Mutagenicity of isoquinoline alkaloids, especially of the aporphine type

Mutation Research, 240 (1990) 267-279 Elsevier

267

MUTGEN 01530

Mutagenicity of isoquinoline alkaloids, especially of the aporphine type Tomio Nozaka 1, Fujio Watanabe l, Shin-ichi Tadaki 1, Masazo Ishino 1, Isao Morimoto 1, Jun-ichi Kunitomo 2, Hisashi Ishii 3 and Shinsaku Natori 4 1 Saitarna Institute of Public Health, 639-1 Kamiokubo, Urawa-shi, Saitarna 338 (Japan), 2 Faculty of Pharmaceutical Sciences, Mukogawa Women's University, 11-68 Koshien Kyuban-cho, Nishinorniya-shi, Hyogo 663 (Japan), 3 Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Chiba-shi, Chiba 260 (Japan)and 4 Meiji College of Pharmacy, 1-22-1 Yato-cho, Tanashi-shi, Tokyo 188 (Japan) (Received 19 September 1989) (Accepted 9 November 1989)

Keywords: Isoquinoline alkaloids; Aporphine alkaloids; Liriodenine; Mutagenicity; Ames assay; Metabolic activation

Summary The mutagenicity of 44 isoquinoline alkaloids was tested in Salmonella typhimurium TA100 and TA98 in the presence or absence of $9 mix. The alkaloids tested included compounds from the isoquinoline, benzylisoquinoline, bisbenzylisoquinoline, monoterpene isoquinoline, berberine, morphinane, hasubanan, benzo[c]phenanthridine and aporphine groups. Among the alkaloids tested, liriodenine was the most potent mutagen for TA100 and roemerine was the most potent for TA98. A clear structure-mutagenicity relationship was observed in a series of aporphine alkaloids (aporphine, dehydroaporphine, 7-oxoaporphine and 4,5-dioxoaporphine), and 10,11-non-substituted aporphines were suggested to exert their mutagenicity through metabolic activation of the 10,11 positions, possibly as the 10,11-epoxides.

In the course of our continuing search for mutagenic principles in crude drugs, we reported the mutagenicity of liriodenine (35), an aporphine alkaloid isolated from sinomeni caulis et rhizoma, which originates from the root, root-stalk and stem of Sinomenium aeutum Rehder et Wilson (Menispermaceae), towards Salmonella typhimurium TA100 and TA98 in the presence of $9 mix (Nozaka et al., 1988). Liriodenine (35) requires microsomal activation for the mutagenicity. The mutagenic potency of liriodenine (35) was comparable to that of the ubiquitous environmental carcinogen, benzo[ a ]pyrene.

Correspondence: Dr. T. Nozaka, Saitama Institute of Public Health, Kamiokubi 639-1, Urawa-shi, Saitama-ken 338 (Japan).

The isoquinoline alkaloids (including aporphine alkaloids) are one of the more numerous groups of natural products and have been isolated from a variety of plants including Papaveraceae, Lauraceae, Magnoliaceae, Menispermaceae and Annonaceae. Some isoquinoline alkaloids such as morphine, papaverine and berberine are of importance because of their medicinal use. However, few studies of the genotoxicity of isoquinoline alkaloids have been reported. Nagao et al. (1977) reported that isoquinoline and 3-methylisoquinoline were n o n - m u t a g e n i c to Salmonella typhimurium TA100 and TA98 in the presence or absence of $9 mix. Suter and Matter-Jaeger (1984) reported the mutagenicity in TA1535 and TA98 of apomorphine, a rearrangement product of morphine, but a clear dose-effect relationship was only found for strain TA1537.

0165-1218/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

268 TABLE 1 MUTAGENICITY MIX

OF ISOQUINOLINE

ALKALOIDS

IN SALMONELLA

T E S T E R S T R A I N S i n T H E P R E S E N C E O F $9

M u t a g e n i c i t y was assayed by the p r e i n c u b a t i o n method. C o m p o u n d (No.)

C A S No.

Mutagenicity Conclusions TA100

Revertants//xg/plate ~ TA98

TAI00

TA98

lsoquinoline-type alkaloids (8-Amino-N- formyl-7-methoxy-6-methyl1 2 , 3 , 4 - t e t r a h y d r o - l - i s o q u i n o l y l ) m e t h a n o l (1) (N-Formyl-5,7,8-trimethoxy-6-methyl-l,2,3,4t e t r a h y d r o - l - i s o q u i n o l y l ) m e t h y l b e n z o a t e (2) ( N-Formyl-5,7,8-trimethoxy-6-methyl1 , 2 , 3 , 4 - t e t r a h y d r o - l - i s o q u i n o l y l ) m e t h a n o l (3) (N-Formyl-7-methoxy-6-methyl-5,8-dioxo-l,2,3,4,5,8hexahydro- 1-isoquinolyl)methanol (4)

*

-

nc

nc

*

-

nc

nc

*

-

nc

DC

*

-

nc

nc

61-25-6 524-20-9 85-64-3

+ -

3 nc nc

nc nc nc

481-49-2 548-40-3 518-34-3 518-94-5 57-94-3

-

316-42-7

-

633-65-8 522-97-4 483-14-7

-

Benzylisoquinoline-type alkaloids P a p a v e r i n e h y d r o c h l o r i d e (5) A r m e p a v i n e (6) L a u d a n i n e (7)

Bisbenzylisoquinoline-type alkaloids C e p h a r a n t h i n e (8) O x y a c a n t h i n e (9) T e t r a n d r i n e (10) C y c l e a n i n e (11) d - T u b o c u r a r i n e chloride (12)

i

nc

nc

nc

nc

nc

nc

nc

nc

nc

nc

nc

fie

Monoterpeneisoquinoline-type alkaloid E m e t i n e h y d r o c h l o r i d e (13)

Berberine-type alkaloids Berberine h y d r o c h l o r i d e (14) T e t r a h y d r o b e r b e r i n e (15) ( - ) - T e t r a h y d r o p a l m a t i n e (16)

nc

tic

nc

nc

nc

nc

4090-18-0 115-53-7

nc

nc

nc

nc

24148-86-5 1805-85-2

nc

nc

nc

nc

I

I

Morphinane-type alkaloids S i n o a c u t i n e (17)

Sinomenine (18) Hasubanan-type alkaloids A k n a d i n i n e (19) H a s u b a n o n i n e (20)

Benzo[c]phenanthridine-type alkaloids C h e l e r y t h r i n e (21) chloride O x y c h e l e r y t h r i n e (22)

34316-15-9 28342-33-8

nc

NC

+

2

nc

+

4

5

nc

nc

nc 2 29 2O8 2O 97

2 4 103 4 17

Aporphine-type alkaloids D i c e n t r i n e (23) D o m e s t i c i n e (24) Laurifoline (25) O - M e t h y l d o m e s t i c i n e (26) O - N o r n u c i f e r i n e (27) R o e m e r i n e (28) Steporphine (29) U s h i n s u n i n e (30)

517-66-8 476-71-5 7224-61-5 2565-01-7 3153-55-7 548-08-3 24191-98-8 3175-89-1

+ + + + +

nc

269 T A B L E 1 (continued) C o m p o u n d (No.)

CAS No.

Mutagenicity Conclusions

Revertants/~g/plate a

TA100

TA98

TA100

TA98

77784-22-6 55898-15-2 111017-06-2 * 475-75-2 15444-20-9 23740-25-2 70403-81-5 * * 96681-50-4

+ + + + + + + -

+

18

23

-

nc

nc

19 nc 330 107 114 37

2 nc 40 nc 10 nc

nc

nc

35 nc

8

88741-67-7 83287-02-9

+

+ +

nc 5

2 5

+

nc

26

Aporphine-type alkaloids Dehydrocrebanine (31) Dehydronantenine (32) N-Demethyl-N- formyldehydronuciferine (33) 1,2,10-Trimethoxydehydroaporphine (34) Liriodenine (35) Lysicamine (36) 9-Methoxy-l,2-methylenedioxy-7-oxoaporphine (37) 1,2,9-Tfimethoxy-7-oxoaporphine (38) 1,2,10-Trimethoxy-7-oxoaporphine (39) 4,5-Dioxodehydrocrebanine (40) Bianfugecine (41) 6-Hydroxy-5,10-dimethoxy-7Hdibenzo[ de, h ]quinolin-7-one (42) Menisporphine (43) 5,6,10-Trimethoxy-7H-dibenzo[ de, h ]quinolin-7-one (44)

88741-68-8

+ -

+ -

+ -

-

+ -

nc

a All results are single points (each point is the average of 3 plates) from the linear region of the dose-response curve and each value is the m a x i m u m number of revertants//~g/plate that a c o m p o u n d induced. Spontaneous revertants have been subtracted and were 113 (TA100, + $9), 36 (TA98, + $9) per plate (average of values in 10 experiments). *, CAS No. not yet available; - , not mutagenic; + , mutagenic (the samples giving over 250 or 100 revertant colonies per plate with strain TA100 or TA98, respectively, were concluded to be mutagenic); nc, not calculated.

The aim of this work was to elucidate the structure-mutagenicity relationship in isoquinoline alkaloids and the possible modes of action of potently mutagenic aporphines such as liriodenine (35), as well as to obtain basic data on the genotoxicity of isoquinoline alkaloids. The mutagenicity of 44 isoquinoline alkaloids, including isoquinoline, benzylisoquinoline, monoterpene isoquinoline, berberine, morphinane, hasubanan, benzo[c]phenanthridine and aporphlne alkaloids, was tested on Salmonella typhimurium TA100 or TA98 in the presence or absence of S9 from rats treated with phenobarbital and fl-naphthoflavone. Materials and methods

d-tubocurarine chloride (12) and emetine hydrochloride (13) were obtained from Tokyo Kasei Kogyo Co., Ltd., Tokyo. Four isoquinoline derivatives (all synthetic) (1-4) were provided by Prof. A. Kubo (Kubo et al., 1986), Meiji College of Pharmacy (Tokyo). Two benzo[c]phenanthridine alkaloids (chelerythrine (21) chloride and oxychelerythrine (22)) were obtained by one of the authors (H.I.) (Ishii et al., 1977). Two benzylisoquinoline (6, 7), 4 bisbenzylisoquinoline (8-11), 2 berberine (15, 16), 1 morphinane (17), 2 hasubanan (19, 20) and 20 aporphine alkaloids (23-32, 34, 36-44) (among these 31 alkaloids, 1,2,9-trimethoxy-7-oxoaporphine (38), 1,2,10-trimethoxy7-oxoaporphine (39), 6-hydroxy-5,10-dimethoxy7H-dibenzo[de,h]quinolin-7-one (42) and 5,6,10-

Chemicals

trimethoxy-7H-dibenzo[ de, h ]quinoline-7-one (44)

Table 1 gives the list of chemicals tested and their Chemical Abstract Services (CAS) registry numbers. The structures of the isoquinoline alkaloids tested are shown in Fig. 1. Papaverine hydrochloride (5), berberine hydrochloride (14),

were synthetic and the others had been isolated from plants) were obtained by one of us (J.K.) (Kunitomo et al., 1970, 1979, 1980, 1981, 1982, 1986). Liriodenine (35), sinomenine (18) and Ndemethyl-N-formyldehydronuciferine (33) were

270 Isoquinoline-type C H 3 ~

0 C H 3 ~

~CH3 C H 3 ~

C H OCH3 3 ~

CH30~N-CHOcH30 ~ N - C H O

~

C H 3 0 ~ , I I N - C H 0 CH30@~f'N-CHO 0 CH20H OCH3 CH20H o~ ~o~o~

~o~

(Z)

(i)

(l)

(i)

Benzylisoquinoline-type CH30~ ~ . ~ CH30~

CH30"~ HCI

N-CH3CH30~ ~ N - C H

HO" ~"

CH30

OCH3 (5_)

(!)

3

I OH (i)

Bisbenzylisoquinoline-type OCH3

~

HO(8)

v

(9)

CH30

CH30~

K

II +N'-c"3

~O~o~~ 0

~o~\~

"'H

~ ~ v

(I__0 )

v

OCH3

(I__i ) Fig.l. Structuresof isoquinolinealkaloidstested.

~

v

(12)

-OCH3

271

Monoterpene isoquinoline-type C H 3 0 ~ H~ ~ " 2

5

H'"L. H CH2

OCH3

2HCI (13)

Berberine-type O- .-C,..A

< 0-.-~/~

A

CH30~ ~

o~~,
v (14)

OCH3 ~

0

C H (15)

3

~.~...~.OCH3 (16)

Morphinane-type

~o7~

~o~

H O ~ CH30" ~

HO ~ -CH3

'•'-N-CH "HH

O~

0

OCH3 (18)

(17) Hasubanan-type CH30

CH30 ~

~.~o 0-w~" ~ OCH3 OCH3

~._~

O~ y

~OCH3 OCH3 (20)

(1_29) Fig. 1 (continued).

3

272

BenzoEc]phenanthridine-type

~

cH3oi

0

o>

CH30

OCH3

0CH3

(21)

(22)

Aporphine-type a) Aporphine-type ( 0 ~

5

o~~-~

C H 3 0 ~

~o

CH30- .. CH30 I ~.,,N-CH

~ .oX ~ + ~

CH30~

""H 3

~Y 0CH3

~___0

(23)

~o~

0

OH

(24)

l__o

(2__5)

o.~

(26)

o~~ 0

-CH3 OH

(2._7)

(28)

(29)

(3.._00)

b) Dehydroaporphine-type


c~3o_~,

c~3o ~ -CH3

OCH3 (3_~i)

c~3°~

W~,~,,~jN-CHO

~ O

~

~

CH30 (32)

(33) Fig. 1 (continued).

(34)

.~_CH3

273 c) 7-Oxoaporphine-type

3

0 2

4

5

CH30~ CH30 ~ N

9 (35)

~o~~

~0 OCH 3

(37)

(3--6) C H 3 0 ~

CH30~ON

CH30

CH30 OCH 3

(39)

(38) d) 4,5-Dioxoaporphine-type

~o~ 0"i~ 10

~ 0

OCH3 e)

~H3

(40)

7-Oxoisoaporphine-type

cH3~~2 c~3o.~ cH3o~ CH30 6~NI Horn cH3o~~ CH30o ~ o~f~bll

o

o

8, io

3

OCH3 (41)

~ O C H

3

OCH3 (42)

Fig.1(continued).

isolated from sinomeni caulis et rhizoma (Nozaka et al., 1987, 1988). All the compounds were checked for chemical purity by thin-layer chromatography

(43)

(44)

(TLC) and high-performance liquid chromatography (HPLC) techniques. No impurities were found. Dimethyl sulfoxide (DMSO), benzo[a]pyrene, /3-

274 naphthoflavone, L-histidine. HC1. H20 and biotin were purchased from Wako Pure Chemical Industries, Ltd. NADH, N A D P H and G-6-P were purchased from Oriental Yeast Co., Ltd. Furylfuramide (trade name AF-2) was provided by Ueno Pharmaceutical Co., Ltd.

Bacteria Salmonella typhimurium TA100 and TA98 were from stocks at the National Cancer Center Research Institute (Tokyo), which had originally been provided by Prof. B.N. Ames (University of California). Overnight cultures of strains TA100 and TA98 were prepared in nutrient broth at 3 7 ° C with shaking. For inoculations, stock cultures that had been stored at - 8 0 ° C were used.

Preparation of $9 mix Male Sprague-Dawley rats (100-120 g) received intraperitoneal injections of phenobarbital 5 days and /3-naphthoflavone 1 day before they were killed. The liver homogenate was centrifuged at 3000 × g for 10 min and then the resulting supernatant solution was collected and centrifuged at 9000 × g for 10 rain. The $9 fraction was stored at - 8 0 ° C in a Revco freezer. The $9 mixture used had the same composition as that used by Yahagi (1975): 50/~mole of sodium phosphate buffer (pH 7.4), 4 /~mole of MgC12, 16.6 /~mole of KC1, 2.5 /~mole of G-6-P, 2 #mole of NADPH, 2 /~mole of N A D H and 150 /~1 of $9 fraction in a total volume of 0.5 ml.

Control Every experiment contained positive controls (benzo[a]pyrene and furylfuramide (AF-2)) to confirm the activity of the metabolic activating system and the susceptibility of the bacteria to mutation, as well as negative controls in the form of sterility controls and incubations without test compounds. Spontaneous revertants amounted to 32 ± 13 (TA98, - $ 9 ) , 36 + 12 (TA98, +$9), 110 ± 12 (TA100, - $9) and 113 ± 11 (TA100, + $9). Furylfuramide 0.2 /~g yielded 395 His + revertants/plate from TA98 without $9 mix and 0.02 /~g yielded 612 His + revertants/plate from TA100 without $9 mix. Benzo[a]pyrene 5 /~g yielded 273 His + revertants/plate from TA98 and 801 His + revertants/plate from TA100 with $9 mix.

Mutation assay The assay was carried out as described by Ames et al. (1975) with some modifications (Yahagi, 1975) using various concentrations of test compounds with strains TA98 and TA100 of Salmonella typhimurium with or without $9 mix. The mixture, consisting of test compound (in 100 /xl of DMSO), 500 /~1 of $9 mix (or 500 #1 of phosphate buffer) and 100 /~I of the bacterial suspension, was preincubated for 20 rain at 37 ° C with gentle shaking in a test tube. 2000/~1 of top agar (0.55% agar, 0.55% NaC1, 50 /~M L-histidine, 50 t~M biotin, pH 7.4, 45°C) was then added in the test tube and poured onto a Petri dish with minimal agar (1.5% agar, Vogel-Bonner E medium, containing 2% glucose). After incubation for 2 days, colonies (His + revertants) were counted. Results

Forty-four isoquinoline alkaloids were tested for mutagenicity to Salmonella typhimurium TA100 and TA98 in the presence or absence of $9 mix. The samples giving over 100 or 250 revertant colonies per plate with strain TA98 or TA100, respectively, were concluded to be positive. Mutagenic potencies of the compounds examined were compared in terms of the numbers of revertants/ plate per/~g.

General results In the presence of $9 mix, 16 alkaloids were mutagenic for Salmonella typhimurium TA100 and 14 for TA98. The results are summarized in Table 1. In the absence of $9 mix, only 3 alkaloids, berberine hydrochloride (14), chelerythrine (21) chloride and 5,6,10-trimethoxy-7H-dibenzo[ de, h ]quinolin-7-one (44) induced mutation in Salmonella typhimurium TA98. Berberine hydrochloride (14) and chelerythrine (21) chloride were weakly mutagenic, whereas 5,6,10-trimethoxy-7H-dibenzo-[de, h]quinolin-7-one (44) was about 5 times more mutagenic than 14 and 21 (Table 2). None of the isoquinoline alkaloids tested showed mutagenicity in Salmonella typhimurium TA100 in the absence of $9 mix. A comparison of mutagenicity data for the 44 isoquinoline alkaloids in Salmonella typhimurium

275 TABLE 2

Berberine-type alkaloids

COMPARISON OF MUTAGENIC ALKALOIDS ON TA98 IN THE ABSENCE OF $9 MIX

In the absence of $9 mix, berberine hydrochloride (14) was weakly mutagenic on TA98 whereas tetrahydroberberine (15) and (-)-tetrahydropalmatine (16) were non-mutagenic. In the presence of $9 mix, all 3 alkaloids tested were nonmutagenic to both TA100 and TA98.

Mutagenicity was assayed by the preincubation method. Mutagenicity (revertants/ /Lg/plate ~)

Compound (No.)

CAS No.

Berberine hydrochloride (14) Chelerythrine (21) chloride 5,6,10-Trimethoxy-7H-dibenzo[ de, h ]quinolin-7-one (44)

633-65-8 34316-15-9

2 3

88741-68-8

13

a All results are single points (each point is the average of 3 plates) from the linear region of the dose-response curve and each value is the maximum number of revertants//~g/plate that a compound induced. Spontaneous revertants have been subtracted and were 32 (TA98, - $ 9 ) per plate (average of values in 10 experiments). The samples giving over 100 revertant colonies per plate were concluded to be mutagenic. No isoquinoline alkaloid tested showed mutagenicity in TA100 in the absence of $9 mix.

TA98 and TA100 in the presence or absence of $9 mix indicated that of the compounds tested liriodenine (35) was the most potent mutagen on TA100 and roemerine (28) on TA98.

Isoquinoline-type alkaloids Four compounds (1-4) tested, which have an N-formyl group in the molecule, were non-mutagenic to both TA100 and TA98 with or without $9 mix.

Benzylisoquinoline-type alkaloids Papaverine hydrochloride (5) proved to be weakly mutagenic only to TA100 and only in the presence of $9 mix, whereas armepavine (6) and laudanine (7), which have a tetrahydro-isoquinoline ring, were non-mutagenic.

Morphinane-type alkaloids Sinoacutine (17) and sinomenine (18) were non-mutagenic to TA100 and TA98 with or without $9 mix.

Hasubanan-type alkaloids Aknadinine (19) and hasubanonine (20) were non-mutagenic to both TA100 and TA98 with or without $9 mix.

Benzo[c]phenanthridine-type alkaloids Chelerythrine (21) chloride showed weak mutagenicity only to TA98 without $9 mix, whereas oxychelerythrine (22) was weakly mutagenic only to TA100 with $9 mix.

Aporphine-type alkaloids Aporphine alkaloids are a major group of isoquinoline alkaloids, and their chemistry, pharmacology and properties have been reviewed (Guinaudeau et al., 1988; Kametani and Honda, 1985). Based upon structural differences, the aporphine alkaloids tested can be divided into 5 subclasses, namely (a) aporphine type, (b) dehydroaporphine type, (c) 7-oxoaporphine type, (d) 4,5-dioxoaporphine type and (e) 7-oxoisoaporphine type.

(1) Mutagenicity of 7-oxoisoaporphine-type alkaloids In the absence of $9 mix, 5,6,10-trimethoxy-

Bisbenzylisoquinoline-type alkaloids

7H-dibenzo[de,h]quinolin-7-one (44) induced mu-

All 5 alkaloids tested, cepharanthine (8), oxyacanthine (9), tetrandrine (10), cycleanine (11) and d-tubocurarine chloride (12) were non-mutagenic to both TA100 and TA98 with or without $9 mix.

tation in TA98. In the presence of $9 mix, 6-hydroxy-5,10-dimethoxy-7H-dibenzo[de, h ]quinolin7-one (42) and 5,6,10-trimethoxy-7H-dibenzo[de, h]quinolin-7-one (44) showed mutagenicity only for TA98 with $9 mix, whereas menisporphine (43) was mutagenic to both TA100 and TA98 with $9 mix. Thus, mutagenicity of 7-oxoisoaporphine alkaloids for TA100 or TA98 was

Monoterpene isoquinoline-type alkaloid Emetine hydrochloride (13) was non-mutagenic to both TA100 and TA98 with or without $9 mix.

276 observed, but a clear structure-activity relationship was not found.

(2) Mutagenicity of aporphine-, dehydroaporphine-, 7-oxoaporphine- and 4,5-dioxoaporphinetype alkaloids The members of these 4 types of aporphine alkaloids tested were mutagenic only with metabolic activation. None of them was found to be mutagenic in either TA100 or TA98 in the absence of microsomal activation. Thirteen alkaloids among 18 of these 4 types were mutagenic for TA100 with $9 mix and 1l of those 13 were also mutagenic for TA98 with $9 mix. A clear structure-activity relationship was found for TA100 with $9 mix, but not for TA98 with $9 mix.

I

fOOD

I--

aporphine-type alkaloids in the presence of $9 mix is shown in Fig. 2. Roemerine (28) was the most mutagenic, followed by ushinsunine (30), Onornuciferine (27) and steporphine (29). Dicentrine (23) and O-methyldomesticine (26) were weakly mutagenic, whereas laurifoline (25) and domesticine (24) were not mutagenic. Of the com-

I

I

I

I

I

I

I

I

I

I

I

I

2000

i000 +

f

---T--

800

600 + 400

200

/v

0

l

I

I

J

5

i0

15

20

Dose

(2.1) Mutagenicity of aporphine-type alkaloids for TAIO0 with $9 mix. The mutagenicity of

r

I

(~g)

()/g/plate)

Fig. 3. Dose-response curve of roemerine (28) in TA98 with $9 mix. Mutagenicity was assayed by the preincubation method. Each point is the average of 3 plates. Spontaneous revertants have not been subtracted.

pounds tested, the former 4 (28, 30, 27, 29), without any substituents on the D ring of the aporphine nucleus, were strong mutagens, while others with substituents at the 9,10 positions were weakly mutagenic or non-mutagenic for TA100 with $9 mix. It is interesting that roemerine (28) is the most potent mutagen for the frameshift strain TA98 with $9 mix among all the isoquinoline alkaloids tested (Fig. 3), and roemerine (28) seems to be more mutagenic than compounds of plant origin previously reported (Brown et al., 1980; Morimoto et al., 1983; Tikkanen et al., 1983; Yamanaka et al., 1979; Mizuta and Kanamori, 1985).

(2.2) Mutagenicity of dehydroaporphine-type alkaloids for TAIO0 with $9 mix. The dose-re0 0

i0

20

30

40

50

60

(~g) Dose

(fig/plate)

Fig. 2. Dose-response curves of aporphine-type alkaloids in TA100 with S9 mix. Mutagenicity was assayed by the preincubation method. Each point is the average of 3 plates. Spontaneous revertants have not been subtracted. © dicentrine (23); • domesticine (24); z~ laurifoline (25); • O-methyldomesticine (26); [] O-nornuciferine(27); • roemerine (28); v steporphine (29); • ushinsunine (30).

sponse plots for dehydroaporphine-type alkaloids on TA100 with $9 mix are shown in Fig. 4. Of the dehydroaporphine alkaloids tested, dehydrocrebanine (31; an 8,9-substituted dehydroaporphine) as well as N-demethyl-N-formyldehydronuciferine (33; an 8,9,10,11-unsubstituted dehydroaporphine) were mutagenic, whereas dehydronantenine (32; a 9,10-substituted dehydroaporphine) was not. As shown in the case of 1,2,10-tri-

277 I

I

I

I

700

I

I

1 2

1 4

I

I

I

I I0

I 12

I

2000

600 1600

500 400

+

1200

m

300

,o

200

-.~

800

I00 400 0

I i0

I 20

I 30

I 40

~g Dose

0

(lag/plate)

0

I 6

Fig. 4. Dose-response curves of dehydroaporphine-type alkaloids in TA100 with $9 mix. Mutagenicity was assayed by

the preincubation method. Each point is the average of 3 plates. Spontaneous revertants have not been subtracted, o dehydrocrebamne (31); • dehydronantenine (32); • N-demethyl-N-formyldehydronuciferine (33); [] 1,2,10-trimethoxydehydroaporphine (34).

methoxydehydroaporphine (34), substitution at C-10 completely abolished the mutagenicity. (2.3) Mutagenicity of 7-oxoaporphine-type alkaloids for TAIO0 with $9 mix. Fig. 5 shows the mutagenic activity of 7-oxoaporphine alkaloids. Liriodenine (35) was most mutagenic for TA100 with $9 mix, followed by 9-methoxy-l,2-methylenedioxy-7-oxoaporphine (37), lysicamine (36) and 1,2,9-trimethoxy-7-oxoaporphine (38), whereas 1,2,10-trimethoxy-7-oxoaporphine (39) was not mutagenic. As evidenced by the comparison of lysicamine (36) and 1,2,10-trimethoxy-7-oxoaporphine (39), substitution at position 10 of the 7-oxoaporphine nucleus abolished the mutagenicity.

I 8

1 14(~g)

Dose (~g/plate)

Fig. 5. Dose-response curves of 7-oxoaporphine-type alkaloids in TA100 with $9 mix. Mutagenicity was assayed by the preincubation method. Each point is the average of 3 plates. Spontaneous revertants have not been subtracted. • liriodenine (35); o lysicamine (36); • 9-methoxy-l,2-methylenedioxy-7oxoaporphine (37); [] 1,2,9-trimethoxy-7-oxoaporphine (38); A 1,2,10-trimethoxy-7-oxoaporphine (39).

oxoaporphine alkaloids. Potently mutagenic compounds were not substituted at the 10,11 positions. As evidenced by comparison of 1,2,9-trimethoxy-7-oxoaporphine (38) and 9-methoxy-l,2methylenedioxy-7-oxoaporphine (37) the 1,2methylenedioxy type was more mutagenic, suggesting that the mutagenicity of 10,11-unsubstituted aporphine alkaloids is influenced by sub!

l

!

I

I

700 600 500 4J

400

(2.4) Mutagenicity of a 4,5-dioxoaporphine-type alkaloid for TAIO0 with $9 mix. 4,5-Dioxodehydrocrebanine (40), an 8,9-substituted 4,5-dioxoaporphine alkaloid, was mutagenic for TA100 with $9 mix as shown in Fig. 6.

+

.~

300 200 i00

I

Discussion A marked structural dependence of potent mutagenicity was found among the aporphine, dehydroaporphine, 7-oxoaporphine and 4,5-di-

I0

20 30 ~g Dose(lag/plate)

Fig. 6. Dose-response curve of 4,5-dioxodehydrocrebanine (40) in TA100 with $9 mix. Mutagenicity was assayed by the preincubation method. Each point is the average of 3 plates, Spontaneous revertants have not been subtracted.

278

stituents at other positions of the aporphine nucleus. Aporphine alkaloids are not mutagenic without $9 mix but are converted to the ultimate mutagens by metabolism. The genotoxic polycyclic aromatic hydrocarbons undergo oxygen insertion reactions at a number of double bonds to form powerful electrophilic reactants, i.e., arene oxides, in the presence of mammalian liver homogenates. Such mono-oxygenase-catalyzed biotransformation of polycyclic aromatic hydrocarbons generates powerful carcinogens and mutagens (WrighL 1980). In the case of aporphine alkaloids having a heterocyclic aromatic ring, the microsomal monooxygenase presumably also plays the key role in their activation. Our data suggest that 10,11-unsubstituted aporphine alkaloids are activated through enzymatic oxidation at the 10,11 positions. In the case of benzo[a]pyrene, the 9,10epoxide is believed to be the ultimate carcinogen (Wright, 1980; Lawley, 1989). Takahashi et al. (1988) also presumed that the mutagenic metabolite of quinoline is an epoxide form. The potent mutagenicity of aporphine alkaloids may be explained by the interaction of electrophilic epoxides with Salmonella DNA. Though the occurrence of aporphine alkaloids in our environment is quite limited, the potent mutagenicity of aporphines such as liriodenine (35) originating from a crude drug suggests that further studies on the metabolic fate and genetic toxicology of aporphine alkaloids in other test systems would be worthwhile. A study along this line is in progress.

Acknowledgement The authors wish to thank Prof. A. Kubo, Meiji College of Pharmacy, for providing samples of isoquinoline alkaloids.

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