PMID- 32798647 OWN - NLM STAT- MEDLINE DCOM- 20210510 LR - 20230301 IS - 1873-6351 (Electronic) IS - 0278-6915 (Print) IS - 0278-6915 (Linking) VI - 145 DP - 2020 Nov TI - Evaluation of pyrrolizidine alkaloid-induced genotoxicity using metabolically competent TK6 cell lines. PG - 111662 LID - S0278-6915(20)30552-4 [pii] LID - 10.1016/j.fct.2020.111662 [doi] AB - Pyrrolizidine alkaloid (PA)-containing plants are among the most common poisonous plants affecting humans, livestock, and wildlife worldwide. A large number of PAs are known to induce genetic damage after metabolic activation. In the present study, using a battery of fourteen newly developed TK6 cell lines, each expressing a single human cytochrome P450 (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C18, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7), we identified specific CYPs responsible for bioactivating three PAs - lasiocarpine, riddelliine, and senkirkine. Among the fourteen cell lines, cells expressing CYP3A4 showed significant increases in PA-induced cytotoxicity, evidenced by decreased ATP production and cell viability, and increased caspase 3/7 activities. LC-MS/MS analysis revealed the formation of 1-hydroxymethyl-7-hydroxy-6,7-dihydropyrrolizine (DHP), the main reactive metabolite of PAs, in CYP3A4-expressing TK6 cells. DHP was also detected in CYP3A5- and 3A7-expressing cells after PA exposure, but to a much lesser extent. Subsequently, using a high-throughput micronucleus assay, we demonstrated that PAs induced concentration-dependent increases in micronuclei and G2/M phase cell cycle arrest in three CYP3A variant-expressing TK6 cell lines. Using Western blotting, we observed that PA-induced apoptosis, cell cycle changes, and DNA damage were primarily mediated by CYP3A4. Benchmark dose (BMD) modeling demonstrated that lasiocarpine, of the three PAs, was the most potent inducer of micronuclei, with a BMD(100) of 0.036 muM. These results indicate that our TK6 cell system holds promise for genotoxicity screening of compounds requiring metabolic activation, identifying specific CYPs involved in bioactivation, and discriminating the genotoxic compounds that have different chemical structures. CI - Published by Elsevier Ltd. FAU - Li, Xilin AU - Li X AD - Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA. FAU - He, Xiaobo AU - He X AD - Office of Scientific Coordination, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA. FAU - Chen, Si AU - Chen S AD - Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA. FAU - Guo, Xiaoqing AU - Guo X AD - Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA. FAU - Bryant, Matthew S AU - Bryant MS AD - Office of Scientific Coordination, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA. FAU - Guo, Lei AU - Guo L AD - Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA. FAU - Manjanatha, Mugimane G AU - Manjanatha MG AD - Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA. FAU - Zhou, Tong AU - Zhou T AD - Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, MD, 20855, USA. FAU - Witt, Kristine L AU - Witt KL AD - Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 27709, USA. FAU - Mei, Nan AU - Mei N AD - Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA. Electronic address: nan.mei@fda.hhs.gov. LA - eng GR - Z99 ES999999/ImNIH/Intramural NIH HHS/United States PT - Journal Article DEP - 20200813 PL - England TA - Food Chem Toxicol JT - Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association JID - 8207483 RN - 0 (Mutagens) RN - 0 (Pyrrolizidine Alkaloids) RN - 9035-51-2 (Cytochrome P-450 Enzyme System) SB - IM MH - Apoptosis/drug effects MH - Cell Line MH - Cytochrome P-450 Enzyme System/metabolism MH - DNA Damage/drug effects MH - G2 Phase Cell Cycle Checkpoints/drug effects MH - Humans MH - Mutagenicity Tests MH - Mutagens/*toxicity MH - Pyrrolizidine Alkaloids/*toxicity PMC - PMC9969979 MID - NIHMS1627935 OTO - NOTNLM OT - Bioactivation OT - Cytochrome P450 OT - DNA damage OT - High-throughput assay OT - Micronuclei OT - TK6-derived cell lines COIS- Conflicts of interest The authors declare that there are no conflicts of interest. The information in this manuscript is not a formal dissemination of information by the U.S. Food and Drug Administration (FDA) or the U.S. National Institutes of Health (NIH) and does not constitute an advisory opinion, does not necessarily represent the formal position of FDA and NIH, and does not bind or otherwise obligate or commit the agency to the views expressed. EDAT- 2020/08/18 06:00 MHDA- 2021/05/11 06:00 PMCR- 2023/02/27 CRDT- 2020/08/18 06:00 PHST- 2020/06/09 00:00 [received] PHST- 2020/07/21 00:00 [revised] PHST- 2020/07/26 00:00 [accepted] PHST- 2020/08/18 06:00 [pubmed] PHST- 2021/05/11 06:00 [medline] PHST- 2020/08/18 06:00 [entrez] PHST- 2023/02/27 00:00 [pmc-release] AID - S0278-6915(20)30552-4 [pii] AID - 10.1016/j.fct.2020.111662 [doi] PST - ppublish SO - Food Chem Toxicol. 2020 Nov;145:111662. doi: 10.1016/j.fct.2020.111662. Epub 2020 Aug 13.