Detection of Mutagenicity for Marmara Sea Pollutants by SOS CHROMOTEST and UmuC-Test

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Executive Summary

Sea water pollution has multidimensional perspectives. The main causes of the pollution include industrial effluents from petrochemical plants, pulp and paper firms, medicine industries, and hospital wastes (Malik, & Grohmann, 2011). In addition, deficiency in wastewater treatment results in many chemical, organic, and inorganic pollutants reaching the adjacent water bodies. Other factors that exacerbate the level and toxicity of pollution include urban sprawl, increase in population density, and use of sea waterways as transportation media (Dokmeci, Yildiz, Ongen, & Sivri, 2014). As a result, numerous hazardous and non-hazardous chemicals accumulate in the sea water. Some of the common pollutants include persistent organic pollutants, heavy metals, toxic elements, biogenic pollutants, and other organic wastes. Hence, this research paper uses an experimental approach to test for DNA-damaging toxins by the use of two main tests; SOS CHROMOTEST and umuC test. The techniques are used to conduct visual and instrumental analyzes of E.coli PQ37, and Salmonella typhimurium TA 1535 pSK1002. Random samples collected from several stations in Marmara Sea to undergo genotoxicity tests. The sample collection should be done twice in varied times (February and May) so as to enhance the detection of seasonal changes in the mutagenicity of the pollutants. SOS CHROMOTEST of the E.coli PQ37 and umuC test on the Salmonella typhimurium TA 1535 pSK1002 are visually analyzed by the use of chromogen solution, which showed varied color developments in order to detect the genotoxic levels. Also, the difference in sensitivities of the specimen against the genomic damaging agents requires the use of a spectrophotometer for instrumental analysis. Empirically, the first samples should provide different results in toxicology, pH, elementary concentrations, and mutagenicity from those collected in the second stage (Tsuzuki, 2014). For example, a study in 2008 showed that in February, 17.6 % of the tested samples which were induced with CIF≥ 1.20 and responded to SOS test at a concentration of one or two. Also, 25% of the samples showed positive results to umuC test at a concentration of one or more. However, in May, 56.2% of the tests were positive to the SOS CHROMOTEST. Hence, time is an integral factor in conducting the tests water pollutants.

Introduction

Marmara Sea is vulnerable to multiple pollutants due to rapid industrialization and a wide spreading spectrum of industrial activities in Marmara region and Turkey as a whole. Numerous industrial wastes evolutions from petrochemical complexes, metal processing, food, paper, foundry, plastic, fertilizer, and pharmaceutical industries aggravate the problem (Malik, & Grohmann, 2011). In addition, domestic and hotel effluents add on to the pollutant, which when combined with the industrial wastes results in superimposition of the toxicity, quantities, and mutagenicity of the waste complexes (Tabrez, & Ahmad, 2011). That is not all; the waste generation has immensely increased due to other factors such as improper waste management and treatment services, rapid urbanization, and over-utilization Marmara Sea as a resource. As a result, it is imperative to conduct tests on the water in order to make an informed decision when using the Marmara Sea.

Considering the complex nature of the pollutants and periodic dynamism of the volumes and lethality of the wastes, toxicology assessment of the wastes is mandatory in order to secure proper public health (Reifferscheid & Buchinger, 2010). Most importantly, genotoxicity analysis is an essential test and a prerequisite mechanism for protecting the marine environment and people. The analysis shows the presence or absence of potentially mutagenic and carcinogenic compounds, which are major health concerns for living organisms (Fletcher & Rise, 2012). Therefore, this paper provides the procedural plan and techniques for conducting the genotoxicity tests using SOS CHROMOTEST and umuC test. Nevertheless, detection of the pollutants’ mutagenicity is apparently complex and intricate because the genotoxic compounds form chemical diversity with a variety of compound (Barceló, Hansen, & Adrián, 2009).

In order to simplify and make the analysis less expensive, in vitro systems of tests are encouraged. SOS chromotest and umuC test are examples of the in vitro tests that can be applied to assess the mutagenicity without providing detailed chemical constituents of the compounds. The SOS chromotest is an effective test for bacteria as the results can be produced within a span of hours. The assay detects the presence of primary mutagents of DNA-damaging agents that are found in E.coli PQ37 (Escher & Leusch, 2012). Similarly, umuC test is another in vitro system that used to detect the DNA-damaging agents that are present in the bacterium called Salmonella typhimurium TA 1535 pSK1002 (Fletcher & Rise, 2012).

Background of the Study

Figure 1: shows the contextual setting of Marmara Sea

Dimensionally, the sea covers a surface area of about 11.137 km2 that binds with bosphorus Canakkale and Istanbul to Aegean Sea and Black Sea. The total area that the Sea covers are estimated to be 70 km by 250 km with an optimum depth of about 1390 km. Notably, the Marmara does not have strong currents or waves that limits pollutant dispersal and transport to other areas (Kirci-Elmas, 2013). As such, most of the industrial and other wastes mentioned above usually accumulate in the sea to complex chemicals and toxicities. Additionally, its salinity is averaged at 22 parts per thousand; particularly at the deepest end near the Dardanelles. Marmara is an inland sea that was formed due to crustal movements that occurred close to 2.5 million years in the past (Carpenter, 2013). Its uniqueness and Inland Sea has seen it attract many activities and features like tourism, trade, hotel industries, residential areas, industrial zones, and summer homes amongst others (Isinibilir, 2010). As a result, the intensity and amount of pollution to the Sea have correspondingly increased.

The pollution level of the sea has reached alarming rates and led to extinction of more than 50 living organisms of the sea. Biological corridors that link the Aegean Sea and Black Sea have revealed a lot of turbidity rates, which are surrogate measures to indicate the levels of pollution. Of the three mentioned Sea, Marmara is the poorest sea in terms of fertility, fish counts, and composition of ecological living organism (Kirci-Elmas, 2013). The plight of the sea is attributed to the immense levels of pollution; particularly by the hazardous carcinogenic and mutagenic substance. Researchers add that every year, the sea turns dirtier to a percentage measure of about 80. Recent studies show the high likelihood of the presence of DNA-altering pollutants due to the nearby industries and services that release waste into the sea (Fletcher & Rise, 2012).

Problem Statement

So far, majority of experimental studies and testing have focused on the application of advanced and various methods to monitor and improve the water quality (Férard & Blaise, 2013). However, such studies have done little or failed to test thoroughly or integrate the idea of toxicity. They have not developed or planned any conventional mechanism for testing the mutagenic agents using assay procedures. Over the last 30 years, studies have pointed out the qualitative levels of increasing pollution, which little information on quantification of the waste (Coskun, 2008). As a result, this study goes a notch higher to evaluate and investigate the potential genotoxic substances and activities that are found on the surface of the sea. Technically, the research uses SOS chromotest and umuC tests to analyze experimentally the presence or absence of mutation causing agents found in E.coli PQ37 and Salmonella typhimurium TA 1535 pSK1002 respectively (Tabrez, & Ahmad, 2011).

Objectives of the Study

The primary objective is to use the techniques mentioned above to test for the presence or absence of mutagenic agents in sampled waters from Marmara Sea. In particular, the research aimed at revealing the genotoxic elements or chemical presents in E.coli PQ37 and Salmonella typhimurium TA 1535 pSK1002. SOS chromotest and umuC tests were respectively used as the best and easiest in vitro system methods to understand the compounds found in the mutagenic bacteria (Xu, Wu, & He, 2013). The two methods use microplate assay technique to investigate the genotoxic activities on the surface of Marmara Sea is order to statistically appraise the potentiality of mutagenicity activities in the samples obtained (In Somasundaran et. al., 2014).

Significance

The overall investigation and evaluation shall act as the reference point for strategizing measures to curb the pollution of Marmara Sea. That is, policy makers, scientist, and environmentalist will use the results of the experiment as the supporting evidences to institute measures and policies concerning the pollution (Lee, 2014). In addition, the results can be used to make an informed decision about how the marine environment can be conserved and managed. For example, the presence of E.coli PQ37 and Salmonella typhimurium TA 1535 pSK1002 as the research shows is a clear indicator of the high levels of carcinogenic wastes present in the sea. Thus, pollution control strategists may trace back the origin of the wastes and make proactive suggestions to avert or curb future pollutions. In addition, genotoxicology assessment and analysis is essential for human health protection and monitoring the health of the marine organism (Leeuwen, & Vermeire, 2007). The research shall also open new avenues for improving the experiments or conduction of other related researches.

Limitations of the Study

First, genotoxicological activities are complex and complicated. The mutagenic substances may form due to a combination of chemical complexes, which deems it intricate to analyze or test for chemical constituents. Secondly, the whole process is expensive since visual, and instrumental analyzes demand expensive instruments, apparatus, and reagents. For example, visual analysis requires chromogen solution and spectrophotometer, which are not only expensive, but also technical to use. Thirdly, mutagenicity study of DNA-altering agents varies with time. As time goes on, the bacteria replicate that makes it not decisively right to use the results at different periods (Mascini, Palchetti, & Royal Society of Chemistry 2011). Lastly, it is very hard to determine the genotoxic potency values of the different wastes and their sources; thus, the applicability of the procedures and results must take note of the determining factors.

Definition of Terms

Pollutants – They are unwanted or harmful chemicals, organic, or inorganic substances that concentrate in the environment (Thomas, Tyrrel, Smith, & Farrow, 2009). In this case, Marmara Sea acts as the reservoirs for the wastes from industrial and other sources.

Mutagenicity – This is the process by which a mutagen that is physical or chemical in nature alters the genetic material (DNA) of living organism (Loibner, Thompson, & Wadhia, 2005). The mutagens results to mutation that may cause cancer. In the research, Salmonella typhimurium TA 1535 pSK1002, and E.coli PQ37 represents the agents of mutagenicity.

SOS CHROMOTEST – It is the biological assay that is used to assess the genotoxic activities or potential of chemical substances or compounds. The colorimetric assay measures the gene induction as expressed in the agent bacteria called E.coli PQ37.

umuC tests – Similar to SOS chromotest, umuC test is used to assess the genotoxic expression of Salmonella typhimurium TA 1535 pSK1002.

Genotoxic Potency Value – The value represents the level of dosage or exposure that would trigger mutagenicity or alteration of the DNA constitution or sequence of the living organisms (Malik, & Grohmann, 2011).

In Vitro Testing – In Vitro is a descriptive term used to test for substances, in this case, the genotoxicity activities of chemical constituents outside the living body.

Potential Hazard – This is the likelihood or probability of a particular chemical substance or compound to cause harm when a living organism sis exposure to the threshold dose. In this research, the toxic materials represent the wastewater pollutants that are evolved into Marmara Sea.

Literature Review

According to Teasdale (2010), SOS chromotest assay is a relevant bacterial test in order to detect the presence of DNA-damaging agents. He added that the assay is based on the induction expression as the agents function. The level of expression is monitored by the use of an operon fusion. The response of the test is always rapid in a matter of hours and does not need the survival of the strain used as tester. Schecter (2012) also added that the dose- response curves, which are analogous to the toxicity potency of most chemicals are represented by a linear region. That is; the slope of the curve is the measure of the inducing potency of SOS. However, different chemical substances differ in the potency values depending on their nature, origin, and chemical composition. As a result, the comparison of the different chemical substances during the SOS chromotest is referred to as genotoxicity of the SOS. The genotoxic chemicals or agents are harmful and pose potential health hazards if organisms are exposed to them beyond the minimum threshold.

Agents that undergo interaction with DNA in vivo always have potential adverse health impacts. That is; they may trigger transmissible cancer and mutations. Therefore, the primary problem of genetic toxicology is to detect, classified the mutagenic agents and to the elucidation of the unknown versions or modes of the agents (Rocco, Peluso, & Stingo, 2012). In testing, bacteria are commonly used as indicators for general toxins because they have a practical advantage and herald the concepts of genecotoxicology and its outcomes. E.coli PQ37 is practically used during the SOS chromotest to indicate the process of mutagenesis (mutatest). Similarly, Salmonella typhimurium TA 1535 pSK1002 is used in the in vitro test using the umuTest technology to reveal the carcinogenicity process.

Although, bacteria represent the simplest life forms that have DNA-containing cells, they exhibit very elaborate mechanism of response to the DNA-damaging agents. In E.coli PQ37, DNA-damaging treatments induce some responses that have a set of functions called SOS responses and “damage-inducible” genes. The bacterium helps to detect the DNA-alteration, which uses the cell’s mechanism to identify the level of genotoxicity. According to Fletcher and Rise (2012), all living cells compose of a sensitive system mechanism to detect any lesions or damages to the genetic material. The cell detection occurs due to a complex enzymatic mechanism or system (SOS repair system), which becomes activated in case of any alteration. The detection is followed by transcription of the genes by the use of SOS promoter. Therefore, the sensitivity, detection, and SOS response forms the basis of applying the SOS chromotest.

Similarly, umuC test is done using genetically engineered bacteria called Salmonella typhimurium TA 1535 pSK1002, which should be a strain of gram-negative and facultative anaerobic enterobacteriaceae. The methodology is commonly used as a reference test system that is essential for analyzing a variety of chemical compounds such as heavy metals. In a highly sensitive range, the test can be used to analyze complex mixtures; for example the industrial effluents (Griffin, Posner, & Barker, 2013). In the analysis, the system reveals the primary damage processes that are caused by mono or single substances or complex environmental contaminants (Teasdale, 2010). Notably, the test has been certified by the international ISO standards and in Germany. The test is relevant due to high sensitivity to mono substances and environmental pollutant complexes. These tests may also discriminate some false positive results from the traditional methodologies.

Evidently, SOS chromotest and umuC tests are considered as simple and short-term techniques used to analyze the level of toxicity in the marine environment. Marmara Sea is subject to complex pollutants from the nearby industrial activities; therefore, an appropriate and reliable test should be applied so as to detect the level of mutagenicity (International Conference on Environmental Toxicology, Popov, & Brebbia, 2010). In addition, the techniques are cost-effective, and their rapidity and simplicity make them useful for conducting screen test for larger genotoxic compounds. Also, the mechanisms can detect genotoxic chemicals or compounds that could not have been possible if the tradition Ames tests were used.

Methodology

Introduction

SOS chromotest can be described as a SOS transcriptional type of fusion that is used as an assay. The methodology involves visual and instrumental analyzes, where the former requires color development while the latter represents the use of instruments. At first, the bacteria are incubated in order to allow the genotoxic material to interact with the DNA of the E.coli PQ37 and Salmonella typhimurium TA 1535 pSK1002. The interaction induces de novo synthesis of an enzyme called b galactosidase. The release of the enzyme depends on the amount of the chromogenic substrate added and the viability of the bacteria used. Bacteria viability is in turn measured using ATP activity. In addition, SOS-chromotest requires visibility, which is aided by the addition of the blue chromogen.

Sampling

Obtaining material from the sea with unpolluted content requires going off shore so to avoid pollutants such as oil spills. Places concentrated with human activity are avoided to reduce the chances of contaminating samples with sediments or any other material solid in nature. To improve the adequacy of the tests, the samples should be collected from different places evenly distributed within the sea. Consideration should be given to the nature of the sampling stations and manner of storage to ensure that the pH value of the samples are maintained at slightly basic as acidic conditions tend to interfere with bacterial activity.

Materials

Growth medium for bacterial strain of SOS-Chromotest

Free-dried bacteria

SOS-chromotest diluents

Standard genotoxic solution, which contains ten µg/mL of 4 Nitro Quinoline Oxide (4NQO)

Blue chromogen solution

Alkaline phosphatase substrate

Stop solution

Bacterial Strains

E. Coli PQ37 is used in the alkaline phosphate analysis for its adaptation against DNA damaging material. S. typhimurium TA1535/pSK1002 is also used whose exposure to DNA damaging material induces the introduction of umu substrate concentrations.

Procedure for SOS Chromotest

I.The test is performed using the SOS chromotest Kit of version 6.3. In order to test all samples from the Sea, same day growth has to be used in relation to the concept of optimization of the results. First, lyophilized bacteria (E.coli PQ37) have to be re-hydrated using an aseptic method with an appropriate medium. The bacteria should be incubated for up to four hours, at a temperature of about 37 degrees Celsius.

II.After four hours, the bacteria have replicated enough to increase their optical density to about 0.05-0.052. Then about 100 μL of the bacteria culture is transferred to 96 well plate that consist of the samples that are diluted, negative, and positive control experiments. The well plate is then incubated for another two hours.

III.A blue chromogen is then added to the bottle which contains p-Nitro-Phenyl-Phophate (pNPP) and then mixed for up to 40 minutes. About 100 μL of the mixture is transferred immediately to the well plate.

IV.After about half an hour, 96 well plate’s absorbance is measured using an ELISA READER in order to determine the AP activity. The incubation must still continue.

V.From the incubation that runs for close to 90 minutes causes the 96 well plate to increase in size, which a determinant of the β-Gal activity.

VI.All the samples must be tested twice at a concentration of four. Positive tests determine the activity of β-Gal while the negative control checks the conditions of non-genotoxic substances.

umuC test

•Materials required

•37oC incubator

•Spectrophotometer that has light-path rectangular Corvettes of about 1 cm and 600nm filter

•A Microplate reader (ELISA READER)

•Micropipettes

•Microcentrifuge

Procedure

The process of umuC assay is similar to that of SOS chromotest in the way it is used to detect DNA damage. However, the 96 well plate absorbent is measured to be averagely 420 and 550 nm. The test detects both the β-Gal activity and alkaline phosphate activity.

Calculations

I.SOSIP EMBED Equation.3

II. C = EMBED Equation.3

Key:(OD is Optic density, C in concentration, Conc is the tested materials’ concentration, Vol is the micro-liters volume of the tested material, and MW is the tested materials’ molecular weight)

Expected Results

Positive and negative tests will show proper results if β-Gal activity and alkaline phosphate activity occur conveniently. If the concentration of the mutagenic matter is high, then there would be a corresponding increase in the induction reaction. In the negative control test, the absorbance results of 0.25 and 0.3 indicate that the tested samples lacked cytotoxic (Lu, 2000). However, SOS chromotest kit has two inbuilt controls which are activated in case the procedures are correctly followed. The controls help when analyzing the results.

If the 4NQO provides a positive standard, then the color appears differently depending on the chemical concentrations. However, when the scale of densities is not obtained or appearing in the column of 4NQO that as graduated reaction to the genotoxic agent, it indicates that the bacteria did not function accordingly (Sharma et. al., 2013). Note that if the SOS-chromotest bacteria do not function properly, then the overall results are invalid. As a result, 4NQO control must be included when testing for mutagenicity in order to facilitate complete and proper analysis of the results.

lefttop

Figure 2: Shows a generalized SOS-CHROMOTEST plot that is obtained with a mutagenic material

In order to calculate the SOS inducing potential (SOSIP), it is first important to identify the positive linear portion of the curve; that is, the OD of the bacteria increases linearly as the concentration of the genotoxic material increases (Sharma et. al., 2015). In figure 1 above, the region represents the line between point 1 and point 4. The SOS inducing potency or SOSIP is represented by the slope of the linear line (OD1-OD3), which is equated as below:

1)SOSIP = 10 X (OD3 OD1)/ (C3 C1)

From the equation, the entity “(C3 C1)” is expressed in terms of nanomoles per well reaction. In addition, the equation 2 below transforms the concentration values in micrograms to required nanomole unitage (Valon, 2006).

(2)C = CONC X VOL / MW

Where MW is the molecular weight of material tested

VOL is the volume of the tested sample in the 96 well plate and is expressed in micro-liters

CONC is the concentration of the material treated in the units of µg/mL

Additionally, spectrophotometric instrumentation can also be used to test quantitatively or visually for the genotoxic activity in the surface of Marmara Sea. It is worth to note that a distinct comparison has to be made between the background culture and color density.

The following table illustrates results from five different sampling stations of the Marmara Sea

Station SOS Chromotest umuC Test

1 – –

2 + –

3 – –

4 – –

5 + –

Discussion

The tests’ results indicate a low level of mutagenity of in the Marmara samples. In as much as the tests indicated immense sensitivity with the reactive compounds, the bacteria used had no cytotoxicity majorly because their adaptations involve modification of the cell walls. The genetic adaptation makes them ‘invincible’ to damaging agents that target the DNA. This adaptive nature makes it possible to run the essential toxicology tests that affect the content of the Marmara sea. The potentially mutagenic and carcinogenic contents of the sea that interfere with the ecosystem are thus easily analyzed.

The value of calculated SOSIP does change from one time to another because of bacteria age, incubation conditions, or material concentrations. As a result, it is wise to correct the figures in accordance with the activity of any known standard. The β-Gal synthesis level are varied in the Marmara sample as an influence of the different water concentrations, a result of varied levels of pollution at different points of the sea and also the sea currents. The toxicology tests give the clearest indication of the toxicology levels in the sea.

Summary

Genotoxicity assessment is essential for samples collected from Marmara Sea in order to analyze the health impacts of the water to the marine organism and human beings (Dokmeci, et. al., 2014). The assessment depends on the change in the genetic materials of the living cells of organisms; especially, bacteria. Bacteria are the simplest forms of life, but their cell function and mechanism can detect any instance of DNA-damaging by mutagenic agents. For this reason, E.coli PQ37 and Salmonella typhimurium TA 1535 pSK1002 are commonly used in SOS-chromotest and umuC test respectively. This study recommends the aforementioned in vivo techniques because they are simple, cost-effective, and produce a faster result. However, the mutagenicity tests are always very complex and complicated since the effluents that are carcinogenic seem to compost of multiple chemical constituents. Luckily, the bacterial genotoxicity tests are now simplified, and the chemical constituents can be detected due to the sensitivity of the bacteria to DNA-damaging.

Genetically engineered bacteria are used for sensor systems or assay particularly on the industrial and municipal wastes. It is important to understand the SOS inducing potency so as to detect the dose-dependent signals. Notably, the genotoxic substances enter the Marmara Sea from a range of municipal and industrial sources the exhibit different potencies. Therefore, in order to carry out the assay, sampling method is imperative. Various homogeny water samples should be obtained from different stations of the Sea. Other parameters to be noted include the possible sources of the pollutants, which can be done through genotoxic backtracking, the incubation period, age of the bacteria, and pH of the samples.

In summation, SOS-CROMOTEST and umuC tests used kits that conveniently detect the genotoxic activity and materials in the wastewater, chemicals, biological fluids, and sediments. Genotoxic substances are hazardous since they are capable of inducing mutation and can cancerously transform normal cells.

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