Correlation between induction of DNA fragmentation in lung cells from rats and humans and carcinogenic activity

Six chemicals, known to induce lung tumors in rats, were examined for their ability to induce DNA fragmentation in primary cultures of rat and human lung cells, and in the lung of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the single-cell gel electrophoresis (Comet) assay, were obtained in primary lung cells from male rats with the following, minimally toxic, concentrations of the six test compounds: N-nitrosodimethylamine (NDMA; 2.5-10mM), hydrazine (HZ; 0.5-4mM), cadmium sulfate (CD; 31.2 and 62.5muM), 4,4'-methylene bis (2-chloroaniline) (MOCA; 31.2-125muM), isobutyl nitrite (IBN; 7.8-31.2muM) and tetranitromethane (TNM; 1.9-15.6muM). Similar degrees of DNA fragmentation were obtained in primary human lung cells; however, due to inter-donor differences, the minimum effective concentrations were in some donors lower and in others higher than in rats, and IBN induced DNA damage only in one of three donors. The DNA-damaging potency of HZ was higher in rats than in humans, and the opposite was true for MOCA. In agreement with these findings, statistically significant increases in the average frequency of DNA breaks were obtained in the lung of rats given a single oral dose (1/2 LD50) of the six test compounds. These findings give evidence that genotoxic lung carcinogens may be identified by use of the DNA fragmentation/Comet assay on rat lung cells as targets cells, and show that the six compounds tested produce in primary cultures of lung cells from human donors DNA-damaging effects substantially similar to those observed in rats.     

Effects of thyroid antagonists on rat embryos cultured in vitro

A literature review of individual pregnancies and recent surveys involving large cohorts reveal an association between congenital malformation and maternal hyperthyroidism, suggesting that some aspect of hyperthyroidism or its treatment might compromise the development of the fetus. Experiments have shown that the thyroid antagonist, ethylenethiourea (ETU), causes fetal malformations when administered to pregnant rats, but it is not known whether it is ETU or the imbalance in maternal thyroid hormone which it causes which is the teratogenic agent. Here we employ in vitro culture to determine the possible direct effects on rat embryos of two thyroid antagonists, ETU and methimazole (MMI), the latter being one which is used for treatment of thyrotoxicosis in humans. It was found that ETU can compromise the development of rat embryos in vitro, confirming that ETU has a direct effect on the rat embryo. It was also found that MMI can cause abnormal development of rat embryos in vitro, although the concentration at which MMI disturbs rat embryogenesis is higher than that which is reached in hyperthyroid patients treated with clinical doses of MMI or carbimazole.     

Induction of repair synthesis of DNA in primary cultures of rat urothelial cells by derivatives of the bladder carcinogen, N-n-butyl-N-(4-hydroxybutyl)nitrosamine

Urinary metabolites of N-n-butyl-N-(4-hydroxybutyl)nitrosamine (BHBN) which have been identified thus far did not induce repair synthesis of DNA in cultured rat urothelial cells. Urine of rats which had been administered with BHBN or BCPN did not induce repair synthesis of DNA either. However, all the synthetic nitrosamines that can produce 1-hydroxyalkylnitrosamine intermediates induced repair synthesis of DNA. The results suggest that rat urothelial cells, at best, may only have very limited capability of activating nitrosamines.     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes.

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells. UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle. Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

Repair replication of opossum lymphocyte DNA: effect of compounds that bind to DNA

Opossum lymphocytes were used for studies of DNA repair. Several compounds were assessed for their capacity to induce repair. Specially interesting was the fact that some intercalators (proflavin, ICR-170, quinacrine and acridine orange) did induce repair, as determined by [³H]thymidine incorporation in the presence of hydroxyurea, CsCl density gradient centrifugation of bromodeoxyuridine-containing DNA and autoradiographically detected unscheduled DNA synthesis.A comparison of the inhibitory effect of several chemicals on DNA replication and DNA repair was also carried out. In this study, repair synthesis was induced by UV irradiation. For most of the compounds, the concentration necessary to inhibit 50% of DNA replication or DNA repair was similar. The most notable exception was cycloheximide which inhibited replication much more effectively than repair. None of the compounds used in this study was found to specifically inhibit repair synthesis.Inhibition of DNA replication and DNA repair was a general effect exhibited by the compounds which bind to DNA. However, only some of these compounds were able to induce repair. As most of these compounds were mutagens it was concluded that the inhibitory effect could be more relevant to mutagenesis that the repair-induction effect.     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells.UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle.Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine.The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

Effects of polyamines on DNA synthesis using various subcellular DNA polymerases extracted from normal rat liver, tumour-bearing rat liver, and tumour cells

The effects of polyamines on DNA synthesis in vitro using various subcellular DNA polymerase fractions from normal and tumour-bearing rat livers, and tumour cells were investigated. When nuclear and mitochondrial DNA polymerase fractions were used, DNA synthesis on activated DNA was increased 3.5-8-fold by the addition of 20 mM putrescine or cadaverine. However, DNA synthesis was not stimulated by the addition of spermidine or spermine at any concentration tested. In contrast, DNA synthesis using the cytoplasmic DNA polymerase fraction was not stimulated at various concentrations of any of the four polyamines tested. The stimulatory effects of putrescine and cadaverine were absent when nuclear fractions from tumour-bearing rat liver or from tumour cells were used. In addition, in vitro DNA synthesis was not stimulated by 20 mM putrescine or cadaverine when nuclear extracts from the livers of rats administered putrescine subcutaneously were used. The specific activities of DNA polymerases extracted from tumour cells and tumour-bearing rat liver were already fully stimulated. These results suggest that DNA polymerases in tumour cells and tumour-bearing liver cells are stimulated by trapped putrescine produced in tumour cells and are thus no longer activated by exogenous putrescine.     

Comparison of the continuous rat hepatoma cell line 2sFou with primary rat hepatocyte cultures for the induction of DNA repair synthesis by nitrosamines, benzo[a]pyrene and hydroxyurea

We have examined the suitability of the continuous rat hepatoma cell line 2sFou for testing the genotoxicity of chemicals in comparison with that of primary rat hepatocyte cultures (HPC). The capacity of the cells for metabolic activation was assessed by measuring induction of DNA-repair synthesis and inhibition of replicative DNA synthesis by the test compounds dimethylnitrosamine (DMN), diethylnitrosamine (DEN), hydroxyurea (HU) and benzo[a]pyrene (BaP), which are substrates for major hepatic and extrahepatic forms of cytochrome P-450 dependent monooxygenases. The cellular capacity for DNA-repair synthesis was assessed using UV-light as a DNA-damaging agent. Repair-specific incorporation of [3H]deoxycytidine (3H-dCyd) caused by UV-light was higher in 2sFou cells than in HPC. In contrast, background repair incorporation of 3H-dCyd in 2sFou cells was only 1/3 that found in HPC. All the test agents induced DNA repair and inhibited DNA synthesis in both 2sFou cells and HPC. The two nitrosamines were more effective in HPC than in 2sFou cells. HU and BaP affected DNA repair and DNA synthesis in the two cell systems at a similar range of concentrations. In general, DNA repair in the 2sFou cells increased near linearly with the concentrations of the test compounds. The data indicate that 2sFou cells are capable of activating hepatotropic pro-mutagens/carcinogens such as dialkylnitrosamines, and are sensitive indicators of DNA damage. In contrast, BaP, a non-hepatotoxic compound, caused only little DNA repair in these cells. Thus, continuously growing cells, such as 2sFou, show a qualitatively similar response to genotoxic chemicals as HPC and offer a potential alternative to HPC for genotoxicity testing.     

Species,sex and inter-individual differences in DNA repair induced by nine sex steroids in primary cultures of rat and human hepatocytes

Sex steroids, due to the generally negative responses observed in routinely employed standard genotoxicity assays, are considered epigenetic carcinogens. Some doubts on this conviction are raised by the results of recent studies providing evidence that cyproterone acetate and two structural analogues, chlormadinone acetate and megestrol acetate, are genotoxic in female rats but only for the liver, and in primary human hepatocytes from donors of both genders. The experimental evidence suggests that the metabolic activation of these molecules to reactive species and the consequent formation of DNA adducts occur only in the intact hepatocyte. Since the possibility that other sex steroids cause a liver-specific genotoxic effect cannot be ruled out a priori, we investigated nine drugs of this family for their ability to induce DNA repair synthesis in primary cultures of rat and human hepatocytes. Each steroid was tested in cultures from at least two male and two female donors of each species. Hepatocytes were exposed for 20h to sub-toxic concentrations ranging from 1 to 50 micro M, and DNA repair induction was measured by quantitative autoradiography. In primary rat hepatocytes, induction of DNA repair indicative of a frankly positive response was detected in cultures from: 2/2 males and 3/3 females with drospirenone, 2/2 males and 1/2 females with ethinylestradiol, 1/2 males and 1/2 females with oxymetholone, 1/2 males with norethisterone, 1/4 females with progesterone, and 1/4 males with methyltestosterone. Consistent negative responses were obtained with testosterone and stanozolol. A few inconclusive responses were observed in rat hepatocytes exposed to progesterone, medroxyprogesterone, norethisterone, methyltestosterone and oxymetholone. In contrast, under the same experimental conditions the nine sex steroids provided frankly negative responses in the large majority of cultures of primary hepatocytes from both male and female human donors; the only exceptions being the inconclusive responses obtained in cultures from two of the donors exposed to norethisterone and to ethinylestradiol, and from one of the donors exposed to testosterone, methyltestosterone, and stanozolol. These results and previous findings concerning cyproterone and its structural analogues suggest that sex steroids differ for their ability to induce DNA repair, and that their genotoxicity may be: (i) different in rat and human hepatocytes, (ii) dependent on the sex of the donor, and (iii) affected by inter-individual variability.     

The effect of aging and dietary restriction on DNA repair

DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.     

Activity of germ-cell mutagens and nonmutagens in the rat spermatocyte UDS assay

The ability of 13 chemicals of known germ-cell mutagenicity to induce unscheduled DNA synthesis (UDS) in rat spermatocytes was examined. At selected times following i.p. injection of test compounds, spermatocytes were isolated from Fischer 344 rats by enzymatic digestion of the seminiferous tubules and cultured for 24 h in the presence of [3H]thymidine. 7 compounds, methyl methanesulfonate, triethylenemelamine, cyclophosphamide, methylnitrosourea, ethylnitrosourea, procarbazine, and dibromochloropropane produced positive UDS responses in spermatocytes. These chemicals are also positive for specific locus mutations, heritable translocations, or dominant lethal mutations when administered to male rodents. Mitomycin C, which produces DNA interstrand crosslinks and induces heritable mutations and translocations in male germ cells, failed to stimulate UDS in rat spermatocytes. Germ-cell nonmutagens N-methyl-N'-nitro-N-nitrosoguanidine, dimethylnitrosamine, 4-nitroquinoline 1-oxide, and ethylene dibromide were negative in the rat spermatocyte UDS assay. Correlation of these results with those of other assays for heritable mutations in germ cells indicates that the in vivo/in vitro spermatocyte DNA repair assay is useful in predicting the mutagenic potential of chemicals in male germ cells.     

ADP effects on bleomycin-induced DNA repair synthesis and adenylate kinase activity in permeable mouse sarcoma cells

DNA repair in bleomycin-pretreated, permeable mouse sarcoma (SR-C3H/He) cells requires ATP for at least two steps, the repair DNA synthesis step and the repair patch ligation step. ADP can apparently replace ATP in both steps. Maximal, 1.5-2 fold stimulation of repair DNA synthesis was observed with 5-10 mM ADP as well as 2.5-5 mM ATP. Repair patch ligation in the presence of 2.5 mM ADP occurred at almost the same high efficiency as it did in the presence of ATP. The ADP effect on DNA repair patch ligation was attributed to ATP formed from ADP by adenylate kinase in permeable cells, however the ADP effect on repair DNA synthesis could not be attributed solely to the formation of ATP in the same manner.     

DNA fragmentation, DNA repair and apoptosis induced in primary rat hepatocytes by dienogest, dydrogesterone and 1,4,6-androstatriene-17beta-ol-3-one acetate

Four steroids that share the 17-hydroxy-3-oxopregna-4,6-diene structure - cyproterone acetate, chlormadinone acetate, megestrol acetate, and potassium canrenoate - have been shown previously to behave with different potency as liver-specific genotoxic agents, the response being markedly higher in female than in male rats, but similar in humans of both genders. In this study, performed to better define the relationship between chemical structure and genotoxicity, dydrogesterone (DGT) with double bonds C4=C5 and C6=C7, dienogest (DNG) with double bonds C4=C5 and C9=C10, and 1,4,6-androstatriene-17beta-ol-3-one acetate (ADT) with double bonds C1=C2, C4=C5 and C6=C7, were compared with cyproterone acetate (CPA) for their ability to induce DNA fragmentation and DNA repair synthesis in primary cultures of hepatocytes from three rats of each sex. At subtoxic concentrations, ranging from 10 to 90 microM, all four steroids consistently induced a dose-dependent increase of DNA fragmentation, which in all cases was higher in females than in males; their DNA damaging potency decreased in the order CPA > DNG > ADT > DGT. Under the same experimental conditions, the responses provided by the DNA repair-synthesis assay were positive or inconclusive in hepatocytes from female rats and consistently negative in hepatocytes from male rats. In the induction of apoptotic cells, examined in primary hepatocytes from female rats, CPA was more active than ADT and DGT, and DNG was inactive. Considered as a whole these findings suggest that a liver-specific genotoxic effect more marked in female than in male rats might be a common property of steroids with two or three double bonds.     

DNA repair synthesis induced by N-hydroxyurea, acetohydroxamic acid, and N-hydroxyurethane in primary rat hepatocyte cultures: comparative evaluation using the autoradiographic and the bromodeoxyuridine density-shift method

N-Hydroxyurea and two structurally related compounds, acetohydroxamic acid and N-hydroxyurethane, were investigated for their potential to induce DNA repair synthesis in primary rat hepatocyte cultures. Repair was determined as repair replication by means of the bromodeoxyuridine density-shift method and, in the same cell preparations, as unscheduled DNA synthesis (UDS) by autoradiography. For all 3 compounds, a clear concentration-dependent induction of DNA repair replication could be demonstrated. Interpretation of the UDS data, however, depended on the mode whereby the results were evaluated. Expression of the results as net grains per nucleus after subtraction of cytoplasmic from nuclear grain counts yielded statistically significant increases over the control values for all compounds. In contrast, no significant changes of the nuclear labeling were obtained when nuclear and cytoplasmic grain counts were plotted separately. These findings demonstrate that the two modes to present UDS data may lead to different conclusions, a consequence of the uncertainty regarding the origin and importance of the cytoplasmic background. The observation that both hydroxyurea and the structurally related compounds acetohydroxamic acid and N-hydroxyurethane induce DNA repair in primary hepatocyte cultures suggests that metabolism-dependent genotoxicity may be a common property of aliphatic hydroxamic acids.     

Comparative genotoxicity studies of the flame retardant tris(2,3-dibromopropyl)phosphate and possible metabolites

Tris(2,3-dibromopropyl)phosphate (Tris-BP) was activated to mutagens in the Salmonella/microsome quantitative test system. Liver microsomes from rats pretreated with phenobarbital (PB) increased the mutagenicity of 0.05 mM Tris-BP to 186% of the activity obtained with liver microsomes from untreated rats. The addition of 0.02 mM Tris-BP to V79 Chinese hamster cells co-incubated with liver microsomes from PB-pretreated rats increased the number of mutants by a factor of 9.7. Tris-BP also caused genotoxic and cytotoxic responses in primary monolayers of rat hepatocytes. The relative increase in unscheduled DNA synthesis after treatment with 0.05 mM Tris-BP was 2.3-fold as measured by scintillation counting of radiolabelled thymidine incorporated into DNA of isolated nuclei. The use of hepatocytes isolated from PB-pretreated rats reduced the increases in DNA repair synthesis relatively to that in control cells. Monolayers of hepatocytes from untreated rats co-cultured with Salmonella typhimurium TA100 activated Tris-BP to mutagenic intermediates which were released into the culture medium. The studies with the V79 and liver-cell systems indicate that the reactive intermediates formed from Tris-BP are sufficiently stable and lipophilic to traverse the various membranes from the site of generation to the respective cellular targets. The relative degree of genotoxic responses of bis(2,3-dibromopropyl)phosphate, 2,3-dibromopropylphosphate, tris(2,3-bromopropyl)phosphate, tris(2-bromopropyl)phosphate and 2,3-dibromopropanol in the systems studied did not indicate that these compounds were proximate or ultimate reactive metabolites of Tris-BP in liver-derived activation systems.     

Bacterial mutagenesis and hepatocyte unscheduled DNA synthesis induced by chrysoidine azo-dye components

5 azo dye components of Gurr chrysoidine 'Y' have been separated, synthesised and identified. Dyes with a methyl substitution (particularly between the two amino groups) were more mutagenic in Salmonella typhimurium strain TA100 with control rat liver S9 than the non-methylated counterpart (range 66-1992 revertants at 50 micrograms/plate). Mutagenicity was also catalysed by human-liver S9 and pre-treatment of rats with either phenobarbitone or beta-naphthoflavone enhanced the activation ability of S9 by greater than 4-fold. Using the most potent promutagenic component (2,4-diamino-3-methylazobenzene), the use of inhibitors of cytochrome P450 (metyrapone: 1.0 mM; alpha-naphthoflavone: 0.075 mM; DPEA: 0.125 mM) and of the flavin monooxygenase (methimazole: 0.75 mM) suggested a major role for cytochrome P448 in the activation of chrysoidine to mutagens. The ability of chrysoidine components to induce unscheduled DNA synthesis in rat hepatocytes in vitro was demonstrated and ranged between 11.92 and 23.5 net nuclear grains at a dose level of 2.5 micrograms/incubation. Since each dye was equi-potent, methyl substitution had little influence on genetic toxicity in hepatocytes.     

An assay system for factors involved in mammalian DNA replication

An assay for cellular factors stimulating DNA synthesis by partially lysed CHO cells is presented. The assay is based on the observation that in highly lysed cells, DNA synthesis, as determined by [3H]dTTP incorporation, was only 2-5% of that in gently lysed cells, and that this low level of DNA synthesis could be increased by a factor of approx. 50 by the addition of CHO cell extract (i.e. supernatant of a cell homogenate subjected to high-speed centrifugation). Highly lysed cells were obtained by treatment with 0.1% Brij-58 and 240 mM KCl, while for the preparation of gently lysed cells, 0.01% Brij-58 and 80 mM KCl were used. Incorporation of [3H]dTTP reflected DNA synthesis qualitatively similar to that in intact cells. It was semiconservative, and no repair synthesis was detected unless cells were irradiated with ultraviolet light prior to parital lysis. DNA molecules of 4 S were synthesized and converted to DNA of more than 25 S via 6-12-S intermediates. DNA synthesis was restricted to nuclei from cells in S phase, and cell extract did not induce DNA synthesis in nuclei from cells in G1 phase. Stimulation of DNA synthesis by cell extract was concentration-dependent. Cell extract activity was recovered to more than 50% after (NH4)2SO4 precipitation. Heat-inactivation experiments suggested that cell extract contained at least tow factors timulating DNA replication. This system may, therefore, be used for the purification and characterization of factors participating in DNA replication of mammalian cells.     

Effects of nicotinamide and structural analogs on DNA synthesis and cellular replication of rat hepatoma cells

The effects of nicotinamide and structural analogs on DNA synthesis were studied in rat hepatoma (HTC) cells. Inhibitory effects of these compounds were observed on DNA synthesis as judged by the incorporation of [3H]-thymidine into DNA. Evidence for a marked effect on DNA integrity after preincubation with 1 mM methyl methanesulfonate was provided by a fluorometric technique with ethidium bromide. There was only a small or insignificant enhancement of this effect when hepatoma cells were incubated with nicotinamide. At concentrations of 2-20 mM, 3-aminobenzamide was observed to cause greater effects than nicotinamide on DNA synthesis and integrity and on cellular proliferation in HTC cells. Comparison of the effects of nicotinamide and 3-aminobenzamide with those of N'-methylnicotinamide suggested that some of the effects on DNA synthesis may not be mediated through inhibition of poly(ADP-ribose) synthetase. Inhibition of HTC cell proliferation was observed at a concentration of 3-aminobenzamide, 2 mM, which has been reported to be nontoxic for other cell types.     

Nicotinamide stimulates repair of DNA damage in human lymphocytes

Nicotinamide stimulates the amount of DNA repair synthesis that occurs when freshly isolated, normal human lymphocytes are treated with UV irradiation, N-methyl-N′-nitro-N-nitroso guanidine, or dimethyl sulfate. Stimulation of DNA repair synthesis is concentration dependent and reaches a maximum between 2 to 5 mM nicotinamide. In contrast, DNA synthesis in cells that have not been subjected to DNA damage is not affected by nicotinamide at concentrations below 2 mM and is inhibited by concentrations between 2 to 5 mM. In the same concentration range, nicotinic acid has no effect on the rate of DNA synthesis in the presence or absence of DNA damage.     

Adenylate cyclase activity of v-ras-k transformed rat epithelial thyroid cells

The regulation of adenylate cyclase has been analyzed in normal rat thyroid cells as well as in the same cells transformed by the v-ras-k oncogene. In both cell types the adenylate cyclase complex consists of the two GTP-binding proteins, Gi and Gs, as demonstrated by the specific ADP-ribosylation induced by pertussis and cholera toxin, respectively. The response of adenylate cyclase of the transformed cells to forskolin, pertussis toxin and cholera toxin is attenuated with respect to the control cell line. The thyrotropic hormone (TSH), that acts on normal thyroid cells in culture as a growth factor by stimulating the adenylate cyclase activity, is not able to induce DNA synthesis nor does it stimulate adenylate cyclase in v-ras-k transformed cells.