Catechol mutagenicity and co-mutagenicity

Information section--Fd Chem. Toxic. Vol. 22, No. 9 in TA100 and the others were not tested in that strain). The methanol extract of the one string-bean cultivar (cv Romore) harvested in 1979 gave positive results in TA98 in the presence of GFE, but not without GFE or in the presence of S-9 mix. No mutagenic activity was observed in TA100 with or without G F E or S-9 mix. However when intercultivar variation was studied using beans harvested the following year, methanol extracts of all four cultivars (including Romore) gave positive results in both TA98 and TA100 in the presence of GFE. No mutagenicity was observed in the other bean extracts or under other conditions. It was noted that the later crop of beans had small areas of necrosis, which were thought to be related to the unfavourable weather conditions. However, these beans were considered to be fit for human consumption and were similar to others being sold that year. Although only two cultivars of paprika were tested, one ripe (red) and one unripe (green) sample of one of the cultivars were tested. Methanol extracts of all three types of paprika tested were mutagenic in TA98 with GFE. No mutagenic activity was observed in the other paprika extracts or under any of the other conditions used. The methanol extracts of two of the cultivars of rhubarb were also mutagenic only in TA98 with GFE. The other two cultivars showed no activity in TA98 or TAI00 under any condition. However, because rhubarb may contain anthraquinones, some of which are mutagenic to TA1537, the extracts of all four cultivars were tested again in this strain, The combined petroleum ether and chloroform extracts of all four cultivars were found to be mutagenic in the presence of S-9 mix. None of the extracts of Brussels sprouts or spinach gave positive results in TA98 or TA 100 under any of the conditions used. For each of the four vegetables that were mutagenic to Salmonella there were considerable differences between cultivars in the level of response. For example, lettuce cv Renate induced seven times

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more revertants than did cv Ravel. The green paprika was about 2.5 times more active than the red. The methanol extracts of one or more cultivars of each of the vegetables that gave a positive response were subjected to the procedure for histidine removal and it was found that in each case at least 75~ of the mutagenic activity was concentrated in the acidic organic fraction obtained after removal of the histidine. For lettuce (cv Romore), string beans (cv Renate) and rhubarb (cv Paragon), an attempt was made to identify the mutagenic compounds. The results indicated that the mutagenicity of lettuce and string beans could be accounted for by the presence of quercetin glycosides, whereas that of rhubarb appeared to be due to the anthraquinone emodin. Quercelin has given positive results in previous bacterial mutagenicity assays and in mammalian cell transformation tests in vitro (Cited in F.C.T. 1982, 20, 976) and was identified as the mutagenic substance in sumac (ibid 1979, 17, 688). It has been reported that quercetin is carcinogenic for the intestinal and urinary-bladder epithelium in rats, but this has not been shown to be the case in other long-term feeding studies in rats, mice or golden hamsters (ibid 1982, 20, 976; ibid 1984, 22, 484). Emodin has previously been shown to be mutagenic to several strains of S. typhimurium in the presence of S-9 mix (Brown & Brown, Mutation Res. 1976, 40, 203; Levin et al. ibid 1982, 94, 1315). van der Hoeven et al. (loc. cit.) suggest that the observed differences in mutagenicity of cultivars grown under the same conditions were related to the intrinsic mutagenic properties of the vegetables. The results of the tests on string beans may indicate the involvement of the mutagenic properties of phytoalexins, chemicals that can be produced in the plants upon fungal infection or under other stress conditions. The extracts from bean cultivars showing necrotic patches were mutagenic in both TA98 and TA100, whereas extracts of the undamaged cultivar were not.

CATECHOL M U T A G E N I C I T Y AND CO-MUTAGENICITY Catechol is used as an antioxidant in a wide range of industries. It also occurs in tobacco smoke, in which it is the most abundant phenolic entity (Mold et al. Analyst, Lond. 1966, 91, 189). Compounds structurally related to catechol occur widely in the diet and in tea and coffee (Yoshida & Fukuhara, Mutation Res. 1983, 120, 7). In skin-painting studies in mice, catechol has proved to be a potent cocarcinogen when applied with benzo[a]pyrene (BaP; Van Duuren et al. J. natn. Cancer Inst. 1973, 51, 703; Van Duuren & Goldschmidt, ibid 1976, 56, 1237), but did not show tumour-promoting activity in two-stage skin-painting studies with BaP (Van Duuren & Goldschmidt, loe. cit.) or in 15-wk skin-painting studies with dimethylbenzanthracene (Boutwell & Bosch, Cancer Res. 1959, 19, 413). Catechol was not mutagenic in Salmonella typhimurium strain TA98 at up to 150#g/plate in the presence of S-9 (the tested

concentrations were toxic without S-9; Yoshikawa et al. Bull. Nat. Lab. Sci., Tokyo 1976 94, 28) but it delayed cell division and was a potent inducer of sister chromatid exchange in cultured human lymphocytes (Morimoto & Wolff, Cancer Res. 1980, 40, 1189). At 0.005 mg/ml, catechol induced chromatid breaks and exchanges in Chinese hamster ovary cells without activation, but these effects were suppressed by the addition of S-9 (Stich et al. Cancer Lett. 1981, 14, 251). Further studies of the mutagenicity of catechol, and its co-mutagenicity with BaP and 4-nitroquinoline-l-oxide (4NQO) have now been reported by Yoshida & Fukuhara (loc. cit,). Catechol was non-mutagenic for S. typhimurium strains TA98, TA 100 and TA 1537 when applied at up to I0 rag/plate in the presence or absence of S-9 mix. Its co-mutagenicity with BaP was investigated in conventional Ames tests in TA98 and TA100 by

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adding 0, 2, 4 or 6 mg catechol to 5/~g BaP in the presence of 10, 25, 50, 100 or 150/~l S-9 per plate. Similar results were obtained with both strains. At 10-25/~1 S-9/plate, the mutagenic response decreased with increasing amounts of added catechol, possibly due to inhibition by the antioxidant of the S-9 catalysed activation of BaP. At 50-100/~1 S-9/plate, catechol had little effect on the mutagenic activity of BaP. At 150~tl S-9/plate (the maximum possible in conventional Ames tests), mutagenic activity increased with the amount of catechol added, although this effect was not statistically significant. To examine the effect at higher levels of S-9, mixtures containing 5 # g BaP, 0, 1, 2, 3 or 4 mg catechol, and 1 ml of S-9 mix containing 20, 50, 100, 200 or 300 ~1 S-9 were incubated with TA98 or TAI00 at 37'~C for 1 hr prior to plating. The level of added catechol had no clear effect on the mutagenic response at 20 50/~1 S-9/ml incubation mix but at 100-300 #1 S-9/ml, activity was

markedly increased with increasing amounts of added catechol. [The mechanism remains obscure but could reflect increases in the degree of activation of BaP or selective changes in the mechanism of activation (e.g. by changing the ratio of secondary to primary activation).] In studies without S-9, increasing amounts of catechol (0-2 rag/plate) enhanced the mutagenicity of 4NQO. The investigators suggested that this finding may have reflected catechol-induced inhibition of the degradation of either 4NQO or active intermediates. ]Although catechol and BaP have been shown to be co-carcinogenic in chronic skin-painting studies, studies in Salmonella using standardized Ames procedures provided little evidence of co-mutagenicity. Co-mutagenicity was only clearly observed following the manipulation of standard Ames procedures. The study demonstrates the importance of a flexible approach to short-term screening assays.]

SPIRIT OF HEALTH . . . . ? A number of recent studies have indicated that moderate alcohol intake may protect against cardiovascular disease and have implicated both beer and wine in this effect (Cited in F.C.T. 1981, 19, 789; Kozararevic et al. Lancet 1980, I, 613; St. Leger et al. ibid 1979, I, 1017; Yano et al. New Engl. J. Med. 1977, 297, 405), although heavy alcohol consumption increases the risk of premature death (Peterson et al. Br. med. J. 1980, 280, 1403), In a recent study in Finland (Salonen et al. J. chron. Dis. 1983, 36, 533) spirits were also found to protect against cardiovascular disease. In this study men aged 30-59 yr in 1972 completed a health questionnaire which included questions about alcohol intake. The intake of spirits was quantified in terms of "frequency of use" (times per year) and beer consumption was recorded as the number of bottles drunk per week. (The alcohol content of Finnish beer does not vary much and the bottle size is standard.) Wine consumption was negligible and was not analysed separately. Regular drinkers who drank spirits at least once a week and/or at least five bottles of beer a week (a group covering both moderate and heavy drinkers) were compared with those who indulged less frequently. Serum 7-glutamyl transferase concentrations were measured in a subsample to check the validity of the reported intakes. Complete data on spirits and beer intake and coronary risk factors were available for 4063 men who had no acute myocardial infarction (AMI) or angina pectoris in the preceding 12 months. During the 7-yr follow-up, 209 subjects had an AMI, 31 had liver cirrhosis or acute pancreatitis and 223 died of any cause. Because of errors in the hospital discharge register, roughly 5~o of the "events" among subjects were lost to follow-up. For 189 men who had an AMI during follow-up, controls were matched one-to-one on the basis of serum cholesterol, tobacco usage, diastolic blood pressure and history of angina pectoris.

Of the 4063 men, 16~ drank at least five bottles of beer a week ('beer drinkers'), 13~ drank spirits at least once a week ('spirits drinkers') and 6 ~ did both. Serum triglyceride levels and mean diastolic blood pressure were significantly raised in regular drinkers of beer and/or spirits. The risk of AMI was significantly reduced in spirits drinkers who were not beer drinkers, even after adjusting for age, serum total cholesterol, triglycerides, diastolic blood pressure and smoking. No such association was observed in beer drinkers. For beer drinkers, the risk of death from any cause was significantly increased, but when adjusted for spirits intake, age, smoking, serum cholesterol, triglycerides and diastolic blood pressure, the association was no longer significant. The relative risk of AMI for spirits drinkers was identical for smokers and non-smokers. The risk of liver cirrhosis or acute pancreatitis was increased in spirits drinkers who smoked compared with non-smokers who did not drink spirits regularly. The data were further analysed with the men divided into two age groups but without adjusting for other confounding factors. In men aged 30~49 yr total mortality rose significantly with increasing alcohol intake from all sources (spirits, beer and wine), but in the 50-59 age group alcohol consumption showed a non-significant inverse relationship with total mortality and a significant inverse relationship with coronary mortality. The incidence of liver cirrhosis or acute pancreatitis was significantly related to alcohol consumption in men aged 30-49 yr but the relationship was not significant in the older group. In the group who died of coronary heart disease or developed a non-fatal AMI during follow-up and in their matched controls, spirits drinkers showed a reduced risk both of death from coronary heart disease and of non-fatal AMI in multiple logistic models allowing for six potential confounding factors. Thus this study shows an inverse association be-