RESUMO
Previous studies suggest that bladder cancer risk may vary with GST genotype but these results are inconsistent. The aim of this study was to explore whether GSTM1, GSTT1 and GSTP polymorphisms were associated with increased bladder cancer risk in an Egyptian population. GSTM1, GSTT1 and GSTP1 genotype frequencies were determined in bladder cancer cases (n=72) and healthy controls with no history of malignancies (n=82) using PCR-based techniques. The GSTT1*2 genotype was particularly associated with increased risk (OR 2.71, 95%CI 1.27-5.73) and the GSTM1*2 genotype to a lesser extent (OR 1.63, 95%CI 0.85-3.10). 18.1% of cases but only 7.3% of controls were GSTP1*B*B homozygotes (OR 2.38, 95%CI 0.83-6.87). The presence of two or more a priori at-risk genotypes was associated with increased bladder cancer risk (OR 2.42; 95%CI 1.47-3.97). These results suggest that polymorphisms in the GST genes are associated with increased risk of bladder cancer among Egyptians.
Assuntos
Predisposição Genética para Doença/genética , Glutationa Transferase/genética , Isoenzimas/genética , Polimorfismo Genético/genética , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Egito , Glutationa S-Transferase pi , Humanos , Pessoa de Meia-IdadeRESUMO
There is increasing evidence that alkylating agent exposure may increase large bowel cancer risk and factors which either alter such exposure or its effects may modify risk. Hence, in a cross-sectional study of 78 patients with colorectal disease, we have examined whether (i) metabolic genotypes (GSTT1, GSTM1, CYP2D6, CYP2E1) are associated with O(6)-methyldeoxyguanosine (O(6)-MedG) levels, O(6)-alkylguanine-DNA alkyltransferase (ATase) activity or K-ras mutations, and (ii) there was an association between ATase activity and O(6)-MedG levels. Patients with colon tumours and who were homozygous GSTT1(*)2 genotype carriers were more likely than patients who expressed GSTT1 to have their DNA alkylated (83 versus 32%, P=0.03) and to have higher O(6)-MedG levels (0.178+/-0.374 versus 0.016+/-0.023 micromol O(6)-MedG/mol dG, P=0.04) in normal, but not tumour, DNA. No such association was observed between the GSTT1 genotype and the frequency of DNA alkylation or O(6)-MedG levels in patients with benign colon disease or rectal tumours. Patients with colon tumours or benign colon disease who were CYP2D6-poor metabolisers had higher ATase activity in normal tissue than patients who were CYP2D6 extensive metabolisers or CYP2D6 heterozygotes. Patients with the CYP2E1 Dra cd genotype were less likely to have a K-ras mutation: of 55 patients with the wild-type CYP2E1 genotype (dd), 23 had K-ras mutations, whereas none of the 7 individuals with cd genotype had a K-ras mutation (P=0.04). No other associations were observed between GSTT1, GSTM1, CYP2D6 and CYP2E1 Pst genotypes and adduct levels, ATase activity or mutational status. O(6)-MedG levels were not associated with ATase activity in either normal or tumour tissue. However, in 15 patients for whom both normal and tumour DNA contained detectable O(6)-MedG levels, there was a strong positive association between the normal DNA/tumour DNA adduct ratio and the normal tissue/tumour tissue ATase ratio (r(2)=0.66, P=0.001). These results indicate that host factors can affect levels both of the biologically effective dose arising from methylating agent exposure and of a susceptibility factor, the DNA repair phenotype.
Assuntos
Neoplasias Colorretais/enzimologia , Citocromo P-450 CYP2D6/genética , Reparo do DNA , DNA de Neoplasias/metabolismo , Glutationa Transferase/genética , Guanina/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Adenosina Trifosfatases/metabolismo , Idoso , Alquilação , Neoplasias Colorretais/genética , Estudos Transversais , Feminino , Predisposição Genética para Doença , Genótipo , Glutationa Transferase/metabolismo , Guanina/análogos & derivados , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
Ivermectin is widely used against parasitic infections in veterinary and human medicine and was found to promote the growth of lesions leading to neoplasia when given continuously in the diet to Wistar rats receiving a single low dose of N-methyl-N1-nitro-N-nitrosoguanidine (MNNG). No tumors or pathological lesions were observed in the forestomach of the control animals or those given ivermectin alone. However, compared to animals receiving MNNG alone, rats maintained on a diet containing ivermectin (2 ppm) and given MNNG (12.5 mg/kg) by gavage showed an increased number of neoplasms (9/26 vs 3/18; p = 0.30) and a statistically significant fourfold increase in the number of pathological lesions (18/26 vs 3/18; p = 0.002), which include preneoplasia in the forestomach. In all cases, the pathological lesions were more severe in the animals receiving ivermectin and MNNG, compared to those receiving MNNG alone.
Assuntos
Inseticidas/efeitos adversos , Ivermectina/efeitos adversos , Metilnitronitrosoguanidina/efeitos adversos , Mutagênicos/efeitos adversos , Lesões Pré-Cancerosas/induzido quimicamente , Neoplasias Gástricas/induzido quimicamente , Administração Oral , Animais , Dieta , Interações Medicamentosas , Inseticidas/administração & dosagem , Ivermectina/administração & dosagem , Masculino , Lesões Pré-Cancerosas/veterinária , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Neoplasias Gástricas/patologia , Neoplasias Gástricas/veterináriaRESUMO
BACKGROUND: The pro-mutagenic lesion O(6)-methyldeoxyguanosine (O(6)-MedG), a marker of exposure to many N-nitroso compounds (NOC), can be detected in normal and tumour DNA isolated from colorectal tissue. The biological significance of this exposure is, as yet, unknown but in situ NOC formation is bacterially catalysed suggesting that NOC formation and potentially DNA alkylation will vary throughout the large bowel. AIMS: To determine if O(6)-MedG levels in colorectal DNA vary within the large bowel. PATIENTS: We studied 62 men and women undergoing surgery for colorectal tumours in the north west of England. METHODS: O(6)-MedG levels were measured in paired normal and tumour DNA samples. DNA was digested to nucleosides, fractionated by HPLC, and purified O(6)-MedG quantified by a radioimmunoassay. RESULTS: O(6)-MedG was detected in 27 out of a total of 62 (43%) normal DNA samples and in 30 of 58 (52%) tumour DNA samples: it was present at concentrations of <0. 01-0.94 and <0.01-0.151 micromol O(6)-MedG/mol deoxyguanosine for normal and tumour DNA, respectively. Levels of O(6)-MedG in normal, but not tumour, DNA from the proximal colon were lower than those found in DNA from either the sigmoid colon (p=0.03) or rectum (p=0. 05). When the analysis was restricted to samples that contained O(6)-MedG, similar results were obtained in that O(6)-MedG levels in normal DNA were lower in the proximal colon than in the sigmoid colon (p=0.04) or rectum (p=0.03). CONCLUSIONS: DNA alkylation varied within the large bowel possibly due to in situ NOC formation and was highest in areas of the colon and rectum where the highest incidence of large bowel tumours occurs, suggesting that DNA alkylation may play a role in the aetiology of colorectal cancer.
Assuntos
Neoplasias Colorretais/metabolismo , Adutos de DNA/metabolismo , Desoxiguanosina/análogos & derivados , Idoso , Alquilação , Cromatografia Líquida de Alta Pressão , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/genética , Desoxiguanosina/metabolismo , Feminino , Humanos , Masculino , RadioimunoensaioRESUMO
DNA damage is a critical factor in the initiation of chemically induced toxicities (including cancer), and the repair of this damage represents the cell's first line of defense against the deleterious effects of these agents. The various mechanisms of DNA repair are reviewed briefly and the actions of the DNA repair protein O6-alkylguanine DNA alkyltransferase (ATase) are used to illustrate how DNA repair can protect cells against alkylating agent-induced toxicities, mutagenesis, clastogenesis, and carcinogenesis. The effectiveness of this repair protein can be measured based on its ability to deplete levels of its promutagenic substrate O6-methylguanine (O6-meG) in the DNA of cells. These studies reveal that the repair of O6-meG from DNA occurs heterogeneously, both intra- and intercellularly. Even in cells that repair O6-meG hyperefficiently, certain regions of chromatin DNA are repaired with difficulty, and in other regions they are not repaired at all; most likely this lack of repair is a result of the location of the lesion in the DNA sequence. When individual cells are compared within a tissue, some cells are clearly repair deficient, because the O6-meG can persist in DNA for many weeks, whereas in other cells, it is removed within a matter of hours. The role of these repair-deficient cells as targets for alkylating agent induced carcinogenesis is considered. The mechanisms of the homeostatic control of DNA repair function in mammalian cells are not yet well understood. Because there are now indications of the mechanisms by which the level of DNA damage may be sensed (and so influence the activity of the ATase repair protein), this is an important area for future study.
Assuntos
Dano ao DNA , Reparo do DNA , Guanina/análogos & derivados , Alquilantes/toxicidade , Animais , Cromatina/efeitos dos fármacos , DNA/efeitos dos fármacos , DNA/metabolismo , Relação Dose-Resposta a Droga , Guanina/metabolismo , Humanos , Fatores de TempoRESUMO
Female SWR mice were treated with 1,2-dimethylhydrazine (DMH: 6.8 mg/kg i.p. injection) once weekly for up to 10 weeks, a dosing regime that produced tumours principally within the distal colon (Jackson et al., 1999. Carcinogenesis 20, 509-513). O(6)-Methylguanine (O(6)-MeG) levels, measured using a simple [3H]-based O(6)-alkylguanine-DNA alkyltransferase (ATase) inactivation assay, ranged from 0.6 to 16.7 fmol/microg DNA with: (i) highest levels in the distal colon; and (ii) higher levels after 68 mg/kg total DMH than 6.8 mg/kg DMH. Basal ATase activity varied between 0.97 and 1.22 fmol/microg DNA within the colon but was not associated with adduct levels or tumour induction. After 6.8 mg/kg DMH, the half life of O(6)-MeG in colonic tissue was 36-42 h whereas after 68 mg/kg DMH, t1/2 was approximately 25, 57 and 96 h in the proximal, mid and distal colon, respectively. Tumour induction was thus associated with the levels and persistence of O(6)-MeG in the distal colon.
Assuntos
Colo/metabolismo , Neoplasias do Colo/metabolismo , Guanina/análogos & derivados , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , 1,2-Dimetilidrazina/farmacologia , Animais , Carcinógenos/farmacologia , Colo/química , Colo/efeitos dos fármacos , Neoplasias do Colo/induzido quimicamente , DNA/química , DNA/metabolismo , Metilação de DNA/efeitos dos fármacos , Feminino , Guanina/análise , Guanina/biossíntese , Camundongos , O(6)-Metilguanina-DNA Metiltransferase/análise , O(6)-Metilguanina-DNA Metiltransferase/antagonistas & inibidores , Reprodutibilidade dos TestesRESUMO
O(6)-Alkylguanine-DNA-alkyltransferase (ATase) is an important modulator of alkylating agent-induced toxicity and carcinogenicity, but those factors which influence the expression of this repair protein in human tissues are poorly characterised. In this study, we have determined ATase levels in macroscopically normal and tumour tissues from 76 individuals with benign or malignant colorectal disease. All tissue samples had detectable ATase activity, with values ranging from 35 to 451 fmol/mg protein. ATase activity in normal rectal tissue was significantly higher than that in normal tissue from the sigmoid colon (148 +/- 76 vs. 100 +/- 40 fmol/mg protein, p = 0.01), whereas ATase levels within different regions of the colon (proximal vs. sigmoid colon) were similar. In normal tissue, inter-individual variation in ATase activity was 4-fold in the colon and 6-fold in the rectum, whereas in tumour tissue the corresponding figures were approx. 13.0- and 7-fold, respectively. There was no detectable difference in normal tissue ATase activity between individuals with benign or malignant disease of the colon. Normal and tumour tissue ATase activities were strongly correlated in the sigmoid colon (r = 0.80) and rectum (r = 0.59) but not the caecum (r = -0.03). In a multivariate analysis, ATase activity in normal colon tissue increased with age (p = 0.01) and current smoking (p = 0.06), whereas tumour ATase activity increased only with use of anti-histamines (p = 0.05). In rectal tumour tissue, activity decreased with age (p = 0.05) and use of anti-muscarinic medications (p = 0.01): in normal rectal tissue, no modulating factors were identified.
Assuntos
Neoplasias do Colo/enzimologia , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Neoplasias Retais/enzimologia , Neoplasias do Colo Sigmoide/enzimologia , Fatores Etários , Idoso , Benzodiazepinas/farmacologia , Colo/enzimologia , Doenças do Colo/enzimologia , Doenças do Colo/genética , Neoplasias do Colo/genética , Ativação Enzimática/efeitos dos fármacos , Feminino , Antagonistas dos Receptores Histamínicos H1/farmacologia , Humanos , Modelos Lineares , Masculino , Análise Multivariada , Antagonistas Muscarínicos/farmacologia , Mutação , O(6)-Metilguanina-DNA Metiltransferase/genética , Neoplasias Retais/genética , Reto/enzimologia , Valores de Referência , Neoplasias do Colo Sigmoide/genética , Fumar/metabolismoRESUMO
In this study we have investigated the relationship between the dose of 1,2-dimethylhydrazine (DMH) and the yield (and location) of tumours in a mouse strain susceptible to colon tumour induction. Female SWR mice were injected with 6.8 mg/kg DMH i.p. once a week for 1, 5, 10 and 20 weeks and the animals were followed for almost 2 years. Administration of increasing doses of DMH resulted in a dose-dependent decrease in survival time. Colon tumours developed in 26, 76 and 87% of mice given a total dose of 34, 68 and 136 mg/kg DMH, respectively: no tumours were detected in animals treated with a total dose of 6.8 mg/kg. Most colon tumours (79%) were located in the distal colon with the remainder being found in the mid colon and none were detected in either the proximal colon or small intestine. As mutations in the K-ras gene are thought to be key events in the pathogenesis of human and rodent colon tumours, we determined the frequency of codon 12 and 13 K-ras mutations in these tumours by restriction site mutation analysis and/or DNA sequencing. A total of 50 colon tumour samples were analysed for codon 12 mutations and of these 29 were also screened for codon 13 mutations. No mutations were detected in either of these codons. The mutational activation of the K-ras gene is not an essential step in the development of DMH-induced colon tumours in female SWR mice and if similar considerations apply to humans, then the aetiological role of alkylating agents may be underestimated from the prevalence of K-ras GC-->AT transitions in human tumours.
Assuntos
1,2-Dimetilidrazina/toxicidade , Adenocarcinoma/induzido quimicamente , Carcinógenos/toxicidade , Neoplasias do Colo/induzido quimicamente , Genes ras , Mutação , 1,2-Dimetilidrazina/administração & dosagem , Adenocarcinoma/genética , Animais , Sequência de Bases , Carcinógenos/administração & dosagem , Neoplasias do Colo/genética , Primers do DNA , Relação Dose-Resposta a Droga , Feminino , CamundongosRESUMO
A previous observation that an N-nitroso-N-carboxymethyl derivative reacts with DNA to give both O6-carboxymethyl-2'-deoxyguanosine (O6-CMdGuo) and O6-methyl-2'-deoxyguanosine (O6-MedGuo) [Shuker, D. E. G., and Margison, G. P. (1997) Cancer Res. 57, 366-369] has been confirmed using a range of nitrosated glycine derivatives [N-acetyl-N'-nitroso-N'-prolylglycine (APNG), azaserine (AS), and potassium diazoacetate (KDA)]. In addition, mesyloxyacetic acid (MAA) was also found to give both O6-adducts in DNA. O6-CMdGuo and O6-MedGuo were assessed in enzymatic hydrolysates of treated calf thymus DNA using a combined immunoaffinity/HPLC/fluorescence procedure. The ratio of O6-CMdGuo to O6-MedGuo varied somewhat between the different compounds with APNG giving the most methylation (O6-CM:O6-Me ratio of 10) and AS the least (39), with KDA and MAA giving intermediate amounts (16 and 18, respectively). The formation of O6-MedGuo by the four compounds probably arises through decarboxylation at various stages in the decomposition pathways, but the exact mechanisms remain to be clarified. The formation of O6-MedGuo from reactions of nitrosated glycine derivatives with DNA in vitro may explain the frequent detection of this adduct in human gastrointestinal DNA, as nitrosation of dietary glycine may occur. O6-CMdGuo is likely to be a useful biomarker of this pathway in vivo and has been detected in human tissues.
Assuntos
Carcinógenos/química , Adutos de DNA/análise , DNA/química , Desoxiguanosina/análogos & derivados , Glicina/química , Compostos Nitrosos/química , Animais , Calibragem , Bovinos , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Desoxiguanosina/química , Imunoadsorventes , Timo/químicaRESUMO
Outbred 7-week old male Wistar rats were exposed for 21 days to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) via the drinking water and N7-methyl deoxyguanosine 3'-monophosphate (N7-MedGp) levels in DNA from the pyloric mucosa (target tissue) and white blood cells (wbc: non-target tissue) were determined by (32)P-postlabelling. Exposure to MNNG resulted in the non-linear, dose-related formation of N7-medGp in both tissues. Adduct levels in the pyloric mucosa were determined to be 1058, 5.4 and 1.1 µmole N7-medGp mole(-1) deoxyguanosine 3'-monophosphate (dGp) after exposure to 4.1, 0.62 and 0.006 mg MNNG kg(-1) day(-1) respectively whereas adduct levels in the wbc DNA were lower at 5.2, 0.52 and 0.68 µmoles N7-medGp mole(-1) dGp after exposure to 4.1, 0.62 and 0.062 mg MNNG kg(-1) day(-1) respectively. In addition, the persistence of N7-medGp was investigated. Loss of adduct occurred rapidly, with a decrease of 87 and 97% respectively in target tissue and wbc DNA by 48 h after cessation of 4.1 mg MNNG kg(-1) day(-1) exposure; 14 days post-MNNG treatment, however, N7-medGp was still detectable (0.46 µmole N7-medGp mole(-1) dGp) in pyloric mucosal DNA. The quantitation of N7-medGp after exposure to low doses of carcinogen, i.e. 0.006 mg MNNG kg(-1) day(-1), approaching environmentally relevant levels has not been previously reported, and indicates that the (32)P-postlabelling assay developed here possesses sufficient sensitivity to quantitate N7- medGp in human DNA arising from environmental exposure to methylating agents.
RESUMO
Alkylpurine-DNA-N-glycosylase (APNG) null mice have been generated by homologous recombination in embryonic stem cells. The null status of the animals was confirmed at the mRNA level by reverse transcription-PCR and by the inability of cell extracts of tissues from the knockout (ko) animals to release 3-methyladenine (3-meA) or 7-methylguanine (7-meG) from 3H-methylated calf thymus DNA in vitro. Following treatment with DNA-methylating agents, increased persistence of 7-meG was found in liver sections of APNG ko mice in comparison with wild-type (wt) mice, demonstrating an in vivo phenotype for the APNG null animals. Unlike other null mutants of the base excision repair pathway, the APNG ko mice exhibit a very mild phenotype, show no outward abnormalities, are fertile, and have an apparently normal life span. Neither a difference in the number of leukocytes in peripheral blood nor a difference in the number of bone marrow polychromatic erythrocytes was found when ko and wt mice were exposed to methylating or chloroethylating agents. These agents also showed similar growth-inhibitory effects in primary embryonic fibroblasts isolated from ko and wt mice. However, treatment with methyl methanesulfonate resulted in three- to fourfold more hprt mutations in splenic T lymphocytes from APNG ko mice than in those from wt mice. These mutations were predominantly single-base-pair changes; in the ko mice, they consisted primarily of AT-->TA and GC-->TA transversions, which most likely are caused by 3-meA and 3- or 7-meG, respectively. These results clearly show an important role for APNG in attenuating the mutagenic effects of N-alkylpurines in vivo.
Assuntos
DNA Glicosilases , Hipoxantina Fosforribosiltransferase/genética , Metanossulfonato de Metila/toxicidade , Mutagênicos/toxicidade , N-Glicosil Hidrolases/fisiologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células Cultivadas , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Eritrócitos/efeitos dos fármacos , Etilnitrosoureia/análogos & derivados , Etilnitrosoureia/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Guanina/análogos & derivados , Guanina/metabolismo , Contagem de Leucócitos/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Mutação , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , TemozolomidaRESUMO
Inbred mice vary in susceptibility to colon carcinogens such as 1,2-dimethylhydrazine (DMH). Differential susceptibility may depend, in part, on formation of promutagenic DNA methyl adducts within target colonic mucosa. The present study was undertaken to evaluate the extent of DNA adduct formation in susceptible (SWR) and resistant (AKR) mice acutely exposed to the colon carcinogen azoxymethane (AOM), a direct metabolite of DMH. In the first experiment, 8-week-old SWR and AKR mice were treated i.p. with 20 mg/kg AOM and sacrificed 6 h later. DNA was isolated from distal colon and liver, and O6-methylguanine (O6-MeGua) adduct levels were assessed by immunoslot blot (ISB) analysis, using a monospecific antibody raised against O6-methyldeoxyguanosine. HPLC-fluorescence detection was also used to quantitate 06-MeGua and 7-methylguanine (7-MeGua), and to generate standard curves. At 6 h, both O6-MeGua and 7-MeGua were significantly higher (2- to 3-fold, p < 0.05) in AKR colon, while an opposite pattern was found in liver. In Experiment 2, mice were injected with AOM (20 mg/kg) and euthanized 12 and 48 h later. At 12 h, O6-MeGua levels were higher in colons (1.4-fold) of SWR mice. Forty-eight hours after treatment, however, adduct levels in colon were markedly (5-fold) reduced in SWR but were unchanged from 12 h in AKR. To further compare activation of AOM in both strains, colon microsomes were incubated with AOM and calf thymus DNA. Comparable levels of O6-MeGua were detected by ISB, demonstrating equivalent metabolic capacity in both SWR and AKR mice. These studies suggest that differential susceptibility to AOM-induced colon carcinogenesis is not based on initial target tissue DNA alkylation and unlikely to depend on differential metabolic capacity.
Assuntos
Azoximetano/toxicidade , Carcinógenos/toxicidade , Adutos de DNA/metabolismo , Neoplasias Experimentais/induzido quimicamente , Animais , Biotransformação , Carcinógenos/farmacologia , Bovinos , Cromatografia Líquida de Alta Pressão , DNA/análise , DNA/efeitos dos fármacos , DNA/isolamento & purificação , Adutos de DNA/efeitos dos fármacos , Guanina/análogos & derivados , Guanina/metabolismo , Immunoblotting , Masculino , Metilnitrosoureia/farmacologia , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos , Valor Preditivo dos TestesRESUMO
O6-alkylguanine DNA-alkyltransferase (ATase) provides protection against the toxic, mutagenic and carcinogenic effects of alkylating agents, principally by removing the promutagenic lesion O6-alkylguanine from DNA. Differences in ATase activity in human tissue may thus determine mutational susceptibility. As GC-->AT transitions, which can be induced by O6-alkylguanine in DNA, are commonly observed in the K-ras oncogene of alkylating agent induced animal tumours and in human colorectal tumours, we have examined whether differences in ATase activity may affect the risk of K-ras mutations in humans with colorectal tumours. NTase activity in normal tissue from individuals with a K-ras mutation in colorectal tissue and more specifically a GC-->AT transition (but not a transversion mutation) was significantly lower than that in individuals without a mutation (P < 0.01). Thus, individuals with low ATase activity in normal tissue (i.e. below the median) were at increased risk of having a transition (OR 10.1; 95% CI 1.9-99.0), but not a transversion mutation (OR 1.7; 95% CI 0.3-12.2). There were no significant differences in tumour ATase activity in individuals with or without a mutation. These results suggest that ATase can protect colorectal tissue against the mutagenic effects of alkylating agents and furthermore, that alkylating agent exposure plays a role in the aetiology of colorectal tumours containing a GC-->AT transition in the K-ras oncogene.
Assuntos
Colo/enzimologia , Neoplasias Colorretais/genética , Genes ras , Metiltransferases/metabolismo , Mutação , Reto/enzimologia , Humanos , O(6)-Metilguanina-DNA MetiltransferaseRESUMO
Most human colorectal cancers arise through the accumulation of a series of genetic alterations such as point mutations within the Ki-ras and p53 genes, but the chemical carcinogens that may be implicated in these events are still unidentified. In a previous study, we showed that DNA from human colorectal tissue contained O6-methyldeoxyguanosine (O6-MedG), a promutagenic lesion arising from exposure to as yet unidentified methylating agents. To address whether such exposure may result in oncogene activation in human colorectal tumors, we examined another series of paired normal and tumor DNA samples from the lower intestinal tract for the presence of O6-MedG in DNA (as a marker of exposure) and for mutations within the Ki-ras gene. After isolation by high pressure liquid chromatography, O6-MedG was quantified by a radioimmunoassay with a limit of detection of 0.01 mumol O6-MedG/mol dG. The frequencies of methylation were 33%, 52%, and 48% for normal DNA and 58%, 32%, and 63% for tumor DNA isolated from the cecum, sigmoid colon, and rectum, respectively. Overall, 35% of the individuals had no detectable O6-MedG in the DNA from both their tumor and normal tissue. Ki-ras mutations were initially identified by a restriction site mutation assay and then sequenced to ascertain the mutations thus detected. The frequencies of mutations in tumor DNA isolated from the cecum, sigmoid colon, and rectum were 28%, 29%, and 42%, respectively. DNA isolated from macroscopically normal tissue was found to contain Ki-ras mutations in 14% of sigmoid colon samples and 12% of rectal samples. Most base mutations were in codon 12 (72%), and 64% were GC-->AT transitions: 28% and 8% were GC-->TA and CG-->CG transversions, respectively. All mutations were at the second base of either codon 12 or codon 13 except for a single GC-->TA transversion at the first base of codon 13 in a rectal tumor sample. There was no association between the presence of O6-MedG in DNA from either normal or tumor tissue or both normal and tumor tissue and the incidence of Ki-ras mutations or GC-->AT transitions in mutated Ki-ras genes. It remains to be determined, however, whether there is a relationship between methylating-agent exposure and Ki-ras mutations, as (i) the presence of O6-MedG in colorectal DNA in these samples may not represent the exposure when Ki-ras mutational activation was occurring (i.e., at some unknown time in the past), (ii) interindividual differences in repair-enzyme activity may alter susceptibility to a mutational event after exposure, (iii) the predominant mutagen in the colon and rectum may not be a methylating agent (e.g., nitric oxide), and (iv) exposure to methylating agents need not result in oncogene activation in human tissues but may perhaps promote the emergence of the mutator phenotype.
Assuntos
Neoplasias Colorretais/genética , Genes ras , Proteínas Proto-Oncogênicas p21(ras)/genética , Sequência de Bases , Colo/química , Dano ao DNA , Primers do DNA/química , Inglaterra , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Metilação , Dados de Sequência Molecular , Mutação PuntualRESUMO
In this study, a combined immunoaffinity purification/32P-postlabelling procedure has been used to quantify O6-methyldeoxyguanosine-3'-monophosphate (O6-MedGp) in human DNA. DNA digests are subjected to a two-stage immunopurification in which the acetone-eluted fraction from the first stage is reapplied to a second immunocolumn, and the O6-MedGp specifically eluted using O6-methylguanosine (O6-MerG). O6-MedGp is then 32P-postlabelled in the presence of deoxyinosine-3'-monophosphate (dIp) as internal standard, separated by two-dimensional TLC and levels of the adduct quantified using storage phosphor technology. The recovery of O6-MedGp at levels between 0.4 and 500 fmol was 61%. Analysis of human DNA samples indicated that < 1 fmol O6-methyldeoxy-guanosine-5'-monophosphate (O6-MepdG) could be detected with a high degree of precision (coefficient of variation < 12%) during a 2 h exposure to a storage phosphor screen. The assay was then applied to 25 human samples from three separate populations, one of which was exposed to methylating agent chemotherapy, for which O6-methyl-deoxyguanosine (O6-MedG) levels had already been quantified by HPLC/radioimmunoassay. The results indicated a high degree of correlation between the two assays (r = 0.99). O6-MedGp was detected in all the samples analysed with levels ranging from 0.026 to 23.2 mumol O6-MedGp/mol dG. The minimum amount of O6-MepdG detected was 0.2 fmol. As there was no detectable signal in the area to which O6-MepdG maps in negative control samples, a detection limit based upon the signal/noise ratio was impossible to quantify. However the limit of detection of the storage phosphor technology itself was estimated by quantifying a visually identifiable compound, which mapped to the same region. The amount of this compound was determined to be 32 +/- 27 amol (n = 5). If a similar amount of O6-MepdG was detected from 50 micrograms of DNA, and assuming that the labelling efficiency and recovery was similar to that found in this study, then this would correspond to an adduct level of approximately 3 nmol O6-MedGp/mol dG.
Assuntos
DNA/análise , Guanina/análogos & derivados , Cromatografia em Camada Fina , Dano ao DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , Feminino , Guanina/análise , Humanos , Marcação por Isótopo/métodos , Masculino , Nucleotídeos/análise , Radioisótopos de Fósforo , Reprodutibilidade dos TestesRESUMO
The 3' and 5'-monophosphates of O6-methyldeoxyguanosine and N7-methyldeoxyguanosine were chemically synthesized. Using these standards with deoxyguanosine-3'-monophosphate (dGp) as an internal standard, conditions were optimized to quantify O6-methyldeoxyguanosine-3'-monophosphate (O6-MedGp) and N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) by 32P-postlabelling. Under optimal conditions, the labelling efficiencies of O6-MedGp and N7-MedGp were respectively approximately 100 and approximately 15%, with detection limits of approximately 1.1 and approximately 6.0 fmol respectively using 10 pmol dGp or 0.8 fmol of O6-MedGp if 2 pmol of dGp was used. The assay developed for O6-MedGp was then applied to the quantitation of [3H]-O6-MedGp and O6-MedGp isolated from DNA digests by immunoaffinity separation. The standard curve generated from the use of [3H]-O6-MedGp, thus isolated, was identical to that generated previously using the chemically synthesized O6-MedGp, indicating that no inhibitory factors co-eluted with the O6-MedGp. After passage through two immunocolumns, recovery of 4 and 40 fmol of O6-MedGp was approximately 30%. Four human stomach samples were analysed by combining this immunoaffinity purification with 32P-post-labelling: levels ranged from 0.21 to 0.86 mumol O6-MedGp/mol dG. Further DNA samples, isolated from the human colon, were fractionated by anion-exchange HPLC and the N7-MedGp and O6-MedGp containing fractions were purified by reverse-phase HPLC and immunoaffinity chromatography respectively. Adduct-containing fractions were dried and 32P-postlabelled. Whereas O6-MedGp was detected at levels between 0.3 and 3.4 mumol O6-MedGp/mol dG, no N7-MedGp was detected in these samples, probably due to depurination of N7-MedGp to N7-methylguanine or reduced assay sensitivity resulting from contaminating nucleotides and/or unidentified radioactivity eluting close to the N7-methyldeoxyguanosine-5'-monophosphate.
Assuntos
DNA/análise , Nucleotídeos de Desoxiguanina/análise , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , HumanosRESUMO
There is increasing experimental evidence to indicate that O6-methyldeoxyguanosine (O6-MedG) formation in DNA is a critical cytotoxic event following exposure to certain antitumor alkylating agents and that the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase) can confer resistance to these agents. We recently demonstrated a wide interindividual variation in the depletion and subsequent regeneration of ATase in peripheral blood lymphocytes of patients treated with 24-h continuous infusion of 1-p-carboxyl-3,3-dimethylphenyltriazene (CB10-277) for metastatic melanoma. We have now measured the formation of O6-MedG in the DNA of peripheral leukocytes of nine patients receiving this treatment regimen. This lesion could be detected in DNA within 1 h and a progressive increase in adduct levels occurred during the CB10-277 infusion and for 24 h after completion. Considerable interindividual variation was observed in the peak O6-MedG levels, with values ranging from 3.0 to 23.8 mumol O6-MedG/mol deoxyguanosine (mean, 12.3 +/- 6.4 mumol O6-MedG/mol deoxyguanosine) following the first treatment cycle, possibly as a consequence of differences in the capacity of patients to metabolize CB10-277 to a methylating agent. There was, nevertheless, a clear temporal relationship between the progressive formation of leukocyte O6-MedG and lymphocyte ATase depletion. Repeated-measures regression showed that this was statistically significant (P < 0.001) during the CB10-277 infusion. A significant inverse correlation was also seen between pretreatment lymphocyte ATase activity and peak O6-MedG levels in leukocyte DNA (r = -0.73) and the area under the leukocyte O6-MedG concentration-time curve (r = -0.76). Metabolism of CB10-277 to a methylating agent could be one factor that combines with DNA repair capacity to determine clinical response, because the two responses observed in this series occurred in the two patients with the highest leukocyte O6-MedG levels and also the lowest pretreatment ATase activity. Hematological toxicity developed in the same two patients.
Assuntos
Antineoplásicos/metabolismo , DNA Nucleotidiltransferases/metabolismo , DNA de Neoplasias/metabolismo , Desoxiguanosina/análogos & derivados , Guanina/análogos & derivados , Leucócitos/metabolismo , Melanoma/metabolismo , Triazenos/metabolismo , Adulto , Idoso , Antineoplásicos/uso terapêutico , Desoxiguanosina/biossíntese , Feminino , Guanina/metabolismo , Humanos , Linfócitos/enzimologia , Masculino , Melanoma/sangue , Pessoa de Meia-Idade , Triazenos/uso terapêuticoRESUMO
There is increasing evidence to indicate that O6-methyldeoxyguanosine (O6-MedG) formation in DNA is a critical cytotoxic event following exposure to certain anti-tumour alkylating agents and that the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) can confer resistance to these agents. We recently demonstrated a wide inter-individual variation in the depletion and subsequent regeneration of ATase in human peripheral blood lymphocytes following sequential DTIC (400 mg m-2) and fotemustine (100 mg m-2) treatment, with the nadir ATase activity occurring approximately 4 h after DTIC administration. We have now measured the formation and loss of O6-methyldeoxyguanosine (O6-MedG) in the DNA of peripheral leucocytes of eight patients receiving this treatment regimen. O6-MedG could be detected within 1 h and maximal levels occurred approximately 3-5 h after DTIC administration. Following the first treatment cycle, considerable inter-individual variation was observed in the peak O6-MedG levels, with values ranging from 0.71 to 14.3 mumol of O6-MedG per mol of dG (6.41 +/- 5.53, mean +/- s.d.). Inter- and intra-individual variation in the extent of O6-MedG formation was also seen in patients receiving additional treatment cycles. This may be a consequence of inter-patient differences in the capacity for metabolism of DTIC to release a methylating intermediate and could be one of the determinants of clinical response. Both the pretreatment ATase levels and the extent of ATase depletion were inversely correlated with the amount of O6-MedG formed in leucocyte DNA when expressed either as peak levels (r = -0.59 and -0.75 respectively) or as the area under the concentration-time curve (r = -0.72 and -0.73 respectively). One complete and one partial clinical response were seen, and these occurred in the two patients with the highest O6-MedG levels in the peripheral leucocyte DNA, although the true significance of this observation has yet to be established.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , DNA/metabolismo , Desoxiguanosina/análogos & derivados , Leucócitos/enzimologia , Nucleotidiltransferases/metabolismo , Adulto , Idoso , Dacarbazina/administração & dosagem , Desoxiguanosina/metabolismo , Feminino , Humanos , Linfócitos/enzimologia , Masculino , Pessoa de Meia-Idade , Compostos de Nitrosoureia/administração & dosagem , Compostos Organofosforados/administração & dosagemRESUMO
O6-Alkylguanine-DNA-alkyltransferase (ATase) activity was measured in extracts of 55 bladder tissue samples (46 tumour and nine uninvolved mucosal tissue) from Egyptian patients with schistosome-associated bladder carcinoma. Activity varied from 2.0 to 16.2 fmole ATase/microgram DNA (mean +/- S.D.; 5.6 +/- 4.0) or from 28 to 351 fmole ATase/mg (117 +/- 71). ATase levels in schistosome-associated bladder cancer tissues (5.6 +/- 4.0 fmole ATase/microgram DNA) tended to be lower than those observed in normal human bladder mucosal tissue (8.5 +/- 4.4 fmole ATase/microgram DNA). In a previous study (Badawi et al., Carcinogenesis, 1992, 13, 877-881) DNA-alkylation damage (O6-methyldeoxyguanosine) was found in 44/46 of these schistosome-associated bladder cancer samples at levels ranging from 0.012 to 0.485 mumole O6-MedG/mole deoxyguanosine. We now report an inverse correlation between the levels of methylation damage and ATase activity (r = -0.67; P < 0.001). These observations encourage further investigations of the possible role of environmental alkylating agents in the aetiology of early bladder cancer associated with schistosomiasis.