DNA lesion in rat hepatocytes induced by in vitro and in vivo exposure to glyoxal

The alkaline elution technique was applied to measure the damage of rat hepatic DNA following exposure to glyoxal. DNA single-strand breaks were induced after exposure of primary-cultured hepatocytes to 0.1-0.6 mg/ml glyoxal for 60 min, while no DNA cross-link was observed. Single-strand breaks were also detected in livers of rats within 2 h following a single oral exposure at 200-1000 mg/kg body weight, and the frequency of the breaks reached a maximum around 9 h after exposure. The breaks were almost fully repaired 24 h after exposure to any dose. However, hardly any DNA lesions were detected in other tissues following exposure to 1000 mg/kg glyoxal. Thus, the present results indicate that glyoxal causes DNA single-strand breaks in rat hepatocytes following in vitro and in vivo exposure.     

DNA damage induced by nitropyrenes in primary mouse hepatocytes and in rat H4-II-E hepatoma cells

The capacity of nitropyrenes to cause DNA damage in primary mouse hepatocytes (C57BL/6N mice) and rat H4-II-E hepatoma cells was studied by estimating single-strand breaks using the alkaline elution technique. 1-Nitropyrene (10-200 microM) caused clear dose-dependent increases in DNA strand breaks in both cell types, whereas no increase in DNA strand breaks was observed in hepatocytes treated with 1.3-, 1,6-, 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene under standard assay conditions (5-20 microM 30-min incubation). However, 1,8-dinitropyrene (1,8-DNP) caused dose-dependent increases in DNA strand breaks when incubated with the H4-II-E cells for 48 h, while no single-strand breaks were observed following treatment with 1,6-dinitropyrene (1,6-DNP) under the same conditions. Neither 1,6-DNP nor 1,8-DNAP induced DNA crosslinks in the H4-II-E cells. These data indicate that substrate specificity exists in the metabolic activation of nitropyrenes in murine liver.     

Daunorubicin-induced DNA lesions in isolated rat hepatocytes and mammary epithelial cells

Daunorubicin, an anticancer drug, induces primarily mammary adenocarcinoma in Sprague-Dawley rats. We investigated daunorubicin-induced DNA lesions in enzymatically isolated mammary epithelial cells and hepatocytes from 7-8-week-old female Sprague-Dawley rats. Differences were observed in the type and quantity of DNA lesions in mammary epithelial cells and hepatocytes as determined by alkaline elution analysis. DNA single-strand breaks and proteinase-K-sensitive cross-linking lesions were observed in mammary epithelial cells. Hepatocytes appeared to have significantly lower relative frequencies of single-strand breaks than mammary epithelial cells when treated with daunorubicin (1.5-10.0 micrograms/10(6) cells). Hepatocytes displayed two types of cross-link. One form was sensitive to proteinase-K digestion, whereas the other form was insensitive. The metabolism of daunorubicin to the aglycone metabolites was substantially lower in mammary cells than in hepatocytes. However, the total uptake of the drug was similar in these two cell types. A metabolite, 7-deoxydaunorubicinol aglycone, was unable to induce single-strand breaks or cross-linking lesions in mammary epithelial cells. Both cell types exhibited a similar ability to repair radiation-induced single-strand breaks of DNA. However, the mammary cells may be less able to repair daunorubicin-mediated DNA damage. These results revealed that mammary epithelial cells are less able to metabolize the active mutagen/carcinogen, daunorubicin, than are hepatocytes. This, coupled with the observations of greater apparent DNA damage in mammary cells, may be of primary importance in the drug-induced carcinogenicity in the rat mammary tissue.     

The genotoxicity of lucidin,a natural component of Rubia tinctorum L., and lucidinethylether,a component of ethanolic Rubia extracts

The genotoxic activity of lucidin (1,3-dihydroxy-2-hydroxymethyl-9,10-anthraquinone), a natural component of Rubia tinctorum L., was tested in a battery of short-term tests. The compound was mutagenic in five Salmonella typhimurium strains without metabolic activation, but the mutagenicity was increased after addition of rat liver S9 mix. In V79 cells, lucidin was mutagenic at the hypoxanthine-guanine phosphoribosyl transferase gene locus and active at inducing DNA single-strand breaks and DNA protein cross-links as assayed by the alkaline elution method. Lucidin also induced DNA repair synthesis in primary rat hepatocytes and transformed C3HI M2-mouse fibroblasts in culture. We also investigated lucidinethylether, which is formed from lucidin by extraction of madder roots with boiling ethanol. This compound was also mutagenic in Salmonella, but only after addition of rat liver S9 mix. Lucidinethylether was weakly mutagenic to V79 cells which were cocultivated with rat hepatocytes. The compound did not induce DNA repair synthesis in hepatocytes from untreated rats, but positive results were obtained when hepatocytes from rats pretreated with phenobarbital were used. We conclude that lucidin and its derivatives are genotoxic.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HA hydroxyanthraquinones - LUE lucidinethylether - PRH primary rat hepatocytes - UDS unscheduled DNA synthesis     

Induction of single-strand breaks and interstrand cross-links in liver DNA after the administration of 2-acetylaminofluorene and trans-4-acetylaminostilbene to rats

2-Acetylaminofluorene (AAF) or trans-4-acetylaminostilbene (AAS) was orally or intraperitoneally administered to female Wistar rats. DNA from liver cells was analyzed for single-strand breaks by the alkaline elution assay. Only borderline effects were observed with doses (100 μMol/kg) used in animal carcinogenesis experiments. Even high doses of AAF (1,000 μMol/kg) were not effective. Methyl methanesulfonate (MMS) in vivo and gamma irradiation in vitro were shown to produce dose-dependent DNA single-strand breaks (positive control). Only a marginal effect was obtained with 100 μMollkg MMS. The elution rate of DNA was increased by a factor of 34 in liver cells in vitro with 400 rad of gamma irradiation. Only a fraction of this rate could be demonstrated immediately after irradiation in vivo, and no lesions were found two hours later. This strongly indicates the rapid repair of single-strand breaks. Additional experiments showed that AAS, a nonhepatocarcinogen, produced more interstrand cross-links in the rat liver DNA than did AAF.     

Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential

We have recently developed an alkaline elution/rat hepatocyte assay to sensitively measure DNA single-strand breaks induced by xenobiotics in non-radiolabeled rat hepatocytes. Here we have evaluated this assay as a predictor of carcinogenic/mutagenic activity by testing 91 compounds (64 carcinogens and 27 non-carcinogens) from more than 25 diverse chemical classes. Hepatocytes were isolated from uninduced rats by collagenase perfusion, exposed to chemicals for 3 h, harvested, and analyzed for DNA single-strand breaks by alkaline elution. DNA determinations were done fluorimetrically. Cytotoxicity was estimated by glutamate-oxaloacetate transaminase release or by trypan blue dye exclusion. The assay correctly predicted the reported carcinogenic/non-carcinogenic potential of 92% of the carcinogens tested and 85% of non-carcinogens tested. The assay detected a number of compounds, including inorganics, certain pesticides, and steroids, which give false-negative results in other short-term tests. Only 2 rat liver carcinogens were incorrectly identified; the other carcinogens incorrectly identified are weakly or questionably carcinogenic (i.e., they cause tumors only in one species, after lifetime exposure, or at high doses). Some chemicals cause DNA damage only at cytotoxic concentrations; of 16 such compounds in this study, 12 are weak carcinogens suggesting a link between DNA damage caused by cytotoxicity and carcinogenesis. Our data indicate that this assay rapidly, reproducibly, sensitively, and accurately detects DNA single-strand breaks in rat hepatocytes and that the production of these breaks correlates well with carcinogenic and mutagenic activity.     

The bioactivation of 1,2-dibromoethane in rat hepatocytes: deuterium isotope effect

The metabolism and genotoxicity of 1,2-dibromoethane (EDB) and its deuterium substituted analog ( d4EDB ) were studied in isolated rat hepatocytes. There was a marked isotope effect on the metabolism of EDB by hepatocytes. This was due to decreased microsomal oxidation of d4EDB . Cytosolic metabolism of EDB, as measured by bromide ion release, was unaffected by deuterium substitution. The genotoxicity of the two analogs was assessed by assaying for the presence of EDB induced single-strand breaks in DNA. As measured by the alkaline elution technique, both compounds caused DNA single-strand breaks when incubated at a concentration of 0.1 mM with hepatocytes. No difference in the degree of DNA damage could be demonstrated between hepatocytes incubated with EDB or d4EDB . These data suggest that the GSH transferase mediated metabolism of EDB is responsible for the genotoxic effects of EDB observed in hepatocytes.     

Accumulation of Copper Induces DNA Strand Breaks in Brain Cells of Long-Evans Cinnamon (LEC) Rats, An Animal Model for Human Wilson Disease.

Copper accumulation and induction of DNA strand breaks were investigated in the brain of Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease that is a heritable disease of copper accumulation and copper toxicity in the liver, kidney and brain. Copper contents in the brain of LEC rats increased from 20 weeks of age and were approximately 3.5 to 6 folds higher than those in the brain of WKAH rats at 24 weeks of age. Hepatic copper contents in LEC rats increased from 4 to 12 weeks of age in an age-dependent manner, and then decreased from 16 to 20 weeks of age. Thus, we consider that copper accumulated in the liver was released from severely damaged hepatocytes and deposited in the brain, although copper contents in the brain were 1/20-fold lower than those in the liver. We also evaluated the amounts of DNA single-strand breaks (SSBs) in the brain by comet analysis. The proportions of nuclei in the cerebrum and cerebellum without DNA damage decreased, and nuclei with severe DNA damage appeared in LEC rats at 24 weeks of age. The comet scores of cerebrum and cerebellum cells significantly increased in LEC rats and were significantly higher than those in WKAH rats at 24 weeks of age. The results show that SSBs in LEC rat brain cells are induced at a lower concentration of copper than are SSBs in hepatic cells.     

Strain differences in in vitro rat hepatocyte unscheduled DNA synthesis (UDS): effect of UV is independent of strain while increased sensitivity is apparent using Fischer-344 instead of Sprague-Dawley rats

The unscheduled DNA synthesis (UDS) assay measures DNA repair following in vitro treatment of rat primary hepatocytes. This report compares the UDS response of primary hepatocytes from 2 widely used rat strains, the Fischer-344 (F344) and Sprague-Dawley (SD) strains. Ultraviolet (UV) light and 5 known genotoxic chemicals were evaluated in each strain in parallel experiments. The chemicals tested were 2-acetylaminofluorene (2-AAF), 4-aminobiphenyl (4-AB), benzidine, dimethylnitrosamine (DMN) and N-propyl-N'-nitro-N-nitrosoguanidine (PNNG). Four of these compounds (2-AAF, 4-AB, benzidine and DMN) require metabolic activation. Benzidine and PNNG were both negative using SD rat hepatocytes, but were weakly positive using F344 rat hepatocytes. In the first of 2 experiments, 4-AB was inconclusive in SD hepatocytes, but strongly positive in F344 cells. In the second experiment, 4-AB was positive in hepatocytes from both strains. 2-AAF was more strongly positive in F344 cells than in SD cells. DMN and UV light induced positive dose responses with little or no differences between strains. It is concluded that hepatocytes from F344 rats may be more sensitive, qualitatively and quantitatively, than hepatocytes from SD rats as indicators of UDS. This difference is not due to intrinsic differences in DNA repair mechanisms but is probably due to differences in drug-metabolizing enzymes between these strains. Thus, for routine screening, F344 rats are preferable for measurement of the in vitro UDS-inducing potential of compounds.     

Acute low-intensity microwave exposure increases DNA single-strand breaks in rat brain cells

Levels of DNA single-strand break were assayed in brain cells from rats acutely exposed to low-intensity 2450 MHz microwaves using an alkaline microgel electrophoresis method. Immediately after 2 h of exposure to pulsed (2 μs width, 500 pulses/s) microwaves, no significant effect was observed, whereas a dose rate-dependent [0.6 and 1.2 W/kg whole body specific absorption rate (SAR)] increase in DNA single-strand breaks was found in brain cells of rats at 4 h postexposure. Furthermore, in rats exposed for 2 h to continuous-wave 2450 MHz microwaves (SAR 1.2 W/kg), increases in brain cell DNA single-strand breaks were observed immediately as well as at 4 h postexposure. © 1995 Wiley-Liss, Inc.     

Interaction between dietary selenium and 2-acetylaminofluorene in the rat

The effect of dietary selenium on the metabolism of 2-acetylaminofluorene (AAF) and on its interaction with hepatic DNA was studied in male, Charles River rats. All studies were commenced at least 3 weeks after placing weanling rats on a tomla yeastbased Se-deficient diet or the same diet supplemented with 0.5 ppm Se as Na₂SeO₃. Analysis of radioactive metabolites generated during in vitro incubation of [9-¹⁴C]-AAF with hepatic microsomes showed that Se-supplemented rats produced greater amounts of noncarcinogenic, phenolic metabolites than did Se-deficient animals. No significant difference was noted between the two dietary groups with respect to the production of the proximate carcinogenic metabolite,N-hydroxy-AAF. Analysis of urinary metabolites excreted during a 24-h period following a single ip injection of [9-¹⁴C]-AAF showed that Se-deficient animals produced 2–3 times as much N-hydroxy-AAF as did the supplemented rats. The increased excretion of the proximate carcinogenic metabolite by Se-deficient rats occurred both as the free and glucuronic acid conjugated forms. In contrast, Se-deficient rats excreted lower amounts of noncarcinogenic AAF metabolites. Taken together, these results suggest that dietary Se alters AAF biotransformation so as to decrease metabolic activation while enhancing detoxification pathways. The effect of dietary Se on AAF-DNA interactions was assessed in two ways. First, it was found that Se had no effect on the total amount of AAF residues covalently bound to hepatic DNA in vivo. This lack of effect was observed both at early (1-24 h) and late (4-7 d) intervals after administering a single ip injection of [9-¹⁴C]-AAF to rats from both dietary groups. In contrast, alkaline sucrose gradient analysis revealed a marked protective.effect of Se against AAF-induced DNA single-strand breaks. Further studies showed that the protective effect of Se was not mediated by a more rapid rate of repair of DNA damage. Accordingly, in addition to its favorable actions on carcinogen metabolism, the ability of Se to protect DNA against reactive metabolites may play a role in its reported anticarcinogenic activity.     

Direct In Vivo Cell Lineage Analysis in the Retrorsine and 2AAF Models of Liver Injury after Genetic Labeling in Adult and Newborn Rats

Backgrounds and Aims

When hepatocyte proliferation is impaired, liver regeneration proceeds from the division of non parenchymal hepatocyte progenitors. Oval cells and Small Hepatocyte-like Progenitor Cells (SHPCs) represent the two most studied examples of such epithelial cells with putative stem cell capacity. In the present study we wished to compare the origin of SHPCs proliferating after retrorsine administration to the one of oval cells observed after 2-Acetyl-Amino fluorene (2-AAF) treatment.

Methodology/Principal Findings

We used retroviral-mediated nlslacZ genetic labeling of dividing cells to study the fate of cells in the liver. Labeling was performed either in adult rats before treatment or in newborn animals. Labeled cells were identified and characterised by immunohistochemistry. In adult-labeled animals, labeling was restricted to mature hepatocytes. Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly. When labeling was performed in newborn rats, results after retrorsine administration were identical to those obtained in adult-labeled rats. In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes. Furthermore, we also observed labeled biliary tracts in 2-AAF treated rats.

Conclusions

Our results srongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin. We also show that hepatic progenitors are labeled in newborn rats suggesting future directions for in vivo lineage studies.     

Cytoskeleton modifications induced by phenobarbital, 2-acetylaminofluorene and 4-acetylaminofluorene in normal and initiated/selected hepatocytes: Relation with the “resistant” phenotype

Initiatedlselected (ISH) and normal (NH) rat hepatocytes were used to study cytoskeleton modifications induced by three liver acting chemicals: 2-AAF, a liver complete carcinogen; PB, a liver tumor promoter; and 4-AAF, a noncarcinogen analogue of 2-AAF. Cytoskeleton alterations were visualized by disappearance of F-actin fibers and tubulin depolymerization. The three drugs induced actin fragmentation in normal hepatocytes; a net loss of actin protein was observed with PB. They also induced varied tubulin depolymerization. The principal difference between chemicals is that 2-AAF led to non-reversible effects, in comparison with PB and 4-AAF which induced reversible damages on cytoskeleton. By contrast to normal hepatocytes, the cytoskeleton of ISH obtained from rats subjected to the resistant hepatocyte protocol was much less susceptible to the effect of the three chemicals. Moreover, we observed a lack of LDH release in the culture medium and a very rapid inducibility of GST activity after exposure of ISH to drugs. The moderate effect of the three chemicals on actin and tubdin in ISH could thus be explained by the resistant metabolic profile of these cells.Abbreviations TPA 12-O-tetradecanoyl-phorbol-13-acetate - PB phenobarbital - 2-AAF 2-acetylaminofluorene - 4-AAF 4-acetylaminofluorene - GSH reduced glutathione - GST glutathione-S-transferase - LDH lactatedehydrogenase - NH normal hepatocytes - ISH initiated/selected hepatocytes - BSA bovine serum albumin     

Changes in DNA strand breaks in non parenchymal cells following hepatocyte regeneration in CCl4-induced rat liver injury

DNA strand breaks (nicks) in non-parenchymal cells (NPCs) in CCl₄-induced acute or chronic liver injury in rats were detected using an in situ nick translation method; their dynamic changes were analysed in relation to the proliferation pattern of hepatocytes and NPCs, as revealed by bromodeoxyuridine (BrdU)-up-take. In acute injury, hepatocyte proliferation started before centrilobular necrosis had occurred, whereas BrdU-labeled sinusoidal NPCs markedly increased only after centrilobular necrosis was apparent. DNA breakages in NPCs paralleled the proliferation pattern of these cells, suggesting that nicks are physiological, and reflect proliferation and activated gene expression. In chronic injury, liver cirrhosis developed after 9 weeks, but BrdU-labeling of hepatocytes was almost the same level as that in untreated liver. The number of BrdU-labeled NPCs showed only a slight increase, while those with DNA breakages were much more frequent in the cirrhotic stage, suggesting a significant role for NPCs in the fibrotic process. These results indicate that DNA strand breaks in NPCs act as a marker for activation states such as proliferation, differentiation and/or activated gene expression.     

Changes in DNA strand breaks in non-parenchymal cells following hepatocyte regeneration in CCl4-induced rat liver injury.

DNA strand breaks (nicks) in non-parenchymal cells (NPCs) in CCl4-induced acute or chronic liver injury in rats were detected using an in situ nick translation method; their dynamic changes were analysed in relation to the proliferation pattern of hepatocytes and NPCs, as revealed by bromodeoxyuridine (BrdU)-uptake. In acute injury, hepatocyte proliferation started before centrilobular necrosis had occurred, whereas BrdU-labeled sinusoidal NPCs markedly increased only after centrilobular necrosis was apparent. DNA breakages in NPCs paralleled the proliferation pattern of these cells, suggesting that nicks are physiological, and reflect proliferation and activated gene expression. In chronic injury, liver cirrhosis developed after 9 weeks, but BrdU-labeling of hepatocytes was almost the same level as that in untreated liver. The number of BrdU-labeled NPCs showed only a slight increase, while those with DNA breakages were much more frequent in the cirrhotic stage, suggesting a significant role for NPCs in the fibrotic process. These results indicate that DNA strand breaks in NPCs act as a marker for activation states such as proliferation, differentiation and/or activated gene expression.     

Mechanisms of butylated hydroxytoluene-mediated modulation of 2-acetylaminofluorene mutagenicity in rat and human hepatocyte/Salmonella assays

Salmonella typhimurium (TA98) mutagenesis assays were used to study the influence of the antioxidant butylated hydroxytoluene (BHT) on 2-acetylaminofluorene (2-AAF) mutagenesis, in search of the mechanism of the anticarcinogenic effects of BHT. Rats pre-treated with BHT in the diet (0.5% w/w for 10 days) provided hepatocytes and hepatocyte S9 which were more efficient in the activation of 2-AAF than were similar preparations from control rats. The increased release of mutagens from hepatocytes might explain the reported increase in the incidence of bladder tumours in BHT-treated rats. In contrast, the mutagenic activity of 2-AAF was inhibited by the in vitro addition of BHT into incubations where human or rat liver S9 and intact hepatocytes were used for metabolic activation. Both competitive and un-competitive inhibition by BHT of 7-ethoxycoumarin O-deethylation was observed in hepatocytes which suggested that the antimutagenic activity may be mediated by one or more mechanisms of cytochrome P-450 inhibition. BHT inhibition of the mutagenicity of N-OH 2-AAF and of rat urinary metabolites of 2-AAF indicated that effects other than those mediated by cytochrome P-450 also occur e.g. scavenging of reactive metabolites. It was concluded that BHT-modulation of 2-AAF metabolic activation and mutagenesis (which may relate to BHT-protection against hepatocarcinogenicity) involves multiple mechanisms.     

Inhibitory effects of trientine, a copper-chelating agent, on induction of DNA strand breaks in hepatic cells of Long-Evans Cinnamon rats

Effects of treatment with trientine, a specific copper-chelating agent, on accumulation of copper and induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. Copper accumulated in the livers of LEC rats in an age-dependent manner from 4 to 13 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, hepatic copper contents did not increase and were maintained at the same levels as those in 10-week-old LEC rats. When the amounts of DNA single-strand breaks (SSBs) were estimated by a comet assay, SSBs of DNA were induced in a substantial population of LEC rat hepatic cells around 8 weeks of age and the amounts of SSBs increased in an age-dependent manner from 8 to 15 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased dramatically, suggesting that induction of SSBs of DNA was inhibited and/or SSBs were repaired during the period of treatment with trientine. The results show that treatment of LEC rats with trientine decreases the number of DNA strand breaks observed, although copper contents remain high in the liver.     

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.     

Induction and repair of double- and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli

Induction and repair of double- and single-strand DNA breaks have been measured after decays of 125I and 3H incorporated into the DNA and after external irradiation with 4 MeV electrons. For the decay experiments, cells of wild type Escherichia coli K-12 were superinfected with bacteriophage lambda DNA labelled with 5'-(125I)iodo-2'-deoxyuridine or with (methyl-3H)thymidine and frozen in liquid nitrogen. Aliquots were thawed at intervals and lysed at neutral pH, and the phage DNA was assayed for double- and single-strand breakage by neutral sucrose gradient centrifugation. The gradients used allowed measurements of both kinds of breaks in the same gradient. Decays of 125I induced 0.39 single-strand breaks per double-strand break. No repair of either break type could be detected. Each 3H disintegration caused 0.20 single-strand breaks and very few double-strand breaks. The single-strand breaks were rapidly rejoined after the cells were thawed. For irradiation with 4 MeV electrons, cells of wild type E. coli K-12 were superinfected with phage lambda and suspended in growth medium. Irradiation induced 42 single-strand breaks per double-strand break. The rates of break induction were 6.75 x 10(-14) (double-strand breaks) and 2.82 x 10(-12) (single-strand breaks) per rad and per dalton. The single-strand breaks were rapidly repaired upon incubation whereas the double-strand breaks seemed to remain unrepaired. It is concluded that double-strand breaks in superinfecting bacteriophage lambda DNA are repaired to a very small extent, if at all.     

Assessment of genotoxic effect of benzo[a]pyrene in endotracheally treated rat using the comet assay

Although benzo[a]pyrene (B[a]P) is a well-known genotoxic agent, little is known about the extent of DNA effects induced by B[a]P in rat tissues after pulmonary exposure. The alkaline single-cell gel electrophoresis (comet assay) was used to measure DNA single-strand breaks in alveolar macrophages, lung cells, peripheral lymphocytes and hepatocytes of OFA Sprague-Dawley rats exposed to a single dose of B[a]P by endotracheal administration.Statistically significant damage was observed in all organs tested after 3, 24 and 48h of pulmonary exposure to 3mg of B[a]P per animal, with a time-dependent relationship. The maximum damage was observed in the four cell types 24h after exposure. The higher level of damage was observed both in lung cells and peripheral lymphocytes; in alveolar macrophages and hepatocytes the level of damage was increased, but at a lower level than in the two other cell types. Furthermore, B[a]P demonstrated a clear dose-related genotoxic activity in the lung cells when tested at doses of 0.75, 1.5 and 3mg.The current study shows that B[a]P caused DNA single-strand breaks in the respiratory tract of endotracheally treated OFA Sprague-Dawley rats. The study also suggests that pulmonary exposure to B[a]P can induce a high level of DNA damage in peripheral lymphocytes. The clear relationship between lung exposure to B[a]P and consequences observed in lymphocytes suggests that the comet assay in peripheral lymphocytes can be used as a sensitive marker in human monitoring studies.