Article

Introduction

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the prototype for a class of halogenated aromatic hydrocarbon (HAH) compounds including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and certain coplanar polychlorinated biphenyls (PCBs). TCDD induces a broad range of biochemical and toxic effects that range from adaptive induction of specific cytochrome P450s (e.g., CYP1A1, CYP1A2) to neoplastic alterations in animals such as development of liver foci, adenomas, and adenocarcinomas (Kociba et al., 1978; Safe, 1990). The mode of action for TCDD and related HAHs involves binding to and activation of the cytosolic aryl hydrocarbon receptor (AHR) ligand-activated transcription factor. The AHR regulates the expression of an expanding list of genes encoding proteins that regulate numerous cellular and tissue processes (Burbach et al., 1992; Okey et al., 1994; Rowlands and Gustafsson, 1997).

Studies in AHR null mice have identified that the ubiquitously expressed AHR plays an important role in normal development (Jain et al., 1998). However, a physiological ligand(s) for the AHR has yet to be confirmed even though a number of naturally occurring exogenous and endogenous chemicals have been identified as AHR ligands (Denison and Nagy, 2003). These include certain indole carbinols and their derivatives, heterocyclic aromatic amines, vitamin A derivatives, tryptophan derivatives, catechins, resveratrol, flavonoids, and carotenoids (Bjeldanes et al., 1991; Denison and Heath-Pagliuso, 1998; Bittinger et al., 2003; Denison and Nagy, 2003; Zhang et al., 2003; Tittlemier, 2004; Oberg et al., 2005). Many of these constituents are present in foods such as vegetables, fruits, nuts, and herbs in milligram quantities. While some of these constituents have been reported to antagonize AHR activation (Fukuda et al., 2004; Park et al., 2004, 2005), many others exhibit AHR agonist activities (Denison and Nagy, 2003) and can be referred to as naturally occurring AHR agonists (NAHRAs). Further, specific indole carbinole derivatives have exhibited AHR activation potencies comparable to TCDD (Vang et al., 1990; Jellinck et al., 1993; Ciolino and Yeh, 1999; Stephensen et al., 2000). The role of TCDD and other PCDDs and PCDFs in competing with the NAHRAs, and how this competition impacts the natural role of the AHR in responding to endogenously formed or dietary-supplied natural AHR ligands have yet to be elucidated.

HAHs are routinely analyzed in blood, lipid, other biological matrices, and environmental media using high-resolution gas chromatography, high-resolution mass spectrometry (HR-GC/MS) after extensive extraction and cleanup steps. More recently, the use of in vitro bioassays based on AHR-regulated genes has increased for estimating HAH concentrations in environmental media, for example, US EPA Method 4425 (Hu et al., 1995; Giesy et al., 1997; Pauwels et al., 2000; US EPA, 2000; Koppen et al., 2001; Covaci et al., 2002; Denison et al., 2004). A commonly used commercially available cell bioassay is the chemical-activated luciferase gene expression (CALUX) bioassay that measures the ability of a chemical mixture to activate AHR-dependent gene expression of the firefly luciferase gene in genetically modified cell lines relative to activation by TCDD (Aarts et al., 1995; Garrison et al., 1996; Murk et al., 1996). The CALUX bioassay typically utilizes an immortalized hepatoma-derived cell line either from rats or mice (Murk et al., 1996; Denison et al., 2004). The results from the CALUX bioassay are often reported in TCDD equivalencies (TEQs) and compared against analytical TEQs determined for the mixture using GC-MS mass data and HAH congener-specific toxicity equivalency factors (TEFs; van den Berg et al., 2006). CALUX and similar reporter gene bioassays have been applied to mixtures from environmental samples as well as to the estimation of serum TEQ in epidemiologic studies (Koppen et al., 2001, 2002; Pauwels et al., 2001; Covaci et al., 2002; Nawrot et al., 2002).

An important limitation of reporter gene bioassays is that they cannot discriminate between anthropogenic AHR agonists (e.g., HAHs) and naturally occurring AHR agonists, and therefore, procedures have been developed to cleanup and extract samples to remove interfering non-HAH agonists prior to testing. Overall, the classical HAH-like chemicals are generally more persistent activators of gene expression and demonstrate well-characterized toxic effects in animals. The NAHRA agonists, on the other hand, appear to demonstrate shorter kinetics of gene expression and only limited toxicological data are available to assess their potential TCDD-like adverse effects. Therefore, in this paper, we refer to the non-PCDD/F and non-PCB AHR activation in the reporter gene bioassay by NAHRAs relative to induction by TCDD, as induction equivalency, or IEQ, rather than TEQ. Whether or not these IEQ measurements have the potential to cause toxicity is yet to be investigated.

It is known that sample processing, choice of cell line, and conditions of bioassay are critical for correlating bioassay-derived TEQ data with HR-GC/MS-derived TEQ data, but under ideal circumstances, very good correlations (r²>0.9) can be achieved. On the other hand, by eliminating specific sample processing procedures, the composite AHR activity of both the HAHs and NAHRAs may theoretically be measured, although this feature is also highly dependent on bioassay conditions (Windal et al., 2005). For example, Schecter et al. (1999) used a limited extraction procedure and relatively short incubation times for estimating IEQ in human blood utilizing a murine CALUX bioassay. Specifically, the usual chromotagraphy cleanup steps for isolating PCDDs/Fs and PCBs were omitted and untreated hexane extracts of blood were directly evaluated in the reporter gene assay. The resulting IEQs ranged from 97 to 823 ppt, levels which were considerably higher than the HR/GC-MS TEQ estimates attributed to HAHs (0.04–0.52 ppt, wet weight basis). The authors attributed this TEQ to IEQ disparity to the presence of "other compounds" in human blood that are known to activate the AHR pathway.

The purpose of this study was to provide needed replication of the results of Schecter et al. (1999) with a more controlled study of human volunteers and to examine the effects of dietary intervention on whole blood IEQ assessed by the reporter gene bioassay. Whole blood and lipid-adjusted TEQs in 10 volunteers were assessed at baseline by HR-GC/MS-derived TEQ and compared with modified whole blood bioassay-derived IEQs determined at baseline and following 4 days of dietary intervention. Two of the volunteers were also examined for the effects of supplementation with indole-3-carbinol (I3C) on whole blood IEQ determined with the bioassay.

Methods

Volunteer Recruitment and Dietary Intervention

Ten volunteers were recruited without preference to race, sex, or age, using e-mail and word-of-mouth to announce the study and offered a modest compensation for participation. Participants were enrolled after establishing that they (1) did not have a medical condition or lifestyle that would preclude adherence to the diets described in this study (e.g., diabetes), (2) were not pregnant or trying to get pregnant, (3) were not taking any regular medication prescribed for an illness, and (4) were not regular users of any tobacco products.

The study was performed in accordance with the principles stated in the Declaration of Helsinki. Each participant gave full informed consent, having been given information about the nature, purpose, and benefits of the study, and informed of the possible risks associated with blood donation. The study protocol was reviewed and approved by an external Institutional Review Board (IRB; Essex, Lebanon, NJ, USA).

Volunteers were asked to maintain specific diets over the course of two 4-day periods and to maintain a food and beverage diary throughout the entire period of dietary intervention, which would be used to record each food item and the approximate amount consumed. Meals were to be consumed at approximately 0800, 1300, and 1900 hours. Basic information on each participant was also collected, including age, height, and daily body weight (Table 2).

Table 2 - Sex, age, and body mass index for study participants.

Full table

A baseline diet was developed that minimized consumption of NAHRA-containing fruits, vegetables, and herbs. A vegetable-free diet described by Lampe et al. (2000) was adopted for this purpose, modified to include a moderate amount of low-fat meat and dairy products (Table 1). These foods were to be consumed in exclusion of all others. Portion sizes were based on a person of average weight (70 kg or 154 pounds) and proportional adjustment for lighter or heavier individuals was allowed. Participants were asked to avoid red meat, fish, and other seafood, and to avoid cooking methods that could char the meat, such as flame-broiling, grilling, or barbequing.

Table 1 - Food items in the baseline and NAHRA diets.

Full table

A NAHRA diet was open to the foods normally consumed by each participant, but with certain NAHRA-containing foods specifically recommended (Table 1). This diet contained specified amounts of cruciferous vegetables (e.g., cabbage, broccoli, Brussels sprouts, and kale), which contain known NAHRAs or their precursors (Bjeldanes et al., 1991; Lampe et al., 2000). A combination of cooked and raw cruciferous vegetables was included in the diet, based on studies that have examined the heat-catalyzed transformations of indole glucosinolates (Bradfield and Bjeldanes, 1987; Slominski and Campbell, 1989). For example, in the case of I3C and its conversion into the active indolomethanes, autolytic formation is known to occur as food is chewed and this process would be reduced in cooked vegetables. However, the indolomethanes are also formed through the cooking process itself. Accordingly, participants were advised to cook vegetables by lightly steaming to minimize the leaching of the NAHRAs into the cooking water. Lastly, participants were asked to avoid certain fruits, vegetables, and herbs, which have been found to contain natural AHR ligands that, on balance, might be expected to be particularly strong antagonists (Casper et al., 1999; Ciolino and Yeh, 1999; Ciolino et al., 1999; Ashida, 2000; Ashida et al., 2000; Allen et al., 2001; Amakura et al., 2002, 2003a), including grapes, grape juice, wine, and red-wine vinegar; and grapefruit, lime, or the peels of any citrus fruits, soybeans and soy products, herbs and beverages, sage and green tea.

Two of the ten volunteers (participants nos. 8 and 9) continued the NAHRA diet for an additional 2-day period, while also taking an I3C extract (Life Extensions™). The participants took three 200 mg tablets per day, in accordance with the recommendations on the container label.

Dietary Schedule and Blood Sample Collection

At the start of the study, one 100-ml sample was taken from each participant before commencing the baseline diet to confirm that the participants had a typical "background" PCDD/F- and PCB-TEQ using HR-GC/MS. All participants consumed the baseline diet over a 4-day period (days 1–4), followed by a 2-day "interim" period during which there were no dietary restrictions (days 5 and 6), followed by 4 days' consumption of the NAHRA diet (days 7–10). Blood samples were collected by a registered nurse at multiple time points throughout the study. Blood was collected in glass Vacutainer™ tubes (10 ml; without anti-coagulant) and immediately packed with frozen "cold packs" and shipped via overnight delivery to the appropriate laboratories. For the bioassay analyses, 30-ml blood samples were collected on days 3 and 4 (samples B1 and B2) of the 4-day baseline diet, on days 8, 9, and 10 (samples E1, E2, and E3, respectively) of the subsequent 4-day NAHRA diet, and on days 11 and 12 (samples ES1 and ES2) of the I3C supplement consumption. All bioassay samples were collected in the afternoon (1400–1600 hours) except sample E2, which was collected in the morning (1000–1100 hours).

Analytical Chemistry

PCDD, PCDF, and PCB concentrations in the blood samples were measured using HR-GC/MS (AXYS Analytical Services Ltd., Sidney, British Columbia, Canada). Seventeen 2,3,7,8-chlorinated PCDD/F congeners and 14 coplanar PCBs were assessed, for which TEFs for mammals and humans have been established by the World Health Organization (WHO-TEF). The percentage lipid for each blood sample was also determined using a gravimetric method as described in EPA Method 1613. The laboratory reported the PCDD/F and PCB data both on a lipid and whole blood basis. In this analysis, the WHO-TEF values were not used to calculate the TEQs, but rather the relative potencies (REPs) provided by Brown et al. (2001) were used; these values were determined using a bioassay very similar to that the one used in this study (REPs provided by George Clark (XDS Inc., Durham, NC, USA) were used for REPs not reported by Brown et al., 2001). Using this approach, the TEQs are more likely to reflect the sum of TEQ values that would be derived from the bioassay. The effects of using the WHO-TEFs in the TEQ calculations, however, would introduce only a minor uncertainty into the comparisons, as the WHO-TEFs are on average within 20% of the REPs from Brown et al. (2001).

Reporter Gene Bioassay

The reporter gene bioassay determination of whole blood IEQs and reference NAHRA activities was carried out at Xenobiotic Detection Systems, Inc. (XDS). This included considerable modification of the XDS–CALUX bioassay and optimization of the sample extraction and assay conditions. Additional experiments were also carried out to determine the recoveries of three reference NAHRAs, daidzein, resveratrol, and indolo[ 3,2-b] carbazole (ICZ) using the chosen method of extraction. The bioassay was initially developed with an aim to replicate the studies by Schecter et al. (1999), which were performed with the murine hepatoma H1L1.1c2 transfected with the TCDD-inducible reporter pGudLuc1.1. This reporter system has in general been replaced by more responsive and reliable systems; however, trial tests were conducted using the H1L1.1c2 cell line, which confirmed that this system would not provide a robust response to dioxin-like chemicals and NAHRAs (data not shown). Therefore, the bioassay analyses were conducted using the mouse hepatoma H1L6.1c3 cell line stably transfected with the TCDD-inducible pGudLuc6.1 reporter plasmid (Han et al., 2002).

Blood samples were shipped on ice and immediately frozen at - 70°C upon receipt by XDS. On the day of assay, samples were slowly thawed and two 0.5-ml volumes of each whole blood sample were extracted three times (at room temperature) with an equivalent volume of optima grade tert-butyl methyl ether (MTBE), using vortexing followed by centrifugation (10 min at 2800 g) with each, according to the method of Anderton et al. (2003). The organic phases recovered from each extraction were combined and the MTBE was evaporated to dryness under a stream of nitrogen gas. Samples were then resuspended in 200 l hexane and this stock extract was used for creating dilutions to be analyzed in the cell-based reporter gene bioassay.

The efficiency of the MTBE extraction for capturing select NAHRAs was determined in a series of spiking experiments. In all experiments, spiked whole blood samples were extracted with MTBE and further processed in the same way as the samples used in the bioassay experiments. Experiment 1 determined the percentage recoveries by measuring extract activity in the reporter gene bioassay in comparison to the activity of the same reference compound placed directly into DMSO. HAH reference compounds for calibrating the bioassay responsiveness, 3-methylcholanthrene (3-MC) and TCDD, were obtained from Accustandard (New Haven, CT, USA). Daidzein and resveratrol (all 98% purity) were obtained from Sigma-Aldrich Inc. (St Louis, MO, USA) and used as reference NAHRA compounds. ICZ was obtained from Dr. Leonard Bjeldanes (University of California at Berkeley) and made up to a concentration of 1.6 mg/ml in DMSO. Stock solutions of each of the other reference NAHRA compounds of 10 mg/ml were prepared in DMSO. The range of maximum attainable (dosing) concentrations were between 438 M for resveratrol and 63 M for ICZ. Standard curves for each compound were established by serial dilution analysis. The range of doses for each NAHRA was chosen based on potency data found in the open literature (Zhang et al., 2003; Amakura et al., 2003b; Guengerich, 2004). The approximate ED₇₅ values (6.9, 13, and 4.1 M, for resveratrol, daidzein, and ICZ, respectively) were determined for each compound and this dose selected as the concentration for spiking whole blood. The spiked, whole blood samples were then extracted (with MTBE) and analyzed as described above for the main study. The percentage recovery was based on the IEQ activity obtained from the spiked, whole blood sample. In experiment 2, whole blood samples (0.5 ml) were spiked with 50 ng radiolabeled [ ¹⁴C] I3C, [ ³H] dihydrodaidzein (American Radiolabeled Chemicals Inc., Saint Louis, MO, USA) or [ ³H] resveratrol (Moravek Biochemicals, Brea, CA, USA). Extraction efficiencies were determined for the MTBE extracts and the final solutions (reconstituted in hexane and DMSO, sequentially) by determining the fraction of radioactively labeled NAHRA remaining relative to the amount originally added to the whole blood sample. Specifically, the extract in DMSO was placed in 0.4 ml RPMI cell culture media and the resulting solution transferred to a 7-ml glass scintillation vial and placed on a scintillation counter for determination of the amount of radiolabel present.

The conditions of the luciferase reporter gene bioassay were as follows: cells were cultured and maintained in 96-well plates in an RPMI-1640 medium supplemented with 8% fetal calf serum and 1% penicillin/streptomycin solution at 36–38°C and in an atmosphere of 5% CO₂. When cells reached 80–90% confluence, they were harvested with trypsin and resuspended in fresh culture medium at a density of 7.5 10⁵ cells/ml, and maintained under the same conditions for 16–24 h, before dosing.

Dosing solutions were prepared by diluting sample extracts in 1 ml of hexane and exchanging into 4 l DMSO by evaporation of the hexane in a vacuum centrifuge concentrator. Each DMSO extract was then added to 400 l culture media and vortexed vigorously. Dosing of cells was initiated by replacing cell culture media covering cells in microtiter plates with 200 l with the media containing the extract (or standard/reference compound).

A standard curve ranging from 1 to 1000 ppt of TCDD and quality control samples were included on each microtiter plate containing sample extracts. In addition, a reference standard of 3-MC was used to estimate recoveries within each bioassay. Plates were incubated for 4 h for induction of firefly luciferase enzyme. Analysis of luciferase activity was carried out with a kit from Promega (Madison, Wisconsin, WI, USA), and light production measured with a Berthold Orion Microplate Luminometer (Oak Ridge, TN, USA). The activity of an extract was estimated from the TCDD standard curve using a four-parameter Hill equation and was expressed as induction equivalents (IEQs). Range-finding experiments were conducted initially, with at least three dilutions of each sample, to identify a sample dilution that would yield an activity within the TCDD standard curve.

Statistical Analysis

Bioassay IEQs measured throughout the dietary study were compared using Student's t-test and Wilcoxon rank-sum paired difference test and differences were considered significant at P<0.05. The day-to-day change in IEQ levels observed in each individual participant was the fundamental measure of comparison for these statistical tests.

Results

The IEQ dose–response curves for daidzein, ICZ, and resveratrol, which were constructed as part of the extraction recovery study (Figure 1), demonstrate the induction by these compounds of a dose-dependent AHR agonism along with that for TCDD after 4 hours of incubation in the reporter gene bioassay. The declining responses at the highest doses, in the case of resveratrol and daidzein, could be due to cellular toxicity. Approximate ED₅₀ values for resveratrol and daidzein were 2.0 and 6.5 M, respectively; while an ED₅₀ for ICZ was not estimated because a clear maximal response was not observed. On the basis of the dose–response IEQ standard curves for these compounds, the average recoveries for resveratrol, daidzein, and ICZ from whole blood were found to be between 47% and 82% at 4 h of incubation. Similarly, from the second recovery experiment, radiolabeled [ ¹⁴C] I3C, [ ³H] dihydrodaidzein, and [ ³H] resveratrol were recovered from spiked whole blood at efficiencies of 54% –78% (Table 4). This demonstrates that these selected NAHRAs are recovered sufficiently with the limited MTBE extraction procedures.

Figure 1.

Dose response of selected NAHRAs in induction of luciferase activity.

Full figure and legend (103K)

Table 4 - NAHRA recovery efficiencies from human blood.

Full table

The 10 study participants recruited into the study consisted of six females ranging in age from 27 to 42 years (mean, 34) and four males ranging in age from 32 to 42 years (mean, 38; Table 2). No adverse symptoms or significant changes in body weight (>2% ) were reported by any of the participants throughout the 2-week study and all participants completed the study. The food diaries indicated reasonable compliance with the diets. Most deviations from the diets were related to the omission of certain items or missing a meal entirely; participants were advised that this was permitted during the baseline diet. During the vegetable-rich NAHRA diet, the sporadic omission of certain vegetables was more common. Additional variability throughout the dietary study included differences in meat consumption, and use of spices and seasonings in cooking the meat.

Whole blood and lipid-adjusted total TEQs were calculated for each sample based on the HR-GC/MS analysis and representing the sum of 28 individual TEQs from each of the 28 PCDD/F and PCB compounds with an assigned relative potency value. On a whole blood basis, the mean of the summed PCDD/F and PCB TEQs was 0.045 ppt, with a range of 0.022- 0.119 ppt; when expressed on a lipid-adjusted basis, the mean was 16.7 ppt with a range of 9.1- 33.1 (Table 3). However, the bioassay IEQs measured during the baseline diet on days 3 and 4 ranged from 13.4 to 66.3 ppt (whole blood basis) with mean IEQ levels of 28.7 (sample B1; day 3) and 34.6 (sample B2; day 4; Table 5). Thus, even the IEQ measured during the baseline diet of 31.7 ppt (the combined means of B1 and B2) is approximately 700-fold greater than the TEQ of 0.045 ppt based on the HR-GC/MS analysis of PCDD/Fs and PCBs.

Table 3 - Whole blood and lipid-adjusted TEQ levels in the study participants.

Full table

Table 5 - Effects of NAHRA diet on whole blood total IEQ levels.

Full table

The HR-GC/MS analyses reported quantifiable values for the vast majority of analyzed PCDD/F and PCB congeners (data not shown); detection limits ranged from 0.01 to 0.22 ppt (whole blood basis). Only PCBs 77, 81, and 169 were frequently below detection limits. The PCDD/F TEQ in each sample was several-fold greater (approximate range of 5- to 20-fold) than the PCB TEQ (Table 3). In any given sample, approximately 80% of the PCDD/F and PCB TEQ was due to six congeners, with 1,2,3,7,8-PeCDD alone contributing approximately 25% and 2,3,4,7,8-PeCDF, 2,3,7,8-TCDD, 1,2,3,6,7,8-HxCDD, and PCBs 126 and 156/157 each contributing between 10 and 15% (data not shown). No significant relationships were observed between the blood TEQ levels and age, sex, estimated percentage body fat, or location of residence. The XDS-CALUX^® assay for dioxin-like chemicals using a cleanup method that removes NAHRAs was used to determine the TEQ associated with anthropogenic dioxins, for example, PCDD/Fs and PCBs, in a blood sample from one of the study participants. The measured TEQ in this sample was 0.4 ppt TEQ, which was very near the limit of detection for this sample due to the use of a limited volume of blood. This is consistent with previous findings, wherein background determinations of blood PCDD/Fs and PCBs from unexposed individuals have been at or near the XDS-CALUX limits of detection (Collins, 2005 (personal communication); Warner et al., 2005). The IEQ level determined for this same individual (during the basic diet) was approximately 40 ppt IEQ. The results confirm that if sample processing is used to remove NAHRAs from a blood sample, then the XDS-CALUX bioassay detects very low (sub-ppt) levels of dioxin-like chemicals in blood, similar to HRGC/MS measurement of TEQ.

IEQs measured with the bioassay increased significantly when the participants began consumption of the NAHRA diet. The mean bioassay IEQ measured on each of the three sample days of the NAHRA diet (E1, E2, and E3) was greater (P<0.05) than the mean IEQ measured during the baseline diet (Table 5). The maximum bioassay IEQ measured during this period (104 ppt) exceeded the maximum PCDD/F and PCB TEQ (0.119 ppt) by approximately 1000-fold; thus, activities measured with the reporter gene bioassay (as IEQ) exceeded the HR-GC/MS-derived TEQs by orders of magnitude. The mean bioassay IEQ measured on day 4 of this diet (sample E3; 79.3 ppt) was greater (P<0.05) than the mean bioassay IEQ value measured on the previous 2 days, E1 and E2, which were 42.9 and 40.5 ppt, respectively. This suggests that NAHRAs may be accumulating in blood a few days after commencing a vegetable-rich diet.

Bioassay IEQ measured in participants 8 and 9 during the I3C ingestion period was as high as 218 ppt and, therefore, among the highest measured in this study. Bioassay IEQs in both participants clearly tended upwards throughout the entire 2-week study. Specifically, mean levels in participant 8 were 21.6 ppt IEQ for the baseline diet (average of B1 and B2), 37.1 ppt IEQ for the NAHRA diet (average of E1, E2 and E3), and 159 ppt IEQ for the I3C consumption (average of ES1 and ES2); mean levels in participant 9 for baseline diet, NHARA diet and I3C supplementation diet were 40.1, 58.2, and 78.6 ppt IEQ, respectively. For participants 8 and 9, the mean of the pooled IEQ values during I3C consumption (118.6 ppt IEQ) was greater than (P<0.05) the mean of their pooled values during the baseline diet (30.8 ppt IEQ) or NAHRA diet (47.6 ppt IEQ; Figure 2).

Figure 2.

Mean bioassay determined whole blood total IEQ levels following NAHRA dietary intervention and supplementation with indole-3-carbinol (I3C; see text for details).

Full figure and legend (12K)

Discussion

In this study of 10 volunteers, the mean lipid-based TEQ levels of 16.7 ppt for 28 PCDD/F and PCB congeners measured by HR-GC/MS, were consistent with "background" mean lipid-based TEQ levels of 25 ppt for the general US population measured in the mid-1990s, especially when considering the substantial decline in TEQ body burdens over the past two decades (US EPA, 2000). The study participants were therefore representative of the general US population with respect to their body burdens of PCDD/Fs and PCBs on a lipid-adjusted basis as determined by HR-GC/MS.

Whole blood IEQ values measured with the reporter gene bioassay were at a level, that was orders of magnitude higher than whole blood TEQs calculated on the basis of HR-GC/MS analysis of PCDD/Fs and PCBs. This observation is consistent with the findings of Schecter et al. (1999) who evaluated unprocessed solvent extracts of blood and also observed human whole blood IEQ levels from reporter gene bioassay results that were >50 ppt. In our study, the mean whole blood bioassay IEQ at baseline was 31.7 ppt, whereas the mean whole blood analytical TEQ at baseline was 0.045 ppt for PCDD/F and PCB congeners. Thus, the results of this study indicate that the reporter gene bioassay of whole blood is measuring a "non-additive" increase in activity that cannot be accounted for by the PCDD/F and PCB TEQ measured by HR-GC/MS. In fact, 99.9% of the activity measured in the reporter gene bioassay of whole blood appears to be attributable to AHR activation from compounds without a currently assigned TEF value. Thus, these compounds are expressed in IEQs rather than TCDD or TEQs.

An alternative explanation for the comparatively high (bioassay) IEQ could be that the PCDD/Fs and/or PCBs are responsible for most or all of this activity via synergistic activation of the AHR. Few reports of synergistic activity among AHR agonists can be identified (Long et al., 1998; Suzuki et al., 2004), wherein the difference between the expected and observed activities was 4-fold. Thus, there is no basis for assigning the approximate 700-fold difference between analytical TEQ and bioassay IEQ observed in this study, to possible synergism. Moreover, it has previously been demonstrated that when assessing purified fractions of PCDD/Fs and PCBs, the TEQ values determined using bioassays similar to the one used in the current study generally agree with those determined using HR-GC/MS analysis (Pauwels et al., 2000; Koppen et al., 2001; Covaci et al., 2002; Warner et al., 2005). It is more likely that the major portion of the activity measured in the reporter gene bioassay of whole blood is from non-PCDD/F or PCB AHR agonists present in the minimally purified extracts.

Bioassays results are very sensitive to the procedures used to cleanup and extract samples (Seidel et al., 2000; Windal et al., 2005). Bioassays that are marketed commercially for the measurement of PCDD/Fs and PCBs in foods, soils, or tissues typically utilize sample cleanup procedures that include an activated-carbon column (e.g., the XDS "XCARB" affinity matrix) (Brown et al., 2001) in series with an acid–silica column. These methods remove most unwanted compounds and selectively retain PCDD/Fs and/or PCBs. Solvents are also typically chosen with the objective of extracting the lipophilic-halogenated hydrocarbons and excluding the more polar compounds. In contrast, the objectives of the present study were to eliminate the solvent extraction cleanup steps with the objective of retaining compounds that might be much more water-soluble. We found that the average recoveries for select radiolabeled NAHRAs, I3C, dihydrodaidzein, and resveratrol were between 54% and 78% using the extraction and cleanup procedures designed for this study (Table 4). Recovery experiments conducted with blood spiked with reference NAHRA compounds (daidzein, resveratrol, and ICZ) and measuring the extract activity in the CALUX assay obtained similar results; recoveries were between 47% and 82% . Therefore, the extraction procedure was clearly capable of retaining the more hydrophilic AHR ligands, whether natural or anthropogenic. At the same time, the extraction and cleanup procedures did result in an appreciable loss of the original blood NAHRA content, and therefore, to the extent that these compounds are contributing to the measured IEQs, the actual IEQ activities in blood may be underestimated in this study. The extraction efficiency of MTBE for PCDDs/Fs and coplanar PCBs was not determined so the contribution of these compounds to the observed IEQ cannot be stated. However, based on the HR-GC/MS TEF-based estimates, they did not contribute significantly to the IEQ.

Examples of anthropogenic compounds other than PCDD/Fs and PCBs that could contribute to the IEQ activity include polybrominated diphenyl ethers (PBDEs), polychlorinated napthalenes (PCNs), and polycyclic aromatic hydrocarbons (PAHs). Giesy et al. (1997) examined the contributions of several non-PCDD/F classes of compounds, such as PBDEs, PCNs, and PAHs, to the IEQ content in fish taken from the Great Lakes (Geisy et al., 1997 referred to the TCDD-like activity as TEQ rather than IEQ). With respect to the net activities of these compounds in cell-based bioassays, these investigators concluded that aside from PCBs, these compounds would at most contribute to an IEQ of comparable magnitude (Giesy et al., 1997). Hence, although other anthropogenic compounds might contribute somewhat to the IEQ activity measured in this study, any such contribution is unlikely to be substantial.

Dietary phytochemicals appear to be the most likely source of the AHR agonistic activity measured in the IEQ bioassay. Commencement of a high-vegetable diet, which would not be expected to increase the consumption of PCDD/Fs and PCBs, in this study caused significant increases in the blood IEQ. Numerous studies have reported that common dietary phytochemicals such as flavanoids, indoles, polyphenolics, and oxidized carotenoids possess AHR agonist activity (Gradelet et al., 1996; Heath-Pagliuso et al., 1998; Denison and Nagy, 2003; Jeuken et al., 2003; Zhang et al., 2003). Many of these natural AHR agonists have been measured in human blood in the low micromolar range (Nakagawa et al., 1997; Paganga and Rice-Evans, 1997; de Vries et al., 1998). One of the best studied NAHRAs is I3C, an indole compound found at high levels in cruciferous vegetables of the Brassica genus (e.g., brussel sprouts, cabbage, and cauliflower). During digestion, I3C is converted into several condensation products, such as ICZ, which is a high-affinity AHR agonist and induces CYP1 gene expression in a manner similar to TCDD (Bjeldanes et al., 1991). In the present study whole blood IEQ levels increased significantly in two subjects who consumed I3C supplements, demonstrating that I3C or more likely one of its biotransformation products (e.g., ICZ) was bioavailable and recovered in the analytical processing of the samples. Participants who closely followed the NAHRA diet in the current study would have consumed an estimated total dose of 90 mg/day of glucobrassicin, the natural form of I3C found in vegetables. This glucobrassicin dose would yield an I3C dose of 36 mg/day, assuming a 40% conversion rate in the GI tract and an ICZ dose of 3.6 g/day, assuming a 0.01% I3C into ICZ conversion rate (Bjeldanes et al., 1991; Connor and Finley, 2003). Applying a relative potency (REP) 0.5 for ICZ (Kleman et al., 1994) yields a blood IEQ of approximately 40–400 ppt for ICZ alone. Despite the obvious uncertainties attached to such estimates, the bioassay IEQ observed in this study are certainly of a magnitude that seems plausible.

It is possible that the increased IEQ activity during vegetable and I3C consumption is due to the enhanced production of endogenous AHR agonists. The current results do not permit an analysis of the relative contribution of dietary NAHRAs vs endogenous agonists.

The results would, however, seem to indicate that at least a major fraction of the bioassay IEQs in this study were due to NAHRAs and/or endogenous ligands. For example, an increase in the bioassay IEQs was observed over the course of 2 or 3 days of consuming a diet rich in NAHRAs. This upward trend in the bioassay IEQs would be consistent with the bioaccumulation of blood NAHRAs over this time period. Further research would be needed to confirm this observed trend and to assess whether it would continue or reach a plateau after several additional days of consumption of a NAHRA diet. It has been estimated that the biological half-lives of the indole carbinoles are on the order of hours or days (Stresser et al., 1995; Arneson et al., 2001). It is therefore interesting to note that the mean total IEQ was statistically significantly greater on day 4 of the NAHRA diet (79.3 ppt) than on day 2 or 3 (42.9 and 40.5 ppt, respectively; Table 5). Further research is needed to confirm this observed trend and to assess whether it would continue or reach a plateau after several additional days of consumption of a NAHRA diet.

While additional pharmacokinetic data could help to predict the disposition in human tissues, there is little doubt that NAHRAs are capable of retaining their biological activities following gastrointestinal absorption in humans. For example, an AHR-inhibitory effect by seven flavonoids (e.g., flavone, apigenin, and luteolin) was virtually unchanged after passing through a human intestinal (Caco-2) cell monolayer — a widely used model of human intestinal (epithelial) cell permeability (Hamada et al., 2006). NAHRAs are clearly biologically active compounds that can influence the risk for certain chronic diseases. For example, consumption of I3C-containing vegetables is associated with a reduced risk of cancer in humans, and experimental studies have demonstrated that orally consumed I3C is an effective chemopreventative agent against a number of carcinogens (NCI, 1996; Verhoeven et al., 1996; Stoner et al., 2002). I3C at doses of 400 and 800 mg/day induced AHR-mediated CYP1A2 expression in women (Reed et al., 2005). I3C also will alter hepatic P450-dependent (CYP1A1, CYP1A2, and CYP3A4) estrogen metabolism (Bradlow et al., 1994; Michnovicz et al., 1997).

For anthropogenic dioxins, AHR activation and AHR-dependent biochemical responses (e.g., CYP1A1 induction) are considered as early biomarkers of the toxicological response to TCDD. Whether such a relationship should be expected to hold true for the NAHRAs is a question receiving increased recent attention. While it might be presumed that "dioxin-like" toxicities are not induced by NAHRAs, some of the data for I3C, at least, indicates that this may not be the case. At high doses, I3C induces a spectrum of toxicological effects in animals (Nishie and Daxenbichler, 1980; NCI, 1996; Wilker et al., 1996; Leibelt et al., 2003; Crowell et al., 2005) some of which resemble TCDD-induced effects (e.g., hepatotoxicity, thymic atrophy, male reproductive toxicity, and tumor promotion). At high dietary levels, I3C has also shown activity as a tumor promoter (Stoner et al., 2002; William et al., 2003) Pohjanvirta et al. (2002) found that the in vivo toxicity of ICZ in comparison to TCDD fall short of that that might be predicted by its in vitro potency; achieving the necessary dose levels at the target tissues may be a barrier for some NAHRAs, due to their relatively rapid metabolism. However, I3C is clearly capable of inducing CYP1A1 in vivo, indicating that certain NAHRAs are quite capable of reaching target tissues in sufficient concentrations. Thus, at present the question of dioxin-like toxicities of NAHRAs is unresolved. Mechanistic differences between natural and man-made AHR agonists are being examined to possibly explain whether toxicological differences should be expected.

The findings of this study suggest that "background" blood levels of PCDD/Fs and PCBs may constitute only a trivial fraction of the total AHR agonist activity in human blood. While the AHR activation by NAHRAs is likely to be relatively transient as compared to that induced by PCDD/Fs and PCBs, the observation in this study of a net agonist activity in human blood of a magnitude that equated to a IEQ of >100 ppt as measured via bioassays clearly warrants further investigation. The high-vegetable diet used in this study included a mix of 5–8 vegetable servings per day. This is well within the latest dietary guidelines in the United States, which call for 5–9 servings of fruits and vegetables a day, and possibly more depending on caloric intake (HHS/USDA, 2005). Therefore, even some of the higher IEQs measured in this study could likely be considered as normal for the general population. In fact, the IEQ levels observed in this study may not necessarily represent the maximum attainable blood IEQ attributable to NAHRAs because of some loss of NAHRA content during the blood extraction procedures and because the consumption of greater amounts of NAHRA-containing foods is possible.

Additional research is required to determine the net activity of NAHRAs in human tissues. Further studies are also clearly needed in order to elucidate the relative contribution of NAHRAs and endogenous AHR ligands to the blood IEQ observed in this study. This work may also provide important clues on normal physiological role of the AHR.

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