S9 fraction

Among common in vitro metabolizing systems, liver microsomes and liver S9 fractions are used more often in metabolite synthesis than other systems. The majority of drug metabolism is mediated by CYPs [8]. Liver microsomes contain a high concentration of CYPs and other... 

Screening is usually carried out with liver microsomes from humans, rats, mice, dogs and monkeys and liver S9 fraction from aroclor 1254-induced rats. The incubation is typically mn with a volume of 0.2-1. OmL in a microcentrifuge or a glass tube. Different incubation conditions are used for CYP and UGT reactions. The incubation mixture for formation of oxidative metabolites and/or GSH conjugates contains ... 

Forti, G.C., Paolini, M., Hrelia, P. et al. (1984) NADPH-generating system influence on microsomal monooxygenase stability during incubation for the liver microsomal assay with rat and mouse S9 fractions. Mutation Research, 129, 291-297. 

Mortelmans et al. 1986 NTP 1991). Negative results were also obtained in mammalian cells, except for one observation of polyploidy in Chinese hamster CHL cells (Ishidate et al. 1984 NTP 1991 Perocco et al. 1983). Only a single report was located on the genotoxicity of 77-hexane metabolites induction of chromosome loss was observed in yeast with 2,5-hexanedione (Mayer and Goin 1994). It is also unclear if incubation with liver microsomes (S9 fraction) in in vitro genotoxicity tests results in similar metabolites to those observed in humans in vivo. 

Some of these problems can be overcome by the use of cell-based systems, in particular, primary hepatocytes. Hepatocytes closely simulate the metabolic systems found in the intact fiver and do not require additional cofactors for optimal enzyme activity. However, apart from greater technical difficulties in obtaining hepatocytes as opposed to S9 fraction, hepatocytes can effectively detoxify particular carcinogens and prevent their detection as mutagens. Despite these difficulties, hepatocytes have a role to play in mutagenicity screening, in both bacterial and mammalian-based systems (Tweats and Gatehouse, 1988). 

In summary, genetic toxicity tests with both bacterial and mammalian cells are normally carried out with rat liver cell-free systems (S9 fraction) from animals pretreated with enzyme inducers. However, investigations should not slavishly follow this regimen there may be sound scientifically based reasons for using preparations from different species or different organs, or for using whole cells such as hepatocytes. 

Standard Method of S9 Fraction Preparation. The following describes the production of hepatic S9 mix from rats induced with a combination of phenobarbitone and jS-naphthoflavone, and is an adaptation of the method described by Gatehouse and Delow (1979). 

S9 Mix. The S9 fraction prepared as described above is used as a component in S9 mix along with buffers and various enzyme cofactors. The amount of S9... 

Phosphate buffer 90.2 M Distilled water to make up to the required volume S9 fraction added at 0.1 ml per ml of S9 mix 100... 

Forster, R., Green, M.H.L. and Priestley, A. (1980). Optimal Levels of S9 fraction in Ames and fluctuation tests apparent importance of diffusion of metabolites from top agar. Carcinogenesis 2 1081-1085. 

S. K. Yang, K. Liu, F. P. Guengerich, Enantioselective Hydrolysis of Oxazepam 3-Acetate by Esterases in Human and Rat Liver Microsomes and Rat Brain S9 Fraction , Chirality 1990, 2, 150-155. 

Glucuronidation of MPA can be performed in high yield with the same technology (also pH 7), except using rabbit liver S9 fraction as the catalyst, which produces exclusively the (9-glucuronide (>95 % conversion). 

S9 fraction Can be frozen and used on demand Contains both cytosolic and membrane-bound enzyme Straighforward to use Closed system so may not be representative of in vivo situation... 

The reasons for identification of metabolites are manyfold but all boil down to human safety of drugs under clinical investigation. Initially metabolites of a drug are characterized with in vitro systems (microsomes, hepatocytes, S9 fractions, etc.) and later lead compounds are assessed using mouse, rat, rabbit, dog, and/or monkey. Subsequently, metabolites in humans are identified following dmg administration to assure that the nonclinical species undergoing safety assessment are adequately exposed to human metabolites of the dmg (Smith and Obach, 2006). 

ADME screening Plasma, urine, CSF liver, brain, S9 fractions, microsomes, PAMPA 4.8 Herman, 2002a... 

Figure 4. Comparison of Sephadex LH-20 column chromatography of DMBA deoxyribonucleoside adducts formed by enzymatic digestion of calf thymus DNA that had been treated for 2 h in the presence of Aroclor-induced rat liver S9 fraction with (a) 74 or (b) 740 nmol [3h]-DMBA per mg S9 fraction protein. The arrow is as defined in Figure 2 and the identities of peaks A-F are discussed in the text.

In model systems reflecting normal cooking conditions, a relatively weak mutagenic effect is exerted compared with that of HAAs. The type of mutagenic effect of these systems reflects base-pair in contrast to frameshift mutation, as caused by HAAs. Moreover, their action is effectively inactivated by liver chromosomal enzymes (S9 fraction), in contrast to HAAs, which require S9 activation to muta-genic/carcinogenic species.31... 

Approximately 106 cells treated with at least three doses of the test article with and without the S9 fraction for two hours at 37 °C in growth medium... 

S9 fraction easy to use,cheap, phase I and II present, whole metabolic profile observed addition of cofactors (complex mixtures), lower enzyme activity than microsomes/supersomes, induction not modeled... 

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). 

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