The beneficial and hazardous effects of simple phenolic compounds

Mutation Research, 259 (1991) 307-324 © 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100066J

307

MUTGEN 00039

The beneficial and hazardous effects of simple phenolic compounds Hans F. Stich Enoironmental Carcinogenesis Unit, British Columbia Cancer Research Centre, Vancouoer, B.C. VSZ 1L3 (Canada) (Received 12 December 1989) (Accepted 23 May 1990)

Keywords: Phenolic compounds, beneficial and hazardous effects

Summary The current emphasis on screening the environment for man-made genotoxic and carcinogenic compounds detracts from studies on the possible health hazard or beneficial effects of naturally occurring agents to which humans are exposed daily. The simple phenolics, which are ubiquitous among plants, used as food additives, and ingested daily in milligram quantities, belong to this category of compounds. They induce double-strand D N A breaks, DNA adducts, mutations and chromosome aberrations in a great variety of test systems. However, they can suppress the genotoxic activity of numerous carcinogenic compounds in both in vitro and in vivo assays. This dual function of dietary phenolics also becomes evident when their carcinogenic or anticarcinogenic potential is examined. Some, but not all, phenolics induce precancerous lesions, papillomas and cancers, act as cocarcinogens, and exert a promoting effect in various rodent assays. On the other hand, phenolics have proved to be potent irdaibitors of carcinogenesis at the initiation and promotion stages induced by carcinogens and promoters of different molecular structures. The extent to which a health hazard or protective activity of complex dietary mixtures is due to their phenolic content remains an unresolved issue. In addition, these multiple, occasionally contradictory functions of simple phenolics make it difficult to propose their use as chemopreventive agents.

In the past, the multiple functions of phenolics to act not only as antimutagens, anticarcinogens and antipromoters, but also as mutagenic, carcinogenic and promoting agents have not attracted the attention they seem to merit. Considering that their distribution is ubiquitous among plants, and that humans ingest milligram quantities of a great variety of naturally occurring and synthetic phenolics daily, a better u~tderstanding of the possible

Correspondence: Dr. Hans F. Stich, B.C. Cancer Research Centre, 601 West 10th Avenue, Vancouver, B.C. V5Z 1L3 (Canada).

hazardous and beneficial effects of these compounds would be desirable. Due to their multiple effects, it is difficult at present to make precise recommendations for preventing their adverse effects or to suggest their use as protective agents. This review focuses primarily on the genotoxic and antigenotoxic effects, the carcinogenic and anticarcinogenic actions, and the promoting or antipromoting properties of simple, naturally occurring plant phenolics. In addition, a discussion of the synthetic butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) appears to be warranted because of their widespread use in the food industry and their relatively large daily ingestion by human population groups.

308 TABLE 1 I N F L U E N C E OF VARIOUS TEST SYSTEMS ON DETECTION OF G E N O T O X I C I T Y OF 2 P H E N O L I C C O M P O U N D S From Stich et al., 1981a, modified. Compound

Salmonellatyphimurium

Saccharomyces cerevisiae

CHO cells

Concentration

His + revertants/plate Concentration

trpt/10 s

Concentration

(mg/plate)

TA100

TA98

(mg/ml)

survivors

(mg/ml)

M a

E b

Chlorogenic acid

19 0

130 136

26 26

40 0

3.7 2.0

0.4 0

22.2 0.6

0.22 0.01

Caffeic acid

10 0

125 116

33 20

40 0

3.5 1.1

0.2 0

33.3 0.6

0.08 0.01

a M, frequency of metaphase plates (%) with at least 1 chromatid break or exchange. b E, average number of chromatid exchanges per cell.

The difficulty of obtaining insight into the biological role of phenolics is due mainly to a lack of precise information on their mechanism of action. It has been suggested that they interfere with exchanges between cells (Williams, 1986), thus leading to hyperplasia, enhance (Das et al., 1985) or inhibit glutathione S-transferase (Spamins et al., 1982; Das et al., 1984) and aryl hydrocarbon hydroxylase (Das et al., 1987b), thus affecting activation and detoxication of carcinogens (Rahimtula et al., 1982), interact with reactive metabolites of carcinogens (M.-T. Huang et al., 1988a,b, 1985), inhibit, covalent binding of ultimate carcinogens to DNA (Dixit et al., 1985; Das et al., 1985; Wang et al., 1989b), scavenge free radical species (Wang et al., 1989a), show strong antioxidant activity (Namiki and Osawa, 1986), form covalent adducts with carcinogens (Sayer et al., 1982), suppress the formation of carcinogeninduced DNA adducts, or inhibit the formation of nitrosamines (Bartsch et al., 1988). Undoubtedly, a better understanding of the mechanism by which phenolics affect cell metabolism should greatly improve the scientific basis for selecting some of them for further exploration as chemopreventive agents, and for introducing preventive measures against those which appear to pose a health hazard.

(I) Genotoxic effects A wide variety of organisms and end points have been used to examine the genotoxic capacity of phenolics. Since the results have depended to a

large degree on the bioassays used, considerable concern has been expressed about the relevance of these data to human population groups. This unresolved issue becomes evident by comparing the degree of genotoxicity of chlorogenic acid and caffeic acid when tested in 3 different test systems (Stich et al., 1981a) (Table 1). Which of these contradictory results can predict the response of human tissues to dietary levels of phenolics remains unknown. However, since considerable evidence from molecular and cellular studies points to the ability of phenolics to interfere with the genome of mammalian cells, a potential health hazard to man cannot be dismissed. At the molecular level, simple phenolics, which are ingested daily with food products, can induce TABLE 2 D N A - B R E A K I N G ACTIVITY OF P H E N Y L C O M P O U N D S IN VITRO From Yamada et al., 1985, modified. Compound

Substitution groups R1

Phenol Pyrocatechol Resorcinol Hydroquinone Pyrogallol Gallic acid Caffeic acid Ferulic acid Chlorogenic acid

R2

OH H OH OH OH H OH H OH OH OH OH OH OH OHC 3 OH

DNA-breaking

R3

R4

activity

H H OH H OH OH H H

H H H OH H H H H

+ + + + + + +

+ + + + + +

+ + + +

309 TABLE 3 CLASTOGENIC ACTIVITY OF PHENOLIC COMPOUNDS IN CHINESE HAMSTER OVARY (CHO) CELLS Compound

Concentration (mg/ml)

Metaphaseswith chromosome aberrations a (~)

Chromatid breaks/cell

Chromatid exchanges/cell

Catechol 4-Methyl catechol Resoreinol Phloroglucinol Pyrogallol p-Hydroxybenzoicacid Protocatechuic acid Vanillic acid Gallic acid Syringic acid Salicylic acid 3,4-Dihydroxyphenylaceticacid p-Coumaric acid Caffeic acid Ferulic acid Eugenol

0.05 0.01 1.6 3.0 0.1 25.0 3.0 25.0 0.05 3.0 25.0 0.1 6.0 0.2 25.0 0.4 0.05

23.5 20.0 14.3 10.1 22.6 0.0 20.5 4.8 24.1 38.6 1.5 18.5 3.0 33.3 7.8 21.5 0.5

0.17 0.10 0.08 0.03 0.14 0.00 0.04 0.02 0.03 0.17 0.02 0.01 0.01 0.08 0.12 0.08 0.01

0.67 0.73 0.95 0.21 1.08 0.00 0.87 0.07 0.70 1.44 0.00 1.23 0.08 3.08 0.15 0.20 0.00

a Concurrent control frequencies for cells receivingculture medium only were 0.7~ metaphase plates with chromosome aberrations, with an average of 0.01 breaks/cell and 0.00 exchanges/cell. double-strand D N A breaks in vitro (Yarlaada et al., 1985) (Table 2). Single- and double-strand breaks also occur in cultured rat cells, and to a lesser degree in H e L a cells following exposure to catecholamines (Murakami et al., 1978, 1979; Y a m a d a et al., 1985). Furthermor0, olectrophilie metabolites of safrole and 0stragolo 10ad to D N A and R N A adducts in hepati¢ cells of mice (J.A. Miller and Miller, 1983), an event which appears to be related to the hepatccarcinogoni¢ activities of these 2 compounds (E,C. Millor et al., 1983). At the ceUular level, several simple phenolics proved to h0 potent inducers of c h r o m o s o m e / chromatid aberrations (Stich et al., 1981b, 1983a) (Table 3) and sister-chromatid exchanges (Morim o t o and Wolff, 1980; Morimoto et al., 1983; Erexson et al., 1985). This strong clastogenic activity of phenolics in mammalian cell systems is not matched by an equally strong mutagenic effect in the Salmonella typhimurium assay (Nestmann et al., 1980; MacGregor and Jurd, 1978; Swanson et al., 1979) or by a high induction of gene conversion in Saccharomyces cerevisiae at acid or neutral p H levels (Rosin, 1984). The observed discrepancy of the results could be caused by various sensitivities of the different bioassays to the electrophilic

metabolites of the phenolics or to reactive compounds generated from the phenolics. The search for a generally valid conclusion is aggravated by the observation that in the same test systems some phenolics, including catechol, pyrogallol and purpurogallin, possess detectable mutagenic properties (Bilimoria, 1975; Ben-Gurion, 1979), whereas most other simple phenolics lack this capacity. The genotoxic activity of phenolics is strongly influenced b y a host of modulating factors. Their chromosome- and chromatid-damaging effects can be modified by the concurrent application of transition metals, including M n 2+, Cu 2+ and Fe 2+, catalase and peroxidase (Stich et al., 1981b; H a n h a m et al., 1983). The capacity to induce mitotic gene conversion is greatly elevated at alkaline p H levels (Table 4). The addition of an $9 preparation m a y inhibit genotoxicity (Stich et al., 1981b) or enhance mutagenicity (T. Yamaguchi, 1981). These results exemplify the importance of various modulating factors which are not always taken into account when phenolics are tested for genotoxicity. Of particular interest is the formation of direct-acting genotoxic compounds when phenolics found in man's diet are nitrosated (Ohshima et

310 TABLE 4 I N D U C T I O N O F M I T O T I C G E N E C O N V E R S I O N IN C U L T U R E S O F Saccharomyces cerevisiae BY P L A N T PHEN O L I C S M A I N T A I N E D A T p H 7 A N D p H 10 From Rosin, 1984, modified. Phenolic

Pyrogallol Gallic acid Phloroglucinol Catechol 4-Methyl catechol Caffeic acid Chlorogenic acid Resorcinol

Concentration

Trp + c o n v e r t a n t s / 105 survivors

(mg/ml)

pH 7

p H 10

0.3 0.1 3.0 0.3 0.3 0.3 1.0 1.0

1.1 (11) a 2.1 (42) 2.3 (86) 1.3 (52) 3.6 (16) 2.5 (89) 2.4 (52) 1.6 (63)

17.7 14.1 4.0 4.6 7.0 8.8 9.1 4.8

(458) (234) (146) (169) (62) (330) (189) (143)

b b b b b b b b

Figures in parentheses are average n u m b e r of trp + convertants/plate. b Significantly different ( P < 0 . 0 1 ; n = 3 ) from conversion frequency of control cultures exposed to Tris buffer at same pH. N o significant difference was observed between frequencies of gene conversion in control cultures at p H 7 (1.3 + 0.5 trp + convertants/105 survivors; n = 10) and at p H 10 (1.4_+ 0.5 trp + convertants/105 survivors; n =10). a

al., 1989). It is likely that the genotoxicity of smoked food products (e.g., smoked fish, ham and bacon) and wood-smoke condensates is due to their nitrosated phenolic content. The appearance of genotoxic diazonium compounds following nitrosation of single phenols and of the complex smoke condensates supports such a conclusion (Ohshima et al., 1989; Kikugawa and Kato, 1988). Phenolics also appear to be responsible for the mutagenic activity of other widely used food products, including regular instant coffee and tea (Nagao et al., 1979), and tannins from apple juice, grape juice, wine (Stich and Powrie, 1982) and areca nuts (Stich et al., 1983a; Panigrahi and Rao, 1986) proved to have potent clastogenic activities. Since humans digest or inhale complex mixtures of compounds, results on the genotoxicity of single compounds could easily lead to erroneous conclusions. To elucidate the possible interactions of two or more compounds present in a chewing mixture (betel quid), the clastogenicity of single agents and various combinations was examined (Stich et al., 1981c). The chromosome/chromatid-damaging effects of the combined application of arecoline plus

eugenol, arecoline plus quercetin, and arecoline plus chlorogenic acid were greater than the sum of each individual action. Similarly, the combination of arecoline, chlorogenic acid and Mn 2÷ induced a frequency of chromosome aberrations which exceeded the sum of each individual clastogenic activity or that of 2 compounds applied jointly (chlorogenic acid plus Mn2÷, arecoline plus chlorogenic acid, or arecoline plus Mn2÷).

(2) Antigenotoxie effects The in vitro bioassays which have been applied to uncover the mutagenic, clastogenic and recombinogenic properties of chemicals can also be used to reveal their antigenotoxic capacities. The S. typhimurium test has proved to be particularly suited to examine the preventive activity of phenolic compounds because it fails to detect their mutagenic potential. Thus by applying this assay, there is no interference in the recognition of inhibitory effects. The objectives of the numerous investigations were to identify phenolics with antigenotoxic capacities, to recognize the types of mutagens which can be blocked, and to learn whether the antimutagenic effect of complex mixtures could be due to their phenolic contents. Further attention has been given to the question of whether the antigenotoxic activity of a phenolic may conceivably reveal its potential as a chemopreventive agent. Phenolic compounds inhibit the genotoxic action of compounds that differ widely in their mode of action. Various phenolics were found to suppress mutagenesis of S. typhimurium or E. coli induced by aflatoxin B1 (AFBa) (Shelef and Chin, 1980; San and Chan, 1987; Francis et al., 1989), benzo[a]pyrene (B(a)P) diol epoxide (Wood et al., 1982; M.-T. Huang et al., 1985; Smart et al., 1986), N-methyl-N '-nitro-N-nitrosoguanidine (MNNG) (Rosin and Stich, 1980; Chan et al., 1986; Francis et al., 1989; Dixit and Gold, 1986), nitrosation products of methylurea (Stich et al., 1982a; Stich and Powrie, 1982; Stich and Rosin, 1984), 4-nitroquinoline 1-oxide (4NQO) and UV (Shimoi et al., 1985), and N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF) (Rosin and Stich, 1980). The quantitative relationships between the doses of phenolics and the degree of inhibition of

311

,ool

~

0

;o?.ii

'

~ 20 40

"

60 80 100

I .-, ,' • 0 20 40 60 DO I00

pI'~NOLICCOKPOU~I~S

I'~OLIC CON]~i'~S

I00"

C ~/C.t,chtn

r.

~~~ChloroKenic

D

7 ~ 100,

IIgB'~

^

~8o-

,10.

i40.

~

o

01' TMOI[C ~%D OIGII, II~TII)

li "°11 m

~

~

Ferul £c A~:~.rl ChZoroseuie ~td

~

2'o ~

~

(n m~l)

~

1;o

E11.glc Acid

o ,bo~o~o~o~o COHI~JND (m~.t)

Fig. 1. (A) Inhibitory effect of several phenolics on the mutagenic activity of aflatoxin B1 (AFB1) (3×10 -5 M) in S. typhimurium TA98. Mutation frequency of AFB1 alone was 64 his + revertants/107 survivors. (B) Inhibitory effect of several phenolics on the mutagenic activity of MNNG (3 x 10 -5 M) in S. typhimurium TA1535. Mutation frequency of MNNG alone was 138 his + revertants/107 survivors. (C) Inhibitory effect of 2 phenolics on mutagenic activity of nitrite-treated fish extracts in S. typhimurium TA1535. (D) Inhibitory effect of tannic acid on UV-induced mutagenesis in E. coil B / r WP2 trp- (Shimoi et al., 1985). (E) Effect of BHT on the mutagenic activity of M N N G (13.6 nmoles) (Francis et al., 1989). (F) Inhibitory effect of phenolics on the mutagenic activity of B(a)P 7,8-diol9,10-epoxide in S. typhimurium TA100 (Wood et al., 1982).

various mutagenic agents are shown in Fig. 1. Many of the results on bacterial test systems have been complemented by studies on cultured mammalian cells. The-reduction in mutations to 8azaguanine in Chinese hamster V79 cells exposed to caffeic acid, ferulic acid, chlorogenic acid and ellagic acid was comparable to the inhibition of revertants in the S. typhimurium test (Wood et al., 1982). Similarly, tannic acid suppresses mutations

in the bacterial assays as well as chromosome aberrations induced by UV in cultured Chinese hamster ovary (CHO) cells (Sasaki et al., 1988). Of particular relevance to man were studies which focused on the inhibitory action of phenolies on the mutagenicity of mixtures to which human population groups are actually exposed. For example, chlorogenic acid, pyrocatechol, quercetin, catechin, tannic acid and propyl gallate reduced the mutagenic activity of albumin pyrolysates to 20-76% in the TA98 strain of S. typhimurium (Fukuhara et al., 1981). Ellagic acid (Terwel and van der Hoeven, 1985) and curcumin (Nagabhushan et al., 1987) suppressed the mutagenie activity of cigarette smoke condensate in Salmonella TA98 tester strain. Curcumin, a widely used tumeric ingredient, can block the mutagenic potential of chili and capsaicin, which are an integral part of an Indian diet (Nagabhushan and Bhide, 1986). In the S. typhimurium TA1535 assay, catechin, chlorogenic acid, gallic acid, pyrogallol and tannic acid inhibited the formation of mutagenic derivatives formed after nitrosation of a salt-preserved Chinese fish extract (Stich et al., 1982b). This antigenotoxic effect of phenolics could be due to their efficacy in inhibiting the formation of N-nitroso compounds by reducing the nitrosating agent to nitrous oxide (NO) (Bartsch et al., 1988). A considerable amount of evidence supports this hypothesis. A variety of phenolics inhibited nitrosamine formation in vitro (Pignatelli et al., 1982a, 1984; Bartsch et al., 1988; Kuenzig et al., 1984 ), in exPerimental animals (Pignatelli et al., 1982a; Stich et al., 1984; Kuenzig et al., 1984), and in humans (Stich et al., 1984). Comparable results were obtained with beer (Pignatelli et al., 1983, 1984), areca nuts (Stich et al., 1983b) and tea (Stich et al., 1984), which are complex mixtures rich in phenolics consumed by a large part of the human population. However, it may be an oversimplification to consider only the inhibitory effect o f phenolics on N-nitrosation. For example, BHA and BHT show no inhibitory action, and other phenolics, including resorcinol, catechin, flavonols and flavanones, exerted a catalysis of nitrosamine formation (Pignatelli et al., 1980, 1982a,b; Walker et al., 1982). Whether a phenolic compound acts as an inhibitor or as a catalyst may depend on several factors, of which

312 the molar ratio of nitrite to phenolics is a major one (Pignatelli et al., 1982b; Stich et al., 1984). Of particular interest and relevance for man is the antimutagenic activity of various teas. Japanese, Chinese and Celanese teas were found to prevent the formation of mutagenic nitrosated fish compounds at doses consumed by humans (Stich et al., 1982b). It is likely that these effects are due to the rich phenolic content of the teas. However, other mechanisms must be involved in the antimutagenic activity of green tea extracts in S. typhimurium exposed to B(a)P, AFB 1 and 2aminofluorene (Wang et al., 1989a), the reduction of high spontaneous mutations in a mutator strain of Bacillus subtilis (Kada et al., 1985; Ramel et al., 1986), and the inhibition of forward mutations, sister-chromatid exchange or chromosome aberrations in V79 cells exposed to AFBa (Wang et al., 1989a). The protective effects of teas could be due to scavenging effects of radicals (Wang et al., 1989a; Namiki and Osawa, 1986), or to inhibition of lipid peroxidation (Namiki and Osawa, 1986). These beneficial functions are in contrast to the mutagenic effects of black, green and roasted teas, which are enhanced by pretreatment of the extracts with hesperidinase (Nagao et al., 1979). Phenolic acids were also tested in vivo for their antigenotoxic properties. In one of the mammalian test systems, B(a)P was applied intrarectally to C57B1/6J mice to induce micronuclei, pyknotic nuclei and karyorhexis in the colonic epithelial cells, whereas the various phenolics were administered through the diet. The order of inhibitory effectiveness was BHA > ferulic acid > caffeic acid > ellagic acid > quercetin (Wargovich et al., 1985). Using another approach, resorcinol and pyrogallol inhibited the formation of micronuclei in polychromatic erythrocytes of mice injected with B(a)P (Paschin et al., 1986), and caffeic acid reduced 7,12-dimethylbenz[ a ]anthracene (DMBA)-induced micronuclei in mouse bone marrow cells (Raj et al., 1983). Considering the hundreds of simple phenolics and flavonoids in edible plant products, the search for a simple screening procedure for their antimutagenic and by implication anticarcinogenic activities has become a necessity. In vitro inhibition of DNA adduct formation seems to represent a sensitive, economic and rapid assay for the detec-

tion of antimutagenic agents. In this connection, it is of interest to note that DNA adduct formation in vitro is comparable to that in vivo following exposure to B(a)P (Alexandrov et al., 1976; Osborne et al., 1976). Thus results from in vitro experiments may become reliable predictors of reactions occurring in the complex tissues of mammals. A significant inhibition of AFB1-DNA adduct formation occurred in vitro when aflatoxin was exposed concurrently with caffeic acid, cinnamic acid, coumaric acid, BHA or BHT (Bhattacharya et al., 1984). Similarly, DNA adduct formation in vitro resulting from B(a)P metabolites was reduced following the addition of ellagic acid (Mukhtar et al., 1984a,b) or flavonoids (Shah and Bhattacharya, 1986; Das et al., 1987a). Covalent binding of polycyclic aromatic hydrocarbon metabolites and DNA adduct formation is also preventable in vivo by BHA (Anderson et al., 1981), ellagic acid (Del Tito et al., 1983; Shugart and Kao, 1984; Dixit et al., 1985), caffeic acid (Shugart and Kao, 1984), tannic acid, quercetin and myricetin (Das et al., 1987b), and green tea polyphenols (Wang et al., 1989b). These short-term in vitro and in vivo studies may conceivably aid in the selection of phenolics with chemopreventive properties. An elevated risk for cancer in 'chromosomal breakage syndromes', including Bloom's syndrome, Fanconi's anemia, and ataxia telangiectasia, and that of xeroderma pigmentosum which is conditional upon UV exposure, has been well established (Ray and German, 1983; Heim et al., 1989). A chromosomal instability also occurs in cells carrying DNA of adenoviruses (Stoltz et al., 1967; Stich and Yohn, 1970), herpes simplex (Stich et al., 1964), human papillomaviruses (Crum et al., 1984) and bovine papillomavirns type 1 (BPV) (H.F. Stich and S.S. Tsang, unpublished data). The question must be raised whether antioxidants, including phenolics, could reduce this high incidence of 'spontaneous' chromatid aberrations. As targets, 2 BPV DNA-transformed cell strains containing approximately 50 BPV D N A copies were chosen, and as endpoints, the frequency of chromatid bridges and fragments in anaphases was used. The results of a 3-day exposure of BPV DNA-carrying cells to several antioxidants showed a reduction of the chromosome instability by el-

313 TABLE 5 EFFECT OF ANTIOXIDANTS ON CHROMOSOME INSTABILITY OF BOVINE PAPILLOMAVIRUS DNA-CARRYING CELLS (C127) Compound

Concentration Chromatidbridges and (M) fragments at anaphase (%)

Ellagic acid Caffeic a c i d Ferulic acid Gallic acid Catechol Propyl gallate Pyrogallol Ascorbic acid r-Carotene Retinol Retinoic acid None

1 xl0 -s 1.6×10 -4 2.1 x 10- 3 1.2 x 10- 4 3.6 × 10- 5 3.3 × 10- 5 5.6 × 10- 5 1 × 10- 3 5 × 10- 7 1 ×10 -6 5 ×10 -6 -

B5

BF3

9.0+3.3 30.4+5.2 14.8 + 4.1 29.0 + 5.1 33.3 4-5.6 27.3 + 5.0 21.4 + 4.6 7.5 + 3.0 10.4 + 3.5 6.34-2.7 6.64-2.7 29.0 + 1.6

7.1-t-2.9 21.6-t-4.7 15.3 + 4.1 19.3 + 4.5 30.1 _ 5.0 24.0 + 4.6 21.3 + 4.6 7.6 + 3.0 11.7 + 3.6 4.14-2.2 7.24-2.9 27.3 4-1.6

lagic acid, which was comparable to that following ascorbic acid, retinol or retinoic acid, but none after exposure to caffeic acid, gallic acid, catechol or propyl gallate (Table 5). On the one hand, phenolics can exert a strong protective effect against the genotoxic and carcinogenic actions of numerous model compounds, and of complex mixtures to which a large segment of the human population is exposed. For example, catechin, protocatechuic acid and chlorogenic acid reduced the mutagenic level of products formed in cooking processes (Fukuhara et al., 1981), chlorogenic acid inhibited the formation of mutagenic products when added prior to the frying of beef patties (Wang et al., 1982), phenolics or phenoliccontaining teas prevented the appearance of mutagenic products when added to the nitrosation reactions of a widely used salted fish (Stich et al., 1982b), and curcumin conveyed an antimutagenic effect against cigarette smoke condensates and tobacco extracts (Nagabhushan et al., 1987). On the other hand, several simple phenolics can induce mutations, chromosome aberrations, sisterchromatid exchanges, hyperplasia and cancer. Furthermore, they can enhance carcinogenesis when added during or after exposure to various carcinogens. These multiple, apparently contradictory functions of naturally occurring and synthetic phenolics, which make it difficult to attribute to

them either beneficial or adverse effects, are by no means unique (Kahl, 1986). Comparable problems have been encountered with ascorbic acid, which can exert genotoxic activities (Stich et al., 1976, 1980), co-carcinogenic effects (Rosin et al., 1980; Banic, 1981), and tumor promotion in a 2-stage urinary bladder model (Fukushima et al., 1983; Mori et al., 1987), but can also exert a preventive effect on carcinogenesis (Cameron et al., 1979; Smart et al., 1987; Lu et al., 1986).

(3) Carcinogenic effects The discovery that the synthetic, widely used antioxidant BHA induces carcinomas in the forestomach of rats and hamsters (Ito et al., 1983) raised the question of whether naturally occurring phenolic compounds, which are ingested daily by man, could also be carcinogenic. Since the development of cancer in experimental animals requires a long time, a search for relevant intermediate endpoints was initiated. The induction of extensive hyperplasia in the gastric epithelium detectable by an increased number of mitoses in the basal cell layer, thickening of the squamous epithelium, and an elevated [3H]thymidine-labeling index was chosen as an early marker to predict an elevated risk for carcinogenesis (Ito et al., 1983; Iverson et al., 1986; Altmann et al., 1985). The similarity between the cancer dose response and the proliferative dose response (Nera et al., 1984) (Fig. 2) and the good correlation between the capacity of some phenolics to induce proliferation in the forestomach of rats and the subsequent development of cancer (Hirose et al., 1988a) (Table 6) make the intermediate endpoints acceptable as predictors of carcinogenesis. Considering the potential health hazard of phenolics, it was necessary to substantiate the original results on the rat forestomach (Ito et al., 1983) with experiments on other animal species. Precancerous lesions o f t h e forestomach were induced by several phenolics in Wistar rats, N M R I mice and Syrian golden hamsters, whereas male guinea pigs which lack a forestomach showed no adverse effects in their stomach epithelium (Altmann et al., 1986). An adverse effect of phenolics is not restricted to the forestomach of rodents, as can be seen from the increased [3H]thymidine labeling

314

90

110

80

100

70

90 ~J

60 -

80

50 -

70

40-

60

30-

,50~

20.

- 40

10°

.30

20

0.5 0/6 ~

1.0 DIET

1.5

2.0

Fig. 2. D o s e - r e s p o n s e curves for effect of B H A o n rat fores t o m a c h : A, c a r c i n o m a ; o , c a r c i n o m a a n d p a p i l l o m a ; × ,

[3H]thymidine-labelingindex (Nera et aL, 1984).

index in the urinary bladder epithelium of Syrian golden hamsters given catechol, tert.-butyl-hydroquinone, methylhydroquinone or propylparabene (Hirose et al., 1986). Diets containing the naturally occurring caffeic acid, sesamol and catechol were found to induce proliferation, papillomas and carcinomas in the forestomach of male F344 rats as effectively as the synthetic BHA or B H T (Hirose et al., 1987a,b). On the other hand, the administration of gallic acid, syringic acid, chloro-

TABLE 6 CAPACITY OF PHENOLICS TO I N D U C E PROLIFERATION, C A R C I N O G E N I C I T Y A N D P R O M O T I O N FOLL O W I N G I N I T I A T I O N BY M N N G IN T H E STOMACH OF RATS From Hirose et al., 1988a, modified. Chemical

Proliferation

Carcinogenicity

Promotion

p-tert.-Butylphenol 2-tert.-Butyl-4-methylphenol Catechol 4-Methoxyphenol Methylhydroquinone Caffeic acid Butylated hydroxyanisole Butylated hydroxytoluene

+ + + + +++ +++ +++

NE a NE NE NE NE + + +

+ ++ ++ +b ++ ++

+ +

+ +

a NE, not examined, or not carcinogenic in 1-year experiment. b DMBA was used for the initiation.

genic acid, ferulic acid and eugenol had no detectable effect on the rat forestomach (Hirose et al., 1987b). Why the latter group of phenolics should be non-carcinogenic despite being active clastogens, whereas BHA is carcinogenic but lacks genotoxic activity when tested in several assays remains an unresolved issue (Williams, 1986). A complex picture emerged when phenolic compounds were tested for their cocarcinogenic potential with several well-known carcinogens. Concurrent treatment of rats, with BHA and the carcinogen DMBA enhanced the incidence of forestomach papillomas, and increased the incidence of urinary bladder carcinomas (Ito et al., 1990). Catechol acted as a cocarcinogen on mouse skin treated with a smoke condensate (Hecht et al., 1981), on the rat esophagus treated with methyl-n-amylnitrosamine (MNAN) (Mirvish et al., 1985), and in subclones of Balb/3T3 cells (Atchison et al., 1982). A particularly worrisome observation was the low dose of BHA that can exert an enhancing effect. In spite of a considerable number of experimental studies aimed at proving or disproving the carcinogenic potential of simple phenolics or flavonoids (MacGregor, 1984), the final word on this issue is still outstanding. The frequent use of herbal teas, which contain a relatively high concentration of phenolics (Kapadia et al., 1976, 1983; Mahboubi and Ghadirian, 1976), the consumption of rice-gruel boiled in tea leaves (Segi, 1975; Hirayama, 1979), the ingestion of fresh and dried persimmons (Yang, 1980), and the eating of various herbs (Morton, 1973) have been associated with an elevated risk for esophageal cancer. However, there is no solid evidence linking carcinogenicity in humans with the phenolic content of these dietary products. The link between the ingestion of smoked food items and an increased risk of stomach cancer (Hill, 1986; Howson et al., 1986) could conceivably be due to the nitrosated derivatives of phenolics (Ohshima et al., 1989). Again, definite proof of the involvement of nitrosated phenolic compounds and derivatives in the etiology of any human cancers is currently outstanding, and requires further examination. Feeding experiments on rats revealed a cocarcinogenic effect of coffee in combination with cycasin (Mori and Hirono, 1977). However, despite the fact that

315 TABLE 7 INHIBITORY EFFECT OF PHENOLICS ON DMBA-INITIATED CARCINOGENESIS IN VARIOUS TISSUES Carcinogen

Species

Site

Phenolic(s)

ReL

DMBA

Mouse Mouse Rat Rat (F344)

Lung Forestomach Breast Liver

BHA BHA, ethoxyquin BHA, BHT, ethoxyquin BHA

Wattenberg (1973) Wattenberg (1972) Wattenberg (1972) Ito et al. (1990)

DMBA + TPA

Mouse (CD-1) Mouse (NMRI) Mouse (SENCAR) Mouse (SENCAR)

Skin Skin Skin Skin

BHA, BHT Ellagic acid Tea phenolics Tannic acid, quercetin

Slaga and Bracken (1977) Lesca (1983) Wang et al. (1989b) Mukhtar et al. (1988)

a relatively large number of individuals drink coffee, which is rich in phenolics (Arnaud, 1988; Challis and Bartlett, 1975), no reproducible evidence links this habit to any type of human cancer (Newell, 1985). Furthermore, some of the phenolics found in cigarette smoke condensate (Tso, 1972) show cocarcinogenic (Heeht et al., 1981; Van Duuren et al., 1973) and tumor-promoting (Van Duuren and Goldschmidt, 1976) activities. Their involvement in carcinogenesis of the respiratory tract of man, however, remains unknown.

(4) Anticarcinogenic effects The multiple, apparently contradictory effects of phenolics were again encountered in studies dealing with their anticarcinogenic or carcinogenic properties. On the one hand, BHA had a carcinogenic effect on the forestomach of mice and rats, and on the other hand, it suppressed carcinogenesis induced by a broad range of chemical carcinogens, including N-2-fluorenylacetamide (UUand et al., 1973), polycyclic aromatic hydrocarbons, 4nitroquinoline 1-oxide, uracil mustard and urethane (Table 7). Several naturally occurring phenolics can also convey protective effects (Newmark, 1984, 1987). Ferulic acid, chlorogenic acid and eUagic acid reduced the incidence of B(a)P-induced pulmonary adenoma in A / J mice (Lesca, 1983). Ellagic acid inhibited B(a)P diol epoxide-induced carcinogenesis in the skin and lung of mice, but had no detectable preventive effect on tumorigenicity of B(a)P in the same test model (Smart et al., 1986). Ellagic acid was also found to be a potent inhibitor of methylcholanthrene (MCA)-

elicited tumor formation when the percentage of mice with skin carcinomas, or the cumulative number of cancers or tumors per mouse are estimated (Mukhtar et al., 1984b). Caffeic acid and ferulic acid reduced the incidence of B(a)P-induced neoplasms of the mouse ( I C R / H a ) forestomach (Wattenberg et al., 1980), chlorogenic acid exerted an inhibitory effect on methylazoxymethanol-induced carcinogenesis of hamster intestine and liver (Mori et al., 1986), and tannic acid, green tea polyphenols and quercetin afforded protection against skin carcinogenesis in mice induced by polycyclic aromatic hydrocarbons (Khan et al., 1988; Chang et al., 1985; Mukhtar et al., 1988). Despite a considerable amount of information, no simple, generally valid conclusion can be drawn. For example, ferulic acid and chlorogenic acid were effective inhibitors against B(a)P-induced pulmonary formation, but lacked protection against DMBA-induced skin cancers (Lesca, 1983), and BHA applied concurrently with dihydroxydi-N-propylnitrosamine (DHPN) suppressed alveolar hyperplasia, whereas this combination increased papillomas in the mouse forestomach (Ito et al., 1990). Through their capacity to reduce nitrosating agents, the phenolics should prove to be good preventive agents when endogenous nitrosation occurs (Mirvish et al., 1975). The suppression of N-nitrosoproline formation in humans who ingested nitrite, proline and caffeic acid or ferulic acid, respectively (Stich et al., 1984), illustrates the efficacy of phenolics to block endogenous nitrosamine formation in man. Comparable effects were obtained with instant coffee and Indian and Chinese teas. Since it is difficult to trace

316 the effect of a complex mixture to a particular compound, the observed inhibition of nitrosamine formation by coffee and tea samples may not necessarily be due to their phenolic content. Epidemiological studies point to a low mortality rate from stomach cancer in Japanese population groups with a high intake of green tea (Oguni et al., 1987). However, this protective effect cannot be conclusively traced to the phenolic compounds, although tea epicatechins exerted a strong inhibitory action on microsomal cytochrome P-450 (Wang et al., 1988), which is required to activate many precarcinogens into the ultimate reactive species. The protective role of common dietary mixtures requires further attention, considering that in an experimental system, coffee showed an inhibitory effect on hepatocarcinogenesis when added to a nitrosamine-forming mixture of aminopyrine and sodium nitrite (Nishikawa et al., 1986).

hyperplasia in the lungs (S. Yamaguchi et al., 1989). Catechol, hydroquinone, pyrogallol, and tannic acid were potent enhancers of thymidine incorporation into cells of the esophagus, suggesting a promoting activity in this particular tissue (Mirvish, 1982). BHA, which has attracted considerable attention due to the necessity of a regulatory decision, strongly promotes forestomach and urinary bladder carcinogenesis initiated by M N N G in rodents (Shirai et al., 1984; Ito et al., 1990), methylnitrosourea (MNU) (Imaida et al., 1984) or Nbutyl-N-(4-hydroxybutyl)nitrosamine (BBN) (Imaida et al., 1983; Miyata et al., 1985), but inhibits the development of cancer in the liver, lung and mammary glands of rats (Ito et al., 1990). In this connection, it is of interest to note that in the same strain of rats (F344), BHA can act as a promoter (urinary bladder) or an inhibitor (liver) of carcinogenesis (Imaida et al., 1983).

(5) Promoting effects

(6) Antipromoting effects

In addition to their genotoxic and carcinogenic effects, some phenolics act as cancer promoters. This conclusion is based on several animal experiments in which well-established carcinogens were applied as initiators and the phenolics subsequently given as cancer promoters. Catechol, ptert.-butylphenol and 2-tert.-butyl-4-methylphenol enhanced forestomach or glandular stomach carcinogenesis when applied to rats that had received an intragastric dose of MNNG in the order of 150 m g / k g body weight (Hirose et al., 1987a,b, 1988a, 1989a,b) (Table 6). The promoting effects of some phenolics appear to be tissue-specific. Caffeic acid enhanced carcinogenesis in the rat forestomach, but not in the glandular stomach following the application of DMBA (Hirose et al., 1988b), whereas catechol increased the formation of carcinomas in the forestomach as well as in the glandular stomach (Hirose et al., 1988b). In F344 rats injected for 2 weeks with the carcinogen MNAN, and fed a phenolic-containing diet commencing 1 week thereafter, catechol and its isomers resorcinol and hydroquinone significantly increased the incidence of tongue papillomas and esophageal carcinomas over that found in MNAN-treated animals, whereas catechol reduced

It has become customary to distinguish chemicals with cocarcinogenic activity (enhancers of carcinogenicity when applied concurrently with a carcinogen) from those with a cancer-promoting effect (enhancers when applied after carcinogen administration). Although this categorization of compounds has proved to be helpful, it does not indicate that the mechanisms of action of cocarcinogens differ radically from those of promoters. Nevertheless, experiments were carried out to explore a possible inhibitory effect of phenolics on the second stage of several 2-step carcinogenesis systems. In vitro, BHA inhibited promoter (TPA)-enhanced transformation of BALB/3T3 cells when added together with TPA after the cultures had been initiated with methylcholanthrene (Saito et al., 1986). In vivo, BHA also proved to be a potent suppressor of TPA-induced skin tumor promotion (Slaga et al., 1982). The naturally occurring catechol reduced alveolar hyperplasia when administered 1 week after initiation by methyl-n-amylnitrosamine (Ito et al., 1990). However, in the same experiment, catechol significantly increased the incidence of tongue papillomas and esophageal carcinomas. These resuits point to an organ-specific effect of phenolics

317 TABLE 8 AMOUNTS OF SELECTED NON-FLAVONOID PHENOLICS IN SEVERAL FOOD PRODUCTS Food product Vegetables Brussel sprouts

Phenolic

Amount

Ref.

Caffeic acid Ferulic acid Coumaric acid Sinapic acid

3.4 mg/100 g 1.0 mg/100 g 1.2 mg/100 g 10.7 rag/100 g

Schmidtlein and Herrmann (1975a)

Potatoes

Caffeic acid Feruhc acid Coumaric acid Sinapic acid

28.0 mg/100 2.0 rag/100 0.4 mg/100 0.3 mg/100

Schmidtlein and Herrmann (1975b)

Lettuce

Caffeic acid Hydroxybenzoic acid

16-90 mg/100 g 20-40 rag/100 g

Herrmann (1978)

Cabbage

Coumaric acid Ferulic acid

20 mg/100 g 22 mg/100 g

H.-M. Huang et al. (1986)

Fruits Apples

g g g g

Caffeic acid Ferulic acid Coumaric acid

8.5-127 mg/100 g 0.4-9.5 mg/100 g 1.5-46 mg/100 g

Mosel and Herrmann (1974)

Strawberries

Caffeic acid Coumaric acid Gallic acid Hydroxybenzoic acid

1.5-3.9 mg/100 g 6.9-17.5 mg/100 g 8.0-12.1 mg/100 g 0.0-10.8 rag/100 g

StiShr and Herrmann (1975)

Stuart pecans

Gallic acid Gentisic acid Vanillic acid Protocatechuic acid p-Hydroxybermoic acid

Beoerages Wine

13 mg/100 g 0.7 mg/100 g 0.6 rag/100 g 0.7 rag/100 g 1.4 mg/100 g

Horvat and Senter (1980)

Caffeoyl tartaric acid Coumaroyl tartaric acid Non-flavonoid phenols

8-116 mg/1 2-33 rag/! 180-320 mg/1

Okamura and Watanabe (1981)

Coffee

Chlorogenic acid Catechol Pyrogallol

260 mg/cup ± 0.7 mg/cup ± 0.4 mg/cup

Challis and Bartlett (1975) Tressl et al. (1978)

Tea (instant)

Gallic acid Coumaric acid Caffeic acid

64 mg/100 g 2.4 mg/100 g 25 mg/100 g

Horvat and Senter (1980)

Guaiacol Catechol Syringol Vanillic acid

3.7 mmoles/l 14 mmoles/I 6.3 mmoles/1 0.4 mmoles/1

Ohshima et al. (1989)

Others Wood smoke condensate

which becomes particularly evident when c a r ci n o g en s with an initiation c a p a c i t y for m a n y diff eren t tissues are used ( T s u d a et al., 1984). In a

2-stage i n i t i a t i o n ( D M B A ) - p r o m o t i o n ( T P A ) ex. p e r i m e n t o n m o u s e skin, c u r c u m i n , ch l o r o g en i c acid, caffeic acid a n d f e r u l i c acid i n h i b i t e d th~

318 number of tumors per mouse by 100, 60, 28 and 35%, respectively (M.-T. H u a n g et al., 1988). The involvement of phenolics in promoter-dominated stages of carcinogenesis was demonstrated by the strong inhibitory effect of BHA, B H T and propyl gallate ( K o z u m b o et al., 1983), and of curcumin, chlorogenic acid, caffeic acid and ferulic acid on the induction of ornithine decarboxylase (ODC) in mouse skin exposed to the cancer promoter TPA. (7) Occurrence The current emphasis on screening the environm e n t for m a n - m a d e carcinogens and genotoxic compounds detracts from studies on the possible health hazard or beneficial effects of naturally occurring agents (Ames, 1989a, b). Frequently one can find a deeper understanding of the toxicity of industrial products to which an average individual is barely exposed than of dietary ingredients which are ingested daily by virtually all humans. The simple phenolics are good examples of widespread agents in our regular diet whose benefits or hazards are difficult to assess. The situation is further aggravated by the multiple actions of phenolics which show genotoxic, carcinogenic, cocarcinogenic and tumor-promoting effects, but which can also convey protection against genetic damage, the formation of precancerous lesions, and the development of cancer induced by a broad variety of carcinogens. These multiple functions of phenolics, a lack of precise data on their metabolism, accumulation and retention in animal (Collings and Sharratt, 1970) or h u m a n (Carmella et al., 1982; Teel and Martin, 1988) tissues, and the ambiguity of extrapolating data on carcinogenesis of the rodent forestomach to humans make it difficult at the present time to support the use of phenolics in chemopreventive trials. The multiple functions of phenolics can also create a dilemma for regulatory agencies which are reluctant to remove useful antioxidants from the market without solid evidence of their health hazard and without having an equally effective replacement. Thus, despite careful deliberations, at its meeting in Geneva, the Joint Expert Committee on Food Additives of the World Health Organization and of the Food and Agriculture Organization of the United Nations was unable to

assess the risk posed by the use of B H A ( F A O / W H O , 1980). The amounts of B H A consumed are not negligible. The dietary intake of 12-19-year-old males (Canada) averages 12.12 mg B H A / p e r s o n / d a y , whereas 1-4-year-old children show the highest ingestion when calculated on a body weight basis, namely 0.39 m g / k g / d a y (Kirkpatrick and Lauer, 1986). The main sources of BHA are cereals (3.48-6.78 m g / p e r s o n / day, depending on the age group), followed by cooking oils (0.2 m g / g ) , peanut butter (0.1 m g / g ) , salad dressing (0.07 m g / g ) , candy and chocolate (0.08 r a g / g ) and margarine (0.08 m g / g ) . The difficulties encountered in attempting to weigh the benefits against the adverse effects of a single, well-defined compound such as B H A indicate the size of the task to be faced when a judgement about the involvement of naturally occurring phenolics in m a n ' s health is required. Simple phenolics are present in considerable amounts in all fruits, leaves and roots of plants which are a part of m a n ' s diet (Table 8). Acknowledgements Research of the author is supported by grants from the National Cancer Institute of Canada and the Natural Sciences and Engineering Research of Canada. Dr. H.F. Stich is a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada. References Alexandrov, K., P. Brookes, H.W.S. King, M.R. Osborne and M.H. Thompson (1976) Comparison of metabolism of benzo[a]pyrene and binding to DNA caused by rat liver nuclei and chromosomes, Chem.-Biol. Interact., 12, 269277. A~mann, H.-J., W. Grunow, P.W. Wester and U. Mohr (1985) Induction of forestomach lesions by butylhydroxyanisole and structurally related substances, Receptors and other targets for toxic substances, Arch. Toxicol., Suppl., 8, 114116. Altmann, H.-J., W. Grunow, U. Mohr, H.B. Richter-Reichhelm and P.W. Wester (1986) Effects of BHA and related phenols on the forestomach of rats, Food Chem. Toxicol., 24, 1183-1188. Ames, B.N. (1989a) What are the major carcinogens in the etiology of human cancer? Environmental pollution, natural carcinogens, and the causes of human cancer: six errors, in: V.Y. De Vita, Jr., S. Hellman and S.A. Rosenberg

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