Fd Chem. Toxic. Vol. 22, no. 2, pp. 177 182, 1984 0278-6915/84 $3.00 +0 .00 Printed in Great Britain Pergamon Press Ltd

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SACCHARIN AND TRYPTOPHAN METABOLISM

The association of saccharin with an increase in the incidence of bladder tumours in male rats fed high dietary saccharm levels for two generations (Arnold et al. Toxic. appl. Pharmac. 1980, 52, 113; Taylor et al. ibid 1980, 54, 57) has recently been the subject of much debate. Saccharin has been termed an epi- genetic carcinogen, since it does not bind covalently to DNA (Lutz & Schlatter, Chemico-Biol. Inter- actions 1977, 19, 253), but there is no satisfactory hypothesis for the in vivo mechanism whereby this metabolically inert molecule exerts its effects in the rat bladder either as a carcinogen or as a promoter. Some moves to fill this gap have recently been reported.

Feeding saccharin to rats at dietary levels that saturate its renal tubular secretion (5-7.5%, w/w) has been found to have a major effect on the microbial metabolism of tryptophan in the gastro-intestinal tract (Sims & Renwick, Toxic. appl. Pharmac. 1983, 67, 132). Preliminary studies indicated that 7.5% saccharin diets fed to rats for 3 months had profound effects on tryptophan metabolism; urinary excretion of indican was increased by 270% and there were other marked changes in the HPLC urine analysis, although the peaks concerned did not correspond exactly to known tryptophan metabolites. These changes were apparent by wk 2 of feeding. The increased excretion of indican was suggestive of increased bacterial metabolism of dietary tryptophan to indole. This could result from the presence in the upper intestine of organisms possessing trypto- phanase activity so that tryptophan is metabolized before it can be absorbed, from an increase in tryptophanase activity in the caecum so that a higher percentage of unabsorbed tryptophan is converted to indole, from inhibition of tryptophan absorption with a consequent increase in its availability to the gut flora, or from inhibition of host proteolytic enzymes, making more protein available for bacterial metabolism. A dose-response study of the effects of saccharin on indican excretion and on the com- position of caecal contents was therefore undertaken.

Groups of male rats maintained for 1 month on diets containing 2-10% saccharin showed increases in the 24-hr urinary excretion of indican despite lower body-weight gains and decreased food consumption. A 3.l-fold increase was observed in rats fed 10% saccharin. A diet containing 2% tryptophan increased indican excretion slightly, while a 2% tryptophan/5% saccharin diet had a simple additive effect without potentiation. The plasma concentration of indican was increased by the higher intakes of saccharin but the effect was not linearly related to the dietary level of saccharin. Neither 2% tryptophan nor the tryptophan/saccharin levels affected the plasma con- centration of indican. High blood-saccharin levels

depressed renal elimination of indican. The renal clearance of indican was reduced also in rats fed 2% tryptophan with or without saccharin.

The caecal contents weighed more in rats fed the saccharin diets for 4-5 wk than in controls and the weight of caecal contents/kg body weight was linearly related to the percentage of saccharin in the diet. The tryptophan diet increased the weight of caecal con- tents slightly and the effect of tryptophan plus sac- charin was additive. The caecal wall of saccharin-fed rats had a more prominent and extensive vasculature and, except with the 1% diet, weighed significantly more than that of the controls, but tryptophan alone did not increase caecal-wall weight significantly.

The caecal concentrations and total amounts of tryptophan, indole and indolelactic acid were markedly higher in saccharin-fed rats than in con- trols. Tryptamine, indoleacetic acid and in- dolepropionic acid did not change significantly in total amount but decreased in concentration because of the caecal enlargement. Correlation between the total amount of indole in the caecum and the excre- tion of indican in the 24-hr urine indicated that the increased indican excretion reflected increased amounts of caecal tryptophan. The 2% tryptophan diet increased the concentration and total amount of tryptophan in the caecum as well as the formation of indole and of indoleacetic, indolelactic and in- dolepropionic acids. Generally the addition of 5% saccharin to the 2% tryptophan diet had an additive effect on the amount of caecal tryptophan and its metabolites.

The effect of saccharin (7.5%) on the weight of the caecal contents was almost immediate, being statisti- cally significant by day 2. By day 14 caecal size had increased to a weight similar to that seen after 5 wk in a parellel study. Indican excretion did not show a consistent increase until 5 days after the start of the saccharin feeding, an earlier decrease being ten- tatively attributed to a loss of tryptophanase activity in the caecal contents.

Microbial-enzyme studies showed only low levels of tryptophanase and nitroreductase activity in the upper intestine of control rats, most activity being present in the caecal, colonic and rectal contents. The almost identical distribution and total amount of nitroreductase, an enzyme possessed by many strains of intestinal bacteria, in the gut contents of rats fed 7.5% saccharin for 2 3 wk indicated that saccharin does not affect the distribution of the gut flora. However, the levels of tryptophanase in these rats were lower than in controls throughout the gastro- intestinal tract, in spite of the observed increase in indole concentration in saccharin-fed rats. The num- ber of viable organisms (both anaerobes and aerobes) per unit weight of faeces was higher in rats fed the

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178 Information section--Fd

7.5% saccharin diet for 3 months than in normal rats, but there was no evidence that saccharin influenced the types of organisms present. Enzyme-distribution studies suggested a saccharin-produced transient de- crease in caecal tryptophanase activity followed by repopulation with organisms possessing this enzyme.

A slight increase in intestinal insoluble protein was observed in rats fed the 7.5~ saccharin diet, but no major treatment-related effect on soluble protein was apparent. However, in the caecal contents, a marked (approximately ninefold) increase was noted for both the concentration and total amount of all protein fractions. Nearly all of the increase in soluble 'pro- tein' (from 54 to 490 mg in total contents) was due to small peptides, not free amino acids, although total tryptophan was increased 30-80 times. This effect was probably due to the decrease in tryptophanase activity, which resulted in the smaller increases in indole (fivefold) in the caecum and in indican (three- fold) in the urine. These findings indicate that the increased formation of indole and indican originate from an increase in the amount of protein present in the caecum.

This study has a number of important implications for the toxicity and carcinogenicity of saccharin. The

Chem. Toxic. Vol. 22, no. 2

disposition of protein and the fate of tryptophan in the gut are of significance since each has been impli- cated in the aetiology of bladder cancer. Indole is a more potent co-carcinogen for the rat bladder than is tryptophan, and the demonstrated increases in the formation of indole and in the renal excretion of its metabolites may be assumed to contribute to the selection of the bladder as the target organ for the effects induced by high levels of saccharin.

A paper presented by Dr Renwick to a Scientific Review Panel that met at Duke University Medical Center last May reported an extension of these animal studies and also gave a brief preliminary account of a human study in which saccharin taken in capsules with meals in divided doses totalling 1 g/day for 4wk was not found to cause any significant change in indican excretion. These data indicate that the profound biochemical and phys- iological changes produced by high doses of saccha- rin in test animals would not occur in man under normal patterns of saccharin usage. This report and the other papers presented to the Scientific Review Panel are scheduled to be published in Food and Chemical Toxicology next year.

CHROMOSOMAL ABNORMALITIES FROM FLAVOURING AGENTS

Surprising results have been produced by a recent Japanese study (Kasamaki et al. Mutation Res. 1982, 105, 387) on the genotoxicity of nine widely used flavouring agents, namely allyl isothiocyanate, anisaldehyde, benzaldehyde, trans-cinnamaldehyde, citronellal, heliotropin, ct-ionone, methyl anthra- nilate and vanillin.

Standard Ames tests on these flavourings using strains TA98 and TA100 of Salmonella typhimurium, with or without rat-liver S-9, produced uniformly negative results, although the positive controls gave positive results. Examination of the ability of these flavourings to induce chromosomal aberrations in Chinese hamster cells produced the surprises. With the exception of vanillin, the flavourings were all associated with dose-related and significant increases in the incidence of chromosomal abnormalities, whether or not metabolic activation was used. Vari- ous types of aberrations were observed, including chromatid and chromosome breaks and ring or di- centric chromosomes, as well as more extreme alter- ations, such as chromosomal pulverization and in- crease in chromosome number. The latter was particularly notable in cells treated with allyl iso- thiocyanate and trans-cinnamaldehyde.

At the concentrations at which maximal frequency of aberrations was observed without visible cyto- toxicity, that is 5 nr~ for allyl isothiocyanate and 10 nM for trans-cinnamaldehyde, the total incidences of abnormal cells were 19.4 and 26~o, respectively. Concentrations of other flavourings at which similar maximal frequencies of aberrations were observed, 25 nM for c~-ionone and 50 nM for anisaldehyde, ben- zaldehyde, citronellal, heliotropin and methyl anthra- nilate, were associated with abnormal-cell incidences of 8.8 21')/O. The incidence of abnormal cells among

those treated with 20 nM vanillin was 6.2~o while the incidence in negative controls was 3.1-3.7~.

[Evidence of mutagenicity in chemicals widely used as food additives does give cause for concern. Allyl isothiocyanate has been shown to possess carcino- genic activity in a 2-yr rat study (Food Chemical News 1981, 23 (16), 59) and thus a cloud of doubt already overshadows this food additive. As far as the present results are concerned, a number of problems must be resolved. The most obvious one is the contrast in the results of the two tests--the Ames test gave negative results despite the fact that the highest concentrations used appear to be considerably higher than those giving positive results in the chromosome aberration test. Interpretation of the results is further compli- cated by incomplete reporting of the Ames data (the background frequencies of revertants are not given and there is no indication of whether or not the range of concentrations tested included toxic treatments), and by the apparent lack of positive controls in the chromosome test. Kasamaki et al. (loc. cit.) intend to continue their work with a study of the growth characteristics of cells treated with trans- cinnamaldehyde and allyl isothiocyanate. A more pressing need would appear to be additional testing of the flavouring agents using a battery of short-term tests. Although there are several reports in the litera- ture on other mutagenicity tests of these flavourings, not all the evidence agrees for any given fiavouring. While vanillin appears to have given uniformly nega- tive results in short-term tests, trans-cinnamaldehyde, for example, has given negative results in two pre- viously reported Ames tests and in a reversion test with Escherichia coli WP2 uvrA (Sasaki & Endo, Mutation Res. 1978, 54, 251; Sekizawa & Shibamoto ibid 1982, 101, 127) but has given positive results in