Environmental and Molecular Mutagenesis 18:2&34 (1 991)

Evaluation of the Mutagenicity of P-Myrcene in Mammalian Cells In Vitro Barbara Kauderer, Helena Zamith, Francisco J.R. Paumgartten, and

Gunter Speit Abteilung Klinische Genetik, Universitat Ulm, Ulm, Federal Republic of Germany

(B.K., G.S.); Fundaqao Oswaldo Cruz, INCQS, Rio de Janeiro, Brasil (H.Z., F.J.R.P.)

The genotoxicity of the terpene (3-myrcene was evaluated in mammalian cells in vitro. Myrcene is the major constituent of oil of bay and hop which are used in the manufacture of alcoholic beverages. Myrcene is also present in lemon grass (Cymbopo on citratus), a plant widely

shown that myrcene is a very potent analgesic substance and might be an alternative to the already available analgesic drugs. Myrcene was tested up to 1,000 kg/ml (limit of solubility) in the presence and absence of S9-mix and did not induce chromosome aberrations and sister chromatid exchanges (SCEs) in human lympho- cytes in vitro. Neither the mitotic index nor the proliferation index was influenced by the myrcene treatment. Myrcene did not cause in- creased mutation frequencies at the hprt-locus

used in Brazilian ? olk medicine. Recently, it was

in V79-cells. Tests with and without S9-mix re- vealed negative results. There was no indication for induced cytotoxicity. However, myrcene re- duced the SCE-inducing effect of cyclophospha- mide in human lymphocytes in a dose dependent manner and also reduced the toxic and muta- genic effect of cyclophosphamide in V79-cells. Under the same test conditions, SCE induction by ethyl methanesulfonate (EMS) and benzo [alpy- rene (BP) was not significantly influenced by simultaneous myrcene treatment. The in vitro results show that myrcene is not mutagenic in mammalian cells, but has antimutagenic prop- erties. The possibility that myrcene exerts its antimutagenic activity by inhibiting certain forms of the cytochrome P-450 isoenzymes re- quired for activation of premutagens and pre- carcinogenes is discussed.

Key words: in vitro chromosome aberration test, in vitro SCE-test, V79-HPRT-gene mutation test, antimutagenicity, cytochrome P-450

INTRODUCTION The terpene P-myrcene is the major constitutent of oil of

bay and hop which are used in the manufacture of alcoholic beverages. Myrcene is also present in lemon grass (Cym- bupogon citrutus), a plant widely used in Brazilian folk medicine. Recently, it was shown that P-myrcene is a very potent analgesic substance and might be an alternative to the already available analgesic drugs and the first member of a new family of such drugs [Lorenzetti et al., 1988; Sarti et al. 19881. In the course of a toxicological study of P-myrcene we tested the mutagenic potential of this substance in mammalian cells in vitro. We performed chromosome aberration tests and sister chromatid exchange (SCE)-tests with human lymphocytes and the V79 HPRT assay.

It has been reported that certain compounds containing carbon-carbon double bonds inactivate liver microsomal cytochrome P-450 (Ortiz De Montellano and Mico, 19801. Since P-myrcene has olifinic double bonds in its structure and an interaction of myrcene with liver microsomal en- zymes has been discussed (Madyastha and Srivatsan, 1987; Austin et al., 19881 we also investigated a possible influence of myrcene on the genotoxocity of SPmix-activated cyclo- phosphamide (CP) and other mutagens. 0 1991 Wiley-Liss, Inc.

MATERIALS AND METHODS In Vitro Chromosome Aberration Test With Human Lymphocytes

The in vitro chromosome aberration test was performed according to published recommendations [Preston et al., 19871. Peripheral blood samples were obtained from two healthy probands (one male, one female, both non- smokers). 0.3 ml whole blood was cultivated in chromo- some medium B (Biochrom, final volume: 2.5 ml) for 72 hr at 37°C. Colcemid (5 X M) was added for the last two hr. The test substances were added after 48 hr either for a period of 24 hr without S9-mix or for 2 hr with SPmix. An S-9-mix was prepared from liver homogenate from Aroclor- 1254-induced rats with an addition of cofactors. Fifty microliters S9-mix was directly added to the cultures and incubated for 2 hr at 37°C in a shaking water bath. After the treatment, the medium was changed. The cultures were

Received July 20, 1990; revised and accepted February 2 I , 199 I .

Address reprint requests to Dr. Gunter Speit. Abteilung Klinische Genetik, Universitat Ulrn, Albert-Einstein-Allee 1 1, D-7900 Ulm, Federal Republic of Germany.

Mutagenicity of P-Myrcene 29

cells were treated in each experiment. The cells were exposed to myrcene or the control substances for 3 hr either in the presence or in the absence of S9-mix. The relative plating efficiency (PE1) was determined by plating 200 cells into 5 replica Petri dishes at the end of the treatment. Cells were transferred as needed during the expression period. After 7 days, 2 X lo5 cells were plated into 5 replica Petri dishes with selective medium (10 pg/ml6-thioguanine). At the time of replating into selective medium, the plating efficiency (PE2) was determined in non-selective medium (five 60 mm replica Petri dishes with 200 cells each). After 1 week, the colonies were fixed with methanol, stained, and counted. The results are expressed as mutants per million surviving cells.

centrifuged for 10 min at 900 rpm and rinsed once with Hank’s solution. The test-substances P-myrcene (MC), ethyl methanesulfonate (EMS), cyclophosphamide (CP), and benzo[a]pyrene (BP) were all obtained from Sigma. Myrcene was dissolved in ethanol and further diluted in Hank’s solution. (The final concentration of ethanol in the culture medium was 0.5%.) Chromosomes were prepared according to standard procedures. Hypotonic treatment was performed with 0.4% KC1 (37°C) for 15 min. The cells were fixed with methanol: acetic acid 3:l and the fixative was changed twice. Air dried slides were stained with Giemsa ( 5 % in Sorensen buffer). Chromosome aberrations were scored in 100 metaphases each according to the following criteria:

I . Gap: acromatic region in one chromatid (chroma- tid-type) or both chromatids (chromosome-type) smaller than the width of a chromatid.

2 . Break: acromatic region in one chromatid (chroma- tid-type) or both chromatids (chromosome-type) greater than the width of a chromatid or a discon- tinuity with displacement.

3 . Exchange: aberrations arising from an exchange between chromosomes or within a chromosome. Chromosome-type exchanges occur as dicentric or ring chromosomes in metaphase cells while chro- matid-type exchanges are recognized as different types of “exchange figures.”

The mitotic index (MI) was determined for 1,000 cells and given as number of mitoses per 1,000 cells.

In Vitro SCE-Test With Human Lymphocytes

The test conditions were essentially the same as in the chromosome aberration test with the following exceptions: BrdUrd (Sigma) was added at the start of the cultures in a concentration of 10 pg/ml. Slides were stained according to a modified fluorescent plus Giemsa technique: at least 1-day-old preparations were covered with Sorensen buffer (pH 6.8) and irradiated for 30 min with UV-light (254 nm) then incubated in 1 X SSC at 60°C for 30 min and after- wards stained with Giemsa (5% in Sorensen buffer). Twenty- five second division metaphases were evaluated for each SCE data point. The MI was determined for 1,000 cells and given as number of mitoses per 1,000 cells. The distribution of first, second, and third division metaphases was counted for 100 mitoses and the proliferation index (PI) was cal- culated according to the formula PI = ((Ml X l) + (M2 X 2) t (M3 X 3)J:lOO.

V79-HPRT-Gene Mutation Test

RESULTS Tables I and I1 summarize the results for the chromosome

aberration test with human lymphocytes in vitro. Neither a 24 hr treatment with myrcene in the absence of S9-mix nor a 2 hr treatment in the presence of S9-mix caused an induction of chromosome aberrations. Concentrations be- tween 100 and 1,000 pg/ml were tested. The maximum concentration was limited by the solubility of myrcene. There was no indication for cytotoxicity and the maximum number of chromatid breaks was 3 as in control cultures. None of these breaks showed a displaced fragment. In 2 cultures treated with myrcene (500 p.g/ml male blood, with and without S9-mix) one chromosome-type aberration was seen. In the test without SPmix, it was an additional small acentric fragment and in the trial with S9-mix, a dicentric chromosome was seen in a metaphase with 45 chromo- somes. Obviously, the dicentric chromosome resulted from a translocation between 2 chromosomes of the C-group. However, these aberrations seem to be insignificant when regarding the data as a whole. There was no dose-related increase in the number of structural chromosome aberrations and no reproducible positive response at any one of the test points. The positive controls showed a clear increase in the frequency of aberrations.

Tables I11 and IV show the results of the SCE-test. The same treatment conditions were used as in the chromosome aberration tests. There was no increase in the frequency of SCEs in the lymphocytes of both donors in the trials with and without S9-mix. Neither the MI nor the PI was influenced by the myrcene treatment. In the positive con- trols, EMS and CP clearly induced SCEs in all assays.

Myrcene in concentrations between 50 and 1,000 pglml was not toxic for V79-cells. We estimated the cytotoxicity by treating 200 cells in 4 replica Petri dishes for 2 hr in the absence and presence of S9-mix (4%) and did not find any reduction of the plating efficiency compared with untreated

I

The V79-gene mutation test was performed according to the guidelines published for CHO-cells by Li et al. [1987]. V79-cells were cultivated in 175 cm2 flasks. About 5 X lo6

control cultures (data not shown): In the V79-gene mutation test (Table V, VI), the plating efficiency was also not reduced (or at best marginally) at the end of the exposure as

30 Kauderer et al.

TABLE 1. The Effect of Myrcene on Chromosome Aberrations in Human Lymphocytes In Vitro

% Cells with Aberrations

Cells With Without Chromatid-typeb Chromosome- type Treatment Donor MI“ scored gaps gaps Gap Break Exchange Gap Break Exchange

Control f 47 100 7 1 5 1 m 48 100 6 3 3 3

Solvent f 19 100 6 3 2 3 m 47 100 12 3 10 4

Myrcene (pglml) 100 f 50 100 4 1 3 1

m 32 100 7 1 7 1 500 f 48 100 2 0 2

m 30 100 8 3 5 2 1 I 1,000 f 31 100 10 3 8 3

m 32 100 10 3 8 3 EMS (5 X lo-’ M) f 14 50 66 48 8 18 12 2 2

m 21 50 46 20 11 12

- - 1

1 I

- - - - -

- - - - - -

- - - - - - -

- - - - - - -

- - 1 - - - -

- - - 3 -

aMitotic index; number of mitoses/ 1,000 cells. bNumber of aberrations observed; for detailed explanation see Materials and Methods.

TABLE 11. The Effect of Myrcene on Chromosome Aberrations in Human Lymphocytes In Vitro in the Presence of SPMix

% Cells with aberrations

Cells With Without C hromatid-type’ C hromosome-type Treatment Donor MIa scored gaps gaps Gap Break Exchange Gap Break Exchange

Control f 41 100 2 0 2 m 60 100 3 0 2

Solvent f n.d.c in 55 100 4 3 1 3

Myrcene (pglml) I00 f 36 100 4 1 2 I

m 51 100 6 0 3 500 f 50 100 4 0 5

m 56 100 6 1 5 1,000 f 65 100 4 0 4

m 55 100 5 0 5 CP (2 x 10-4 M) f 54 50 22 12 5 5 1 3

m 59 50 30 16 5 6 1 1

- - - - - - - - - 1

- - - -

- - 1 3

-

- - - - - - - - -

I - - - - - - - - - - - - - -

- - 1 -

“Mitotic index; number of mitoses/ 1,000 cells. ’Number of aberrations observed; for detailed explanation see Materials and Methods. ‘No data due to contamination of the culture.

well as at the end of the expression time. No increase of thioguanine-resistant colonies was found compared with the control after myrcene treatment, whereas EMS and BP clearly increased the mutation frequency.

Tables VII, VIII, and IX show the influence of a combined treatment with myrcene on SCE induction by the mutagens CP, BP, and EMS in human lymphocytes. For a better demonstration of an effect, the induced SCEs (ASCE) are compared. The “calculated” value represents the SCE count minus control. The “expected” value is the sum of the ASCE values of the 2 independent treatments.

Myrcene reduced SCE-induction by CP in human lym- phocytes (Table VII). The induced SCEs (ASCE) were significantly lower after combined treatment with myrcene and CP than would be expected for an “additive” effect. This effect was dose dependent as 500 pgiml myrcene had a stronger effect than 100 Fglml and it could be demonstrated for 2 CP concentrations. Myrcene also reduced the toxic and mutagenic effect of CP in V79-cells. CP is not a very good mutagen in the HPRT test due to its toxic and clastogenic effects. However, a reproducible increase in the mutation frequency compared with the parallel control culture can be

Mutagenicity of P-Myrcene 31

TABLE IV. The Effect of Myrcene on the Frequency of SCEs In Human Lymphocytes In Vitro: Experiments With S9-Mix

Treatment MIa PIb SCE k S.E.M.

Female blood Control 43 2.0 6.0 f 0.5 S9-mix (2%) 45 2.1 7.0 zk 0.5

100 45 2 .o 6.4 zk 0.4 5 00 41 2.1 8.1 -t 0.6 1,000 47 2.2 7.0 f 0.6 CP (5 X lo-’ M) 28 1.8 37.6 zk 2.0

Control 52 2.3 9.0 zk 0.7 S9-mix (2%) 19 2.0 8.5 f 0.7

100 26 1.9 9.0 f 0.6 500 41 2.0 7.2 f 0.5 1,000 37 1.9 9.6 f 0.7 CP ( 5 x 10-5 M) 23 1.8 48.9 f 2.6

Myrcene (pg/ml)

Male blood

Myrcene (pg/ml)

aMitotic index. bProliferation index.

TABLE 111. The Effect of Myrcene on the Frequency of SCEs In Human Lymphocytes In Vitro

Treat rnent MIa PIb SCE k S.E.M.

Female blood Control 24 2.0 8.9 f 0.7 Solvent 20 1.9 8.0 zk 0.6

100 22 1.9 8.8 k 0.7 500 27 2.0 8.3 f 0.6 1,000 21 1.8 7.9 zk 0.7 EMS M) 18 1.8 26.3 -t 1.7

Control 45 2.0 7.9 f 0.6 Solvent 23 2.0 8.5 f 0.5

100 24 1.8 8.7 f 0.9 5 00 23 2.0 7.8 zk 0.6 1,000 23 1.9 8.2 k 0.6 EMS (10-3 M) 25 2.2 20.8 f 0.9

Myrcene (pg/rnl)

Male blood

Myrcene ( p g i m l )

aMitotic index. bProliferation index.

TABLE V. The Effect of Myrcene on Gene Mutations in the In Vitro V79 HPRT Assay Without S9-Mix

Plating efficiency Mutation frequency/ Treatment PE1 (%) PE2 (S) 106 survivors

Control 100 100 10 Solvent 100 97 0 Myrcene (pg/ml)

100 92 103 10 500 94 95 13 1,000 90 99 5 EMS (5 x 10-3 M) 90 92 218

TABLE VI. The Effect of Myrcene on Gene Mutations in the In Vitro V79 HPRT Assay With S9-Mix

Plating efficiency Mutation frequency/ Treatment PE1 (%) PE2 (%) 1 O6 survivors

Control 100 100 6 S9-mix (4%) 84 50 0 Myrcene (pg/ml)

100 90 I I7 0 5 00 101 94 0 1,000 108 98 4 BP (5 X lo-’ M) 78 100 262

demonstrated. Table X shows that the acute toxicity of CP summarizes the results for BP and shows that BP induced (PE1) was clearly reduced when myrcene was added simul- SCEs in human lymphocytes without S9-mix added. There taneously. The frequency of induced mutations at the HPRT was no dose effect response and S9-mix did not enhance locus was reduced to control level. In contrast to the results SCE induction. This effect had been published earlier with CP (Table VII) myrcene had only a marginal effect on [Mehnert et al., 19841. Neither in the absence nor in the SCEs induced by BP in human lymphocytes. Table VIII presence of S9-mix, a clear effect of rnyrcene on BP-

32 Kauderer et al.

TABLE VII. The Effect of Myrcene on CP-Induced SCEs In Human Lymphocytes

ASCE' Treatment MIa P P SCE & S.E.M. Calculated Expected

Control 24 2.1 6.9 f 0.7 MC 100 pgiml 45 2.0 6.4 f 0.4 -0.5

CP 2 x 10-5 M 34 2.0 15.2 5 1.1 8.3 CP 5 x 10-5 M 39 I .9 33.4 & 1.6 26.5

MC 500 pg/ml 41 2.1 8.1 f 0.6 1.2

CP 2 x 1 0 - ~ M

CP 2 x 10-5 M

CP 5 x 10-5 M

CP 5 x 10-5 M

+ MC 100 pgiml 30 2.1 11.2 f 0.8 4.3 7.8

+ MC 500 pglml 31 2.4 9.3 f 0.8 2.4 9.5

+ MC 100 pg/ml 39 2.1 16.9 * 1.1 10.0 26.0

+ MC 500 pgiml 37 2.1 10.5 k 0.8 3.6 27.7

"Mitotic index. bProliferation index. 'Induced SCEs; for detailed explanation see the text.

TABLE VIII. The Effect of Myrcene on BP-Induced SCEs In Human Lymphocytes

A S C E ~ Treatment S9-mix PI" SCE f S.E.M. Calculated Expected

Control - 2.4 7.1 k 0.5 - MC 500 pg/ml - 2.0 6.4 fi 0.4 -0.7 BP 10-4 M - I .7 17.2 k 1.0 10.1 BP 2 X lop4 M - 2. I 17.5 k 1.2 10.4 BP M + MC 500 p/ml - 1.9 14.1 k 0.6 7.0 9.4 BP 2 X M + MC 500 pgiml - I .4 16.4 k 9.3 9.3 9.7 Control + 2.0 6.6 k 0.5 - MC 500 pg/ml + 2.2 7.3 f 0.5 0.7 BP M + 1.8 14.6 f 1.0 8.0 BP 2 x 10-4 M + 1.7 12.9 k 0.8 6.3 BP M + MC 500 p/ml + 2.0 15.6 f 1.2 9.0 8.7 BP 2 X M + MC 500 pg/mI + 1.9 15.5 f 1.0 8.9 7.0

"Proliferation index. hInduced SCEs; for detailed explanation see the text.

induced SCEs could be demonstrated. In trials without S9-mix, the induced SCE-frequency was somewhat lower than expected whereas in experiments with S9-mix they were even higher. However, in a second independent experiment, SCE frequencies induced by M BP were slightly lower in the presence of myrcene while those induced by 2 X lop4 M BP were slightly higher when myrcene was present. Myrcene did not reduce SCE induc- tion by EMS, a directly acting mutagen (Table IX).

DISCUSSION The terpene 0-myrcene was investigated for mutagenic

properties in mammalian cells in vitro. Our results clearly

show that myrcene does neither induce chromosome aber- rations nor gene mutations in cultivated mammalian cells under generally accepted test conditions (Li et al., 1987; Preston et al., 1987). We used 2 different cell culture systems-human lymphocytes for the chromosome aberra- tion test and V79 Chinese hamster cells for the gene mutation test. Aroclor-induced rat liver S9-mix was used as an extrinsic metabolic activation system. It is known that myrcene is metabolized by rat liver microsomal enzymes [Madyastha and Srivatsan, 19871, but obviously no muta- genic metabolites are produced. In addition, myrcene had no effect on the frequencies of SCEs which are a very sensitive indirect indicator for mutagens. These findings support the negative results from the 2 tests mentioned above. Up to a

Mutagenicity of p-Myrcene 33

TABLE IX. The Effect of Myrcene on EMS-Induced SCEs In Human Lymphocytes

ASCE' Treatment MIa PIb SCE * S.E.M. Calculated Expected

Control 42 2.4 8.5 f 0.6 MC 100 pglml 22 1.9 8.8 f 0.7 0.3

EMS 10-3 M 29 2.2 24.9 f 1.1 16.4 EMS 2 x 10-3 M 45 2.2 36.7 f 1.5 28.2

MC 500 pglml 27 2.0 8.3 k 0.6 -0.2

EMS 10-3

EMS 10-3 M

EMS 2 x 10-3 M

EMS 2 x 10-3 M

+ MC 100 pglml 35 2.2 27.6 f 1.5 19.1 16.7

+ MC 500 pglml 32 2.2 23.8 + 1.2 15.3 16.2

+ MC 100 pglml 31 1.9 42.6 k 1.1 34.1 28.5

+ MC 500 pg/ml 26 2.1 41.4 + 1.8 32.9 28.0

aMitotic index. bProliferation index. 'Induced SCEs; for detailed explanation see the text.

TABLE X. The Effect of Mvrcene on CP-Induced Mutations in V79-Cells

Treatment Induced HPRTl

PEI (%) PE2 (%) I O6 survivorsa

CP 2 X M 65 96 15 CP 2 X + MC (500 pg/ml) 92 97 0 CP 5 X IOP5 M 18 87 22 CP 5 X + MC (500 pg/ml) 93 93 I

aSpontaneous mutation frequency: 3 / 1 06.

concentration of I mg/ml which represented the limit of solubility there were no indications for genotoxic or cyto- toxic properties of myrcene. To finally state that myrcene is non-mutagenic, one in vivo test should be performed to pay attention to the mammalian metabolism in the intact animal.

One interesting aspect of our study was the antimutagenic effect caused by myrcene. Myrcene very efficiently reduced CP-induced SCEs in human lymphocytes but did not effect the genotoxicity of EMS and BP. Our results furthermore indicate that myrcene can also reduce the cytotoxic and mutagenic action of S9-mix-activated CP in V79 Chinese hamster cells. It is known that certain compounds containing carbon-carbon double bonds inactivate liver microsomal cytochrome P-450 [Ortiz De Montellano and Mico, 19801. Since P-myrcene has olefinic double bonds in its structure P-myrcene may interfere with the enzymatic process of CP activation. In this regard, our results show striking similar- ities to the antimutagenic action of vitamin A. Vitamin A (retinol) strongly inhibited the genotoxicity of the premuta- gens CP and aflatoxin B1 (AFB), but not those of BP or dimethylbenzanthrazene (DMBA) in the Ames-test and in SCE and chromosome aberration studies. Since the former

and the latter 2 mutagens are believed to be activated by different microsomal cytochrome P-450 isoenzymes it was proposed that retinoids may inhibit only certain forms of the cytochrome P-450 [Qin and Huang, 1985; Qin et al., 19851. Furthermore, it was reported that concurrent treatment of retinol with dimethylnitrosamine (DMN) or diethylnitro- samine (DEN) in the presence of Aroclor- 1254-induced S9-mix resulted in a significant reduction of SCEs and mutations induced by these 2 mutagens [Huang, 19871. The above mentioned experiments were performed either in bacteria or in mammalian cells in vitro with an S9-mix as an activation system. These in vitro findings were supported by an in vivo study [Qin and Huang, 19861. It could be shown, that mice fed a vitamin A supplemented diet had signifi- cantly fewer SCEs in bone marrow cells than mice fed a vitamin A deficient diet, after both groups were treated with AFB. S9-mix prepared from livers of mice fed a vitamin A supplemented diet were consistently less potent than those prepared from the livers of mice fed a vitamin A dificient diet in activating AFB to its mutagenic metabolits in the Ames-test. However, no such difference of activation potential in the two S9-preparations were observed when the

34 Kauderer et al.

premutagen BP was used in the Ames-test [Qin and Huang, 19861.

Of the many isoenzymes related to the “cytochrome P-450 complex,” two major inducible species can be distinguished: the phenobarbital (PB)-inducible P-450 (PB- P-450) and the 3-methylcholanthren (MCA)-inducible P- 448 (MCA-P-448). Some premutagens such as CP and AFB seem to require PB-P-450 for metabolic activation, while others like polycyclic aromatic hydrocarbons (PAH) are premutagens activated mainly by MCA-P-448 [Huang, 19871. The antimutagenic effect of retinol might thus be due to its ability to inhibit the PB-P-450 forms of the activating liver enzymes.

The terpene P-myrcene is structurally related to retinoids and seems to behave similarly with respect to its antimuta- genic properties. It reduced the genotoxic effects of CP but had no effect on BP or the directly acting mutagen EMS. In the meantime we have found that myrcene has also no effect on SCEs induced by BP or DMBA in V79-cells but clearly reduced SCEs induced by CP or AFB (manuscript in preparation). These findings strongly support the idea that myrcene specifically inhibits PC-P-450 of the S9-mix. Further studies have to show whether the antimutagenic activity of myrcene is also relevant for the in vivo situation. For vitamin A and dietary carrot it could be demonstrated that an antimutagenic effect also exists in vivo [Qin and Huang, 1986; Darroudi et al., 19881. However, adminis- tration of P-myrcene to rats did not result in any significant effect on the hepatic drug metabolizing enzymes and in particular no significant change in the amount of PB-P-450 was found [Madyastha and Srivatsan, 1987; Austin et al., 19881. Thus, the present results give interesting indications for a possible antimutagenic activity of P-myrcene but further studies are needed to support these findings and to elucidate the underlying mechanism(s).

ACKNOWLEDGMENTS We are grateful to Mrs. Sabine Haupter for skillful

technical assistance and Mrs. Barbel Tabib Khoie for secretarial assistance. This project was supported by the Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ) .

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I7 1539-549.

Accepted by- H.E. Holden