678 Information section Fd Chem. Toxic. Vol. 21, no. 5

lease that results from caffeine exposure is likely to be more relevant than that resulting from sucrose ex- posure, since caffeine exposure enhanced sensitivity within the plasma-glucose range observed in the neonate, whereas the effects of sucrose were evident only at higher glucose concentrations. In contrast, the biosynthetic responsiveness of the neonatal pancreas to glucose was not enhanced by caffeine ingestion by the mother. Therefore, the progressive depletion of pancreatic insulin stores and the hypoinsulinaemia observed in the offspring of caffeine-treated rats may be explained by the enhanced sensitivity of insulin release to glucose without a change in the bio- synthetic proinsulin process.

The authors consider that the high concentration of amino acids used in the incubation medium, which

was chosen to replicate the physiological conditions of the neonate, may have produced an enhanced sensitivity of insulin release to glucose in all groups in the study, compared to other studies. (The half- maximal response for glucose-stimulated insulin re- lease was 4.1 mmol/litre in the control group in comparison with 7 8 mmol/litre quoted for other studies.) The mechanism of the caffeine effect was not determined in this study, but the authors comment that it may be due to direct effects on the pancreas by inhibition of cyclic adenosine 5'-monophosphate (AMP) phosphodiesterase causing cyclic AMP el- evation or by cyclic AMP effects on calcium mobil- ization. Alternatively, caffeine may affect maternal tissue lipolysis and thereby indirectly affect the foetus or neonate by altering its food supply.

THEOBROMINE

Theobromine (3,7-dimethylxanthine) is struc- turally related to caffeine (1,3,7-trimethylxanthine), occurs naturally in a number of plants used in the manufacture of food products and is present in cocoa-based beverages, tea and chocolate. It has been used as a diuretic, as a cardiac stimulant and for the dilation of arteries (Timson, Mutation Res. 1975, 32, 169), although its diuretic effect and its effect on sleep are reported to be much weaker than those due to caffeine (Dorfman & Jarvik, Clin. Pharmac. Ther. 1970, 11, 869). Theobromine has been shown to induce testicular atrophy in rats (Cited in F.C.T. 1981, 19, 790), while in man large doses may cause nausea and anorexia ( Martindale The Extra Phar- macopoeia, Edited by N. W. Blacow, p. 360; The Pharmaceutical Press, London, 1972) and gastric irritation (Dorfman & Jarvik, loc. cir).

In an early metabolic study, Cornish & Christman (J. biol. Chem. 1957, 228, 315) investigated the uri- nary excretion of single 1-g doses of theobromine given to two individuals and found that about 62~o of the dose appeared as metabolites in the urine within 48 hr of ingestion, with 28-301Yo of the dose appearing as 7-methylxanthine, 14-21To as 3-methylxanthine, 11-12% as unchanged theobro- mine and 3~','/o as 7-methyluric acid. The authors suggest that demethylation of theobromine occurs more readily at the 3 position than at the 7 position, since more 7- than 3-methylxanthine appeared in the urine. They also speculate that the small amount of 7-methyluric acid was formed by the oxidation of 7-methylxanthine or by demethylation of 3,7-di- methyluric acid formed by the direct oxidation of theobromine (although no 3,7-dimethyluric acid was detected in the urine). Drouillard et al. (Clin. Phar- mac. Ther. 1978, 23, 296) found that the mean metabolic clearance rate of a single oral dose of 6 mg theobromine/kg, given to male volunteers on methyl- xanthine-free diets, was 61.6 ml/min (about 0,93 ml/ min/kg) and reported that this clearance rate was reduced to a mean of 52.7 ml/min (about 0.80 ml/ min/kg) after these men had received a further four doses of 6 mg theobromine/kg on the next 4 days.

METABOLISM

This impairment of theobromine clearance was re- versible within 4 days of the last of the five con- secutive daily doses. Each dose given was equivalent to the theobromine content of five or six cups of hot chocolate, assuming 65 mg theobromine/5 oz. serving (Zoumas et al. J. Fd Sei. 1980, 45, 314).

Arnaud & Welsch (J. agric. Fd Chem. 1979, 27, 524) administered [7-methyl-UC]theobromine to rats by gavage and found that almost all of the dose was absorbed. Only 1~o of the theobromine was collected unchanged in the faeces in the 24 hr after adminis- tration. Only 6~o of the administered radioactivity was expired as HCO2 within 24hr, showing that demethylation at position 7 is not a qualitatively important route of theobromine metabolism in the rat. The faeces collected within 24 hr of dosing con- tained 11 ~o of the administered radioactivity; labelled faecal metabolites included 3,7-dimethyluric acid and very small amounts of a dimethylallantoin and N- methylurea. The bulk of the radioactivity was recov- ered in the urine (840/o in 24 hr) while less than 3~o remained in the body after 24 hr. labelled compounds appearing in the urine within 24hr included unchanged theobromine (49~o of the administered radioactivity), 4-amino-5-(N-methylformylamino)-3-

o / methyluracil (36~o), 7-methylxanthine (6,o), 7-methyluric acid (3.9~) and 3,7-dimethyluric acid (2.7~). The uracil derivative was also found in the urine of a human volunteer fed 1 g theobromine.

These authors concluded that metabolism of theo- bromine to 3- and 7-methylxanthines (and sub- sequently to 3- and 7-methyluric acids) was less important in the rat than in man (as indicated by the study of Cornish & Christman, loc. cir). The uracil derivative was assumed to result, via an unstable (and undetected) intermediate, from the direct hydration of theobromine. The direct oxidation of theobromine to 3,7-dimethyluric acid (found in the faeces and urine of the rats and in the urine of the human volunteer) was considered to be a minor metabolic pathway in both species.

Recently, Miners et al. (Drug Metab. Dispos. 1982, 10, 672) have investigated the role of the xanthine

Information section--Fd Chem. Toxic. Vol. 21, no. 5 679

oxidase system in theobromine metabolism in four male nonsmokers. Subjects were given 200 mg theo- bromine orally (in capsules) every 8 hr throughout two 4-day periods 2 wk apart. Two subjects also took 100 mg allopurinol (an inhibitor of xanthine oxidase) every 8 hr for 7 days before and during the first 4-day theobromine-treatment period, and the other two subjects received this allopurinol treatment during the second 4-day theobromine-treatment period. Pre- dose blood samples showed a constant theobromine concentration by day 2 of each 4-day period. Plasma samples collected on the last day of each 4-day period (at intervals over the 8 hr after the first theobromine dose of the day) showed that plasma theobromine clearance (0.746 ml/min/kg in men taking theobro- mine only) was unaffected by allopurinol treatment (0.744ml/min/kg). Urine analyses showed that urinary excretions of unchanged theobromine, 4- amino-5-(N-methylformylamino)-3-methyluracil and 3-methylxanthine were 14, 8 and 22~, respectively, of the recovered theobromine-derived metabolites when men took theobromine only. These percentages did not differ significantly from those calculated for the men taking allopurinol also. However, when the men took theobromine only, they excreted 44~ of the recovered dose as 7-methylxanthine and 11~o as 7-methyluric acid; when they took allopurinol as well,

no 7-methyluric acid was found in their urine but their excretion of 7-methylxanthine was significantly higher (59~o of the recovered dose). Nevertheless, the combined metabolic clearance of theobromine to 7-methylxanthine plus 7-methyluric acid was un- changed by allopurinol. Furthermore, the total uri- nary excretion of theobromine and its metabolites was unaffected by allopurinol and represented about 90-93~o of the administered dose. No 3,7-dimethyl- uric acid or 3-methyluric acid was found in the urine of any subject.

Miners et al. (loc. tit.) consider that the observed mean plasma clearances of theobromine are similar to those seen by Drouillard et al. (loc. cir.). Unlike the earlier study, however, their work provides no evi- dence of reduced theobromine clearance developing over the course of repeated doses. The general lack of effect of allopurinol treatment is thought to indicate that theobromine metabolism is dependent on en- zyme systems other than xanthine oxidase, probably the heptatic mixed-function oxidases. However, the effect of allopurinol on the recovery of 7-methylxanthine and 7-methyluric acid in the urine indicates that the latter is formed by the xanthine oxidase-catalysed oxidation of 7-methylxanthine, rather than via the demethylation of 3,7-dimethyluric acid.

MUTAGENIC VINTAGE?

As part of a general survey of the mutagenic potential of a wide variety of food products, the Canadian Food Directorate has tested a range of beverages (Stoltz et al. Envir. Mutagen. 1982, 4, 477). The 28 beverages selected for study were from the following 13 general categories: tap-water, bottled carbonated soft drinks, powdered soft drinks, frozen concentrated fruit juices, canned or bottled fruit juices, vegetable juices, dairy products, tea, coffee, beer and ale, wine, spirits and liqueurs. Sample treatment by evaporation or extraction resulted in seven fractions of each beverage. These fractions were subjected to initial mutagenicity screening (stage I) by the Salmonella/microsome plate incorporation test using Salmonella typhimurium strains TA98 and TAI00 with and without S-9 mix. Fractions were retested at the same or a lower concentration (stage II) if they showed evidence either of mutagenicity (i.e. if the mean number of revertants per plate exceeded 1.5 times that of the solvent control) or of toxicity (visually or by a mean count per plate no more than 0.67 times the control count). The stage II results were assessed statistically for mutagenic potential.

Samples of grape juice, red wine and instant coffee produced positive results in several fractions and concentrations at stage II. Results were strongly positive with grape juice and red wine and moderately positive with instant coffee. High-fat milk, a choco- late drink, light beer and a white wine demonstrated some mutagenic potential, but this was not confirmed in a second sample. The remaining beverages showed no marked mutagenic activity when tested at stage II.

The fractionation procedure and screening strategy were validated by spiking with known mutagens. However, the potential limitations of this study are numerous. For many of the products only one sample was tested and there was no simulated digestive process prior to the extraction. Partition of a mu- tagenic compound between fractions, loss of volatile compounds during concentration and of labile com- pounds during storage, the use of only two Sal- monella strains and the absence of any dose-response assessment are all recognized as reducing the sensi- tivity of the method. Nevertheless a low false-positive rate for non-mutagens and moderate false-negative rates for strong mutagens are claimed. Histidine, which could interfere with the results of the Sal- monella assay, was not removed from the test sam- ples, but analysis of the fractions did not indicate that histidine contributed to the positive results obtained with red wine.

This study, the initial phase of a long-term project, was used primarily to establish the test strategy to be used for more extensive testing of multiple samples and brands of products. While no firm conclusions can be drawn from the initial screening studies, some parallels with earlier reports on the beverages are indicated and the authors speculate on the possible involvement of certain specific components in the positive results obtained. Development of the latter aspect is anticipated in the final phase of the project which is scheduled to include the chemical character- ization of the active components of products confirmed as showing mutagenic potential.