Aryl Hydrocarbon Receptor Activation and Developmental Toxicity in

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Aryl Hydrocarbon Receptor Activation and Developmental Toxicity in Zebrafish in Response to Soil Extracts Containing Unsubstituted and Oxygenated PAHs Emma Wincent,†,‡ Maria E. Jönsson,†,‡ Matteo Bottai,† Staffan Lundstedt,§ and Kristian Dreij*,† †

Institute of Environmental Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden Department of Environmental Toxicology, Uppsala University, 751 05 Uppsala, Sweden § Department of Chemistry, Umeå University, 901 87 Umeå, Sweden ‡

S Supporting Information *

ABSTRACT: Many industrial sites are polluted by complex mixtures of polycyclic aromatic compounds (PACs). Besides polycyclic aromatic hydrocarbons (PAHs), these mixtures often contain significant amounts of more polar PACs including oxygenated PAHs (oxy-PAHs). The effects of oxyPAHs are, however, poorly known. Here we used zebrafish embryos to examine toxicities and transcriptional changes induced by PAC containing soil extracts from three different industrial sites: a gasworks (GAS), a former wood preservation site (WOOD), and a coke oven (COKE), and to PAH and oxy-PAH containing fractions of these. All extracts induced aryl hydrocarbon receptor (Ahr)-regulated mRNAs, malformations, and mortality. The WOOD extract was most toxic and the GAS extract least toxic. The extracts induced glutathione transferases and heat shock protein 70, suggesting that the toxicity also involved oxidative stress. With all extracts, Ahr2-knock-down reduced the toxicity, indicating a significant Ahr2-dependence on the effects. Ahr2-knock-down was most effective with the PAH fraction of the WOOD extract and with the oxy-PAH fraction of the COKE extract. Our results indicate that oxy-PAH containing mixtures can be as potent Ahr activators and developmental toxicants as PAHs. In addition to Ahr activating potency, the profile of cytochrome P4501 inhibitors may also determine the toxic potency of the extracts.

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INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants formed during incomplete combustion of organic material. Exposure to PAHs is a well-established risk for human health and wildlife.1 PAHs are, however, rarely the only compounds of concern in PAH-contaminated environments. Another group of polycyclic aromatic compounds (PACs) that are of toxicological importance is the oxygenated PAHs (oxy-PAHs).2 These include polycyclic aromatic quinones and ketones, and are frequently found at similar levels as PAHs in the environment.3,4 Oxy-PAHs are emitted from the same primary sources as PAHs but may also be formed through secondary oxidation of PAHs in the environment.2,5 The presence of carbonylic oxygen in the oxyPAHs results in higher water solubility and thereby higher bioavailability compared to the PAHs, which may impact their biological activity.6,7 It has been shown that the oxy-PAH containing fraction of soil extracts in many cases is as toxic as the fraction containing the PAHs.2,8 A recent in vivo study performed in Japanese medaka showed that oxy-PAHs can induce the same levels of DNA damage as its parent PAH.9 Many of the biological effects of PAHs are postulated to be mediated through activation of the aryl hydrocarbon receptor © XXXX American Chemical Society

(AHR) and the subsequent induction and action of cytochrome P450 monooxygenases (CYP) and other downstream targets.10 Studies have emphasized the role of Ahr signaling in the observed developmental effects in zebrafish embryos exposed to PAHs.11 Typically, AHR-activating PAHs are planar and unsubstituted12,13 and, in agreement with that, oxy-PAHs have been found to be less potent activators of the AHR.14−17 However, Knecht et al. (2013) recently reported that environmental oxy-PAHs caused developmental toxicity in zebrafish including mortality and malformations with differing Ahr-dependency and induction of genes associated with oxidative stress.18 In line with this report, reactive oxygen species (ROS) generation through redox cycling has previously been suggested to be an important mode of action for the observed effects of oxy-PAHs.19,20 PAHs are naturally present as mixtures, and previous studies have shown that interactions between PAHs can cause nonadditive effects,11,21 but mixture effects from oxy-PAHs Received: November 15, 2014 Revised: February 24, 2015 Accepted: February 25, 2015

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DOI: 10.1021/es505588s Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology have not been studied in detail. We have previously shown that oxy-PAH containing extracts of contaminated soil induced a prolonged DNA damage response in mammalian cells, consistent with persistent DNA damage most likely due to interactions.22,23 To improve the understanding of the mechanism of toxicity and the importance of mixture effects of oxy-PAHs, we studied activation of Ahr signaling and developmental toxicity in zebrafish embryos in response to environmental PAC mixtures with different profiles. Using PAH- and oxy-PAH containing extracts from contaminated soils, we studied time- and dose-dependent induction of Ahr regulated genes including the CYP1 family. Furthermore, we investigated effects on zebrafish development in response to the soil extracts and the role of Ahr by employing morpholinos targeting the zebrafish Ahr gene ahr2. The potency of the soil extracts was compared to the PAH benzo[a]pyrene (B[a]P).

Figure 1. Overall exposure and sampling regime. Zebrafish embryos were exposed to DMSO, soil extracts (equivalent to 6.25−800 μg soil/ mL corresponding to 0.000 14−0.725 μg PAC/mL), B[a]P (0.001−1 μg/mL), or PCB126 (20 nM) at 1 dpf for up to 96 h. RNA was sampled at 6 and 72 h after treatment, as described in the Materials and Methods section. Morphology and mortality were scored 96 h after treatment with or without morpholino (MO) injection against ahr2, which was performed at the 2−4 cells stage, as described in the Materials and Methods section.

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MATERIALS AND METHODS Reagents. Benzo[a]pyrene (B[a]P) (CASRN 50-32-8) and dimethyl sulfoxide (DMSO) were purchased from SigmaAldrich (Stockholm, Sweden). Gibco (Invitrogen, Paisley, UK) supplied growth media, fetal bovine serum, and cell culture reagents. 3,3′,4,4′,5-Pentachlorobiphenyl (PCB126) (CASRN 57465-28-8) was purchased from Larodan Fine Chemicals (Malmö, Sweden). Soil Extracts. The soil samples used originated from three contaminated sites in Sweden, including one former gas works site (GAS; Husarviken, Stockholm), one former wood preservation site (WOOD; Holmsund, Umeå), and one coke oven site still in use (COKE; SSAB, Luleå), together representing the most important categories of PAC contaminated sites in Sweden.2 The samples were pretreated as described previously,24 and extracted with acetone/hexane (1:1) using pressurized liquid extraction (PLE) at 150 °C with three extraction cycles of 5 min each. The extracts were fractionated on open silica gel columns as described previously,24 producing three fractions from each extract: a pre-fraction containing aliphatic compounds, a nonpolar fraction containing PAHs, and a semipolar fraction containing oxy-PAHs (hereafter referred to as the PAH and oxy-PAH fractions, respectively). The prefraction was discarded, whereas the other two fractions were evaporated into small volumes of DMSO for subsequent in vivo and in vitro exposures. The contents of PACs were determined using a similar extraction and fractionation procedure as above but with gas chromatography (GC) high resolution mass spectrometry (HRMS) analysis similar to that previously described.6 Dioxins and dioxin-like PCBs were also analyzed in the extracts using an accredited method similar to one previously described.25 Zebrafish and Exposure. Zebrafish (Danio rerio) embryos of AB type were used in the experiments. Fertilized eggs were obtained from the Zebrafish Core Facility at Comparative Medicine, Karolinska Institutet and held at 28 °C in E3 embryo medium (5.0 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4). Groups of 24−40 embryos (in 24−40 mL of medium) were exposed to soil extracts (equivalent to 6.25−800 μg soil/mL corresponding to 0.000 14−0.725 μg PAC/mL), B[a]P (0.001−1 μg/mL), PCB126 (20 nM), or solvent control (0.08−0.1% DMSO) in E3 medium starting at 1 day postfertilization (dpf) and up to 5 dpf. Overall exposure and sampling regime is depicted in Figure 1. All exposures were performed in glass Petri dishes and all given concentrations are nominal concentrations. Compounds were added directly to

the E3 medium and immediately mixed by gently swirling the dish. For gene transcription analysis group replicates composed of 8−10 pooled embryos were collected at indicated time points and stored in RNAlater (Ambion Inc., Austin, TX, USA) at 4 °C until RNA extraction. Ahr2 Knock-down in Zebrafish by Morpholinos. Knock-down of Ahr2 was performed using a fluorescein-tagged morpholino antisense oligonucleotide targeting the transcriptional start site of ahr2 (ahr2-MO; 5′-TGTACCGATACCCGCCGACATGGTT-3′), thereby blocking its translation26 and a negative control morpholino (CT-MO; 5′-CCTCTTACCTCAGTTACAATTTATA-3′) (Gene Tools, Philomath, OR, USA). Both morpholinos were diluted in sterile deionized water to a final concentration of 0.15 mM and injected into zebrafish embryos at 2- to 4-cell stage using an Eppendorf FemtoJet injector (Eppendorf, Hamburg, Germany). At 6−8 h postfertilization (hpf), embryos were screened by fluorescence microscopy to verify incorporation of morpholinos. Damaged embryos or those not displaying homogeneous fluorescence were removed. Morphological Assessment. At 5 dpf, embryos were photographed using a Leica MZ16F microscope and Leica Application Suite V4.1. The incidence of edema (pericardialand/or yolk sac) and swim-bladder inflation was determined as the percentage of embryos showing those effects compared to the total number at exposure start. Dead embryos were discarded every day and cumulative mortality was calculated at 5 dpf. Other effects such as craniofacial malformations and reduced body length was noted but not scored for. RNA Purification and Real-Time-PCR. RNA from zebrafish was purified using the Aurum Total RNA Fatty and Fibrous Tissue kit followed by cDNA synthesis using the iScript cDNA Synthesis kit, both from Bio-Rad (Hercules, CA, USA) according to protocol. Quantification of gene expression was performed using Maxima SYBR Green qPCR Master Mix (Thermo Fisher, Stockholm, Sweden) with detection on an Applied Biosystem 7500 Real-Time PCR system (Applied Biosystem, Foster City, CA, USA). The reaction cycles used were 95 °C for 2 min, and then 40 cycles at 95 °C for 15 s and 60 °C for 1 min followed by melt curve analysis to ensure single PCR products. Determination of relative gene expression levels compared to DMSO vehicle control was based on the comparative threshold cycle method (2−ΔΔCt) using l13 and arnt2 as reference genes. Primer sequences and gene bank B


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Environmental Science & Technology accession numbers are given in Table S1 of the Supporting Information. Statistics. Differences in the proportions of fish that died, developed edema, or noninflated swim-bladder, across all combinations of exposure and soil extract were tested by the Fisher’s exact test. The exact inference was required because of the small expected counts in some of the considered combinations. Calculated p-values for all effects are reported in Tables S2 and S3 of the Supporting Information. Statistical significance of the gene expression results was determined by one-way ANOVA with Bonferroni post-test. Gaussian distribution was analyzed by plotting the residuals from the ANOVA model (n = 455), which showed large kurtosis but nearly no skewness. All data are provided as mean ± standard error of the mean (SEM), n = 2−6. The p-values less than 0.05 were considered statistically significant.

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RESULTS PAH and oxy-PAH Containing Extracts Are Strong Activators of Ahr Signaling. A summary of the levels of PACs and dioxin-like compounds (DLCs) in the soils investigated is presented in Table 1 and in more detail in Tables S4 and S5 of the Supporting Information. Table 1. Summary of the Amounts of PACs and Dioxin-like Compounds (DLCs), including Toxic PCDDs and PCDFs and Dioxin-like PCBs in 1 g of Soila ∑PAHs (μg) ∑oxy-PAHs (μg) ∑PAC (μg) ∑DLCs (μg)

WOOD

GAS

COKE

1559 253 1811 0.61

144 22 166 2.2

410 107 517 0.65

Figure 2. PAH and oxy-PAH soil extracts are potent inducers of Ahr signaling in zebrafish embryos. QRT-PCR analysis of Ahr downstream genes after exposure to the WOOD, GAS, or COKE soil extracts or their PAH and oxy-PAH containing fractions corresponding to 0.1 mg soil/mL for 6 and 72 h. Results are shown as relative gene expression levels compared to DMSO vehicle control (DMSO = 1), *p < 0.05 and >2-fold change compared to vehicle control, #p < 0.05 compared to 6 h, n = 3, with 10 pooled embryos in each sample.

a

More detailed information about the contents of the extracts is shown in Tables S4 and S5 of the Supporting Information.

Zebrafish embryos (1 dpf) exposed either to total PAC (a combination of the PAH and oxy-PAH fractions) or to the PAH or oxy-PAH containing fractions (corresponding to 0.1 mg soil/mL) for 6 or 72 h showed statistical significant effects on gene expression levels. These genes included the cytochrome P450 (CYP) 1 family members (CYP1A, CYP1B1, CYP1C1, CYP1C2), the Ahr isoform 2 (ahr2), the Ahr repressor isoforms a and b (ahrra and ahrrb), and cyclooxygenase 2b (cox2b) (Figure 2 and Figure S1 of the Supporting Information). The strongest response was observed after exposure to the total WOOD PAC extract with CYP1A, CYP1C1, and ahrra mRNAs displaying up to about 750-, 80-, and 85-fold increases of expression levels compared to those of DMSO controls (p < 0.05), respectively. This is in agreement with this extract having the highest PAC content (see Table 1). Although the PAC level in the total COKE extract was about 3fold higher than in the total GAS extract, similar responses on gene expression levels were observed. Overall, the induction levels were generally lower after 72 h than after 6 h of treatment (Figure 2), indicating transient Ahr activation. However, embryos exposed to the total GAS and COKE PAC extracts displayed significantly higher levels of ahr2 expression at 72 h than at 6 h (p < 0.05). Comparing the effects of the two fractions of the WOOD extract revealed that the oxy-PAH fraction was a weaker inducer of Ahr regulated genes than the PAH fraction (Figure 2 and Figure S1 of the Supporting Information). As observed with the

WOOD total PAC extract, the two fractions elicited a strong but transient response of most genes with the exception of cox2b, which, in response to the PAH fraction, displayed significant higher level of induction after 72 h than after 6 h (p < 0.05) (Figure S1 of the Supporting Information). For the GAS and COKE extracts, the two fractions were generally as potent CYP1 inducers per milligram soil, both in terms of level and duration of induction (Figure 2 and Figure S1 of the Supporting Information), even though the oxy-PAH levels were 4- to 6-fold lower than the PAH levels (Table 1). Furthermore, the GAS and COKE extracts all caused significantly higher induction levels of ahr2 at 72 h compared to 6 h (p < 0.05), and the GAS PAH fraction also caused significantly increased expression level of ahrrb at 72 h compared to 6 h (p < 0.05). Soil Extracts Are More Potent Activators of Ahr Signaling than B[a]P. To investigate the dose dependency of the induction of Ahr signaling after exposure to soil extracts, 1 dpf zebrafish embryos were exposed to the total GAS PAC extract or its fractions using doses equivalent to 0.000 14−0.134 μg PAC/mL for 6 h. Normalizing the extracts to PAC content allowed for comparison with the response to B[a]P. Accordingly, 1 dpf zebrafish embryos were exposed to 0.001−1 μg B[a]P/mL for 6 h. As can be seen in Figure 3, C


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B[a]P caused dose dependent increases of CYP1A and CYP1B1 expression with up to 250- and 5-fold induction compared to DMSO control, respectively (Figure 3). Comparison of the B[a]P dose−response curves with those obtained with the GAS extracts, it is clearly evident that the latter were much more potent inducers of CYP1 induction and resulted in much higher magnitudes of induction. Soil Extracts Induce Dose-Dependent Developmental Toxicity. To evaluate if exposure to the soil extracts induced developmental toxicities in zebrafish, embryos were exposed to increasing concentrations of extracts up to 0.8 mg soil/mL. Here we chose to normalize the exposure to amount of soil to be able to compare the effects of the different soil extracts. The results showed a clear dose-dependent toxic effect on zebrafish embryonic development leading to malformations and increased mortality. The malformations included pericardialor yolk sac edemas, absent swim-bladder inflation, body shortening, small eyes, and cranio-facial malformations (Figure S3 of the Supporting Information). The total PAC extracts from the WOOD (Figure 4A) and COKE (Figure 4C) samples were much more toxic to the zebrafish embryos than the GAS extract (Figure 4B), in agreement with the higher PAC content in the former extracts (compare Figure 4 and Table 1). The WOOD extract and its fractions showed the highest toxicity regarding cumulative mortality, incidence of edema, and swim-bladder inflation (Figure 4A). The total WOOD PAC extract caused 100% mortality at both doses, and a comparison of effects with the PAH and oxy-PAH fractions showed that the former was significantly more potent than the latter, in agreement with the

Figure 3. PAH and oxy-PAH soil extracts induce CYP1 expression more strongly than B[a]P. Dose−response curves of relative CYP1A (A) and CYP1B1 (B) gene expression levels in 1 dpf zebrafish exposed to PAC containing soil extract (or fractions) from a gas works site or B[a]P for 6 h. Relative expression levels are compared to DMSO vehicle control (DMSO = 1), n ≥ 3, with 10 pooled embryos in each sample.

CYP1A and CYP1B1 showed a clear dose-dependent increase of expression levels in response to all treatments. A comparison of the total PAC extract and the two fractions showed that the oxy-PAH fraction was the most potent activator with a steep dose−response curve resulting in maximums of 950- and 35fold increased levels of CYP1A and CYP1B1 expression compared to DMSO control, respectively. The potency level of the total PAC extract was in between that of the oxy-PAH and PAH fractions. The same effect was observed for the expression levels of CYP1C1, CYP1C2, ahrra, ahrrb, ahr2, and cox2b (Figure S2 of the Supporting Information). Exposure to

Figure 4. Soil extracts were more toxic than B[a]P. Developmental toxicity was compared in zebrafish embryos exposed to increasing levels of the three soil extracts (total PAC), their PAH and oxy-PAH containing factions or B[a]P-solutions. Exposure started at 1 dpf, and incidence of noninflated swim-bladder (-SB and no edema), edema, and cumulative mortality was scored at 5 dpf (A−C). Exposure to B[a]P is included for comparison (D). The results are presented as percentage of embryos with the different effects based on n = 2−4 with 24−34 embryos per experiment. Results from statistical analysis are shown in Table S2 of the Supporting Information. D


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Figure 5. Induction of developmental toxicity is partly mediated through Ahr2. The role of Ahr2 in mediating the toxic effects was investigated in zebrafish treated with morpholinos (MO) targeting ahr2 (ahr2-MO) in comparison with noninjected (NI) and control-MO injected (CT-MO) zebrafish (A−D). At 1 dpf, embryos were exposed to soil extracts (or fractions) or to PCB126 up to 5 dpf. The results are presented as percentage of embryos with the different effects based on n = 2, with 30 embryos per experiment. Results from statistical analysis are shown in Table S3 of the Supporting Information.

< 0.05). In agreement with the gene expression data, the oxyPAH fractions from the COKE extract showed higher potency than its corresponding PAH fraction even though the total PAC content was 4- to 6-fold lower. Soil Extracts Are More Potent Developmental Toxicants than B[a]P. The relative potency of the soil extracts compared to B[a]P was investigated by exposing zebrafish embryos to a dilution series of B[a]P ranging from 0.004 to 0.5 μg B[a]P/mL. An increased incidence of mortality and edema was observed at the two highest doses (20% mortality at 0.5 μg B[a]P/mL; p < 0.05) and the highest dose (p < 0.05), respectively, while noninflated swim-bladder was observed at all doses (p < 0.01) (Figure 4D). B[a]P equivalent (B[a]Peq) doses were calculated for the soil extracts using published Toxic Equivalency Factors (TEFs) (Table 6 of the Supporting Information). Because the range of B[a]Peq doses (0.0032−0.0256 μg/mL) coincided with the range of B[a]P concentrations used, direct comparisons could be made. The results reveal that the WOOD and COKE extracts and fractions caused a much higher developmental toxicity than what current TEF scales can predict. At the lower dose of the WOOD PAH fraction (0.1 mg soil/mL corresponding to 0.0064 μg B[a]Peq/ mL), the incidence of edema was higher than at the highest B[a]P dose (0.5 μg/mL, p = 0.001), and the mortality rate was higher than at 0.1 μg B[a]P/mL (p < 0.001). The higher dose of the COKE PAH fraction (corresponding to 0.0127 μg B[a]Peq/mL) induced higher rates of mortality and edema than 0.1 μg BP/mL (p < 0.05). The results obtained with the total

gene expression results. The PAH fraction showed 100% and 40% mortality at the two doses (p < 0.001), with all surviving embryos having edema or noninflated swim-bladder. The oxyPAH fraction caused 60% mortality and 40% edema at 0.4 mg soil/mL (both p < 0.001), whereas the lower dose increased the incidences of edema (p = 0.003) and noninflated swim-bladder (p < 0.001), all relative to vehicle controls. The least toxic extract was from the GAS site, which also contained lowest level of PACs. The total PAC extract did not cause significantly increased levels of mortality, but a dose-dependent incidence of edema was observed (p < 0.001) (Figure 4B). Neither doses of the PAH and oxy-PAH fractions cause increased mortality, and only low levels of edema and noninflated swim-bladder were observed. Notably, the total GAS extract caused much stronger effects than the two subfractions, suggesting interactions and nonadditive effects. The total COKE PAC extract showed increased incidence of all endpoints examined at the lower dose (0.1 mg soil/mL) compared to control (all p < 0.01) (Figure 4C). The mortality rate increased dose-dependently from 20% to 88% at the low and high dose respectively (p < 0.001). Dose dependent effects were also observed with the PAH and oxyPAH fractions with the latter being significantly more potent in inducing mortality at both doses (p < 0.05). The high potency of the COKE oxy-PAH fraction was also shown as an increased incidence of edema at both doses versus the control (p < 0.01), whereas the PAH fraction caused increased edema only at the highest dose (p < 0.001). Swim-bladder inflation was abrogated at the lowest dose for both fractions compared to the control (p E


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Environmental Science & Technology GAS sample was more in line with B[a]P when comparing toxic potency at B[a]Peq doses. However, lower mortality rates were observed at both doses of the PAH fraction than with the highest B[a]P dose (p < 0.05). Since the oxy-PAH fraction does not contain B[a]P and the oxy-PAHs lack TEF values, a direct comparison with this fraction cannot be made. However, our results strongly indicate that oxy-PAHs make an important contribution to the biological activity of the soil PAC extracts. Developmental Toxicity Is Partly Mediated through the Ahr2. The role of the Ahr in mediating the observed developmental effects after exposure to the soil extracts was investigated by employing morpholino construct against zebrafish ahr2 (ahr2-MO). No significant difference was observed between the NI and CT-MO injected embryos, thus all reported results are a comparison between NI and ahr2-MO exposed embryos. Exposure to PCB126 was included as positive control because our previous studies have shown that the developmental effects of PCB126 are mainly mediated through an Ahr2-dependent mechanism in zebrafish.27,28 Knock-down of Ahr2 abolished the mortality and attenuated malformations from PCB126 (Figure 5A). The level of toxicity in response to the soil extracts was reduced to different extents when ahr2 was knocked-down and was mainly observed as reduced mortality. This was most evident with the WOOD samples (0.1 mg soil/mL) where the mortality of embryos exposed to the total PAC extract and the PAH fraction was reduced from 100% and 67%, respectively, to 0% (p ≤ 0.001) (Figure 5B). However, although all embryos survived, the incidence of edema was 100% and 97%, respectively, in response to these exposures. Ahr2-MO embryos exposed to the WOOD oxy-PAH fraction showed a reduction in incidence of edema (p = 0.024). The ahr2-MO did not appear to affect swim-bladder inflation in embryos exposed to the WOOD oxyPAHs. With regard to the GAS extract, only the total PAC extract at 0.8 mg soil/ml was used to investigate the role of Ahr2 because the fractions showed low levels of toxic potency (Figure 5C). Knock-down of Ahr2 abolished the mortality in response to the GAS extract and also showed indications of reduced incidence of edema, although this was not statistically significant. In the COKE samples, Ahr2 knock-down resulted in a more efficient rescue of the toxicity by the oxy-PAH fraction (p < 0.001 and p < 0.01 respectively) than by the PAH fraction (no significant rescue) or by the total PAC extract (mortality, p < 0.001) (Figure 5D). Together, these results show partial and profile dependent Ahr2-dependency of the observed effects. Soil Extracts Induce Expression of Cellular Stress Markers. To further investigate possible additional mechanisms underlying the observed toxicities, the effects of the soil extracts on gene expression of important cellular stress markers were examined. The results showed significant effects on the expression of glutathione transferases p1 and p2 (gstp1 and gstp2), which are important in cellular detoxification and protection against oxidative stress, and of heat shock protein 70 (hsp70), a marker of cellular stress response (Figure 6). In embryos exposed to the total WOOD extract, gstp2 was the most strongly induced transcript (89-fold, p < 0.05) followed by gstp1 (4.8-fold, p < 0.05) and hsp70 (2.5-fold, p < 0.05), all compared to the DMSO solvent control. Similar induction patterns were observed with the other extracts. The mRNA levels of thioredoxin reductase 1, glutamate-cysteine ligase and catalase were also measured but no significant effects of any of the treatments were observed (data not shown). In general, the total PAC extracts and PAH fractions elicited the strongest

Figure 6. Soil extracts induce stress response markers in zebrafish embryos. QRT-PCR analysis of stress responsive genes after exposure to soil extracts or their fractions corresponding to 0.1 mg soil/ml for 6 h. Results are shown as relative gene expression genes compared to DMSO vehicle controls (DMSO = 1), *p < 0.05 and >2-fold change compared to vehicle control (n ≥ 3, with 10 pooled embryos in each sample).

inductions, consistent with both the Ahr signaling data and results from the developmental toxicity study. However, the GAS oxy-PAH fraction induced higher levels of gstp1 compared to both the total PAC extract and the PAH fraction of this soil.

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DISCUSSION Activation of AHR Signaling. The ability of oxy-PAHs to activate the AHR has not been investigated in detail. Available data from cellular luciferase reporter assays indicate that oxyPAHs are, in general, weaker AHR activators than their parent compound, and as observed with PAHs, 4-ringed or larger oxyPAHs such as benzo[a]fluorenone, benz[a]anthracene-7,12dione, and napthacene-5,12-dione show the strongest activation.15−17 Here we exposed embryonic zebrafish to PAC containing soil extracts and demonstrated that both the PAH and oxy-PAH containing fractions are potent inducers of Ahr signaling in zebrafish. Activation of Ahr in fish exposed to PAH containing contaminated sediment extracts has been shown before.29−32 Our data represent the first demonstration to our knowledge that oxy-PAH containing extracts from contaminated sites are also potent inducers of Ahr signaling. Moreover, our data comply with those of Knecht et al. (2013), showing CYP1A induction in zebrafish vasculature or liver by 13 of 38 tested oxy-PAHs.18 In addition to induction of CYP1A, our results showed significant induction of other genes involved in Ahr signaling including other CYP1s, the ahrrs and ahr2 of which the latter mediates dioxin-like toxicity in fish.33,34 Notably, all assayed genes except ahr2 displayed transient induction of expression levels. The regulation of Ahr is not well understood but can involve several pathways, including Sp1, Notch, and Nrf2.35−37 Developmental Toxicity and Ahr-Dependence. We found that all soil extracts had dose-dependent effects on F


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is 42:1, which is close to the 50:1 ratio used in earlier studies showing synergistic developmental effects in fish,29,49 whereas the ratios in the GAS and COKE extracts are around 3:1. Notably, the WOOD sample contains high levels of 4Hcyclopenta[def ]phenanthrenone and the COKE sample high levels of 9-fluorenone which both have been shown to be very weak AHR activators.15 Though numerous PAHs are known CYP1 inhibitors,55 less is known regarding enzyme inhibiting properties of oxy-PAHs and, to the authors’ knowledge, no data is available about the possible contribution of CYP1-AHR interaction to the toxic effects of oxy-PAHs. To the authors’ knowledge, this is also the first study to investigate the impact on developmental effects in zebrafish of PAC profile using environmentally relevant samples including oxy-PAHs. Clearly, to better understand the importance of contamination profile and interaction effects, further investigation is needed. An important issue to consider when studying effects of organic extracts from environmental samples is the possible contribution from “unknowns” in the extract. The method used here will for example also extract other PACs including alkylated PAHs that will end up in the nonpolar fraction, and nitro- and hydroxy-PAHs that will end up in the semipolar fraction. In addition, other lipophilic contaminants, such as dioxins and PCBs, will be coextracted and could possibly contribute to the toxicity. Consequently, dioxins and PCBs were analyzed and detected at very low levels in the extracts, i.e., expressed as toxic equivalents (TEQs) 1.3−13 pg TEQ/g soil. At the highest dose of soil extract used in the study, this corresponds to a TCDD concentration of 30 fM (in the worst case), which is at least 3 orders of magnitude below previously reported levels causing developmental effects in zebrafish.56,57 Furthermore, about 50% of the PCBs and dioxins are coeluted with the alkanes in the discarded prefraction. Thus, in this study the contribution from dioxins and PCBs to the observed effects was most likely negligible. Toxicological relevance of oxy-PAHs. In this study, we show that the soil extracts and their fractions were more potent inducers of CYP1 expression and developmental effects than B[a]P, or what is predicted by applying TEF values. We also found that the oxy-PAH fractions are potent inducers of Ahr signaling and toxicity in zebrafish embryos, an established in vivo model for developmental effects of chemicals.58,59 Our results have important implications for current risk assessment and abatement strategies of PAC contaminated sites. During health risk assessment, only B[a]P or a small subset of 16 priority PAHs are generally monitored while polar PACs, including the oxy-PAHs, rarely are taken into account, and so far lack relative potency factors. The present results and previous work by us and others have shown that the levels of oxy-PAHs often are in the same range as the PAH levels, and that the oxy-PAHs can be as potent as PAHs in inducing detrimental effects. Because of that, the oxy-PAHs are most likely contributing significantly to the risk posed by contaminated sites. Despite these facts, the oxy-PAHs are generally not included in monitoring programs or in current risk assessment models.

survival and morphology in zebrafish embryos. The morphological effects included pericardial- or yolk sac edemas, absent swim-bladder inflation, body shortening, small eyes, and craniofacial malformations. Similar effects have previously been shown to occur with PACs and dioxins and signify hallmark phenotypic effects of Ahr mediated developmental toxicity in fish.38,39 In general, the effects correlated well with the levels of PACs determined in the extracts, with strongest effects observed in response to WOOD > COKE > GAS extracts. However, when comparing the two subfractions this correlation was less evident; the GAS and COKE oxy-PAH fractions were as toxic as or even more toxic than the corresponding PAH fractions, even though the oxy-PAH levels were 4- to 6-fold lower. These data represents the first demonstration that oxyPAHs can be more potent developmental toxicants than PAHs in contaminated environments. Furthermore, our results showed a profile specific Ahr2dependence of the observed effects. This is in agreement with previous studies indicating that PAHs may induce developmental toxicity through both AHR-dependent and AHRindependent pathways.40−43 Profile specific Ahr2-dependency was most evident comparing the two fractions from the WOOD and COKE extracts. In the WOOD extract, ahr2knock-down was much more efficient in protecting against toxicity by the PAH fraction than by the oxy-PAH fraction, and with the COKE extract the reverse was observed. These data are in agreement with previous studies showing a compound specific role of Ahr signaling in mediating developmental toxicities in zebrafish in response to PACs11,18,42,43 and confirm that also oxy-PAHs may elicit their biological effects through Ahr signaling. In addition, some PACs are bioactivated by CYP1 enzymes to reactive intermediates that may covalently bind to and alter the function of proteins or DNA, causing developmental anomalies, and also to induce oxidative stress resulting in oxidative damage and/or altered signal transduction. Accordingly, ROS formation has been shown to be an important mechanism behind the adverse effects of both PAHs and oxyPAHs.19,44−46 In the present study, induction of gstp1 and gstp2 together with the cellular stress marker hsp70 was observed in embryos exposed to oxy-PAH fractions, further confirming the role of oxidative stress in the mode of action of these compounds. Possible Contribution of Mixture Effects. Our results also show that interactions between the different PACs in the extracts are likely to be important for the toxicity. The oxy-PAH fractions potently induced both Ahr signaling and developmental effects at lower PAC levels than the corresponding PAH fractions. Furthermore, the total GAS extract was significantly more potent in causing developmental toxicity than the two subfractions suggesting interactions and nonadditive effects. These findings comply with earlier studies showing that PAHs may interact synergistically causing severe developmental effects in fish embryos. These results have mainly been ascribed to combined effects of CYP1A inhibiting PAHs (e.g., fluoranthene (FL) or α-naphtoflavone) in combination with AHR activating PAHs (e.g., B[a]P or βnaphtoflavone).29−32,40,47−49 Although the mechanism behind these effects is largely unknown, oxidative stress appears to be an important factor.50 The combination of FL and B[a]P is reported to elicit synergistic effects in mammalian in vitro and in vivo systems causing higher levels of DNA adducts compared to B[a]P alone.51−54 The FL:B[a]P ratio in the WOOD extract

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ASSOCIATED CONTENT

S Supporting Information *

Tables S1−S6 (chemical analysis of soil samples, B[a]Peq levels, qRT-PCR primers and statistical results) and Figures S1−S3 (qRT-PCR results and examples of phenotypic effects). This G


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material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*K. Dreij. Tel: +46-8-52487566. E-mail: [email protected]. Funding

This work was supported by the Swedish Research Council Formas (E.W. and M.J.), Carl Tryggers Foundation (M.J.), the Swedish EPA through the Snowman Network project PACMAN (S.L.), and Cancer and Allergy foundation, ÅForsk and EU/FP7Marie Curie IRG (K.D.). Notes

The authors declare no competing financial interest.

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REFERENCES

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