Longer period of oral administration of aspartame on cytokine response in Wistar albino rats

Abstract

Introduction

Aspartame is a non-nutritive sweetener particularly used in ‘diet’ and ‘low calorie’ products and also in a variety of foods, drugs and hygiene products. Aspartame is metabolized by gut esterases and peptidases to three common chemicals: the amino acids, aspartic acid and phenylalanine, and small amounts of methanol. The aim of the present study was to assess potential changes in molecular mediators of aspartame as a chemical stressor in rats.

Materials and methods

The effects of long-term administration of aspartame (40 mg/kg body weight/day) were tested in Wistar Albino rats. The treatment effects were assessed in different conditions, including control groups. After 90 days of treatment, circulating concentrations of different parameters were assessed: corticosterone, lipid peroxidation, antioxidant activity, nitric oxide, reduced glutathione and cytokines (interleukin 2, interleukin 4, tumor necrosis factor-α and interferon-γ).

Results

The results show that there was a significant increase in plasma corticosterone, serum lipid peroxidation and nitric oxide level along with a decrease in enzymatic and non-enzymatic antioxidant as well as significant decrease in interleukin 2, tumor necrosis factor-α and interferon-γ. There was also a significant increase in interleukin 4 irrespective of whether the animals were immunized or not.

Conclusion

The findings clearly point out that aspartame acts as a chemical stressor because of increased corticosterone level and increased lipid peroxidation and nitric oxide level induce generation of free radicals in serum which may be the reason for variation of cytokine level and finally results in alteration of immune function. Aspartame metabolite methanol or formaldehyde may be the causative factors behind the changes observed.

Resumen

Introducción

El aspartamo es un edulcorante no nutritivo que se utiliza especialmente en productos «dietéticos» y «bajos en calorías», así como en gran diversidad de alimentos, fármacos y productos de higiene. Las esterasas y peptidadas intestinales metabolizan el aspartamo a 3 compuestos químicos comunes: los aminoácidos, el ácido aspártico y la fenilalanina, y cantidades pequeñas de metanol. El objetivo de este estudio fue valorar los posibles cambios de los mediadores moleculares del aspartamo como estresante químico en ratas.

Material y métodos

Se estudiaron los efectos de la administración crónica de aspartamo (40 mg/kg de peso/día) en ratas albinas Wistar. Los efectos del tratamiento se valoraron en distintas condiciones, incluyendo grupos de control. Tras 90 días de tratamiento se determinaron las concentraciones circulantes de distintos parámetros: corticosterona, peroxidación lipídica, actividad antioxidante, óxido nítrico, glutatión reducido y citocinas (interleucina 2, interleucina 4, factor de necrosis tumoral α e interferón γ).

Resultados

Los resultados mostraron aumentos importantes de la corticosterona plasmática, la peroxidación lipídica y el nivel de óxido nítrico, junto con un descenso de antioxidante enzimático y no enzimático y una reducción importante de la interleucina 2, el factor de necrosis tumoral α y el interferón γ. Había también un aumento importante de la interleucina 4 con independencia de si los animales estaban o no inmunizados.

Conclusión

Los hallazgos indican claramente que el aspartamo actúa como un estresante químico debido al aumento del nivel de corticosterona, y el aumento de los niveles de peroxidación lípidica y óxido nítrico induce la generación de radicales libres en el suero, lo que puede ser la causa de la variación del nivel de citocinas y origina finalmente la alteración de la función inmunológica. Los metabolitos del aspartamo metanol o formaldehído pueden ser los factores causales de los cambios observados.

Introduction

Nowadays consumers are increasingly concerned about the quality and safety of many products of industrialized countries, in particular the use of artificial sweeteners, flavorings, colorings, preservatives and dietary supplements. General apprehension also exists regarding the possible long-term health effects of the raw materials and technologies used for the packaging, sterilization and distribution of foods. Many non-nutritive sweeteners have been used in foods and beverages to allow people to enjoy the sweet taste without consuming sugar-associated calories. One of these sweeteners is aspartame. This sweetener is incorporated into a number of foodstuffs (drinks, desserts, sweets, etc.) and in table sweeteners, under different brand names and into some 600 medicines.¹ Its sweetening power is 180–200 times greater than that of sucrose.² Because it contains no calories, aspartame is considered a boon to health-conscious individuals everywhere; a recent observation indicated that aspartame is slowly making its way into ordinary products used every day, which do not carry any indication as being for people on diets or diabetic patients. Aspartame in dry products is fairly stable even at high temperatures. However, in solution, its stability is a function of time, temperature, pH and available moisture. Aspartame is most stable between pH values of 3 and 5, even with increasing temperature.³ However, it breaks down and loses its sweetness in normal cooking or baking. Thus its use is limited to a table-top sweetener (Equal^®TM – The NutraSweet Co., Deerfield, IL) and as NutraSweet in dry foods, soft drinks, and frozen foods like ice cream. It is slightly soluble in water (about 1.0% at 25 °C), sparingly soluble in alcohol and insoluble in fats and oils. Being a peptide, it is amphoteric and is metabolized extensively to release its constituent amino acids and methanol.³ Upon ingestion, this artificial sweetener is immediately absorbed from the lumen and metabolized by gut esterases and peptidases to phenylalanine, aspartic acid and methanol. Orally ingested aspartame components are immediately absorbed from the lumen and reach the portal blood in a manner similar to that of amino acids arising from dietary protein or polysaccharides.4, 5 Their concentrations are found increased in the blood stream.⁶ This sweetener and its metabolic breakdown products (phenylalanine, aspartic acid and methanol) have been a matter of extensive investigation for more than 20 years, including experimental animal studies. Ten per cent of aspartame consists of methanol. Methanol is a toxicant that causes systemic toxicity.⁷ The primary metabolic fate of methanol is the direct oxidation to formaldehyde and then into formate. The severity of clinical findings in methanol intoxication correlated better with formate levels.⁸ Methanol is gradually released in the small intestine when the methyl group of aspartame encounters the enzyme chymotrypsin,⁴ but methanol is more readily generated by the body (thus becoming even more dangerous) when it is heated above 30 °C before being ingested. This occurs when soft drinks are left out in the sun or foods containing aspartame are heated. Methanol breaks down into formaldehyde and formic acid in the body. Formaldehyde is an embalming fluid, as a preservative in vaccines and a deadly neurotoxin. Formic acid causes cells to become too acidic, thereby producing metabolic acidosis. Acidosis damages cellular health by causing enzymes to stop functioning. Oxidative stress arises from the imbalance between pro-oxidants and antioxidants in favor of the former, leading to the generation of oxidative damage.⁹ Generation of free radicals is an integral feature of normal cellular functions, in contrast, excessive generation and/or inadequate removal of free radical results in destructive and irreversible damage to the cell.¹⁰ A stressor is a stimulus that is either internal or external, which activates the hypothalamic pituitary adrenal axis and the sympathetic nervous system resulting in a physiological change¹¹ Corticotropin-releasing hormone is released during stress and stimulates the release of adrenocorticotropic hormone,¹² which in turn releases corticosterone from the adrenal cortex. Elevation in the corticosterone level accelerates the generation of free radicals¹³ and alters the normal homeostasis of organ.¹⁴

Cytokines belong to the family of signaling molecules. They are released by specific cells of the immune system. Cytokines are small glycoprotein chemical structures that act in a paracrine and endocrine fashion as soluble signals between cells and play a pivotal role in the immune response. They are the hormonal messengers responsible for most of the biological effects in the immune system, such as cell-mediated immunity and humoral immunity. T lymphocytes are a major source of cytokines. Immune cells produce mediators of inflammatory and immune reactions called cytokines. These low-molecular weight glycoproteins in small concentrations are indispensable for normal functioning of the immune system. Their excessive secretion, however, leads to immune cell dysfunctions. Inflammatory cytokines may be produced by mononuclear cells of the immune system in response to numerous agents, such as microorganisms and their products (e.g., lipopolysaccharide – LPS)15, 16 as well as some xenobiotics.¹⁷ It is postulated that xenobiotics can influence the concentrations of inflammatory cytokines through oxidative stress mechanisms.18, 19, 20 The experimental and epidemiological data currently available to evaluate the above toxigenic risks of aspartame are insufficient and often unreliable, due to the inadequate planning and conduct of previous experiments. Recently from previous studies on aspartame and its metabolite, altering the oxidative status of the cells was investigated. Oral aspartame (75 mg/kg body weight/day) consumption causes oxidative stress in brain²¹ and its (40 mg/kg bw/day) consumption caused oxidative stress in brain,²² liver and kidney,²³ and also in immune organs.²⁴ This inadequacy, combined with the general limited knowledge about the safety/potential toxigenic effects of substances widely present in the industrialized diet, motivated the design of the current study. However, little is known about the effects of aspartame on cytokine expression. The detailed mechanisms of the effects of aspartame on cytokines are still unclear; therefore, the present study aimed at clarifying whether longer time of aspartame consumption has any effect on cytokine expression in serum of Wistar albino rats.