RESUMO
Currently, all soft tissue sarcomas (STS) are irradiated by the same regimen, disregarding possible subtype-specific radiosensitivities. To gain further insight, cellular radiosensitivity was investigated in a panel of sarcoma cell lines. Fourteen sarcoma cell lines, derived from synovial sarcoma, leiomyosarcoma, fibrosarcoma and liposarcoma origin, were submitted to clonogenic survival assays. Cells were irradiated with single doses from 1-8 Gy and surviving fraction (SF) was calculated from the resulting response data. Alpha/beta (α/ß) ratios were inferred from radiation-response curves using the linear-quadratic (LQ)-model. Cellular radiosensitivities varied largely in this panel, indicating a considerable degree of heterogeneity. Surviving fraction after 2 Gy (SF2) ranged from 0.27 to 0.76 with evidence of a particular radiosensitive phenotype in only few cell lines. D37% on the mean data was 3.4 Gy and the median SF2 was 0.52. The median α/ß was 4.9 Gy and in six cell lines the α/ß was below 4 Gy. A fairly homogeneous radiation response was observed in myxoid liposarcoma cell lines with SF2 between 0.64 and 0.67. Further comparing sarcomas of different origin, synovial sarcomas, as a group, showed the lowest SF2 values (mean 0.35) and was significantly more radiosensitive than myxoid liposarcomas and leiomyosarcomas (P = 0.0084 and 0.024, respectively). This study demonstrates a broad spectrum of radiosensitivities across STS cell lines and reveals subtype-specific radiation responses. The particular cellular radiosensitivity of synovial sarcoma cells supports consideration of the different sarcoma entities in clinical studies that aim to optimize sarcoma radiotherapy.
Assuntos
Tolerância a Radiação , Sarcoma/radioterapia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Humanos , Sarcoma/patologiaRESUMO
Topotecan- or mitoxantrone-selected cell lines (T8 and MX3, respectively), derived from the human IGROV1 ovarian cancer cell line, were resistant to the topoisomerase I inhibitors topotecan, SN-38 (the active metabolite of irinotecan), and 9-aminocamptothecin, as well as to the topoisomerase II drug mitoxantrone. In both resistant cell lines, decreased accumulation of topotecan and mitoxantrone was observed, caused by enhanced energy-dependent efflux of the drugs involved. In both cell lines, we found that the breast cancer resistance protein/mitoxantrone resistance/placenta-specific ATP binding cassette (BCRP/MXR/ABCP) gene was overexpressed. Furthermore, BCRP/MXR/ABCP expression levels in various partially revertant T8 cells correlated with the levels of resistance to topotecan, SN-38, and mitoxantrone, strongly suggesting BCRP/MXR/ABCP to be the transporter responsible for the enhanced efflux. Pharmacodynamic analysis demonstrated that BCRP/MXR/ABCP is a very efficient transporter of topotecan; in vitro, 70% of the intracellular topotecan pool was transported out of the T8 or MX3 cells within 30 s. In conclusion, we report for the first time that BCRP/MXR/ABCP can also be up-regulated upon exposure of tumor cells to the clinically important drug topotecan, and that BCRP-mediated efflux of topotecan is very efficient. This highly efficient efflux of topotecan by BCRP/MXR/ABCP may have clinical relevance for patients being treated with topotecan.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antineoplásicos Fitogênicos/toxicidade , Resistência a Múltiplos Medicamentos , Regulação Neoplásica da Expressão Gênica , Mitoxantrona/toxicidade , Proteínas de Neoplasias , Topotecan/toxicidade , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Camptotecina/análogos & derivados , Camptotecina/toxicidade , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Irinotecano , Cinética , Neoplasias Ovarianas , Células Tumorais CultivadasRESUMO
We have studied the formation and repair of cisplatin-DNA adducts in wild-type mouse leukemia L1210/0 cells and in the sublines L1210/2 and L1210/5, which differ in cisplatin sensitivity. In a colony-formation assay these sublines were 9- and 22-fold more resistant compared to L1210/0, respectively. Cisplatin-induced DNA modification was studied at the cellular level by immunocytochemistry with antiserum NKI-A59 raised against cisplatin-treated DNA. Levels of nuclear staining immediately after a 1-h treatment were similar to those seen after a 24-h post-incubation in drug-free medium. Clear differences in DNA platination were found between the cell lines: immediately after exposure, L1210/2 and L1210/5 showed only 32 and 14%, respectively, of the nuclear staining observed in L1210/0, and 48 and 13% after 24 h. In these experiments, adduct-specific nuclear staining was quantified as the area under the adduct versus concentration curves (AUC). The formation and repair in these cell lines of the bifunctional adducts cis-Pt(NH3)2d(pGpG) (Pt-GG), cis-Pt(NH3)2d(pApG) (Pt-AG) and cis-Pt(NH3)2(dGMP)2 (G-Pt-G) were studied with an enzyme-linked immunosorbent assay (ELISA). No relation between repair and resistance was observed. The results suggest that differences in induced DNA platination levels, rather than in repair, are responsible--at least in part--for the differences in cisplatin resistance. A mechanism such as an increased tolerance of the resistant cells to plantinum-DNA damage may also be involved.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/metabolismo , Cisplatino/farmacologia , Adutos de DNA/metabolismo , Reparo do DNA , DNA de Neoplasias/metabolismo , Leucemia L1210/metabolismo , Animais , Bicarbonatos/química , Divisão Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica/métodos , Leucemia L1210/tratamento farmacológico , Camundongos , Sensibilidade e Especificidade , Tioureia/químicaRESUMO
Binding of the cytostatic drug carboplatin to DNA was studied in solution, in RIF-1 and CHO cell lines and in human buccal cells after in vitro or in situ drug exposure. Results were compared with DNA adduction by cisplatin. The rate of binding in solution, determined by atomic absorption spectroscopy, was 35 times lower for carboplatin than for cisplatin. Adduct formation in cells in vitro was determined in a quantitative immunostaining assay. Staining intensities after carboplatin treatment were at least 29 times lower than after an equimolar dose of cisplatin. For RIF-1 and CHO cells, maximum levels of carboplatin-induced DNA modification were obtained 24 h after treatment; these levels correlated with cell killing. Adduct-specific staining in buccal cells from two carboplatin-treated patients increased 5-7 fold between 0 and 14 h after infusion, reaching a maximum at 10-14 h. This strongly contrasts with buccal cells from a cisplatin-treated patient, in which the adduct-specific staining signal increased by only 23% between 0 and 6 h after infusion, and then declined. This difference in the rate of adduct formation in vivo is consistent with the in vitro data.
Assuntos
Carboplatina/metabolismo , DNA/metabolismo , Neoplasias/metabolismo , Animais , Carboplatina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cisplatino/metabolismo , Humanos , CamundongosRESUMO
Cytotoxic effects of cis-diamminedichloroplatinum-(II) (cis-DDP) are thought to be mediated by binding to DNA. Studies on binding of cis-DDP to cellular DNA rely heavily on the availability of specific antibodies. We therefore raised and characterized four rabbit antisera: one against cis-DDP-modified DNA (antiserum NKI-A59) and three others against the cis-DDP-modified (di)nucleotides cis-Pt(NH3)2d(pApG) (NKI-A68), cis-Pt(NH3)2d(GMP)2 (NKI-A10), and Pt(NH3)3dGMP (NKI-A39). Reactivities to platinum compounds were determined in an enzyme-linked immunosorbent assay (ELISA) and in a quantitative immunocytochemical assay. In the ELISA, NKI-A59 showed a high affinity for DNA heavily substituted with either cis-DDP or CBDCA [cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)]; amounts of platinum per well giving 50% inhibition (IA50) were as low as 15 and 76 fmol, respectively. NKI-A59 also showed affinity to cis-DDP-modified poly[d(G-C)].poly[d(G-C)], poly(dC), and poly(dG). No affinity was found for trans-DDP [trans-diamminedichloro-platinum(II)]-modified DNA, enzymatically digested cis-DDP-DNA, or cis-DDP-DNA, or cis-DDP-modified poly(dA).poly(dT), oligo(dA)15.oligo(dT)15, oligo(dG)21, oligo(dG)42, or oligo(dAAAG)10. The efficiency of binding to cis-DDP-DNA decreased with decreasing DNA modification levels. Although other cis-DDP-DNA- and cis-DDP-(di)nucleotide-specific antisera have been identified, NKI-A59 is the first antiserum described that is suitable for the in situ detection of cis-DDP-DNA adducts at clinically relevant platinum levels. Adduct-specific immunostaining signals in cultured RIF-1 cells or rat liver paralleled platinum-DNA binding as measured by atomic absorption spectroscopy. The antisera NKI-A68, NKI-A10, and NKI-A39 showed high affinity for their corresponding haptens and varying affinity for non-hapten cis-DDP-DNA adducts. Their affinity for digested cis-DDP-modified DNA was up to 30 times that for intact cis-DDP-DNA. Neither NKI-A68 nor NKI-A10 resulted in specific immunocytochemical staining of cis-DDP-DNA adducts. We conclude that NKI-A68, NKI-A10, and NKI-A39 are suitable for platinum-DNA adduct analysis of digested DNA in ELISA and that NKI-A59 is suitable for platinum-DNA adduct detection at the single-cell level using immunocytochemical methods.
Assuntos
Anticorpos/isolamento & purificação , Cisplatino/farmacologia , DNA/efeitos dos fármacos , DNA/imunologia , Oligonucleotídeos/imunologia , Polinucleotídeos/imunologia , Animais , Anticorpos/análise , Especificidade de Anticorpos/efeitos dos fármacos , Especificidade de Anticorpos/imunologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/imunologia , DNA/análise , DNA/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Soros Imunes/análise , Soros Imunes/isolamento & purificação , Imuno-Histoquímica , Masculino , Camundongos , Oligonucleotídeos/análise , Polinucleotídeos/análise , Coelhos , Ratos , Ratos EndogâmicosRESUMO
The relationship between cell killing and the binding of the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) to DNA was studied in six mammalian cell lines. Two of the human cell lines (COV413B) were of the same origin, comprising one sensitive to cis-DDP and the other with induced resistance to the drug. The four other lines, two rodent (RIF-1, Chinese hamster ovary) and two human (A2780, A1847), were unrelated. The cell lines differed in their sensitivity to cis-DDP, as tested in a clonogenic assay. cis-DDP-DNA binding was determined by quantitative immunocytochemistry using an antiserum against cis-DDP-modified DNA. The resistance factors relative to RIF-1, calculated from full survival curves for cis-DDP, were 3.8 +/- 0.4 for Chinese hamster ovary cells and 8.8 +/- 0.7 for both A2780 and A1847 lines. Using quantitative immunocytochemistry, the levels of the adduct-specific nuclear staining density compared with RIF-1 cells were 4.8 +/- 0.2 for Chinese hamster ovary cells, 9.1 +/- 0.2 for A2780, and 10.0 +/- 0.1 for A1847 cells, i.e., in good agreement with the resistance factors. In studies with the COV413B cells and their cis-DDP-resistant counterpart COV413B-PtR, immunologically detected adduct levels again correlated closely with resistance factors (correlation coefficient = 0.97). The kinetics of cis-DDP-DNA adduct formation and loss was investigated in RIF-1, A2780, and A1847 cells by the immunocytochemistry technique. Adduct levels after a 1-h incubation with approximately equitoxic doses of cis-DDP increased by 18 to 32% (average, 27%) between 0 and 6.5 h after treatment and then declined. Adduct half-lives in this latter phase did not correlate with the sensitivities of the cells for cis-DDP. These results indicate that the initial level of cis-DDP-DNA binding measured by quantitative immunocytochemistry may be a reasonable predictor of sensitivity to this chemotherapeutic drug.
Assuntos
Cisplatino/metabolismo , Reparo do DNA , DNA de Neoplasias/metabolismo , Fibrossarcoma/metabolismo , Neoplasias Ovarianas/metabolismo , Animais , Anticorpos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/imunologia , Cisplatino/farmacologia , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/imunologia , Resistência a Medicamentos , Feminino , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/genética , Humanos , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
The in situ binding of the anticancer drug cis-diamminedichloroplatinum(II) (cisDDP) to DNA was studied in the rat dorsal root spinal ganglion (DRG), using an antiserum against cisDDP-modified calf thymus DNA in a quantitative immunocytochemical assay. Rats received a dose of cisDDP (1 mg/kg), two times a week, up to a cumulative dose of 15 mg/kg (group I) or 34 mg/kg (group II). Rats of group III were given a single dose of 15 mg/kg. Rats were killed 48 hr (groups I and II) or 6 hr (group III) after the last injection. In groups I and II cisDDP-induced neurological damage was assessed by measuring both motor and sensory nerve conduction velocities (MNCV and SNCV). Whereas the MNCV was not influenced by the treatment with cisDDP, the SNCV decreased significantly. The level of cis-DDP-DNA binding in DRG satellite cells equalled that in liver cells, but binding could not be shown in DRG neuron nuclei. The level of cisDDP-DNA binding in spinal cord and brain was very low. The neuroprotecting peptide ORG.2766, an ACTH4-9 analog, was given sc (10 micrograms/rat) four times a week concomitantly with cisDDP to some rats of groups I and II. ORG.2766 prevented the decrease of the SNCV, but did not change the extent of cisDDP-DNA binding in satellite or liver cells. It is concluded that the amelioration of cisDDP toxicity by ORG.2766 is not directly related to the cisDDP-DNA binding in satellite cells.
Assuntos
Hormônio Adrenocorticotrópico/análogos & derivados , Cisplatino/metabolismo , DNA/metabolismo , Gânglios Espinais/metabolismo , Fragmentos de Peptídeos/farmacologia , Hormônio Adrenocorticotrópico/farmacologia , Animais , Encéfalo/metabolismo , Cisplatino/toxicidade , Interações Medicamentosas , Feminino , Gânglios Espinais/efeitos dos fármacos , Técnicas Imunoenzimáticas , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The formation and stability of interaction products between the anti-cancer drug cis-diamminedichloroplatinum(II) (cis-DDP) and DNA were studied in buccal epithelial and urinary cells from ten cancer patients who received cis-DDP-based therapy. Buccal cells were collected 1 h before and 1-2 h after i.v. infusions with cis-DDP. The interaction products were visualized in an immunocytochemical peroxidase assay, using an antiserum against cis-DDP-modified calf thymus DNA. The nuclear staining density was measured by microdensitometry. Nuclear staining densities in buccal cells after infusions of greater than or equal to 20 mg/m2 cis-DDP were always higher than pretreatment values. Repeated sampling from individual patients treated for 2-5 consecutive days with daily doses of 20-70 mg/m2 cis-DDP indicated that cis-DDP-DNA binding in buccal cells increased in proportion to the cumulative total dose of cis-DDP. The variation in dose-density response between patients was 17%. Apparent adduct loss in buccal cells from four patients, as measured 8-17 days after the last infusion, amounted to 67-86%. Platinum-induced DNA modifications could also be detected in buccal cells from two cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)-treated patients. In vitro experiments with human buccal cells and lymphocytes indicated linear relationships between DNA modification and either cis-DDP concentration or incubation time. Nuclear staining densities in pretreatment buccal cells from ten cancer patients treated in vitro with 33 microM cis-DDP for 1 h revealed that interpatient variation in in vitro DNA modification by cis-DDP was low. No quantitative correlation was found between in situ and in vitro DNA modification.
Assuntos
Cisplatino/metabolismo , DNA de Neoplasias/metabolismo , Compostos Organoplatínicos/metabolismo , Carboplatina , Feminino , Humanos , Imuno-Histoquímica , Masculino , Neoplasias/genética , Bexiga Urinária/metabolismoRESUMO
Calf thymus DNA was modified in vitro by cis-diamminedichloroplatinum(II) (cisDDP), complexed with methylated bovine serum albumin and used to immunize rabbits. The anti-cisDDP-DNA antiserum obtained was applied in a double peroxidase-antiperoxidase staining procedure to localize cisDDP-DNA and cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA)-DNA interaction products in cryostat tissue sections of mice and rats. Rats received cisDDP (0-10 mg/kg) and were killed after 24 h. Mice received cisDDP (0-15 mg/kg) or CBDCA (200 mg/kg), and were killed after 2 h-162 days. For each time-dose combination two mice or one rat were used; agents were given i.p. Specific nuclear staining was observed in all tissues examined from cisDDP- or CBDCA-treated animals. No significant nuclear staining could be observed in tissue sections from control rats and mice. The extent of staining after cisDDP was dose and time dependent. The lowest dose of cisDDP after which specific nuclear staining could be detected varied from tissue to tissue [e.g., 0.1 mg/kg, pancreas (mouse); 0.5 mg/kg, liver, kidney (mouse, rat)]. The longest time interval after a single dose of 6 mg/kg cisDDP in which adducts could be visualized also depended on the tissue and varied between 9 days (spleen, testis) and 162 days (kidney). The staining intensity in liver and kidney, measured microdensitometrically, decreased relatively fast in the first days after treatment, but much slower thereafter. In the kidney, cisDDP-induced DNA modification showed regional variation: inner cortex greater than outer cortex greater than medulla (rat) and cortex greater than medulla (mouse). In the mouse kidney, a small subpopulation of tubular cells in close association with the renal corpuscles showed a remarkably high staining intensity after both cisDDP and CBDCA administration. Tissues that showed clear cisDDP-induced histological alterations (kidney, pancreas, testis, and duodenum) also showed moderate to high levels of cisDDP-DNA interaction products. A correlation between cell damage (measured histologically) and cisDDP-DNA binding within one tissue type was demonstrated in the rat inner renal cortex, the murine renal cortex, and in duodenal epithelial cells of both mice and rats.
Assuntos
Cisplatino/análise , DNA/análise , Compostos Organoplatínicos/análise , Animais , Carboplatina , Imuno-Histoquímica , Rim/análise , Masculino , Músculos/análise , Pâncreas/análise , Ratos , Ratos EndogâmicosRESUMO
Rats were pretreated for a number of weeks with the liver tumour promoters phenobarbital and 2,3,7,8-tetrachlorodibenzo-p-dioxin, the direct alkylating agents N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea, and the hepatocarcinogens ethionine and diethylnitrosamine. A subsequent challenge with a single, low dose of radioactively labelled dimethylnitrosamine was given to assay the capacity of the liver for O6-methylguanine repair. Pretreatment with 0.05% phenobarbital in the diet for 8 weeks (a promoting regimen) resulted in significantly enhanced O6-methylguanine repair; shorter pretreatment periods (3 days or 2 weeks) had no significant effect. Repeated injection of another liver tumour promoter, 2,3,7,8-tetrachlorodibenzo-p-dioxin, also resulted in enhancement of O6-methylguanine repair. Pretreatment for 2 weeks with N-ethyl-N-nitrosourea resulted in strongly enhanced O6-methylguanine repair, as did a similar pretreatment with diethylnitrosamine, which was included as a positive control. The same pretreatment scheme which was highly effective in the case of N-ethyl-N-nitrosourea, was found to be totally ineffective in the case of N-methyl-N-nitrosourea. When N-methyl-N-nitrosourea was administered for 8 weeks instead of 2, a small but statistically significant increase in O6-methylguanine repair was observed. It is concluded that two factors are responsible for the low effectivity of N-methyl-N-nitrosourea. The first is the relatively low extent of liver DNA methylation by this compound when compared with dimethylnitrosamine. The second is the low efficiency of methylating agents (expressed per extent of DNA alkylation) to induce O6-methylguanine repair in rat liver when compared with ethylating agents. Pretreatment for 2 weeks with a diet containing DL-ethionine also resulted in a substantially increased O6-methylguanine repair capacity. Neither this enhancement, nor that induced by a pretreatment with diethylnitrosamine, could be inhibited by simultaneous feeding of a methionine-enriched diet. Our results indicate that neither increased hepatocellular proliferation nor direct interaction with DNA are necessary for the induction of O6-methylguanine repair enhancement. It is concluded that the capacity of an agent to enhance O6-methylguanine repair in rat liver reflects the hepato(co)carcinogenic capacity of that agent.
Assuntos
Reparo do DNA/efeitos dos fármacos , Dioxinas/farmacologia , Etionina/farmacologia , Guanina/análogos & derivados , Fígado/metabolismo , Compostos de Nitrosoureia/farmacologia , Fenobarbital/farmacologia , Dibenzodioxinas Policloradas/farmacologia , Alquilação , Animais , DNA/metabolismo , Guanina/metabolismo , Masculino , Metionina/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Rat-liver DNA alkylation by diethylnitrosamine (DEN), dimethylnitrosamine (DMN) and ethyl methanesulphonate (EMS) was studied in an attempt to relate chromosome-damaging effects of these agents (the formation of micronuclei in hepatocytes; see preceding paper) to specific alkylation patterns. No correlation was observed between the induction of micronuclei and liver DNA N-alkylation, measured as 3- and 7-alkyl-purines. O6-Alkylguanine is probably not involved in micronucleus induction because it is lost from DNA too rapidly to explain the much more persistent clastogenic effects. In contrast, both the initial amounts of alkylphosphotriesters and the persistencies of these products roughly paralleled the respective effects on micronucleus induction. The possible involvement of alkylphosphotriesters or other O-alkylation products of comparable stabilities is discussed. Results with DMN suggest that part of the primary DNA methylation damage is converted into a secondary (DNA) lesion and that both the primary and secondary lesion(s) contribute to the process of micronucleus formation.
Assuntos
Carcinógenos , Aberrações Cromossômicas , Dietilnitrosamina/farmacologia , Dimetilnitrosamina/farmacologia , Metanossulfonato de Etila/farmacologia , Fígado/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Nitrosaminas/farmacologia , Alquilação , Animais , DNA/metabolismo , Dietilnitrosamina/metabolismo , Dimetilnitrosamina/metabolismo , Metanossulfonato de Etila/metabolismo , Fígado/fisiologia , Linfócitos/fisiologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
In vivo alkylation of DNA leads to DNA fragmentation in alkaline sucrose gradients. In a previous paper (Chem.-Biol. Interact., 19 (1977) 111) we presented evidence that, depending on the experimental conditions, a major fraction of the single-stranded breaks observed might be derived from alkali-labile alkylphosphotriesters. Using alkaline gradients the present paper shows that injection of ethyl methanesulphonate (EMS) into Sprague-Dawley female rats results in significantly increased liver DNA fragmentation up to at least 56 days after injection. Accumulation of single-strand breaks was indicated by experiments in which at 6 days after the last of a series of 5 weekly EMS injections (5 X 110 mg/kg) 11.4 breaks/10(9) Dalton were found, being 3 times more than the number of breaks observed at 6 days after a single injection of 110 mg/kg EMS (3.8 breaks/10(9) Dalton). In animals treated with methyl methanesulphonate (MMS) single-strand breaks were observed at 4 h, 1 day and 2 days, but not at 6 days after injection (40 mg/kg). Repeated weekly injections of MMS (5 X 40 mg/kg) did not result in increased numbers of breaks when compared with animals receiving a single injection of this agent (1 X 40 mg/kg; animals were killed 1 day after (the last) injection). It is suggested that MMS-induced breaks are derived, either on the gradient or in situ, from apurinic sites, whereas persistent EMS-induced breaks reflect the presence of ethylphosphotriesters. The results are discussed in relation to the lacking capacity of EMS to induce foci of precancerous lesions in rat liver and the non-hepatocarcinogenic properties of both MMS and EMS.
Assuntos
DNA/metabolismo , Metanossulfonato de Etila/toxicidade , Fígado/metabolismo , Alquilação , Animais , Reparo do DNA , DNA de Cadeia Simples/metabolismo , Feminino , Fígado/efeitos dos fármacos , Metanossulfonato de Metila/toxicidade , RatosRESUMO
Effects of diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) on the sedimentation pattern of [3H]thymidine-labelled Sprague-Dawley female rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. In experiments at 1--56 days after a single injection it was observed that (potential) single strand breaks induced by DEN were repaired at a low rate. At 56 days the sedimentation pattern was still grossly abnormal. Half-life values of 27 and 46 days were observed after 134 mg/kg DEN (approx. 45% of the LD50) and 13.4 mg/kg DEN, respectively. Identical experiments after DMN (10 mg/kg, corresponding to about 35% of the LD50) showed return to (almost) completely control sedimentation patterns within 56 days after injection (t 1/2 = 8 days). Experiments at 6 or 56 days after the last of a series of 5 or 10 weekly injections of DEN (13.4 mg/kg) showed that a major part of DEN-induced damage (measured as single strand breaks) is of a persistent and accumulating character. No accumulation of DMN-induced rat liver lesions was observed. It is concluded that DNA fragmentation and lack of DNA repair is not a consequence of hepatotoxicity. Since at equimolar doses DEN gives appreciably less DNA alkylation (including O6-alkylguanine) but is much more effective both as an inducer of preneoplastic liver lesions and as a hepatocarcinogen when compared with DMN, we believe that the formation of persistent (and accumulating) DNA damage after DEN administration might be relevant in the process of liver tumour formation.