Cancer Letters, 22 (1984) 275-280 Elsevier Scientific Publishers Ireland Ltd.

275

URINARY MUTAGENS IN HUMANS AFTER FRIED PORK AND BACON MEALS

PIER0 DOLARA, GIOVANNA CADERNI, MADDALENA SALVADORI, LUCIA TRINGALE and MAURA LODOVICI

Institute of Pharmacology and Toxicology, University of Florence, Viale Morgagni, 65, 50134 Florence (Italy)

(Received 16 May 1983) (Revised version received 20 January 1984) (Accepted 31 January 1984)

SUMMARY

Urinary mutagenic activity on Salmonella typhimurium strain TA1538 with S9 was determined after morning meals of fried pork and bacon. Both in fasting and non-fasting subjects a very marginal elevation of urinary mutagenic activity was observed, accounting for a small fraction only of the total amount of mutagens ingested.

INTRODUCTION

A recent study by Baker et al. reports the occurrence of high mutagenic activity with Salmonella typhimurium TA1538 in the presence of S9 by testing human urine after ingestion of fried pork or bacon. The urinary muta- genic activity accounted for about 30% of the total content of mutagens in the ingested meat [ 21.

In a previous study in rats we observed [4] that about 2.5% of beef extract mutagens were recovered in the urine after its systemic administration p.0. or i.p.

Moreover, in studies on human populations previously carried out in our laboratory [ 51 we observed very low values of mutagenic activity with strain TA1538 and S9 in non-smoking subjects, although the great majority of them were on a diet comprising roasted or fried meats. We decided, there- fore, to carry out urine analyses with the protocol of Baker et al. [2] to ascertain whether we could reproduce their results.

MATERIALS AND METHODS

We basically followed the procedure of Baker et al. [2]. Bacon and pork

0304-3835/84/$03.00 o 1984 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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were purchased in a local meat shop and cooked in a frying pan with a surface temperature of about 200°C. The mutagens were extracted after cooking with the procedure of Felton et al. [7] and tested with Salmonella typhimurium strain TA1538 with S9 [l] . Urine samples were collected in polyethylene containers, frozen for one day at -20°C and analyzed the next day with the method of Yamasaki and Ames [ 91. Urine was processed ac- cording to this procedure and its dry residual was re-suspended in 0.4 ml of DMSO for each 100 ml of urine. Aliquots of 20,50 and 100 ~1 of the final suspension were tested in duplicate, with strain TA1538 in the presence of PCB-induced rat liver S9.

In some experiments, the day before the test, meat was excluded from the diet. But no fasting was observed during the experiment itself (this is termed non-fasting experiment in the results section). In some others, meat was excluded from the diet the day before, and only liquids were consumed during the experiment (milk, water and fruit juices) (this is termed semi- fasting experiment). In one experiment a 24-h fast, in which only liquids were consumed, preceded and followed the pork morning meal (termed total fasting experiment).

After collecting the night urine the meat was eaten for breakfast at a dose of 2 g fresh wt/kg by academic people working in the Institute. Four urine samples were collected afterwards at various intervals.

The hourly baseline mutagen excretion was calculated by dividing the total excretion during the night (mutagenic activity/ml X voided volume) by the number of hours of the night collection (8 h). By multiplying this value of hourly baseline by the number of hours of the collection period after the morning meal (8 in two cases and 11 in all others), we obtained an estimate of the excretion of mutagens not related to the meat meal. This last value was subtracted from the sum of the total mutagenic activity of the samples collected after the meal (C: (revertants,/ml) X volume,), and is indicated in the results as total meat-related mutagenic activity.

RESULTS

We followed urinary mutagenic activity after a pork or bacon morning meal in conditions of non-fasting and semi-fasting (Fig. 1) and in one case, with the total fasting protocol (Fig. 2). It can be observed that there was a moderate elevation of mutagenic activity in some urine samples after the morning meal, although of an order of magnitude lower than reported by Baker et al. [2]. Since variations in the nature of diet before and after the meat morning meal did not seem to have an effect on the excretion of mutagens, we obtained additional data on different subjects, using the non- fasting protocol. The results are shown in Fig. 3, in which we report the values of urinary mutagenic activity/25 ml in the first sample collected after the morning meal, that was often the highest. In Fig. 4 we calculated the overall excretion of mutagens after the meal, after detracting the hourly

277

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mutagenic activity background, as described in Methods. The variability was high, but most samples showed a modest or null elevation of mutagenic activity over the background, with the exclusion of sample DI, that was clearly out of range. The mean meat-related mutagenic activity was 107 f 30 or 201 * 98 (S.E.) excluding or including sample D1, respectively.

We then obtained an estimate of the total intake of mutagens with the meat meal, by measuring the mutagenic activity of meat samples from the same batch used in the experiments, after cooking them in the same ap- pliance for the same time and at the same temperature. The results are shown in Fig. 5. From the data of Figs. 4 and 5 we calculated that the mutagens recovered in the urine after the meal were about 0.6% of the ingested dose.

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Fig. 5. Mutagenic activity of fried pork samples cooked at 200°C for 7 min. Strain TA1538 with S9. Means 2 S.E. (n = 10).

DISCUSSION

Our results qualitatively confirm the increase of urinary mutagenic activity after a fried meat meal described by Baker et al. [ 21. However, the overall excretion of mutagens in the urine was much lower in our study, and ac- counted for only a small percentage of the ingested dose.

These discrepancies are hard to explain, unless one assumes that the mutagenic activity of the meat used in the experiments of Baker et al. was actually higher than that reported. It is, in fact, relatively easy to have a low recovery in the laborious extraction procedure, and equally easy to increase mutagenic activity by using even a slightly higher temperature in the cooking. It has, for example, been demonstrated that cooking tempiratures increase exponentially the production of mutagens [ 3,8] . In our study the meat was cooked for 7 min at 2OO”C, and was medium well- done, but without charred parts.

The results of the present study agree with what we reported when using beef extract in rats [6] and seem to indicate that a very small fraction of the meat-related mutagens can be recovered in body fluids. The reasons for this phenomenon are not clear, but poor absorption, inactivation, or binding to cellular structures are probable causal mechanisms. We presented some evidence of the last two mechanisms by studying one of the mutagens of this group (2-amino-3-methyl[4,5$]imidazoquinoline, or IQ) that seems to be partially inactivated and bound to components of the intestinal mucosa

E61. Whatever the cause may be, the urinary mutagenic activity related to the

exposure to meat mutagens is marginal, and much lower than that reported

280

after exposure to cigarette smoke or mutagenic chemicals in the work environment [4]. If meat mutagens have a genetic effect in humans this effect should not occur in organs and tissues removed from the digestive tract.

ACKNOWLEDGEMENT

This work was supported by a Grant from C.N.R. “Control Of Toxic Risk.”

REFERENCES

1 Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the SaZmoneZZa/mammalian microsome mutagenicity test. Mutat. Res., 31, 347-364.

2 Baker, R., Arlauskas, A., Bonin, A. and Angus, D. (1982) Detection of mutagenic activity in human urine following fried pork and bacon meals. Cancer Letters, 16, 81-89.

3 Dolara, P., Commoner, B., Vithaiathil, A., Cuca, G., Tuley, E., Madyastha, P. and Nair, S. (1979) The effect of temperature on the formation of mutagens in heated beef stock and cooked ground beef, Mutat. Res., 60, 231-237.

4 Dolara, P., Barale, R., Mazzoli, S. and Benetti, D. (1980) Activation of the mutagens of beef extract in vivo and in vitro. Mutat. Res., 79,213-221.

5 Dolara, P., Mazzoli, S., Rosi, D., Buiatti, E., Baccetti, S., Turchi, A. and Vanucci, V. (198 1) Exposure to carcinogenic chemicals and smoking increases urinary excretion of mutagens in humans. J. Toxicol. Environ. Health, 8,95-103.

6 Caderni, G., Lodovici, M. and Dolara, P. (1983) Inhibition of the mutagenic activity of 2-amino-3-methyl-imidazo (4,5-f) quinoline by rat intestinal mucosa. In: Extra- hepatic Drug Metabolism and Chemical Carcinogenesis, pp. 309-310. Editors: S. Rydstrom, J. Montelius and M. Bengtsson. Elsevier, New York, Amsterdam.

7 Felton, J.S., Healy, S., Stuermer, D., Berry, C., Timourian, H. and Hatch, F.T. (1981) Mutagens from the cooking of food. I. Improved extraction of mutagenic fractions from cooked ground beef. Mutat. Res., 88, 33-44.

8 Lin. J.Y., Lee, H. and Huang, H.I. (1982) Formation of mutagens in boiled pork extract. Food Chem. Toxicol., 20,531-533.

9 Yamasaki. E. and Ames, B.N. (1977) Concentration of mutagens from urine by absorption with the non-polar resin XAD-2: Cigarette smokers have mutagenic urine. Proc. Natl. Acad. Sci. U.S.A., 74,3555-3559.