CALUX

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Chemical Activated LUciferase gene eXpression (CALUX) is a ligand-dependent nuclear receptor-based bioassay used in the detection of specific chemicals or classes of chemicals in samples. It consists of a modified cell line that has been stably transfected with a DNA construct with a luciferase reporter gene under control of receptor-specific DNA response elements that can stimulate transcription of the inserted luciferase gene and produce the light-generating enzyme which can be easily measured. The DNA response elements can be varied in order to provide binding sites for other receptors that are regulated by a chemical or class of chemicals of interest that want to be detected. Thus, numerous CALUX bioassays have been developed for detection of diverse chemicals of interest. Most applications have been directed toward the detection of environmentally harmful chemicals, such as those affecting the endocrine system (environmental hormones).

Contents

CALUX is an effect based screening method as it measures the total effect ligands (from a sample) have on a specific receptor. Unlike chemical analysis, CALUX is thus able to measure total activity on the receptor of interest. This includes both identified and unidentified activators (agonists) and inhibitors (receptor antagonists).

Applications

The aryl hydrocarbon receptor (AhR)-responsive (AhR-CALUX) bioassay is commonly used in the detection of dioxins and dioxin-like compounds in sample extracts. It is based on the molecular mechanism by which the AhR activates gene expression. [1] AhR-CALUX is used by researchers and companies that want to screen for the presence of dioxins and dioxin-like compounds in a wide variety of biological and environmental matrices, commercial and consumer products and in food and feed in order to evaluate their safety and/or level of contamination. [2] While the measurement of dioxin and related dioxin-like chemicals in a sample extract using the AhR-CALUX bioassay is significantly cheaper than the chemical analysis requiring gas chromatography - high-resolution mass spectrometry (GC-HRMS), it only provides a measurement of the total level of AhR-active dioxins and related dioxin-like chemicals in a sample extract and determination of the specific congeners requires analysis any GC-HRMS. Additionally, given the recently established role of the AhR in human health and disease, the AhR-CALUX bioassay is also being used widely as a high-throughput screening method to identify and characterize AhR-active chemicals as potential human therapeutic drugs. [3]

Environmental chemicals having sex hormone-like activity are detected by similar bioassays as well, including the Estrogen Receptor-Responsive CALUX (ER-CALUX) and Androgen Receptor-Responsive CALUX (AR-CALUX).

See also

Related Research Articles

Aryl Molecular groups or substituents derived from an aromatic ring

In organic chemistry, an aryl is any functional group or substituent derived from an aromatic ring, usually an aromatic hydrocarbon, such as phenyl and naphthyl. "Aryl" is used for the sake of abbreviation or generalization, and "Ar" is used as a placeholder for the aryl group in chemical structure diagrams, analogous to “R” used for any organic substituent. “Ar” is not to be confused with the elemental symbol for argon.

Methylcholanthrene Chemical compound

Methylcholanthrene is a highly carcinogenic polycyclic aromatic hydrocarbon produced by burning organic compounds at very high temperatures. Methylcholanthrene is also known as 3-methylcholanthrene, 20-methylcholanthrene or the IUPAC name 3-methyl-1,2-dyhydrobenzo[j]aceanthrylene. The short notation often used is 3-MC or MCA. This compound forms pale yellow solid crystals when crystallized from benzene and ether. It has a melting point around 180 °C and its boiling point is around 280 °C at a pressure of 80 mmHg. Methylcholanthrene is used in laboratory studies of chemical carcinogenesis. It is an alkylated derivative of benz[a]anthracene and has a similar UV spectrum. The most common isomer is 3-methylcholanthrene, although the methyl group can occur in other places.

In the biological context of organisms' production of gene products, downregulation is the process by which a cell decreases the quantity of a cellular component, such as RNA or protein, in response to an external stimulus. The complementary process that involves increases of such components is called upregulation.

Aryl hydrocarbon receptor Vertebrate transcription factor

The aryl hydrocarbon receptor is a protein that in humans is encoded by the AHR gene. The aryl hydrocarbon receptor is a transcription factor that regulates gene expression. It was originally thought to function primarily as a sensor of xenobiotic chemicals and also as the regulator of enzymes such as cytochrome P450s that metabolize these chemicals. The most notable of these xenobiotic chemicals are aromatic (aryl) hydrocarbons from which the receptor derives its name.

Aryl hydrocarbon receptor nuclear translocator Protein-coding gene in the species Homo sapiens

The ARNT gene encodes the aryl hydrocarbon receptor nuclear translocator protein that forms a complex with ligand-bound aryl hydrocarbon receptor (AhR), and is required for receptor function. The encoded protein has also been identified as the beta subunit of a heterodimeric transcription factor, hypoxia-inducible factor 1 (HIF1). A t(1;12)(q21;p13) translocation, which results in a TEL-ARNT fusion protein, is associated with acute myeloblastic leukemia. Three alternatively spliced variants encoding different isoforms have been described for this gene.

The aryl-hydrocarbon receptor repressor also known as AHRR is a human gene.

Polychlorinated dibenzofurans

More thorough treatise of all groups with similar actions and binding to aryl hydrocarbon receptor is given in Dioxins and dioxin-like compounds.

Response elements are short sequences of DNA within a gene promoter or enhancer region that are able to bind specific transcription factors and regulate transcription of genes.

AH receptor-interacting protein Protein-coding gene in the species Homo sapiens

AH receptor-interacting protein (AIP) also known as aryl hydrocarbon receptor-interacting protein, immunophilin homolog ARA9, or HBV X-associated protein 2 (XAP-2) is a protein that in humans is encoded by the AIP gene. The protein is a member of the FKBP family.

NCOA7

Nuclear receptor coactivator 7 is a protein that in humans is encoded by the NCOA7 gene.

Ecdysone receptor

The ecdysone receptor is a nuclear receptor found in arthropods, where it controls development and contributes to other processes such as reproduction. The receptor is a non-covalent heterodimer of two proteins, the EcR protein and ultraspiracle protein (USP). It binds to and is activated by ecdysteroids. Insect ecdysone receptors are currently better characterized than those from other arthropods, and mimics of ecdysteroids are used commercially as caterpillar-selective insecticides.

Bioreporter Genetically engineered microbial cells

Bioreporters are intact, living microbial cells that have been genetically engineered to produce a measurable signal in response to a specific chemical or physical agent in their environment. Bioreporters contain two essential genetic elements, a promoter gene and a reporter gene. The promoter gene is turned on (transcribed) when the target agent is present in the cell’s environment. The promoter gene in a normal bacterial cell is linked to other genes that are then likewise transcribed and then translated into proteins that help the cell in either combating or adapting to the agent to which it has been exposed. In the case of a bioreporter, these genes, or portions thereof, have been removed and replaced with a reporter gene. Consequently, turning on the promoter gene now causes the reporter gene to be turned on. Activation of the reporter gene leads to production of reporter proteins that ultimately generate some type of a detectable signal. Therefore, the presence of a signal indicates that the bioreporter has sensed a particular target agent in its environment.

Dioxins and dioxin-like compounds Class of chemical compounds

Dioxins and dioxin-like compounds (DLCs) are a group of chemical compounds that are persistent organic pollutants (POPs) in the environment. They are mostly by-products of burning or various industrial processes - or, in case of dioxin-like PCBs and PBBs, unwanted minor components of intentionally produced mixtures.

Chlorinated polycyclic aromatic hydrocarbons (Cl-PAHs) are a group of compounds comprising polycyclic aromatic hydrocarbons with two or more aromatic rings and one or more chlorine atoms attached to the ring system. Cl-PAHs can be divided into two groups: chloro-substituted PAHs, which have one or more hydrogen atoms substituted by a chlorine atom, and chloro-added Cl-PAHs, which have two or more chlorine atoms added to the molecule. They are products of incomplete combustion of organic materials. They have many congeners, and the occurrences and toxicities of the congeners differ. Cl-PAHs are hydrophobic compounds and their persistence within ecosystems is due to their low water solubility. They are structurally similar to other halogenated hydrocarbons such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs). Cl-PAHs in the environment are strongly susceptible to the effects of gas/particle partitioning, seasonal sources, and climatic conditions.

2,3,7,8-Tetrachlorodibenzodioxin Polychlorinated dibenzo-p-dioxin, chemical compound

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a polychlorinated dibenzo-p-dioxin (sometimes shortened, though inaccurately, to simply 'dioxin') with the chemical formula C12H4Cl4O2. Pure TCDD is a colorless solid with no distinguishable odor at room temperature. It is usually formed as an unwanted product in burning processes of organic materials or as a side product in organic synthesis.

Toxic equivalency factor (TEF) expresses the toxicity of dioxins, furans and PCBs in terms of the most toxic form of dioxin, 2,3,7,8-TCDD. The toxicity of the individual congeners may vary by orders of magnitude.

A bioanalytical equivalent (BEQ) is a unit of measure in the field of bioassays. It is used for example in the bioassay CALUX for testing dioxins and dioxin-like compounds.

E-SCREEN is a cell proliferation assay based on the enhanced proliferation of human breast cancer cells (MCF-7) in the presence of estrogen active substances. The E-SCREEN test is a tool to easily and rapidly assess estrogenic activity of suspected xenoestrogens. This bioassay measures estrogen-induced increase of the number of human breast cancer cell, which is biologically equivalent to the increase of mitotic activity in tissues of the genital tract. It was originally developed by Soto et al and was included in the first version of the OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupters published in 2012. However, due to failed validation, it was not included in the updated version of the framework published in 2018.

Heptachlorodibenzo-<i>p</i>-dioxin Chemical compound

1,2,3,4,6,7,8-Heptachlorodibenzo-para-dioxin (often referred to as 1,2,3,4,6,7,8-HpCDD) is a polychlorinated derivative of dibenzo-p-dioxin and can therefore be categorized as polychlorinated dibenzo-p-dioxin (PCDD), a subclass of dioxins which includes 75 congeners. HpCDD is the dibenzo-p-dioxin which is chlorinated at positions 1, 2, 3, 4, 6, 7, and 8. It is a polycyclic heterocyclic organic compound, since HpCDD contains multiple cyclic structures (two benzene rings connected by a 1,4-dioxin ring) in which two different elements (carbon and oxygen) are members of its rings. HpCDD has molecular formula C12HCl7O2 and is an off-white powder, which is insoluble in water.

6-Formylindolo(3,2-b)carbazole Chemical compound

6-Formylindolo[3,2-b]carbazole (FICZ) is a chemical compound with the molecular formula C19H12N2O. It is a nitrogen heterocycle, having an extremely high affinity (Kd = 7 x 10−11M) for binding to the aryl hydrocarbon receptor (AHR).

References

  1. ↑ Murk, A.J.; J. Legler; M.S. Denison; J.P. Giesy; C. van de Guchte; A. Brouwer (1 September 1996). "Chemical-Activated Luciferase Gene Expression (CALUX): A Novel in-Vitro Bioassay for Ah Receptor Active Compounds in Sediments and Pore Water". Fundamental and Applied Toxicology. 33 (2): 149–160. doi:10.1006/faat.1996.0152. PMID   8812260.
  2. ↑ Denison, M.S.; B. Zhao; D.S. Baston; G.C. Clark; H. Murata; D.-H. Han (8 August 2004). "Recombinant cell bioassay systems for the detection and relative quantitation of halogenated dioxins and related chemicals". Talanta. 63 (5): 1123–1133. doi:10.1016/j.talanta.2004.05.032. PMID   18969542.
  3. ↑ Esser, C.; B.P. Lawrence; D.H. Sherr; G.H. Perdew; A. Puga; R. Barouki; X. Coumoul (15 November 2018). "Old Receptor, New Tricks-The Ever-Expanding Universe of Aryl Hydrocarbon Receptor Functions. Report from the 4th AHR Meeting, 29-31 August 2018 in Paris, France". International Journal of Molecular Sciences. 19 (11): E3603. doi: 10.3390/ijms19113603 . PMC   6274973 . PMID   30445691.
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