Mutation Research, 267 (1992) 229-241 © 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00

229

MUT 0031 !

Antimutagcnicity of secondary metabolites from higher plants L e s t e r A. M i t s c h e r , H a n u m a i a h Telikepalli, Polly B.-B. W a n g , S i m o n K u o a, D e l b e r t M. S h a n k e l a a n d G e o r g e Stewart a Departments of Medicinal Chemistry and '~Microbiology, The Unit'ersity of Kansas, Lawrence, KS 66045 (U.S.A.) (Received 1 August 1991) (Revision received 26 November 1991) (Accepted 26 November 1991)

Keywords: Antimutagenesis; Anticarcinogenesis; Higher plants; Screening methods; Fractionation

Summary Higher plants contain both mutagens and antimutagens and are susceptible to mutagenesis but screening programs for detection of antimutagenesis rarely employ higher plant systems. Short-term bacterial and mammalian tissue culture systems are the norm. Using modified screening tests for detecting antimutagenic agents, higher plants have been shown to contain a variety of structurally novel antimutagenie agents. Systematic bioassay-directed methodology resulted in the isolation in pure form and biological and chemical characterization of the responsible individual active components from various plants. The methodology in use is illustrated by the isolation of cinnamic acid, cinnamyl cinnamate and einnamyl ricinoleate as the active constituents of the classic medicinal plant product, Styrax asiatica. The methods which may be used to reveal structure-activity relationships and to explore putative molecular modes of action arc illustrated with excerpts from the same study.

Detection of antimutagens using plant systems The science of genetics began with the study of higher plants and the mention of Gregor Mendel and his studies of Pisum saticum fixes this in our minds. Until the 1940s higher plant studies continued to dominate the field. Soon thereafter, however, the manifold advantages of bacterial systems were recognized and the emphasis of the

Correspondence: Dr. L A . Mitscher, Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045 (U.S.A.).

field shifted to prokaryotic studies and has largely remained there ever since. Nonetheless it is clear that higher plants contain a number of agents capable of causing mutations as well as a number of antimutagenic agents. It is curious, therefore, that the reviewer should have great difficulty in finding studies of antimutagenicity in which higher plants are the organisms to be protected. Only one laboratory appears to have published work in this area. Gichner et al. (1985, 1986) have utilized the plant Arabidopsis thaliana to screen the effects of various antimutagens (e.g., 9-hydroxyellipticine, thiourea, cysteine, phenolic acids) against chemically induced embryonic and chloro-

230

phyll mutatiops. Thc presence of the thiol-containing compounds thiourea or cysteine before and during treatment with dimethylnitrosamine (DMN) or N-methyl-N-nitrosourea (MNU) resulted in a decline of mutation frequency (Gichner et al., 1985). In similar studies with phenolic acids, they demonstrated that gallic and tannic acids reduced the frequency of mutations induced by the direct-acting mutagen N-methyl-N'-nitroN-nitrosoguanidine (MNNG), but had no effect on the mutagenicity of the promutagen DMN (Gichner et al., 1986). This would appear to be a potentially fruitful area for scientific enquiry. Almost all of the higher plant work in the contemporary literature deals with the detection of mutagens and antimutagens and the effects that these agents exert on prokaryotic and mammalian systems. This, then, will provide the substance of this review.

Antimutagenicity screening of agents isolated from plants In recent years increasing numbers of scientists have become interested in antimutagenesis (Fiala eta[., 1985; Hartman and Shankel, 1990; Ramel et al., 1986; Wall et al., 1988, 1990). Natu. ral products chemists, in particular, have taken up the challenge of devising suitable screening programs to detect the presence of these agents in higher plant mixtures and, using bioassay.directed methodologies, to isolate the responsible agents in pure form, to determine their chemical structures, to pursue structure-activity experiments and to undertake studies directed toward revealing their molecular modes of action (Kada and Shimoi, 1987; De Flora and Ramel, 1988). Modified bacterial mutation assays are most commonly employed in these studies because they are rapid, inexpensive, technically simple, sensitive and generally accepted by the scientific community as being meaningful. Two general classes of screens arc employed, those directed toward detection of complete mutagens and those involving metabolic activation steps so as to detect promutagens. Either of these can produce meaningful results. The question of which assay to use in one's work is not trivial. It is estimated that over 100

short-term in vitro test methods have been developed from which one can choose (Mortelmans, 1985). In principle, assays based upon mutagenic insult by alkylating agents, ultraviolet light, ionizing radiation and other complete mutagens are simple to perform and to interpret because there are putatively fewer steps involved between insult and expression of injury than is the case when assaying for promutagens. There is, thus, a lesser chance of detecting intercepting agents (desmutagens) instead of antimutagens. Desmutagens are phenomenologicaUy interesting agents relevant to chemoprevention of mutagenesis but since these frequently involve mutually annihilative interaction between antimutagen and agent these may not invoke interesting cellular responses and, so, reduce the animal or bacterium to bystander status, a sort of 'living' or even 'furry' test tube, and consequently may reveal rather less about underlying cell biology than is desired. Assays dependent upon induction of oxidative enzymes, usually P-450, and, following activating oxidation of the promutagen, mutagenic insult, are quite meaningful as many important mutagenic agents fall into this class and so they require careful study and analysis. Nonetheless, plants contain many antioxidative agents and it is possible that an assay not set up with particular care could represent an excellent screen for repetitive discovery of ascorbic acid, toeopherols and other quinones, sulfhydrjl-containing compounds, and so on, as desmutagens and not for finding what is desired. Many groups have taken up this challenge successfully. Recent illustrative results from various laboratories A number of complete studies have been published recently describing results employing short-term tests. One can cite in this respect several papers by the Wall group in which they utilized a screen based upon the metabolic activation (mouse S9 microsome enzymes) of 2aminoanthracene (2.5 p.g/plate) (Wall et al., 1988). For screening purposes 600 p,g/plate of test substance was the initial concentration employed and two-fold dilutions of this solution

231 Me H HO

O

~

O

v

~

~

H

"'Br Beta-Me [li Alpha-Me 121

~

OH O X = Y = Z = H I51 X=OH, Y = Z = H [61 X = Z = OH, Y = H 171 X=Y--OH, Z = H [81

OR O, z

O

W

[3] were subsequently used in case of toxicity. This has led to the isolation and characterization of the very potent brominated arylmonoterpenes cymobarbatol [1] and 4-isocymobarbatol [2] from the marine alga Cymopolia barbara (Wall et al., 1989); the coumarin psoralen [3] and the phenolic bakuchiol [4] from Psoralea corylifolia (Wall et al., 1988b); the flavones apigenin [5], gahmgin [6], morin [7], quercetin [8], rhamnetin [9], tangerctin [10], nobiletin [11] and rutin [12] (from Psoralea corylifolia and a variety of other plants) (Wall et al., 1988a) and, later from various plants, the isoflavones daidzein [13], biochanin A [14] (from Cicer arietinum), and neobavaisoflavone [15], the flavanones bavachin [16], naringenin [17] and hesperidin [18] and related phenolics isobavachalcone [19] and bakuchinol [4] (Wall et al., 1988a);

[41

x" T

~ [

"~OR2

y-v

OR 1 0 R =Me;W= X = R1 = R2=H; Y = Z = O H [91 R=R i =R2=Me;W=X=OMe; Y = Z = H II01 R=R I = R 2 = M e ; W - - X = Z = O M e ; Y = H 111] R=R I = R 2 = H ; W = X = H ; Y = Rutinoside; Z --- OH 1121

I

R

R = X = Y = H 1131 R= Y = H,X = OH 1141 R= Pr, X = Y = H [15]

the isoflavones fremontin [20] and fremontonc [21] from Psorothamnus fremontii (they were also active against EMS-induced mutagenicity) (Manikumar et al., 1989); the homoisoflavinoids intricatin [22] and intricatinol [23] from Hoffmanosseggia intricata (intricatinol was also active against EMS- and metabolically activated acetylaminofluorine-induced mutagenesis) (Wall et al., 1989a). In order to pursue structure-activity rela-

~2

X

O [241

R=RI=X=Y=H [161 R = W = X = Y = H; R ! = Ncohespcridoside [17] R = Me; R l = Rutinoside; X = Y = OH; W = H [18]

C

H

O

~

H

3

0

~

O

OH

[2sl

OH

0

tionshipsfurther,Wallet al. then investigatedthe antimutagenic properties of a variety of known coumarinsleft overfromother studiesand found coumarin, imperatorin[24],osthol [25],8-methoxypsoralen[26]and 5-methox3,psoralen[27]to be stronglyantimutagenicagainstmetabolicallyactivated benzo[a]pyrene(Wall et al., 1988b).Cassady et al., usingan assaybased upon the recta-

1191

HO

~

0

HO

OH 0

R

.,~__

R -- H [201 R - Pr 121] OCH3 1+61

OH

R

O

~

0 R -- Me 1221 R = H 1231

OCH3

~

0 [271

233

bolism by Syrian hamster embryo cell cultures and subsequent interaction of the metabolites of benzo[a]pyrene with DNA, isolated the highly active isoflavone daidzein [13] from Trifolium pratense (Cassady et al., 1988). Many other papers could be cited but these are sufficient to document the point that a variety of assays are in use in a variety of laboratories and that their use results in the isolation of a significant variety of antimutagens belonging to a number of different secondary metabolic structural types. In summary, it would appear that a testing triad is necessary at present: toxicity, mitogenesis and antimutagenesis.

Methodological aspects Choice of assays In our own laboratory we have opted to work with direct-acting mutagens (Sega, 1984; Saffhill et al., 1985) and our approach will be described in greater detail below. Many particulars of antimutagenic screen development are either matters of individual preference or common to all sources of antimutagens and having been discussed elsewhere in this volume need not be recapitulated in this paper. The following paragraphs will emphasize those aspects of greatest importance to detection of antimutagenic agents from higher plants.

Control of toxicity One problem which must be dealt with stems from the fact that many higher plants produce antimicrobial agents and that these can kill the tester strains. Dead cells do not mutate and do not perpetuate DNA damage (Lee et al., 1987; Thiily, 1985). The tester strains are more sensitive to antibiosis than other Salmonella species, such as Salmonella gaUinamm, ATCC 9184, widely used in industrial screens for antibiotics. Thus the viability of the tester strain itself must be ascertained using histidine-containing media at doses such as are to be employed in the experiments to detect antimutagens. If toxicity, as detected by decreased colony-forming units, is found, lower doses must be employed. Using these precau-

tions, we have found examples of antimicrobial substances without antimu~:agenic action and antimutagenic agents causing no measurable decrease in colony-forming uaits at the doses employed.

Testing at forcing doses There has also been a considerable recent discussion relating to the meaning of testing data obtained using forcing doses. Briefly, with respect to mutagenicity assays in bacterial systems, mitogenesis markedly increases mutagenesis (Ames and Gold, 1990; Cohen and Ellwein, 1990). This has led to concerns about the meaning of animal tests for carcinogenesis at high doses unless DNA damage can be shown to have occurred (Ames and Gold, 1990, 1991, a; Brusick, 1991; Cogliano et al., 1991; Scott et al., 1991; Weinstein, 1991; Wilson,,1991). For our purposes we need to focus on the bacterial systems and it is important to point out that antimutagenic assays would be rendered more complex to perform and to interpret without consideration of the possible effects of drug-induced mitogenesis on the assays, if the addition of a plant extract were toxic to the cells but not toxic enough to kill them, then mitogenesis might well be enhanced and mutagenesis increased. This would put a greater burden on the antimutagenic agent and the cell to reverse the process. This, however, presumes that the underlying antimutagenic process itself would be intact. This may not he so. If these mechanisms were also impaired, the resulting data would be misleading. In some cases the antimutagenic effect could be diminished, in others overridden, in yet others exaggerated. One would still, by definition, often be measuring an antimutagenic effect, but the quantitative aspects of the data would be impossible to interpret in a comparative manner. Consequently, we have performed mitogenesis assays in the presence and the absence of plant extracts and individual antin..tagens, in the cases measured to date, we find t h . t the antimutagenic doses employed do not lead to a significant alteration in either the rate of increase in cell number or its ultimate magnitl,de. The possible influence of this factor must be established experimentally for each class of antimutdgenic compound and, perhaps, for each compound as well until a con-

sensus is reached based on the cumulative weight of data. One of the most relevant ways to approach this important contemporary question is to isolate individual antimutagens detected under these conditions and to elucidate the molecular interactions responsible for the antimutagenic effect.

Choice of plant material The choice of plant material to be tested is an important consideration. There are several studies in the literature in which individual plants or groups of plants have been found to have active constituents but in which the active principle has not been identified in pure form. These are excellent places to start but, given the variability in biological material, the investigator should not be surprised if h e / s h e is unable to reproduce some of these findings. The plant, after all, may be gotten at the wrong season or location, belong to a different chemical race, etc. Species belonging to families frequently associated with antimutagenie activity are also an excellent source of material. For screening with or without guidance from published accounts it is also fruitful to use plants with a venerable association with human or animal consumption. These may well not be antimutage;~ic but they arc not likely to be ferociously toxic or distasteful either. For compliance and motivational reasons, agents which are toxic, have significant side effects, disagreeable taste, etc., are not likely to be consumed as chemopreventive agents by persons who do not feel particularly sick or threatened.

Means of extraction The means of producing the plant extract must be addressed. Some investigators prefer the use of freshly expressed juices. These are certainly representative of fresh plant material and are of special interest because of this. Such work, however, puts severe temporal and positior~al coustraints on the experiments and has not ~'oven to be particularly necessary in our ha~,'.,s. We have chosen to dry the plants wilheu) the application of superambient heat as quickly as possible after collection to prevent ,:omposting. The plant material is then powdered and stored for future use when convenient.

Various means of extraction of these powders may be used. The most gentle process involves steeping the plant material in a suitable solvent at room temperature and decantation, filtration or centrifugation. This is a discontinuous method. Percolation at room temperature is more efficient than decantation since it is continuous. In most cases, use of heat and a Soxhlet apparatus is satisfactory and this is the most efficient process of all. Of course the active constituents are at highest risk in Soxhlet extraction because of the prolonged heating involved. Extraction is continued until it is judged that no more material is being extracted. This can be established experimentally by evaporating a portion of the most recent extractive (usually 1 ml) and weighing the residue, if any. The most common extraction solvents are ethanol and methanol. As the plant material is not anhydrous, the use of absolute alcohol is neither necessary nor desirable. In some cases, notably seeds and waxy leaves, there is much lipid material present and the investigator will usually prefer to extract first with nexane and then dry the fat-free plant residue before following with an alcohol extraction. Both extracts must, of course, be assayed separately. Occasionally, the active constituents are too reactive to survive this treatment and the investigator will then use an eluotropic series (a series of solvents of increasing solvent power) instead for the extraction. A suitable series would start with hexane and then employ in turn benzene, ether, methylene chloride, ethyl acetate, chloroform, alcohol and then water. Each extract must be assayed. Generally speaking, about 10% of the dry powdered weight of the plant material will extract under these conditions and will be unevenly distributed among these various solvents.

Choice of screening doses The screening doses to be employed will generally be established by trial and error in each individual laboratory as these are highly dependent upon the viability of the tester strains in the individual assay systems involved and it is necessary to determine in advance the doses which will be free from production of toxic artifacts. At present we are using I m g per plate in our assays.

235

Scoring of activity The level of antimutagenicity which nmst be present in order to score the extract as active differs depending upon a variety of considerations. The primary consideration is the precision and the variability of the assay results themselves. A pragmatic consideration is the number of positives needed. Obviously an assay in which no positive results are ever obtained is useless and it indicates either that the phenomenon does not exist under the assay conditions chosen or that the doses are too low to demonstrate activity. On the other hand an assay in which a very high percentage of leads is generated indicates either that one has a very general phenomenon or that the test is too sensitive to be meaningful. Screeners generally conclude the latter. In most screening systems, including those for antimutagenesis, a level of 1-10% actives makes investigators comfortable. The real value of a screen cannot be judged until a few cycles have been run and the results can be evaluated. A complicating factor which cannot be evaluated a priori is that the activity may be generated by a small amount of a very potent constituent, a large amount of a very weakly active agent, by the cumulative effect of many components or none of the above. In our hands all of these things have occurred.

Exemplification by studies on Styrax asiatica With these general considerations in mind, we now proceed to describe recent results with Styrax asiatica as specifically illustrative of the above points. Styrax (also known as storax) is a balsam obtained from the trunk of Liquidambar orientails Miller, a tree from Asia Minor. The balsam is obtained by bruising or puncturing the bark in the early summer and thus stimulating formation of balsam-secreting ducts. In autumn the balsam-saturated bark is peeled off and pressed. The residual bark is boiled in water and pressed again to obtain a second quantity of balsam. The balsam has seen medicinal use as a stimulant, exrectorant and as an antiseptic since at least the 12til century. It is now primarily used as a component in aromatic mixtures employed in steam vaporizers for inhalant therapy (Claus and Tyler, 1965).

In our antimutagenic assay employing Salmonella typhimurium tester strain TAI0O grown in Oxoid nutrient broth, the various plant extracts are dissolved or suspended in 25/zl of dimethyl sulfoxide and added to 100/zl of culture. This is allowed to stand for 20 min to allow for uptake of the various constituents and at least preliminary induction of defensive mechanisms if any. It is then mixed with 2 ml of top agar containing 5/zi of ethyl methanesulfonate (EMS). Mixing the agent to be tested separately from the mutagenic agent and allowing them to become dilute before mixing minimizes the chance that a desmutagenic effect independent of the tester strain will be encountered. The top agar is then placed on the minimal glucose bottom agar and incubated at 37°C for 3 days and the revertant colonies are counted. Use of the solvent alone in this assay gives a measure of spontaneous revertants, in our hands, this is usually about 120 colonies. Solvent plus mutagen gives a measure of the mutagen-induced revertants. In our hands this number is usually about 500-600 colonies. The use of the solvent and extract provides the effect of the extract on spontaneous revertants. This is often a measure of toxicity. Finally, the use of solvent, mutagen and extract provides a measure of the antimutagenic effect of the extract. If the effect is less than 20% decrease, then the extract is graded as inactive and is not pursued. Styrax asiatica was found to decrease survival levels in our hands at doses of 1.5/~g/plate even though it was inactive at 1000/zg/ml in standard antibiotic assays against Salmonella gallinarum ATCC 9184. An antimutagenic effect against EMS was seen in the assay system at doses as low as 0.0625/zg/plate but no significant effect was seen against the spontaneous mutation rate. These doses are far below those at which any measurable toxic effect was seen.

Isolation of acidic constituents The assay was then used to monitor fractionation into individual components. The balsam was found to release its active constituents into methylene chloride. When this was partitioned into acids and non-acids by washing the methylene chloride solution with 5% aqueous sodium bicarbonate solution, both fractions were active and so

~6 O

I281 TOXICITY = 10 mcg/ml AMAat5 = 25%

O

[311 TOXICITY = 0.125 mcg/ml AMA at 0.0625 mcg/ml = 27%

antimutagenic activity in our hands at the maximum tolerated non-antibiotic dose.

Isolation of lipoidal constituents

COOH

[29]

The agents present in the non-acidic fraction were obtained in crude form by evaporation of the methylene chloride layers. This was partitioned between 90% aqueous methanol and hexane. The polar lipids extracted into the wet methanol layer and produced, following silica gel chromatography, the active constituents. Cinnamyi cinnamate [31] was already known to be a styrax constituent (Claus and Tyler, 1965) but its antimutagenic activity was unsuspected previously. The other constituent, cinnamyl ricinoleate [32], was new to the literature and had to be characterized. Cinnamyl ricinoleate was shown to have a molecular weight of 414 by negative ion fast atom bombardment mass spectrometry and the base peak, at m / z 297, corresponds to the presence of a ricinoleic acid [33] moiety. The proton magnetic resonance spectrum was completely in agreement as all of the anticipated peaks were present along

[301 were processed separately. The acids were isolated from the aqueous bicarbonate layer by acidification and crystallization. This produced an impressive quantity (15%) of cinnamic acid [28] as the active constituent. Cinnamic acid was known already to be a constituent of styrax (Claus and Tyler, 1965) but its antimutagenic activity was not previously known. Interestingly, processing the mother liquors led to the isolation of abietic acid [29] and flehydroabietic acid [30]. These compounds are antibiotically active but display no

0

[321 TOXICITY = 5 mcg/ml AMA at 2.5 mcg/ml = 26%

237 H

~

COOH

[35]

1331 TOXICITY = 10 mcg/ml AMAat 5 = 9 %

with peaks associated with a cinnamyl alcohol [34] moiety. These inferences were easily confirmed by saponification and isolation of the individual fragments. The absolute configuration of the ricinoleic acid component was shown to be R by measurement of the optical rotation. Thus the structure is clearly established. For comparative purposes, each of these agents was tested for antimutagenic activity at half of its toxic level. These doses were not only too low to show acute toxicity (no decrease in colony-forming units) but were also too low to affect the growth curve significantly, so they were neither pro- nor anti-mitogenic. Cinnamyl cinnamate was the most potent (0.0625/~g/ml), followed by cinnamyl ricinoleate (2.5 ~g) and then cinnamic acid (5 ~g). All were equally efficacious causing 2527% decrease in EMS-induced mutations. It is interesting to note that cinnamates have been shown to induce phase II metabolizing enzymes in mammals without significant induction of phase ! enzymes (Meyer et al., 1991). This phenomenon may be therapeutically advantageous as this property would in most cases lead to clearance of mutagens from the body without at the same time stimulating activation of metabolism to new or additional mutagens or causing DNA damage. The styrax compounds have not yet been tested for these properties.

TOXICITY = 20 mcg/ml AMA at 10=37% The efficacy levels of the styrax components are not particularly impressive but given their novel and simple structures and the unknown nature of the underlying biology, it was felt worthwhile to pursue at least limited structureactivity studies.

Preliminary structure-activity relationships The field of antimutagenesis is so comparatively new that there are very few systematic structure-activity studies available which are devoted to optimization of a given lead compound by synthesis. The following summarizes the present status of one of these. Cinnamyl cinnamate [31] was examined first as it contained both common units, a cinnamate ester and a cinnamyl alcohol moiety. Both cinnamic acid [28] and cinnamyl alcohol [34] were active individually. Interestingly, cinnamic acid was markedly less active than cinnamyl cinnamate and cinnamyl alcohol and, indeed, cinnamyl alcohol alone was as potent and as efficacious as cinnamyl cinnamate itself. Converting the alcohol to cinnamyl acetate [35] dramatically reduced its potency (and toxicity) but somewhat enhanced its efficacy. A similar effect on efficacy was seen by converting cinnamic acid to methyl cinnamate [36]. Thus it can be concluded that both halves of

CH 3

~

OH

[34] TOXICITY = 10 mcg/ml AMA at 5=34%

O

[36] TOXICITY = 20 mcg/ml AMA at 10=41%

238 H

~

COOCH3

[371 TOXICITY = 0.3 rncg/ml AMA at 0.15 = 16%

H

~

[40] TOXICITY= 20 mcg/ml AMA at 10 -- 45 %

COOCH3

~

OH

[41]

[38l TOXICITY = 0.3 mcg/ml AMA at 0.15 = 19 %

TOXICITY = 10 mcg/ml AMA at 5 = 24 %

this molecule contribute to the activity but that the alcohol component seems more important than the acid half. A similar analysis of the contributions of the constituent parts of cinnamyl ricinoleate [32] leads to a somewhat different conclusion. The ricinoleate portion-contributes at best a trivial amount to the potency and to the efficacy of this molecule. This contrasts strongly with the results obtained with cinnamyl cinnamate and follows

~

O°H O

COOH

[391 TOXICITY = 0,16 mcg/m] AMA at0.08 = 13%

C/:x

[42]

from examination of ricinoleic acid [33], its methyl ester [37], its dihydro analog [38] and from stearic acid [39], which has neither the olefinic linkage nor the hydroxyl group. In the cinnamic acid moiety, reduction of the

olefinic linkage (dihydrocinnamic acid [40]) actually enhances efficacy even though it decreases potency. In the intrinsically more active alcohol portion, however, after reduction to dihydrocinnamyl alcohol [41] the potency remains the same but efficacy decreases substantially. Thus the double bond is much more important to the alcohol portion than to the acid portion of the molecule. The latter finding is particularly interesting. Whereas cinnamic acid and cinnamyl alcohol have not been examined previously for their antimutagenic activity, cinnamaldehyde [42, X = H] has been studied fairly thoroughly (Ohta et al., 1983, a; Kakinuma et al., 1984; De Silva and Shankel,

~

n

O "~X

[43]

239 1986; Ishibashi et al., 1987; Rutten and Gocke, 1988). It not only has long been known to be antimutagenic, it has been speculated that this activity stems from its ability to be an allq,lating agent based upon its ability to add nucleophiles such as DNA bases in a conjugate sense (1, 4) to produce adducts such as 43 (Neudecker et al., 1983). This sort of reaction would be chemically unlikely for cinnamic acid but would be possible for its esters. The alcohol, on the other hand, would not participate in such a reaction. The lack of need for the olefinic linkage for the alcohol component shown in our work indicates that the effect which we see is highly unlikely to take place through a mechanism like that suggested for cinnamaldehyde. Other reactions (SN-2 or SN-2' come to mind) can readily he invoked but these are entirely speculative with the evidence in hand. Analogy can be quite dangerous. One might reason by analogy that cinnamic acid and its derivatives could operate by a mechanism like that of cinnamaldehyde and the apparent similarity of structure could be invoked to support this idea. The evidence from the dihydro analogs and, particularly, with the more potent alcohol analogs lends no support to this idea. The danger is enhanced when one considers the antimutagenic properties of Meldrum's acid analog [44] and its diethyl analog [45]. These compounds were synthesized as they would be expected to be outstanding reactants in Michael processes. They are not particularly potent but they are reasonably efficacious as antimutagens. They could be operating by a Michael-based mechanism but: if so,

O

Hs

[45] TOXICITY = 20 mcg/ml AMAat 10 = 37%

this would seem not to be relevant to the underlying responses of cells to the natural products discovered by screening Styrax asiatica. Conclusions While much more remains to be accomplished in this very young field, basically useful methodology has been devised and reduced to practice and as a result it is no longer acceptable for work on antimutagenic agents to be devoted to examination of crude mixtures. In most instances these studies can now be pursued to molecular purity and the focus should shift to discoveD" of as many disparate structural types as possible and elucidation of the underlying cell biology and genetics using pure compounds. One other point should be stressed. In view of the large numbers of mutagens and antimutagens that occur in plants it is surprising that there is no accepted plant system for the study of antimutagenesis. This could be a fruitful area of research requiring initially only a plant tissue culture system in which mutant plant cells could be scored for mutagenic and antimutagenic effects.

Acknowledgement This work was supported in part by the National Cancer Institute, USA (Grant, CA-43713). [44] TOXICITY = 20 mcg/ml AMAat 10 = 32%

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