{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,6]],"date-time":"2026-03-06T05:08:43Z","timestamp":1772773723293,"version":"3.50.1"},"reference-count":46,"publisher":"MDPI AG","issue":"23","license":[{"start":{"date-parts":[[2024,11,22]],"date-time":"2024-11-22T00:00:00Z","timestamp":1732233600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Cytochrome P450 3A (CYP3A) enzymes catalyze the metabolism of a wide range of endogenous and exogenous compounds. Genetic variations in the 3 CYP3A isoforms (CYP3A28, CYP3A74, and CYP3A76) may influence their expression and activity, leading to inter-individual differences in xenobiotic metabolism. In domestic cattle, understanding how genetic variations modulate CYP3A activity is crucial for both its therapeutic implications (clinical efficacy and adverse drug effects) and food safety (residues in foodstuff). Here, we updated the variant calling of CYP3As in 300 previously sequenced Piedmontese beef cattle, using the most recent reference genome, which contains an updated, longer sequence for CYP3A28. All but one previously identified missense variants were confirmed and a new variant, R105W in CYP3A28, was discovered. Through computational mutagenesis and molecular docking, we computationally predicted the impact of all identified CYP3A variant enzymes on protein stability and their affinity for aflatoxin B1 (AFB1), a potent carcinogen and food contaminant. For CYP3A28, we also computationally predicted its affinity for the probe substrate nifedipine (NIF). We found that CYP3A28 with R105W variant cannot accommodate NIF nor AFB1 in the binding pocket, thus affecting their metabolism. Our work provides computational foundation and prioritized ranking of CYP3A variants for future experimental validations.<\/jats:p>","DOI":"10.3390\/ijms252312529","type":"journal-article","created":{"date-parts":[[2024,11,22]],"date-time":"2024-11-22T04:21:36Z","timestamp":1732249296000},"page":"12529","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Impact of Missense Mutations on AFB1 Metabolism in Bovine Cytochrome P4503A Isoforms: A Computational Mutagenesis and Molecular Docking Analysis"],"prefix":"10.3390","volume":"25","author":[{"given":"Ludovica","family":"Montanucci","sequence":"first","affiliation":[{"name":"Department of Neurology, McGovern Medical School, UTHealth\u2014University of Texas Health Science Centre at Houston, Houston, TX 77030, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-2130-0161","authenticated-orcid":false,"given":"Silvia","family":"Iori","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedicine and Food Science, University of Padua, 35020 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maija","family":"Lahtela-Kakkonen","sequence":"additional","affiliation":[{"name":"School of Pharmacy, University of Eastern Finland, 70210 Kuopio, Finland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2424-7484","authenticated-orcid":false,"given":"Marianna","family":"Pauletto","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedicine and Food Science, University of Padua, 35020 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0015-3800","authenticated-orcid":false,"given":"Mery","family":"Giantin","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedicine and Food Science, University of Padua, 35020 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3637-5643","authenticated-orcid":false,"given":"Mauro","family":"Dacasto","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedicine and Food Science, University of Padua, 35020 Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,11,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"690","DOI":"10.1006\/bbrc.1999.1887","article-title":"Forty Years of Cytochrome P450","volume":"266","author":"Omura","year":"1999","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"413","DOI":"10.3109\/03602539709037591","article-title":"Human Cytochrome P450 Enzymes: A Status Report Summarizing Their Reactions, Substrates, Inducers, and Inhibitors","volume":"29","author":"Rendic","year":"1997","journal-title":"Drug Metab. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1081\/DMR-120001392","article-title":"Summary of Information on Human CYP Enzymes: Human P450 Metabolism Data","volume":"34","author":"Rendic","year":"2002","journal-title":"Drug Metab. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1124\/dmd.111.042259","article-title":"Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities","volume":"40","author":"Ohtsuki","year":"2012","journal-title":"Drug Metab. Dispos."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0009-8981(01)00419-3","article-title":"Pharmacogenomics: Will it change the field of medicine?","volume":"308","author":"Wieczorek","year":"2001","journal-title":"Clin. Chim. Acta"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1038\/86882","article-title":"Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression","volume":"27","author":"Kuehl","year":"2001","journal-title":"Nat. Genet."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"47","DOI":"10.2217\/pgs.12.187","article-title":"CYP3A4* 22: Promising newly identified CYP3A4 variant allele for personalizing pharmacotherapy","volume":"14","author":"Elens","year":"2013","journal-title":"Pharmacogenomics."},{"key":"ref_8","first-page":"274","article-title":"Intronic polymorphism in CYP3A4 affects hepatic expression and response to statin drugs","volume":"11","author":"Wang","year":"2011","journal-title":"Pharm. J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1044","DOI":"10.1038\/clpt.2011.336","article-title":"PPARA: A novel genetic determinant of CYP3A4 in vitro and in vivo","volume":"91","author":"Klein","year":"2012","journal-title":"Clin. Pharm. Ther."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Khalifa, H.O., Shikoray, L., Mohamed, M.-Y.I., Habib, I., and Matsumoto, T. (2024). Veterinary Drug Residues in the Food Chain as an Emerging Public Health Threat: Sources, Analytical Methods, Health Impacts, and Preventive Measures. Foods, 13.","DOI":"10.3390\/foods13111629"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1007\/s12550-016-0265-7","article-title":"Mycotoxins as human carcinogens\u2014The IARC Monographs classification","volume":"33","author":"Ostry","year":"2017","journal-title":"Mycotoxin Res."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"EFSA (European Food Safety Authority) Panel on Contaminants in the Food Chain (CONTAM), Schrenk, D., Bignami, M., Bodin, L., Chipman, J.K., del Mazo, J., Grasl-Kraupp, B., Hogstrand, C., Hoogenboom, L., and Leblanc, J.C. (2020). Risk assessment of aflatoxins in food. EFSA J., 18, e06040.","DOI":"10.2903\/j.efsa.2020.6040"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"528","DOI":"10.1111\/j.1365-2885.2010.01173.x","article-title":"Proposed New Nomenclature for Bos Taurus Cytochromes P450 Involved in Xenobiotic Drug Metabolism","volume":"33","author":"Zancanella","year":"2010","journal-title":"J. Vet. Pharmacol. Ther."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1016\/j.tvjl.2014.07.028","article-title":"Absolute Quantification and Modulation of Cytochrome P450 3A Isoforms in Cattle Liver","volume":"202","author":"Zancanella","year":"2014","journal-title":"Vet. J."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Giantin, M., K\u00fcblbeck, J., Zancanella, V., Prantner, V., Sansonetti, F., Schoeniger, A., Tolosi, R., Guerra, G., Ros, S.D., and Dacasto, M. (2019). DNA Elements for Constitutive Androstane Receptor- and Pregnane X Receptor-Mediated Regulation of Bovine CYP3A28 Gene. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0214338"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Giantin, M., Rahnasto-Rilla, M., Tolosi, R., Lucatello, L., Pauletto, M., Guerra, G., Pezzato, F., Lopparelli, R.M., Merlanti, R., and Carnier, P. (2019). Functional Impact of Cytochrome P450 3A (CYP3A) Missense Variants in Cattle. Sci. Rep., 9.","DOI":"10.1038\/s41598-019-56271-8"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1023\/A:1010686508307","article-title":"In vitro metabolism of 14C-moxidectin by hepatic microsomes from various species","volume":"25","author":"Dupuy","year":"2001","journal-title":"Vet. Res. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1053\/rvsc.1998.0239","article-title":"In vitro complex formation and inhibition of hepatic cytochrome P450 activity by different macrolides and tiamulin in goats and cattle","volume":"66","author":"Horbach","year":"1999","journal-title":"Res. Vet. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1046\/j.1365-2885.2001.00362.x","article-title":"Oxidative monensin metabolism and cytochrome P450 3A content and functions in liver microsomes from horses, pigs, broiler chicks, cattle and rats","volume":"24","author":"Nebbia","year":"2001","journal-title":"J. Vet. Pharmacol. Ther."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1111\/jvp.13395","article-title":"Effects of Acaricides on the Activities of Monooxygeases in Bovine Liver Microsomes","volume":"46","author":"Larsen","year":"2023","journal-title":"J. Vet. Pharmacol. Ther."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3097","DOI":"10.1007\/s00204-024-03799-y","article-title":"New insights into aflatoxin B1 mechanistic toxicology in cattle liver: An integrated approach using molecular docking and biological evaluation in CYP1A1 and CYP3A74 knockout BFH12 cell lines","volume":"98","author":"Iori","year":"2024","journal-title":"Arch. Toxicol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"EFSA (European Food Safety Authority) (2007). Opinion of the scientific panel on contaminants in the food chain (CONTAM) related to the potential increase of consumer health risk by a possible increase of the existing maximum levels for aflatoxins in almonds, hazelnuts and pistachios and derived products. EFSA J., 5, 446.","DOI":"10.2903\/j.efsa.2007.446"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1016\/j.canlet.2005.11.018","article-title":"Aflatoxin B1-induced DNA damage and its repair","volume":"241","author":"Bedard","year":"2006","journal-title":"Cancer Lett."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Wang, L., Huang, Q., Wu, J., Jiang, J., Yan, H., Huang, J., Sun, Y., and Deng, Y. (2022). The metabolism and biotransformation of AFB1: Key enzymes and pathways. Biochem. Pharmacol., 199.","DOI":"10.1016\/j.bcp.2022.115005"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"EFSA (European Food Safety Authority) (2004). Opinion of the scientifc panel on contaminants in the food chain (CONTAM) related to Afatoxin B1 as undesirable substance in animal feed. EFSA J., 2, 239.","DOI":"10.2903\/j.efsa.2004.39"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1080\/02652030701823142","article-title":"Mycotoxins in cattle feeds and carry-over to dairy milk: A review","volume":"25","year":"2008","journal-title":"Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.tvjl.2007.12.034","article-title":"The role of mycotoxins in the health and performance of dairy cows","volume":"176","year":"2008","journal-title":"Vet. J."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Jiang, Y., Ogunade, I.M., Vyas, D., and Adesogan, A.T. (2021). Aflatoxin in dairy cows: Toxicity, occurrence in feedstufs and milk and dietary mitigation strategies. Toxins, 13.","DOI":"10.3390\/toxins13040283"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2892","DOI":"10.1002\/jsfa.6225","article-title":"Mycotoxin Occurrence in Feed and Feed Raw Materials Worldwide: Long-Term Analysis with Special Focus on Europe and Asia","volume":"93","author":"Streit","year":"2013","journal-title":"J. Sci. Food Agric."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2773","DOI":"10.1080\/10408398.2019.1658570","article-title":"Worldwide Contamination of Food-Crops with Mycotoxins: Validity of the Widely Cited \u2018FAO Estimate\u2019 of 25%","volume":"60","author":"Eskola","year":"2020","journal-title":"Crit. Rev. Food Sci. Nutr."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Leggieri, M.C., Toscano, P., and Battilani, P. (2021). Predicted Aflatoxin B1 Increase in Europe Due to Climate Change: Actions and Reactions at Global Level. Toxins, 13.","DOI":"10.3390\/toxins13040292"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e13850","DOI":"10.7717\/peerj.13850","article-title":"Research Progress in Toxicological Effects and Mechanism of Aflatoxin B1 Toxin","volume":"10","author":"Li","year":"2022","journal-title":"Peer J."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"EFSA Scientific Committee (SC), More, S., Bampidis, V., Benford, D., Bragard, C., Hern\u00e1ndez-Jerez, A., Bennekou, S.H., Koutsoumanis, K.P., Lambr\u00e9, C., and Machera, K. (2023). Guidance on protocol development for EFSA generic scientific assessments. EFSA J., 21, e08312.","DOI":"10.2903\/j.efsa.2023.8312"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"699","DOI":"10.1080\/03602530701690374","article-title":"Drugs as CYP3A probes, inducers, and inhibitors","volume":"39","author":"Liu","year":"2007","journal-title":"Drug Metab. Rev."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Hlavica, P. (2023). Key regulators in the architecture of substrate access\/egress channels in mammalian cytochromes P450 governing flexibility in substrate oxyfunctionalization. J. Inorg. Biochem., 241.","DOI":"10.1016\/j.jinorgbio.2023.112150"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Liu, J., Tawa, G.J., and Wallqvist, A. (2013). Identifying Cytochrome P450 Functional Networks and Their Allosteric Regulatory Elements. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0081980"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Saikatikorn, Y., Lertkiatmongkol, P., Assawamakin, A., Ruengjitchatchawalya, M., and Tongsima, S. (2010, January 3\u20135). Study of the structural pathology caused by CYP2C9 polymorphisms towards flurbiprofen metabolism using molecular dynamics simulation. Proceedings of the International Conference on Computational Systems-Biology and Bioinformatics, Bangkok, Thailand.","DOI":"10.1007\/978-3-642-16750-8_3"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1042\/bj20040062","article-title":"Phe120 contributes to the regiospecificity of CYP2D6:\u2009 Mutation leads to the formation of a novel dextromethorphan metabolite","volume":"380","author":"Flanagan","year":"2004","journal-title":"Biochem. J."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1016\/j.bcp.2018.08.030","article-title":"The critical role of porcine cytochrome P450 3A46 in the bioactivation of aflatoxin B1","volume":"156","author":"Jiang","year":"2018","journal-title":"Biochem. Pharmacol."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Sevrioukova, I.F. (2022). Crystal Structure of CYP3A4 Complexed with Fluorol Identifies the Substrate Access Channel as a High-Affinity Ligand Binding Site. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms232012591"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1016\/j.tiv.2005.08.001","article-title":"Metabolism of Coumarin by Human P450s: A Molecular Modelling Study","volume":"20","author":"Lewis","year":"2006","journal-title":"Toxicol. Vitr."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Xu, Y., Ye, Y., Gao, Y., Sun, X., Shao, J., and Sun, X. (2023). Cytochrome P450 mediates the formation of four new citrinin metabolites. Food Biosci., 53.","DOI":"10.1016\/j.fbio.2023.102663"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"McLaren, W., Gil, L., Hunt, S.E., Riat, H.S., Ritchie, G.R.S., Thormann, A., Flicek, P., and Cunningham, F. (2016). The Ensembl Variant Effect Predictor. Genome. Biol., 17.","DOI":"10.1186\/s13059-016-0974-4"},{"key":"ref_44","first-page":"11.10.1","article-title":"From FastQ data to high confidence variant calls: The Genome Analysis Toolkit best practices pipeline","volume":"43","author":"Carneiro","year":"2013","journal-title":"Curr. Protoc. Bioinform."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1073\/pnas.1616198114","article-title":"Structural basis for regiospecifc midazolam oxidation by human cytochrome P450 3A4","volume":"114","author":"Sevrioukova","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1021\/ct600085e","article-title":"Generalized Born model with a simple, robust molecular volume correction","volume":"1","author":"Mongan","year":"2007","journal-title":"J. Chem. Theory Comput."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/23\/12529\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:37:10Z","timestamp":1760114230000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/23\/12529"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,11,22]]},"references-count":46,"journal-issue":{"issue":"23","published-online":{"date-parts":[[2024,12]]}},"alternative-id":["ijms252312529"],"URL":"https:\/\/doi.org\/10.3390\/ijms252312529","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,11,22]]}}}