METHODS




	Materials and Methods *Aspartame. Aspartame labelled ("C) in the methanol carbon was custom-prepared by Amersham (Amersham, uk). The product had a specific activity of 433 MBq/mmol, and a chromatographic purity >98%. The standard dose given orally to the rats was 4.5 Mbq per kg of rat weight, always supplementing unlabelled aspartame (Sigma, St Louis, MO USA) to give a specific activity of 55 Mbq/mmol. Acute and chronic administration of aspartame to normal rats.* Sixteen week-old healthy adult male Wistar rats, weighing initially 380-460 g, were used. The rats were housed in collective cages in a controlled environment (21-22'C; 70-75 % relative humidity; lights on from 08:00 to 20:00), and were fed a standard chow pellet (B&K, Sant Vicent dels Horts, Spain) and tap water ad libitum. Two groups of rats were selected. The first group NC (Normal-Chronic, N=5) received a daily oral gavage of 0.68 mmol per kg of rat weight (200 mg per kg) of a water suspension (2.5 mL/kg) of non-radioactive aspartame (Sigma). This treatment was continued for I 0 days. On day I 1, the rats were administered a gavage of 4.5 Mbq per kg of rat weight of labelled aspartame in 68 µmol of cold aspartame per kg, in the same volume of the standard gavage. The second group NA (Normal-Acute, N=l2) was given a single dose of 4.5 Mbq per kg of rat weight of labelled aspartame in 68 µmol of cold aspartame per kg of rat weight. Prior to the administration of the last (or only) dose, blood was extracted from the tail vein and used for the measurement of biochemical parameters using a Spotchem dry strip (panel I and 2) analysis system (Menarini, Milano, Italy). The rats chronically treated (NC group) were killed by decapitation 6 hours after the administration of the labelled aspartame gavage. The rats in the NA group were killed by decapitation at 15 or 30 min and at 1, 2, 6 or 24 hours after the administration of the final labelled aspartame load. All animals were dissected, and samples of blood plasma (heparinized), liver, kidneys, brain, cornea, retina, hind leg striated muscle, epididymal fat pads and interscapular brown adipose tissue were cut, weighed (blotted when necessary), and frozen in liquid nitrogen. The samples were preserved at - 20'C until processed. Tissue samples were homogenized in water: methanol (4: 1) in order to limit the losses of free methanol, using an all-glass Tenbroek homogenizer. Aliquots of the homogenates were immediately counted for radioactivity using a water-miscible scintillation cocktail (Ecolite, from ICN, Costa Mesa, CA USA. Plasma samples were counted directly after mixing with the scintillation cocktail. In all cases, two countings, 24-hours apart were performed. In all cases we obtained the same countings; there were no samples showing a significant loss of radioactivity (purportedly due to the eventual evaporation of methanol to the head space of the vial). Thus it was assumed that no significant amounts of labelled methanol were present in the final homogenates. Aliquots of the homogenates were precipitated with trifluoroacetic acid to remove the protein from supernatants, and the two fractions were then counted separately. Acute and chronic administration of aspartame to liver-damaged rats Six week-old healthy adult male Wistar rats weighing initially 100-120 g were used. The rats were housed and fed under the same conditions described above for the controls. The rats were made cirrhotic by means of three i.p. injections per week of carbon tetrachloride diluted 1:1 with corn oil (36). The rats received 0.4 mL injections during the first 2 weeks, then 0.6 mL until week 6 and finally 0.8 mL until week 10, when the period of treatment was considered finished, when the rats weighed 340-380 g. Two groups of liver-damaged rats were selected. The first group CC (Cirrhotic-Chronic, N=5) received a daily oral gavage of non-radioactive aspartame for 10 days, and on day I I they received 4.5 Mbq/kg of labelled aspartame as in the NC group. The second group CA (Cirrhotic-Acute, N=1 1) was given a single dose of 4.5 Mbq/kg of labelled aspartame in 68 µmol of cold aspartame per kg as in the NA group. Tail vein blood was sampled from these animals, and its plasma stored frozen; this was later used to measure biochemical parameters as in group NA. The CC chronically treated rats were killed by decapitation -as in the control series- 6 hours after the administration of the labelled oral bolus of aspartame, and those in the CA group were killed at 15 or 30 min and at 1, 2, 6 or 24 hours after receiving the labelled aspartame load. Samples of blood plasma and tissues were weighed, frozen and stored at -20'C until processed. Some samples of liver were preserved in 4 % formaldehyde and later used for the preparation of stained tissue sections in order to determine the degree of hepatic alteration (37). Blood and tissue samples were processed as described for normal rats. Statistical comparison between means was determined with standard two-way anova programs, as well as with the Student's t test. *Nucleic acids analysis.* Two additional adult rats were treated as in group NC, but they received the gavage for only three days. The last gavage contained 37 Mbq of radioactive aspartame. After killing, blood plasma and liver samples were obtained and frozen. Liver tissue was used for the extraction and purification of total RNA and DNA using the Tripure (Boehringer Mannheim, Germany) isolation reagents system. These preparations yielded pure fractions of DNA, RNA and protein. Nucleic acids content was determined by uv light absorption at 260/280 nm (38), and protein with the Bradford method (39). The radioactivity of these fractions was measured and used for the estimation of their specific radioactivity. The pooled DNA samples of the two rats used were hydrolysed with 88 % formic acid at 170'C in a sealed glass ampoule (40), and the corresponding constituting bases separated through thin layer chromatography on 0. I mm thick cellulose plates (5716 Merck, Darmstadt, Germany), run against standards of 14C-labelled adenosine, guanine and thymine (all from ICN, Costa Mesa, CA USA) containing their cold counterparts (from Sigma, St Louis, MO USA). The mobile phases used were isopropanol: 25% ammonium hydroxide (4:1 by volume) and butanol: acetic acid: water (4: 1:1 by volume) (4 1). Spot radioactivity was measured by exposure of the chromatograms with the Bio-Rad Molecular Imaging Screen-BI (Bio-Rad, Hercules, CA USA) for several days. The plates were later read with a Bio-Rad Molecular Imager System GS-525 two-dimensional array radioactivity counter; this instrument provided a printed "photographic plate" of the bidimensional distribution of radioactivity in the chromatogram. Labelled standards of DNA bases were used to determine whether the hydrolysed sample presented any radioactivity in their spots. Cytosine was not included as standard since no carbon from IC pool participates in its structure through the whole process of pyrimidine synthesis. The DNA digest from the liver of rats exposed to labelled aspartame was also analysed through HPLC, using a Kontron (Milano, Italy) UPLC fitted on line with a diode array detector 440 (Kontron) and an eluate scintillation detector LB 507 A (Beckman, Fullerton CA USA). The instrument was run with the Data System 450-MT2/DAD (Kontron) software. We used a sex cationic interchange column (Kontron) (250x4 mm, 10 µm), maintained at 25ºC, and total flow was 0.8 mL/min. An isocratic gradient of 100 % 10 mM ammonium phosphate buffer pH 5.56 was used. The scintillation detector used a cocktail ultima-flo M (Packard, Meriden IL USA) with a mixture ratio of 3:1. A series of standards of adenine, thymine and guanine were run under the same conditions. In all cases the radioactivity in the fractions was recorded. *Protein analysis.* The rats used for nucleic acid analysis provided enough plasma samples for protein analysis; plasma proteins were selected because they could not be contaminated with nucleic acids. The plasma proteins (0.100 mL aliquots) were precipitated with 10% trifluoroacetic acid. Aliquots of the precipitated proteins were then hydrolyzed for 48 h at 110°C in 6N HCl in Teflon-sealed tubes with occasional shaking (42). The digests were filtered to remove the black Maillard adducts (which retained part of the radioactivity). The amino acids in the digests were derivatized with dinitrofluorobenzene, and the DNP-amino acids were separated by bidimensional thin layer chromatography (43) on 0. 1 5 mm thick silicagel plates (Polygram Sil G/UV254, Mocherey-Nagel, Düren, Germany). The presence of label in amino acid spots was measured as in the case of nucleic acids using the Bio-Rad Molecular Imager. In separate runs, 14C_Iabelled methionine (NEN, Boston, MA< USA) diluted with cold methionine (Sigma) was added to rat plasma, digested, derivatized and processed as indicated above. Thus, the DNP-methionine spot was identified; in any case, the position of standard amino acids in the bidimensional chromatogram was known (43). The derivatization method used prevented the contamination of the plates by radioactive materials different from amino acids, since only the DNP-derivatized compounds were recovered. An aliquot of 0.2 mL of blood serum albumin (Sigma) dissolved in water (100g/L) was incubated for 2 h at 37ºC with 0.02 mL of a labelled substrate preparation, containing I nmol and 5 kBq of 14C-labelled: a) aspartame, b) formaldehyde (Amersharn), c) formic acid (Sigma), or d) methanol (Amersham)@ The samples were then precipitated, washed with 10% trifluoroacetic acid and the precipitates counted for radioactivity. The protein exposed to formaldehyde retained a large proportion of the initial radioactivity added. In the cases of aspartame, formic acid and methanol, only background values were obtained in the washed protein precipitates, showing that none of these procedures resulted in stable label attachment to proteins. The samples of albumin exposed to formaldehyde label were processed in parallel to the sample of plasma (i.e. hydrolysis, derivatization and thin layer chromatography).