Considerations for potency equivalent calculations in the Ah receptor-based CALUX bioassay: normalization of superinduction results for improved sample potency estimation

The chemically activated luciferase expression (CALUX) system is a mechanistically based recombinant luciferase reporter gene cell bioassay used in combination with chemical extraction and clean-up methods for the detection and relative quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like halogenated aromatic hydrocarbons in a wide variety of sample matrices. While sample extracts containing complex mixtures of chemicals can produce a variety of distinct concentration-dependent luciferase induction responses in CALUX cells, these effects are produced through a common mechanism of action (i.e. the Ah receptor (AhR)) allowing normalization of results and sample potency determination. Here we describe the diversity in CALUX response to PCDD/Fs from sediment and soil extracts and not only report the occurrence of superinduction of the CALUX bioassay, but we describe a mechanistically based approach for normalization of superinduction data that results in a more accurate estimation of the relative potency of such sample extracts.     

The CALUX bio-assay: analytical comparison between mouse hepatoma cell lines with a low (H1L6.1c3) and high (H1L7.5c1) number of dioxin response elements

Dioxins, furans and polychlorinated biphenyls are contaminants of high concern and as such, sensitive tools are needed to detect these persistent organic compounds in a variety of matrices. Due to the large amount of samples that need to be investigated for example for food and feed control, the CALUX bioassay (H1L6.1 clone) was developed allowing rapid and cost-efficient analysis of biological and environmental samples. Recently, a new and more sensitive clone (H1L7.5) was constructed as the third generation CALUX bioassay. This new cell line was subject of an amplification of dioxin response elements (DREs), allowing lower concentrations of target compound to be analyzed. A comparison is made between the previous, well-defined H1L6.1c3 cell line and the new H1L7.5c1 cell line: it appears that the bioassay making use of the higher number of DREs is more stable and robust, shows better repeatability and reproducibility and is; on average, 3 times more sensitive.     

Challenges encountered in the analysis of phthalate esters in foodstuffs and other biological matrices

Phthalate esters are ubiquitous environmental pollutants and are recognized as environmental endocrine disruptors because of their potential to elicit reproductive and developmental toxicity. Several phthalate esters have been listed by the US Environmental Protection Agency (EPA) as chemicals of concern. Determination of concentrations of phthalate esters in foodstuffs, typically present at sub to low nanogram-per-gram concentrations (between 0.1 and 100?ng?g^?1), is essential for assessment of human dietary exposure. However, phthalate esters are commonly present as contaminants in several laboratory products, including organic solvents, that are used in sample preparation and analysis. Therefore, accurate analysis of phthalates in food samples is a challenging task. In this review, we summarize the methods available for the determination of phthalate esters in foodstuffs and report on concentrations of phthalates in foodstuffs and potential sources of contamination by phthalates in the analysis of foodstuffs. We offer suggestions to eliminate and/or reduce background levels of contamination by phthalates in the analysis of food and other biological samples. We also introduce methods that are suitable for trace analysis of phthalates in a variety of liquid and solid food samples, in particular, a liquid–liquid extraction method for removal of lipids from food samples, because these can substantially reduce background levels of phthalates in the analytical procedure.     

Uses of speciation techniques in biomonitoring for assessing human exposure to organic environmental chemicals

Speciation analysis has been used for many years to identify and measure different forms of a given chemical in environmental and human samples. Although the term speciation is generally applied to the measurement of inorganic chemicals, the term can also be applied to many measurements of organic chemicals in complex samples, such as environmental media and biological matrices. We present several examples of achieving speciation analysis by selecting the appropriate biological matrix in which to measure a specific chemical(s), by a given analytical method, for the most accurate assessment of human exposure to the environmental chemical. Much of this information and many of these techniques are transferable to the measurement of inorganic elements in environmental and biological samples.     

Solid phase extraction of food contaminants using molecular imprinted polymers

Food contamination from natural or anthropogenic sources poses severe risks to human health. It is now largely accepted that continuous exposure to low doses of toxic chemicals can be related to several chronic diseases, including some type of cancer and serious hormonal dysfunctions.Contemporary analytical methods have the sensitivity required for contamination detection and quantification, but direct application of these methods on food samples can be rarely performed. In fact, the matrix introduces severe disturbances, and analysis can be performed only after some clean-up and preconcentration steps. Current sample pre-treatment methods, mostly based on the solid phase extraction technique, are very fast and inexpensive but show a lack of selectivity, while methods based on immunoaffinity extraction are very selective but expensive and not suitable for harsh environments. Thus, inexpensive, rapid and selective clean-up methods, relaying on “intelligent” materials are needed. Recent years have seen a significant increase of the “molecularly imprinted solid phase extraction” (MISPE) technique in the food contaminant analysis. In fact, this technique seems to be particularly suitable for extractive applications where analyte selectivity in the presence of very complex and structured matrices represents the main problem. In this review, several applications of MISPE in food contamination analysis will be discussed, with particular emphasis on the extraction of pesticides, drugs residua, mycotoxins and environmental contaminants.     

Monitoring dioxins in food and feedstuffs using accelerated solvent extraction with a novel integrated carbon fractionation cell in combination with a CAFLUX bioassay

The concentrations of dioxins in fish oil and fish meal were determined with accelerated solvent extraction, using a novel integrated carbon fractionation extraction cell followed by a miniturized multilayer silica column and bioanalysis on a recently-developed chemically-activated fluorescent gene expression cell bioassay. The developed method allows for simultaneous gravimetric lipid weight determination, which was shown for both matrices under study (about 100% lipid recovery of each sample). Initial results practically meet the quality criteria on screening methods for control of dioxins in food and feedstuffs laid down in the EU Commission Directives 2002/69/EC (food) and 2002/70/EC (feed). This demonstrates that the developed method can be used as a screening tool for monitoring dioxins in food and feed after some additional improvements and testing on a greater number of matrices.     

Wastewater-based epidemiology to assess human exposure to personal care and household products – A review of biomarkers,analytical methods,and applications

Humans are nowadays exposed to numerous chemicals in our day-to-day life, including parabens, UV filters, phosphorous flame retardants/plasticizers, bisphenols, phthalates and alternative plasticizers, which can have different adverse effects to human health. Estimating human’s exposure to these potentially harmful substances is, therefore, of paramount importance. Human biomonitoring (HBM) is the existing approach to assess exposure to environmental contaminants, which relies on the analysis of specific human biomarkers (parent compounds and/or their metabolic products) in biological matrices from individuals. The main drawback is its implementation, which involves complex cohort studies. A novel approach, wastewater-based epidemiology (WBE), involves estimating exposure from the analysis of biomarkers in sewage (a pooled urine and feces sample of an entire population). One of the key challenges of WBE is the selection of biomarkers which are specific to human metabolism, excreted in sufficient amounts, and stable in sewage. So far, literature data on potential biomarkers for estimating exposure to these chemicals are scattered over numerous pharmacokinetic and HBM studies. Hence, this review provides a list of potential biomarkers of exposure to more than 30 widely used chemicals and report on their urinary excretion rates. Furthermore, the potential and challenges of WBE in this particular field is discussed through the review of pioneer WBE studies, which for the first time explored applicability of this novel approach to assess human exposure to environmental contaminants. In the future, WBE could be potentially applied as an “early warning system”, which could promptly identify communities with the highest exposure to environmental contaminants.     

An extraction/concentration procedure for analysis of low-level explosives in soils

The methods traditionally used for explosives analysis in soil matrices have inherent data quality limitations for low-level samples. The traditional methods employ a soil-dilution extraction of the sample prior to analysis by high performance liquid chromatography with UV absorption detection. Another concern with the traditional analysis is that energetics contamination in environmental samples is often very heterogeneous in nature, usually requiring a large number of samples and multiple testing. The technique presented here addresses these data quality limitations by using a concentrative extraction procedure which produces a small volume of extract from a large soil sample. A concentration factor of 60-fold is achieved in this manner and energetics detection limits for soils are lowered by two orders of magnitude. The larger soil sample size also helps reduce the error associated with sample heterogeneity. The ability to detect explosive-based contaminants at levels of environmental interest enables a more accurate assessment of the transport pathways and treatment options for explosives contamination.     

Rosmarinus officinalis L. Leaf Extracts and Their Metabolites Inhibit the Aryl Hydrocarbon Receptor (AhR) Activation In Vitro and in Human Keratinocytes: Potential Impact on Inflammatory Skin Diseases and Skin Cancer

Aryl hydrocarbon receptor (AhR) activation by environmental agents and microbial metabolites is potentially implicated in a series of skin diseases. Hence, it would be very important to identify natural compounds that could inhibit the AhR activation by ligands of microbial origin as 6-formylindolo[3,2-b]carbazole (FICZ), indirubin (IND) and pityriazepin (PZ) or the prototype ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Five different dry Rosmarinus officinalis L. extracts (ROEs) were assayed for their activities as antagonists of AhR ligand binding with guinea pig cytosol in the presence of [³H]TCDD. The methanolic ROE was further assayed towards CYP1A1 mRNA induction using RT-PCR in human keratinocytes against TCDD, FICZ, PZ, and IND. The isolated metabolites, carnosic acid, carnosol, 7-O-methyl-epi-rosmanol, 4′,7-O-dimethylapigenin, and betulinic acid, were assayed for their agonist and antagonist activity in the presence and absence of TCDD using the gel retardation assay (GRA). All assayed ROE extracts showed similar dose-dependent activities with almost complete inhibition of AhR activation by TCDD at 100 ppm. The methanol ROE at 10 ppm showed 99%, 50%, 90%, and 85% inhibition against TCDD, FICZ, IND, and PZ, respectively, in human keratinocytes. Most assayed metabolites exhibited dose-dependent antagonist activity. ROEs inhibit AhR activation by TCDD and by the Malassezia metabolites FICZ, PZ, and IND. Hence, ROE could be useful for the prevention or treatment of skin diseases mediated by activation of AhR.     

Holographic QSAR of environmental estrogens

Environmental endocrine disrupting chemicals(EDCs) or environmental estrogens pose severe healthhazard to wildlife and humans. They are believed to bethe main cause of the weakening activity and secretionof sex hormone, sperm declination, abnormal repro-d…     

Assessment of total effective xenoestrogen burden in adipose tissue and identification of chemicals responsible for the combined estrogenic effect

Test systems to screen for estrogenicity and appropriate biomarkers of human exposure are required for epidemiological studies of endocrine disruption. We addressed these issues by developing and standardising a method to assess the total estrogenic xenobiotic burden in human adipose tissue. In this study, which is the continuation of a previous work, we have improved the protocol for extensive fractionation of a higher number of tissue samples in order to investigate bioaccumulated xenoestrogens that are candidates for estrogenicity and to assess their combined estrogenic effect. This was achieved by extensive HPLC separation of xenoestrogens from endogenous hormones followed by testing of individual fractions in the E-Screen test for estrogenicity. Organochlorine pesticides, PCBs and halogenated bisphenols and alkylphenols were collected in the most lipophilic fractions, followed by progestins, androgens and estradiol esters, and then by steroidal estrogens; phyto- and myco-estrogens were collected around the end of the run. These results were confirmed by exhaustive chemical analysis. In 458 human adipose tissue samples, the total effective xenoestrogen burden was positive in 75% of samples in the pooled fraction that contained organohalogenated xenoestrogens (mean 515.3 pM Eeq/g lipid; range 0–14.5 nM) and in 82% of samples in the pooled fraction where natural estrogens eluted (mean 696.6 pM Eeq/g lipid; range 0–12.9 nM). Organochlorine pesticides emerged as candidate chemicals for the estrogenicity of the first pooled fraction, because DDT and derivatives were present in 98.3% of the samples. However, no correlation was found between the concentration of any single chemical and the estrogenicity determined in the bioassay. There may be several reasons for this lack of concordance: (i) the estrogenic effects depicted in the E-Screen bioassay are a consequence of the combined effect of several organohalogens or (ii) the proliferative effect is due to other chemicals not measured. Because additive, synergistic or antagonistic mechanisms may account for the final effect observed in the pooled fractions, the approach proposed in this work is more appropriate for exposure assessment in epidemiological studies than the determination of individual chemicals in human samples.     

Biological matrices for the evaluation of exposure to environmental tobacco smoke during prenatal life and childhood

The measurement of nicotine and its major metabolites cotinine and trans-3´-hydroxicotinine together with other minor metabolites (e.g., cotinine N-oxide, cotinine, and trans-3´-hydroxicotinine glucuronides) in conventional and nonconventional biological matrices has been used as a biomarker to assess the exposure to environmental tobacco smoke during childhood. The determination of these substances in matrices such as amniotic fluid, meconium, and fetal hair accounts for prenatal exposure to cigarette smoking at different stages of pregnancy. Nicotine and its metabolites in cord blood, neonatal urine, and breast milk are useful for determining acute exposure to drugs of abuse in the period immediately before and after delivery. Cotinine measurement in children’s blood and urine and nicotine and cotinine measurements in children’s hair constitute objective indexes of acute and chronic exposure during infancy, respectively. However, for monitoring and categorizing cumulative exposure to environmental tobacco smoke during the entire childhood, including the prenatal period, the assessment of nicotine in teeth has been proposed as a promising noninvasive tool. This article reviews the usefulness of measurement of nicotine and its metabolites in different fetal and pediatric biological matrices in light of noninvasive collection, time window of exposure detection, and finally clinical application in pediatrics.     

Analytical methods for the determination of bisphenol A in food

Food constitutes the primary route for human exposure to bisphenol A (BPA), one of the highest volume chemicals produced worldwide. The estrogenic properties of BPA, its wide dispersive use and the recent extensive literature describing low-dose BPA effects in animals, have raised concerns about its possible adverse effects on human health. A reliable health risk assessment of BPA relies basically on its unambiguous identification and accurate quantification in food, and the aim of the present review is to give an overview of the analytical methods reported so far for the determination of BPA in these matrices. Emphasis is placed on the main strategies developed for sample treatment, which usually consists of several laborious and time-consuming steps in order to achieve the required sensitivity and selectivity. Separation, identification and quantitation of BPA is today reliably made with mass spectrometric methods, namely liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), and thus main attention is devoted to these techniques, but other methods using LC coupled to fluorescence or electrochemical detection, as well as immunochemical methods are also covered. Recent and expected future developments are discussed.     

How suitable are scalp hair and toenail as biomonitors?

This paper describes the assessment of exposure levels to metals and possible workers' contamination in three galvanizing factories applying the same processes. Concerning the elements determined in air filters, 92.3% of them were also determined in hair and toenail samples: Ag, Al, As, Au, Cl, Cr, Cu, Fe, Mn, Na, Sb and Zn. These result point out that hair and toenail reflect the influence of the polluted environment on workers' health and can be useful as bioindicators in epidemiological studies. Instrumental neutron activation analysis was applied to all matrices which confirms its status as one of the most versatile analytical techniques.     

TEQ-value determinations of animal feed; emphasis on the CALUX bioassay validation

Polyhalogenated aromatic hydrocarbons, such as polychlorinated dibenzo-p-dioxins are a large and diverse group of environmental pollutants. Their tendency to accumulate in the food chain and their toxicity make monitoring necessary. The reference analysis method is laborious and very expensive, therefore cheap and rapid bioassays have been developed. The chemical-activated luciferase bioassay (CALUX) bioassay uses a recombinant cell line, which responds to dioxins and dioxin-like molecules with Ah receptor (AhR)-dependent induction of firefly luciferase in a dose related response. The CALUX was tested for its use in the screening of feed. Aliquots of 20 g of enriched feed were extracted with a toluene:methanol mixture (20:4 v/v) and extracts were defatted on 33% H₂SO₄ silica columns and purified on carbon columns. Only the dioxin and furan fraction was analysed, the PCB fraction was discarded. The precision of the method is acceptable and in compliance with an R.S.D. <30% as suggested for cell-based bioassays in the Commission Directive 2002/70/EC of July 2002. The results evidence good agreement between TEQ-values obtained by either CALUX or GC–HRMS. The method is now routinely in use for a feed screening programme designed by the Federal Agency for the Safety of the Food chain. Approximately, 25 samples are analysed weekly. From the obtained results approximately 10% was confirmed by GC–HRMS. The false positive ratio is 1% and no false negatives were found, making the use of the CALUX technology advantageous.     

Challenges in determining perchlorate in biological tissues and fluids: implications for characterizing perchlorate exposure

The ability to measure environmental contaminants in biological tissues and fluids is important in the characterization of exposure. However, the analysis of certain contaminants in these matrices presents significant challenges. Perchlorate (ClO₄⁻) has emerged as a potential contaminant of concern primarily in drinking water and also in contaminated food. Significant advances have been made in the analysis of perchlorate in environmental matrices (water, soil) by ion chromatography (IC). In contrast, the analysis of perchlorate in extracts of biological tissues and fluids (vegetation, organs, milk, blood, urine, etc.) presents several challenges including small sample sizes, extracts with high matrix conductivity, and co-elution of other ions during IC analysis. To be able to detect low concentrations of perchlorate in biological samples, interferences must be removed or minimized, such as through the use of preparative chromatography cleanup techniques and/or alternative analytical methods less susceptible to common interferences (preconcentration or mass spectrometric detection). We present discussion and examples of the challenges encountered in the analysis of tissue extracts and fluids for perchlorate by IC and how some of those analytical challenges have been overcome.     

Application of Biomarkers to Assessment of Risk to Human Health from Exposure to Mycotoxins

Mycotoxins, the toxic compounds produced by mold secondary metabolism, represent a relevant source of danger to humans through alimentary channels. Efforts have been made by researchers and by national authorities to assess mycotoxin incidence in food, but often results are to be considered approximate or inaccurate due to the huge difficulties posed by sampling procedures. More recently the evaluation of mycotoxins in biological fluids have been given increasing attention since the results may offer valuable indications, although general on the overall status of mycotoxin contamination in food and feed. The assessment of the degree of exposure to these contaminants in the population or in specific groups can also be pursued. Researches on mycotoxins in biological fluids greatly contribute to clarify the mechanism of health impairment attributable to these toxic compounds and to elucidate the dose–response relationship. Despite the considerable efforts devoted to mycotoxin research in the past few decades, improvements in methodology has to be achieved mainly in sampling procedures and in quality assurance of the laboratories involved in mycotoxin analysis, as well as in the selection of appropriate biomarkers.     

Elucidating the aryl hydrocarbon receptor antagonism from a chemical-structural perspective

ABSTRACT

The aryl hydrocarbon receptor (AhR) plays an important role in several biological processes such as reproduction, immunity and homoeostasis. However, little is known on the chemical-structural and physicochemical features that influence the activity of AhR antagonistic modulators. In the present report, in vitro AhR antagonistic activity evaluations, based on a chemical-activated luciferase gene expression (AhR-CALUX) bioassay, and an extensive literature review were performed with the aim of constructing a structurally diverse database of contaminants and potentially toxic chemicals. Subsequently, QSAR models based on Linear Discriminant Analysis and Logistic Regression, as well as two toxicophoric hypotheses were proposed to model the AhR antagonistic activity of the built dataset. The QSAR models were rigorously validated yielding satisfactory performance for all classification parameters. Likewise, the toxicophoric hypotheses were validated using a diverse set of 350 decoys, demonstrating adequate robustness and predictive power. Chemical interpretations of both the QSAR and toxicophoric models suggested that hydrophobic constraints, the presence of aromatic rings and electron-acceptor moieties are critical for the AhR antagonism. Therefore, it is hoped that the deductions obtained in the present study will contribute to elucidate further on the structural and physicochemical factors influencing the AhR antagonistic activity of chemical compounds.