Modulation of rat liver cytochrome P450 by protein restriction assessed by biochemical and bacterial mutagenicity methods [corrected].

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.

S9 induction by the combined treatment with cyclohexanol and albendazole.

Cyclohexanol (CH) is an industrial solvent capable of inducing cytochrome P450 (CYP) enzymes including the CYP2E and CYP2B subfamilies. S9 from CH treated rats is able to activate several N-nitrosamines that are poorly activated by Aroclor 1254, phenobarbital/beta-naphthoflavone (PB/NF) or 3-methylcholanthrene S9 fractions into mutagens detected by the Salmonella typhimurium Ames test. Additionally, albendazole (ABZ) is a widely used anthelmintic drug and a potent inducer of the CYP1A subfamily. Since CYP1A, -2B and -2E subfamilies are implicated in the activation of several environmental mutagens/carcinogens, we studied CYP induction in the rat liver by the combined effect of these two compounds, and used S9 derived from it in the Salmonella/microsome assay to compare with S9 obtained from Aroclor or PB/NF treated rats. Total CYP content in hepatic microsomes was induced by Aroclor, but not by any of the other chemical combinations. Western blot and enzymatic activity analysis revealed quantitative but not qualitative differences in the CYP subfamilies present in the different microsomal fractions; all of the chemicals used increased the levels of CYP1A1/2, CYP2B1/2 and CYP2E1 with respect to control microsomes. CYP3A was not modified by the different treatments. When tested in the Ames test, Aroclor S9 and PB/NF S9 were the most effective in the activation of benzo[a]pyrene and 3-methylcholanthrene which are metabolized mainly by CYP1A1; additionally, the highest mutagenic potency of 2-aminofluorene and N-nitrosodipropylamine, which are activated by CYP1A2 and CYP2B, respectively, were obtained with PB/NF S9. All these compounds were also activated when CH/ABZ S9 was used as the exogenous source of metabolism. Mutagens like N-nitrosopyrrolidine and N-nitrosodimethylamine, activated by CYP2E1, were detected only when CH/ABZ S9 was used, and the effectiveness of the different S9 fractions in activating cyclophosphamide decreased in the following order: Aroclor = PB/NF > CH/ABZ > control. From these experiments we can conclude that the individual CYP- inducing properties of ABZ and CH complement each other when the two compounds are administered in conjunction and that the resulting S9 fraction is able to activate several known mutagens in the Ames test.

Inhibitory effect of naringin on the micronuclei induced by ifosfamide in mouse, and evaluation of its modulatory effect on the Cyp3a subfamily.

Naringin (Nar) is a flavonone found in high amount in grapefruit. In in vitro studies to determine its antimutagenicity results have been both positive and negative. On the other hand, an increase in the bioavailability of some medicaments have been observed when these are ingested together with grapefruit. It has been suggested that the effect may be related to the inhibition of the human enzyme Cytochrome P450 (CYP) 3A4 by Nar, an enzyme with a high aminoacid sequence homology with the Cyp3a in mouse. The present study was designed for three main purposes: (1) to determine whether Nar has a genotoxic effect in mouse in vivo. This was evaluated by measuring the rate of micronucleated polychromatic erythrocytes (MNPE); (2) to determine its antigenotoxic and its anticytotoxic potential on the damage produced by ifosfamide (Ifos). The first study was done by scoring the rate of MNPE, and the second one by establishing the index polychromatic erythrocytes/normochromatic erythrocytes (PE/NE); and (3) to explore whether its antigenotoxic mechanism of action is related to an inhibitory effect of Nar on the expression of the Cyp3a enzyme, an effect which could avoid the biotransformation of Ifos. A single oral administration was used for all groups in the experiment: three groups were given different doses of Nar (50, 250, and 500 mg/kg), other groups received the same doses of Nar plus an administration of Ifos (60 mg/kg), another group treated with distilled water and another with Ifos (60 mg/kg) were used as negative and positive controls, respectively. The micronuclei and the cell scoring were made in blood samples taken from the tail of the animals at 0, 24, 48, 72, and 96 h. The results showed that Nar was neither genotoxic nor cytotoxic with the doses tested, but Ifos produced an increase in the rate of MNPE at 24 and 48 h. The highest value was 24+/-1.57 MNPE per thousand cells at 48 h. The index PE/NE was significantly reduced by Ifos at 24 and 48 h. Concerning the antigenotoxic capacity of Nar, a significant decrease was observed in the MNPE produced by Ifos at the three tested doses. This effect was dose-dependent, showing the highest reduction in MNPE frequency (54.2%) at 48 h with 500 mg/kg of Nar. However, no protection on the cytotoxicity produced by Ifos was observed. Immunoblot analysis was used to assess the Cyp3a expression in liver and intestinal microsomes from mouse exposed orally to Nar. An induction in the Cyp3a protein was observed in both intestinal and hepatic microsomes from treated mice. This induction correlated with an increase in erythromycin N-demethylase activity. These data suggest that other mechanism(s) are involved in the antigenotoxic action of naringin.

Bacterial mutagens in the urine of patients under tinidazole treatment.

Tinidazole is an antiparasitic drug belonging to the 5-nitroimidazole family. It is prescribed against protozoal infestations and is widely used in Mexico as well as other underdeveloped countries where infectious diseases are the first cause of children mortality. The drug is a direct mutagen in Salmonella typhimurium TA100 strain and the presence of S9 mixture did not modify its mutagenic effect. At low doses no mutagenicity was detected with strains TA100NR, TA98 or UTH8414 (Uvr+ derivative of TA100). Urine from four patients under tinidazole treatment exhibited a mutagenic activity on strain TA100, greater than the expected from the tinidazole concentrations determined by high-performance liquid chromatography (HPLC). Components from the urine samples were separated on thin-layer chromatography (TLC) plates, and their mutagenic effects tested by direct application of the Salmonella assay onto sections of the developed chromatoplate. The Rf of one component (0.62) corresponded to the one obtained for a tinidazole standard and showed the expected mutagenicity, while a second component with an Rf=0.39, exhibited a mutagenic potency slightly greater than the observed for tinidazole; however, as in the case of the drug itself, reduction of the nitro group was necessary for a mutagenic activity.