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Development of a sensitive in vitro assay to quantify the biological activity of pro-inflammatory phorbol esters in Jatropha oil

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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.

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Acknowledgments

The authors wish to thank Sheila Masson and Annamaria Halasz for technical assistance and Drs. Phil Shwed and Subramanian Karthikeyan for critical review of this manuscript. This work was supported by the Program of Energy Research and Development (PERD project C24.001) from Natural Resources Canada (NRcan).

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Authors and Affiliations

  1. 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, Bhaja K. Padhi & Raymond Poon

  2. National Research Council of Canada, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, QC, H4P 2R2, Canada

    Jalal Hawari & Geoffrey I. Sunahara

  3. Department of Civil Engineering, Geology and Mining, Polytechnique Montréal, 2500 Chemin dePolytechnique, C.P. 6079, succ. Centre-ville, Montreal, QC, H3C 3A7, Canada

    Jalal Hawari

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  1. Guillaume Pelletier
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  2. Bhaja K. Padhi
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  3. Jalal Hawari
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  4. Geoffrey I. Sunahara
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  5. Raymond Poon
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Correspondence to Guillaume Pelletier.

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Editor: T. Okamoto

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Pelletier, G., Padhi, B.K., Hawari, J. et al. Development of a sensitive in vitro assay to quantify the biological activity of pro-inflammatory phorbol esters in Jatropha oil. In Vitro Cell.Dev.Biol.-Animal 51, 644–650 (2015). https://doi.org/10.1007/s11626-014-9861-z

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  • DOI: https://doi.org/10.1007/s11626-014-9861-z

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