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A Study of the Effects of Allura Red AC (Red 40) and Tartrazine (Yellow 5) Individually

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A Study of the Effects of Allura Red AC (Red 40) and Tartrazine (Yellow 5) Individually 1 A study of the effects of Allura Red AC (Red 40) and Tartrazine (Yellow 5) individually and in combination, on development of Xenopus laevis (African clawed frog). By Brittanie L. Fils Spring 2019 A thesis Submitted in partial fulfillment Of the requirements For a baccalaureate degree In Biology In cursu honorum Reviewed and approved by: _______________________________ Dr. Laura Twersky Thesis Supervisor Submitted to The Honors Program, Saint Peter’s University 2 Acknowledgements I would like to thank my family, especially my older brother, Steven for being part of an amazing support system. I would also like to thank my close friends including but not limited to, Kayla Murray, Jack Payne, Sanket Kumar, and Naomi Dawkins. Most of all, I would like to thank Dr. Laura Twersky for her patience, understanding, and advice throughout the years and work on this project. Finally I would like to thank the TriBeta Research Foundation for supporting this project through a grant. 3 Table of Contents Cover Page ...................................................................................................................... 1 Acknowledgments ........................................................................................................... 2 Table of Contents ............................................................................................................ 3 Abstract ........................................................................................................................... 4 Introduction..................................................................................................................... 5-12 Materials and Methods ................................................................................................... 12-15 Results ............................................................................................................................. 15-18 Discussion ........................................................................................................................ 18-19 References ....................................................................................................................... 20-23 4 ABSTRACT The consumption of artificial food dyes is a topic of controversy in the United States. In contrast, many European nations opted for natural alternatives after studies suggested that they cause hypersensitivity in children. Two of the most prevalent food dyes, Red 40 and Yellow 5, are heavily debated as carcinogens and are solidly linked to ADHD in children. The purpose of this study is to use the model organism, Xenopus laveis, the African clawed frog, to observe the effects of these dyes separately and together. Specimens will be incubated in different concentrations of Red 40, Yellow 5, or both at either 2.5μg/ml or 5μg/ml. There will be one control group and eight experimental groups. These groups will be exposed to the dyes from metamorphosis stage 48 (Nieuwkoop and Faber) until adulthood. They were observed by determining how long movement lasted once it began. The results suggest a strong link to hyperactivity and possible effects on development, such as different mortalities among the groups. Tadpole length was in terms of length, and any malformations were documented. A review was also conducted on dyes present in common products available to consumers, which will be discussed. 5 INTRODUCTION Food dyes are used by large corporations to create a uniform and appealing product to the masses. These synthetic dyes have been used by modern, Western nations for at least a hundred years. Dyes are organic (carbon-containing) chemicals. These dyes were originated from coal tar, and are now made from petroleum (Kobylewski & Jacobson, 2012). In the United States, since 1955, the consumption of food dyes has increased five times over. Only three of these dyes, Allura Red AC (Red 40), Yellow 5 (Tartrazine), and Yellow 6 (Sunset Yellow FCF) make up ninety percent used in foods. These additives are not necessary and only serve aesthetic purposes. In recent times, there have been evidence suggesting food dyes have carcinogenic, neurological and hypersensitivity effects, but more specifically, there is evidence suggesting that they causes both hypersensitivity and attention deficit hyperactivity disorder (ADHD) (Bateman et al., 2004). Xenopus laevis is being used as a model organism for a few reasons. For one, during the tadpole stage, the organism is transparent making it ideal for this experiment. It allows observation and documentation of the specimen. The adverse effects will be documented through all the stages, as well as mortality. This assessment will show us which dye is most likely to cause cancer. Thus, Xenopus laevis, as a model organism, will show which dye, Yellow 5 or Red 40, most likely causes cancer in pre-pubescent children. That would allow any possible carcinogenic effects of Red 40 and/or Yellow 5 to be documented and observed. Tartrazine or Yellow 5 is an orange-colored water soluble powder (Tanaka et al., 2008). A search through the common American household shows that it is used in products ranging from sodas to shampoos. The prevalence of this color is troubling considering the fact that it causes allergic reactions. Like Red 40, Yellow 5 is associated with hyperactivity with children 6 and it causes DNA damage in the colons of mice. Damage occurs at close to the acceptable intake value (Rowe and Rowe, 1994). Yellow 5 also causes other changes such as restlessness and issues with sleep (Tanaka, 2006). Interestingly, other food dyes do not cause damage in the colon in a statistically significant manner (Poul et al., 2009). All of this information makes Yellow 5 pertinent to this study and further studies of food dyes. ADHD is a childhood-onset neurodevelopmental disorder that affects about 1.4-3.0 percent of children. It is more common in males. ADHD is primarily inherited, but other factors such as exposure to lead play into it as well. ADHD is treatable, but will most likely lead to other mental and social issues later on in life. For that reason, if there is a way to cut down on the number of children suffering from it, steps should be taken. (Thapar and Cooper, 2015). The diagnosis of ADHD requires the symptoms to be present in a variety of settings. The symptoms must be debilitating in some way including but not limited to academics or social situations. The age of diagnosis must be made before the age of twelve according to the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders 5th edition (Thapar and Cooper, 2016). The unnecessary increase led to concerted efforts to curb the use of food dyes. While the association of food additives with hyperactivity is accepted, there is no conclusive evidence to prove that these additives have a negative effect on human development (Lau et al., 2005). In 2008, the Center for Science in the Public Interest (CSPI) petitioned the Food and Drug Administration to ban artificial food dyes. Food dyes are used heavily in foods marketed and consumed by children. Examples are breakfast cereal, candy, and vitamins. Even when parents feed their children fresh fruit, such as oranges, there is a strong possibility that food dyes were used to enhance its physical appearance. The most common way to ingest dyes is through 7 beverages. (Batad and Jacobson, 2016). The CPSI were successful in influencing the United States government to ban Violet 1 in 1973 because it was linked to cancer in animals. On the other side of the argument, the Internal Association of Color Manufacturers (a trade association for food dye makers and users) claim that food dyes correct natural colors and give an identity to food through color. It is also used to give color to food that have been stored long term. In terms of vitamins, the argument is that the food dye colors allow for an easy, efficient way to identify the pills (Potera, 2010). However, it should be noted that these dyes are possibly used because they are less expensive, easier to preserve and are able to make brighter colors (Kobylewski & Jacobson, 2012). There are currently nine FDA approved dyes. Besides Allura Red AC, Tartrazine, and Yellow 6, the other dyes include, Brilliant Blue (Blue 1), Indigo Carmine (Blue 2), Citrus Red 2, Green 3, Orange B, Erythrosine (Red 3). There are several banned dyes such as Green 1 which was found to cause liver cancer in animal studies, and Red 2 which is a possible carcinogen (Kobylewski & Jacobson, 2012). In terms of United States, there are a few, albeit, antiquated laws concerning dyes. Before 1960, the law required that dyes could not be used at all if there were not completely harmless. However, that standard is impossible to live up to. In 1960, Congress passed the 1960 Color Additives Amendment that simultaneously lessened the standards of acceptable dyes and made the regulations more clear. The law included provisions including testing each batch of food to test if it was an acceptable level of contamination. Preservatives and flavorings are not tested. Due to a loophole, companies are allowed to label their products as “generally safe for use” even if it has not been subject to toxicity testing (Kobylewski & Jacobson, 2012). The Federal Drug Association has legal limits for carcinogens in dyes. Within a lifetime of consumption, these dyes can’t result in cancer in one person out of a 8 million. However, these standards are based on consumption rates of Americans in 1990, but the consumption
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