Introduction

It was brought to the attention of the Orange County (OC) Department of Health that the Dole Whip® being sold to park visitors at Disneyland may have been altered in a way that was potentially life threatening to consumers via an anonymous tip. Knowing the popularity of the park itself and the amount of people that visit everyday, the OC Department of Health decided to investigate into this matter. 
Dole Whip® is a soft serve dessert that is sold at Disneyland at the Tiki Juice Bar in Adventureland \cite{bar}. Although there is a menu for the Tiki Juice Bar showing that Dole Whip™ soft serve is being sold  there \cite{resort}, there is no official ingredient list or table of nutritional facts for this specific food item. However, the company that is the official producer of this dessert does have this information available \cite{information}. Despite this, it cannot be guaranteed that Disneyland uses this exact same recipe in the soft serve they give to guests at the park. Correspondence with Disneyland Director of Dining was attempted. However, when asked about the ingredient list for the Pineapple Dole Whip®, they declined to comment and replies to e-mails and phone calls stopped. As a result, this caused suspicion on what Disneyland put in their Dole Whip®. Consequently, this study is meant to explore the possible differences in content between the Dole Whip™ produced by the original company and the Dole Whip™ being sold to park goers at Disneyland. 
Aspartame, an artificial sweetener, was first approved for use in food in 1981 by the Food and Drug Administration (FDA) and has become one of the most widely used sweeteners worldwide \cite{Soffritti_2007} with the allowed daily intake for aspartame in the US being 50 mg/kg \cite{Soffritti_2005}. When ingested, aspartame is broken down and metabolized into aspartic acid, phenylalanine, and methanol \cite{Soffritti_2007}. In addition, several studies were done on the carcinogenic effects of aspartame in which the findings showed that aspartame caused an increased incidence of lymphoma within their test subjects. For example, a study in which 20% of the mice were treated with amounts of aspartame well below the daily limit were found to have developed some form of lymphocytic tumor \cite{Soffritti_2005}.  
What exactly causes these tumors to develop and grow larger in size are antigens and growth factors that are specific to the specific cancer cell type. For lymphocytic cells found within the lymph nodes and spleen, a differentiated B-cell antigen known as CD45R \cite{Ward_2006} as well as basic fibroblast growth factors (bFGFs) \cite{al2000} are responsible. These bFGFs are what promote cell growth via angiogenesis, a process that causes new blood vessels to form around the tumor cell which enables it to utilize the increased amount nutrients to become larger in size \cite{al2000}.  
 

Material and Methods

    2.1 - Sample Collection 

One unit of the Dole Whip™ soft serve was obtained from the Tiki Juice Bar in Adventureland in Disneyland. In addition, another unit of the Dole Whip™ soft serve was obtained straight from the manufacturer. 

    2.2 - Centrifugation of Samples 

To make analysis of what is inside the soft serve easier, small samples of each Dole Whip™ were be placed within a centrifuge to separate the compounds within the actual soft serve itself.  An Eppendorf Centrifuge 5920R was used to perform this task. The different layers were then extracted out of the centrifugal tube and into separate vials for spectroscopy analysis. 

    2.3 - Spectroscopy of Each Sample

Before any toxicological tests were conducted with the samples, infrared (IR) and nuclear magnetic resonance (NMR) spectra were taken for each of the layers previously extracted. IR spectroscopy was performed using a Nicolet iS5 FTIR Spectrometer and NMR spectra were taken using an NMReady-60PRO compact spectrometer. 

    2.4 - Toxicological Testing

A 90-day sub-chronic oral toxicity study was conducted using 18 mice (Mickeylius myceus). One control group was established in which 2 mice (one male, one female) did not receive any dosage of Dole Whip™ and were instead given deionized water and mouse pet food twice a day. A group of 6 mice (3 male, 3 female) were set up to receive high doses of Dole Whip™ in which their diet consisted only of deionized water and Dole Whip™ for the duration of the study. The third and final experimental group had 10 mice (5 male, 5 female) that were fed deionized water and mouse pet food twice a day but every other day, one of their meals would be replaced with Dole Whip™ instead. Another sub-chronic oral toxicity study was conducted under the same conditions but instead of the Dole Whip™ from Disneyland being administered, the Dole Whip™ obtained straight from the manufacturer was given to the subjects. So in total, 36 mice were involved in this experiment. 
During the course of this study, the physical condition of the mice were regularly observed and recorded. At the end of each week, blood samples were taken and physical examinations that involved weighing the mice and assessing any changes in physical appearance were also performed. Any mice that died prior to the completion of the study underwent histopathological analysis. The mice that were alive by the end of the study were humanely sacrificed under anesthesia and were also subject to histopathological analysis. 

     2.5 - Immunohistochemical (IHC) Analysis of Affected Tissues

This analysis involved the use of anti-CD45R antibodies [RA3-6B2] (FITC) from AbCam in order to visualize the antigen in question, CD45R. The steps for this analysis were done as outlined in the manufacturer's IHC-Paraffin Protocol (IHC-P)\cite{6hg46} in which the tissues of the retrieved organs were prepped for staining via tissue fixation (in 10% neutral buffered formalin), cut with a microtome to be placed into slides, dried, and then deparaffinated using various washes of Xylene and varying concentrations of Ethanol (100%, 95%, 70%, and 50%). The antigen retrieval was performed using a pressure cooker with Tris/EDTA pH 9.0 as the buffer solution. For the staining process, the tissue from the livers obtained from the necropsy of the mice were used as a negative control since the antigen in question is not known to be synthesized within the liver.