Mutation Research, 280 (1992) 29-34 0 1992 Elsevier Science Publishers B.V. All rights reserved 01651218/92/$05.00
29
MUTGEN 01783
Evaluation of the genotoxic effects of a folk medicine, Petiveria alliacea (Anamu) Luz Stella Hoyos a, William W. Au b, Moon Y. Heo ‘, Debra L. Morris b and Marvin S. Legator b ’ University of Cauca, Department of Biology, Papayan, Colombia, ’ Uniuersity of Texas Medical Branch, Department of Preventive Medicine and Community Health, JIO, Galueston, TX 77550, USA and ’ College of Pharmacy, Kangweon National University, Chuncheon, South Korea (Received 23 May 1991) (Revision received 28 January 1992) (Accepted 29 January 1992)
Keywords: Petiueria alliacea; Anamu, genotoxicity
Summary Crude extract from a plant known as Petiveria alliacea (Anamu) is used extensively as folk medicine in developing countries like Colombia, South America. Although the plant is known to contain toxic ingredients potential adverse health effects from its use have not been adequately evaluated. We investigated its genotoxic activities by conducting a sister chromatid exchange (SCE) assay using cells in vitro and in vivo. Lymphocytes from humans were treated at 24 h after initiation of culture for 6 h with alcohol extract from the folk medicine. Concentrations of 0, 10, 100, 250, 275, 500, 750, and 1000 pg/ml of the extract were used. Significant dose-dependent increase of SCE (3.7-7.4 SCE per cell) were observed (analysis of variances, p < 0.011. Delay in cell proliferation but not inhibition of mitosis was also observed. In another experiment, mice were exposed once orally to 1 x , 200 x , 300 x and 400 x the human daily consumption dose of Anamu. The induction of sister chromatid exchanges in bone marrow cells were investigated. We observed a significant dose dependent increase of SCE compared with the saline control (2.15-4.53; p < 0.01) and compared with the solvent control (3.04-4.53; p < 0.01). Our data suggest, therefore, that the folk medicine contains mutagenic and potentially carcinogenic agents although the medicine is not a potent mutagen. Individuals who consume large amounts of this drug may be at risk for development of health problems. Further studies with cells from exposed individuals and from experimental animals should be conducted to provide a better evaluation of health risk from the use of this drug.
Plants are routinely used by humans for food and for preparation of drugs. So far, more than 150,000 species of plants have been studied and
Correspondence: Dr. William W. Au, University of Texas Medical Branch, Department of Preventive Medicine and Community Health, JlO, Galveston, TX 77550, USA.
some of them contain ingredients that have therapeutic properties (Miller et al., 1979; Ishii et al., 1984; Milscher et al., 1986). These therapeutically active ingredients are, therefore, extracted and used for preparation of drugs. Alternately, some plants are used directly as medicine. This practice is particularly popular in developing countries like Colombia. However, the directly consumed
30
plants have not usually been evaluated for potential genotoxic side-effects or health consequences, therefore, botanic folk medicine is frequently used without any safety evaluation. It is known that plants synthesize toxic chemicals to defend themselves against infections, and from consumption by insects and herbivorous animals (Jad hov et al., 1981). Many of these natural chemicals from plants are known mutagens and indiscriminate consumption of some plants by humans have been linked to development of health problems. For example, Bababunmi (1978) reported that the high prevalence of hepatocellular carcinoma in Nigeria may be caused by exposure to food contaminants, herbal teas and environmental chemicals. Lin et al. (1979) showed that one of the etiologic factors for development of nasopharyngeal carcinoma in China is the use of herbal drugs. Beier’s review (1990) indicates that ‘although crucifers may provide some protection from cancer when taken prior to a carcinogen, when taken after a carcinogen they may act as promoters of carcinogenesis’. In the same review, Beier reported that some herbs like Bishop’s week seed contains known carcinogens. Therefore indiscriminate use of plants or plant extracts without full knowledge of their possible side-effects to consumers may subject the exposed populations to increased risk of developing long-term health consequences. Petirleria alliacea (Anamu) is a commonly used plant in Colombia, South America as folklore medicine. The same plant is used in other countries with names like Zorrillo, Pipi, Guinea, Mapurite, and Ave. Crude extracts and capsules containing powder from dry leaves and roots of Anamu have been used widely to treat a large number of diseases such as paralysis, rheumatism (De Sousa et al., 19871, fever, diarrhea, headache, as well as cancer (Lopez, 1983). Some cancer patients supplement Anamu with traditional chemotherapy and, in some cases, even replace chemotherapeutic drugs with the folk medicine. In addition, many disease-free individuals take 2 tablets or capsules (0.76 mg per capsule) daily and drink tea prepared from leaves as a preventive procedure against health problems. On the other hand, the leaves of the plant are used as an insecticide (Lopez, 1983) and the roots are used
as drugs to induce abortion (Chirinos, 19061. This brief summary shows that the drug has therapeutic as well as toxic activities. The risk and benefits from the use of Anamu, especially among disease-free individuals, have not been carefully evaluated yet. Experimentally, Naundorf and Zambrano (1982) demonstrated that germination of Phaseolus vulgaris seeds was inhibited by extracts from the plant. Nunez (1983) reported that sheep fed with Anamu exhibited excessive salivation, excessive lacrimation, bradycardia, diarrhea and posterior limbs ataxia. In addition, the sheep had marked inhibition of blood cholinesterase, muscular atrophy and muscular system lesion. With its known toxic activities, it is possible that some undesirable side-effects can develop in humans who consume the plant. The undesirable effects may be cancer or genetic diseases if the plant contains genotoxic chemicals. We have conducted studies to determine the genotoxicity of extracts from Anamu in human lymphocytes in vitro and in bone marrow cells of mice in vivo. We observed that the plant induced a significant dose-dependent increase of sister chromatid exchanges (SCE) in both systems. Materials
and methods
Chemicals Bromodeoxyuridine (BrdU), Colcemid, dimethyl sulfoxide (DMSO) and ethyl nitrosourea (ENU) were purchased from Sigma. The drug, Anamu, was obtained as capsules from Naturcol Laboratory, Colombia, South America (Lie. 653 MS). To prepare a stock solution of the drug, 1 g of powder from several capsules was mixed with 100 ml of ethanol. The suspension was maintained in a water bath at 70°C overnight in order to extract chemical compounds from the powder. Then, the solution was filtered and the alcohol was evaporated. The extract of the drug was redissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution. The dose equivalence of this stock solution per capsule based on the weight of the powder used and on the number of capsules used is calculated. Different dilutions were made from the stock and.used immediately in experiments.
31
Cell culture conditions Blood samples were obtained from an adult volunteer. Blood was drawn using a lo-ml sterile syringe which contained 0.8 ml heparin for each 20 ml of blood. Blood cultures were set up by putting 1 ml of blood into 9 ml of medium per culture tube. The medium was made up of RPM1 1640 (Gibco No. 380-2400),25% heat inactivated fetal bovine serum (inactivated at 60°C for 15 min; Gibco No. 200-6140), 1% sodium heparin (Invenex No. 33-101, 1% Pen-Strep antibiotics (Gibco No. 600-51451, and 2% phytohemagglutinin (PHA; reagent grade, 9 mg/ml; Wellcome No. HA15). Then the culture tubes were placed in a 37°C incubator. Treatment conditions and cell harvest At 6 h after initiation of the culture,
10 PM BrdU was added to each blood culture. At 24 h after initiation, cultures were centrifuged to pack cells. Medium was removed and saved for later use. Cultures were treated with concentrations of 0, 10, 100, 250, 275, 500, 750, and 1000 @g/ml of extract. The inoculation volume was limited to 0.1 ml in 10% DMSO per culture for the low doses and to 1.1 ml in 10% DMSO for the high dose (1000 pgg/ml). Control cultures include untreated controls and cultures that were treated with 0.1 ml of 10% DMSO; 1.1 ml of 10% DMSO, or 50 pg/ml of ENU in culture medium. 6 h later, cultures were spun down to remove chemicals, washed once with plain RPM1 medium and resuspended in the complete medium that was saved earlier. At 70.5 h after initiation, colcemid was added to cultures at a final concentration of 1 pg/ml. At 72 h after initiation, the cultures were harvested. Slides were prepared and stained with the fluorescent-plus-Giemsa technique to document SCE frequencies and cell proliferation. Treatment of mice and bone marrow cell assay
Male NIH Swiss albino mice of 4-8 weeks old were purchased from Harlan Sprague-Dawley Laboratory (Indianapolis, IN). They were maintained in our standard Animal Care Facilities for at least one week before they were used for our experiments. Mice were administered once orally to extracts of the drug in 10% DMSO. The concentrations used were based on human consump-
tion doses of two tablets per day. Our doses used in mice were 1 X , 200 X , 300 X and 400 X human dose and they were equivalent to 0.51, 102, 153 and 204 mg/kg body weight of the drug respectively. In addition, mice were also treated with saline as negative control, DMSO as solvent control and with 50 mg/kg cyclophosphamide (CTN) as positive control. The volume of administration was limited to 0.5 ml per mouse. Due to the labor intensiveness of in vivo assays, not all doses of chemicals were tested in every experiment. However, certain representation doses of Anamu and appropriate controls were conducted in each experiment. Mice were implanted each with a 50 mg paraffin-coated bromodeoxyuridine tablet subcutaneously in the abdominal regions at 1 h before the administration of chemicals. At 23 h after treatment, mice were each injected with colcemid to block cells in mitosis. 1 h later, mice were sacrificed by cervical dislocation. Femurs were removed, bone marrow cells were flushed out and processed for cytogenetic analyses. Air-dried cytological preparations were made. They were stained with the fluorescent-plus-Giemsa technique as mentioned earlier. Cytological analysis
The frequency of SCE was determined from the analysis of 30 sequential metaphase cells per experimental condition. Each metaphase cell showed the second cell cycle staining pattern and contained 46 chromosomes. For the cell proliferation assay using human lymphocytes, 100 metaphase cells were analyzed and classified as cells having the first, second, or third cell cycle staining patterns. A proliferation index was calculated by using the equation PI = % MI X 1 + % MI1 X 2 + % MI11 X 3 (Schneider et al., 1981). Statistical analyses
Statistical evaluations of the cytogenetic data included: (1) the use of Analysis of Variance with and without the Scheffe procedures to determine changes among specified groups of subjects, and (2) the use of Linear Regression analysis to determine the significance of dose-related trends.
32
1
TABLE
SISTER-CHROMATID TRACT Anamu
EXCHANGE
FREQUENCIES
IN HUMAN
LYMPHOCYTES
EXPOSED
TO ALCOHOLIC
EX-
OF ANAMU Expt. 1 mean SCE/cell
(~g/ml)
0 DMSO DMSO
10%
0.1 ml 1.1 ml
1 10 100 250 275 500 750 1000 ENU 50
4.3 b
2.5
3.4 h.c 4.6 ’ 6.0 b.c
2.5 2.9 2.6
5.1 b
2.3
5.9 h.c
2.2
Expt. 2 mean SCE/cell
f SD
3.1 kd 4.4 h 4.5 h
2.2 3.3 2.5
3.5 5.1 1.3 5.0 1.4 6.5
h h kd h h.d ’
1.8 2.8 2.7 2.5 3.0 2.6
16.4 b.d
6.8
a Lymphocyte cultures were treated during the 24th and 30th h of cell culture time. metaphases were analyzed. h Analysis of variance shows significant dose-dependent increases in SCE (p < 0.01). ’ Significantly different from the low dose as determined by the Scheffe test (p < 0.02). d Significantly different from the control as determined by the Scheffe test (p < 0.05).
TABLE
Cells were
f SD
harvested
at 72 h and
2
CELL PROLIFERATION
OF HUMAN
LYMPHOCYTES
TREATED
Expt. 1 Anamu
% of cells in different 1st
2nd
replication 3rd
PI a
34
14
1.6
DMSO 10% 1.1 ml 1 10 100 250 215 500 750 1000
50 58 49
43 33 40
7 9 11
1.6 1.5 1.6
59
36
5
1.4
71
24
5
1.3
ENU 50 ’ PI, proliferation
EXTRACT
OF ANAMU
cycles
0
52
ALCOHOLIC
Expt. 2
kg/ml)
DMSO 0.1 ml
WITH
index; calculated
by the equation:
1st
2nd
3rd
PI a
56
29
15
1.6
56
35
9
1.5
55
34
11
1.5
62 58 63 75 13 19
33 38 32 23 22 20
5 4 5 2 5 1
1.4 1.4 1.4 1.3 1.3 1.2
74
23
3
1.3
PI = % MIX 1+ % MI1 X 2 + % MI11
X
3
30
33
Result As shown in Table 1, the alcoholic extract from Anamu is able to induce DNA damage leading to the formation of SCE in human lymphocytes. There were significant differences in SCE frequencies among different treated groups and from control groups in both experiments. In experiment 1, the frequency of SCE shows a significant dose-dependent increase (F ratio = 4.4; p < 0.01). Use of the Scheffe test confirms that significantly more SCE were induced in the high dose groups (100 and 1000 pg/ml), than the low dose group (1 pg/ml; p < 0.02). In experiment 2 of Table 1, analysis of variance shows that the drug induced significant dose-dependent increase of SCE (F ratio = 38.5; p < 0.01). The frequency of SCE increased from 3.7 t_ 2.2 from negative control to 7.3 + 2.7 at 250 pg/ml of the extract. Scheffe test confirms that they are significantly different from each other with a p value < 0.04. Exposure to higher doses of the drug did not cause any further increase in SCE frequencies. The proportion of metaphase cells in their first, second, and third divisions at each different treatment is shown in Table 2. Delay in cell proliferation is shown in cells treated with more than 100 pug/ml of the extract. However, no toxicity as shown in mitotic inhibition was observed. A summary of the in vivo data is shown in Table 3. Analysis of Variance of the data shows that ENU (positive control) induced significantly more SCE than any other treatment conditions (P < 0.001). In addition, the solvent control is different from the DMSO control (P = 0.06). Linear regression analysis of the data shows that there is a significant increase of SCE among Anamu-treated mice compared with the saline control (P < 0.01) and compared with the DMSO control (P < 0.01). If we assume that 1 mg/kg in vivo dose is roughly equivalent to 1 ,ug/ml in vitro dose, the maximum Anamu concentrations used in our mice is 204 mg/kg which is much less than our 1000 pug/ml lymphocyte concentrations. Since we did not observe cytotoxicity and significant cell cycle delay in lymphocytes nor any obvious toxicity in bone marrow cells of treated mice, we did not
TABLE
3
SISTER-CHROMATID EXCHANGE BONE-MARROW CELLS OF MICE COHOL EXTRA’CT OF ANAMU Treatment
a
C DMSO Anamu
CTN
Dose
Mice
FREQUENCIES IN EXPOSED TO AL-
Mean SCE /cell
S.D.
0
4
2.15 b.d
0.36
10%
7
3.04 h,d
0.71
1 200 300 400
3 7 3 7
3.39 3.41 3.43 4.53
d d Ii d
0.39 1.07 1.27 0.96
50
4
12.68 ’
3.52
a Mice were injected each orally with chemicals of < 0.5 ml per mouse. C, saline control; DMSO, dimethyl sulfoxide; CTN, cyclophosphamide. b Anova shows significance of difference p = 0.06. ’ Anova shows significance of difference from the rest, p < 0.001. d Linear regression analysis shows significant dose-dependent difference from C ( p < 0.01) and from DMSO ( p < 0.01).
conduct mice.
a cell cycle proliferation
analysis with
Discussion Our data suggest that a folk medicine commonly used in Colombia, South America, can induce significant dose-dependent increases of SCE in human lymphocytes in vitro and in mouse bone marrow cells in vivo. The increase was detected at doses that did not cause extensive delay of cell proliferation nor cytotoxicity. The increase after exposure to Anamu is, however, much less than those induced by potent mutagens such as ENU and CTN. Although the biological significance of an SCE event is not determined, the SCE test is a simple assay to identify mutagens and carcinogens. Therefore, we suggest that Anamu contains some mutagenic and potentially carcinogenic agents. Individuals who consume the drug extensively may be at increased risk of developing health problems. Our conclusion is based on studies using alcohol extract of the crude medicine. Effects from exposure to non-alcohol soluble extracts or from the crude medicine itself is unknown. In order to
34
confirm and extend our investigation, studies with exposed populations and with experimental animals should, therefore be conducted. Induction of chromosome aberrations, DNA alterations etc in cells of different organs and their consequences should be investigated. Acknowledgements
We would like to thank Dr. James A. Hokanson and Dr. Elbert Whorton for their generous assistance for statistical analysis, We also thank Dr. Jonathan Ward Jr. and Mr. Lance Hallberg for their assistance to Mrs. Luz Stella Hoyos. Mrs. Hoyos is a visiting scientist to the Department of Preventive Medicine and Community Health, University of Texas Medical Branch. Her work is supported by funds from The Andean Peace Scholarship Project of The United States Agency for International Development. References Bababunmi, E.A. (1978) Toxins and carcinogens in the environment: an observation in the tropics. J. Toxicol. Environ. Health, 4, 691-699. Beier, R.C. (1990) Natural pesticides and bioactive components in foods. Rev. Environ. Contam. Toxicol., 113, 47137. Chirinos, D.N. (1983) El Anamu Serrano Legitimo Mapurite, Ediciones de Lavoz de San Luis.
DeSousa, J.R., Demuner, A.J., Pedersoli, J.P. and Alfonso, A.M. (1987) Medicinal or poisonous herb? Cienc. Cult. (Sao Paulo), 39, 645-646. Ishii, R., Yoshikawa, H., Minakata, N.T., Komura, K. and Kada, Y. (1984) Specificity of bio-antimutagens in the plant kingdom. Agric. Biol. Chem., 48, 2587-2591. Jad hov, S.F., Sharma, R.P. and Salunkhe, D.K. (1981) Naturally occurring toxic alkaloids in foods. CRC Crit. Rev. Toxicol., 21, 231-240. Lin, T.M., Yang, C.S., Tu, S.M., Chen, C.J., Juo, K.C. and Hirayama, T. (19791 Interaction of factors associated with cancer of the nasopharynx. Cancer, 44, 1419-1423. Lopez Palacios, S. (1983) Notas bibliograficas sobre una planta posiblemente auticancerigena, el mapurite a Anamu. Rev. Fat. Farm. ULA N 23, Merida, Mexico. Miller, EC., Miller, J.A., Hirona, I. and Takayama, S. (1979) Naturally occurring carcinogens, in: Mutagens and Modulators of Carcinogenesis, University Park Press, Baltimore, MD. Milscher, L.A., Drake, S., Gollapudi, S.R., Harris, J.A. and Shankel, D.M. (1986) Isolation and identification of higher plant agents active in antimutagenic assay system, Glycyrrhiza glabra, in: D.M. Shankel, Hartman, Y. Kada and A. Hollaender (Eds.), Antimutagenesis and Anticarcinogenesis Mechanisms, Plenum, New York, pp. 153-165. Naundorf, G. and Zambrano, L. (1982) Efects del extracts crude de raiz y hojas en la germination de semillas de Phaseolus uulgaris e indice mitotico en celulas de raices de Alliutn cepa. Nunez, V. (1983) Caquexia muscular distrofica y su relation clinico-patologica con la neurotoxicidad retardada. Rev. ICA Bogota (Colombia), XVIII, 345-353. Schneider, E.L., Sternberg, H. and Tice, R.R. (1977) In vivo analysis of cellular replication. Proc. Natl. Acad. Sci. (USA), 74, 2041-2044.
29
MUTGEN 01783
Evaluation of the genotoxic effects of a folk medicine, Petiveria alliacea (Anamu) Luz Stella Hoyos a, William W. Au b, Moon Y. Heo ‘, Debra L. Morris b and Marvin S. Legator b ’ University of Cauca, Department of Biology, Papayan, Colombia, ’ Uniuersity of Texas Medical Branch, Department of Preventive Medicine and Community Health, JIO, Galueston, TX 77550, USA and ’ College of Pharmacy, Kangweon National University, Chuncheon, South Korea (Received 23 May 1991) (Revision received 28 January 1992) (Accepted 29 January 1992)
Keywords: Petiueria alliacea; Anamu, genotoxicity
Summary Crude extract from a plant known as Petiveria alliacea (Anamu) is used extensively as folk medicine in developing countries like Colombia, South America. Although the plant is known to contain toxic ingredients potential adverse health effects from its use have not been adequately evaluated. We investigated its genotoxic activities by conducting a sister chromatid exchange (SCE) assay using cells in vitro and in vivo. Lymphocytes from humans were treated at 24 h after initiation of culture for 6 h with alcohol extract from the folk medicine. Concentrations of 0, 10, 100, 250, 275, 500, 750, and 1000 pg/ml of the extract were used. Significant dose-dependent increase of SCE (3.7-7.4 SCE per cell) were observed (analysis of variances, p < 0.011. Delay in cell proliferation but not inhibition of mitosis was also observed. In another experiment, mice were exposed once orally to 1 x , 200 x , 300 x and 400 x the human daily consumption dose of Anamu. The induction of sister chromatid exchanges in bone marrow cells were investigated. We observed a significant dose dependent increase of SCE compared with the saline control (2.15-4.53; p < 0.01) and compared with the solvent control (3.04-4.53; p < 0.01). Our data suggest, therefore, that the folk medicine contains mutagenic and potentially carcinogenic agents although the medicine is not a potent mutagen. Individuals who consume large amounts of this drug may be at risk for development of health problems. Further studies with cells from exposed individuals and from experimental animals should be conducted to provide a better evaluation of health risk from the use of this drug.
Plants are routinely used by humans for food and for preparation of drugs. So far, more than 150,000 species of plants have been studied and
Correspondence: Dr. William W. Au, University of Texas Medical Branch, Department of Preventive Medicine and Community Health, JlO, Galveston, TX 77550, USA.
some of them contain ingredients that have therapeutic properties (Miller et al., 1979; Ishii et al., 1984; Milscher et al., 1986). These therapeutically active ingredients are, therefore, extracted and used for preparation of drugs. Alternately, some plants are used directly as medicine. This practice is particularly popular in developing countries like Colombia. However, the directly consumed
30
plants have not usually been evaluated for potential genotoxic side-effects or health consequences, therefore, botanic folk medicine is frequently used without any safety evaluation. It is known that plants synthesize toxic chemicals to defend themselves against infections, and from consumption by insects and herbivorous animals (Jad hov et al., 1981). Many of these natural chemicals from plants are known mutagens and indiscriminate consumption of some plants by humans have been linked to development of health problems. For example, Bababunmi (1978) reported that the high prevalence of hepatocellular carcinoma in Nigeria may be caused by exposure to food contaminants, herbal teas and environmental chemicals. Lin et al. (1979) showed that one of the etiologic factors for development of nasopharyngeal carcinoma in China is the use of herbal drugs. Beier’s review (1990) indicates that ‘although crucifers may provide some protection from cancer when taken prior to a carcinogen, when taken after a carcinogen they may act as promoters of carcinogenesis’. In the same review, Beier reported that some herbs like Bishop’s week seed contains known carcinogens. Therefore indiscriminate use of plants or plant extracts without full knowledge of their possible side-effects to consumers may subject the exposed populations to increased risk of developing long-term health consequences. Petirleria alliacea (Anamu) is a commonly used plant in Colombia, South America as folklore medicine. The same plant is used in other countries with names like Zorrillo, Pipi, Guinea, Mapurite, and Ave. Crude extracts and capsules containing powder from dry leaves and roots of Anamu have been used widely to treat a large number of diseases such as paralysis, rheumatism (De Sousa et al., 19871, fever, diarrhea, headache, as well as cancer (Lopez, 1983). Some cancer patients supplement Anamu with traditional chemotherapy and, in some cases, even replace chemotherapeutic drugs with the folk medicine. In addition, many disease-free individuals take 2 tablets or capsules (0.76 mg per capsule) daily and drink tea prepared from leaves as a preventive procedure against health problems. On the other hand, the leaves of the plant are used as an insecticide (Lopez, 1983) and the roots are used
as drugs to induce abortion (Chirinos, 19061. This brief summary shows that the drug has therapeutic as well as toxic activities. The risk and benefits from the use of Anamu, especially among disease-free individuals, have not been carefully evaluated yet. Experimentally, Naundorf and Zambrano (1982) demonstrated that germination of Phaseolus vulgaris seeds was inhibited by extracts from the plant. Nunez (1983) reported that sheep fed with Anamu exhibited excessive salivation, excessive lacrimation, bradycardia, diarrhea and posterior limbs ataxia. In addition, the sheep had marked inhibition of blood cholinesterase, muscular atrophy and muscular system lesion. With its known toxic activities, it is possible that some undesirable side-effects can develop in humans who consume the plant. The undesirable effects may be cancer or genetic diseases if the plant contains genotoxic chemicals. We have conducted studies to determine the genotoxicity of extracts from Anamu in human lymphocytes in vitro and in bone marrow cells of mice in vivo. We observed that the plant induced a significant dose-dependent increase of sister chromatid exchanges (SCE) in both systems. Materials
and methods
Chemicals Bromodeoxyuridine (BrdU), Colcemid, dimethyl sulfoxide (DMSO) and ethyl nitrosourea (ENU) were purchased from Sigma. The drug, Anamu, was obtained as capsules from Naturcol Laboratory, Colombia, South America (Lie. 653 MS). To prepare a stock solution of the drug, 1 g of powder from several capsules was mixed with 100 ml of ethanol. The suspension was maintained in a water bath at 70°C overnight in order to extract chemical compounds from the powder. Then, the solution was filtered and the alcohol was evaporated. The extract of the drug was redissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution. The dose equivalence of this stock solution per capsule based on the weight of the powder used and on the number of capsules used is calculated. Different dilutions were made from the stock and.used immediately in experiments.
31
Cell culture conditions Blood samples were obtained from an adult volunteer. Blood was drawn using a lo-ml sterile syringe which contained 0.8 ml heparin for each 20 ml of blood. Blood cultures were set up by putting 1 ml of blood into 9 ml of medium per culture tube. The medium was made up of RPM1 1640 (Gibco No. 380-2400),25% heat inactivated fetal bovine serum (inactivated at 60°C for 15 min; Gibco No. 200-6140), 1% sodium heparin (Invenex No. 33-101, 1% Pen-Strep antibiotics (Gibco No. 600-51451, and 2% phytohemagglutinin (PHA; reagent grade, 9 mg/ml; Wellcome No. HA15). Then the culture tubes were placed in a 37°C incubator. Treatment conditions and cell harvest At 6 h after initiation of the culture,
10 PM BrdU was added to each blood culture. At 24 h after initiation, cultures were centrifuged to pack cells. Medium was removed and saved for later use. Cultures were treated with concentrations of 0, 10, 100, 250, 275, 500, 750, and 1000 @g/ml of extract. The inoculation volume was limited to 0.1 ml in 10% DMSO per culture for the low doses and to 1.1 ml in 10% DMSO for the high dose (1000 pgg/ml). Control cultures include untreated controls and cultures that were treated with 0.1 ml of 10% DMSO; 1.1 ml of 10% DMSO, or 50 pg/ml of ENU in culture medium. 6 h later, cultures were spun down to remove chemicals, washed once with plain RPM1 medium and resuspended in the complete medium that was saved earlier. At 70.5 h after initiation, colcemid was added to cultures at a final concentration of 1 pg/ml. At 72 h after initiation, the cultures were harvested. Slides were prepared and stained with the fluorescent-plus-Giemsa technique to document SCE frequencies and cell proliferation. Treatment of mice and bone marrow cell assay
Male NIH Swiss albino mice of 4-8 weeks old were purchased from Harlan Sprague-Dawley Laboratory (Indianapolis, IN). They were maintained in our standard Animal Care Facilities for at least one week before they were used for our experiments. Mice were administered once orally to extracts of the drug in 10% DMSO. The concentrations used were based on human consump-
tion doses of two tablets per day. Our doses used in mice were 1 X , 200 X , 300 X and 400 X human dose and they were equivalent to 0.51, 102, 153 and 204 mg/kg body weight of the drug respectively. In addition, mice were also treated with saline as negative control, DMSO as solvent control and with 50 mg/kg cyclophosphamide (CTN) as positive control. The volume of administration was limited to 0.5 ml per mouse. Due to the labor intensiveness of in vivo assays, not all doses of chemicals were tested in every experiment. However, certain representation doses of Anamu and appropriate controls were conducted in each experiment. Mice were implanted each with a 50 mg paraffin-coated bromodeoxyuridine tablet subcutaneously in the abdominal regions at 1 h before the administration of chemicals. At 23 h after treatment, mice were each injected with colcemid to block cells in mitosis. 1 h later, mice were sacrificed by cervical dislocation. Femurs were removed, bone marrow cells were flushed out and processed for cytogenetic analyses. Air-dried cytological preparations were made. They were stained with the fluorescent-plus-Giemsa technique as mentioned earlier. Cytological analysis
The frequency of SCE was determined from the analysis of 30 sequential metaphase cells per experimental condition. Each metaphase cell showed the second cell cycle staining pattern and contained 46 chromosomes. For the cell proliferation assay using human lymphocytes, 100 metaphase cells were analyzed and classified as cells having the first, second, or third cell cycle staining patterns. A proliferation index was calculated by using the equation PI = % MI X 1 + % MI1 X 2 + % MI11 X 3 (Schneider et al., 1981). Statistical analyses
Statistical evaluations of the cytogenetic data included: (1) the use of Analysis of Variance with and without the Scheffe procedures to determine changes among specified groups of subjects, and (2) the use of Linear Regression analysis to determine the significance of dose-related trends.
32
1
TABLE
SISTER-CHROMATID TRACT Anamu
EXCHANGE
FREQUENCIES
IN HUMAN
LYMPHOCYTES
EXPOSED
TO ALCOHOLIC
EX-
OF ANAMU Expt. 1 mean SCE/cell
(~g/ml)
0 DMSO DMSO
10%
0.1 ml 1.1 ml
1 10 100 250 275 500 750 1000 ENU 50
4.3 b
2.5
3.4 h.c 4.6 ’ 6.0 b.c
2.5 2.9 2.6
5.1 b
2.3
5.9 h.c
2.2
Expt. 2 mean SCE/cell
f SD
3.1 kd 4.4 h 4.5 h
2.2 3.3 2.5
3.5 5.1 1.3 5.0 1.4 6.5
h h kd h h.d ’
1.8 2.8 2.7 2.5 3.0 2.6
16.4 b.d
6.8
a Lymphocyte cultures were treated during the 24th and 30th h of cell culture time. metaphases were analyzed. h Analysis of variance shows significant dose-dependent increases in SCE (p < 0.01). ’ Significantly different from the low dose as determined by the Scheffe test (p < 0.02). d Significantly different from the control as determined by the Scheffe test (p < 0.05).
TABLE
Cells were
f SD
harvested
at 72 h and
2
CELL PROLIFERATION
OF HUMAN
LYMPHOCYTES
TREATED
Expt. 1 Anamu
% of cells in different 1st
2nd
replication 3rd
PI a
34
14
1.6
DMSO 10% 1.1 ml 1 10 100 250 215 500 750 1000
50 58 49
43 33 40
7 9 11
1.6 1.5 1.6
59
36
5
1.4
71
24
5
1.3
ENU 50 ’ PI, proliferation
EXTRACT
OF ANAMU
cycles
0
52
ALCOHOLIC
Expt. 2
kg/ml)
DMSO 0.1 ml
WITH
index; calculated
by the equation:
1st
2nd
3rd
PI a
56
29
15
1.6
56
35
9
1.5
55
34
11
1.5
62 58 63 75 13 19
33 38 32 23 22 20
5 4 5 2 5 1
1.4 1.4 1.4 1.3 1.3 1.2
74
23
3
1.3
PI = % MIX 1+ % MI1 X 2 + % MI11
X
3
30
33
Result As shown in Table 1, the alcoholic extract from Anamu is able to induce DNA damage leading to the formation of SCE in human lymphocytes. There were significant differences in SCE frequencies among different treated groups and from control groups in both experiments. In experiment 1, the frequency of SCE shows a significant dose-dependent increase (F ratio = 4.4; p < 0.01). Use of the Scheffe test confirms that significantly more SCE were induced in the high dose groups (100 and 1000 pg/ml), than the low dose group (1 pg/ml; p < 0.02). In experiment 2 of Table 1, analysis of variance shows that the drug induced significant dose-dependent increase of SCE (F ratio = 38.5; p < 0.01). The frequency of SCE increased from 3.7 t_ 2.2 from negative control to 7.3 + 2.7 at 250 pg/ml of the extract. Scheffe test confirms that they are significantly different from each other with a p value < 0.04. Exposure to higher doses of the drug did not cause any further increase in SCE frequencies. The proportion of metaphase cells in their first, second, and third divisions at each different treatment is shown in Table 2. Delay in cell proliferation is shown in cells treated with more than 100 pug/ml of the extract. However, no toxicity as shown in mitotic inhibition was observed. A summary of the in vivo data is shown in Table 3. Analysis of Variance of the data shows that ENU (positive control) induced significantly more SCE than any other treatment conditions (P < 0.001). In addition, the solvent control is different from the DMSO control (P = 0.06). Linear regression analysis of the data shows that there is a significant increase of SCE among Anamu-treated mice compared with the saline control (P < 0.01) and compared with the DMSO control (P < 0.01). If we assume that 1 mg/kg in vivo dose is roughly equivalent to 1 ,ug/ml in vitro dose, the maximum Anamu concentrations used in our mice is 204 mg/kg which is much less than our 1000 pug/ml lymphocyte concentrations. Since we did not observe cytotoxicity and significant cell cycle delay in lymphocytes nor any obvious toxicity in bone marrow cells of treated mice, we did not
TABLE
3
SISTER-CHROMATID EXCHANGE BONE-MARROW CELLS OF MICE COHOL EXTRA’CT OF ANAMU Treatment
a
C DMSO Anamu
CTN
Dose
Mice
FREQUENCIES IN EXPOSED TO AL-
Mean SCE /cell
S.D.
0
4
2.15 b.d
0.36
10%
7
3.04 h,d
0.71
1 200 300 400
3 7 3 7
3.39 3.41 3.43 4.53
d d Ii d
0.39 1.07 1.27 0.96
50
4
12.68 ’
3.52
a Mice were injected each orally with chemicals of < 0.5 ml per mouse. C, saline control; DMSO, dimethyl sulfoxide; CTN, cyclophosphamide. b Anova shows significance of difference p = 0.06. ’ Anova shows significance of difference from the rest, p < 0.001. d Linear regression analysis shows significant dose-dependent difference from C ( p < 0.01) and from DMSO ( p < 0.01).
conduct mice.
a cell cycle proliferation
analysis with
Discussion Our data suggest that a folk medicine commonly used in Colombia, South America, can induce significant dose-dependent increases of SCE in human lymphocytes in vitro and in mouse bone marrow cells in vivo. The increase was detected at doses that did not cause extensive delay of cell proliferation nor cytotoxicity. The increase after exposure to Anamu is, however, much less than those induced by potent mutagens such as ENU and CTN. Although the biological significance of an SCE event is not determined, the SCE test is a simple assay to identify mutagens and carcinogens. Therefore, we suggest that Anamu contains some mutagenic and potentially carcinogenic agents. Individuals who consume the drug extensively may be at increased risk of developing health problems. Our conclusion is based on studies using alcohol extract of the crude medicine. Effects from exposure to non-alcohol soluble extracts or from the crude medicine itself is unknown. In order to
34
confirm and extend our investigation, studies with exposed populations and with experimental animals should, therefore be conducted. Induction of chromosome aberrations, DNA alterations etc in cells of different organs and their consequences should be investigated. Acknowledgements
We would like to thank Dr. James A. Hokanson and Dr. Elbert Whorton for their generous assistance for statistical analysis, We also thank Dr. Jonathan Ward Jr. and Mr. Lance Hallberg for their assistance to Mrs. Luz Stella Hoyos. Mrs. Hoyos is a visiting scientist to the Department of Preventive Medicine and Community Health, University of Texas Medical Branch. Her work is supported by funds from The Andean Peace Scholarship Project of The United States Agency for International Development. References Bababunmi, E.A. (1978) Toxins and carcinogens in the environment: an observation in the tropics. J. Toxicol. Environ. Health, 4, 691-699. Beier, R.C. (1990) Natural pesticides and bioactive components in foods. Rev. Environ. Contam. Toxicol., 113, 47137. Chirinos, D.N. (1983) El Anamu Serrano Legitimo Mapurite, Ediciones de Lavoz de San Luis.
DeSousa, J.R., Demuner, A.J., Pedersoli, J.P. and Alfonso, A.M. (1987) Medicinal or poisonous herb? Cienc. Cult. (Sao Paulo), 39, 645-646. Ishii, R., Yoshikawa, H., Minakata, N.T., Komura, K. and Kada, Y. (1984) Specificity of bio-antimutagens in the plant kingdom. Agric. Biol. Chem., 48, 2587-2591. Jad hov, S.F., Sharma, R.P. and Salunkhe, D.K. (1981) Naturally occurring toxic alkaloids in foods. CRC Crit. Rev. Toxicol., 21, 231-240. Lin, T.M., Yang, C.S., Tu, S.M., Chen, C.J., Juo, K.C. and Hirayama, T. (19791 Interaction of factors associated with cancer of the nasopharynx. Cancer, 44, 1419-1423. Lopez Palacios, S. (1983) Notas bibliograficas sobre una planta posiblemente auticancerigena, el mapurite a Anamu. Rev. Fat. Farm. ULA N 23, Merida, Mexico. Miller, EC., Miller, J.A., Hirona, I. and Takayama, S. (1979) Naturally occurring carcinogens, in: Mutagens and Modulators of Carcinogenesis, University Park Press, Baltimore, MD. Milscher, L.A., Drake, S., Gollapudi, S.R., Harris, J.A. and Shankel, D.M. (1986) Isolation and identification of higher plant agents active in antimutagenic assay system, Glycyrrhiza glabra, in: D.M. Shankel, Hartman, Y. Kada and A. Hollaender (Eds.), Antimutagenesis and Anticarcinogenesis Mechanisms, Plenum, New York, pp. 153-165. Naundorf, G. and Zambrano, L. (1982) Efects del extracts crude de raiz y hojas en la germination de semillas de Phaseolus uulgaris e indice mitotico en celulas de raices de Alliutn cepa. Nunez, V. (1983) Caquexia muscular distrofica y su relation clinico-patologica con la neurotoxicidad retardada. Rev. ICA Bogota (Colombia), XVIII, 345-353. Schneider, E.L., Sternberg, H. and Tice, R.R. (1977) In vivo analysis of cellular replication. Proc. Natl. Acad. Sci. (USA), 74, 2041-2044.
