S~ie~tt~qae et Tecbnologique, B. P. 7192, ~u~udougou ~Bur~ia~t Fuse /. (Accepted May ..... Krishna Rao and Ganapaty, 1983) and by clinical trials (Martin et al.
Journal o/ Efhnopharmacology,
235
34 (1991) 235-246
Elsevier Scientific Publishers Ireland Ltd.
Use of an Amoeba proteus model for in vitro cytotoxicity testing in phytochemical research. Application to ~~~~~rbia hirta extracts P. Dueza, A. Livaditis”, P.I. Guissoub, M. Sawadogob and M. Hanocqa uUni~e de Chimie
Bioanaiytique.
Triomphe. 1050 Bruxeiles
de To.uicologie et de Chimir Physique Appliqutk.
Universitc; Libre dc i3ru.wNe.s. C. P. 205/i.
{Belgium} and b~astitut de Reeherches sur les Substances S~ie~tt~qae et Tecbnologique, B. P. 7192, ~u~udougou
Nature~ie.~, Centre rational ~Bur~ia~t Fuse /
Bd. du
de la Recherche
(Accepted May 23. 1991)
Amoeba proteus is proposed as a low-cost multi-purpose biochemical tool for screening and standardizing cytotoxic plant extracts with possible application in the laboratories of developing countries. Advantages and ljmitations of this test are examined and different mathematical treatments (probit analysis versus curve fitting to Von Bertalanffy and Hill functions) are investigated. Known anti-cancer (doxorubicine, daunorubicine, dacarbazine, S-fluorouracil) and antiparasitic (emetine, dehydroemetine, metronidazole, cucurbitine, chloroquine) drugs were tested using this method and only metronidazole appeared inactive. Application of this model to Euphorbia hirta established that a 100°C aqueous extraction of fresh aerial parts allows eficient extraction of active constituents and that drying the plant material before extraction considerably reduces activity.
lvey words: antiparasitic; cytotoxic; emetine; dehydroemetine; cucurbitine.
Introduction Following recommendations from the World Health Organization (1977), African, Asian and South American medicinal plants are being actively investigated in a search for potentially useful drugs, mainly antiparasitic drugs for local use. Such an approach proceeds through multidisciplinary teams and includes ethnobotanic surveys, phytochemical studies and biological testing in addition to the investigation of natural ecosystems and cultivation techniques for raw material production. A large part of these research programs has been undertaken in Third World countries where laboratory infrastructures are rapidly developing but are still often incomplete and suffer from drastic Correspondence fo: M. Hanocq. Unit& de Chimie Bioanalytique, de Toxicologic et de Chimie Physique Appliquke, Universit6 Libre de Btuxelles, C.P. 205/l, Bd. du Triomphe, 1050 Bruxelles, Belgium,
0378-8741/$03.50 0 1991 Elsevier Scientific Publishers Ireland Ltd Published and Printed in Ireland
budget constraints. Notably, strict sterility conditions such as those required for cell culture work are costly, difficult to implement and demand specialized technical care. A simple general cytotoxicity test could allow prescreening of plant extracts and sorting of active fractions; more sophisticated tests could then be performed on a considerably reduced number of fractions. Moreover, biological standardization of crude extracts would allow planning of production by optimization of cultivation, harvesting, drying and extraction as well as control of raw material near production sites. Amoeba proteus, a free-living protozoa widely studied for fundamental information on cytoplasm, or- nucleus functions, organelle cytochemistry and cytokinesis (Hirschfield, 1959; Chapman-Andresen, 1964; Prescott and Carrier, 1964), was chosen as the organism for a cytotoxicity test with possible applicatjons in deveIoping countries. This non-pathogenic organism is not difficult to culture, is a complete eukaryotic cell
236
and has been previously used as a cell model in toxicological (Ord, 1970, 1979) and environmental (Al-Atia, 1980) research. A. proteus is a cell lacking specialization and therefore offers an overall picture of many cell activities (Ord, 1979). Material and Methods Chemical sources
All tested chemicals, (+)-emetine dihydrochloride (Aldrich, U.S.A.), (*)-dehydroemetine dihydrochloride (Roche, Switzerland), chloroquine diphosphate and metronidazole (Deschamps-Barnett, Belgium), tetracycline hydrochloride (Aldrich, U.S.A.), atropine sulfate (Merck, R.F.A.), doxorubicine hydrochloride and daunorubicine hydrochloride (gift from RhonePoulenc, France), dacarbazine citrate (gift from Dome Laboratories, U.S.A.), 5-fluorouracil (gift from the National Cancer Institute, U.S.A.) and ( A))-cucurbitine hydrobromide (synthesized according to the method of Sun et al., 1961) were solubilized and diluted in the amoebae inorganic culture medium. Euphorbia hirta extraction Euphorbia hirta L. (Syn. E. piM$era
Chev.) of the family Euphorbiaceae was either harvested in Burkina Faso (Farako BB) or grown under glass in Belgium (ULB Systematic Botany Laboratory) from seeds harvested in Zaire (Kisangani) or in Burkina Faso (Farako B1 and Barn Lake) (voucher specimen Lejoly 841029 and Lejoly 5428, BRLU Herbarium). Plant aerial parts were either cut up and extracted immediately after harvesting (“fresh plant”) or dried for 3 days at ambient temperature in a shady ventilated room (Burkina) or at 35°C in a ventilated oven (Belgium) and powdered to pass a 3 15pm sieve. Samples of 5 g were extracted by agitation in 100 ml of the amoebae inorganic medium at 20°C or at 100°C followed by centrifugation; obtained extract solutions were diluted in the amoebae inorganic medium and tested. Owing to relatively small amounts of sample available, notably for fresh plant material, dry weight yields of the extracts could not be measured. All results were expressed in terms of dried starting material weights, as deter-
mined using aliquots desiccated in a 105°C ventilated oven. Maintenance of cultures
All glassware was washed with glass-distilled water without the use of detergent. A binocular microscope (magnification 60 x to 400x) was used for every operation. The Amoeba proteus strain, originally obtained from Carlsberg laboratories (Copenhagen, Denmark) and maintained for 30 years in the ULB Molecular Biology Laboratories (infusion cultures in Chalkey’s medium) (Chalkey, 1930) was adapted to Tetrahymena feeding according to the method of Griffin (1960). Cultures were grown in covered 200-ml glass bowls in an inorganic medium containing 6 mg KCl, 4 mg CaHP04 and 2 mg MgSO, in 1 1 total volume of glass-distilled water. They were kept in the dark at 23-27°C. Tetrahymena pyriformis (ATCC strain 30203) were axenically grown in a medium containing 18 g proteose peptone (Difco, U.S.A.) and 2 g yeast extract (Difco, U.S.A.) in 1 1total volume of glass-distilled water. Twice a week, the amoebae inorganic medium was renewed and cultures were fed with harvested Tetrahymena (centrifugation at 350 x g, 4°C 2 min, followed by three washing/centrifugation operations in the amoebae inorganic medium containing 30 mg% agar). The agar (Difco, U.S.A.) was used to prevent cell clumping. No particular optimization of the feeding schedule (Griffin, 1960) was practiced but overfeeding was avoided. Every 2 weeks, amoebae were transferred to new culture vessels. Cytotoxicity test protocol
Tests were performed in covered glass microscopy chambers (40 x 40 mm with a circular cavity of 30 mm diameter and 8 mm in height) maintained at 23-27°C in the dark. Washing operations were effected by careful sucking up of supernatant, agitation with 2 ml of the inorganic culture medium and decantation (2 min) of the amoebae. Approximately 50 amoebae were sucked up from culture bowls with capillary tubing, transferred to an assay chamber, washed three times and left fasting in 2 ml inorganic medium for 3 days.
237
(ii) Curve fitting using the Von Bertalanffy function (VB). Data were analysed by means of an original algorithm (Abi Khalil et al., 1986; Dubois et al., 1989) which minimizes a non-linear cost function by the simplex method; experimental results were fitted to the VB parametric ‘function with data equiweighted. In the following equation (Dubois et al., 1989), C = concentration or time, N = percent of living amoebae at concentration (or time) C, No = percent of living amoebae at concentration (or time) 0, W is the asymptotical value of N, and k = parameter. N I No and W = No when the curve intercepts the x-axis. In the original Von Bertalanffy function, a = 3; according to Dubois et al. (1989), a was considered as a parameter in order to obtain a better lit.
Supernatant was sucked up, amoebae were counted three times and 2 ml of the solution to be tested was added and left in contact for the indicated time. Amoebae were washed three times, the last supernatant sucked up, two drops of a trypan blue (Aldrich, U.S.A.) solution (0.2% in inorganic culture medium) added and diluted after 1 min with 2 ml of the inorganic medium. After decantation and sucking up of the su~rnatant, living amoebae (those excluding trypan blue) were counted three times.
Analysis
of remits
Three different compared:
mathematical
treatments
were
N = No - W (1 - e-kC)a
(i) Probit analysis. In the manner of Finney (1971), data were transformed into probits and plotted versus the logarithm of time or of concentration. Linear relationships were computed by the least-square method allowing the determination of Efficient Times,, (ET& or Efficient Concentrationso (EC&).
(iii) Curve fitting using the Hill function, Experimental results were fitted to the Hill parametric function as in (ii) with data equiweighted. In the following equation (Hill, 1910), C, N and No are as in the VB function, EC
TABLE I TIME/EFFECT
RELATIONSHIP
FOR EMETINE DIHYDROCHLORIDE
(1 mg/ml)
N = number of points; ET,, = efficient time 50; r = correlation coefficient; s = absolute standard deviation; So/o= relative standard deviation. “Pooled results” were computed from data of all three experiments simultaneously treated; this value takes within and without experimental variations into account. In paren t.J ses, for probit analysis, ET, 95% confidence limits and, for curve fitting, ETss vahres range as determined from the two curves cakzulated with the extreme statistical values of regression parameters banters + standard deviations” and “parameters - standard deviations”) (Dubois et al., 1989). Experiment
N
Probit analysis
Curve fitting VB function
I 2 3
23 21 24
r
ET,, (h)
r
0.923 0.968 0.949
5.79 6.01 5.05
0.993 0.950 0.970
Mean S s% Pooled
5.6 0.5 9 68
0.913
ETso (h) 4.52 5.68 3.98
r
0.994 0.952 0.971
4.7 0.9 19 0.956
t;Bg-5.9)
Hill function
4.9 (4.7-5.1)
ETso (h) 4.49 5.92 4.26 4.9 0.9 18
0.957
4.8 (4.7-5.0)
238
X Living
Cells
125.0
100.0
75.0
50.0
25.0
0.0
Probit
I
I Factwau-/X
:
10.000
7.5 -
5.0 -
2.5 -
i? d
, --l--T1rrl-rn-----T8 .i
-l--l l--rn-ll-1-----l--T--T-rl-r-l8 i Tim@ M
8 8
Fig. 1. Time/effect relationship: cytotoxicity of emetine dihydrochloride (1 mg/ml) on Amoeba proteus. Three experiments are represented (68 points). Top diagram represents curve fitting of experimental data using the Von Bertalanffy (VB) and Hill functions. Bottom diagram represents classic probit analysis.
239
= Efficient Concentration n = parameter. N=NO
l-
50 or EfIicient Time 50,
C” EC” + C”
Results
The complex dose/time/effect relationship has been classically (Ord, 1979) investigated by dissociation using either a study of time/effect for a Iixed dose (Table 1, Fig. 1) or of dose/effect for a fixed time (Table 2, Fig. 2). Although the former relationship more clearly approaches in vivo conditions, the latter was found to be much more manageable and was retained as the in vitro test basis. Determination of ET,, or ECSo values were realized by probit analysis and by fitting ex-
X Living
perimental data to the parametrical functions of “on Bertalanffy (VB) or Hill. Values computed by these three methods were very similar. Fitting of curves to experimental points was excellent (high values for the correlation coefficients) for the two investigated functions. In some cases (Fig. 2) differences were observed in the upper and lower sections of the sigmoid curves which would result in different EClo, ETlo, ECgOor ETgO, if calculated. This problem has been previously discussed (Dubois et al., 1989). Table 2 reveals constant significantly lower efticient concentrations for (*)-dehydroemetine than for (+)-emetine; in vitro Entamoeba histolytica tests show a variable efficient concentrations ratio for these two compounds (Neal, 1978; Cedeno et al., 1983; Keene et al., 1986) and the relationship between stereochemistry and activity was investigated with contradictory results (Neal, 1978). Using the A. proteus model here, these two com-
Cells
125.0
F~ctmJr/X
:
10.000
25.0
((IO/d
1
,o
E
0”
5:
Fig. 2. Concentration/effect relationship: cytotoxicity of emetine dihydrochloride on Amoeba proteus. 0, Contact time, 3 h (5 experiments, 55 points); a, contact time, 24 h (4 experiments, 28 points). Curve fitting of experimental data using the Von Bertalanffy (VB) and Hill functions.
2HCI
Emetine
24
3
2HCl
Emetine
I
k Pooled
Mean
2 3 4
I
3 4 5 Mean s tix Pooled
1 2
Experiment
28
0.968
0.982 0.995 0.989 0.913
0.921
55
I 7 7 7
0.978 0.972 0.989 0.992 0.991
r
Probit
analysis
0.22 0.25 0.24 0.21 0.23 0.02 9 0.23 (0.21-0.25)
fitting
0.992
1.000 1.000 0.997 1.000
0.987
0.991 0.994 0.985 1.000 1.000
r
VB function
Curve
DEHYDROEMETINE
1.72 1.46 1.55 I.54 I.5 0.3 17 1.20 (1.15-1.26)
1.05
(mg/ml)
EC,,
50; ND = not determined.
DIHYDROCHLORIDE.
II II II II II
N
concentration
FOR EMETINE
of terms; ECse = efficient contact time (h) ,
I for definition
RELATIONSHIP
Compound
See Table
CONCENTRATION/EFFECT HYDROBROMIDE
TABLE 2
0.19 0.25 ND 0.25 0.23 0.03 13 0.19 (0.17-0.21)
0.3 23 1.23 (1.19-1.28)
1.04 1.74 1.20 1.39 ND 1.3
ECso (mg/ml)
DIHYDROCHLORIDE
0.993
l.ooo l.ooo 0.996 1.000
0.988
0.996 0.994 0.993 1.000 1.000
r
0.17 0.24 ND 0.23 0.21 0.04 I9 0.17 (0.14-O. 19)
(1.26-1.27)
1.27
1.04 1.70 1.36 1.40 ND I.4 0.3 21
EGO (mg/ml)
Hill function
AND CUCURBITINE
3
24
3
‘2HCl
Dehydroemetine ‘2HCI
Cucurbitine . HBr
Dehydroemetine
1
2 3 Mean s A% Pooled
I
S% Pooled
2 3 4 Mean s
I
Mean s s% Pooled
3 4 5 6
2
0.997
0.898 0.979 0.926
0.943
IO 7 I
24
0.993 0.999 0.999 1.000
0.939
28
66
0.966 0.972 0.968 0.979 0.986 0.998
13.7 13.2 12.9 13.3 0.4 3 12.1 (11.6-12.6)
0.16 0.17 0.17 0.18 0.17 0.01 4 0.17 (0.15-0.20)
0.9 0.1 15 0.81 (0.77-0.88)
0.79 0.77 0.39 1.09
0.89 0.87
0.995
0.995 0.999 0.997
0.999
1.000 l.ooo l.ooo 1.000
0.986
1.ooo 1.000 1.000 1.000 1.000 1.000
0.5 4 11.4 (11.1-11.8)
12.2 11.2 11.5 11.6
12 0.14 (0.14-O. 14)
0.12 0.13 0.13 0.16 0.14 0.02
0.9 0.2 26 0.86 (0.81-0.91)
1.12 0.96 0.77 0.77 0.5 I .05
0.994
0.995 0.999 0.997
0.999
l.ooo 1.ooo l.ooo 1.000
0.986
1.ooo 1.000 l.ooo 1.ooo l.ooo l.ooo
11.9 11.3 11.1 11.4 0.4 4 II.1 (10.7-11.5)
7 0.12 (0.12-0.12)
0.11 0.11 0.11 0.12 0.11 0.01
(0.85-0.89)
1.11 0.96 0.81 0.80 0.50 1.04 0.9 0.2 26 0.87
242 X Living
Cells
125.0 Fmctmur/X
Fig. 3. Concentration/effect periments, 24 points).
relationship:
cytotoxicity
of cucurbitine
pounds present logically lower efficient concentrations at 24 h contact as compared to 3 h contact. Cucurbitine was found active at high concentrations (Fig. 3). Although curve fitting methods lead to better tit and more complete information than probit analyTABLE
hydrobromide
:
10.000
on Amoeba proteus.
Contact
time, 3 h (3 ex-
sis, they require a larger number of points in the steepest part of the curve (determination of 3 or 4 parameters) which is singularly complicated by the generally observed narrow activity ranges. Probit analysis was thus chosen as an estimation tool in rapid screenings for comparison of different com-
3
CONCENTRATION/EFFECT (N = 6) Tested compound
RELATIONSHIP
FOR DIFFERENT
Contact time (h)
COMPOUNDS
AS ESTIMATED
Concentration range for 0 to 100% killing
ECso (r&ml)
BY PROBIT
95% Confidence interval (m&d)
(mgml) Dacarbazine Daunorubicine Doxorubicine 5-Fluorouracil Metronidazole
3 5 4 24 72
0.1 to 10.0 0.01 to 0.1 0.01 to 0.1 0.5 to 1.0 > 10.0”
I.19 0.037 0.042 0.73 > 10.0
0.95-1.50 0.03-0.05 0.04-0.05 0.54-0.99 -
Atropine sulfate Chloroquine phosphate Tetracycline HCI
72 I6 -
> 10.0” 0.1 to 10.0 _b
> 10.0 0.24
0.21-0.2s
“No toxicity
up to maximum
concentrations
tested of IO mg/ml.
bPrecipitates
with calcium
ANALYSIS
ions of the medium.
TABLE 4 CONCENTRATION/EFFECT RELATIONSHIP FOR VARIOUS EXTRACTS OF EUPHORBIA TACT TIME, 24 h) AS ESTIMATED BY PROBIT ANALYSIS (A’ = 6)
HIRTA
AERIAL PARTS (CON-
Origin of sample
Treatment Before extraction
Residual” water content (W
Extraction methodology
EC,, b (ms/ml)
95% Contidence interval (mgml)
Zaire seeds grown in Belgium
Fresh Fresh Fresh 35°C
plant plant plant oven desiccated
85.0 85.0 85.0
10.0
20°C; 5 h 20°C; I6 h IOO’C; 15 min IOO’C; I5 min
4.1 3.0 2.3 18.0
(3.4-5.0) (2.4-3.7) (1.6-3.1) (14.4-22.5)
Burkina seeds (Farako-BB) grown in Belgium
Fresh plant 35°C oven desiccated
81.0 9.2
100°C; I5 min IOO’C; 15 min
5.5 27.6
(4.8-6.3) (24.4-31.0)
Burkina seeds (Barn) grown in Belgium
Fresh plant Fresh plant
85.0 85.0
100°C; I5 min 100°C; I5 min and lyophilisation 100°C; I5 min
11.9 14.9
(9.8-14.4) (13.0-16.9)
29.6
(26.0-33.7)
20°C; I6 h IOO’C; 5 min 100°C; 15 min 100°C; 30 min IOO’C; 45 min 100°C; 60 min
16.1 25.8 18.6 19.4 19.9 25.1
(13.2-19.8) (23.8-27.9) (16.3-21.2) (17.1-21.9) (17.7-22.3) (23.2-27. I)
35°C oven desiccated Harvested in Burkina (Farako-BP)
Air Air Air Air Air Air
9.7
10.0 10.0 10.0 10.0 10.0 10.0
desiccated desiccated desiccated desiccated desiccated desiccated
‘Determined using aliquots desiccated in a 105°C ventilated oven. bExpressed in terms of lO5“C dried starting material X Living
Cells
125.0 -
75.0
-
50.0
-
25.0
-
Fmcteur/X : 10.000
0.0 E: d
Fig. 4. Concentration/effect (2 experiments, I4 points).
g d
(mQ/ml)
8
8
E:
8
relationship: cytotoxicity of an aqueous Euphorbiu hirta extract on Amoeba proteus. Contact time, 24 h
244
pounds (Table 3) or for study of extraction methods (Application to Euphorbiu hirta, Table 4) while curve fitting was reserved for exact quantification of an extract’s activity (Fig. 4). Discussion The proposed cellular model is intended as a general bioassay for active plant constituents. Ideally, such a model should enable the detection and quantification of all cytotoxic and antiparasitic compounds thereby reducing the costs of maintaining several different cell and parasite cultures. Specific bioassays for the specific activities represent the ideal situation but costs are usually out of reach especially in Third World countries. The present authors believe that the proposed Amoeba proteus model could be substituted for more sophisticated models in many cases with good results. A. proteus is a complete eukaryotic aerobic cell; indeed a sensitivity to known cytotoxic compounds has been demonstrated here (Table 3) and a non-cytotoxic molecule (atropine) found to be inactive. These preliminary results are only indicative and many more experiments are required before establishing the suitability of this test as a means of detecting cytotoxic agents. Predictive values should be compared with the usually used murine or human cell models for hundreds of compounds before drawing any tentative conclusions. It can, however, already be predicted that certain pharmacological classes of cytotoxic agents will not be detected by the described test. Under the present test conditions, no cellular division is observed, so several phases of the cell cycle are not accessible to testing. This is probably due to the fasting condition that was originally specified so as to improve test reproducibility (digestive vacuoles are resorbed and pinocytic and endocytic activities are stabilized) and to avoid ~etrahymena metabolisation of the test chemicals (Ord, 1970, 1979; Al-Atia, 1980). Study of phase-specific agents would require modifications of test conditions, namely strict feeding schedules both before and during the test and longer incubation times. A general antiparasitic screen may seem an anomaly because a search for an antiparasitic drug
is really a search for an extremely specific compound, i.e. a drug active on a specific parasite and innocuous to the host organism. Therefore, specific tests can never be fully replaced, but a socalled general cytotoxicity test can be useful for a certain number of operations. Among the tested antiparasitics were three amoebicides (emetine, dehydroemetine and metronidazole), an anthelmintic (cucurbitine) and an antimalaric (chloroquine). All molecules were found active with the notable exception of metronidazole. This amoebicide, a blocker of anaerobic respiratory chains, is extremely specific for strict anaerobic organisms such as Entamoeba histolytica and therefore cannot be detected with the aerobic A. proteus model. Any compound interfering with the culture medium such as tetracycline will also be found inactive on this model. While the extent of A. proteus use in the screening of chemicals and plant extracts has yet to be assessed, a plant study methodology conscious of the test limitations can be proposed: (i) confirmation by literature and/or ethnobotanical survey of the reputed uses of the investigated plant (anticancer or antiparasitic); (ii) test of a total extract on A. proteus; (iii) if active, biological standardization of raw extracts; (iv) checking of various fractions using A. proteus to identify the potentially active drug(s); (v) testing of the active principles using more specific bioassays. At any stage, extracts, fractions and subfractions can be checked for desired activity using more specific in vitro models (murine or human cells, Entamoeba hist~lytica, ~eisseri~ spp., Acanthamoeba spp. or helminths) or even by in vivo methods. Nevertheless, the use of A. proteus should considerably reduce the number of these tests, thus allowing cost reductions and easier labto-lab collaborations. The proposed bioassay assumes that activity will be mediated on the different models by the same molecules, but this is the general problem of any in vitro assay. The first steps of this methodology were applied to an Euphorbia hirtu extraction study. Aqueous decoctions of E. hirta aerial parts are frequently used in many African countries as an anti-amoebic drug (Agence de Cooperation Culturehe et
245
Technique, 1986). Previous workers have conlirmed .this activity by in vitro tests (Ridet et al, 1964; Krishna Rao and Ganapaty, 1983) and by clinical trials (Martin et al., 1964) but so far no active compound has been isolated and cultivation, harvesting and extraction conditions have not been investigated. In the present study, aqueous extracts were found cytotoxic on A. proteus (Table 4) which allows some fundamental observations: (i) activity is strongly reduced by plant desiccation: (ii) ambient temperature extraction of up to 16 h preserves activity and such long extraction times seem necessary; (iii) 100°C extraction of up to 1 h preserves activity and an extraction time of 5 min is sufficient; (iv) lyophilisation of the extract preserves activity; and (v) active concentrations show large sample-to-sample variations. It appears that the traditional practice (Agence de Cooperation Culturelle et Technique, 1986) of decoction or infusion of principally fresh plants is justified; however, the sometimes recommended (Fernandez de la Pradilla, 198 1) reduction of doses in case of dried plant material is clearly a mistake. Due to the observed sample-to-sample variation in activity, biological standardization will be essential in case of raw extract use. Large-scale extractions will necessitate enzymatic stabilization of harvested material. The use of the A. proteus assay to study these last two points seems very appropriate. The culturing of amoebae in an aqueous medium makes treatment with water-soluble chemicals or aqueous extracts simple and methods for testing water-insoluble chemicals or extracts have been described, e.g. injection into single cells, use of phagocytosis or induction of pinocytosis (Hirschlield, 1959; Ord, 1979). Further investigations are currently under way in order to test the activities of organic extracts, to compare extract activities using Entamoeba histolytica and to assess the toxicological safety of Euphorbia hirta raw extracts by the study of cocarcinogenic phorbol esters that have been described as present (Baslas et al., 1980) or absent (Atallah et al., 1979). Conclusion
of an Amoeba proteus bioassay makes it an interesting low-cost alternative to more sophisticated models. Whenever a screening is found positive, the test is particularly useful as a preliminary tool to define optimal plant harvesting and drying as well as active fraction extraction, notably in the laboratories of developing countries. With the appropriate mathematical treatment of results, exact quantification of activity leads to precise biological standardization of raw extracts. The Hill function appears particularly suited to fit experimental data. Furthermore, techniques such as micrurgy (Hirschfield, 1959; Ord, 1979) are available for indepth studies of toxicity mechanisms and for differentiating cytoplasmic and nuclear effects or changes in metabolic activities of the Amoeba proteus cell. Such techniques can complement simple screening and standardization and allow fairly sophisticated research with this easy-to-culture and low-cost organism. Acknowledgments We thank Dr. J. Lejoly for supplying and identifying plant material, Dr. M. Devleeschouwer for the Tetrahymena cultures and Dr. J. Dubois for helpful discussion. This work was supported by the General Administration for Development Cooperation of the Belgian Ministry of Foreign Affairs, Foreign Trade and Development Cooperation. References Abi Khalil,
F., Dubois,
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the generality
the parameters
of linear
models in pharmacokinetics.
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et Technique
(1986)
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G.R.
(1980) Toxicity
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approach.
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of cadmium
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to Amoebu proteus:
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A.M.
and Nicholas,
H.J.
steroids of Euphorhia pihdfira. Baslas,
its limitations,
M. and Atassi. G. (1986)
Europeun Journal of Drug Metabolism and Pharmacokineiics 1 I, 5I-59.
compartment
R.K.
and
characterization
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for computing
Agarwal,
of different
(1972)
Triterpenoids
Phyrochemisrrj~ R.
(1980)
constituents
Linn. Current Science 49. 31 I-312.
I 1,
Isolation
and
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