301
Mutation Research, 68 (1979) 301--305 © Elsevier/North-Holland Biomedical Press
THE MUTAGENICITY OF NATURAL PRODUCTS FROM MARINE ALGAE
J.V. LEARY 1, R. KFIR 2, j.j. SIMS I and D.W. FULBRIGHT 3 1 Department of Plant Pathology, University o f California, Riverside, CA 92521 (U.S.A.), 2 National Institute for Water Resources, Pretoria 0001 (South Africa) and 3 Department of Botany and Plant Pathology, Michigan State University, East Lansing, M148824 (U.S.A.) (Received 6 March 1979) (Revision received 19 July 1979) (Accepted 30 July 1979)
Summary 5 polyhalogenated hydrocarbon natural products isolated from the marine red alga Plocamium spp. were tested for mutagenicity in the Ames reversion assay. All 5 of the compounds induced revertants in Salmonella typhimurium strains TA100 and TA1535, indicating the mutational events involved base substitutions. One of the compounds, designated cross,conjugated ketone, was shown to be almost 200 times more effective as a mutagen than was ethyl methanesulfonate.
The use of natural products from plants as medicinals in the treatment of human disease is a centuries-old practice. Such common drugs as quinine and strychnine are natural metabolic products of plants. It is also well known that many plant products are biological hazards and can cause disease and/or death of animals, including man. Examples of such hazardous compounds are aflatoxin, which is a potent carcinogen causing angiosarcoma, and cycasin, found in the fruits of Cycas circinalis, which was shown to induce kidney and liver tumors in mammals [6]. The marine red algae are a rich source of natural products with a wide range of biological activity [2--4]. We have been studying a number of natural products from species of the marine red algae Plocamium with the aim of identifying those with antimicrobial activity [8]. Among recently isolated and structurally characterized compounds are 5 acyclic halogenated products. These structures, shown in Fig. 1, have been reported previously [9]. It was noted that these 5 natural products have a marked structural resemblance to halogenated hydrocarbons known to have high mutagenic and carcinogenic activity. Two examples of such carcinogens are (1)chloropren (2-chloro-2,3butadiene), which has been reported to induce liver injury in several roam-
302
••--Br C
l
~
Acyclic
Cl Acyclic
. CI ,* B Cl 65
| Cl 6,5
r
C
I
Br
Cl
~
C l Cl 64-
Acychc
Cl
Cl
Acyclic 6 6 Br
0
el
Cl
Cross--Conj ugoted
Ketone
Fig. 1. Chemical structures of the P l o c a m i u m spp. natural p r o d u c t s tested for m u t a g e n i c l t y .
malian species, and is suspected as a cause of skin and lung cancer in humans [10]; and (2) DBCP (1,2-dibromo-3-chloropropane) which induced stomach cancer in test rats and mice [7]. The structural similarity of the 5 new natural products we isolated to these known mutagens indicated that the algal products could have similar activities. Therefore, before continuing our studies on the possible antimicrobial activity of these compounds, we decided it would be prudent to ascertain whether they did demonstrate mutagenicity. We used the Ames reversion assay [1] with 3 strains o f Salmonella typhimurium with and w i t h o u t the liver homogenate. Thus, we could get a direct indication of the degree of mutagenic activity, the t y p e o f mutation (base substitution vs. frameshift), and whether liver enzyme induction was required for activity. Since there is evidence that approx. 90% of the substances which show positive mutagenic activity in the Ames test are also active as carcinogens, the results from these tests would also provide some indication of the possible carcinogenicity of these algal natural products. We report here the results of the Ames test on the 5 acyclic halogenated hydrocarbons from the marine red algae, Plocamium spp. Materialsand methods The 5 algal natural products tested for mutagenicity were dissolved in DMSO to the desired concentrations and added to the molten overlay agar containing the test bacteria. The S. typhimurium strains used were TA100, which detects mutagens causing DNA damage including base-pair substitutions and frameshift mutations, and TA98 which will detect frameshift mutations specifically. Both strains contained an antibiotic resistance plasmid and have been shown repeatedly to be extremely sensitive in detecting mutagenicity [1]. The c o m p o u n d s were first tested over a wide range of concentrations, usually 0.5-250 ug/plate. If a c o m p o u n d showed any increased reversion activity, a narrower range o f concentrations was then tested. The rat-liver homogenate fraction ($9 fraction) was prepared essentially by
303 the method of Garner et al. [5] as described by Ames et al. [1]. The $9 mix was constituted as described by Ames et al. [1] and 0.5 ml added to the top agar containing 0.1 ml of the bacterial tester strain and varying concentrations of the natural product being tested. A range of concentrations (5--50 #g/ml) of $9 fraction was used in all cases. Each experiment routinely included a positive control. In the tests for basesubstitution mutations, 0.1 ml of undiluted ethyl methanesulphonate (EM8) was added to the overlay agar. The positive control in the experiments involving induction of frameshift mutations by the $9 mix was 2-aminofluorene (2-AF). The other controls are described with the results.
Results When the 5 compounds were tested over a range of concentrations (0.5--250 ~g/plate), each showed mutagenic activity significantly above the controls at at least one concentration (Table 1). The most active compound appeared to be the cross-conjugated ketone, and this is dramatically obvious when the activity is calculated on a molar basis. The EMS induced 0.0438 revertants/nanomole, whereas the cross-conjugated ketone induced 8.22 revertants/nanomole. These data indicate that the cross-conjugated ketone is 187 times more effective than the known mutagen EMS. The dose--response for each of the compounds tested was different, but linear in all cases. All of the compounds showed a toxic effect by reducing the number of revertants at the higher concentrations. It appears that all of the compounds induce base-substitution mutations and not frameshift mutations. None of the compounds was active when tested with strain TA98, whereas all were active with strain TA100 (Table 2). That these were indeed base-substitution reversions was confirmed by spotting each compound onto a lawn of S. typhimurium strain 1535, specific for base substitutions. All of the compounds induced revertants significantly above the controls.
TABLE 1 R E V E R S I O N O F S. T Y P H I M U R I U M S T R A I N T A 1 0 0 I N D U C E D BY V A R I O U S C O N C E N T R A T I O N S OF THE NATURAL PRODUCTS COMPOUNDS ConcenW~ion (#g/pla~)
0.5 3 5 8 10 15 20 50 100 250
Co~nies/pla~ a AcycHc 63
AcycHc 64
AcycHc 65
AcycHc 66
CCK
38 78 126 148 154 207 207 272 213 59
60 90 164 215 267 211 189 253 119 Toxic
105 59 108 112 132 113 154 104 To~c To~c
27 69 101 133 135 57 98 50 Tox~ Toxic
57 171 173 239 370 260 175 To~c Toxic
a T h e assay w a s d o n e in t r i p l i c a t e . T h e flgures g i v e n are averages o f 2 E x p t s . D a t a g i v e n is t h e n u m b e r o f r e v e r t a n t s a b o v e t h o s e o n t h e s p o n t a n e o u s reversion c o n t r o l plates w h i c h a v e r a g e d 1 2 4 / p l a t e .
304 TABLE 2 C O M P A R I S O N OF THE A C T I V I T Y OF T H E N A T U R A L PRODUCTS IN I N D U C I N G BASE S U B S T I T U TION (TAIO0) AND FRAMESHIFT MUTATIONS (TA98) a
TA100 TA98
Acychc 63
Acyclic 64
Acyclic 65
Acyclic 66
CCK
132 4
267 1
154 2
135 0
239 2
a All o f the c o m p o u n d s w e r e used at 10 p g / p l a t e . T h e c o l o n i e s / p l a t e is t h e average o f 2 e x p t s . All assays w e r e d o n e in t m p l i c a t e . T h e d a t a g i v e n is t h e n u m b e r o f r e v e r t a n t s a b o v e t h o s e o n t h e s p o n t a n e o u s r e v e r s i o n c o n t r o l p l a t e s w h i c h averaged 1 3 8 / p l a t e .
TABLE 3 A C T I V I T Y O F $ 9 M I X I N T H E A S S A Y W I T H S. T Y P H I M U R I U M S T R A I N T A 1 0 0 $9 m i x concentration
CCK (15 #g/plate)
A c y c l i c 63 (10 pg/plate)
Acychc 64 (1O/~g/plate)
5 1O 25 50 $9 m i x m m u s m m r o s o m e s No S 9 r m x
177 a 174 119 76 224 370
105 151 102 98 176 154
253 297 265 212 293 267
a The n u m b e r s g i v e n are t h e average o f triplicate assays in 2 E x p t s . and are s h o w n as t h e n u m b e r o f c o l o nies a b o v e t h e s p o n t a n e o u s r e v e r t a n t s w h i c h averaged 1 1 7 / p l a t e .
The inclusion of $9 mix did not activate reversions of TA98 by any of the 5 compounds. However, the $9 mix appeared to reduce the reversion frequency of TA100 only slightly (Table 3), indicating that the liver homogenate has a negligible effect on the activity o f these halogenated hydrcarbons. Discussion The greatest significance of the results reported here is the finding that a natural product from marine algae has mutagenic activity much greater than a known powerful mutagen (EMS). At present, we cannot speculate as to either why such compounds would be synthesized and stored by the algal species or as to what role such compounds might have in the normal physiology of the algae. What is apparent is that natural products which are easily recoverable from marine organisms and may be of interest because of their "beneficial" properties as antimicrobial agents should be screened carefully for potential harmful properties such as mutagenicity/carcinogenicity. References 1 A m e s , B.N., J. M c C a n n a n d E. Y a m a s a k i , M e t h o d s for d e t e c t i n g c a r c i n o g e n s and m u t a g e n s w i t h t h e S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i e i t y t e s t , M u t a t i o n R e s . , 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 3 . 2 B a s l o w , M . H . , Marine P h a r m a c o l o g y . Williams a n d Wilkins, B a l t i m o r e , 1 9 6 9 . 3 F a u l k n e r , D . J . , I n t e r e s t i n g a s p e c t s o f m a r i n e natural p r o d u c t s c h e m i s t r y , T e t x a h e d z o n , 3 3 ( 1 9 7 7 ) 1421--1433.
305 4 Fenical, W., Halogenation in the R h o d o p h y t a -- A review, J. Phycol., 11 (1975) 245--259. 5 Garner, R.C., E.C. Miller and J.A. Miller, Liver microsomal m e t a b o l i s m of a fl a t oxi n B 1 t o a reactive derivative toxic to Salmonella typhimurium TA1530, Cancer Res., 32 (1972) 2058--2066. 6 Magee, P.M., and J.M. Barnes, Carcinogenic nitroso compounds , Adv. Cancer Res., 10 (1967) 163-246. 7 0 l s o n , W.A., R.T. Habermann, E.K. Weisburger, J.M. Ward and J.H. Weisbuxger, I n d u c t i o n of s t o m a c h cancer in rats and mice by halogenated aliphatic fumigants, J. Natl. Cancer Inst., 51 (1973) 1993-1996. 8 Sims, J.J., M.S. Donnel, J.V. Leafy and G.H. Lacy, Ant i mi c robi a l agents from marine algae, Antimicrob. Agents Chemother., 7 (1975) 320--321. 9 Stierle, D. Halogenated m o n o t e r p e n e s from the red alga Plocamium, Ph.D. Thesls, University of Calif., Riverside, 1977. 10 Unsigned article, Chem. Eng. News, 53 (1975) 4--5.
Mutation Research, 68 (1979) 301--305 © Elsevier/North-Holland Biomedical Press
THE MUTAGENICITY OF NATURAL PRODUCTS FROM MARINE ALGAE
J.V. LEARY 1, R. KFIR 2, j.j. SIMS I and D.W. FULBRIGHT 3 1 Department of Plant Pathology, University o f California, Riverside, CA 92521 (U.S.A.), 2 National Institute for Water Resources, Pretoria 0001 (South Africa) and 3 Department of Botany and Plant Pathology, Michigan State University, East Lansing, M148824 (U.S.A.) (Received 6 March 1979) (Revision received 19 July 1979) (Accepted 30 July 1979)
Summary 5 polyhalogenated hydrocarbon natural products isolated from the marine red alga Plocamium spp. were tested for mutagenicity in the Ames reversion assay. All 5 of the compounds induced revertants in Salmonella typhimurium strains TA100 and TA1535, indicating the mutational events involved base substitutions. One of the compounds, designated cross,conjugated ketone, was shown to be almost 200 times more effective as a mutagen than was ethyl methanesulfonate.
The use of natural products from plants as medicinals in the treatment of human disease is a centuries-old practice. Such common drugs as quinine and strychnine are natural metabolic products of plants. It is also well known that many plant products are biological hazards and can cause disease and/or death of animals, including man. Examples of such hazardous compounds are aflatoxin, which is a potent carcinogen causing angiosarcoma, and cycasin, found in the fruits of Cycas circinalis, which was shown to induce kidney and liver tumors in mammals [6]. The marine red algae are a rich source of natural products with a wide range of biological activity [2--4]. We have been studying a number of natural products from species of the marine red algae Plocamium with the aim of identifying those with antimicrobial activity [8]. Among recently isolated and structurally characterized compounds are 5 acyclic halogenated products. These structures, shown in Fig. 1, have been reported previously [9]. It was noted that these 5 natural products have a marked structural resemblance to halogenated hydrocarbons known to have high mutagenic and carcinogenic activity. Two examples of such carcinogens are (1)chloropren (2-chloro-2,3butadiene), which has been reported to induce liver injury in several roam-
302
••--Br C
l
~
Acyclic
Cl Acyclic
. CI ,* B Cl 65
| Cl 6,5
r
C
I
Br
Cl
~
C l Cl 64-
Acychc
Cl
Cl
Acyclic 6 6 Br
0
el
Cl
Cross--Conj ugoted
Ketone
Fig. 1. Chemical structures of the P l o c a m i u m spp. natural p r o d u c t s tested for m u t a g e n i c l t y .
malian species, and is suspected as a cause of skin and lung cancer in humans [10]; and (2) DBCP (1,2-dibromo-3-chloropropane) which induced stomach cancer in test rats and mice [7]. The structural similarity of the 5 new natural products we isolated to these known mutagens indicated that the algal products could have similar activities. Therefore, before continuing our studies on the possible antimicrobial activity of these compounds, we decided it would be prudent to ascertain whether they did demonstrate mutagenicity. We used the Ames reversion assay [1] with 3 strains o f Salmonella typhimurium with and w i t h o u t the liver homogenate. Thus, we could get a direct indication of the degree of mutagenic activity, the t y p e o f mutation (base substitution vs. frameshift), and whether liver enzyme induction was required for activity. Since there is evidence that approx. 90% of the substances which show positive mutagenic activity in the Ames test are also active as carcinogens, the results from these tests would also provide some indication of the possible carcinogenicity of these algal natural products. We report here the results of the Ames test on the 5 acyclic halogenated hydrocarbons from the marine red algae, Plocamium spp. Materialsand methods The 5 algal natural products tested for mutagenicity were dissolved in DMSO to the desired concentrations and added to the molten overlay agar containing the test bacteria. The S. typhimurium strains used were TA100, which detects mutagens causing DNA damage including base-pair substitutions and frameshift mutations, and TA98 which will detect frameshift mutations specifically. Both strains contained an antibiotic resistance plasmid and have been shown repeatedly to be extremely sensitive in detecting mutagenicity [1]. The c o m p o u n d s were first tested over a wide range of concentrations, usually 0.5-250 ug/plate. If a c o m p o u n d showed any increased reversion activity, a narrower range o f concentrations was then tested. The rat-liver homogenate fraction ($9 fraction) was prepared essentially by
303 the method of Garner et al. [5] as described by Ames et al. [1]. The $9 mix was constituted as described by Ames et al. [1] and 0.5 ml added to the top agar containing 0.1 ml of the bacterial tester strain and varying concentrations of the natural product being tested. A range of concentrations (5--50 #g/ml) of $9 fraction was used in all cases. Each experiment routinely included a positive control. In the tests for basesubstitution mutations, 0.1 ml of undiluted ethyl methanesulphonate (EM8) was added to the overlay agar. The positive control in the experiments involving induction of frameshift mutations by the $9 mix was 2-aminofluorene (2-AF). The other controls are described with the results.
Results When the 5 compounds were tested over a range of concentrations (0.5--250 ~g/plate), each showed mutagenic activity significantly above the controls at at least one concentration (Table 1). The most active compound appeared to be the cross-conjugated ketone, and this is dramatically obvious when the activity is calculated on a molar basis. The EMS induced 0.0438 revertants/nanomole, whereas the cross-conjugated ketone induced 8.22 revertants/nanomole. These data indicate that the cross-conjugated ketone is 187 times more effective than the known mutagen EMS. The dose--response for each of the compounds tested was different, but linear in all cases. All of the compounds showed a toxic effect by reducing the number of revertants at the higher concentrations. It appears that all of the compounds induce base-substitution mutations and not frameshift mutations. None of the compounds was active when tested with strain TA98, whereas all were active with strain TA100 (Table 2). That these were indeed base-substitution reversions was confirmed by spotting each compound onto a lawn of S. typhimurium strain 1535, specific for base substitutions. All of the compounds induced revertants significantly above the controls.
TABLE 1 R E V E R S I O N O F S. T Y P H I M U R I U M S T R A I N T A 1 0 0 I N D U C E D BY V A R I O U S C O N C E N T R A T I O N S OF THE NATURAL PRODUCTS COMPOUNDS ConcenW~ion (#g/pla~)
0.5 3 5 8 10 15 20 50 100 250
Co~nies/pla~ a AcycHc 63
AcycHc 64
AcycHc 65
AcycHc 66
CCK
38 78 126 148 154 207 207 272 213 59
60 90 164 215 267 211 189 253 119 Toxic
105 59 108 112 132 113 154 104 To~c To~c
27 69 101 133 135 57 98 50 Tox~ Toxic
57 171 173 239 370 260 175 To~c Toxic
a T h e assay w a s d o n e in t r i p l i c a t e . T h e flgures g i v e n are averages o f 2 E x p t s . D a t a g i v e n is t h e n u m b e r o f r e v e r t a n t s a b o v e t h o s e o n t h e s p o n t a n e o u s reversion c o n t r o l plates w h i c h a v e r a g e d 1 2 4 / p l a t e .
304 TABLE 2 C O M P A R I S O N OF THE A C T I V I T Y OF T H E N A T U R A L PRODUCTS IN I N D U C I N G BASE S U B S T I T U TION (TAIO0) AND FRAMESHIFT MUTATIONS (TA98) a
TA100 TA98
Acychc 63
Acyclic 64
Acyclic 65
Acyclic 66
CCK
132 4
267 1
154 2
135 0
239 2
a All o f the c o m p o u n d s w e r e used at 10 p g / p l a t e . T h e c o l o n i e s / p l a t e is t h e average o f 2 e x p t s . All assays w e r e d o n e in t m p l i c a t e . T h e d a t a g i v e n is t h e n u m b e r o f r e v e r t a n t s a b o v e t h o s e o n t h e s p o n t a n e o u s r e v e r s i o n c o n t r o l p l a t e s w h i c h averaged 1 3 8 / p l a t e .
TABLE 3 A C T I V I T Y O F $ 9 M I X I N T H E A S S A Y W I T H S. T Y P H I M U R I U M S T R A I N T A 1 0 0 $9 m i x concentration
CCK (15 #g/plate)
A c y c l i c 63 (10 pg/plate)
Acychc 64 (1O/~g/plate)
5 1O 25 50 $9 m i x m m u s m m r o s o m e s No S 9 r m x
177 a 174 119 76 224 370
105 151 102 98 176 154
253 297 265 212 293 267
a The n u m b e r s g i v e n are t h e average o f triplicate assays in 2 E x p t s . and are s h o w n as t h e n u m b e r o f c o l o nies a b o v e t h e s p o n t a n e o u s r e v e r t a n t s w h i c h averaged 1 1 7 / p l a t e .
The inclusion of $9 mix did not activate reversions of TA98 by any of the 5 compounds. However, the $9 mix appeared to reduce the reversion frequency of TA100 only slightly (Table 3), indicating that the liver homogenate has a negligible effect on the activity o f these halogenated hydrcarbons. Discussion The greatest significance of the results reported here is the finding that a natural product from marine algae has mutagenic activity much greater than a known powerful mutagen (EMS). At present, we cannot speculate as to either why such compounds would be synthesized and stored by the algal species or as to what role such compounds might have in the normal physiology of the algae. What is apparent is that natural products which are easily recoverable from marine organisms and may be of interest because of their "beneficial" properties as antimicrobial agents should be screened carefully for potential harmful properties such as mutagenicity/carcinogenicity. References 1 A m e s , B.N., J. M c C a n n a n d E. Y a m a s a k i , M e t h o d s for d e t e c t i n g c a r c i n o g e n s and m u t a g e n s w i t h t h e S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i e i t y t e s t , M u t a t i o n R e s . , 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 3 . 2 B a s l o w , M . H . , Marine P h a r m a c o l o g y . Williams a n d Wilkins, B a l t i m o r e , 1 9 6 9 . 3 F a u l k n e r , D . J . , I n t e r e s t i n g a s p e c t s o f m a r i n e natural p r o d u c t s c h e m i s t r y , T e t x a h e d z o n , 3 3 ( 1 9 7 7 ) 1421--1433.
305 4 Fenical, W., Halogenation in the R h o d o p h y t a -- A review, J. Phycol., 11 (1975) 245--259. 5 Garner, R.C., E.C. Miller and J.A. Miller, Liver microsomal m e t a b o l i s m of a fl a t oxi n B 1 t o a reactive derivative toxic to Salmonella typhimurium TA1530, Cancer Res., 32 (1972) 2058--2066. 6 Magee, P.M., and J.M. Barnes, Carcinogenic nitroso compounds , Adv. Cancer Res., 10 (1967) 163-246. 7 0 l s o n , W.A., R.T. Habermann, E.K. Weisburger, J.M. Ward and J.H. Weisbuxger, I n d u c t i o n of s t o m a c h cancer in rats and mice by halogenated aliphatic fumigants, J. Natl. Cancer Inst., 51 (1973) 1993-1996. 8 Sims, J.J., M.S. Donnel, J.V. Leafy and G.H. Lacy, Ant i mi c robi a l agents from marine algae, Antimicrob. Agents Chemother., 7 (1975) 320--321. 9 Stierle, D. Halogenated m o n o t e r p e n e s from the red alga Plocamium, Ph.D. Thesls, University of Calif., Riverside, 1977. 10 Unsigned article, Chem. Eng. News, 53 (1975) 4--5.
