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FEATURE
Investigating the Embryotoxic Effects of Caffeine and MSG using Mouse Embryonic Stem Cells
Dr. Suchitra Derebail, Chun Su Yee Judy, Melvin Tan Poh Leng, Ngo Yun Xuan, Koh Wee Kiat
Republic Polytechnic, School of Applied Sciences, Singapore

Stem cells of mouse or human origin are well known for their capacity to self-renew and proliferate into various specialized cell types. Stem cells are classified broadly according to their origin (1);

  1. Embryonic stem cells (derived from the inner cell mass of blastocysts or growing embryos)

  2. Adult stem cells (derived from various adult tissues like bone marrow, eye, hair, adipose tissue etc.)

Further, stem cells can be classified in two categories based on either their potency or potential to differentiate into various cell types; i) The embryonic stem cells which are either totipotent or pluripotent and ii) somatic or adult stem cells which are multipotent. Totipotent cells have the ability to form both embryonic and extra-embryonic tissues (placenta and umbilical cord) that can develop into a viable, complete embryo while pluripotent cells are localised to the internal cell mass of the blastocyst and form only embryonic tissues. As stem cells differentiate, they gradually lose pluripotency and differentiate into multiple-cell types representative of all 3 germ layers, which are the ectoderm, mesoderm and endoderm. In adults, multipotent stem cells have limited potential to develop into various cell types. For example, bone marrow stem cells can differentiate mainly into white blood and red blood cells, but not into liver or skin cells.

The usage of stem cells is rapidly becoming popular in research, medical biotechnology, and in regenerative medicine. Embryonic stem cells remain in an undifferentiated state in vitro, when maintained in optimum conditions (for example, on feeder cells), appropriate cell culture media and growth factors. They can be harvested and made to grow as clusters of suspension bodies called ‘embryoid bodies’. These embryoid bodies are representative of early post-implantation embryos. These aggregates have the ability to grow into all 3 germ layers (Figure 1). The differentiation and developmental potential of these embryoid bodies closely mimic those of growing embryos. This property makes embryoid bodies an ideal testing ground of various drugs and chemicals that have varying intensities of embryotoxicity.

During the course of living, many chemicals and potential toxins enter the human serum through food additives, pharmaceuticals, household cleaning reagents, etc. Most of these chemicals can have adverse effects on the reproductive ability of an individual or on the developmental and differentiation potential of a growing foetus. Usually, embryotoxicity tests are tests done to establish these unknown toxicities of a wide range of chemicals and drugs using mouse or rat whole embryos cell culture. But these kinds of tests are time-consuming, expensive and also involve a lot of animal testing using mice, rats and rabbits. Due to the economical and ethical concerns of conventional embryotoxicity testing, many government aided agencies like ECVAM, ICCVAM and JCVAM (the European, Interagency Committee and Japanese Centres for Validation of Alternative Methods, respectively) are encouraging the development and validation of alternative methods to animal testing. These agencies have promoted the use of embryonic stem cell derived embryoid bodies as an in vitro alternative method. This type of testing, called EST or embryonic stem cell testing, has already successfully classified many chemicals according their toxic severity into: non-embryotoxic, weakly embryotoxic and strongly embryotoxic reagents.

Some of the endpoints tested in a typical EST are the assessment (2-3) of inhibition of differentiation into various cell types like cardiomyocytes, endothelial cells, chondrocytes and osteoblasts, etc., cytotoxicitiy effects on murine stem cells and murine fibroblasts. These assays generate vital information for pharmaceutical companies, the food industry and the cosmetic industry on chemicals with developmental toxicities. It has been shown in many reports that the EST prediction of embryotoxicity ranges from 78 – 100% accuracy depending on whether the chemicals are strongly or weakly embryotoxic (2-5).

The purpose of this project is to investigate whether mouse embryonic stem cells (mESCs) could be a replacement and a simulative model for embryotoxicity in human cells. In order to determine the embryotoxic potential of chemicals like pharmaceutical drugs or food additives, two endpoints were assessed: the cytotoxicity of the chemicals and effects on pluripotent potential of mESCs. A cell titre determining assay (called MTS assay) was conducted to assess the cytotoxicity level of the chemicals on mESCs cells. Alkaline phosphatase staining was used to determine whether the chemical-treated mESCs retain their pluripotent properties. It is shown that cells that were exposed to chemicals like caffeine and monosodium glutamate (MSG) in concentrations below 150µm have maintained cell viability and retained pluripotency, while cells that were exposed to concentrations higher than 150µm were observed to become necrotic or apoptotic. The tested chemicals were compared against hydrogen peroxide and glucose as positive and negative controls, respectively. The project has shown promising results in the use of mESCs in determining embryotoxic effects of chemicals. By including differentiation assays and chemical recovery assays, we can further develop this project into a complete testing ground for therapeutic drugs and food additives.

Methodology

Based on information garnered from the literature, the effects of two food-related compounds; caffeine and monosodium glutamate or MSG, on mouse embryonic stem cells were carried out. The effects of these two compounds were tested against a negative control (glucose) and a positive control (hydrogen peroxide).

It has been shown that caffeine levels can be easily quantified in blood, plasma, or serum. Plasma caffeine levels are usually in the range of 2–10 mg/L in coffee drinkers, and 40–400 mg/L in cases of acute over dosage (6). There are varying ranges of caffeine in many beverages; for example, 40 to 180 mg/150 ml for coffee, 24 to 50 mg/150 ml for tea, 15 to 29 mg/180 ml for cola, 2 to 7 mg/150 ml for cocoa, and 1 to 36 mg/28 g for chocolate. Caffeine absorption from the gastrointestinal tract is rapid and reaches 99% in humans in about 45 min after ingestion. The hydrophobic properties of caffeine allow its passage through all biological membranes. There is no blood-brain barrier to caffeine in the adult or the fetal animal. Studies on high-dose caffeine exposure in developing embryos showed that higher doses of human maternal caffeine exposure decreases the body weight of newborn offspring, as well as the sizes of their head and upper limb (7).

Monosodium glutamate (MSG) has been shown to penetrate the placental barrier. The uptake of MSG in the fetal brain is twice as much as that in the maternal brain in Kunming mice (8). The adverse effects of MSG increase with higher concentrations. High doses (100 μg/ml) of MSG may cause endocrine disruption and may also stunt the skeletal development of the growing embryos. MSG added to 12-day chick embryo retinas in culture causes severe morphologic damage to the retina (as judged by light microscopic examination). Damage is evident after a few hours with concentrations as low as 0.3 mM (9). Based on the aforementioned results, MSG may harm human off springs if they are exposed to high dose of MSG-laced foods.

To test for embryotoxicity, we cultured mESCs on 0.1% gelatin-coated dishes for 48 hours using standard culturing protocols. The cells were then harvested and counted, and 1X105 cells/well was plated onto 12-well plates. After 24 hours of incubation, each plate was treated with each chemical compound in varying concentrations for another 24 hours of incubation, as shown in Figures 2 and 3 for caffeine and MSG, respectively. Two ranges were tested: 50 – 500µM with increments of 50µM, and 50-500mM with increments of 50mM. Cells were analyzed pre- and post-treatment with all four chemicals. Two end-points were tested; cytotoxic effects using MTS assay and inhibitory effects on pluripotency using alkaline phosphatase assay.

The MTS assay is a colorimetric, cell proliferation assay that helps to quantify viable cells. The assay is composed of two solutions; a novel tetrazolium compound [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), and an electron coupling reagent (phenazine methosulfate; PMS). In viable cells, dehydrogenase enzymes can convert MTS into formazan product, whereas in dead cells, MTS remains unchanged. Absorbance measurement of formazan at 490nm is a direct indicator of number of viable cells. The alkaline phosphatase assay is a qualitative test of pluripotency and differentiation in stem cells. In this method, cells are stained with a mixture of napthol and fast red violet. The presence of alkaline phosphatase enzyme in pluripotent cells results in a dark pink color. Lighter shades indicate that the cells are losing pluripotency and are tending towards differentiation. In the case of embryotoxicity, lack of dark staining in cells implies that the chemicals have an inhibitory effect on pluripotency.

Results and Discussion

This investigation on the embryotoxic effects of caffeine and monosodium glutamate was conducted for a period of six months as a final year project that was supported by Republic Polytechnic. We have successfully established mESCs cultures, derived embryoid bodies and conducted relevant assays like MTS and AP stain. We analysed the morphology of cells before and after 24 hours of treatment. Our results show that glucose has a growth promoting effect on the mESCs at all concentrations tested. Cells did not show any adverse cytotoxic effects, as expected for glucose. In contrast, our positive control of hydrogen peroxide showed extreme cytotoxic effects at concentrations as low as 100µM. The morphological abnormalities like presence of vacuoles, apoptotic bodies and cell death are supported by our cell counts, estimated using MTS assay. In the micromolar range of chemical concentrations, the glucose treated cells showed a healthy increase in cell counts from an initial count of 1X105 cells/well to a maximum count of 20 X 105 cells/well. In the case of caffeine and MSG, there was a drop in cell proliferation as evidenced by the cell counts of 5 X 105 cells/well in both cases. This drop is comparable to a similar drop in the case of hydrogen peroxide (<5 X 105 cells/well). A more pronounced morphological effect and drop in cell proliferation was seen at the higher range of millimolar concentrations. In addition, cells treated with caffeine and MSG showed the presence of necrosis. AP staining was done at only two concentrations for each chemical; 50mM and 500mM. The results indicate that the chemicals do not affect pluripotency at 50mM concentrations. But cytotoxicity kills most cells at 500mM, hence, it is difficult to estimate if there is any real inhibitory effect on pluripotency.

The assays and experiments designed in this project have provided some preliminary information on the cytotoxic effects of common food compounds like caffeine and monosodium glutamate. However, it is still premature to classify these chemicals as non-embryotoxic, weakly embryotoxic or strongly embryotoxic reagents. These experiments have only tested the effects on cells after a brief exposure to the chemicals for 24 hours. There are still many questions that need to be addressed; for example what is the recovery rate of the cells after removal of chemicals; what is the effect of the chemicals on ES cell differentiation into cardiomyocytes or into cells of ectodermal and endodermal lineages; what is the safe limit for consumption of these chemicals; do these chemicals cause accelerated differentiation in stem cells, etc. Nevertheless, these assays offer an attractive alternative to the laborious task of animal testing. They are also easily adaptable to the high-throughput testing of chemicals. In conclusion, this methodology shows good potential for development into a complete testing ground for pharmaceuticals and food additives.

About the Author

Ms. Chun Su Yee Judy graduated from RP in the year 2013. She graduated with a Diploma in Biomedical Science and studied topics like Molecular Cell Biology, Cell Cycle and Oncology, Proteomics, Genetics and Genomics. Her Final Year Project was on investigating the Embryotoxic effects of Caffeine and MSG using mouse embryonic stem cells. In future, she wants to be able to work and excel in the Research and Development sector of Food and Human nutrition to improve consumer’s health.

Mr. Melvin Tan Poh Leng graduated from RP in the year 2013. He graduated with a Diploma in Biomedical Sciences and studied topics like Genetics, Proteomics, Cell cycle and Oncology, Structural Biology and many more. His Final Year Project was on Development and Validation of Embryotoxicity Assays using Mouse Embryonic Stem cells. In future, he wishes to do clinical/medical diagnostics in the biomedical field.

Mr. Koh Wee Kiat is a graduand of RP in the year 2013. He graduated with a Diploma in Biomedical Sciences and he has studied topics like Genetics, Proteomics, Cell cycle and Oncology, Structural Biology and many more. His Final year project was on Development and Validation of Embryotoxicity Essays. In the future, he wishes to be a doctor or a lab researcher in the biomedical field.

Mr. Ngo Yun Xuan, graduated from Republic Polytechnic in the year 2013. On the academic front, Yun has obtained a Merit Award for his outstanding academic achievements in 2011, having since been inducted into the Roll of Honours list every academic semester since AY2011/12. Yun is also very active in planning and execution of several events for School of Applied Sciences: Chiron Club and was appointed the position of Vice president from 2011 to 2012.

Dr. Suchitra Derebail is an experienced Scientist with over 11 years of experience in the fields of Stem cell Biology, Virology and Molecular Biology. Following a Master’s degree in Zoology, she completed her PhD in Molecular Biology from the University of Maryland, College Park in the US. She has a diverse experience which is a result of her research work in both academic and industrial fields. Her doctoral work was focused on the chaperone effects of nucleocapsid protein during HIV recombination. She continued her interests in AIDS research while doing her post-doctoral work at the well-renowned National Institutes of Health, in Bethesda, USA. She then pursued her research interests in an industrial setting at Invitrogen (Lifetech) where she worked on several areas like baculoviral and lentiviral transfection kits, stem cell reprogramming and other patented technologies like Gateway, Vector NTI, BlockIT miRNA etc. She is a self-motivated professional with a consistent track record of achievements. Her findings have been published in several journals and cited by her peers in the field. Currently, she is working as Academic Staff at Republic Polytechnic in the School of Applied Sciences, where she facilitates several modules like Genetics and Cell Culture and is involved in training students through Final Year projects.

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