Primer sets for analyzing fatty acyl-CoA oxidase expression

Details Primer sets for analyzing fatty acyl-CoA oxidase expression Primer sets for analyzing fatty acyl-CoA oxidase expression

2000-07-19

2001-05-08

Campbell, Eggerton A. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S091510, C536S024300, C536S024330

Reexamination Certificate

active

06228595

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method of detecting expression of isoenzymes of cytochrome P450 (CYP450) and Phase II conjugating enzymes in the rat. More specifically, this invention relates to specific 5′ and 3′ specific oligonucleotide primers, as well as a method of using the same with reverse transcriptase-polymerase chain reaction (RT-PCR) to detect mRNA expression of the major isoenzymes of CYP450 and fatty acyl-CoA oxidase in the rat. This invention includes the development of an in vitro culture system using rat hepatocytes which has been optimized for expression of both cytochrome P450 and Phase II conjugating enzymes.
2. Description of the Prior Art
The cytochrome P450 mixed function oxidases (MFO) are a group of enzymes which are predominantly expressed within the liver, kidney, lung and intestine of mammalian species where they play an important role in the oxidative metabolism of both endogenous and exogenous (xenobiotic) compounds. The role of the various members of this enzyme superfamily in the metabolism of drugs and chemicals, as well as their potential role in the generation of toxic metabolites and chemical-induced carcinogenesis, is well established.
In particular, induction of specific CYP450 isoforms has been associated with drug-drug interactions in humans and increases in liver weight, proliferation of the endoplasmic reticulum and non-genotoxic liver carcinogenicity and tumorigenicity in rodents. Guengerich,
Cancer Res
48:2946-2954, 1988; Lake,
Ann Rev Pharmacol Toxical
35: 483-507, 1995; Hankinson,
Ann Rev Pharmacol Toxicol
35:307-340, 1995; Parkinson,
Toxicol Pathol
24:45-57, 1996; and Kirby et al.,
Toxicol Pathol
24:458-467, 1996; and Burchell et al.,
Pharmacol Ther
43:261-289, 1989.
In addition to CYP450 enzymes, Phase II enzymes, which are involved in the conjugation of xenobiotics and their metabolites for excretion, have also been associated with the bioactivation of xenobiotics to toxic and tumorigenic metabolites. Parkinson,
Casarett
&
Doull's Toxicology, The Basic Science of Poisons
(Ed., Klaasen CD), 5th Edn, pp 113-186, McGraw-Hill, Inc., New York, 1996; Boberg,
Cancer Res
. 43:5163-5173, 1983; Curran et al.,
Endocrine Rev
12:135-150, 1991; Bock,
Pharmacogenetics
4:209-218, 1994; and Monks, et al.,
Toxicol Appl Pharmacol
106:1-19, 1990.
Examples of these Phase II enzymes include uridine diphosphate-glucuronosyltransferases (UDPGT), glutathione-S-transferases (GST), and sulfotransferases (ST). Historically, in vivo models are commonly used for the study of chemical-induced enzyme expression and hepatotoxicity. However, this approach is costly, time consuming and requires large quantities of test material.
Several approaches have been used to monitor the regulation of cytochrome P450 (CYP450) enzymes following exposure to xenobiotics. The approach most often employed is to measure the enzymatic profiles of microsomal protein fractions using enzyme selective substrates. Although this technique is useful for the study of substrate specificities, enzyme kinetics, and metabolism of chemicals there are several disadvantages which can limit the application of this technique in assessing the biochemical regulation of these enzymes. Two important disadvantages are that CYP450 enzyme activity requires additional cofactors (e.g., requirement for the presence of heme) and can show non-selectivity for, or be inhibited by certain chemical substrates (e.g., ketoconazole and metyrapone), resulting in potentially misleading or inaccurate assessments of enzyme activity.
Today, the chemical industry (Pharmaceutical and Chemical manufacturers) recognize the value in developing in vitro techniques to assess the safety and efficacy of drugs and chemicals at an early stage of development. In vitro techniques most commonly used to study the expression of hepatic metabolizing enzymes and cytotoxicity include precision-cut liver slices (Brendel et al.,
Methods in Toxicology
(Ed. Tyson and Frazier), Vol 1 A. pp 222-243, Academic Press Inc. NY., 1993; and Gandolfi et al.,
Toxicol. Pathol
. 24:58-61, 1996), primary cultures of hepatocytes and immortalized cell lines. Donato et al.,
In Vitro Cell Develop Biol
30A:574-580, 1994; and MacDonald et al.,
Human and Exp Toxicol
13:439-444, 1994.
However, without exception, these in vitro systems have limitations in their applications. For example, continuously dividing cell lines fail to preserve their ability to express or induce specific Phase I and II metabolizing enzymes, resulting in enzymatic activities which are either absent or too low to be measured. In addition, a loss of more than 50% of the total metabolizing enzyme levels have been reported within 24 hr of culture using non-dividing whole cell systems (e.g., precision-cut tissue slices and primary cell culture systems). Guzelian et al.,
Drug Metabol Rev
10:793-809, 1989; Waxman et al.,
Biochem J
. 271:113-119, 1990; Paine,
Chem Biol Interac
747:1-31, 1990; and Dunn et al.,
Biotechnol Prog
7:237-245, 1991.
A number of reports have demonstrated that primary hepatocytes cultured under conditions which restore normal cell's morphology and liver specific gene expression, can respond to xenobiotics with induction of specifically inducible CYP450 enzymes to levels comparable with those achieved in vivo. Bissel et al.,
Ann NY Acad Sci
349:85-98, 1980; Isom et al.,
J Cell Biol
105:2877-2885, 1987; Ben-Ze'ev et al.,
Proc Natl Acad Sci
85:2161-2165, 1988; Schuetz et al.,
J Cell Physiol
134:309-323, 1988; Musat et al.,
Hepatology
18:198-205, 1993; Arterburn et al.,
Hepatology
21:175-187, 1995; Kocarek et al.,
Mol Pharmacol
43:328-334, 1992; Sidhu et al.,
In Vitro Toxicol
7:225-242, 1994; and Zurlo et al.,
In Vitro Cell Develop Biol
32:211-220, 1996.
Examples of these cell culture conditions include the use of an extracellular matrix (ECM), Schuetz et al.,
J Cell Physiol
134:309-323, 1988, chemically defined culture media conditions and hepatocytes co-cultured with non-parenchymal cells. Begue et al.,
Hepatol
4:839-842, 1984; Rogiers et al.,
Biochem Pharmacol
40:1701-1706, 1990; Donato et al.,
In vitro Cell Develop Biol
30A:825-832, 1994; Guzelian et al.,
Proc Natl Acad Sci
85:9783-9787, 1988; Kocarek et al.,
Mol Pharmacol
38:440-444, 1990; Kocarek,
In Vitro Cell Develop Biol
29A:62-66, 1992; and Kocarek et al.,
Biochem Pharmacol
48:1815-1822, 1994.
More recently, polyclonal and monoclonal antibodies have been generated against various isoenzymes of CYP450 found in rat, thereby allowing for a more selective and defined analysis of CYP450 expression, Parkinson et al.,
Meth. Enzymol
. 206: 233-245, 1991. This approach to monitoring changes in CYP450 isoenzymes has many advantages over those which monitor enzyme activity, particularly with regard to enzymes which are regulated post-translationally (e.g., CYP2E1). However, the success of this approach is dependent on the quality and availability of reagents (e.g., polyclonal versus monoclonal antibodies) and may lack the specificity for determining enzyme subtype expression (e.g., CYP3A1 and CYP3A2). Moreover, the generation of antibodies and the measurement of proteins using Western immunoblot analysis are both labor intensive and time consuming and cannot be readily implemented when new enzymes are identified.
A number of reports have described use of ECM systems in studying the expression of liver-specific gene regulation in primary rat hepatocytes, Kocarek et al.,
Drug Metabol Dispos
23:415-421, 1995, Waxman et al.,
Biochem J
271:113-119, 1990, Sidhu et al.,
In Vitro Toxicol
7:225-242, 1994, and Zurlo et al., In Vitro
Cell Develop Biol
32:211-220, 1996.
These reports indicate that 1) hepatocyte genes expression decreases markedly after cell isolation, 2) hepatocyte isolation procedures result in altered expression of CYP450 mRNAs, 3) hepatocyte gene expression is restored after several days in culture, 4) the maintenance and/or inducibility of one or more P450 isoenzymes

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