Lack of mutations in DNA polymerase beta of estradiol-induced hamster kidney tumors: sequence of hamster DNA polymerase beta cDNA.

We examined the effects of estradiol (E2), the natural estrogenic hormone, on the structure and expression of DNA polymerase beta (DNA pol beta), a DNA repair gene, from E2-induced primary kidney tumors of twelve Syrian hamsters, their metastases, and from kidney tissues surrounding the tumors. We sequenced the coding region of the hamster DNA pol beta and found it to differ from that of the human by 11%. No mutations were detected in the entire coding region including the catalytic domain of the DNA pol beta from E2-induced primary kidney tumors, their metastases, or from kidney tissues surrounding the tumors. The expression of the DNA pol beta mRNA was also not significantly altered in E2-induced kidney tumors or in kidney tissues surrounding the tumors compared to that of control kidney tissues. These results suggest that mutations in the DNA pol beta gene may not be involved in the induction or malignant progression of hamster kidney tumors induced by E2. The nucleotide sequence of the hamster DNA pol beta described here will be useful for the study of the structure and expression of this gene.

Estrogen-induced microsatellite DNA alterations are associated with Syrian hamster kidney tumorigenesis.

Exposure to estrogens is associated with an increase in cancers, including malignancies of the breast and uterus in humans, and of the kidney in hamsters. DNA damage induced by metabolic activation of estrogen has been postulated to result in gene mutations critical for the development of estrogen-induced kidney tumors in hamsters. As part of our examination of the genetic consequences of estrogen-induced DNA damage, we searched for estrogen-induced alterations in microsatellite DNA, a frequent site of mutation in tumors. Genomic DNA isolated from kidney of hamsters treated with estradiol, from estrogen-induced kidney tumors and from untreated age-matched controls, was examined by Southern blot analysis with three multi-locus oligonucleotide probes: (GACA)4, (CAC)6 and (CAG)6. Alterations in DNA fragments containing GACA and CAC tandem repeats were detected in kidney DNA of hamsters treated with hormone for 3 and 4 months, whereas no such effects were seen in control animals. In estrogen-induced tumors, microsatellite alterations were observed in fragments that contain these same two repeat sequences and also CAG repeat sequences. The induction of microsatellite alterations by estradiol in kidney DNA preceding estrogen-induced renal malignancy may play a role in hormone-induced tumorigenesis.

Quantitation of FAD-dependent cytochrome P450 reductase activity by photoreduction.

NADPH cytochrome P450 reductase binds two flavin cofactors, FMN and FAD, per molecule of reductase. We have developed an assay to quantitate the reduction activity of FMN-bound flavoprotein. This Trislight assay system takes advantage of the ability of photoactivated flavins to release electrons to acceptors. In turn, electrons derived from Tris buffer restore the flavin to the unexcited, ground state which can again undergo photoactivation to release another electron. FMN-bound reductase, supplied with reducing equivalents from a Tris-light electron generating system, reduces ferricyanide at a rate of 1.8 mumol/min/ nmol reductase. Holoreductase in this system is able to catalyze ferricyanide reduction at a rate of 1.6 mumol/ min/nmol reductase, while FAD-bound reductase has no activity. The 8-NH2-FAD and 8-OH-FAD analog-reconstituted FMN-bound reductase catalyzes the reduction of ferricyanide at rates of 0.43 and 0.28 mumol/min/ nmol reductase, respectively. The riboflavin-reconstituted FMN-bound reductase catalyzes ferricyanide reduction at a rate of 1.1 mumol/min/nmol reductase. FAD or its analogs at the concentrations used to reconstitute enzymatic activity do not support the reduction of ferricyanide in the Tris-light system in the absence of reductase protein. The free flavins, i.e., FMN, 8-OH-FAD, 8-NH2-FAD, and riboflavin, are able to support ferricyanide reduction at a rate of 0.40, 0.52, 0.87, and 0.16 mumol/min/nmol flavin, respectively. This is the first report of an enzymatic assay specific for FMN-bound NADPH cytochrome P450 reductase activity in the absence of its FAD cofactor. Moreover, this report describes the use of an assay procedure based on the provision of reducing equivalents by a Tris-light system which may be useful for other flavin redox enzymes in the absence of reduced pyridine nucleotides or biopterin cofactors.

Characterization of the FAD binding domain of cytochrome P450 reductase.

The redox potentials of FAD and FMN of Cytochrome P450 reductase (reductase) are equivalent in solution but differ by 138 mV when bound to reductase. The interaction of each flavin with its flavin binding domain confers the unique electron transferring abilities to each flavin. In order to determine flavin binding properties and activity of the FAD binding domain, we have expressed in pTrcHis three fragments (1161, 1244, and 1556 bp) of rat liver reductase cDNA encompassing the proposed FAD and NADPH binding domain. The FAD binding fragments from cells harboring the 1161- and 1556-bp-containing vectors were stable and bound 0.66 and 0.71 mol FAD/mol enzyme, respectively. Both fragments reduce ferricyanide (54 and 104% of FMN-less reductase/mol bound flavin, respectively) and participate in the transhydrogenation reaction of 3-AcPy-ADP (41 and 65% of FMN-less reductase/mol bound flavin, respectively). FAD-less fragments were purified and reconstituted with 8-amino-FAD and 8-chloro-FAD to determine binding efficiencies.

Expression of multiple forms of brain cytochrome P450.

Multiple forms of cytochrome P450 (P450) in brain tissue have been demonstrated to be expressible in brain tissue using polymerase chain reaction (PCR) techniques, Northern blotting, hydroxylation activity assessment and cloning approaches. The antidepressant drug imipramine is metabolized by brain microsomes to multiple products by pathways inhibitable by quinidine, 7,8-benzoflavone, and ketoconazole, well-known inhibitors of P450-catalyzed reactions. Moreover, PCR studies revealed that a number of P450s are expressible in brain tissue and in glioma C6 cells. Quantitative PCR studies further demonstrated the response of many of these forms to induction in agreement with hydroxylation activity results.

Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction.

Metabolism of therapeutic drugs in the body by the mixed function oxidase system is an important consideration in the analysis of a drug's effectiveness. P450-dependent metabolism within the brain of a neuro-specific drug may affect the drug's course of action. To determine whether cytochrome P450 was expressed in brain, RNA was isolated from the whole brains of rats treated with a variety of known hepatic P450 inducers, including amitriptyline, imipramine, isosafrole, phenobarbital, and beta-naphthoflavone. The RNA was analyzed for the presence of P450 isozymes by the PCR technique. Differential expression of P450IA1, P450IIB1, P450IIB2, P450IID, and P450IIE1 was detected in the brain samples, depending on the treatment. Cytochrome P450 reductase expression was also detected in the brain samples, giving strong evidence that the brain contains a competent mixed function oxidase system under all conditions studied.