SOS chromotest

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Overview of the use of the SOS response for genotoxicity testing Genotoxic Damage.png
Overview of the use of the SOS response for genotoxicity testing

The SOS chromotest is a biological assay to assess the genotoxic potential of chemical compounds. The test is a colorimetric assay which measures the expression of genes induced by genotoxic agents in Escherichia coli, by means of a fusion with the structural gene for β-galactosidase. The test is performed over a few hours in columns of a 96-well microplate with increasing concentrations of test samples. This test was developed as a practical complement or alternative to the traditional Ames test assay for genotoxicity, which involves growing bacteria on agar plates and comparing natural mutation rates to mutation rates of bacteria exposed to potentially mutagenic compounds or samples. The SOS chromotest is comparable in accuracy and sensitivity to established methods such as the Ames test and is a useful tool to screen genotoxic compounds, which could prove carcinogenic in humans, in order to single out chemicals for further in-depth analysis. [1] [2]

Ames test biological testing method

The Ames test is a widely employed method that uses bacteria to test whether a given chemical can cause mutations in the DNA of the test organism. More formally, it is a biological assay to assess the mutagenic potential of chemical compounds. A positive test indicates that the chemical is mutagenic and therefore may act as a carcinogen, because cancer is often linked to mutation. The test serves as a quick and convenient assay to estimate the carcinogenic potential of a compound because standard carcinogen assays on mice and rats are time-consuming and expensive. However, false-positives and false-negatives are known.

In genetics, genotoxicity describes the property of chemical agents that damages the genetic information within a cell causing mutations, which may lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, whereas not all genotoxic substances are mutagenic. The alteration can have direct or indirect effects on the DNA: the induction of mutations, mistimed event activation, and direct DNA damage leading to mutations. The permanent, heritable changes can affect either somatic cells of the organism or germ cells to be passed on to future generations. Cells prevent expression of the genotoxic mutation by either DNA repair or apoptosis; however, the damage may not always be fixed leading to mutagenesis.

Contents

As with other bacterial gentoxicity and mutagenicity assays, compounds requiring metabolic activation for activity can be investigated with the addition of S9 microsomal rat liver extract.

Mechanism

The SOS response plays a central role in the response of E. coli to genotoxic compounds because it responds to a wide array of chemical agents. Triggering of this system can and has been used as an early sign of DNA damage. Two genes play a key role in the SOS response: lexA encodes a repressor for all the genes in the system, and recA encodes a protein able to cleave the LexA repressor upon activation by an SOS inducing signal (caused in this case by the presence of a genotoxic compound). Although the exact mechanism of the SOS response is still unknown, it is induced when DNA lesions perturb or stop DNA replication. . [3]

SOS response An error-prone process for repairing damaged microbial DNA.

The SOS response is a global response to DNA damage in which the cell cycle is arrested and DNA repair and mutagenesis is induced. The system involves the RecA protein. The RecA protein, stimulated by single-stranded DNA, is involved in the inactivation of the repressor (LexA) of SOS response genes thereby inducing the response. It is an error-prone repair system that contributes significantly to DNA changes observed in a wide range of species.

Various end-points are possible indicators of the triggering of the SOS system; activation of the RecA protein, cleavage of the LexA repressor, expression of any of the SOS genes, etc. One of the simplest assays consists of monitoring the expression of an SOS gene by means of fusion with lacZ, the structural gene for E. coli β-galactosidase.

Procedure

Overview of the use of the SOS response for genotoxicity testing Sos results.png
Overview of the use of the SOS response for genotoxicity testing

The SOS chromotest consists of incubating the E. coli with increasing concentrations of the chemical to be tested. After allowing time for protein synthesis, β-galactosidase activity is assayed using a simple colorimetric assay. By including a lactose analog which yields a colored compound upon degradation, an easily observable or quantifiable change in colour is used as a metric. Since the chemical tested may inhibit protein synthesis at higher concentrations, which would lead to an underestimation of B-galactosidase induction, alkaline phosphatase is assayed simultaneously with β-galactosidase in order to scale the data to survivability of the cells.

The assay can easily be completed in a number of hours. If using a micro-plate reader, the test is quantitative and dose-response curves have an initial linear region. The slope of this linear region allows unequivocal association of each compound with a single parameter, the SOS-inducing potency (SOSIP), which reflects the inducing activity of the compound. [4]


This assay provides both a qualitative (visible observation of colour gradient) for screening purposes, or quantitative measurement (spectrophotometry) for calculation of commonly accepted metrics. In the quantitative assay, the dose-response (colour production linked to beta-galactosidase production) of a compound is plotted, with the slope of the initial linear region used as a universal parameter, the SOS-inducing potency (SOSIP), which reflects the ability of a compound to induce the SOS response (measured indirectly through production of beta-galactosidase and the breakdown of a lactose analog). Typically, the lactose analog is X-Gal, which produces a blue colour when cleaved by beta-galactosidase. The dose response is also scaled by the survival of cells, measured through the breakdown of alkaline phosphatase (which produces a yellow colour), allowing for the calculation of the SOSIP.

Although the SOSIP is a concentration-based metric, the same method can be used for complex environmental mixtures where the concentration or even compounds of interest are unknown. Through the calculation of the intermediate SOS-induction factor (SOSIF), which can be plotted against dilution in the same manner in order to give an illustration of the dose-response without analytical measurements of samples beforehand.

Advantages

THE SOS chromotest is considered to be the most simple and rapid short-term test for genotoxicity. It serves as a useful and cost effective complement to the traditional Ames test for a number of reasons. Firstly, because of its simplicity and rapidity, the SOS chromotest may be used as a screening test for a large number of potentially genotoxic compounds. Secondly, it may allow the detection of genotoxic chemicals which yield false negatives in the Ames test (such as estradiol, a compound of growing concern). Thirdly, it can prove an effective method to discriminate false positive results in the Ames test.

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Enterobacteria phage λ is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. It was discovered by Esther Lederberg in 1950 when she noticed that streaks of mixtures of two E. coli strains, one of which treated with ultraviolet light, was "nibbled and plaqued". The wild type of this virus has a temperate lifecycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase ; mutant strains are unable to lysogenize cells – instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.

Mutagen Chemical agents that increase the rate of genetic mutation by interfering with the function of nucleic acids. A clastogen is a specific mutagen that causes breaks in chromosomes

In genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level. As many mutations can cause cancer, mutagens are therefore also likely to be carcinogens, although not always necessarily so. All mutagens have characteristic mutational signatures with some chemicals becoming mutagenic through cellular processes. Not all mutations are caused by mutagens: so-called "spontaneous mutations" occur due to spontaneous hydrolysis, errors in DNA replication, repair and recombination.

Beta-galactosidase hydrolase enzyme

β-galactosidase, also called lactase, beta-gal or β-gal, is a glycoside hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides through the breaking of a glycosidic bond. β-galactosides include carbohydrates containing galactose where the glycosidic bond lies above the galactose molecule. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins.

François Jacob French biologist

François Jacob was a French biologist who, together with Jacques Monod, originated the idea that control of enzyme levels in all cells occurs through regulation of transcription. He shared the 1965 Nobel Prize in Medicine with Jacques Monod and André Lwoff.

<i>lac</i> operon Set genes encoding proteins and enzymes for lactose metabolism

The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of beta-galactosidase. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize. Their work on the lac operon won them the Nobel Prize in Physiology in 1965.

Auxotrophy

Auxotrophy is the inability of an organism to synthesize a particular organic compound required for its growth. An auxotroph is an organism that displays this characteristic; auxotrophic is the corresponding adjective. Auxotrophy is the opposite of prototrophy, which is characterized by the ability to synthesize all the compounds needed for growth.

Allolactose disaccharide similar to lactose

Allolactose is a disaccharide similar to lactose. It consists of the monosaccharides D-galactose and D-glucose linked through a β1-6 glycosidic linkage instead of the β1-4 linkage of lactose. It may arise from the occasional transglycosylation of lactose by β-galactosidase.

Isopropyl β-<small>D</small>-1-thiogalactopyranoside chemical compound

Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a molecular biology reagent. This compound is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, and it is therefore used to induce protein expression where the gene is under the control of the lac operator.

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X-gal chemical compound

X-gal is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl galactoside. The X from indoxyl may be the source of the X in the X-gal contraction. X-gal is often used in molecular biology to test for the presence of an enzyme, β-galactosidase. It is also used to detect activity of this enzyme in histochemistry and bacteriology. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo as a result of enzyme-catalyzed hydrolysis.

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Blue–white screen

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Micronucleus test

A micronucleus test is a test used in toxicological screening for potential genotoxic compounds. The assay is now recognized as one of the most successful and reliable assays for genotoxic carcinogens, i.e., carcinogens that act by causing genetic damage and is the OECD guideline for the testing of chemicals. There are two major versions of this test, one in vivo and the other in vitro.

Carbon catabolite repression, or simply catabolite repression, is an important part of global control system of various bacteria and other micro-organisms. Catabolite repression allows micro-organisms to adapt quickly to a preferred carbon and energy source first. This is usually achieved through inhibition of synthesis of enzymes involved in catabolism of carbon sources other than the preferred one. The catabolite repression was first shown to be initiated by glucose and therefore sometimes referred to as the glucose effect. However, the term "glucose effect" is actually a misnomer since other carbon sources are known to induce catabolite repression.

The Umu Chromotest, first developed and published by Oda et al., is a biological assay (bioassay) to assess the genotoxic potential of chemical compounds. It is based on the ability of DNA-damaging agents to induce the expression of the umu operon. In connection with the damage inducible (din) genes recA, lexA and umuD, the umuC gene is essentially involved in bacterial mutagenesis through the SOS response.

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 reliable tool to easily and rapidly assess estrogenic activity of suspected xenoestrogens. This bioassay that 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, was originally developed by Soto et al.

References

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