Dioxin concentration in human milk in Hebei province in China and Tokyo, Japan: potential dietary risk factors and determination of possible sources.

Very limited information is available on body burdens and environmental levels of dioxins and dioxin-like PCBs (dl-PCBs) in mainland China. In the current studies, human milk samples were collected from 30 breastfeeding mothers in Shijiazhuang city (industrialized) and 11 in the Tanshan countryside (agricultural) of Hebei Province in northern China. An additional 20 samples were obtained from mothers in Tokyo, Japan. PCDDs, PCDFs, and dl-PCBs in human milk were analyzed by high-resolution gas chromatography/high-resolution mass spectrometry. Our results show that arithmetic means for body burdens of PCDDs/Fs and dl-PCBs in Hebei were 3.6 and 1.9 pg TEQg(-1) fat, respectively, which were only about one fourth of the levels in Japan. In addition, no difference was found in the chemical levels except dl-PCBs between the urban and rural areas. Based on the results of an in-person interview of the Chinese mothers using a 59-item questionnaire, freshwater fish consumption was found to correlate with the body burden of dioxins. Principal component analysis of dioxin congeners revealed that the patterns of dioxins in the Hebei urban and rural areas are quite similar; however, they are clearly different from those in Japan. Collectively, our results suggest that the lower body burdens of dioxin in Hebei may be due in part to the relatively slow industrialization and a lower consumption of marine foods. Finally, the results indicate that comprehensive monitoring of dioxins and dl-PCBs in humans as well as in the environment and foods is necessary in China.

Evidence of aryl hydrocarbon receptor ligands in Presque Isle Bay of Lake Erie.

The purpose of the present studies was to use a biomarker approach to examine xenobiotic exposure of brown bullhead in Presque Isle Bay, Lake Erie (USA). In particular, the presence of compounds that act through the aryl hydrocarbon receptor (AhR) was of interest due to its central role in gene regulation and carcinogenesis of dioxins and certain polycyclic aromatic hydrocarbons (PAHs). Initial screening of Presque Isle Bay sediment samples by gene expression microarray in mouse hepatocytes revealed prototypical dioxin-response genes such as cytochrome P450 1A1 and 1B1 (CYP1A1 and CYP1B1). The presence of AhR ligands in sediment samples was confirmed and quantified using an in vitro assay, the Chemical Activated Luciferase Expression (CALUX) assay. The CALUX assay system, by using different incubation times, allows for determination of total dioxin induction equivalents (IEQ) for less persistent compounds such as PAHs as well as for stable compounds such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and certain polychlorinated biphenyls (PCBs). Parts of Presque Isle Bay have significant concentrations of AhR ligands in sediment ranging from 200 to 1400 parts per trillion (ppt) dioxin IEQ equivalents (dry weight). This is much higher than levels of dioxin equivalents found in similar sediment samples (approximately 10 ppt). Cascade Creek appears to be a major source of dioxin-like contaminants as IEQs in sediments taken from various regions of this tributary ranged from 1300 to 42000 ppt IEQ. In addition, the CALUX assay indicated that the majority of the IEQs (>90%) in PIB samples were in fact derived from less stable compounds. To determine if brown bullhead are exposed and respond to these high levels of AhR ligands, CYP1A cDNA was cloned from this species and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine mRNA levels. The CYP1A mRNA concentration was lower and less variable in fish taken from Presque Isle Bay than from a body of water with much lower AhR ligand concentration. Taken together, these studies show that sediment in Presque Isle Bay is highly contaminated with AhR ligands including dioxins and PAHs, but the brown bullhead are either not exposed or are non-responsive to these carcinogenic compounds.

Analysis of Ah receptor pathway activation by brominated flame retardants.

Brominated flame-retardants (BFRs) are used as additives in plastics to decrease the rate of combustion of these materials, leading to greater consumer safety. As the use of plastics has increased, the production and use of flame-retardants has also grown. Many BFRs are persistent and have been detected in environmental samples, raising concerns about the biological/toxicological risk associated with their use. Most BFRs appear to be non-toxic, however there is still some concern that these compounds, or possible contaminants in BFRs mixtures could interact with cellular receptors. In this study we have examined the interaction of decabromodiphenyl ether, Firemaster BP4A (tetrabromobisphenol A), Firemaster PHT4 (tetrabromophthalic anhydride), hexabromobenzene, pentabromotoluene, decabromobiphenyl, Firemaster BP-6 (2,2',4,4',5,5'-hexabromobiphenyl) and possible contaminants of BFR mixtures with the Ah receptor. Receptor binding and activation was examined using the Gel Retardation Assay and increased expression of dioxin responsive genes was detected using the reporter gene based CALUX assay. The results demonstrate the ability of BFRs to activate the AhR signal transduction pathway at moderate to high concentrations as assessed using both assays. AhR-dependent activation by BFRs may be due in part to contaminants present in commercial/technical mixtures. This was suggested by our comparative analysis of Firemaster BP-6 versus its primary component 2,2',4,4',5,5'-hexabromobiphenyl. Some technical mixtures of brominated flame-retardants contain brominated biphenyls, dioxins or dibenzofurans as contaminants. When tested in the CALUX assay these compounds were found to be equivalent to, or more active than their chlorinated analogues. Relative effective potency values were determined from dose response curves for these brominated HAHs.

Determination of relative assay response factors for toxic chlorinated and brominated dioxins/furans using an enzyme immunoassay (EIA) and a chemically-activated luciferase gene expression cell bioassay (CALUX).

Determination of toxic activity requires knowledge of both the concentration and toxicity to evaluate the risk for adverse human health and environmental effects. A chemically-activated luciferase gene expression cell bioassay system (CALUX) and an antibody-based method enzyme immunoassay (EIA) were used to detect the dioxin-like response of several polybrominated, polychlorinated, and polybrominated/chlorinated dibenzo-p-dioxins/furans (PBDDs/Fs, PCDDs/Fs, and PBCDDs/Fs, respectively). It has been suggested that the biological activity of the brominated and mixed bromo/chloro compounds is similar to their chlorinated analogues (measured by binding to the Ah receptor). PBDD/F, PCDD/F, and PBCDD/F laboratory standards exhibited biological activity ranging over three orders of magnitude. The highest relative potency (REP) values from CALUX analysis, when compared to 2,3,7,8-TCDD, were 2,3,7,8-TBDD at 0.99 (+/-0.07), 1,2,3,7,8-PeCDD at 0.69, and 2-Br-3,7,8-TriCDD at 0.72 (+/-0.02). Cross-reactivities were calculated using EIA for several PBDDs/Fs and PBCDDs. The highest percent cross-reactivity was found for 2,3,7,8-TBDD at 138 (+/-34%), and 2,3,7-TriBDD at 84 (+/-36%).

AH receptor agonist activity in human blood measured with a cell-based bioassay: evidence for naturally occurring AH receptor ligands in vivo.

In the present study, an aryl hydrocarbon receptor (AHR)-driven reporter gene bioassay was used to measure the activity, measured as an induction equivalent (IEQ) as compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or IEQ concentration in human blood samples from 10 volunteers under different dietary regimens. Blood concentrations of polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) and polychlorinated biphenyls (PCBs), as determined by analytical chemistry (HR-GC/MS), and expressed as toxic equivalents (TEQs) with the use of TCDD equivalency factors (TEFs), were within a range that has been reported in the general US population, ranging from 0.022 to 0.119 ppt (whole blood basis). However, the human blood IEQ measured directly via bioassay ranged from 13.4 to 218 ppt (whole blood basis). These order of magnitude greater IEQs compared to the TEQs for dioxins, furans, and certain PCBs suggests that human blood contains a relatively high level of AHR agonists able to activate the CYP1A1 dioxin response element (DRE)-linked reporter gene bioassay and that this AHR activity is not accounted for by PCDDs/Fs and dioxin-like PCBs based on standard HR-GC/MS and TEF analysis. When study participants switched from a "baseline" to a high-vegetable diet, increases in bioassay IEQ were observed that were statistically significant (P<0.05). In addition, IEQ activity was elevated above levels observed following dietary intervention in two subjects given indole-3-carbinol (I3C) supplements. We conclude that a substantial portion of the IEQ activity occurred as a result of the increased intake of natural AHR agonists (NAHRAs) present in many fruits, vegetables. and herbs. Our findings also suggest that dietary NAHRAs constitute a substantial daily dietary intake of AHR-active compounds, and these NAHRAs could influence AHR status in humans and play a role in a basal level of AHR activation.

Mathematical model developed for environmental samples: prediction of GC/MS dioxin TEQ from XDS-CALUX bioassay data.

Remediation of hazardous waste sites requires efficient and cost-effective methods to assess the extent of contamination by toxic substances including dioxin-like chemicals. Traditionally, dioxin-like contamination has been assessed by gas chromatography/high-resolution mass spectrometry (GC/MS) analysis for specific polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyl congeners. Toxic equivalency factors for these congeners are then used to estimate the overall dioxin toxic equivalency (TEQ) of complex mixtures found in samples. The XDS-CALUX bioassay estimates contamination by dioxin-like chemicals in a sample extract by measuring expression of a sensitive reporter gene in genetically engineered cells. The output of the XDS-CALUX assay is a CALUX-TEQ value, calibrated based on TCDD standards. Soil samples taken from a variety of hazardous waste sites were measured using the XDS-CALUX bioassay and GC/MS. TEQ and CALUX-TEQ from these methods were compared, and a mathematical model was developed describing the relationship between these two data sets: log(TEQ) = 0.654 x log(CALUX-TEQ) + 0.058-(log(CALUX-TEQ))2. Applying this equation to these samples showed that predicted and GC/MS measured TEQ values strongly correlate (R2 = 0.876) and that TEQ values predicted from CALUX-TEQ were on average nearly identical to the GC/MS-TEQ. The ability of XDS-CALUX bioassay data to predict GC/MS-derived TEQ data should make this procedure useful in risk assessment and management decisions.

Persistent organic pollutants in human milk in women from urban and rural areas in northern China.

Human milk specimens from 55 women in Shijiazhuang urban and Tangshan rural areas in Hebei Province in northern China were collected and analyzed for persistent organic pollutants, such as p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE), hexachlorobenzene (HCB), beta-hexachlorocyclohexane (beta-HCH), and dioxins. We administered a questionnaire to milk donors at collection time, asking about lifestyle factors that potentially influence organochlorine pesticide (OCP) levels in human milk. We found that the concentrations of p,p'-DDE and beta-HCH in human milk from the primiparous mothers in Shijiazhuang (3330 and 108.8 ng/g fat, respectively) were higher than in samples from mothers from Tangshan (1916 and 21.2 ng/g fat, respectively). In addition, we found the level of p,p'-DDE correlated positively with a dietary history of meat consumption or consumption of internal organs of pigs and sheep (r=0.38 and r=0.52, respectively), but not with intake frequencies of vegetables and fruits in primiparous mothers. The higher level for p,p'-DDE may be due to a greater intake of contaminated fatty foods in the industrialized areas. We also showed that the body burden of p,p'-DDE was higher in the urban group compared to the values in other regions in China after 1998. However, there was no difference in dioxin levels between the two regions. Comprehensive monitoring of persistent organic pollutants in human milk and food is needed in China.

Validation of the CALUX bioassay for the screening of PCDD/Fs and dioxin-like PCBs in retail fish.

The chemical-activated luciferase expression (CALUX) assay is a reporter gene assay that detects dioxin-like compounds based on their ability to activate the aryl hydrocarbon receptor (AhR) and thus expression of the reporter gene. In this paper, the CALUX assay was examined for its application in the screening of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dioxin-like PCBs) in retail fish. The sample extracts were cleaned up on a sulfuric acid-silica gel column followed by an activated carbon column, and the AhR activity of the separated PCDD/F and dioxin-like PCB fractions was determined using the assay. The quantitative limit for 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was 0.98 pg ml(-1) (0.19 pg assay(-1) in the standard curve, corresponding to 0.16 pg g(-1) of CALUX-based toxic equivalency (2,3,7,8-TCDD equivalents) in the tested sample. Recovery tests in which dioxins were added to fish samples resulted in acceptable recoveries (77-117%). The CALUX assay performed well in the analysis of dioxins in fish samples and a comparative study revealed a strong correlation between the CALUX assay and high-resolution gas chromatography-high-resolution mass spectrometry analysis for the determination of PCDD/Fs (r = 0.89) and dioxin-like PCBs (r = 0.91) in retail fish (n = 22). These data revealed that the CALUX assay would be a useful screening method for PCDD/Fs and dioxin-like PCBs in retail fish.

Recombinant cell bioassay systems for the detection and relative quantitation of halogenated dioxins and related chemicals.

Proper epidemiological, risk assessment and exposure analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related halogenated aromatic hydrocarbons (HAHs) requires accurate measurements of these chemicals both in the species of interest and in various exposure matrices (i.e. biological, environmental, food and feed). High-resolution instrumental analysis techniques are established for these chemicals, however, these procedures are very costly and time-consuming and as such, they are impractical for large scale sampling studies (i.e. for epidemiological studies and assessment of areas with widespread contamination). Accordingly, numerous bioanalytical methods have been developed for the detection of these chemicals in extracts from a variety of matrices, the majority of which take advantage of the ability of these chemicals to activate the aromatic hydrocarbon receptor (AhR) and the AhR signal transduction pathway. Here we review the currently available in vitro AhR-based cell bioassay systems with a focus on recent recombinant reporter gene cell lines that have been developed for detection and relative quantitation of TCDD and related HAHs. Comparison of the relative sensitivities of the various cell bioassays and examples of their use in screening and analysis of environmental, biological, and food and feed samples are presented. Currently available experimental results and validation studies demonstrate the utility of these cell bioassay systems to provide a relatively rapid, accurate, and cost effective screening approach for the detection of TCDD and related HAHs in a variety of environmental, biological, food and feed samples. The availability of these cell bioassay systems will not only facilitate the large scale sampling studies needed for accurate assessment of contamination and exposure to these environmental chemicals, but they provide avenues for the identification of novel classes of TCDD-like chemicals.