The high label presence in plasma and liver is in agreement with the carriage of the label from the intestine to the liver via the portal vein. The high label levels in kidney and, to a minor extent, in brown adipose tissue and brain are probably a consequence of their high blood flows (45). Even in white adipose tissue, the levels of radioactivity found 6 hours after oral administration were 1/25th those of liver. Cornea and retina, both tissues known to metabolize actively methanol (21,28) showed low levels of retained label. In any case, the binding of methanol-derived carbon to tissue proteins was widespread, affecting all systems, fully reaching even sensitive targets such as the brain and retina.

In all groups studied, the label bound found in plasma and tissues corresponds to that injected with aspartame, since there is no other source of radioactivity available. The lack of changes in plasma radioactivity from 1 to 24 h suggests that the half life of this newly added carbon was quite long, thus precluding the possibility that the label detected would simply correspond to unattached methanol. The label bound to plasma proteins was not aspartame either, since the latter is a non-reactive molecular species fully hydrolysed in the intestine (1-2); the peptide never arrived to be in contact with the rat tissues or its components. We were not able to reproduce any direct labelling of protein exposed either to aspartame, methanol nor formic acid.

 Most of the label found in the tissues is the result of the formation of formaldehyde or (in smaller proportion in any case, because of its lower reactivity) formate adducts. Methanol is highly volatile and, eventually, its radioactivity could hardly be taken into account, since the counting method already eliminates this possibility. In addition, the stabilized maintenance of the plasma radioactivity levels could not belong to formate nor methanol, since these unattached substrates are easily taken up and oxidized by tissues, filtered by the kidney or even lost through respiration as occurs with short chain volatile alcohols. The shape of the time-course graph representing the changes in tissue label supports the hypothesis assuming that the label is firmly bound at least for 6 hours after administration of aspartame. This behaviour is also found in formaldehyde-protein adducts (3 1), long lived species difficult to eliminate, in which the protein is denatured and its original function altered.

 The transfer of "one-carbon" units from aspartame to plasma and tissue proteins has been known for a time (35). Its nature, and the mechanism of attachment, however, were assumed to be due to the incorporation of the methanol carbon to normal amino acid structures (essentially forming the methyl group of methionine) through the "one-carbon " tetrahydrofolate and S-adenosyl-rnethionine pathways (35). The lack of radioactivity in the methionine spot from aspartme-treated labelled rat proteins, however, shows that this assumption could no longer be maintained. The finding of other -different- labelled DNP-derivatized amino acid(s) in the exposed protein hydrolysates confirms that the label was not carried into protein through the one-carbon pool metabolism labelling of methionine, i.e. prior to the synthesis of the protein. The coincidence of this labelled DNP-amino acid residue with that obtained from protein experimentally exposed to formaldehyde confirms that the label fixed to rat proteins after labelled aspartame exposure was derived from formaldehyde adducts, and definitely proves that the label in tissue proteins does not correspond to methionine.

 This agrees with the incorporation of the label into the fully synthesized protein at a remarkably uniform rate of label distribution between different molecular species in spite of their eventually different turnover (synthesis) rates.

 The analysis of label distribution in the nucleic acids shows a remarkable uniformity in the specific activities of DNA and protein, with RNA showing somewhat lower values in the same range. This distribution is in agreement with a fairly uniform exposure to the same reacting species, and could not be explained through incorporation of one-carbon pathways into molecules which show widely different half lives, as is the case with the highly recycled RNA and some proteins and long-lived DNA and proteins. The finding of large amounts of label in DNA, higher than in RNA, could be only explained through direct reaction, since its slow turnover would require inordinately long exposure times to achieve the observed specific activities. The additional existence of different labelled bases, probably formed by the binding of formaldehyde and the "normal" bases not coinciding with any of the other bases. The thin layer chromatograius show a single spot, resolved in at least three peaks, none of which coincided with adenine, guanine nor thymine. The lack of label in these spots is incompatible with the "one-carbon" pathways hypothesis of label incorporation, since two "IC" units are needed for the synthesis of adenine and guanine and one for that of thymine. The presence of label in other different molecules strongly supports the adduct formation postulate, attributing to formaldehyde the main responsibility for the appearance of aspartame- methanol label in tissue components. The evidence presented, then, proves that a significant portion of the methanol carbon of aspartame finds its way into adducts of proteins and nucleic acids under the conditions tested, both in normal and cirrhotic rats. The results presented show that the carbon adducts of protein and DNA could have been generated only from formaldehyde derived from aspartame methanol, since all the otherbiochemical forms in which this carbon may be found could not produce adducts with protein and nucleic acids.

The doses of aspartame given to the rats in this experiment were high, higher at least than that any human may receive daily with normal consumption of the additive -in the range of 2-6 mg/kg-day (46)-, but were similar to those used in comparable tests on rodents in which no ill-effects were detected. These doses were in the same range as the adi for humans established for Canada and the EC (40 mg/kg-day) (46). The dose administered was also lower than that used for toxicity studies, which have shown that even at very high doses aspartame does not produce immediately appreciable harm (I 7). Most of these studies, however, refer to direct acute toxicity effects, which were not observed either in the rats used in the present study (except, perhaps, for softer droppings in those subjected to the chronic treatment with aspartame gavages).

The amount of label recovered in tissue components was quite high in all the groups, but especially in the NA rats. In them, the liver alone retained, for a long time, more than 2 % of the methanol carbon given in a single oral dose of aspartame, and the rest of the body stored an additional 2 % or more. These are indeed extremely high levels for adducts of formaldehyde, a substance responsible of chronic deleterious effects (33) that has also been considered carcinogenic (34,47). The repeated occurrence of claims that aspartame produces headache and other neurological and psychological secondary effects -more often than not challenged by careful analysis- (5,9,10,15,48) may eventually find at least a partial explanation in the permanence of the formaldehyde label, since formaldehyde intoxication can induce similar effects (49).

The cumulative effects derived from the incorporation of label in the chronic administration model suggests that regular intake of aspartame may result in the progressive accumulation of formaldehyde adducts. It may be further speculated that the formation of adducts can help to explain the chronic effects aspartame consumption may induce on sensitive tissues such as brain (6,9,19,50). In any case, the possible negative effects that the accumulation of formaldehyde adducts can induce is, obviously, long-term. The alteration of protein integrity and function may needs some time to induce substantial effects. The damage to nucleic acids, mainly to DNA may eventually induce cell death and/or mutations. The results presented suggest that the conversion of aspartame methanol into formaldehyde adducts in significant amounts in vivo should to be taken into account because of the widespread utilization of this sweetener. Further epidemiological and long-term studies are needed to determine the extent of the hazard that aspartame consumption poses for humans.

Acknowledgements