Changes of AhR-mediated activity of humic substances after irradiation M. Bittner a, ⁎ , K. Hilscherová a , J.P. Giesy b,c,d a RECETOX, Masaryk University, Kamenice 126/3, 62500 Brno, Czech Republic b Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada c Zoology Department, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University, E. Lansing, MI 48823, United States d Biology and Chemistry Department, City University of Hong Kong, Kowloon, Hong Kong, SAR, China Received 30 November 2006; accepted 22 March 2007 Available online 30 April 2007 Abstract Humic substances (HS) and natural organic matter (NOM) are natural organic compounds ubiquitous in the environment. However, some studies indicate that both HS and NOM can act as xenobiotics, e.g. induce hormone-like effects in fish, amphibians and invertebrates. Molecules of these substances contain a number of aromatic rings and conjugated double bonds — the so called chromophores. Irradiation of dissolved HS and NOM can lead to a series of photochemical reactions which can act on these substances itself, or on other substances present in aquatic environment along with HS and NOM such as e.g. xenobiotics. In our previous study, we have found significant interactions of five humic acids (HA) with cytosolic aryl hydrocarbon receptor (AhR) in an in vitro bioassay based on H4IIE-luc cells. In the present study, we have studied the changes in AhR-mediated activities both of HS and NOM after irradiation that simulated natural solar light. Nine different HS and two NOM samples were irradiated in Pyrex tubes with a medium-pressure mercury lamp for a duration of 0 to 52 h (which corresponds to 0–52 d natural solar radiation). Original concentrations of the samples were 50 mg L - 1 , and the greatest concentration of HS and NOM photoproducts subsequently tested in the bioassay was 17 mg L - 1 , which is an environmentally relevant concentration. After irradiation the absorbances of all the samples were less than the original materials. The AhR-mediated activity of the HA-Fluka and HA Sodium Salt were partially decreased by irradiation. The activities of other HS and NOM, that were either AhR-active or -inactive were not changed by irradiation. The results of the study demonstrate that AhR-mediated activities of two active HA is caused by both photo-stable and photo-labile AhR activators, while the other three active HA contain only photo-stable AhR activators. Potential mechanisms of the observed irradiation-induced changes in AhR-mediated activities are discussed. © 2007 Elsevier Ltd. All rights reserved. Keywords: Humic acids; Ah receptor; H4IIE-luc; Absorbance; NOM 1. Introduction Humic acids (HA) and fulvic acids (FA) are important fractions of the group of organic compounds called humic substances (HS) that are ubiquitous natural products of decomposition of dead organic matter. In the aquatic environ- ment, HS form approximately 50–70% of dissolved/natural organic matter (D/NOM, Timofeyev et al., 2004) found in most natural freshwaters at concentrations of 0.5 to 50 mg L - 1 , but can occur at concentrations as great as 100 mg L - 1 in raised peat bogs (Steinberg, 2003), where the HA fraction forms about 6–8% of the total NOM (Yamamoto et al., 2004). Although both HS and NOM have been regarded to be without any direct biological activity, recently it has been found that these substances alone can act as xenobiotic chemicals. One of the rather non-specific mechanisms of action is NOM- dependent modulation of heat shock proteins 70 in carp and Daphnia magna, and induction of biotransformation enzymes glutathione-S-transferases, oxidative stress enzymes peroxidase and glutathione peroxidase in amphipods (Wiegand et al., 2003). A more specific mechanism of action is both HS and NOM-dependent modulation of photosynthetic oxygen release in alga Scenedesmus armatus, water moss Vesicularia dubyana Environment International 33 (2007) 812 – 816 www.elsevier.com/locate/envint ⁎ Corresponding author. Tel.: +420 549491462; fax: +420 549492840. E-mail address: bittner@recetox.muni.cz (M. Bittner). 0160-4120/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2007.03.011