ABSTRACT
Effects of a flavor enhancer, monosodium glutamate (MSG) was evaluated in female albino rats during the gestational period. Thirty two rats were divided into 4 equal groups (each having 8 rats), Group I, control; Group II, treated with 6 g/kg/day of MSG, Group III, treated group with 8 g/kg/day and Group IV received 10 g/kg/day of MSG. Different doses of MSG (6 g/kg, 8 g/kg and 10 g/kg body weight) were administered orally from 7 -15th day of gestational period on completion of the treatment period. The Dams were allowed to deliver their pups. Sixty four pups were used for histology and the remaining were used for motor activity. Morphometric results of the fetal weight ,crown rump length, head of the pups, brain weight, weight of the cerebral cortex, cerebral width (narrow area) and cerebral width (broad area) of the developing cerebral cortex showed that high dose group rats demonstrated significant reduction in all the parameters when compared to the control. MSG also induced some abnormal changes in gestation such as tincture in the stomach of group III animals, dead of the pups, macrocephaly as well as ulceration in the thoracic region among the group IV animals. Cerebral tissues were obtained and processed to prepare sections stained with H&E and toluidine blue. Histological examination of group II showed degenerative changes present on the stellate cells. However, group III showed pyramidal cells, stellate cells and degenerated pyramidal cells with disintegrated nuclei while group IV showed a lot of vacoulation and clumped cells with some degenerating cells. The glial fibrillary acidic protein (GFAP) was detected immunohistochemically in the developing cerebral cortex of the pups. The result from montoya staircase test showed reduced exploratory motor activity in rats exposed to high concentration of monosodium glutamate that was statistically significant in the second and third week. There was significant motor
xix
exploratory activity impairment due to monosodium glutamate consumption even at low doses. The present study suggested that MSG has neurotoxic effect leading to degenerative changes in neurons and adversely affected the fetal development of the offsprings.
TABLE OF CONTENTS
Title Page ………………………………………………………………………………..i Declaration…………………………………………………………………….…………ii Certification………………………………………………………………………………iii Dedication……………………………………………………………………..………..iv Acknowledgements……………………………………………………………………….v Table of contents……………………………………………………………………….vii List of figures …………………………………………………………………………………………..xi List of Tables……………………………………………………………………………xii List of Plates……………………………………………………………………………..xiii List of Abbreviations…………………………………………………………………..xiv Abstract……………………………………………………………..………..……….xvii CHAPTER ONE
1.0 INTRODUCTION…………………………….……….……………………….1
1.1 BACKGROUND……………………………………………………………….1
1.2 STATEMENT OF THE RESEARCH PROBLEM.….…………………………4
1.3 JUSTIFICATION OF THE STUD………………………………………………5
1.4 AIM AND OBJECTIVES OF THE STUDY……………………………………5
1.4.1 Aim of the study………………………….…………………………………………5 1.4.2 Objectives of the study………………………………………………………….6 1.5 RESEARCH HYPOTHESIS…….………………………………………………6
viii
CHAPTER TWO 2.0 LITERATURE REVIEW………………………………………….…….…….7 2.1 CHEMISTRY OF MONOSODIUM GLUTAMATE.…………….……………7 2.2 MSG IN FOOD..……….…………………………………….………………….11 2.2. 2 Stability and chemical properties….…………….………………………………12 2.2. 3 Metabolism of monosodium glutamate…………………………………………12 2.3 DEVELOPMENT OF THE CENTRAL NERVOUS SYSTEM………………13 2.3.1 Pre-differentiation stage….…………………………………….………………13 2.3.2 Embryonic stage…………………………………….………………………….14 2.3.3 Development of the neural tube………………………………………………..14 2.3.4 Start of neurogenesis……………………………………………………….…..15 2.3. 5 Feotal stage……………………………………………………………………….15 2.3 .6 Pre and postnatal age……………….………………………………….…….…16 2.3.7 Impact of neuroteratogen during neurogenesis………………….………………16 2.3.8 Impact during migration and settling of neurons………….…………………….18 2.3.9 Impact on the development of neurotransmitter systems……….……………..19 2.4 Fundamentals embryonic layers of the cerebral cortex………………………..19 2.4 ANATOMY OF THE CEREBRUM..…………………………………………..21 2.4.1 Morphology of the cerebrum……………………………….……….……..……20 2.4.2 Cellular layers of the cerebrum…….………………….………….……………23 2.4. 3 Functions associated with cerebrum……………………………………………27 2.5 Estrous cycle of the rat………………………………………………………….29
ix
CHAPTER THREE 3.0 MATERIALS AND METHOD …………………………………………..……31 3.1 MATERIALS…………………………………………………………………..31 3.2 Experimental design……………………………….……………… ……………31 3.3 EXPERIMENTAL PROCEDURE………………….…….………………..…..32 3.4 DETERMINATION OF THE ESTROUS CYCLE……………………………34 3.4.1 Mating of rats and confirmation of pregnancy ….……………………………..35 3.5 ANIMALS SACRIFICE……………………………………………………….35 3.6 MORPHOMETRIC STUDIES……………………… ………………………..35 3.7 Tissue processing ………………………………………………………………36 3.7.1 H and E staining Method……………………………………………………….36 3.7.2 Special stain Toluidine blue method….………………………………….…….36 3.8 IMMUNOHISTOCHEMICAL METHOD…………………………………….37 3.8.1 Peroxidase linked avidin biotin complex (ABC) method……………………… 37 3.9 NEUROBEHAVIORAL STUDIES……………………………………………38 3.9.1 Montoya staircase Test Method…………………………………………………..38 3.9.2 Training time……………………………………………………………………38 3.10 STATISTICAL ANALYSIS…..……………………………………………….40
x
CHAPTER FOUR 4.0 RESULTS………………….……..……………………………………………41 4.1 Physical observation …………..…….…………………………………………41 4.2 MORPHOMETRIC CHANGES…..…………………….…………………….42 4.3 HISTOLOGICAL CHANGES…………………………………………………51 4.4 PHYSICAL OBSERVATION OF ANIMALS IN MONTOYA STAIRCASE BOX……………………………………………………………………………52 4.5 MOTOR ACTIVITY TEST USING MONTOYA STAIRCASE TEST………53 CHAPTER FIVE 5.0 DISCUSSION ………………………………………………………………….77 5.1 MORPHOMETRIC STUDY………………………………………………….. 77 5.2 HISTOLOGICAL STUDY……………………………………………………..78 5.3 NEUROBEHAVIORAL STUDY……..……..…………………………………79 CHAPTER SIX 6.0 CONCLUSION AND RECOMMENDATIONS………..……….….…………81 6.1 CONCLUSION………………………………………………………………..82 6.2 RECOMMENDATION………………………………………………………..82 References………………………………………………………………………………82 Appendix……………………………………………………………………………….92
xi
CHAPTER ONE
1.0 INTRODUCTION 1.1 BACKGROUND Food additives have been used to keep the quality, texture, consistency, taste, colour, alkalinity or acidity of foods. Humans are daily exposed to these chemical substances in their foods. Monosodium glutamate (MSG) is a food additive widely used as flavour enhancer of many foods like meats, poultry, seafood, snacks, soups and stews (Fuke and Shimizu, 1993). MSG is sodium salt of the amino acid glutamate and provides a flavouring function similar to naturally occurring free glutamate in foods (Yamaguchi and Ninomiya, 2000). Glutamate is a major component of most natural protein foods such as meat, fish, milk and some vegetables and plays an essential role in human metabolism (Filer and Stegink, 1994; Fernstorm and Garattini, 2000; Freeman, 2006). MSG is manufactured industrially by a fermentation process of molasses from sugar cane, sugar beets, starch and corn sugar. The breakdown and change of natural bound glutamate into various free forms of glutamate leads to the production of a white crystalline powder, when present in the free form, it has a flavour enhancing effect in food (Food Standard Australia New Zealand) (FSANZ, 2003). This distinctive taste is known as “Umami”, a word coined by the Japanese to describe the taste imparted by glutamate. Westerners often describe this flavour as savory, broth-like or meaty (Fuke and Shimizu, 1993). The optimal palatability concentration for MSG is between 0.2 and 0.8% with the highest palatable dose for humans being about 60 mg/kg body weight (Yang et al., 1997). Since 1960s, MSG has received great opposition against its use as a flavour enhancer. There is a general belief that it has harmful health effects. The high consumption of MSG, has led to the description of a variety of discomforts described by some non-oriental people after eating at a Chinese restaurant as “Chinese restaurant syndrome” (example flushing, tightness of the chest or difficulty in breathing) after the consumption of Chinese foods (Morseli and Garattini, 1970). There are considerable reports about various adverse effects intake of MSG as food additive (Stevenson, 2000; Hermanussen et al., 2006; Farombi and Onyema, 2006; Ortiz et al., 2006; Pavlovic and Cekic, 2006). However, there are claims that there has been suppression of information on the toxicity/safety of MSG (Samuels, 1999). Also, there has not been a general acceptance that MSG could be toxic to the humans. With all the controversies surrounding the safety of MSG, it is still being consumed in large quantity in fast and packaged foods.
Several studies assert that the average consumption of MSG in Chinese adults is upwards of 3.6 grams (Zhou et al., 2003; He et al., 2008). However, other studies suggest between 1 and 5 grams (Shi et al., 2010). This makes MSG an important part of the human diet. MSG is a salt derivation of the amino acid glutamic acid, or glutamate, that is commonly found in Asian
2
cuisine associated with Chinese restaurants and now frequently found in the Western diet (Shi et al., 2010). Because MSG is both an additive and a chemical in natural foods, exposure to this substance is worldwide. It exist in atypically high concentration in brain regions that are critical in the mediation of cognitive performance such as cerebral cortex, dentate gyrus of hippocampus and striatum (Park et al., 2000) indicating that the amino acid plays an important role in higher cognitive functions including memory which has been linked to activation of excitatory amino acid receptors. This stimulation leads to an enzymatic cascade of events ultimately resulting in cell death (Park et al., 2000). Monosodium glutamate is one of the many food ingredients used by human in homes and food industries. The use of monosodium glutamate in food goes back to the oriental cooks of antiquity, who used seaweed called sea Tangle to make starch. The link between the seaweed (also Laminaria Japonica) is flavour important, and Glutamate, (First isolated in 1866) was discovered by Professor Kikunae Ikeda of the University of Tokyo in 1980. Monosodium glutamate is a fine white crystal, easy to dissolve and very similar to salt and sugar in appearance. It is called “Kafi Zaboˮ in Hausa, Magi Funfun in Yoruba and Magi Ocha in Igbo all in Nigeria. It is widely used in food processing and cooking as a flavor enhancer in soups, sauces and spices blends- as well as in the wide variety of canned and frozen meats, poultry, vegetables and combination dishes (Martin, 2004). Today MSG is produced in the United States from sugar beet molasses in a fermentation process similar to that used in making yeast. The production of MSG in United State had grown to about 40 million pound per year in 1966. It is now produced in about 15 countries throughout the world. The volume of MSG produced by these countries is about 200, 000 tons per year. It is found in abundance in plants and animal tissues. The human body typically has 4.4 pounds of naturally occurring glutamate in its system. High levels found in mushrooms, peas and tomatoes which help to suggest the effectiveness of their food flavours (Jordan, 2005)
In addition to the multiple symptoms mentioned above, there are five most common symptoms; heartburn, diarrhea, abdominal cramps, unsual thirst and nausea (Kwok, 1968). Since 1968 a reaction was identified after a Korean-American physician wrote a letter to the editor of the New England Journal of Medicine describing an unusual physiological reaction he often experience while dinning in a chinese restaurant which he called as ‟ Chinese restaurant syndromeˮ. In most cases the symptoms go away or are reversed by treatment (Cameron et al., 1976). In rare cases, symptoms can be prolonged and may lead to death. It has been proven that asthma can be precipitated by MSG (Little, 2000). MSG was included among the
3
list of carcinogens (WHO, 2004). Many research works have been done on MSG in relation to its effect on tissues of certain organs, or system such as effect of MSG on some gastrointestinal function (Martins, 2004). Park et al. (2000) observed that systemic administration of MSG could impair memory and damage hypothalamic neurons in the adult mice. Ali et al. (2000), studied the behavioural effects in rats treatment with sub-neurotoxic doses of MSG which they found that there was no significant change in any of the components of spontaneous locomotory activity, but after apomorphine challenge, a marked decreased in the distance travelled was observed. They concluded that exposure to MSG in early life in rats could lead to subtle behaviour aberrations in late adulthood (Ali et al., 2000) 1.2 STATEMENT OF THE RESEARCH PROBLEM MSG is used widely in West Africa and many put 10-20 g/l in their food. The permissible amount of 5 g/l of foodstuff is mostly exceeded (Martins, 2004). There is limited knowledge about the toxic effects of MSG and particularly as regards to its effects when administered prenatally (Martins, 2004). Considering this fact there is need to evaluate the effect of prenatal exposure to MSG on the developing cerebrum. There is introduction of many brands of MSG in Nigerian markets with many brand names such as Ajinomoto, vedan, kings, Sandoz, Kawasaki among others. More than 20 brands can be found nowadays in shops in Nigeria (Oska, 2005).
1.3 JUSTIFICATION OF THE STUDY
1. There is evidence that MSG has toxic effects on the nervous system of adult mice and rats but little is known of its prenatal effects despite the fact that it is consumed during pregnancy.
2. This study seeks to provide information on the prenatally administered effects of MSG on the developing cerebrum in rats.
3. The study outcome could serve as a basis on which further studies could be carried out.
1.4 AIM AND OBJECTIVES OF THE STUDY 1.4.1 Aim
4
The aim of the present study was to study the effect of prenatal administration of monosodium glutamate on the developing cerebrum of Wistar rat and possibly behavioural changes in the dams. 1.4.2 Objectives The specific objectives of the study were:
1. To determine the morphometrical changes due to MSG administration in the cerebrum of the offsprings.
2. To determine the histological changes due to MSG administration in the neurons and glial cells in the cerebrum of the offsprings of adult Wistar rats using Haematoxylin and Eosin (H and E) and Toluidine blue
3. To determine the effect of MSG on calcium binding neurons in the cerebral b
cortex using anti-calbindin D28K immunohistochemically. 4. To evaluate the effect of MSG administration on the motor activity of the Wistar rat using Montoyastaircase test. 1.5 RESEARCH HYPOTHESIS Prenatal exposure of Wistar rats during pregnancy to monosodium glutamate results in toxic changes in the developing cerebrum and behavioural changes in the offsprings.
IF YOU CAN'T FIND YOUR TOPIC, CLICK HERE TO HIRE A WRITER»
