S9 mix is a mixture of several enzymes and liver (that is, not in a subject but also, for example, in laboratory vessels ) are used in in vitro experiments to simulate a liver. Since the liver by means of enzymes can change substances in the blood tests would be of potentially toxic substances without these enzymes are not very meaningful ( see, eg, Ames test). Most S9 derived from rats and used.
The abbreviation " S9 " comes from the supernatant (supernatant) and centrifugation at 9.000G.
A problem should be noted: In mammalian cells, the S9 mix is also effective cytotoxic ( such as a cell poison).
For preparation: Rats pretreated with enzyme inducers (eg 1 x 500 mg / kg bw Aroclor1254 five days before killing). After isolation of the liver is homogenised in 0.25M sucrose and centrifuged at 9,000 g for 10 minutes. The supernatant is by definition the S9 mix. Centrifuged these S9 mix at 100,000 g for one hour, you get a microsomal pellet and a cytosolic supernatant.
Enabled various cytochrome P450 enzymes ( CYP). Are strongly activated: CYP1A1, 2B1, 2B4. Be activated Moderate: CYP2A1, 2B1, 2C5, 2C6, 3A2, 3A4, 4B1, P450 reductase, microsomal epoxide hydrolase ( mEH ), UDP -GT, GST isoenzymes
In the Ames test following S9- Standardmix is used: 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.
- Heterogeneous protein preparation