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Metabolic Activation
Cytochrome P450-dependent mixed function
oxygenases (MFO’s) play a central role in the
metabolism of physiologic substrates such as
steroids (Nebert, D., Nature, 347:709, 1990) as
well as xenobiotics and natural products such as
polycyclic hydrocarbons and aflatoxins,
respectively (Guengerich, F., Mammalian
Cytochromes P450, Vols. 1&2, CRC Press,1987).
P450-mediated metabolism of xenobiotics as well
as of natural products may result in the
generation of metabolites that exhibit
activities, such as toxicities, that are not
observed in the parental material. Most in vitro
systems utilize prokaryotic target cells or
mammalian cells that lack significant MFO
activities and therefore supplementation with an
exogenous source of P450 and appropriate
cofactors is required if the in vitro biological
activities of potential metabolites are to be
evaluated.
Post mitochondrial supernatants (S9’s) can be
readily prepared from various animal tissues and
are a convenient source of P450 activities. S9’s
contain families of P450’s that are
characterized, in purified form, by broad
substrate affinities (Oguri, et al., Annu Rev.
Pharm. Tox. 34:251, 1994) and that are present
in amounts that are species-, sex-,and tissue
dependent. The P450 activities in S9’s can be
altered by treatments of donor animals with pure
chemicals (e.g. phenobarbitol; dexamethasone)
that may act to increase the abundance of
specific P450 gene products (Soucek & Gut,
Xenobotica, 1:83,1992). In contrast, treatments
with Aroclor 1254, a mixture of PCB’s result in
a more general increase in P450-dependent
activities (Alvarles, et al., PNAS, 70:1321,
1973). Following the observations of Mal ing (Mutat.
Res., 13:425, 1971) and Ames, et al.,
(PNAS,70:2281, 1973), metabolic “activation”
systems based on S9 derived from the livers of
Aroclor 1254 induced male Sprague Dawley rats
have been widely applied in in vitro studies;
e.g., in the Salmonella mutagenicity test (Ames,
et al., Mutat. Res., 31:347, 1975; Maron & Ames,
Mutat. Res., 113:173, 1983),
L5178Y mouse lymphoma cell assay (Clive, et al.,
in: Chemical mutagens: Principles and methods
for their detection. A. Hollaender, eds., Plenum
Press, N.Y., 1973, pp. 79-103) and cytogenetic
assays (Perry, P., in: Chemical mutagens:
Principle and methods for their detection. J.
deSerres & A. Hollaender, eds., Plenum Press,
N.Y., 1980, pp. 1-39).
S9’s derived from various laboratory rodents and
utilizing several inducing agents are available.
Except as specified, our S9’s and cofactor (NADPH
regenerating) systems are prepared as described
by Maron & Ames (Mutat. Res., 113:173, 1983).
MOLTOX S9 preparations and cofactor reagents for
use in metabolic activation studies
quality and
performance and are used by leading industrial,
government and academic laboratories worldwide.
Each lot of MOLTOX Rat Liver S9 is analyzed for
each of the following key properties:
• Total Protein.
• P450 Isozyme Activities: Resorufin dealkylase
assays (EROD, MROD, BROD and PROD) measuring
isoforms such as 1A1, 1A2, and 2B1 are
performed.
• Bioassay: Salmonella mutagenesis tests – dose
responses in TA98 with ethidium bromide (1A1)
and in TA1535 with cyclophosphamide (2B) and S9
titrations using benzo(a)pyrene and
2-aminoanthracene with TA100.
MOLTOX S9’s and activation system products are
accompanied by GLP-compliant Production &
Formulation and Quality Assurance Certificates –
your assurance of quality and performance.
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Moltox © 1986 |