Supernatant S9 Fractions

Otsuka M, Mine T, Ohuchi K, Ohmori S (1996) A detoxication route for aldehyde metabolism od diacetyl, acetoin and 2,3-butanediol in liver homogenate and perfused liver of rats. J Biochem 119 246-251 

Zhang J, Dean RA, Brzezinski MR, Bosron WF (1996) Gender-specific differences in activity and protein levels of cocaine corboxylesterase in rat tissue. Life Sci 59 1175-1184 

The same considerations as for other subcellular systems with respect to solvent influences have to be considered in tests in 9000g fractions because organic solvents deactivate cytochrome P450 isozymes and others concentration dependently (Busby 1999 Easterbrook 2001). Solvent influences are lowest for methanol and acetonitrile ( 10% inhibition at 0.3 % solvent) but higher for ethanol and DMSO. Therefore, as low as possible solvent amounts should be used. 

Besides applications in studying the metabolism of compounds the S9 liver fractions of human or aroclor induced rat are in use as metabolic activation of the Ames Test for mutagenicity of chemicals, in most instances without addition of Phase II cofactors (Maron and Ames 1983). S9 fractions are also used for activation in reportergene assays (Sumida et al. 2001). 

Brandon EFA, Raap CD, Meijerman I et al. (2003) An update on in vitro test methods in human hepatic drug biotransformation research pros and cons. Toxicol Applied Pharmacol 189 233-246 

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Microsomal activation for bacterial assays typically has involved crude supernatant fractions (S9) from livers of rats induced by polychlorobiphenyl mixtures, usually Aroclor 1254. These S9 mixtures contain a spectrum of mixed function oxidases and other enzymes active in biotransformation. In such mixtures, a given compound might be activated to become more mutagenic, may be inactivated, or may remain unaffected. All three types of response have been observed with various water residues (9, 14). 

Microsomes contain membrane-bound DMEs and cytosol has soluble ones, but another available product, liver subcellular fraction S9, the supernatant obtained from centrifugation at 9000 g, contains both. When all of the necessary co-factors are added, a more complete representation of the hepatic metabolism of a compound can be obtained. However, an even better one could come from the use of whole cells, hepatocytes, as discussed below. Consequently, S9 most often finds its use in conjunction with the Ames test for mutagenicity (Chapter 10). A metabolite rather than the parent compound can often be the genotoxic species, and incorporating S9 incubation allows both to be evaluated. " ... 

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