Rhône-Poulenc Rorer, Nonclinical Safety Assessment, 13 Quai Jules Guesde, BP 14, 94403 Vitry sur Seine Cedex and
1 Institut Curie-Biologie, 91405 Orsay Cedex, France
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Abstract |
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Abbreviations: DBC, 7H-dibenzo[c,g]carbazole; DMDBC, 5,9-dimethyldibenzo[c,g]carbazole; DMN, dimethylnitrosamine; FBS, fetal bovine serum; MF, mutant frequency; NER, nucleotide excision repair; NNG, net nuclear grain; TM, tail moment; UDS, unscheduled DNA synthesis; WME, William's medium E.
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Introduction |
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As it is well known that the initial amount of primary DNA damage, as well as the rates and efficiencies of DNA repair mechanisms, also influence the mutagenic potential of a chemical, these parameters were examined to gain an insight into the mechanisms underlying the non-linear mutagenicity of DMDBC in vivo (8,9). The induction and removal of DNA adducts were monitored in mouse hepatocytes over 4144 h after a single skin application of 10 or 90 mg/kg DMDBC. DNA adducts were measured using 32P-post-labeling analysis and DNA repair in individual cells was assessed by means of: (i) the unscheduled DNA synthesis (UDS) assay, which measures nucleotide excision repair (NER) of bulky DNA adducts through incorporation of [3H]thymidine during DNA resynthesis (10); (ii) the Comet or the single cell gel electrophoresis assay, which detects a wide range of DNA damage, including DNA strand breaks transiently produced between the initial incision and the terminal rejoining steps of the excision repair process (11,12).
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Materials and methods |
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Experiments were conducted with 7- to 9-week-old male CD2F1 mice (Charles River, Saint-Aubin-Lès-Elbeuf, France).The animals were housed in plastic cages under standard conditions (20 ± 2°C, 50 ± 10% relative humidity, 12 h light/dark cycle) and were allowed to acclimatize for 7 days prior to treatment.
Experimental design
An area of ~10 cm2 was shaved on the animals' backs with an electric clipper 2 days before treatment. DMDBC was synthesized as previously described (14), dissolved in acetone immediately before administration and applied in a volume of 200 µl. Treated animals received a single skin application of DMDBC at 10 or 90 mg/kg, i.e. 0.84 or 7.62 µmol/mouse. The negative control group received the vehicle alone. As a positive control for the UDS and Comet assays, animals received a single oral administration of 2.5 or 10 mg/kg dimethylnitrosamine (DMN) in water in a volume of 10 ml/kg.
Hepatocyte isolation
At various times after treatment (4144 h), animals were anesthetized with 150 mg/kg pentobarbital in aqueous solution (Imalgène 1000; Rhône-Mérieux, Lyon, France). Hepatocytes were prepared by using a two-step in situ collagenase perfusion method. The liver was initially perfused with HEPES buffer (10 mM HEPES, 136 mM NaCl, 2.7 mM KCl, 0.4 mM Na2HPO4·12H2O, pH 7.65) at 40°C at a flow rate of 10 ml/min for 5 min, and then with 0.025% w/v collagenase A (Boehringer Mannheim, Mannheim, Germany) plus 0.075% w/v CaCl2 (Sigma, St Louis, MO) for 5 min. Hepatocytes were collected from the perfused liver, filtered through a 60 µm sterile mesh and suspended in William's medium E (WME) (Gibco BRL, Cergy Pontoise, France) supplemented with 10% fetal bovine serum (FBS) (Gibco). The percentage of viable cells, measured using the Trypan blue exclusion test, always exceeded 75% (data not shown). Isolated hepatocytes were either stored at 80°C until DNA isolation for 32P-post-labeling assay or immediately used for the UDS or Comet assay. Each experiment included vehicle-, DMN- and DMDBC-treated mice.
32P-post-labeling analysis
DNA from frozen hepatocytes was isolated by enzymatic digestion of protein and RNA and solvent extraction as described by Gupta (15). Using the nuclease P1 enhancement procedure of the 32P-post-labeling assay (16), DMDBCDNA adducts were labeled, separated and quantified as described by Périn-Roussel et al. (17). Data were expressed as total DMDBC-related DNA adducts/108 nucleotides.
UDS assay
Approximately 3x105 viable hepatocytes were seeded onto 18 mm diameter round coverslips placed in each well of 6-well culture plates in WME medium supplemented with 50 U/ml penicillin (Gibco BRL), 50 µg/ml streptomycin (Gibco BRL), 2 mM L-glutamine (Gibco BRL), 1 U/ml insulin (Novo Nordisk Pharmaceutics, Boulogne, France), 106 M hydrocortisone (Roussel, Paris, France) and 10% FBS. Non-attached cells were removed after 2 h. Attached hepatocytes were then incubated in serum-free medium containing 0.37 MBq/ml [3H]thymidine (sp. act. 185 MBq/ml; Amersham, Créteil, France) in a humidified atmosphere at 37°C, 5% CO2. After 4 h incubation the hepatocytes were rinsed and incubated for 1518 h in serum-free WME medium containing 0.25 mM unlabeled thymidine (Sigma). After the cold chase period the cultures were washed with phosphate-buffered saline at 37°C (Gibco), swollen in 1% sodium citrate solution and fixed in ethanol:acetic acid (3:1 v:v). The dried coverslips were mounted on microscope slides, dipped in Kodak NTB2 emulsion (Integrabioscience, Cergy le Haut, France) diluted 1:1 with deionized water and exposed for 10 days at 4°C. After development, the slides were stained with Harris hematoxylin solution (Merck, Darmstadt, Germany). One hundred randomly selected nuclei were analyzed on duplicate slides. Silver grains were quantified using image analysis systems (Grain v.1.6; York Electronic Research, Huntington, UK and Domino, Perceptives Instruments, Haverhill, UK) coupled to a Leitz microscope (x100 magnification, oil immersion) and a video camera. All the slides were coded and analyzed by the same operator. The net nuclear grain (NNG) value, defined as the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm adjacent to the nucleus, was recorded. Data were expressed as means ± SD obtained from 100 hepatocytes. The percentage of cells undergoing repair, i.e. cells with an NNG exceeding the threshold value of 5, was also determined (18). Hepatocytes undergoing replicative DNA synthesis can be easily distinguished from hepatocytes undergoing DNA repair by the heavily labeled nuclei. The percentage of hepatocytes in S phase was determined by scoring 3000 randomly selected hepatocytes per animal.
Comet assay
As described by Singh et al. (19), freshly isolated hepatocytes were resuspended at 5x104 cells/ml in 0.5% low melting point agarose (Sigma). Seventy-five microliters of the suspension was embedded in an agarose sandwich on fully frosted microscope slides. The slides were immersed in freshly prepared cold lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris base, 1% sodium sarcosinate, pH 10, supplemented with 1% Triton X-100 and 10% dimethylsulfoxide) for at least 1 h at 4°C, then transferred to a horizontal electrophoresis tank filled with electrophoresis buffer (1 mM Na2EDTA, 300 mM NaOH, pH 13.5) for 20 min at 4°C in the dark to allow DNA to unwind before electrophoresis for 15 min at 25 V and 300 mA. After electrophoresis the slides were washed in neutralization buffer (0.4 M Tris, pH 7.5) to remove excess alkali and detergent and then stained with 50 µl of aqueous propidium iodide (20 µl/ml). One-hundred and fifty randomly selected cells were analyzed on duplicate slides using an image analysis system (Komet v.3.0; Kinetic Imaging Ltd, Liverpool, UK) coupled to a Zeiss fluorescence microscope (x250 magnification) and a CDD camera. All the slides were processed simultaneously, coded and analyzed by the same operator. The tail moment (TM), defined as the product of the relative amount of DNA in the comet tail and the length of the comet measured from the trailing edge of the cell, was recorded. Data were expressed as the median of the 150 TM values obtained per animal. Additional parameters, such as the percentage of tail DNA and tail length were also recorded, and were in keeping with the TM values (data not shown).
Statistical analysis
Student's t-test was used to analyze the UDS response and the percentage of S phase hepatocytes. The MannWhitney test was used to analyze the Comet assay data.
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Results |
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A continuous increase in the amount of DNA adducts was observed from 4 to 24 h after skin application of 10 mg/kg (Table I). At 24 h post-treatment, a steady-state level of 423 adducts/108 nucleotides was reached and persisted over the entire observation period. In the liver of mice treated with 90 mg/kg, the number of DNA adducts increased sharply from 4 to 24 h, reaching a maximum of 8814 adducts/108 nucleotides at 24 h. The number of DNA adducts then fell strongly at 48 and 72 h. After a post-application period of 144 h, 481 adducts/108 nucleotides, representing 5% of the peak value, still persisted.
UDS assay
The NNG of hepatocytes from control animals ranged from 4.7 to 3.6 and the proportion of cells undergoing DNA repair ranged from 0.10 to 0.15% during the study. These results are consistent with previous data (20).
DMN, used as a positive control, significantly increased UDS activity. Two hours after oral administration of 2.5 or 10 mg/kg, NNG counts reached +29.6 and +76.3, respectively (P < 0.001). The proportion of hepatocytes undergoing excision repair was 77.8 and 95.7%, respectively. These results are in good agreement with published data (18).
Following skin application of 10 mg/kg, a clear UDS response was observed at the 15 h sampling time (+6.2 NNG, P < 0.001) (Table II). The induction of UDS was maximal 24 h after treatment. The NNG count reached +7.7 (P < 0.001) and 60.8% of hepatocytes were undergoing repair. Seventy-two hours after exposure the NNG count declined to +0.7, a value no longer statistically different from the control. However, 17.0% of hepatocytes were still undergoing repair. Following exposure to 90 mg/kg DMDBC, UDS peaked as early as 15 h; the NGG count reached +24.5 (P < 0.001) and 64% of cells were undergoing repair. NNG counts then fell steadily to +15.9 and +6.6 at 24 and 72 h (P < 0.001), respectively, returning to a value no longer statistically different from the control at 144 h (0.1 NNG). However, 22.3% of hepatocytes were still undergoing repair at 144 h.
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Comet assay
The TM in the Comet assay of control hepatocytes ranged from 0.5 to 0.9 between 4 and 144 h (Table III). When observed at 250x magnification, the nuclei of untreated hepatocytes showed compact fluorescence, no DNA fragments were visible in the tail and baseline DNA damage remained at a low level over time.
As expected, DMN induced a significant increase in TM. Two hours after oral administration of 2.5 mg/kg the TM was increased 14-fold (P < 0.001), while at 10 mg/kg the DNA was so fragmented that the nucleus was reduced to a single spot, most of the DNA having migrated to the tail, which hindered measurement. Therefore, 2.5 mg/kg DMN was used as a positive control in the rest of the study.
No changes in TM were observed at any sampling time after skin application of 10 mg/kg DMDBC. Following exposure to 90 mg/kg DMDBC, the level of DNA strand breaks remained similar to that observed in controls during the first 15 h. TM values were increased 4.2- and 2.0-fold relative to the relevant controls at 24 and 48 h (P < 0.01), respectively. The TM declined at 72 h and returned to near control values at 144 h.
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Discussion |
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On exposure to 10 mg/kg DMDBC, DNA adduct levels reached a plateau as early as 24 h and then remained unchanged until the last sampling time of 144 h (Figure 1). UDS activity was rapidly induced, reaching a maximum at 24 h and then falling to a near baseline level at 72 h. This time course of DNA repair induction, with an early peak and a decline to baseline after 2448 h, is consistent with that described for other liver carcinogens, like 2-acetylaminofluorene and quinoline, and confirmed that UDS is a transient phenomenon (2124). Taken together, these results suggest that following skin application of a low dose of DMDBC, DNA repair mechanisms were rapidly and efficiently induced to prevent the formation of large numbers of DNA adducts. However, an apparent steady-state of ~400 DNA adducts/108 nucleotides persisted.
Interestingly, no DNA strand breaks, as measured by the Comet assay, were detected during the same study period. The lack of increase in TM was somewhat surprising and contrasted with the recent report of a reliable concordance between the in vivo UDS and Comet assays in rat hepatocytes (25). However, the negative response observed in the Comet assay at a dose that induced UDS in mouse hepatocytes may reflect efficient removal of bulky DMDBC-induced DNA damage by NER. Indeed, under physiological conditions, this multistep biochemical pathway produces dual incisions of the DNA backbone flanking the lesion and forms short-lived gaps that are rapidly filled by a DNA polymerase using the undamaged complementary strand as template (26,27). Therefore, following skin application of 10 mg/kg, DNA strand breaks were probably too short-lived to be detected by the Comet assay under our experimental conditions, while DNA resynthesis occurred efficiently and yielded marked incorporation of [3H]thymidine in the UDS assay.
Following skin application of 90 mg/kg DMDBC, the level of DNA adducts increased sharply to a maximum at 24 h that was ~20-fold higher than in the liver of mice treated with 10 mg/kg. The number of DNA adducts then fell strongly to ~500/108 nucleotides at 144 h. These results, which are in agreement with our previous observations, showed that 90 mg/kg DMDBC induced far higher levels of DNA adducts at early sampling times than did 10 mg/kg (7).
The UDS activity peaked 15 h after skin application of 90 mg/kg DMDBC and persisted at a very substantial level until 72 h. This suggested that in response to larger numbers of DNA adducts, DNA repair mechanisms were more strongly and more persistently induced than after exposure to 10 mg/kg. However, comparison of the kinetics of 32P-post-labeling with UDS data gave unexpected findings, as the levels of DNA adducts in the 90 mg/kg group still increased between 15 and 24 h post-dosing, whereas UDS had already reached a maximum. Moreover, the magnitude of the UDS response was not related to the level of DNA adducts in the two dose groups. Namely, 24 h after exposure to 10 mg/kg DMDBC, the NNG value was +7.7 and the corresponding total binding was 423 adducts/108 nucleotides, while after dosing with 90 mg/kg the NNG value was +15.9 and the corresponding binding was 8814 adducts/108 nucleotides. As enzymatic processes like NER are subject to saturation, these results suggest that although DNA repair mechanisms were substantially induced following skin application of 90 mg/kg DMDBC, they were rapidly overwhelmed and consequently unable to efficiently remove DNA primary damage (28,29). Therefore, the sharp decrease in the global DNA adduct level may partly result from cell proliferation and elimination of badly damaged cells instead of active DNA repair. The significant increase in S phase cells observed at 72 and 144 h, which is in line with the liver necrosis and regenerative hepatocellular proliferation previously reported (7), supported this hypothesis.
The slight but significant increases in TM observed in the Comet assay 24 and 48 h after exposure to 90 mg/kg DMDBC could reflect the accumulation of incomplete repair intermediates in response to the impairment of DNA repair mechanisms. Similar results have been produced in vitro using DNA repair inhibitors to reveal or amplify the effects of several polycyclic aromatic hydrocarbons when tested with the Comet assay (3032). The slight increases in TM could also reflect DNA strand breaks occurring secondarily through the formation of depurinating DNA adducts and resulting alkali-labile abasic sites, as already described for benzo[a] pyrene and 7,12-dimethylbenz[a]anthracene, and more recently reported for DBC (3335). Finally, as DBC metabolites are known to bind not only to nuclear DNA but also to mitochodrial DNA, RNA and proteins, we cannot rule out the possibility that the appearance of DNA strand breaks could result from oxidative DNA damage in response to acute cellular defects (5,36).
It is noteworthy that following skin application of 10 or 90 mg/kg, UDS activity fell markedly and returned to a near baseline level at late sampling times despite the persistence of similar significant levels of DNA adducts (~400500/108 nucleotides). However, unusually high percentages of hepatocytes undergoing DNA excision repair were observed in both dose groups at these late time points. The reason why, after an initial induction of DNA repair, DMDBCDNA adducts were no longer repaired remained unclear. This could reflect heterogeneous DNA repair activity in the mammalian genome, which rapidly removes DNA damage from the transcribed strand of expressed genes, whereas lesions located in inactive genomic regions are repaired slowly (27,37). A second possible explanation is that the repair efficiency of the numerous DNA adducts formed by DMDBC depends on the conformation they adopt in the double-stranded DNA helix, as already shown for benzo[a]pyrene and dibenzo[a,l]pyrene in mouse skin DNA (38,39).
In conclusion, our results suggest that following exposure to 10 mg/kg DMDBC, a dose previously shown to slightly increase the MF in the liver of the MutaTMMouse, DNA repair mechanisms were efficiently induced, which avoided the formation of a large number of DNA adducts. In contrast, following exposure to 90 mg/kg DMDBC, despite a strong initial induction, DNA repair mechanisms were overwhelmed and unable to prevent the formation of large numbers of DNA adducts. This, together with the induction of cell proliferation, probably accounted for the 50-fold increase in MF previously observed in the MutaTMMouse liver.
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Acknowledgments |
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Notes |
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References |
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