Papain—Allergenic tendencies

Information section Fd Chem. Toxic. Vol. 22, no. 6 of this difficulty, both of these studies have managed to achieve an approximately similar bottom line, adding two more citations to the already crowded column marked 'equivocal positive result'. The allimportant link between exposure and degree of risk remains elusive. Case-control studies must be interpreted with caution. Memory in normal circumstances is known to be imperfect. A patient who has recently been made aware of the presence of a potentially life-threatening disease is unlikely to be in the same emotional state as an apparently healthy individual. Therefore, even when cases and controls are being asked about an uncontentious detail of their lifestyle, there is a danger of marked differences in the accuracy of recall. With a widely criticized habit, such as cigarette smoking, there is scope for a more conscious distortion of the truth on the part of the interviewee. The validity of the findings from any of the coffee studies would be almost as seriously undermined by systematic errors in the cigarette history as by inaccuracies in the record of coffee exposure. With complications such as these, the difficulty of generating a neat conclusion from a series of case-control studies is hardly surprising. Taken as a whole, particularly now that the whole includes this large study of Hartge et al. (loc. tit.), the data suggest that coffee drinkers are marginally more likely to develop bladder cancer than are abstainers. However, because the search for evidence of a dose response relationship is seldom successful, the causal link--that coffee drinking is responsible for the increased number of bladder cancers--must still be considered unsupported by the epidemiology. The ambiguity of the epidemiology is mirrored experimentally. Coffee itself, although fairly poorly tested in animal carcinogenicity studies, is at present unconvicted as a carcinogen (Wfirzner et al. Fd Cosmet. Toxicol. 1977, 15, 289), a status shared by caffeine (Johansson, Int. J. Cancer 1981, 27, 521).

483

Coffee, nevertheless, has been shown to be a direct mutagen in Ames tests (Aeschbacher et al. Fd Cosmet. Toxieol. 1980, 18, 605) and caffeine is mutagenic in a broad spectrum of in vitro systems but not apparently in the living mammal (Tarka, C R C Crit. Rev. Toxicol 1982, 9, 275). Where does the seeker-after-truth go from here? Hartge et al. (loc. cit.) felt that future epidemiology should concentrate on populations either with a low prevalence of coffee drinking, such as the Mormons, or with a very high consumption of coffee, as is the case in Scandinavia. Retrospective case-control studies of even an atypical community seem, however, unlikely to unravel the mystery. Prospective cohort studies, in which the health (and death) of groups with defined coffee exposures are monitored over a number of years, have surely earned by now a higher priority. Exposures, whether to coffee, tobacco or other risk factors, can be ascertained more accurately than is possible in any retrospective study. There is even scope for independent verification of the information offered by the direct participants. All of those involved, moreover, are questioned at a time of similar healthy optimism. On the other side of the balance sheet, with new cases of bladder cancer occurring in a typical, ageing, male population at an annual rate of about 100/100,000 (Waterhouse et al. (Eds), 1 A R C Scient. Publ. no. 42, p. 207, IARC, Lyon, 1982), a cohort study would realistically need to involve something of the order of 10,000 people and to continue for close on a decade if the epidemiologists are to have sufficient data (that is cancer cases) to manipulate. The many published case-control studies eliminate the possibility that coffee is anything other than, at worst, a weak bladder carcinogen. The leisurely (but more effective) cohort approach would seem to hold the best prospects of differentiating further between the remaining two credible verdicts. J. Hopkins--BIBRA

P A P A I N - - A L L E R G E N I C TENDENCIES Papain, a proteolytic enzyme obtained from unripe papaya fruit, has many industrial applications and, in particular, is used as an agent for tenderizing meat and clarifying beer. It is also a common ingredient in cosmetic, food and drug products. There have been several publications describing allergic reactions (expressed mainly as respiratory illness) which have been attributed to papain exposure (Cited in F.C.T. 1981, 19, 791; Baur et al. Clin Allergy 1982, 12, 9). However, despite the potentially considerable exposure of the general public to this enzyme, reports of papain allergy outside the workplace are rare. A recent paper by Mansfield & Bowers (J. Allergy clin. Immun. 1983, 71, 371) helps to fill this particular gap and is especially interesting since ingestion, rather than inhalation, was the chief route of exposure involved. The patient described by Mansfield & Bowers (Ioc. cit.), a 31-yr-old man, had a history of allergic reactions, including childhood asthma, but suffered a particularly severe response following the ingestion of

a beefsteak that had been liberally treated with a meat tenderizer containing an unspecified amount of papain. Within 20-30min of his meal, the patient experienced generalized itching, swelling of hands, feet, lips and eyelids, tightness of chest and throat, and wheezing. Initial laboratory data indicated a serum IgE level of 6000 IU/ml, but no IgG antibody was detected. The food and drink taken during the meal, other than the tenderized steak, had been previously tolerated without any recognized problems. Severe symptoms had occurred on two previous occasions, thought to be following the ingestion of large amounts of German draft beer, but had not occurred when different brands of meat tenderizer (at least one of which was known not to contain papain) had been used. In an attempt to determine whether papain was the causative agent, direct skin testing was carried out on the patient with the offending brand of tenderizer (0.1 g/ml) and with pure papain (0.1, 1.0 or

484

Information section--Fd Chem. Toxic. Vol. 22, no. 6

10mg/ml). All of these tests were positive. In a further test, using serum obtained from the patient, 0.1 ml heated serum (56C for 2 hr, to render serum IgE non-active) or unheated serum was intracutaneously injected into different skin sites of a non-allergic volunteer. After 48 hr, these sites were challenged with a nonqrritant concentration of papain (0.02 ml of 0.1 mg papain/ml), whereupon a large whealing response was observed at the unheated-serum site, with only minimal reactions at the untreated site and heated-serum site. In a third test, ingestion of a hamburger containing 10rag papain provoked an allergic response in the patient

whereas hamburgers that were papain-free caused no adverse reaction. Pretreatment with cromolyn, an antiqnflammatory agent, appeared to block the patient's adverse response to papain ingestion. The authors conclude that the severe systemic allergic reaction observed in this patient was mediated by IgE antibody to papain. They also postulate that papain may be a significant unrecognized cause of allergic symptoms in the general public, particularly since papain exposure is probably increasing due to the growing popularity of natural health food supplements and meat tenderizers.

RUTIN CAUSES ORAL C A N C E R - - H A R D TO SWALLOW'? The flavonoid quercetin and its glycoside rutin are commonly found in tobacco leaves and in a wide variety of edible plant products. While orally ingested rutin has been shown to be non-carcinogenic in rats, quercetin has been found to be carcinogenic in one strain of rats but not in hamsters, mice or other strains of rats (Cited in F.C.T. 1982, 20, 976; IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans 1983, 31, 213). The possibility that rutin may be hydrolysed in the gut to the genotoxic and potentially carcinogenic quercetin had caused some concern (Food Chemical News 1981, 22 (44), 45; ihid 1981, 23 (10), 34: Cited in F.C.T. 1982, 211, 976) but now it may not be necessary to look further than the tips of our tongues to find the site of hydrolysis. Parisis & Pritchard (Archs oral Biol. 1983, 28, 583) have reported that oral streptococci isolated from human subjects can perform this reaction in vitro. They speculate, furthermore, that the strong association of excessive alcohol consumption and tobacco-chewing/snuff-dipping with intra-oral cancer may be linked, by this mechanism, to the presence of quercetin glycosides in alcoholic beverages and tobacco leaves. Plaque and soft-tissue scrapings from the mouths of two healthy individuals were cultured anaerobically in broth, containing tryptone yeast extract and resazurin medium, and on an agar plate, both media containing 0.4,~0 rutin. The dominant organism was isolated from these cultures on blood agar and the bright yellow sediment formed by the isolates in the liquid and solid rutin-containing media was identified as quercetin by thin-layer chromatography and spectrophotometry, The isolated strain was identified as Streptococcus milleri, though not necessarily all strains of that species are capable of hydrolysing rutin and other rutinolytic oral microorganisms may, of course, exist. In order to study the enzyme system responsible for rutinolysis, cell-free extracts (CFEs) were prepared from anaerobic bulk cultures of the isolated streptococci grown in tryptone-yeast extract medium with or without 0.025'~; rutin. Cells from both cultures were disrupted and CFEs were prepared by centrifugation (10,000 g). Enzyme activity in the CFEs was determined by using the Ames test in Sahnonella typhimurium TA98 as a bioassay. Rutin was

effectively treated as the test compound while the CFEs replaced the S-9 liver homogenate component of the standard Ames test: quercetin was the positive control. CFEs from both cultures converted rutin to a mutagen and mutagenic activity was directly related to protein concentration in the CFEs and to rutin concentration. The CFE from the rutin-grown isolates produced significantly higher mutagenicity at any given protein concentration than that t¥om the culture without rutin, indicating that the rutinolytic enzyme is partially inducible. When a CFE was further centrifuged at 100,000 g the entire activity was recovered in the supernatant, indicating that the enzyme is present in the cell cytosol or, alternatively, is loosely associated with the cell membrane. The enzyme was active over the pH range 5.5 7.0: the bright yellow precipitate formed at optimum pH (6.5) was again shown to be quercetin. Although activity of the enzyme preparation was lost by incubation for 3 min at pH 4.5, it was restored by increasing the pH. The enzyme retained full activity after dialysis, showing the absence of any dialysable co-factor. Failure to produce quercetin on rutin-agar under aerobic conditions may indicate that the enzyme is synthesized at reduced oxygen tensions; however, a similar examination of activity in an aerobic broth culture was 11ot made. CFEs of broth cultures containing isolates and quercitrin, another glycoside of quercetin, were tested in Ames assays as beR)re to determine the extract's ability to activate quercitrin. No changes were observed in the quercitrin cultures and quercitrin was not activated by the CEE to a mutagem which indicates that the enzyme hydrolysing rutm is a fi-D-glycosidic enzyme, not an ~-rhamnosidase~ fl-glucosidase system. The authors propose that the enzyme should be called rutinase by analogy to a rutinolytic glycosidase produced by Asper~,illa.s .flavus; these rutinolytic enzymes differ from fi-glucosidase, which hydrolyses only terminal glucose units, in their ability to hydrolyse internal glucose units. Parisis & Pritchard (lot. oil.) conclude that the pH range of rutinase activity is perfectly suited for oral functioning if the enzyme is liberated through autolysis. This is serious news indeed considering the ubiquity of rutin in edible plant products: the authors literally provide further lk)od for thought by sug-