Chemical-activated Luciferase Gene Expression Assay Research Articles

The role of receptor-mediated activities of 4- and 5-ring unsubstituted and methylated polycyclic aromatic hydrocarbons (PAHs) in developmental toxicity.

The present study evaluated the aryl hydrocarbon receptor (AhR), estrogen receptor-α (ER-α), and retinoic acid receptor (RAR) mediated activities of nine 4- and 5-ring unsubstituted and monomethylated polycyclic aromatic hydrocarbons (PAHs) using a series of Chemical-Activated LUciferase gene eXpression (CALUX) assays. The potential role of these aforementioned receptors in relation to the developmental toxicity of these PAHs was further assessed in the zebrafish embryotoxicity test (ZET). The results show that all nine tested PAHs were AhR agonists, benz[a]anthracene (BaA) and 8-methyl-benz[a]anthracene (8-MeBaA) were ER-α agonists, and none of the tested PAHs induced ER-α antagonistic or RAR (ant)agonistic activities. In the AhR CALUX assay, all the methylated PAHs showed higher potency (lower EC50) in activating the AhR than their respective unsubstituted PAHs, implying that the addition of a methyl substituent on the aromatic ring of PAHs could enhance their AhR-mediated activities. Co-exposure of zebrafish embryos with each individual PAH and an AhR antagonist (CH223191) counteracted the observed developmental retardations and embryo lethality to a certain extent, except for 8-methyl-benzo[a]pyrene (8-MeBaP). Co-exposure of zebrafish embryos with either of the two estrogenic PAHs (i.e., BaA and 8-MeBaA) and an ER-α antagonist (fulvestrant) neutralized embryo lethality induced by 50 μM BaA and the developmental retardations induced by 15 μM 8-MeBaA. Altogether, our findings suggest that the observed developmental retardations in zebrafish embryos by the PAH tested may partially be AhR- and/or ER-α-mediated, whereas the RAR seems not to be relevant for the PAH-induced developmental toxicity in the ZET.

Detection of Aryl Hydrocarbon Receptor Activation by Some Chemicals in Food Using a Reporter Gene Assay.

The purpose of this study was to examine whether a simple bioassay used for the detection of dioxins (DXNs) could be applied to detect trace amounts of harmful DXN-like substances in food products. To identify substances with possible DXN-like activity, we assessed the ability of various compounds in the environment to bind the aryl hydrocarbon receptor (AhR) that binds specifically to DXNs. The compounds tested included 19 polycyclic aromatic hydrocarbons (PAHs), 20 PAH derivatives (nitrated, halogenated, and aminated derivatives), 23 pesticides, six amino acids, and eight amino acid metabolites. The AhR binding activities (AhR activity) of these compounds were measured using the chemical activated luciferase gene expression (CALUX) reporter gene assay system. The majority of the PAHs exhibited marked AhR activity that increased in a concentration-dependent manner. Furthermore, there was a positive link between AhR activity and the number of aromatic rings in the PAH derivatives. Conversely, there appeared to be a negative correlation between AhR activity and the number of chlorine residues present on halogenated PAH derivatives. However, there was no correlation between AhR activity and the number and position of substituents among nitrated and aminated derivatives. Among the pesticides tested, the indole-type compounds carbendazim and thiabendazole showed high levels of activity. Similarly, the indole compound tryptamine was the only amino acid metabolite to induce AhR activity. The results are useful in understanding the identification and characterization of AhR ligands in the CALUX assay.

Aryl hydrocarbon receptor mediated activities in road dust from a metropolitan area, Hanoi—Vietnam: Contribution of polycyclic aromatic hydrocarbons (PAHs) and human risk assessment

Dioxin-Responsive Chemical-Activated LUciferase gene eXpression assay (DR-CALUX) was applied to assess the total toxic activity of the mixture of PAHs and related compounds as well as dioxin-related compounds in road dust from urban areas of Hanoi, Vietnam. Road dust from Hanoi contained significantly higher DR-CALUX activities (3 to 39, mean 20ng CALUX-TEQ/g dw) than those from a rural site (2 to 13, mean 5ng CALUX-TEQ/g dw). The total concentrations of 24 major PAHs (Σ24PAHs) in urban road dust (0.1 to 5.5, mean 2.5μg/g dw) were also 6 times higher than those in rural road dust (0.08 to 1.5, mean 0.4μg/g dw). Diagnostic ratios of PAHs indicated vehicular engine combustion as the major PAH emission source in both sites. PAHs accounted for 0.8 to 60% (mean 10%) and 2 to 76% (mean 20%) of the measured CALUX-TEQs in road dust for Hanoi the rural site, respectively. Benzo[b]-/benzo[k]fluoranthenes were the major TEQ contributors among PAHs, whereas DRCs contributed <0.1% to CALUX-TEQs for both rural and urban sites. These results suggest TEQ contribution of other aryl hydrocarbon receptor agonists in road dust. Significant PAH concentrations in urban dust indicated high mutagenic and carcinogenic potencies. Estimated results of incremental life time cancer risk (ILCR) indicated that Vietnamese populations, especially those in urban areas such as Hanoi, are potentially exposed to high cancer risk via both dust ingestion and dermal contact. This is the first study on the exposure risk of AhR agonists, including PAHs and DRCs, in urban road dust from a developing country using a combined bio-chemical analytical approach.

The in vivo estrogenic and in vitro anti‐estrogenic activity of permethrin and bifenthrin

Pyrethroids are highly toxic to fish at parts per billion or parts per trillion concentrations. Their intended mechanism is prolonged sodium channel opening, but recent studies reveal that pyrethroids such as permethrin and bifenthrin also have endocrine activity. Additionally, metabolites may have greater endocrine activity than parent compounds. The authors evaluated the in vivo concentration-dependent ability of bifenthrin and permethrin to induce choriogenin (an estrogen-responsive protein) in Menidia beryllina, a fish species known to reside in pyrethroid-contaminated aquatic habitats. The authors then compared the in vivo response with an in vitro assay--chemical activated luciferase gene expression (CALUX). Juvenile M. beryllina exposed to bifenthrin (1, 10, 100 ng/L), permethrin (0.1, 1, 10 µg/L), and ethinylestradiol (1, 10, 50 ng/L) had significantly higher ng/mL choriogenin (Chg) measured in whole body homogenate than controls. Though Chg expression in fish exposed to ethinylestradiol (EE2) exhibited a traditional sigmoidal concentration response, curves fit to Chg expressed in fish exposed to pyrethroids suggest a unimodal response, decreasing slightly as concentration increases. Whereas the in vivo response indicated that bifenthrin and permethrin or their metabolites act as estrogen agonists, the CALUX assay demonstrated estrogen antagonism by the pyrethroids. The results, supported by evidence from previous studies, suggest that bifenthrin and permethrin, or their metabolites, appear to act as estrogen receptor (ER) agonists in vivo, and that the unmetabolized pyrethroids, particularly bifenthrin, act as an ER antagonists in cultured mammalian cells.

Identification of nonregulated pollutants in north sea‐produced water discharges

Produced water collected from oil and gas platforms on the United Kingdom Continental Shelf was characterized for nonregulated pollutants through an effects-directed analysis procedure. Produced water samples were characterized for the presence of aryl hydrocarbon receptor (AhR) and estrogen receptor (ER) agonists using the dioxin-responsive, chemical-activated luciferase gene expression assay (DR-CALUX) and yeast estrogen screen (YES) bioassays. The AhR and ER agonists were then isolated by normal-phase, high-performance liquid chromatography and identified using gas chromatography coupled to mass spectrometry in a number of formats. The identified compounds were cross-referenced with those compounds routinely analyzed and regarded by the Oslo and Paris (OSPAR) Commission for the North East Atlantic as priority hazardous substances. The occurrence in produced water of a number of nonregulated compounds with demonstrable potential environmental effects is presented, to our knowledge for the first time. These include persistent organic contaminants, such as hexachlorobenzene, decachlorobiphenyl, and octachlorodibenzofuran.

AhR agonist and genotoxicant bioavailability in a PAH-contaminated soil undergoing biological treatment

Degradation of the 16 US EPA priority PAHs in soil subjected to bioremediation is often achieved. However, the PAH loss is not always followed by a reduction in soil toxicity. For instance, bioanalytical testing of such soil using the chemical-activated luciferase gene expression (CALUX) assay, measuring the combined effect of all Ah receptor (AhR) activating compounds, occasionally indicates that the loss of PAHs does not correlate with the loss of Ah receptor-active compounds in the soil. In addition, standard PAH analysis does not address the issue of total toxicant bioavailability in bioremediated soil. To address these questions, we have used the CALUX AhR agonist bioassay and the Comet genotoxicity bioassay with RTL-W1 cells to evaluate the toxic potential of different extracts from a PAH-contaminated soil undergoing large-scale bioremediation. The extracts were also chemically analyzed for PAH16 and PCDD/PCDF. Soil sampled on five occasions between day 0 and day 274 of biological treatment was shaken with n-butanol with vortex mixing at room temperature to determine the bioavailable fraction of contaminants. To establish total concentrations, parts of the same samples were extracted using an accelerated solvent extractor (ASE) with toluene at 100 degrees C. The extracts were tested as inducers of AhR-dependent luciferase activity in the CALUX assay and for DNA breakage potential in the Comet bioassay. The chemical analysis of the toluene extracts indicated slow degradation rates and the CALUX assay indicated high levels of AhR agonists in the same extracts. Compared to day 0, the bioavailable fractions showed no decrease in AhR agonist activity during the treatment but rather an up-going trend, which was supported by increasing levels of PAHs and an increased effect in the Comet bioassay after 274 days. The bio-TEQs calculated using the CALUX assay were higher than the TEQs calculated from chemical analysis in both extracts, indicating that there are additional toxic PAHs in both extracts that are not included in the chemically derived TEQ. The response in the CALUX and the Comet bioassays as well as the chemical analysis indicate that the soil might be more toxic to organisms living in soil after 274 days of treatment than in the untreated soil, due to the release of previously sorbed PAHs and possibly also metabolic formation of novel toxicants. Our results put focus on the issue of slow degradation rates and bioavailability of PAHs during large-scale bioremediation treatments. The release of sorbed PAHs at the investigated PAH-contaminated site seemed to be faster than the degradation rate, which demonstrates the importance of considering the bioavailable fraction of contaminants during a bioremediation process. It has to be ensured that soft remediation methods like biodegradation or the natural remediation approach do not result in the mobilization of toxic compounds including more mobile degradation products. For PAH-contaminated sites this cannot be assured merely by monitoring the 16 target PAHs. The combined use of a battery of biotests for different types of PAH effects such as the CALUX and the Comet assay together with bioavailability extraction methods may be a useful screening tool of bioremediation processes of PAH-contaminated soil and contribute to a more accurate risk assessment. If the bioremediation causes a release of bound PAHs that are left undegraded in an easily extracted fraction, the soil may be more toxic to organisms living in the soil as a result of the treatment. A prolonged treatment time may be one way to reduce the risk of remaining mobile PAHs. In critical cases, the remediation concept might have to be changed to ex situ remediation methods.

Dioxin-Like and Transthyretin-Binding Compounds in Indoor Dusts Collected from Japan: Average Daily Dose and Possible Implications for Children

Many researchers are increasingly interested in human exposure to house dust containing household compounds such as polybrominated diphenylethers (PBDEs). Nevertheless, no investigations have addressed the endocrine-disrupting potencies of compounds contained in indoor dust. This study specifically addresses endocrine-disrupting potencies such as dioxin-like activity and human transthyretin (TTR)-binding potencies. Using in vitro bioassays, we investigated these activity levels in indoor dusts. We performed exposure assessments of active compounds that are contained in house dust for subsequent evaluation of house dusts' risks to humans. Dioxin-like and TTR-binding activities in sulfuric acid treatment extracts of house and office dust were investigated using Dioxin-Responsive Chemical-Activated LUciferase gene eXpression assay (DR-CALUX) and TTR-binding assay (in vitro competitive human TTR-binding assay). Dioxin-like activities in indoor dust were 38-1400 pg CALUX-TEQ (2,3,7,8-TCDD equivalent)/g (median 160 pg CALUX-TEQ/g) and TTR-binding potencies were 300-5000 pmol T4EQ (thyroxine (T4) equivalent)/g (median 1000 pmol T4EQ/g), which are higher values than those in other environmental samples, e.g., contaminated sediments. These exposure results suggest that children might be affected by both dioxin-like compounds and TTR-binding compounds via house dust. When the ingestion rate, CALUX-TEQ, and T4EQ for house dust are high, thyroid hormone homeostasis in children may be disrupted.

Time-course changes of mixture effects on AhR binding-dependent luciferase activity in a crude extract from a compost sample

Crude extracts of environmental samples contain stable and labile aryl hydrocarbon receptor (AhR) ligands, and show huge activities in the cell-based bioassay, and these activities are higher than the chemically calculated induction equivalent values. It is thought that not only unidentified AhR ligands but also mixture effect among compounds might contribute to these activities. In the previous work, we have indicated that hydrophobic compounds in household sewage sludge (HSS) compost may interact synergistically with 2,3,7,8-TCDD in the CALUX (DR-CALUX ®: Dioxin-Responsive Chemical-Activated Luciferase gene eXpression) assay [Suzuki, G., Takigami, H., Kushi, Y., Sakai, S., 2004. Evaluation of mixture effects in a crude extract of compost using the CALUX bioassay and HPLC fractionation. Environ. Int. 30, 1055–1066]. In this study, we focused on co-existing stable compounds such as halogenated aromatic hydrocarbons (HAHs) and labile compounds such as polyaromatic hydrocarbons (PAHs) in the crude extract and investigated the time-course changes of mixture effects among compounds in environmental samples using the CALUX assay and normal-phase high-performance liquid chromatography (NP-HPLC) fractionation. We confirmed that CYP1A-inducing PAHs and HAHs could be separated by NP-HPLC on a nitrophenylpropylsilica (NITRO) column. To determine whether the activities of AhR ligands in environmental samples (including the HSS compost) could be assessed by the additivity theory, we compared the CALUX activity of the crude extract with the arithmetical sum of the activities of all the fractions separated by NP-HPLC. We confirmed a potentiation of CALUX activity at 12-, 24- and 48-h exposure durations. In contrast, CALUX activity increased additively at 6- and 72-h exposure durations. CALUX activity was potentiated when the CALUX activity of the HPLC fractions showed a remarkable reduction resulting in a change of activity profiles. In contrast, additivity was observed at a 72-h exposure duration when the CALUX activity of the HPLC fractions showed neither remarkable reduction nor a change in profile. Our results suggest that differences in the metabolic decomposition of compounds affected mixture effects on CALUX activity in a crude extract from HSS compost.

Chemical-Activated Luciferase Gene Expression (CALUX): A Novelin VitroBioassay for Ah Receptor Active Compounds in Sediments and Pore Water

This study demonstrates that the novelin vitroCALUX (chemical-activated luciferase expression) assay is a rapid, sensitive assay for assessing the toxic potency of (mixtures of) aryl hydrocarbon receptor (AhR)-active compounds in sediments and pore waters. A rat hepatoma (H4IIE) cell line, stably transfected with a construct containing the dioxin-responsive element (DRE) sequence and the luciferase reporter gene, was used to determine the relative potency or the total activities of AhR-active compounds in sediment and pore water extracts. This novel CALUX assay had a detection limit of 0.5 fmol of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The sensitivity and linear working range was slightly better than for the ethoxyresorufinO-deethylase (EROD) assay in H4IIE wild type cells. The primary improvement of the CALUX assay compared to the EROD assay, however, is that the CALUX assay is insensitive to substrate inhibition. The CALUX activity induced by organic extracts from 450-mg aliquots of sediment or 250-μl aliquots of pore water corresponded with the instrumentally analyzed degree of pollution of the sediment. Using pore water, only a simple and rapid extraction procedure was needed, without additional clean-up to prevent cell death. The response from pore water samples in an 8-day early life stage test with zebra fish (Branchydanio rerio) corresponded with the CALUX induction, although the correlation was sometimes disturbed by heavy metals. Two polychlorinated terphenyl mixtures, the PCB-substitute Ugilec 141, polybrominated diphenylethers, and the PCB-mixture Clophen A50 were tested in the CALUX assay and had induction potencies that were 10−4–10−7compared to TCDD.