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The efficient preparation of corn oil solutions
Mutation Research, 206 (1988) 127-128 Elsevier
127
MTR 01294 L e t t e r to t h e E d i t o r
The efficient preparation of corn oil solutions Bologna, 23 December 1987
Dear Sir, In an interesting letter, Ashby (1987) has recently reported a simple method to bypass the problems encountered in conducting experiments with laboratory animals using water-insoluble materials. In fact, with these highly lipophilic chemical compounds, problems from 'blocked needles' and from 'different dose levels injected' are often encountered when non-uniform corn oil suspensions are given by oral gavage or intraperitoneal injection. According to Ashby, with the use of the Ultra-Turrax homogenizer a 'perfect milky suspension is produced'. So, following this method from a 'macro-suspension' in corn oil it is possible to obtain a 'micro-suspension'. Here, w e report an alternative and simple method to obtain, from the same macro-suspension in corn oil, a perfect and 'limpid solution' that can be easily administered to animals. The insoluble material and an equal amount of arabic gum, as emulsifying agent, in the presence of about 150 #1 of dimethylsulphoxide, are sonicated for 1-2 min to obtain a complete dissolution. This operation is best carried out by sonicating the mixture at intervals no longer than 1 min and maintaining the sample on ice. The subsequent addition of the appropriate amount of corn oil and the last sonication (as mentioned before) give a perfect corn oil solution. It is recommended that this solution is injected as soon as possible because the stability is strongly dependent upon the molecular structure of the highly lipophilic compounds (no more than 30-40 rain, in our experience). As an example of this simple and suitable procedure, we reported the application of this
protocol in the preparation of liver $9 fraction from phenobarbital (PB)- and fl-naphthoflavone (fl-NF)-induced rodents as metabolic activation system in in vitro short-term genotoxicity testing. The general schedule of this cytochrome P450/P448-type induction foresees the repeated i.p. injection of PB combined with a single dose of fl-NF 80 mg/kg b.w. (in corn oil) 48 h before the sacrifice (Matsushima et al., 1976). Using the standard procedure, where the last inducer is given by means of 'corn oil suspension', and that proposed here using the final 'corn oil solution' so obtained, we compared the increased enzyme activity after 48 h, when the maximal activity is reached. To compare the traditional procedure to the proposed one, the rates of ethoxyresorufin O-deethylation, a P448-specific activity (Lubet et al., 1985), induced by fl-NF were significantly different (P<0.01) in liver $9 fraction from treated male Swiss albino mice, CD1 strain. In particular, the observed values were 1.51 + 0.22 and 1.97 + 0.20 nmole × mg-1 X min-l, respectively. Apart from the advantage in the routine injection practice when a corn oil solution is used, the outcome of the experiments is certainly influenced positively by a major quantity of adsorbed compound and difference in animal response should be reduced (Ashby, 1987). To dissolve compounds with limited solubility in water in a perfect corn oil solution, we recommend the proposed simple operative protocol. The usefulness of this procedure could be great in the field of biomedical research where administration to laboratory animals is needed.
Istituto di Farrnacologia, Universitd degli Studi, Via lrnerio 48, 40126 Bologna ( ltaly) 0165-1218/88/$03.50 © 1988 ElsevierSciencePublishersB.V. (BiomedicalDivision)
Moreno Paolini Gian Luigi Biagi Giorgio Cantelli Forti
128
References Ashby, J. (1987) The efficient preparation of corn oil suspen~ sions, Mutation Res., 187, 45. Lubet, R.A., R.T. Mayer, J.W. Cameron, W.N. Raymond, D.M. Burke, T. Wolff and F.P. Guengerich (1985) Dealkylation of pentoxyresorufin: a rapid and sensitive assay for measuring induction of cytochromes P-450 by pheno-
barbital and other xenobiotics in the rat, Arch. Biochem. Biophys., 238, 43-48. Matsushima, T., M. Sawamura, K. Hara and T. Sugimura (1976) A safe substitute for polychlorinated biphenyls as an inducer of metabolic activation system, in: F.J. de Serres, J.R. Fouts, J.R. Bend and R.M. Philpot (Eds.), In Vitro Metabolic Activation in Mutagenesis Testing, Elsevier, Amsterdam, pp. 85-88.
127
MTR 01294 L e t t e r to t h e E d i t o r
The efficient preparation of corn oil solutions Bologna, 23 December 1987
Dear Sir, In an interesting letter, Ashby (1987) has recently reported a simple method to bypass the problems encountered in conducting experiments with laboratory animals using water-insoluble materials. In fact, with these highly lipophilic chemical compounds, problems from 'blocked needles' and from 'different dose levels injected' are often encountered when non-uniform corn oil suspensions are given by oral gavage or intraperitoneal injection. According to Ashby, with the use of the Ultra-Turrax homogenizer a 'perfect milky suspension is produced'. So, following this method from a 'macro-suspension' in corn oil it is possible to obtain a 'micro-suspension'. Here, w e report an alternative and simple method to obtain, from the same macro-suspension in corn oil, a perfect and 'limpid solution' that can be easily administered to animals. The insoluble material and an equal amount of arabic gum, as emulsifying agent, in the presence of about 150 #1 of dimethylsulphoxide, are sonicated for 1-2 min to obtain a complete dissolution. This operation is best carried out by sonicating the mixture at intervals no longer than 1 min and maintaining the sample on ice. The subsequent addition of the appropriate amount of corn oil and the last sonication (as mentioned before) give a perfect corn oil solution. It is recommended that this solution is injected as soon as possible because the stability is strongly dependent upon the molecular structure of the highly lipophilic compounds (no more than 30-40 rain, in our experience). As an example of this simple and suitable procedure, we reported the application of this
protocol in the preparation of liver $9 fraction from phenobarbital (PB)- and fl-naphthoflavone (fl-NF)-induced rodents as metabolic activation system in in vitro short-term genotoxicity testing. The general schedule of this cytochrome P450/P448-type induction foresees the repeated i.p. injection of PB combined with a single dose of fl-NF 80 mg/kg b.w. (in corn oil) 48 h before the sacrifice (Matsushima et al., 1976). Using the standard procedure, where the last inducer is given by means of 'corn oil suspension', and that proposed here using the final 'corn oil solution' so obtained, we compared the increased enzyme activity after 48 h, when the maximal activity is reached. To compare the traditional procedure to the proposed one, the rates of ethoxyresorufin O-deethylation, a P448-specific activity (Lubet et al., 1985), induced by fl-NF were significantly different (P<0.01) in liver $9 fraction from treated male Swiss albino mice, CD1 strain. In particular, the observed values were 1.51 + 0.22 and 1.97 + 0.20 nmole × mg-1 X min-l, respectively. Apart from the advantage in the routine injection practice when a corn oil solution is used, the outcome of the experiments is certainly influenced positively by a major quantity of adsorbed compound and difference in animal response should be reduced (Ashby, 1987). To dissolve compounds with limited solubility in water in a perfect corn oil solution, we recommend the proposed simple operative protocol. The usefulness of this procedure could be great in the field of biomedical research where administration to laboratory animals is needed.
Istituto di Farrnacologia, Universitd degli Studi, Via lrnerio 48, 40126 Bologna ( ltaly) 0165-1218/88/$03.50 © 1988 ElsevierSciencePublishersB.V. (BiomedicalDivision)
Moreno Paolini Gian Luigi Biagi Giorgio Cantelli Forti
128
References Ashby, J. (1987) The efficient preparation of corn oil suspen~ sions, Mutation Res., 187, 45. Lubet, R.A., R.T. Mayer, J.W. Cameron, W.N. Raymond, D.M. Burke, T. Wolff and F.P. Guengerich (1985) Dealkylation of pentoxyresorufin: a rapid and sensitive assay for measuring induction of cytochromes P-450 by pheno-
barbital and other xenobiotics in the rat, Arch. Biochem. Biophys., 238, 43-48. Matsushima, T., M. Sawamura, K. Hara and T. Sugimura (1976) A safe substitute for polychlorinated biphenyls as an inducer of metabolic activation system, in: F.J. de Serres, J.R. Fouts, J.R. Bend and R.M. Philpot (Eds.), In Vitro Metabolic Activation in Mutagenesis Testing, Elsevier, Amsterdam, pp. 85-88.