RESUMO
Protein restriction (PR) significantly inhibits spontaneous and chemical carcinogenesis. Several factors seem to be involved in this effect, including a decrease in body weight, cellular proliferation and DNA damage and an increase in antioxidant defenses. The current study was designed to determine modifications in some hepatic cytochromes P450 (CYPs) due to a hypoproteic diet and to investigate its implications on chemical mutagenesis. Western blot analysis showed decreases of 73, 40 and 74% in CYP1A, CYP2B and CYP2E1 protein concentrations in hepatic microsomes from animals fed a protein-restricted (6% protein) diet for 6 weeks in comparison with microsomes from rats fed a 24% protein diet during the same period. In the same way, low protein fed animals showed a 3.5-fold decrease in hepatic CYP1A1-associated ethoxyresorufin O-deethylase activity, a 6-fold decrease in CYP1A2-associated methoxyresorufin O-demethylase activity, a 1.7-fold decrease in CYP2B1-associated penthoxyresorufin O-dealkylase activity, a 9-fold decrease in CYP2B2-associated benzyloxyresorufin O-dealkylase and, finally, a 3.4-fold decrease in CYP2E1-associated 4-nitrophenol hydroxylase activity. As a result of decreased CYP hepatic protein concentrations and enzymatic activities, liver S9 from rats fed a hypoproteic diet was less efficient in activating promutagens than S9 prepared from rats fed a 24% protein diet in the Ames test. Mutagenic potency obtained with protein-restricted S9 was reduced 25-fold for 2-aminoanthracene, 1.5-fold for N-nitrosodipropylamine, 12.5-fold for N-nitrosodibutylamine, 2-fold for cyclophosphamide and N-nitrosopyrrolidine and 71-fold for N-nitrosodimethylamine. However, the mutagenic potency of benzo[a]pyrene was the same (4 revertants/ microg) with S9 derived from rats fed either a 6 or 24% protein diet.
Assuntos
Biotransformação/efeitos dos fármacos , Carcinógenos/farmacocinética , Sistema Enzimático do Citocromo P-450/biossíntese , Dieta com Restrição de Proteínas , Proteínas Alimentares/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Testes de Mutagenicidade , Mutagênicos/farmacocinética , Animais , Hidrocarboneto de Aril Hidroxilases/biossíntese , Hidrocarboneto de Aril Hidroxilases/genética , Peso Corporal/efeitos dos fármacos , Carcinógenos/toxicidade , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/biossíntese , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2E1/biossíntese , Citocromo P-450 CYP2E1/genética , Sistema Enzimático do Citocromo P-450/genética , Dano ao DNA , Proteínas Alimentares/administração & dosagem , Indução Enzimática/efeitos dos fármacos , Hidroliases/biossíntese , Hidroliases/genética , Masculino , Microssomos Hepáticos/enzimologia , Mutagênese , Mutagênicos/toxicidade , Oxazinas/farmacocinética , Oxazinas/toxicidade , Oxirredutases/biossíntese , Oxirredutases/genética , Ratos , Ratos Wistar , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Esteroide Hidroxilases/biossíntese , Esteroide Hidroxilases/genética , Especificidade por SubstratoRESUMO
In a previous report we described the ability of cyclohexanol to induce CYP activity. In order to characterize this induction we tested the capacity of liver S9 from rats orally treated with cyclohexanol for 5 days, to activate several carcinogenic nitrosamines into mutagens in the Salmonella typhimurium TA100 test system. Additionally, Western blot analysis of hepatic microsomes from the same treated animals were analysed with specific antibodies against P450 protein families 1A1/A2, 2B1/B2 and 2E1. Cyclohexanol-S9 mixture was more efficient in activating the following nitrosamines: N-nitrosodimethylamine (NDMA), N-nitrosodipropylamine (NDPA), N-nitrosomethylpropylamine (NMPA), N-nitrosodibutylamine (NDBA), and N-nitrosopyrrolidine (NPYR) into bacterial mutagens than S9 from non-treated animals. The mutagenicity of N-nitrosodiethylamine (NDEA) was not modified in the presence of S9 from cyclohexanol-treated animals. Since the main metabolic pathway leading to the production of mutagenic intermediates of NDMA and NPYR is catalysed by isozyme CYP2E1 and that of NDPA, NMPA and NDBA by CYP2B1/B2, mutagenicity experiments predicted that cyclohexanol induces these two P450 isozyme families. Western blot analysis confirmed the results of the mutagenicity assay, showing an increase in the intensity of CYP2E1 and CYP2B1/B2 protein bands in hepatic microsomes from cyclohexanol treated rats in comparison with non-treated controls. Bacterial mutagenicity tests with specific pro-mutagens were good predictors of the P450 induction properties of cyclohexanol.
Assuntos
Cicloexanóis/toxicidade , Sistema Enzimático do Citocromo P-450/biossíntese , Isoenzimas/biossíntese , Mutagênicos/toxicidade , Animais , Indução Enzimática/efeitos dos fármacos , Técnicas In Vitro , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Testes de Mutagenicidade , Mutagênicos/metabolismo , Nitrosaminas/metabolismo , Nitrosaminas/toxicidade , Ratos , Ratos Wistar , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Solventes/toxicidadeRESUMO
The S9 fraction obtained from rats orally pretreated for 3 days with cyclohexanol was able to activate the pro-mutagen N-nitrosodimethylamine (NDMA) into highly mutagenic metabolite(s) detected in the TA100 strain of Salmonella typhimurium. NDMA was not mutagenic when uninduced S9 was used as metabolic source but was approximately twice more mutagenic with cyclohexanol-induced S9 compared to ethanol-induced S9. Separation of microsomal proteins by sodium dodecylsulfate gel electrophoresis, displayed protein bands situated in the range of 50,000 to 52,000 molecular weight induced by both, ethanol and cyclohexanol. These results are evidence of the induction properties of cyclohexanol.
