Connection between EROD and CYP1 activity

In mammals, CYP1A and CYP1B1 oxidize and activate PAH’s (Shimada & Fujii-Kuriyama 2004) and EROD activity is catalyzed by both (Chang & Waxman 2006). However, the activity towards EROD catalyzed by CYP1B1 is less than that catalyzed by CYP1A1 (Shimada et al. 1997). Contributions of the single CYP1 enzymes to EROD activity vary among species (Jönsson et al. 2009a). Substrates for the CYP1C enzymes in zebrafish have not yet been identified, but they are likely also involved in xenobiotic metabolism (Goldstone et al. 2009, Goldstone et al. 2010). Rates of EROD activity of zebrafish CYP1D1 expressed in yeast were lower than that of CYP1A, but it is proposed that in basal conditions CYP1D1 might contribute to EROD activity usually attributed to CYP1A (Goldstone et al. 2009). In the present study, basal EROD activity measured was quite low, as was transcript abundance of cyp1a, while transcript abundance of the cyp1c’s was higher. Basal transcript abundance of cyp1d1 in the zebrafish embryo is reported to be higher than that of cyp1a (Goldstone et al. 2009). Therefore, it is possible that in basal conditions EROD activity could in part be attributed to the cyp1c’s or cyp1d1. After exposure to AhR-agonists EROD activity was first significantly up-regulated compared to basal EROD activity at 96 hpf. Cyp1a transcripts were induced the strongest at 48 hpf, at 96 and 120 hpf cyp1b1 was similarly expressed. This leads to the conclusion that EROD activity measured at 96 and 120 hpf might be primarily related to activity of CYP1A and to a lesser extent CYP1B1.

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Conclusion 5.6.

This study provides a comprehensive analysis of the time-dependent development and fluctuation of basal and induced CYP1 gene expression and enzyme activity in early-life stages of the zebrafish embryo. The results show that each gene has a distinct pattern of expression in basal conditions during embryonic development. All four CYP1 genes were readily up-regulated after exposure to potent AhR-agonists and showed a temporal decline in up-regulation at 72 hpf compared to 48 and 96 hpf. Activity of EROD was measured as a biomarker of CYP1A activity. It could be shown that a stable measurement of EROD activity was possible at 96 hpf. Therefore, this time-point is recommended for future investigations of EROD activity in zebrafish embryos.

Acknowledgments

The authors acknowledge financial support by the German Federal Ministry of Education and Research (grant nos. 02WU1053). The authors also want to thank the BfG and the Hamburg Port Authority for providing the sediments and its related parameters.

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Chapter 6

6. Quantitative assessment of the embryotoxic potential of NSO-

heterocyclic compounds using zebrafish (Danio rerio)

Sabrina Peddinghaus1, Markus Brinkmann1, Kerstin Bluhm1, Anne Sagner2, Gunnar Hinger3, Thomas

Braunbeck3, Adolf Eisenträger,4,5 Andreas Tiehm2, Henner Hollert1* & Steffen H. Keiter1

1

Department of Ecosystem Analysis, Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany

2

Water Technology Center, Department of Environmental Biotechnology, Karlsruher Strasse 84, 76139 Karlsruhe, Germany 3

Aquatic Ecology and Toxicology, Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230,

69120 Heidelberg, Germany 4

Dept. IV 2: Pharmaceuticals, Chemicals, Experimental Studies, Federal Environment Agency, Woerlitzer Platz 1, 06844 Dessau, Germany

5

Institute of Hygiene and Environmental, Medicine, Medical Faculty, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany

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Abstract 6.1.

Heterocyclic aromatic compounds (NSO-HET) have frequently been detected in the environment. Several studies have concluded that NSO-HETs pose a threat to organisms in waters, sediments and soils. Consequently, several heterocyclic compounds are under discussion to be included in the priority list of the European Water Framework Directive. However, few publications are available assessing the ecotoxicology of NSO-HETs. The present study aims to assess the embryotoxicity of heterocycles using Danio rerio. A combination of the Fish Embryo Toxicity test (FET) and analytical quantification should aid to determine the hazard potential. Changes of the total concentrations due to sorption or volatility were quantified by GC/MS. Loss of compounds during the test was observed primarily for volatile or hydrophobic NSO-HETs. The LC50 calculated with nominal

concentrations underestimates the toxicity by a factor up to 16 (2 h), demonstrating that a chemical analysis for comparing nominal and measured concentrations is essential for such investigations.

Keywords: heterocyclic aromatic compounds, NSO-HET, zebrafish, embryotoxicity, Fish Embryo Toxicity test

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Introduction 6.2.

Heterocyclic aromatic compounds are widely distributed pollutants in soil, air, sediments, surface water and groundwater, as well as in animal and plant tissues (Brack & Schirmer 2003). They may be of natural origin (e.g. alkaloids), but high environmental concentrations mainly result from human activities. In particular, industrialized areas, such as creosote contaminated sites, represent important sources of tar oil pollutants (Blum et al. 2011, Weber et al. 2008). Creosote represents a complex mixture of over 10.000 single organic substances which are formed by thermal processes related to coal and fossil fuels (Blotevogel et al. 2008). Beside technical and chemical processes that involve tar oil, heterocyclic compounds are also present in dyestuff (Cripps et al. 1990), pesticides and pharmaceuticals (Broughton & Watson 2004, Fernández-Alba et al. 2002).

While creosote contains only 5 – 13% heterocyclic compounds (Dyreborg et al. 1997, Meyer 1999), up to 40% of their water-soluble fraction consists of these heterocyclic compounds (Licht et al. 1997). The higher polarity and water solubility of the heterocyclic substances is based on the substitution of one carbon atom by nitrogen, sulfur or oxygen (NSO-HET; Meyer & Steinhart 2000). These chemical properties lead to increased bioavailability and mobility as compared to the homologous polycyclic aromatic hydrocarbons (PAH). Several studies using the concept of effect-directed analysis and mass balance calculations concluded that NSO-HETs contribute significantly to the ecotoxicological hazard of water, sediment and soil samples (Brack et al. 2007, Keiter et al. 2008, Wölz et al. 2008). NSO-HETs are known to show a large range of ecotoxic effects, e.g. acute toxicity, developmental and reproductive toxicity, cytotoxicity, photo-induced toxicity, mutagenicity, and carcinogenicity (Barron et al. 2004, Brack & Schirmer 2003, Bundy et al. 2001, Eisentraeger et al. 2008, Robbiano et al. 2004). Moreover, some studies have shown that NSO-HETs bioaccumulate in aquatic organisms, and acute toxicity has been reported for Daphnia, midge, and algae (Eisentraeger et al. 2008, Jung et al. 2001, Robbiano et al. 2004). Only a few publications are available comparing the toxicology of different groups of NSO-HETs (Eisentraeger et al. 2008, Feldmannová et al. 2006, Hinger et al. 2011, Sovadinová et al. 2006). Even though heterocyclic compounds have frequently been detected in the environment, there are no publications about the toxic effects on embryos of Danio rerio and the knowledge of their occurrence, the environmental fate, biological metabolism and toxic effects is limited (Bleeker et al. 1999, Blum et al. 2011, Feldmannová et al. 2006). Thus, further investigations are needed to evaluate the toxicity of these compounds with a special focus on the

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development of aquatic organisms. In ecotoxicological testing, fish are an indispensable component of integrated toxicity testing strategies for the aquatic environment.

In the present study the embryotoxicity of NSO-HETs was investigated by a vertebrate-based test system using embryos of zebrafish (Danio rerio). For this purpose, several NSO-HETs typically found at creosote-contaminated sites (Blotevogel et al. 2008; 2,3-dimethyl- benzofuran, 2,4,6-trimethylpyridine, 2-methylbenzofuran, 6-methylquinoline, acridine, benzofuran, benzothiophene, carbazole, quinoline, dibenzofuran, dibenzothiophene, pyridine, xanthene) were selected for testing.

Acute toxicity in fish embryos shows a high correlation with acute toxicity in adults (Braunbeck & Lammer 2005, Scholz et al. 2008). For some chemicals the Fish Embryo Toxicity (FET) test proved to be more sensitive than the acute fish test (Lange et al. 1995), and Nagel (2002) documented that the FET is a very promising tool to replace the acute fish test. Furthermore, since the embryos will not hatch during the test period of 48 h, the 48 h version of the FET is regarded an alternative for animal testing in the German federal state of North Rhine Westphalia (Nagel 2002).

Previous studies demonstrated that loss of compounds due to processes such as volatilization and sorption to plastic surfaces may lead to an underestimation of the toxicity and have to be taken into account (Eisentraeger et al. 2008, Riedl & Altenburger 2007, Schreiber et al. 2008, Sverdrup et al. 2002). In order to evaluate the influence of sorption, precipitation or volatilization of the compounds during the test procedure, the total medium compounds were quantified by chromatographic analysis. LC50-values were calculated for both nominal and

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Material and methods 6.3.

For detailed information see chapter 2. Brief description of modifications of the procedures are listed below.

6.3.1. Chemicals

All chemicals were purchased from Merck (Darmstadt, Germany) with exception of 2-methylbenzofuran, 2,3-dimethylbenzofuran, 2,4,6-trimethylpyridine (Fluka, Buchs, Switzerland) and xanthene (Sigma-Aldrich, Deisenhofen, Germany). Molecular structures and properties are given in Table 6.1.

Stock solutions of the NSO-HETs were prepared in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored in darkness at 4°C. The maximum content of DMSO during the test was below 0.25%. The DMSO concentration in the treatments was applied as a serial dilution together with the test substances. In order to exclude adverse effects caused by DMSO a solvent control of 0.25% was used.

Dissolved concentrations of the tested substances were measured by means of GC/MS or HPLC/DAD-FLD, respectively.

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Substance CAS-no. Purity (%) Log Kowa

Water solubility (mg/L)a Vapor pressure (Pa)a HLC (Pa m³/mole)a N -H ET Quinoline 91-22-5 >96 2.14 6110 8.00 0.169 6-Methylquinoline 91-62-3 >98 2.69 631 1.18 0.077 Acridine 260-94-6 >98 3.32 38.4 0.02 0.040 Pyridine 110-86-1 >99.5 0.65 1 x 106 2773.1 1.115 Carbazole 86-74-8 ~95 3.23 1.8 1 x 10-4 0.012 2,4,6-Trimethylpyridine 108-75-8 > 98 1.88 35,000 265.31 0.893 2-Methylpyridine 109-06-8 >98 1.11 1 x 106 1493.2 1.009 S -H ET Benzothiophene 95-15-8 >98 2.99 130 31.73 28.979 Dibenzothiophene 132-65-0 ~97 4.38 1.47 0.03 3.425 O -H ET 2-Methylbenzofuran 4265-25-2 >95 3.09 160 64.93 56.667

Chapter 6 – Material and methods 99 Benzofuran 271-89-6 >99 2.54 678 58.66 53.196 Dibenzofuran 132-64-9 ~97 3.71 3.1 0.33 21.582 Xanthene 92-83-1 99 4.31 1.02 0.14 4.833 2,3-Dimethylbenzofuran 3782-00-1 >95 3.63 62.2 7.09 ---b

a _{Data from EpiWin 4.1 (US-EPA 2009)} b

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