- Email: [email protected]
phenobarbital-induced rat-liver homogenate: comparison of enzymatic activities and metabolic potency
261
Robertson, J.A., W.J. Harris and D,B. McGregor, Inveresk Research International Limited, Inveresk Gate, Musselburgh, EH21 7UB (Scotland) The detection of azo compounds by Ames test Azo dyes are poorly detected in the standard Ames test. It has been reported that pre-incubation and supplementation of $9 mix with riboflavin (1 pmole/ ml), ATP (5 pmoles/ml) and NADH (4 /zmoles/ml) improve their detection. With these experimental conditions, however, it was not possible to demonstrate, in our laboratory, that Trypan Blue and Congo Red were mutagens. $9 mix supplementation with either FMN (1 pmole/ml) or FAD (1 pmole/ml) and static pre-incubation in tubes of I.D. 11 m m for 30 min allowed detection of these dyes. Efficient aeration of the liquid incubation mixtures prevented detection and decolourisation (azo reduction) of these dyes. For example, in S. typhimurium TA1538, the revertants/plate (mean of 3) with Trypan Blue were: Pre4ncubation(min) Static Aerated
0 25 21
10 22 16
15 28 18
20 44 17
25 75 15
30 62 20
40 78 23
50 67 22
60 57 27
Concentrations of FMN in $9 mix greater than 1 pmole/ml reduced and eventually abolished detection of Trypan Blue, Evans Blue and Congo Red. In our pre-incubation protocol, activation of these bis-azo dyes involves (1) flavinmediated azo reduction, i n the tubes, releasing the premutagenic components o-tolidine (from Trypan and Evans Blue) or benzidine (from Congo Red), followed by (2) oxidative metabolism, on t h e plates, to mutagenic products. Response to the mono-azo dye, dimethylaminoazobenzene, was n o t improved by any of the above modifications; it also varied according to the $9 batch used and was related to the efficiency of enzyme induction.
Surer, W., and I. Jaeger, Preclinical Research Division, Sandoz Ltd., CH-4002 Basle (Switzerland) Aroclor 1254 and 5,6-benzoflavone/phenobarbital.induced rat4iver homogenate: comparison of enzymatic activities and metabolic potency The aim of this study was to compare enzymatic activities and metabolic potency of $9 rat-liver homogenates induced either by Aroclor 1254 or by its safe substitute, a combination of 5,6-benzoflavone and sodium phenobarbital. Livers from male rats (strain OFA SANDOZ) treated either with Aroclor 1254 or with 5,6-benzoflavone/phenobarbital, and livers from untreated control rats were used to prepare $9 homogenates. All 5 enzymatic activities determined in these preparations were marginally higher in the liver homogenate from Aroclor-1254-pretreated animals than in the 5,6-benzoflavone/phenobarbital-induced preparation. The activities in both were several times greater than
262
the activities in the control $9 homogenate. The mutagenic potency in the Ames test of 16 procarcinogens of a variety of chemical classes was determined using 3 different concentrations of the 2 induced and the control liver homogenates. Enzyme induction improved the sensitivity of the assay for many types of procarcinogen (e.g. benzo[a]pyrene, aminoazotoluene, 2-naphthylamine) while in the case of some aromatic amines {e.g. 6-aminochrysene) the control homogenate was clearly more active. A third group of compounds (e.g. benzidine) caused no clear differences. Van Bladeren, P.J., D.D. Breimer, G.R. Mohn and A. van der Gen, Departments of Pharmacology, Chemical Mutagenesis and Organic Chemistry, University of Leiden, Leiden (The Netherlands)
The role of the glutathione-S-transferases in the mutagenicity of vicinal dihalogeno compounds The glutathione (GSH)~-transferases, a group of cytosolic enzymes possessing overlapping substrate specificities, catalyse the conjugation of certain electrophiles to glutathione. These conjugation products are often excreted as mercapturic acid derivatives into urine, which is considered to be an important detoxication mechanism of xenobiotics. However, when compounds possessing 2 vicinal halogen atoms are conjugated with GSH, 2-halogenothioethers (sulfur half-mustards) are formed, which are reactive and mutagenic compounds [1]. This then represents an activation to mutagens by the GSH-S-transferases. GSH~-transferase activity was assessed for a series of 1,2-dihalogeno compounds. The specific activity followed the halide order C1 < Br < I and substituents on the carbon atoms bearing the halogen atoms have a decreasing effect. The same series was tested for mutagenic activity, using 100 000 g supernatant (cytosol) as activating system and Salmonella typhimurium TA100 as indicator bacteria. Two effects were clear. There is an optimum leaving group (C1 < Br I) as far as mutagenicity is concerned, because 1,2-dibromoethane is more mutagenic than 1,2~lichloroethane and 1,2-diiodoethane. And substituents again diminish mutagenic activity, e.g. 1,2-dibromopropane is less mutagenic than 1,2-dibromoethane. 1 V a n B l a d e r e n , P . J . , A. v a n d e r G e n , D . D . B r e i m e r a n d G . R . M o h n ( 1 9 7 9 ) B i o c h e m . P h a r m a c o l . , 28, 2521--2524.
Solymosy, F., Institute of Biochemistry, Department 1, Semmelweis University Medical School, Puskin 9, Budapest (Hungary)
The role of metabolic activation in sister-chromatid exchange inducing activity of ethyl carbamate (urethane) and vinyl carbamate Ethyl carbamate (EC) at 10 mM concentration induced more sister-chromatid exchanges (SCEs) in cultured human blood lymphocytes in the absence of
Robertson, J.A., W.J. Harris and D,B. McGregor, Inveresk Research International Limited, Inveresk Gate, Musselburgh, EH21 7UB (Scotland) The detection of azo compounds by Ames test Azo dyes are poorly detected in the standard Ames test. It has been reported that pre-incubation and supplementation of $9 mix with riboflavin (1 pmole/ ml), ATP (5 pmoles/ml) and NADH (4 /zmoles/ml) improve their detection. With these experimental conditions, however, it was not possible to demonstrate, in our laboratory, that Trypan Blue and Congo Red were mutagens. $9 mix supplementation with either FMN (1 pmole/ml) or FAD (1 pmole/ml) and static pre-incubation in tubes of I.D. 11 m m for 30 min allowed detection of these dyes. Efficient aeration of the liquid incubation mixtures prevented detection and decolourisation (azo reduction) of these dyes. For example, in S. typhimurium TA1538, the revertants/plate (mean of 3) with Trypan Blue were: Pre4ncubation(min) Static Aerated
0 25 21
10 22 16
15 28 18
20 44 17
25 75 15
30 62 20
40 78 23
50 67 22
60 57 27
Concentrations of FMN in $9 mix greater than 1 pmole/ml reduced and eventually abolished detection of Trypan Blue, Evans Blue and Congo Red. In our pre-incubation protocol, activation of these bis-azo dyes involves (1) flavinmediated azo reduction, i n the tubes, releasing the premutagenic components o-tolidine (from Trypan and Evans Blue) or benzidine (from Congo Red), followed by (2) oxidative metabolism, on t h e plates, to mutagenic products. Response to the mono-azo dye, dimethylaminoazobenzene, was n o t improved by any of the above modifications; it also varied according to the $9 batch used and was related to the efficiency of enzyme induction.
Surer, W., and I. Jaeger, Preclinical Research Division, Sandoz Ltd., CH-4002 Basle (Switzerland) Aroclor 1254 and 5,6-benzoflavone/phenobarbital.induced rat4iver homogenate: comparison of enzymatic activities and metabolic potency The aim of this study was to compare enzymatic activities and metabolic potency of $9 rat-liver homogenates induced either by Aroclor 1254 or by its safe substitute, a combination of 5,6-benzoflavone and sodium phenobarbital. Livers from male rats (strain OFA SANDOZ) treated either with Aroclor 1254 or with 5,6-benzoflavone/phenobarbital, and livers from untreated control rats were used to prepare $9 homogenates. All 5 enzymatic activities determined in these preparations were marginally higher in the liver homogenate from Aroclor-1254-pretreated animals than in the 5,6-benzoflavone/phenobarbital-induced preparation. The activities in both were several times greater than
262
the activities in the control $9 homogenate. The mutagenic potency in the Ames test of 16 procarcinogens of a variety of chemical classes was determined using 3 different concentrations of the 2 induced and the control liver homogenates. Enzyme induction improved the sensitivity of the assay for many types of procarcinogen (e.g. benzo[a]pyrene, aminoazotoluene, 2-naphthylamine) while in the case of some aromatic amines {e.g. 6-aminochrysene) the control homogenate was clearly more active. A third group of compounds (e.g. benzidine) caused no clear differences. Van Bladeren, P.J., D.D. Breimer, G.R. Mohn and A. van der Gen, Departments of Pharmacology, Chemical Mutagenesis and Organic Chemistry, University of Leiden, Leiden (The Netherlands)
The role of the glutathione-S-transferases in the mutagenicity of vicinal dihalogeno compounds The glutathione (GSH)~-transferases, a group of cytosolic enzymes possessing overlapping substrate specificities, catalyse the conjugation of certain electrophiles to glutathione. These conjugation products are often excreted as mercapturic acid derivatives into urine, which is considered to be an important detoxication mechanism of xenobiotics. However, when compounds possessing 2 vicinal halogen atoms are conjugated with GSH, 2-halogenothioethers (sulfur half-mustards) are formed, which are reactive and mutagenic compounds [1]. This then represents an activation to mutagens by the GSH-S-transferases. GSH~-transferase activity was assessed for a series of 1,2-dihalogeno compounds. The specific activity followed the halide order C1 < Br < I and substituents on the carbon atoms bearing the halogen atoms have a decreasing effect. The same series was tested for mutagenic activity, using 100 000 g supernatant (cytosol) as activating system and Salmonella typhimurium TA100 as indicator bacteria. Two effects were clear. There is an optimum leaving group (C1 < Br I) as far as mutagenicity is concerned, because 1,2-dibromoethane is more mutagenic than 1,2~lichloroethane and 1,2-diiodoethane. And substituents again diminish mutagenic activity, e.g. 1,2-dibromopropane is less mutagenic than 1,2-dibromoethane. 1 V a n B l a d e r e n , P . J . , A. v a n d e r G e n , D . D . B r e i m e r a n d G . R . M o h n ( 1 9 7 9 ) B i o c h e m . P h a r m a c o l . , 28, 2521--2524.
Solymosy, F., Institute of Biochemistry, Department 1, Semmelweis University Medical School, Puskin 9, Budapest (Hungary)
The role of metabolic activation in sister-chromatid exchange inducing activity of ethyl carbamate (urethane) and vinyl carbamate Ethyl carbamate (EC) at 10 mM concentration induced more sister-chromatid exchanges (SCEs) in cultured human blood lymphocytes in the absence of