title: Development of a sensitive in vitro assay to quantify the biological activity of pro-inflammatory phorbol esters in Jatropha oil.
abstract: New health safety concerns may arise from the increasing production and use of Jatropha oil, a biodiesel feedstock that also contains toxic, pro-inflammatory, and co-carcinogenic phorbol esters. Based on the exceptional sensitivity of Madin-Darby canine kidney (MDCK) cells to the model phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a robust bioassay was developed to quantify the biological activity of Jatropha phorbol esters directly in oil, without sample extraction. We first verified that the characteristic response of MDCK cells to TPA was also observed following direct exposure to phorbol esters in Jatropha oil. We further confirmed that similarly to TPA, Jatropha oil's phorbol esters can activate protein kinase C (PKC). We then assessed the transcriptional response of MDCK cells to Jatropha oil exposure by measuring the expression of cyclooxygenase-2 (COX-2), a gene involved in inflammatory processes which is strongly upregulated following PKC activation. Based on the parameterization of a TPA dose-response curve, the transcriptional response of MDCK cells to Jatropha oil exposure was expressed in term of TPA toxic equivalent (TEQ), a convenient metric to report the inflammatory potential of complex mixtures. The sensitive bioassay described in this manuscript may prove useful for risk assessment, as it provides a quantitative method and a convenient metric to report the inflammatory potential of phorbol esters in Jatropha oil. This bioassay may also be adapted for the detection of bioactive phorbol esters in other matrices.
authors.affiliation: Hazard Identification Division, Environmental Health Science and Research Bureau, Health Canada, Environmental Health Centre, 50 Colombine Driveway, P.L. 0803B, Tunney's Pasture, Ottawa, ON, K1A 0L2, Canada, guillaume.pelletier@hc-sc.gc.ca.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Pelletier
authors.forename: Bhaja K
authors.initials: BK
authors.lastname: Padhi
authors.forename: Jalal
authors.initials: J
authors.lastname: Hawari
authors.forename: Geoffrey I
authors.initials: GI
authors.lastname: Sunahara
authors.forename: Raymond
authors.initials: R
authors.lastname: Poon
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D048430:Cell Shape / Q000187:drug effects; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018836:Inflammation Mediators / Q000494:pharmacology*; D031286:Jatropha / Q000737:chemistry*; D061985:Madin Darby Canine Kidney Cells; D010938:Plant Oils / Q000737:chemistry*; D011493:Protein Kinase C / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D018836:Inflammation Mediators; D010938:Plant Oils; D051546:Cyclooxygenase 2; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate