Formation and persistence of O6-(2-hydroxyethyl)-2′-deoxyguanosine in DNA of various rat tissues following a single dose of N-nitroso-N-(2-hydroxyethyl)urea. An immuno-slot-blot study

Rabbit antibodies against O6-(2-hydroxyethyl)-2′-deoxyguanosine (O6-HEdG) were used to develop a highly sensitive immuno-slot-blot assay for this promutagenic base which enabled the quantitation of ≥ 3.6 μmol O6-HEdG/mol deoxy-guanosine, corresponding to ≥ 5 fmol in a 3-μg DNA sample. This assay was used to study DNA hydroxyethylation by N-nitroso-N-(2-hydroxyethyl)urea (HENU) in adult male F344 rats. Initial amounts of O6-HEdG 2 h after a single i.v. dose of 50 mg/kg were highest in kidney (81 μmol O6HEdG/mol deoxyguanosine), followed by lung and liver (67 and 55 μmol/mol dG respectively). Formation of O6-HEdG in cerebral DNA was considerably lower (18 μmol O6-HEdG/mol deoxyguanosine), probably reflecting delayed crossing of the blood—brain barrier by HENU due to its hydrophilicity. The formation of O6-HEdG in liver and kidney was strictly proportional to dose over a range of 5-50 mg HENU/kg. Repair of O6-HEdG was very rapid in liver (apparent half-life, 12 h), and somewhat slower in kidney and lung (approximate half-life, 40 h and 48 h respectively). In contrast, 62% of the initial amount of O6-HEdG in cerebral DNA was still present after 7 days. Saturation of the hepatic O6-alkyl-guanine-DNA alkyltransferase by pretreatment with N-nitrosodimethylamine (20 mg/kg) almost completely inhibited the removal of O6-HEdG, indicating that O6-HEdG is predominantly repaired by this repair enzym

p53 mutations in phenacetin-associated human urothelial carcinomas

Chronic abuse of the analgesic drug phenacetin is associated with an increased risk of development of transitional cell carcinomas of the urinary tract. It is unclear whether phenacetin acts through chronic tissue damage (phenacetin nephropathy) or via a genotoxic metabolite causing promutagenic DNA lesions. In the present study, we investigated 15 urothelial carcinomas from 13 patients with evidence of phenacetin abuse. Tumors were screened for p53 mutations in exons 5-8 by single-strand conformation polymorphism (SSCP) analysis, followed by direct sequencing of PCR-amplified DNA. p53 Mutations were detected in 8/14 primary tumors (57%). All except one were missense mutations located in exon 5 (three mutations), exon 6 (one), exon 7 (two) and exon 8 (one). The type of mutation varied, with a preference for CpG sites. A frameshift mutation resulting from the insertion of a single cytosine at codons 151/152 was detected in a bladder tumor and its lung metastasis. Urothelial carcinomas located in the renal pelvis and in the ureter of the same patient exhibited two different mutations, strongly suggesting that they developed independently. Another patient had tumors in the renal pelvis and bladder, both of which contained the same p53 mutation, indicating intracavitary metastatic spread. This demonstrates that screening of p53 mutations allows the clonal origin of tumors in patients with multiple primary and metastatic lesions to be determined. None of the tumors investigated contained mutations in codons 12, 13 or 61 of H-ras or K-ras protooncogene

Modulation of N-nitrosomethylbenzylamine bioactivation by diallyl sulfide in vivo

Diallyl sulfide (DAS), a major component of garlic oil, is an inhibitor of tumorigenesis by various metabolcally activated carcinogens. In rats, pretreatment with DAS has been observed to suppress completely the induction of oesophageal neoplasms by N-nitrosomethylbenzylamine (NMBzA) (Wargovich et al. (1988) Cancer Res., 48, 6872-6875). This communication reports the effects of DAS on overall NMBzA metabolism and on DNA methylation of NMBzA in vivo under conditions equivalent to a single treatment of the chemoprevention assay. Male Fischer 344 rats received a single i.g. dose of DAS (200 mg/kg body wt) followed by an s.c. injection of [methyl-14C]NMBzA (3.5 mg/kg). In controls, exhalation of 14CO2 was complete within 5 h (t½max = 1.2 h). with 50% of the injected radioactivity recovered as 14CO2. When DAS was given 3 h prior to [methyl-14C]NMBzA, 49% of the injected radioactivity was released within 10 h (t½max = 3 h). When DAS was administered 18 h before the carcinogen, 42% of [methyl-14C]NMBzA was converted to 14CO2, with exhalation complete after 6 h (t½max = 1.8 h). We further examined the effects of acute doses of 10—200 mg/kg of DAS on DNA methylation by a single dose of NMBzA (3.5 mg/kg; survival time, 6 h) administered 3 h later. At 200 mg/kg, DAS inhibited the formation of O6-methyldeoxyguanosine (O6-MEdG) in oesophagus (−26%), nasal mucosa (−51%), trachea (−68%) and lung (−78%). In liver, levels of 7-MEdG were reduced by 43%. Decreases in DNA methylation were proportional to dose for >25 mg/kg of DAS in oesophagus, liver and nasal mucosa, for 25—200 mg/kg in trachea and 10—50 mg/kg in lung. The dose—activity relationship for inhibition by DAS of DNA methylation by NMBzA suggests that short-term modulation of carcinogen bioactivation in situ contributes to but may not be sufficient for the chemoprevention of nitrosamine tumorigenesis by DAS. Diallyl sulfide (DAS), a major component of garlic oil, is an inhibitor of tumorigenesis by various metabolcally activated carcinogens. In rats, pretreatment with DAS has been observed to suppress completely the induction of oesophageal neoplasms by N-nitrosomethylbenzylamine (NMBzA) (Wargovich et al. (1988) Cancer Res., 48, 6872-6875). This communication reports the effects of DAS on overall NMBzA metabolism and on DNA methylation of NMBzA in vivo under conditions equivalent to a single treatment of the chemoprevention assay. Male Fischer 344 rats received a single i.g. dose of DAS (200 mg/kg body wt) followed by an s.c. injection of [methyl-14C]NMBzA (3.5 mg/kg). In controls, exhalation of 14CO2 was complete within 5 h (t½max = 1.2 h). with 50% of the injected radioactivity recovered as 14CO2. When DAS was given 3 h prior to [methyl-14C]NMBzA, 49% of the injected radioactivity was released within 10 h (t½max = 3 h). When DAS was administered 18 h before the carcinogen, 42% of [methyl-14C]NMBzA was converted to 14CO2, with exhalation complete after 6 h (t½max = 1.8 h). We further examined the effects of acute doses of 10—200 mg/kg of DAS on DNA methylation by a single dose of NMBzA (3.5 mg/kg; survival time, 6 h) administered 3 h later. At 200 mg/kg, DAS inhibited the formation of O6-methyldeoxyguanosine (O6-MEdG) in oesophagus (−26%), nasal mucosa (−51%), trachea (−68%) and lung (−78%). In liver, levels of 7-MEdG were reduced by 43%. Decreases in DNA methylation were proportional to dose for >25 mg/kg of DAS in oesophagus, liver and nasal mucosa, for 25—200 mg/kg in trachea and 10—50 mg/kg in lung. The dose—activity relationship for inhibition by DAS of DNA methylation by NMBzA suggests that short-term modulation of carcinogen bioactivation in situ contributes to but may not be sufficient for the chemoprevention of nitrosamine tumorigenesis by DA

Formation and persistence of O6-(2-hydroxyethyl)-2′-deoxyguanosine in DNA of various rat tissues following a single dose of N-nitroso-N-(2-hydroxyethyl)urea. An immuno-slot-blot study

Rabbit antibodies against O6-(2-hydroxyethyl)-2′-deoxyguanosine (O6-HEdG) were used to develop a highly sensitive immuno-slot-blot assay for this promutagenic base which enabled the quantitation of ≥ 3.6 μmol O6-HEdG/mol deoxy-guanosine, corresponding to ≥ 5 fmol in a 3-μg DNA sample. This assay was used to study DNA hydroxyethylation by N-nitroso-N-(2-hydroxyethyl)urea (HENU) in adult male F344 rats. Initial amounts of O6-HEdG 2 h after a single i.v. dose of 50 mg/kg were highest in kidney (81 μmol O6HEdG/mol deoxyguanosine), followed by lung and liver (67 and 55 μmol/mol dG respectively). Formation of O6-HEdG in cerebral DNA was considerably lower (18 μmol O6-HEdG/mol deoxyguanosine), probably reflecting delayed crossing of the blood—brain barrier by HENU due to its hydrophilicity. The formation of O6-HEdG in liver and kidney was strictly proportional to dose over a range of 5-50 mg HENU/kg. Repair of O6-HEdG was very rapid in liver (apparent half-life, 12 h), and somewhat slower in kidney and lung (approximate half-life, 40 h and 48 h respectively). In contrast, 62% of the initial amount of O6-HEdG in cerebral DNA was still present after 7 days. Saturation of the hepatic O6-alkyl-guanine-DNA alkyltransferase by pretreatment with N-nitrosodimethylamine (20 mg/kg) almost completely inhibited the removal of O6-HEdG, indicating that O6-HEdG is predominantly repaired by this repair enzym

p53 mutations in phenacetin-associated human urothelial carcinomas

Chronic abuse of the analgesic drug phenacetin is associated with an increased risk of development of transitional cell carcinomas of the urinary tract. It is unclear whether phenacetin acts through chronic tissue damage (phenacetin nephropathy) or via a genotoxic metabolite causing promutagenic DNA lesions. In the present study, we investigated 15 urothelial carcinomas from 13 patients with evidence of phenacetin abuse. Tumors were screened for p53 mutations in exons 5-8 by single-strand conformation polymorphism (SSCP) analysis, followed by direct sequencing of PCR-amplified DNA. p53 Mutations were detected in 8/14 primary tumors (57%). All except one were missense mutations located in exon 5 (three mutations), exon 6 (one), exon 7 (two) and exon 8 (one). The type of mutation varied, with a preference for CpG sites. A frameshift mutation resulting from the insertion of a single cytosine at codons 151/152 was detected in a bladder tumor and its lung metastasis. Urothelial carcinomas located in the renal pelvis and in the ureter of the same patient exhibited two different mutations, strongly suggesting that they developed independently. Another patient had tumors in the renal pelvis and bladder, both of which contained the same p53 mutation, indicating intracavitary metastatic spread. This demonstrates that screening of p53 mutations allows the clonal origin of tumors in patients with multiple primary and metastatic lesions to be determined. None of the tumors investigated contained mutations in codons 12, 13 or 61 of H-ras or K-ras protooncogene

Modulation of N-nitrosomethylbenzylamine bioactivation by diallyl sulfide in vivo

Diallyl sulfide (DAS), a major component of garlic oil, is an inhibitor of tumorigenesis by various metabolcally activated carcinogens. In rats, pretreatment with DAS has been observed to suppress completely the induction of oesophageal neoplasms by N-nitrosomethylbenzylamine (NMBzA) (Wargovich et al. (1988) Cancer Res., 48, 6872-6875). This communication reports the effects of DAS on overall NMBzA metabolism and on DNA methylation of NMBzA in vivo under conditions equivalent to a single treatment of the chemoprevention assay. Male Fischer 344 rats received a single i.g. dose of DAS (200 mg/kg body wt) followed by an s.c. injection of [methyl-14C]NMBzA (3.5 mg/kg). In controls, exhalation of 14CO2 was complete within 5 h (t½max = 1.2 h). with 50% of the injected radioactivity recovered as 14CO2. When DAS was given 3 h prior to [methyl-14C]NMBzA, 49% of the injected radioactivity was released within 10 h (t½max = 3 h). When DAS was administered 18 h before the carcinogen, 42% of [methyl-14C]NMBzA was converted to 14CO2, with exhalation complete after 6 h (t½max = 1.8 h). We further examined the effects of acute doses of 10—200 mg/kg of DAS on DNA methylation by a single dose of NMBzA (3.5 mg/kg; survival time, 6 h) administered 3 h later. At 200 mg/kg, DAS inhibited the formation of O6-methyldeoxyguanosine (O6-MEdG) in oesophagus (−26%), nasal mucosa (−51%), trachea (−68%) and lung (−78%). In liver, levels of 7-MEdG were reduced by 43%. Decreases in DNA methylation were proportional to dose for >25 mg/kg of DAS in oesophagus, liver and nasal mucosa, for 25—200 mg/kg in trachea and 10—50 mg/kg in lung. The dose—activity relationship for inhibition by DAS of DNA methylation by NMBzA suggests that short-term modulation of carcinogen bioactivation in situ contributes to but may not be sufficient for the chemoprevention of nitrosamine tumorigenesis by DAS. Diallyl sulfide (DAS), a major component of garlic oil, is an inhibitor of tumorigenesis by various metabolcally activated carcinogens. In rats, pretreatment with DAS has been observed to suppress completely the induction of oesophageal neoplasms by N-nitrosomethylbenzylamine (NMBzA) (Wargovich et al. (1988) Cancer Res., 48, 6872-6875). This communication reports the effects of DAS on overall NMBzA metabolism and on DNA methylation of NMBzA in vivo under conditions equivalent to a single treatment of the chemoprevention assay. Male Fischer 344 rats received a single i.g. dose of DAS (200 mg/kg body wt) followed by an s.c. injection of [methyl-14C]NMBzA (3.5 mg/kg). In controls, exhalation of 14CO2 was complete within 5 h (t½max = 1.2 h). with 50% of the injected radioactivity recovered as 14CO2. When DAS was given 3 h prior to [methyl-14C]NMBzA, 49% of the injected radioactivity was released within 10 h (t½max = 3 h). When DAS was administered 18 h before the carcinogen, 42% of [methyl-14C]NMBzA was converted to 14CO2, with exhalation complete after 6 h (t½max = 1.8 h). We further examined the effects of acute doses of 10—200 mg/kg of DAS on DNA methylation by a single dose of NMBzA (3.5 mg/kg; survival time, 6 h) administered 3 h later. At 200 mg/kg, DAS inhibited the formation of O6-methyldeoxyguanosine (O6-MEdG) in oesophagus (−26%), nasal mucosa (−51%), trachea (−68%) and lung (−78%). In liver, levels of 7-MEdG were reduced by 43%. Decreases in DNA methylation were proportional to dose for >25 mg/kg of DAS in oesophagus, liver and nasal mucosa, for 25—200 mg/kg in trachea and 10—50 mg/kg in lung. The dose—activity relationship for inhibition by DAS of DNA methylation by NMBzA suggests that short-term modulation of carcinogen bioactivation in situ contributes to but may not be sufficient for the chemoprevention of nitrosamine tumorigenesis by DA