a ratio of 2:1 (d-DAHKCu), completely protected DNA in Raji cells exposed to copper/ascorbate. Southern blots of DNA treated with copper/ascorbate showed severe depletion and shortening of telomeres and Raji cell treated samples showed some conservation of telomere sequences. d-DAHK provided complete telomere length protection at a ratio of 2:1 (d-DAHKCu). The human albumin N-terminus analog, d-DAHK, protects DNA and telomeres against copper-mediated ROS damage and may be a useful therapeutic adjunct in ROS disease processes.     

Simple cryoprotection and cell dissociation techniques for application of the comet assay to fresh and frozen rat tissues

The single-cell gel electrophoresis (comet) assay has been widely used for genotoxicity studies in cell cultures, but its use in solid tissues is hindered by problems in isolation of cells and in cryopreservation techniques. Here, we used minced liver tissues from rats to compare a homogenization technique for isolation of nuclei with a collagenase digestion method (300 λunits/g liver at 37°C for 20 λmin) for isolation of intact cells for subsequent comet assay. We found that collagenase digestion was preferred to the homogenization technique in fresh tissues, but neither method prevented the extensive DNA damage caused by cryopreservation ( -85°C for 72 λh). To minimize this damage, minced liver (1.0 λg) and kidney (0.5 λg) tissues were added to 20 λml of pre-cooled 10% glycerol or 10% dimethylsulfoxide (DMSO). We showed that cryoprotection with DMSO ( -85°C for 72 λh and 3 weeks), and to a slightly lesser extent with glycerol (72 λh), followed by collagenase digestion led to satisfactory recovery of liver cells with little or no DNA strand breakage. We then used DMSO as a cryoprotective agent to optimize the amount of collagenase and its incubation time in frozen liver and kidney tissues. We showed that the collagenase digestion at 150 λunits/g liver and 300 λunits/g kidney for 10 λmin produced highest cell numbers and minimal DNA strand breaks. We also validated these procedures by injection (i.p.) of rats with a known renal carcinogen, ferric nitrilotriacetate (Fe/NTA). We showed that Fe/NTA strongly induced DNA strand breaks in both rat liver and kidney, while no DNA strand breakage occurred in these tissues from the control rats. In addition, no significant differences in strand breaks were found between fresh tissues and tissues treated with DMSO during freezing at -85°C for 72 λh. Thus, the cryoprotection and the cell dissociation techniques developed here are satisfactory for preparing both fresh and frozen tissues for comet assay. These simple techniques are expected to expand greatly the usefulness and efficacy of the assay.