In Vivo and In Vitro Mixed-Function Oxygenase Activity and Vitellogenin Induction in Fish and in Fish and Rat Liver Cells by Stilbenes Isolated from Scotch Pine (Pinus sylvestris) Joanne L. Parrott • Jagmohan Kohli • James P. Sherry • L. Mark Hewitt Received: 21 May 2009 / Accepted: 20 April 2010 / Published online: 13 May 2010 Ó Her Majesty the Queen in Right of Canada 2010 Abstract Many types of pulp and paper mill effluents have the ability to induce mixed-function oxygenase (MFO) activity and vitellogenin (VTG) protein in exposed male fish. The search for the compounds responsible for MFO induction has led to several classes of compounds, among them retene and stilbenes. The objective of this study was to investigate the biological activities of candi- date stilbene compounds. Three stilbenes, 3,5-dihydroxy- stilbene (pinosylvin; P1), 3-hydroxy-5-methoxystilbene (P2), and 3,5-dimethoxystilbene (P3), were extracted from Scotch pine (Pinus sylvestris) and purified to evaluate their ability to induce MFO activity in vitro using ethoxyres- orufin-O-deethylase (EROD) activity in a rat hepatoma cell line (H4IIE). As these compounds may be chlorinated during pulp bleaching, chlorination of P2 was undertaken, producing di- and trichlorinated isomers (Cl-P2), which were also tested. Compounds were tested for EROD- inducing ability in vivo by exposing juvenile rainbow tout (Oncorhynchus mykiss) to waterborne concentrations (0.010 to 1.0 mg/L) for 4 days. Compounds were also tested for their ability to induce VTG in trout primary liver cells in vitro. The stilbenes were weak inducers of EROD and VTG. H4IIE EROD was induced by all four com- pounds, with the most potent induction by P3, followed by P1, the Cl-P2 mixture, and then P2. Induction for all four stilbenes was from 3.13 9 10 -3 to 3.57 9 10 -4 as potent as retene and about 1.11 9 10 -5 to 1.20 9 10 -6 as potent as TCDD. Juvenile rainbow trout did not show EROD induction after exposures to P1, P2, or the Cl-P2 mixture, whereas P3 caused activity fourfold above that of controls. P1, P3, and Cl-P2 all weakly induced VTG in rainbow trout hepatocytes. The most potent inducer of VTG was Cl-P2, followed by P3 and P1. The results show the ability of wood-derived stilbenes to cause weak MFO induction in fish and in rat liver cells and to weakly induce vitellogenin in fish liver cells. Pulp mill effluents (PMEs) contain compounds that induce mixed-function oxygenase (MFO) enzymes in fish. Initially, in the early 1990s, the induction was caused by high con- centrations of known MFO inducers, tetrachlorodibenzo- p-dioxin (TCDD) and tetrachlorodibenzofuran (TCDF), by products of chlorine bleaching of wood fibers and defoamer contaminants. However, limiting the use of elemental chlo- rine and strict regulations related to discharge of TCDD and TCDF have not eliminated the MFO-inducing compounds in PMEs. Fish MFOs are induced by exposure to PMEs that contain no detectable TCDD or TCDF (reviewed by Parrott et al. 2006). Although dioxins and furans contributed to the MFO induction seen on fish downstream of PMEs, they are not the sole cause of the observed MFO induction and several studies have attempted to identify additional effluent com- ponents associated with induction since the early 1990s (reviewed by Hewitt et al. 2006). Because of effluent complexities and changes in effluent compositions over the last two decades, the pulp mill effluent matrix has represented one of the greatest analytical challenges to address in identifying bioactive substances in complex mixtures. As a result, few studies have been able to definitively attribute cause and effect in any of the biolog- ical responses that have been investigated, be it MFO induction or the spectrum of reproductive responses in fish. In the absence of definitive structural identifications, J. L. Parrott (&) Á J. Kohli Á J. P. Sherry Á L. M. Hewitt Water Science and Technology Directorate, Environment Canada, National Water Research Institute, 867 Lakeshore Road, Burlington, ON L7R 4A6, Canada e-mail: joanne.parrott@ec.gc.ca 123 Arch Environ Contam Toxicol (2011) 60:116–123 DOI 10.1007/s00244-010-9539-9