Assessment of the mutagenic and genotoxic potential of indomie noodle seasoning using bacterial (Salmonella) reverse mutation and SOS Chromo tests
Okunola A. Alabi1*, Edward B. Esan1, Oluwatobi C. Odunukan1, Olutayo S. Shokunbi2
1Biosciences and Biotechnology Department, School of Science and Technology, Babcock University, Ilisan Remo, Ogun state, Nigeria 2Department of Biochemistry, Benjamin Carson School of Medicine, Babcock University, Ilisan Remo, Ogun state, Nigeria
J Innov Biol (2014) Volume 1, Issue 4: Pages: 210-214
Abstract: Indomie noodles are consumed in large quantities worldwide. There is a public health concern about the possible mutagenic and genotoxic effects the seasoning might have on the consumers, especially on children of different developmental stages. In this study, an evaluation of the genotoxic and mutagenic potential of indomie noodle’s seasoning was carried out using Save Our Soul (SOS) chromotest on Escherichia coli PQ37 and the Ames Salmonella fluctuation test on Salmonella typhimurium strains TA98 and TA100 without metabolic activation. The seasoning was subjected to Ames test at concentrations of 0.005, 0.0175, 0.035 and 0.065 g/mL (v/v, seasoning/ dimethyl sulfoxide); and SOS Chromotest at concentrations of 0.065, 0.0325, 0.01625, 0.008125, 0.0040625 and 0.0020313 g/mL (v/v, seasoning/dimethyl sulfoxide) according to standard procedures. The result of the Ames test showed mutagenicity of the indomie seasoning in both strains utilized. However, the TA100 strain was more responsive in terms of mutagenic index in the absence of metabolic activation. The SOS Chromotest result showed the genotoxicity of indomie noodle’s seasoning. Nevertheless, the E. coli PQ37 system of the SOS Chromotest was slightly more sensitive than the Salmonella assay for detecting genotoxins. The study showed that indomie noodle’s seasoning is genotoxic and mutagenic in in vitro assays and should be consumed with caution.
Received: 05 October 2014
Accepted: 16 December 2014
Published: 31 December 2014
Biosciences and Biotechnology Department, School of Science and Technology, Babcock University, Ilisan Remo, Ogun state, Nigeria
Keywords: Mutagenic, Genotoxic, Indomie noodle seasoning, Ames test, SOS Chromotest
Indomie noodles are consumed in large quantities worldwide. It is sold throughout Indonesia, Malaysia, Australia, Nigeria, United States of America, and other countries. Indomie noodle is very nutritious, easy to make and can be eaten as snacks and major meal (Sanni et al., 2013). It is very versatile and this makes it attracts the patronage of majority of people both at work, in school, and at home. Because of its versatility, there is an increase rate of consumption among children and young adults. Over the past 17years, Indomie has made an impact in the Nigerian market with the different brands appealing to several people as well as becoming a household name across countries. It has been estimated that one in two Nigerians have tasted indomie noodles and that up to 15million people eat them regularly (Sanni et al. 2013). Indomie noodles is usually prepared for consumption using its seasoning, therefore, the rate at which indomie noodles is consumed is proportional to the rate at which the seasoning is consumed. The major constituents of indomie noodle are wheat flour, vegetable oil, iodized salt, sodium polyphosphate, sodium carbonate, potassium carbonate, guargum and tartrazine while that of the seasoning powder are iodized salt, monosodium glutamate (MSG), hydrolyzed vegetable protein, soy powder, garlic powder, chicken flavor and chili powder (Sanni et al. 2013).
In spite of the large consumption of indomie noodles especially among children of different developmental stages, knowledge about the possible genotoxic and mutagenic potential of indomie noodles seasoning is scarce. When humans are directly exposed to a potentially
genotoxic substance, as with food, there is an imperative need to evaluate diverse types of
DNA alterations in order to thoroughly determine the health hazard. This evaluation
requires the use of a battery of tests. In vitro genotoxicity test batteries recommended by regulatory agencies to detect genotoxic carcinogens include at least two or three test procedures, such as bacterial reverse mutation test (Ames test) and Save Our Soul (SOS) Chromotest (Kirkland et al. 2005).
Genotoxic substances induce deoxyribonucleic acid (DNA) damage and mutations. A set of responses from a group of genes known as the SOS (save our soul) genes has been used to determine genotoxicity (Quillardet and Hofnung, 1985). The SOS chromotest is based on the detection of DNA-damaging agents. It involves incubation of a specially developed Escherichia coli (E. coli) strain (PQ37) with the test substance of concern. If a SOS response occurs, lacZ operon is expressed and is measured photometrically by measuring β-galactosidase (Sundermann et al. 1996). The SOS chromotest has been used to determine the genotoxicity of a variety of chemicals, metal compounds, hospital effluents, and complex environmen¬tal extracts (Jolibois et al. 2003; Lantzsch and Gebel, 1997; White et al. 1996; Mersch-Sundermann et al. 1991). On the other hand, Ames test is based on detecting reverse mutations in two histidine auxotrophic mutants of the Salmonella typhimurium bacterium rendering them histidine prototrophs (Ames et al. 1973). One mutant strain, TA100, allows the detection of base substitution mutation, while another strain, TA98, allows the detection of frameshift mutation. This test, a reference in chemical mutagenicity detection, has been extensively validated (Kubo et al. 2002).
Thus, in this present study the genotoxic and mutagenic effect of indomie noodles seasoning was evaluated using the bacterial reverse mutation (Ames) test and SOS Chromotest.
Indomie noodles seasoningCartons of two mostly consumed varieties (normal size containing 7g seasoning; and super pack containing 13g seasoning) of a popular Indomie noodles brand in Nigeria were obtained from a supermarket in Lagos, Nigeria.
In vitro testing
Ames (Salmonella) fluctuation test
Salmonella typhimurium strains TA98 and TA100 obtained from Environmental Bio-Detection Products Inc. (EBPI, Canada) were used in the Ames test conducted according to the method described by Maron and Ames (1983) and Alabi et al. (2014). Tests were conducted under aseptic conditions according to the method described by Rao and Lifshitz (1995). Four concentrations of 0.005, 0.0175, 0.035 and 0.065 g/mL, which are equivalent of 1.0, 3.5, 7.0 and 13.0 g of the indomie seasoning dissolved in 200 mL of distilled water, the amount of water specified by the producer of the indomie on the indomie sachet, and was sterilized by filtration through a 0.22 µm pore-size cellulose nitrate filter (Millipore). Then 200 µL of each of the concentration was mixed with 19.8 mL of the reaction mixture (Davis-Mingioli salts composed of D-glucose [ICN Biomedicals, Aurora, OH, USA; CAS 50-99-7], D-biotin [ICN Biomedicals, Aurora, OH; CAS 22879-79-4], L-histidine [Sigma, St. Louis, MO, USA; CAS 7048-02-4], and bromocresol purple [Fisher Scientific, Nepean, Ont.; CAS 115-40-2]). Reagents were added to sterile culture tubes in the following order: (1) reaction mixture, (2) indomie seasoning (3) bacteria. The culture tubes were vortexed after each addition and a 200 µL portion was transferred into 96-well flat-bottomed microplates. The microplates were sealed in plastic bags and incubated for five days at 37°C. At the end of this period, the plates were examined for color: all yellow, partially yellow and turbid wells were considered positive, whereas purple wells were deemed negative. The number of positive wells per plate was recorded and compared to the controls. Chi-square analysis (Gilbert, 1980) was used for statistical evaluation of the treated plates versus the control plates. A sample is considered mutagenic when there is a significant increase in the number of positive wells in treated plates over the negative control plates (i.e. mutagenic index (MI)). The results were expressed as Mutagenicity Ratio (MR=number of positive wells in treated plates/number of positive wells in the negative control plates) and are an average of at least three experiments. Sodium azide (NaN3) and 2-Nitrofluorene (2-NF) were used as positive controls for TA100 and TA98 respectively, while dimethyl sulfoxide (DMSO) was used as negative control.
The tester strain E. coli PQ37 was obtained from Environmental Bio-Detection Products Inc. (EBPI, Canada). The SOS Chromotest was performed without metabolic activation as described by Quillardet and Hofnung (1985) with modifications provided by Kevekordes et al. (1999) and Alabi et al. (2014). Thirteen grams of the seasoning was dissolved in 200 mL distilled water (an equivalent of 0.065 g/mL) and five additional two-fold serial dilutions: 0.0325, 0.01625, 0.008125, 0.0040625 and 0.0020313 g/mL (v/v, seasoning/dimethyl sulfoxide) were prepared. A 600 µL volume of an appropriate overnight culture dilution were added to a tube containing 20 µL sample volume, and incubated with agitation for 2 h at 37°C and subsequently centrifuged at 700 g for 20 min. The supernatant was discarded and the bacterial pellets were resuspended with 200 µL of SOS Chromogen [p-nitrophenyl phosphate (PNPP, Boehringer Mannheim, Laval, Que.; CAS 4264-83-9) for alkaline phosphatase (AP) and 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal, Vector Biosystems, Toronto, Ont.; CAS X100) for Beta-galactosidase (βgal). Plates were re-incubated (10 min for AP and 60 min for βgal), after which optical density readings were taken at 620 (βgal) and 405 nm (AP) respectively. 4 Nitro-Quinoline Oxide (4 NQO) was used as positive control.
AP reduction factors (RF), βgal induction factors (IF) and corrected induction factors (CIF-IF/RF) were calculated as described by Legault et al. (1996):
RF - XOD405t/XOD405c
IF - XOD620t/XOD620c
CIF - IF/RF
where X is the mean of four OD readings and t and c refer to test and control dilutions, respectively. As shown above, the RF and IF values account for the background activity of the control. The ratio of IF to RF units yields an estimate of βgal activity corrected for toxicity. A normalized induction factor of 1.5 or more was considered to represent significant genotoxic activity (Legault et al. 1996).
SPSS 15.0 statistical package was utilized for the analysis and significance was considered at 0.05 and 0.01 levels.
Bacterial reverse mutation (Ames) test
The indomie noodle seasoning was subjected to Ames fluctuation test to study if it is mutagenic and able to induce a reverse mutation in the Salmonella TA98 and TA100. The result is shown in Table 1. Three of the four concentrations tested with the two strains were mutagenic. Compounds were considered as mutagenic if the value of the Mutagenicity Ratio (MR) was high enough and significant as evaluated by the chi-square analysis (Gilbert, 1980). The observed mutagenicity was concentration dependent with the highest concentration of the seasoning (0.065g/mL) inducing the highest MR value. The two strains (TA98 and TA100) of S. typhimurium used were sensitive to the seasoning, with both showing mutagenicity at 0.0175, 0.035 and 0.065 g/mL of the seasoning. However, TA100 was more sensitive with higher MR value at each of the concentration. The MR value obtained for 0.035 and 0.065 g/mL of the seasoning in both TA98 and TA100 were higher than the values obtained in the respective positive controls, 2-NF and NaN3. The MI of >1 was observed in the tested sample in both bacteria strains with the highest induction recorded in the highest concentrations of the seasoning (Figure 1).
The induction factor (IF) values of the SOS Chromotest of the indomie noodles seasoning are summarized in Table 2. The tester strain, E. coli PQ37, was exposed to different concentrations of the seasoning. Compounds were considered non genotoxic if the IF remains <1.5, as slightly genotoxic if the IF ranges between 1.5 and 2, and as moderately or strongly genotoxic if the IF>2 (Kevekordes et al. 1999; Abdel-Massih et al. 2013). Out of the six concentrations tested, four were positive with the SOS Chromotest. The genotoxicity was concentration dependent with the highest concentration (0.065g/mL) being the most genotoxic. The values of the genotoxic concentrations were higher than the genotoxic value obtained for the highest concentration of 4-Nitro-Quionoline Oxide (10 µg/mL) used as positive control.
The consumption of Indomie noodles as well as the seasoning is on the increase. It is especially a food of choice for children and teenagers. Much concern has been raised because some of the constituents have been implicated as substances that have the ability to induce various degree and types of toxicity. However, there is little information in literature about the potential genotoxic and mutagenic effects of indomie noodle consumption especially on children within the developmental stage. This study therefore aimed at studying the possible genotoxicity and mutagenicity of indomie noodles seasoning. Objective of genotoxicity testing of food additives and other food ingredients is the genotoxic hazard identification, with the purpose of minimizing the health risk for consumers through the primary prevention of the exposure to genotoxic substances. The results of a single test cannot provide a completely accurate assessment of the geno¬toxicity of food substances. Therefore, it is necessary to employ a variety of genotoxic tests such as SOS Chromotest and bacterial reverse mutation (Ames) test.
These tests showed that the tested indomie noodle seasoning induced significant genotoxic and mutagenic effect. The seasoning was mutagenic in the Ames test in both the TA98 and TA100 strains used. Ames test had been extensively used to help evaluate the mutagenic and carcinogenic risks for a number of chemicals. The mutagenic effects of the indomie seasoning detected by the Ames fluctuation test include at least two different molecular mechanisms: base pair substitution mutation (TA100 positive) and frameshift mutation caused by nucleotide insertion or deletion (TA98 positive). When these bacteria are exposed to the indomie seasoning, reverse mutation from amino acid (histidine) auxotrophy to prototrophy occurs.
Genotoxicity was confirmed at concentration as low as 0.008125 g/mL in the SOS Chromotest. The SOS Chromotest employs the error-prone DNA repair pathway of E. coli PQ37, also known as the SOS response, a complex regulatory network that is induced by DNA-damaging substances (Walker, 1987). The SOS Chromotest allows the detection of primary DNA damaging agents on E. coli. The observed result is an indication that the constituents of the indomie seasoning are capable on activating the E. coli SOS repair system. The SOS chromotest indicates potential DNA-damaging agents present in the seasoning.
The result showed mutagenicity and genotoxicity at 0.0175 and 0.008125 g/mL in the Ames and SOS Chromo tests respectively, concentrations lower than the quantity present in the indomie smallest pack (7g seasoning). This is an indication of the high mutagenicity and genotoxicity of indomie seasoning and a possible effect on the general populace who are exposed to it.
There are no genotoxicity or mutagenicity of indomie seasoning in literature, however, the observed results in this study further clarifies the study of Sanni et al. (2013) where indomie noodles spiced with the seasoning reduced the activities of alanine aminotransferase, an important antioxidant enzyme. This observed genotoxicity and mutagenicity of indomie seasoning is believed to be as a result of its constituents that might have worked synergistically to cause the observed genetic damage. Indomie noodle’s seasoning contains: salt, sugar, flavour enhancers (monosodium glutamate: 621, 631, 627), garlic powder, onion powder, yeast extract, flavours (hydrolyzed vegetable protein), white pepper and anti-caking agent (551), as written on the indomie noodle wrapper by the manufacturer. Monosodium glutamate, a major constituent of indomie seasoning, has been reported to have a toxic effect on the testes of male wistar rats by causing a significant oligozoospermia and increased abnormal sperm morphology in a dose-dependent manner (Onakewhor et al. 1998). It has also been implicated in male infertility by causing testicular hemorrhage, degeneration and alteration of sperm cell population and morphology (Oforofuo et al. 1997). More studies have examined other metabolic and toxic effects of MSG, with a number of the reports showing the induction of oxidative stress in different tissues of experimental animals after administration of chronic doses of MSG (Onyema, et al. 2006; Diniz et al. 2004; Singh et al. 2003).
Also, the seasoning is composed of hydrolyzed vegetable protein which is a savory flavoring agent that brings out the natural flavors in food. A chemical process called acid hydrolysis breaks down protein into amino acids from various food sources (Samojlik and Chang, 1970). The acid hydrolysis can result in the production of the toxic glycerol chlorohydrins (3MCPD and 3DCP). 3-MCPD (3-monochloropropane-1,2-diol or 3- chloropropane-1,2-diol) is an organic chemical compound which is carcinogenic and highly suspected to be genotoxic in humans, has male anti-fertility effects, able to cross the blood testis barrier and blood brain barrier (Ericsson and Baker, 1970).
In conclusion, the results of this study therefore showed that indomie seasoning is genotoxic and mutagenic in vitro using Ames Salmonella test and SOS Chromotest. More studies are required to have holistic information about the potential harmful effects of the consumption of this food item and also to understand the mechanism of action of its constituents. The findings of this study may be significant in Nigeria where there is high rate of consumption of Indomie noodles due to its versatility.
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