S9 mix
The S9-Mix is a mixture of several liver enzymes and is used in in-vitro experiments ( i.e. not in a living being, but e.g. in laboratory vessels) to simulate a liver . Since the liver can change substances in the blood with the help of enzymes, tests of possibly toxic substances would not be very meaningful without these enzymes (see e.g. Ames test ). Most S9 is obtained from rats and used.
The abbreviation "S9" comes from S upernatant (supernatant) and centrifugation at 9 g 000th
One problem should be considered: the S9-Mix acts like a cell poison on mammalian cells .
Extraction
For preparation: rats are pretreated with enzyme inducers (e.g. 1 x with 500 mg / kg body weight Aroclor 1254 five days before sacrifice). After isolating the liver, it is homogenized in 0.25 M sucrose and centrifuged at 9000 g for 10 minutes. The supernatant is by definition the S9 mix. If this S9 mix is centrifuged at 100,000 g for one hour, a microsome pellet and a cytosol supernatant are obtained.
Metabolic activation
Various cytochrome P450 enzymes (CYP) are activated . Strongly activated: CYP1A1, 2B1, 2B4. Moderately activated: CYP2A1 , 2B1, 2C5, 2C6, 3A2, 3A4, 4B1, P450 reductase, microsomal epoxide hydrolase (mEH), UDP-GT, GST isoenzymes
The following S9 standard mix is used in the Ames test : 10% S9 fraction, 4 mM NADP , 5 mM glucose-6-phosphate , 10 mM Na / K phosphate buffer (pH 7.4), 8 mM Mg- aspartate , 33 mM KCl.