title: Prototype Systems Containing Human Cytochrome P450 for High-Throughput Real-Time Detection of DNA Damage by Compounds That Form DNA-Reactive Metabolites.
abstract: The formation of reactive metabolites through biotransformation is the suspected cause of many adverse drug reactions. Testing for the propensity of a drug to form reactive metabolites has increasingly become an integral part of lead-optimization strategy in drug discovery. DNA reactivity is one undesirable facet of a drug or its metabolites and can lead to increased risk of cancer and reproductive toxicity. Many drugs are metabolized by cytochromes P450 in the liver and other tissues, and these reactions can generate hard electrophiles. These hard electrophilic reactive metabolites may react with DNA and may be detected in standard in vitro genotoxicity assays; however, the majority of these assays fall short due to the use of animal-derived organ extracts that inadequately represent human metabolism. The current study describes the development of bacterial systems that efficiently detect DNA-damaging electrophilic reactive metabolites generated by human P450 biotransformation. These assays use a GFP reporter system that detects DNA damage through induction of the SOS response and a GFP reporter to control for cytotoxicity. Two human CYP1A2-competent prototypes presented here have appropriate characteristics for the detection of DNA-damaging reactive metabolites in a high-throughput manner. The advantages of this approach include a short assay time (120-180 min) with real-time measurement, sensitivity to small amounts of compound, and adaptability to a microplate format. These systems are suitable for high-throughput assays and can serve as prototypes for the development of future enhanced versions.
authors.affiliation: Centre for Toxicogenomics and Human Health (ToxOmics), Genetics, Oncology and Human Toxicology, NOVA Medical School/FCM, Universidade Nova de Lisboa , CEDOC II Building, Rua Câmara Pestana 6, room 2.23, 1150-082 Lisbon, Portugal.
authors.forename: Bernardo
authors.initials: B
authors.lastname: Brito Palma
authors.affiliation: School of Pharmacy, Texas Tech University , 1300 Coulter Avenue, Amarillo, Texas 79106, United States.
authors.forename: Charles W
authors.initials: CW
authors.lastname: Fisher
authors.affiliation: Centre for Toxicogenomics and Human Health (ToxOmics), Genetics, Oncology and Human Toxicology, NOVA Medical School/FCM, Universidade Nova de Lisboa , CEDOC II Building, Rua Câmara Pestana 6, room 2.23, 1150-082 Lisbon, Portugal.
authors.forename: José
authors.initials: J
authors.lastname: Rueff
authors.affiliation: Centre for Toxicogenomics and Human Health (ToxOmics), Genetics, Oncology and Human Toxicology, NOVA Medical School/FCM, Universidade Nova de Lisboa , CEDOC II Building, Rua Câmara Pestana 6, room 2.23, 1150-082 Lisbon, Portugal.
authors.forename: Michel
authors.initials: M
authors.lastname: Kranendonk
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage; D057166:High-Throughput Screening Assays; D006801:Humans
chemical_list: D004247:DNA; D003577:Cytochrome P-450 Enzyme System