SUMMARY




	FORMALDEHYDE DERIVED FROM DIETARY ASPARTAME BINDS TO TISSUE COMPONENTS IN VIVO C. Trocho, R. Pardo, I. Rafecas, J. Virgili, X. Remesar, J.A. Fernández-López and M. Alemany Departament de Bioquimica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona Spain. (Received in final form May 13, 1998) Summary Adult male rats were given an oral dose of 10 mg/kg aspartame 14C-labelled in the methanol carbon. At timed intervals of up to 6 hours, the radioactivity in plasma and several organs was investigated. Most of the radioactivity found (>98 % in plasma, >75 % in liver) was bound to protein. Label present in liver, plasma and kidney was in the range of 1-2 % of total radioactivity administered per g or mL, changing little with time. Other organs (brown and white adipose tissues, muscle, brain, cornea and retina) contained levels of label in the range of 1/12 to 1/10th of that of liver. In all, the rat retained, 6 hours after administration about 5 % of the label, half of it in the liver. The specific radioactivity of tissue protein, RNA and DNA was quite uniform. The protein label was concentrated in amino acids, different from methionine, and largely coincident with the result of protein exposure to labelled formaldehyde. DNA radioactivity was essentially in a single different adduct base, different from the normal bases present in DNA. The nature of the tissue label accumulated was, thus, a direct consequence of formaldehyde binding to tissue structures. The administration of labelled aspartame to a group of cirrhotic rats resulted in comparable label retention by tissue components, which suggests that liver function (or its defect) has little effect on formaldehyde formation from aspartame and binding to biological components. The chronic treatment of a series of rats with 200 mg/kg of non-labelled aspartame during 10 days resulted in the accumulation of even more label when given the radioactive bolus, suggesting that the amount of formaldehyde adducts coming from aspartame in tissue proteins and nucleic acids may be cumulative. It is concluded that aspartame consumption may constitute a hazard because of its contribution to the formation of formaldehyde adducts. Aspartame is one of the most widely used artificial sweeteners. Its peptide nature: aspartyl- phenylalanine methyl-ester facilitates its intestinal hydrolysis and the absorption (I -3) of innocuous amino acids together with small amounts of free methanol, far away from the lower limits of toxicity for that compound (4). The use of large amounts of aspartame in the diet, however, has been claimed to be the cause of a number of ailments, like headaches (5) and other symptoms (6-7), which are difficult to explain (8) from its known composition and the easy blending of its building components in the overall host metabolism. A number of studies have linked aspartame with neurologic pathologies, but most of the results yielded negative or inconclusive correlations (9-16). The acute toxicity of aspartame is believed to be low (I7), which has promoted a wide distribution of the product as a potent hypocaloric and safe substitute of sugar (I 8-19). Methanol is primarily oxidized in several tissues to formaldehyde and formic acid (20-2 1), the latter being considered the main metabolite responsible for the deleterious effects of acute methanol intoxication in man (22), but also in experimental animals (23), in spite of the marked resistance of the rat to formate (24-25). The enzymes involved in methanol metabolism are alcohol dehydrogenase (EC 1. 1. 1. 1) and aldehyde dehydrogenase (EC 1.2.1.3), as well as the microsomal oxidase pathway (26). Acute methanol intoxication may produce blindness and hepatic loss of function (27-28), since the retina, cornea and liver contain the highest alcohol dehydrogenase activity (29-30). These tissues are, thus, where one can expect, eventually, the largest accumulation of their byproducts: formaldehyde and formate, in the event of intoxication. It may be assumed that liver functional failure due to cirrhosis could result in the loss of its role as barrier to intestinal methanol, and thus, the effects of methanol intoxication on other tissues (i.e. the retina) would be more marked. The cirrhotic rat may be, then, used as a model of acute or chronic methanol toxicity. Formaldehyde is a highly reactive small molecule which strongly binds to proteins (3 1) and nucleic acids (32) forming adducts which are difficult to eliminate through the normal metabolism pathways. As a result, formaldehyde induces severe functional alterations (33), including the development of cancer (34). The small amounts of formaldehyde which can be potentially produced from dietary use of aspartame have been often overlooked in its potential toxicity precisely because of the limited amount eventually produced. However, the administration of labelled aspartame to experimental animals results in the incorporation of a significant proportion of the label to proteins (35). The accumulation of label has been postulated to be the consequence of label drift into amino acids (essentially in the methionine methyl group) through the one-carbon pool (35). This aspect has not been, however, proved nor further investigated. We have intended here to determine the extent of conversion of aspartame methanol to formaldehyde and its eventual effect on the overall physiologic function of the rat. In addition we have probed whether the aspartame methanol carbon presence in tissue components is due to the eventual drift of label into methionine and nucleic acid components through the one-carbon pool, or is the consequence of a direct reaction with free formaldehyde forming stable adducts.