Correlation between induction of DNA fragmentation and micronuclei formation in kidney cells from rats and humans and tissue-specific carcinogenic activity

Five chemicals, known to induce kidney tumors in rats, were assayed for their ability to induce DNA damage and formation of micronuclei in primary cultures of rat and human kidney cells and in the kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, and of micronuclei frequency were obtained in primary kidney cells from both rats and humans with the following concentrations of the five test compounds: lead acetate (not tested for micronuclei induction) and potassium bromate from 0.56 to 1.8 mM, phenacetin from 1 to 3.2 mM, and 1, 4-dichlorobenzene and nitrilotriacetic acid from 1.8 to 5.6 mM. In terms of DNA-damaging potency all the five chemicals were more active in rat than in human kidney cells, whereas the potencies in inducing micronuclei formation were similar in the two species with the exception of 1,4-dichlorobenzene, which was slightly more potent in human than in rat cells. Consistently with the results observed in vitro, statistically significant increases in the average frequency of both DNA breaks and micronucleated cells were detected in the kidney of rats given po a single (12 LD50) or three successive daily doses (13 LD50) of the five test compounds. 4, 4'-Methylenedianiline, a carcinogen which does not induce kidney tumors in rats, gave negative responses in both in vitro and in vivo assays. These findings give evidence that kidney carcinogens may be identified by short-term genotoxicity assays using as target kidney cells and show that the five chemicals tested produce in primary cultures of kidney cells from human donors effects similar to those observed in rats.     

DNA fragmentation and DNA repair synthesis induced in rat and human thyroid cells by four rat thyroid carcinogens

Four chemicals that are known to induce in rats thyroid follicular-cell adenomas and carcinomas were assayed for their ability to induce DNA damage and DNA repair synthesis in primary cultures of human thyroid cells. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measures by the Comet assay, were obtained after a 20-h exposure to the following subtoxic concentrations of the four test compounds: 2,4-diaminoanisole (DAA) from 0.10 to 1.0 mM, 4,4'-methylene-bis(N,N-dimethyl)benzenamine (MDB) from 0.32 to 1.8 mM, propylthiouracil (PTU) from 1.8 to 5.6 mM, and 4,4'-thiodianiline (THA) from 0.032 to 0.18 mM. Under the same experimental conditions, DNA repair synthesis, as evaluated by quantitative autoradiography, was present in thyreocytes exposed to DAA but absent after treatment with MDB, PTU, and THA. Consistent with their thyroid-specific carcinogenic activity, all the four chemicals, administered p.o. in rats in a single dose corresponding to 1/2 LD50, induced a statistically significant degree of DNA fragmentation in the thyroid, whereas any substantial evidence of DNA lesions was absent in liver, kidney, and lung, which, with the exception of liver tumors caused by THA, are not targets of the carcinogenic activity of the four test compounds. These findings indicate that the DNA damage observed in thyroid cells was consistent with the carcinogenicity of the four test compounds, and suggest that DAA, MDB, PTU, and THA might be carcinogenic to thyroid in humans.     

Correlation between induction of DNA fragmentation in urinary bladder cells from rats and humans and tissue-specific carcinogenic activity

Seven chemicals, six of which are known to induce epithelial neoplasms of the urinary bladder in rats, were assayed for their ability to induce DNA damage in primary cultures of rat and human cells from urinary bladder mucosa, and in urinary bladder, liver and kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, were obtained in cells from both rats and humans with the following concentrations of five test compounds: 2-naphthylamine and N-nitrosodi-n-butylamine 0.5 and 1 mM, phenacetin 2 and 4 mM, cyclophosphamide from 2 to 8 mM, and o-toluidine 16 and 32 mM. Nitrilotriacetic acid (1-4 mM), a rat bladder carcinogen, and 4-aminobiphenyl (0.125-0.5 mM), a bladder carcinogen in humans but not in rats, gave a weak positive response in rats cells and a more marked response in humans cells. In terms of DNA-damaging potency, 4-aminobiphenyl, cyclophosphamide, phenacetin and 4 nitrilotriacetic acid were more active in human than in rat cells, whereas the converse occurred with 2-naphthylamine. Consistently with the results observed in vitro statistically significant dose-dependent increases in the average frequency of DNA breaks were detected in the urinary bladder mucosa of rats given p.o. single doses corresponding to 14 and 12 LD50 of six of the seven test compounds; the only one which gave a substantially negative response was 4-aminobiphenyl. With the exception of N-nitrosodi-n-butylamine which caused DNA damage in liver and of phenacetin and nitrilotriacetic acid which caused damage in kidney in agreement with their tumorigenic activity, any substantial evidence of DNA lesions in these two organs was absent in rats treated with 12 LD50 of the other 4 test compounds. These findings give evidence that urinary bladder genotoxic carcinogens may be identified by the DNA damage/Comet assay using as targets cells of urinary bladder mucosa, and show that the effect may be quantitatively different in cells from rats and from human donors.     

Genotoxicity of styrene-7,8-oxide and styrene in Fisher 344 rats: a 4-week inhalation study

The cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules.     

Salubrious effects of lipoic acid against adriamycin-induced clastogenesis and apoptosis in Wistar rat bone marrow cells

Adriamycin (ADR), an anthracycline antibiotic, which is widely used as an antineoplastic drug in the treatment of various solid tumors, has been shown to induce genotoxicity in erythropoietic system. The aim of the present study was to investigate the protective efficacy of DL-alpha-lipoic acid (LA) on ADR-induced clastogenicity and apoptosis in the bone marrow of rats. The animals were randomly divided into eight groups consisting of six rats each. Five groups were administered ADR (20 mg/kg body weight, i.v.) to induce genotoxicity; four of these groups received a single intraperitoneal injection of LA at a dose of either 100 or 200 mg/kg body weight, and either 30 or 60 min prior to ADR administration. A vehicle treated control group and LA control groups were also included. The beneficial effects of LA were monitored by DNA strand breaks, chromosomal aberrations, micronucleus assay and apoptotic studies in the bone marrow cells of rats after 24 h following single dose of ADR treatment. ADR treatment caused significant clastogenicity and apoptosis in rat bone marrow cells. The treatment with LA showed significant reduction in the frequency of chromosomal aberrations, DNA strand breaks and apoptosis in bone marrow cells as well as decreased the micronuclei formation in bone marrow and peripheral blood of rats treated with ADR. The protective effect of LA was found to be stronger at a dose of 200 mg/kg body weight than 100 mg/kg body weight dosage with respect to the above results, indicating the dose dependent effect of LA. However, the protection by LA was not dependent on the time intervals between LA and ADR administration. The results of this study illustrate the protective effect of LA on ADR-induced clastogenicity and apoptosis in the erythropoietic system of rats.     

DNA methylation analysis in rat kidney epithelial cells exposed to 3-MCPD and glycidol

3-Monochloropropane-1,2-diol (3-MCPD) is a well-known food processing contaminant that has been regarded as a rat carcinogen, which is known to induce Leydig-cell and mammary gland tumors in males, as well as kidney tumors in both genders. 3-MCPD is highly suspected to be a non-genotoxic carcinogen. 2,3-Epoxy-1-propanol (glycidol) can be formed via dehalogenation from 3-MCPD. We aimed to investigate the cytotoxic effects of 3-MCPD and glycidol, then to demonstrate the possible epigenetic mechanisms with global and gene-specific DNA methylation in rat kidney epithelial cells (NRK-52E). IC₅₀ value of 3-MCPD was determined as 48?mM and 41.39?mM, whereas IC₅₀ value of glycidol was 1.67?mM and 1.13?mM by MTT and NRU test, respectively. Decreased global DNA methylation at the concentrations of 100?μM and 1000?μM for 3-MCPD and 100?μM and 500?μM for glycidol were observed after 48?h exposure by using 5-methylcytosine (5-mC) ELISA kit. Methylation changes were detected in promoter regions of c-myc and Rassf1a in 3-MCPD and glycidol treated NRK-52E cells by using methylation-specific PCR (MSP), whereas changes on gene expression of c-myc and Rassf1a were observed by using real-time PCR. However, e-cadherin, p16, VHL and p15 genes were unmethylated in their CpG promoter regions in response to treatment with 3-MCPD and glycidol. Alterations in DNA methylation might be key events in the toxicity of 3-MCPD and glycidol.     

A comparative study of chemically induced DNA damage in isolated human and rat testicular cells

Testicular cells prepared from human organ transplant donors or from Wistar rats were used to compare 15 known reproductive toxicants with respect to their ability to induce DNA damage, measured as single-strand DNA breaks and alkali labile sites (ssDNA breaks) with alkaline filter elution. The compounds tested included various categories of chemicals (i.e., pesticides, industrial chemicals, cytostatics, and mycotoxins) most of which are directly acting genotoxicants (i.e., reacting with DNA either spontaneously or via metabolic activation). In addition, a few indirect genotoxic and nongenotoxic reproductive toxicants were included. Six of the chemicals induced no significant levels of ssDNA breaks in human and rat testicular cells: methoxychlor (10 to 100 μM, human and rat), benomyl (10 to 100 μM, human and rat), thiotepa (10 to 1000 μM, human and rat), cisplatin (30 to 1000 μM, human; 100 to 1000 μM, rat), Cd²⁺ (30 to 1000 μM, human; 100 to 1000 μM, rat), and acrylonitrile (30 to 1000 μM, human; 30 to 300 μM, rat). Four chemicals induced significant levels of ssDNA breaks in testicular cells from both species: styrene oxide (≥ 100 μM, rat and human), 1,2-dibromoethane (EDB) (≥ 100 μM, rat; 1000 μM human), thiram (≥ 30 μM, rat; ≥ 100 μM, human), and chlordecone (300 μM, rat; ≥ 300 μM, human). Finally, five chemicals induced ssDNA breaks in one of the two species. Four chemicals induced significant ssDNA breaks in rat testicular cells only: 1,2-dibromo-3-chloropropane (DBCP) (≥ 10 μM), 1,3-dinitrobenzene (1,3-DNB) (≥ 300 μM), Cr⁶⁺ (1000 μM), and aflatoxin B₁ (≥ 100 μM), the last two of these produced only a minor positive response. One chemical, acrylamide, induced a marginal increase in ssDNA breaks in human at 1000 μM, but not in rat testicular cells. Although based on a limited number of donors, the data indicate a close correlation between the induction of DNA damage in human and rat testicular cells in vitro. For some chemicals, however, there appears to be differences in the susceptibility to chemically induced ssDNA breaks of isolated testicular cells from the two species. The data indicate that the parallel use of human and rat testicular cells provides a valuable tool in the assessment of human testicular toxicity.     

DNA damage in tissues of rat treated with potassium canrenoate

Potassium canrenoate (PC), a competitive aldosterone antagonist previously found to increase tumor incidence in rats and to produce genotoxic effects in in vitro systems, was examined in rats to acquire information on its genotoxic activity in vivo. Intragastric administration of 1/2 LD50 produced, as revealed by the Comet assay, a modest but statistically significant increase in the frequency of DNA lesions in liver but not in thyroid and bone marrow of male rats, and in thyroid and bone marrow but not in liver of female rats. In contrast with the frankly positive responses observed in primary cultures of rat hepatocytes (Martelli et al., Mutagenesis 14 (1999) 463-472) any evidence of DNA repair and micronuclei formation was absent in liver of rats treated with 1/2 LD50, and initiation of enzyme-altered liver preneoplastic lesions did not occur in the liver of rats given 100 mg/kg PC once a week for 6 successive weeks. A high and dose-dependent frequency of DNA lesions was found to occur in testes and ovaries of rats given single doses ranging from 1/8 to 1/2 LD50.     

Evaluation of the genotoxic potential of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites, glycidol and beta-chlorolactic acid, using the single cell gel/comet assay.

3-monochloropropane-1,2-diol (3-MCPD) is a member of a group of chemicals known as chloropropanols. It is found in many foods and food ingredients as a result of food processing. 3-MCPD is regarded as a rat carcinogen known to induce Leydig-cell and mammary gland tumours in males and kidney tumours in both genders. The aim of our study was to clarify the possible involvement of genotoxic mechanisms in 3-MCPD induced carcinogenicity at the target organ level. For that purpose, we evaluated DNA damages in selected target (kidneys and testes) and non-target (blood leukocytes, liver and bone marrow) male rat organs by the in vivo alkaline single cell gel electrophoresis (comet) assay, 3 and 24 h after 3-MCPD oral administration to Sprague-Dawley and Fisher 344 adult rats. 3-MCPD may be metabolised to a genotoxic intermediate, glycidol, whereas the predominant urinary metabolite in rats following 3-MCPD administration is beta-chlorolactic acid. Therefore, we also studied the DNA damaging effects of 3-MCPD and its metabolites, glycidol and beta-chlorolactic acid, in the in vitro comet assay on CHO cells. Our results show the absence of genotoxic potential of 3-MCPD in vivo in the target as well as in the non-target organs. Glycidol, the epoxide metabolite, induced DNA damages in CHO cells. beta-Chlorolactic acid, the main metabolite of 3-MCPD in rats, was shown to be devoid of DNA-damaging effects in vitro in mammalian cells.     

Regucalcin down-regulation in rat kidney tissue after treatment with nephrotoxicants

Gene expression of regucalcin (Rgn), a calcium-binding protein, was investigated in kidney of male Wistar rats treated with proximal tubule segment-specific nephrotoxicants, namely hexachloro-1:3-butadiene (HCBD), specific for S(3) segment (pars recta) and potassium dichromate (chromate) specific for S(1)-S(2) segments (pars convoluta), according to age of animals and dose of chemicals. In the age-dependent study, male Wistar rats were treated with a single injection of HCBD (100mg/kg b.w. i.p.) or chromate (25 mg/kg b.w. s.c.) at 5 weeks or 12 weeks of age; in dose-response study, rats were treated with a single injection of three doses of HCBD (25, 50, and 100 mg/kg b.w. i.p.) or chromate (8, 12.5, and 25mg/kg b.w. s.c.) at 8 weeks of age. Forty-eight hours after treatment, Rgn and glutamine synthetase (GS) activity in kidney cortex, blood urea nitrogen (BUN) and plasma creatinine were measured; light microscopy was performed also. The results show that young rats are less susceptible to chromate (severe necrosis is evident only in adult rats), whereas age does not influence HCBD nephrotoxicity. Rgn is down regulated by HCBD at both age points, but not by chromate at 5 weeks of age. In addition, HCBD causes down-regulation of Rgn from the low dose in 8-week-old rats, whereas chromate causes the same effect at the high dose only. GS activity in kidney cortex shows a similar behavior, even if sensitive to low doses of chromate also, whereas BUN and creatinine increase after the high dose of both chemicals only. Accordingly, light microscopy shows a segment-specific, dose-dependent increase of severity of damage caused by the chemicals. Rgn gene expression appears a sensitive genomic marker to evaluate the renal impairment caused by chemicals and its down-regulation seems to be related to damage, early or already established, to S(3) segment of the proximal tubule.     

In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24?h after a single pharyngeal aspiration of MWCNT-S (1–200?μg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2–10.8?mg/m³) for 4 d, 4?h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1–200?μg/mouse) and in MNPCEs after inhalation exposure (17.5?mg/m³). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10?μg/cm²), while MWCNT-T increased strand breakage only at 200?μg/cm². Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.     

阿司匹林,对乙酰氨基酚和布洛芬对整体大鼠着床前胚泡的微核诱导作用

采用一种灵敏,准确,简便的致突变研究方法,收集ig阿司匹林(Asp)对乙酰氨基酚(Par)及布洛芬(Ibu)的妊娠d 4大鼠胚泡进行微核试验,结果表明,孕后d3给Asp为微核产生高峰期;Asp>0.5g·kg~(-1),Par>0.25g·kg~(-1)时呈显著的胚泡微核诱导作用,而Ibu0.5g·kg~(-1)则无此效应,Asp各组微核呈依剂量增加,三药对同体孕鼠骨髓均无显著微核诱导作用,结果提示Asp和Par具遗传毒性,并证实整体大鼠着床前胚泡对某些化学物质的染色体断裂作用较骨髓细胞更为敏感。     

Assessment of unscheduled and replicative DNA synthesis in rat kidney cells exposed in vitro or in vivo to unleaded gasoline

Unleaded gasoline (UG) induces renal toxicity and neoplasia in male but not female rats after chronic inhalation exposure. Before a meaningful determination of the potential human health risk of UG can be made, it is imperative that the mechanism responsible for its carcinogenic action be understood. The purpose of the present investigation was to determine whether the induction of kidney tumors by UG is related to genotoxic or to cell-proliferative effects. Unscheduled DNA synthesis (UDS), as an indicator of genotoxicity, was measured autoradiographically as the incorporation of [3H]thymidine in isolated rat kidney cells following in vivo or in vitro exposure to UG. As an indicator of proliferative activity, cells in S-phase were quantitated in isolated cell preparations obtained from exposed rats. UG was administered to rats by inhalation (2000 ppm) or by gavage (up to 5000 mg/kg). The ability of the in vivo/in vitro kidney cell UDS assay to detect genotoxicants was verified using a variety of compounds. No UDS activity was elicited by UG under any of the conditions employed, including inhalation exposure to a concentration that produced kidney tumors in the 2-year bioassay. A five- to eightfold increase in the percentage of cells in S-phase was observed in male rats exposed to UG for 18 days either by inhalation or by gavage. Cell turnover was not markedly enhanced in identically treated female rats. These data indicate that UG does not evoke UDS in the rat kidney even after exposures that, in all probability, resulted in greater tissue concentrations of UG components than was realized in the long-term inhalation bioassay. The sex-specific induction of replicative DNA synthesis in the kidney paralleled the carcinogenic activity of UG, suggesting that induced cell turnover may be an important factor in the carcinogenic action of this motor fuel.     

Evaluation of the genotoxic potential of sorbic acid and potassium sorbate.

The genotoxic potential of sorbic acid and potassium sorbate was investigated in vivo and in vitro. Oral administration of sorbic acid (up to 5000 mg/kg body weight) did not induce sister chromatid exchanges or the formation of micronuclei in bone marrow cells of mice. Intraperitoneal treatment of rats with 400-1200 mg potassium sorbate/kg body weight did not alter the elution profile of DNA from isolated liver cells in the in vivo alkaline elution assay. Sorbic acid did not induce DNA repair in cultured human A549 cells in the unscheduled DNA synthesis (UDS) assay. In vitro incubation of the cells with 1-1000 micrograms potassium sorbate/ml, in the absence or presence of rat liver homogenate, did not result in the formation of DNA single-strand breaks in the alkaline elution assay. These results demonstrate that sorbic acid and its potassium salt are not genotoxic in vivo or in vitro. In contrast to sorbic acid and potassium sorbate, sodium sorbate is very sensitive to oxidative degradation; the main oxidation product was identified to be 4,5-oxohexenoate, which was mutagenic in the Ames test.     

Investigation of micronuclei and other nuclear abnormalities in peripheral blood and kidney of marine fish treated with crude oil

The induction of micronuclei and other nuclear abnormalities (nuclear buds, bi-nucleated and fragmented-apoptotic cells) was analyzed in the erythrocytes of peripheral blood and cephalic kidney of turbot (Scophthalmus maximus) and Atlantic cod (Gadus morua), treated with crude oil (Statfjord B, Norway) and with nonylphenol. Significant increase in MN was observed in turbot kidney and blood after exposure to 30 ppb of nonylphenol, 0.5 ppm of oil, and after co-exposure to 0.5 ppm of oil spiked with additional mixture of alkylphenols and PAHs (P varied between 0.0054 and <0.0001). The induction of micronuclei was observed only in cod kidney after exposure to spiked oil (P=0.0317). Significant inter-specific differences after the exposure to 0.5 ppm of oil (P=0.0385) and after treatment with spiked oil (P=0.0067) were observed. In turbot cephalic kidney, the elevated levels of bi-nucleated cells were observed in all treatment groups (P values varied in a range from 0.05 to 0.0025) while the increase in cells with nuclear buds was noted after the exposure to 0.5 ppm of oil (P=0.05). The fragmented-apoptotic cells appeared after the exposure to nonylphenol (P=0.0039) and to spiked oil (P<0.0001). In turbot blood, only the significant induction in nuclear buds was detected. Statistically significant inter-tissue differences were found only in the induction of fragmented-apoptotic cells after the exposure to nonylphenol and to spiked oil.     

Genotoxicity of ochratoxin A in mice: DNA single-strand break evaluation in spleen, liver and kidney

Ochratoxin A, a natural contaminant of feed and food, has been shown to induce experimental liver and kidney tumours. In vitro experiments on mice spleen cells showed evidence of DNA single-strand breaks induced by ochratoxin A. We measured single-strand breaks in the DNA of spleen, liver and kidney of ochratoxin A-treated animals. Ochratoxin A induced DNA damage in vivo. This damage reversed with time. The appearance and extent of the damage varied in different tissues. Except for spleen our data correlate with the tumours induced by ochratoxin in mouse liver and kidney. With regard to the spleen, there has been no report to date of experimental leukemia induced by ochratoxin A. Thus our results indicate that this possibility has to be considered.     

DNA strand breaks induced by hydrogen peroxide in isolated rat hepatocytes

It has been proposed that increased rates of hepatic hydrogen peroxide (H2O2) production may initiate or promote the liver tumors that appear following chronic exposure of rodents to chemicals that cause peroxisome proliferation. However, the effect of H2O2 on the structural integrity of DNA in parenchymal hepatocytes, the target cells of peroxisome proliferator-induced carcinogenesis, is largely uncharacterized. Furthermore, oxidant-induced cellular damage has been invoked as causal of a number of hepatotoxic effects associated with exposure to chemicals other than peroxisome proliferators. For these reasons, alkaline elution analysis was used to study the action of H2O2, added exogenously, on DNA of intact, isolated rat hepatocytes. Addition of a bolus of H2O2 (0.01-1.0 mM) to monolayer cultures of hepatocytes caused concentration-dependent increases in single-strand DNA breaks (SSDB), which were maximal within 30 min of exposure. Cytotoxicity, as measured by lactate dehydrogenase (LDH) release, was minimal during a 1-h exposure to H2O2 concentrations less than 1 mM, but the efflux of oxidized glutathione was increased. Formation of SSDB was nearly linear with respect to H2O2 concentration in the range 0.1-1.0 mM. No double-strand DNA breaks or DNA-protein crosslinks were identified at H2O2 concentrations of 1 mM or less. Repair of SSDB in H2O2-free medium occurred in a rapid, linear manner only for the first 15 min, resulting in disappearance of 65% of the SSDB. A second, slower phase of SSDB rejoining occurred between 20 and 60 min of incubation in H2O2-free media; at 60 min rejoining was maximal (80% repair). These results define a specific type of DNA damage associated with H2O2 exposure of hepatocytes and suggest that primary cultures of rat hepatocytes are a suitable model for characterizing the potential genotoxic effects of oxidants, particularly excess H2O2 that may occur in the livers of animals exposed chronically to peroxisome proliferators.     

The activation of DNA damage detection and repair responses in cleavage-stage rat embryos by a damaged paternal genome

Male germ cell DNA damage, after exposure to radiation, exogenous chemicals, or chemotherapeutic agents, is a major cause of male infertility. DNA-damaged spermatozoa can fertilize oocytes; this is of concern because there is limited information on the capacity of early embryos to repair a damaged male genome or on the fate of these embryos if repair is inadequate. We hypothesized that the early activation of DNA damage response in the early embryo is a critical determinant of its fate. The objective of this study was to assess the DNA damage response and mitochondrial function as a measure of the energy supply for DNA repair and general health in cleavage-stage embryos sired by males chronically exposed to an anticancer alkylating agent, cyclophosphamide. Male rats were treated with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females. Pronuclear two- and eight-cell embryos were collected for immunofluorescence analysis of mitochondrial function and biomarkers of the DNA damage response: γH2AX foci, 53BP1 reactivity, and poly(ADP-ribose) polymer formation. Mitochondrial activities did not differ between embryos sired by control- and cyclophosphamide-exposed males. At the two-cell stage, there was no treatment-related increase in DNA double-strand breaks; by the eight-cell stage, a significant increase was noted, as indicated by increased medium and large γH2AX foci. This was accompanied by a dampened DNA repair response, detected as a decrease in the nuclear intensity of poly(ADP-ribose) polymers. The micronuclei formed in cyclophosphamide-sired embryos contained large γH2AX foci and enhanced poly(ADP-ribose) polymer and 53BP1 reactivity compared with their nuclear counterparts. Thus, paternal cyclophosphamide exposure activated a DNA damage response in cleavage-stage embryos. Furthermore, this damage response may be useful in assessing embryo quality and developmental competence.     

Induction of strand breaks in DNA by trichloroethylene and metabolites in rat and mouse liver in vivo

The ability of trichloroethylene (TCE) and selected metabolites to induce single-strand breaks in hepatic DNA of male B6C3F1 mice and Sprague-Dawley rats in vivo was evaluated using an alkaline unwinding assay. Doses of TCE of 22-30 mmol/kg were required to produce strand breaks in DNA in rats, whereas a dose of 11.4 mmol/kg was sufficient to increase the rate of alkaline unwinding in mice. To assess the importance of TCE metabolism to this response, rats were subjected to pretreatments of ethanol, phenobarbital, TCE, or the appropriate vehicle for 4 days prior to challenge doses of TCE. Phenobarbital and TCE, but not ethanol pretreatments, reduced the dose of TCE required to produce significant increases in single-strand breaks. In another series of experiments, mice and rats were treated with metabolites of TCE. Trichloroacetate, dichloroacetate, and chloral hydrate induced strand breaks in hepatic DNA in a dose-dependent manner in both species. Strand breaks in DNA were observed at doses that produced no observable hepatotoxic effects as measured by serum aspartate aminotransferase and alanine aminotransferase levels. The slopes of the dose-response curves and the order of potency of these metabolites differed significantly between rats and mice, suggesting that different mechanisms of single-strand break induction may be involved in the two species. These data provide a potential explanation for the different sensitivity of mice and rats to the hepatocarcinogenic effects of TCE.     

Characterization of the Na+, K+-ATPase activity of basolateral plasma membranes of kidney proximal tubular cells from young and old rats

Several characteristics of the Na+, K+-ATPase activity of basolateral plasma membranes of kidney proximal tubular cells from young (3 months) and old (24 months) rats were studied. In both cases, the ATPase activity reached optimum values under the following conditions: Mg2+:ATP concentrations (mM) 5:5 (apparent Km 0.5 mM); Na+ concentration 50 mM (apparent Km 18 mM); K+ concentration 20 mM (apparent Km 2.5 mM); pH 7.2; temperature 52 degrees. The values of the apparent energy of activation of the system were similar for young and old rats in the temperature range 20-52 degrees but were 55% higher for the old rats in the temperature range 10-20 degrees.