Hereditas 85: 105-112 (1977)
Mutagenicity of Bacillus thuringiensis exotoxin I. Mammalian tests TYTTI MERETOJA, GUNNEL CARLBERG,' ULLA GRIPENBERG, KAIJA LINNAINMAA AND MARJA SORSA Department of Genetics and Department of Microbiology,' University of Helsinki, Finland
MERETOIA,T., CARLBERG, G., GRIPENBERG, U.,LINNAINMAA, K.and SORSA,M. 1977. Mutagenicity of Bacillus thuringiensis exotoxin 1. Mammalian tests. - Hereditas 85: 105-1 12. Lund, Sweden. ISSN 0018-0661. Received December 12, 1976
The mutagenicity of B. thuringiensis preparations was tested in different mammalian systems. Human blood cultures were used for studying cytological effects on the cells after in vitro treatment with autoclaved supernatant on B. fhuringiensis serotype I (producer of exotoxin), serotype 3 (non-producer) and purified exotoxin. Bone-marrow cells and cultured blood cells of rats were studied after long-term feeding with varying concentrations of supernatant of serotype I . Cytological effects of acute exposure to lethal concentrations of exotoxin were studied in the same cell systems. In human blood cultures toxic concentrations of exotoxin induced a significant increase in the incidence of chromosomal aberrations. Also the treatment with supernatant of serotype 3 revealed a slight clastogenic effect, although no mitotic arrest could be observed. In in vivo experiments with rats no clastogenic effects could be demonstrated in bone-marrow cells. Low concentrations of serotype 1 supernatant did not affect blood metaphases of the treated animals either. Clastogenic effects appeared when the drinking water was substituted by 50 or 100% of the agent during three months. Similarly, clastogenic effects were observed in blood cells of rats given lethal doses of serotype 1 supernatant. Marja Sorsa, Department of Genetics. P . Rautatiekatu 13, SF-00100 Helsinki 10, Finland
The two main insecticidal toxines synthetized by Bacillus thuringiensis are: the proteinaceous endotoxin widely used in the control of Lepidopterous pest insects, and the exotoxin, a nucleotide. The latter is nevertheless produced only by certain serotypes. During the last decade commercial exotoxin containing B. thuringiensis preparations have been used for microbial control of flies. The exotoxin is structurally related to ATP, and inhibits the RNA synthesis in vitro (DEBARJAC and DEWNDER1965, 1968; BONDet al. 1969; SEBFSTAet al. 1967, 1969a; FARKAS et al. 1969; BEEBEEet al. 1972; SMUCKLER and HADJIOLOV 1972; MACKEDONSKI et al. 1972; MACKEWNSKI 1975). In addition, inhibition of DNA synthesis has been demonstrated (KIMet al. 1972; CARLBERG 1973). It is also toxic for mammals by injection (DEBARJACand RIOU1969; SEBESTAet al. 1969b), and therefore the use of exotoxin is no longer recommended. Recently exotoxin has been shown to be a competitive inhibitor of adenyl cyclase (GRAHAME-SMITR et al. 1975) and at high concentrations it inhibits cellfree protein synthesis (SOMERVILLE and SWAIN19f5). Exotoxin is able to cross cell and nuclear membranes
in mammalian systems (SMUCKLER and HADJIOLOV 1972; BEEBEE et al. 1972; MACKEWNSKI et al. 1972). It is known to cause severe teratological effects in susceptible insects (e.g. DUNN1960; BRIGGS1960; CANTWELL et al. 1964, BURGERJON et al. 1969; WOLFENBERGER et al. 1972; LAMand WEBSTER 1972). The only evidence so far, concerning mutagenesis of exotoxin, is based on the observations of inherited deformations in Leptinotarsa (BURGERJON 1974). Thus an effective search on the possible mutagenic properties of exotoxin is well motivated. The object of this paper is to study the effects of B. thuringiensis preparations on rats in vivo after long-term feeding and acute exposure, and on human blood cells in vitro.
Material and methods 1. Source of exotoxin Previous experiments have shown that purified exotoxin has a biological effect different from the crude autoclaved culture supernatant (CARLBERG 1973).
106
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AL.
Because the crude culture is the preparation generally used in the microbial control of pest insects, both broth culture of B. thuringiensis and pure exotoxin were used in the experiments. B. thuringiensis serotype 1, known as a constant exotoxin producer, was grown on nutrient broth containing 1.O% glucose, and on a modified Conner and Hansen’s medium (CONNER and HANSEN1967) containing glutaminate instead of casamino acids. Growth was continued at 28°C in shake flasks (Gyrotory shaker, 200 rpm) for one week. The growth medium was centrifuged and the supernatant autoclaved for 20 min at 120°C. B. thuringiensis serotype 3, which does not produce exotoxin, was grown and treated as serotype 1. The purified exotoxin was kindly supplied by Dr. R. Bond, Shell Research Ltd., Borden Microbiological Laboratory, Sittingbourne, Kent, UK. In the present work the concepts “ASI” and “AS3” are used for autoclaved supernatants, and “ET Bond” for the purified substance. The media per se, used as controls, are called nutrient medium and glutaminate medium.
injected with an equal volume of pure nutrient medium. In control group 2, the animals were untreated. From previous studies (CARLBERG 1973) exotoxin-treated mice were known to die on the third day after injection. In these experiments the rats were sacrificed on the second day. Bone-marrow tissue of the femur as well as heart-blood were sampled for chromosome studies. Bone-marrow cells were suspended in Hanks’ solution, incubated one hour with Velbe (Lilly)0.02 m1/3ml, followed by hyptr tonic treatment with 0.075 M KCl solution, and fixed with methanol-glacial acetic acid (3:1). The slides were air-dried and stained with 10% solution of Giemsa. The blood of rats was cultured according to the microculture technique used for human cells. In addition, the animals were submitted to pathological and bacteriological examinations. The following organs were included in the histological screening: heart, liver, kidneys (Scharlach Rot, haematoxylin-eosin, PAS), lungs, spleen, adrenals, thymus, thyroid gland, pancreas, abdomen, intestine, bladder, ovary, womb, cerebrum, cerebellum arid parotis (haematoxylin-eosin, PAS).
2. Human blood cultures
Whole blood microcultures, established according to conventional methods, were used to test the effects of exotoxin on human cells in vitro. To minimize the known individual variation in cell growth and reactivity, blood from the same healthy female donor was used throughout the experiments, thus allowing a strict comparison of results in successive test systems. Separate cultures which were to be compared were always established from the same blood sample and cultured at the same time. Each test included several parallel cultures and was repeated two or more times. The agents to be tested were added to the cell suspensions for the entire culture period. Air-dried slides were stained with 10% solution of Giemsa stain. Inhibition of mitotic activity was used as a measure of cytotoxicity. To estimate the mitotic activity or mitotic index, the number of mitoses per 10,OOO cells was counted in each culture. Metaphase plates were selected for detailed chromosome analysis. The number of aneuploid metaphases and the occurrence of structural chromosome and chromatid aberrations including breaks and fragments were recorded. 3. Acute exposure experiments with rats
Female rats of the same age were injected intraperitoneally with a lethal dose of “ASI” (0.02 ml/g, concentrated ten times). In control group 1 rats were
4. Long-term feeding experiments with rats Different concentrations of “ASI” grown in glutaminate medium were given to female rats of the same age for three months in drinking water. “ASI” grown in glutaminate medium instead of nutrient medium was chosen because of its greater toxicity to house flies (CARLBERC, unpubl.) in order to elicit the most striking effects of the agent. Pure medium was given to the control animals. The animals were thereafter sacrificed, the bone-marrow cells and blood sampled, and pathological examinations performed as above.
Results 1. Human lymphocyteculture tests
Cytotoxic effects of exotoxin. -The effect of varying concentrations of “ASl” grown in nutrient medium, affecting the cells during the entire culture time, was tested. In preliminary cultures polymorphonuclear leukocytes were shown to be affected so that in higher concentrations (over 10%) no polymorphs could be seen on the slides, while they were frequently present on the controls. The same effect was noted in cultures containing supernatant of B. thuringiensis serotype 3, which does not produce exotoxin. However, purified exotoxin seemed to have
MUTAGENICITY OF EXOTOXIN I
Hereditas 85 (1977)
107
\ I-
.1
1
2
I
4
I
8
10
15
2 0 %(v/v)
CONCENTRATION OF EXOTOXIN
Fig. I . The effect of "ASI" on the mitotic activity of human lymphocytes. The mitotic index ratio gives the ratio of mitotic indices in "AS1" and control cultures.
Table 1. The Occurrence of aneuploidy and chromosomal aberrations in metaphase cells from cultures containing toxic concentration of exotoxin In each case 100 metaphases examined Conc.
Aneuploid cells
Cells with chromosomal aberrations Gaps alone
AS1 nutrient medium
Breaks
Total
a
20%-v/v
3
4
26
30
Control nutrient medium
20%-v/v
1
6
3
9
AS1 glutaminate medium
20%-v/v
3
2
16
18
Control glutaminate medium
20%-v/v
1
2
3
5
Control AS3 nutrient medium
20%-v/v
2
1
7
8
'ET-Bond'
20~g/ml
1
-
10
10
1
-
2
2
Control redest. water
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Table 2. The occurrence of chromosomal aberrations in bone-marrow cells of rats injected with lethal doses of exotoxin Metaphases examined
Cells with chromosomal aberrations Gaps alone
Test group animal I animal 2 animal 3 animal 4
I00 100 100
Control group 1 animal 5 animal 6
100
Control group 2 animal I
2 1 1 2
I00
100
Total
4 9
6 10 12 10
II
8 9 4(=7/100)
9
2(=3/100) 1
I
2
~
60
Breaks
6(=10/100)
Table 3. The occurrence of aneuploidy and chromosomal aberrations in cultured rat lymphocytes after injection with lethal doses of exotoxin In each case 100 metaphases examined Aneuploid cells
Cells with chromosomal aberrations Gaps alone
Breaks
Total
2
3 6
24 25
21
5
Control group I animal 5
1
I
10
11
Control group 2 animal 7
3
7
14
21
Test group animal I animal 3
31
no effect of this kind. When considering the toxicity however, significant. Autoclaved supernatant of of B. thuringiensis preparations, the comparison of the , “ASI” shows more pronounced effects than does the effects of the three agents is inevitable. On the other purified exotoxin. The slight increase in the frehand, the quantity of polymorphs is known to vary, quency of breaks in “AS3” cultures indicates a thus complicating the interpretation of the results in possible influence of metabolic factors other than successive sets of experiments. Therefore, in order to exotoxin in the medium. Even though 4% v/v “ASI” remove polymorphonuclear leukocytes, the blood in the cultures had no effect on cell growth, it seemed was allowed to sediment overnight in a glass bottle to induce chromosome breakage. At this concentrabefore establishing cultures. tion the frequency of metaphases with aberrations Fig. 1 shows the relation between the mitotic index was 17% (cells with gaps alone 3/100, cells with and the concentration of exotoxin. Total inhibition breaks 14/100). In control cultures the aberration of cell growth could not be demonstrated. frequency was 6% (all breaks). Clastogenic effects of exotoxin. - The distinctly toxic concentration of 20% v/v was selected when comparing the effects of different B. thuringirnsis 2. Acute exposure experiments with rats preparations. Table I gives the results of a detailed A test group of four female rats was given a lethal chromosome analysis of the metaphase plates. The dose of “ASl” as an i.p. injection. Two rats injected occurrence of aneuploidy is not statistically signifi- with the same volume of pure nutrient medium and cant. The incidence of chromosomal aberrations is, one rat with no treatment served as controls. The
MUTAGENICITYOF EXOTOXINI
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Table 4. The occurrence of chromosomal aberrations in bone-marrow cells of rats fed with exotoxin during three months In each case 100 metaphases examined Conc. of AS1
Cells with chromosomal aberrations Gaps alone
Breaks
Total
Control animal 1 animal 2
6 4
7 4
Test animal 3 animal 4 animal 5
2 9 9
3 I1
5
6
3
3
2
3
-
4
5 4
animal 6 animal 7 animal 8 animal 9
animals were sacrificed on the second day after treatment and examined for pathologic changes. Macroscopically the animals were normal. In histological screening the only changes observed in the test group animals were liver changes, including fatty liver and haemorrhagia, and calcification of kidneys. Bacteriological findings were negative. The results of the chromosome analysis of bonemarrow cell metaphases are listed in Table 2. As shown, “ASl” had no effect on induction of chromosomal aberrations. Table 3 gives the results of a detailed analysis of rat lymphocyte cultures. In the occurrence of aneuploidy no difference could be demonstrated between test and control animals. Instead, the incidence of chromosomal aberrations is significantly higher in treated animals. 3. Long-term feediig experiments with rats
Three test groups of young female rats were given in drinking water l000/,, 50% and 5% concentrations of “ASI” grown in glutaminate medium. After 3 months of treatment all animals were in good condition. Veterinary examination revealed no pathological changes except calcification in tubuli renales observed both in test and control animals. Bacteriologic findings were negative. Altogether 100 bone-marrow metaphases were analysed in detail for the occurrence of chromosomal aberrations. The results are presented in Table 4. According to the results exotoxin had no effect on the incidence of aneuploidy or chromosomal aberrations
5% 5%
10
in bone-marrow cells. However, observation of bonemarrow cells revealed a lack of megakaryocytes in animals of the 100% group with none or very few megakaryocytes on the slides. The blood picture of the rats was not examined prior to culturing, thus permitting only limited conclusions on the Occurrence of different cell types. Lack of polymorphonuclear leukocytes in animals of the 100% group was, however, obvious. In addition, disintegration of cells and a very low number of mitoses could be seen on the slides. In blood cultures of the 50% group, cell debris was obvious, as well. In the 5 % group, however, no effect of the treatment could be observed. In the control group, the substitution of drinking water totally by glutaminate medium induced a strong inhibition of mitotic activity in cultured blood cells. Thus only a few mitoses were available for the analysis. No disintegration of cells could be observed. The experiment was repeated later and identical results were obtained. The results of the chromosome analysis of rat blood cells are presented in Table 5 . In the 5 % group no effect on the incidence of chromosomal aberrations could be demonstrated. However, when taking into account the relatively low frequency of chromosomal aberrations in the control group, the chromosomal damage (cf. Fig. 2) and cell debris observed in the 50% and 100% groups can largely be attributed to the “ASI” feeding, although the possibility of some effect of the glutaminate medium itself cannot be excluded.
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Herediras 8.5 (1977)
MUTAGENICITY OF EXOTOXIN I
11 1
Table 5. The occurrence of chromosomal aberrations in cultured lymphocytes of rats fed with exotoxin during three months ~
Conc. of AS1
Metaphases examined
~~
Aneuploid
~
% Cells with chromosomal aberrations
cells Gaps alone
Breaks
Total
Control animal 1 animal 2 animal 3 Test animal 4 animal 5 animal 6
100 5;; 100% 100%
10 24
30 12.5
-
40 66.7
40 66.7
animal 7 animal 8
50 % 50 %
II
9.1 2.9
-
34
2.9
54.5 61.8
54.5 64.7
animal 9 animal 10
5% 5%
5 12
5 13
No mitoses
100
100
Discussion According to the results presented in this paper, the long-term feeding experiments with rats revealed no difference between test and control animals in pathological examinations. Earlier long-term feeding studies with mice were also negative (CARLBERG 1973). SEBFSTAand HORSKA(1972) administered purified exotoxin intraperitoneally to mice and showed it to be rapidly excreted in urine in an unaltered form. In addition, exotoxin was dephosphorylated to a non-toxic form in the tissue. On the other hand, DE BARJAC and LECADET (1975) demonstrated that mice excrete orally given exotoxin rapidly in feces. The urine was totally negative. No accumulation of exotoxin in the tissue could be demonstrated in either of the studies. The substitution ofdrinking water totally by “ASI” did not, however, remain without effects. Lack of megakaryocytes in the bone marrow and polymorphs in the blood cultures could be demonstrated. Many chemicals, radiation and infections are known to induce similar changes. As for chromosomal effects and possible mutagenicity, in in vivo experiments even the total substitution of drinking water by “ASl” for three months had no effect on the incidence of chromosomal aberrations in the bone-marrow cells. The results of the bone-marrow cell analysis of rats given a lethal dose of “ASI” were also negative. Instead, a clear clastogeniceffect was observed in vivo in the blood cells of rats in the 50% and 100% groups of the long-term feeding experiment as well as in the acute exposure test and in human blood cultures
2 2
-
I
in vitro. Hence, the cell debris and the resulting decrease in the number of cells observed in blood cultures of the 50% and 100% groups of rats in the long-term feeding experiment is at least partially explained by the Occurrence of chromosomal aberrations. Because all the animals were sacrificed at the same time we cannot ascertain whether the effects observed were transient, as was the case with toxic changes in pullets induced by B. thuringiensk preparations (BARKER and ANDERSON 1975). While considering the relatively high incidence of chromosomal aberrations in the blood cells of even the control animals, the white blood count in rats should be taken into account. It differs strikingly 1967) with possibly from that of humans (SCHERMER quite different functions. Generally the clastogenic effects, observed only in blood cells, were caused by high concentrations of exotoxin, much above the amounts normally used in the control of pest insects. Chromosomal aberrations have been demonstrated in connection with bacterial and viral products including commonly used vaccines (NICHOLS et al. 1962; NICHOLS 1963; HARNDEN1964; MIKHAIL~VA and GORSHUNOVA 1975). Further studies, taking into account the sensitive reactivity of the immunocompetent cells in blood, are needed for critical consideration of the actual harm of the effects observed, and for drawing final conclusions on the safety of B. thuringiensis preparations. Acknowledgments. -We wish to thank Drs. A . 4 . Andersson and P. Andersson (State Veterinary Medical Institute) for the histological and pathological examination of the rats used in
112
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the experiments. The Academy of Finland and Kemira Oy have financially supported this study.
Literature cited BARJAC,H. and DEDONDER,R. 1965. Isolement d'un nucleotide identifiable d la "toxin thermostable" de Bacillus rhuringiensis var. Berliner. - C . R. Acad. Sci. (Paris) 260: 7050-7053 DE BARJAC, H. and DEWNDER, R. 1968. Purification de la toxine thermostable de Bacillus thuringiensis var. thuringiensis et analyses complementaires. - Bull. Soc. Chim. Biol. 50: 941 -944 DE BARJAC,H. and LECADET, M.-M. 1975. Elimination de I'exotoxine thermostable de B. thuringiensi.9 apres ingestion chez la Souris. - C. R. Acad. Sci. (Paris) 286: 677-679 DE BARJAC, H. and RIOU.J.-Y.1969. Action de la toxine thermostable de Bacillus thuringiensis var. thuringiensis administree a des souris. - Rev. Pathol. Comp. Mid. E.xp. 6 : 367-374 BARKER, R. J. and ANDERSON, W. F. 1975. Evaluation of p-exotoxin of Bacillus thuringiensis Berliner for control of flies in chicken manure. - J . Med. Entomol. 1 2 103-1 10 BEEBEE, T., KORNER,A. and BOND,R. P. M. 1972. Differential inhibition of mammalian ribonucleic acid polymerases by an exotoxin from Bacillus Ihuringiensi.9. -- Biochem. J . 127: 619-624 BOND,R. P. M., BOYCE,C. B. C. and FRENCH, S. J. 1969. A purification and some properties of an insecticidal exotoxin from Bacillus thuringiensis Berliner. - Biorhem. J. 114 477-488 BRIGGS,J . D. 1960. Reduction of adult housefly emergence by the effects of Bacillus spp. on the development of immature forms. - J..Insect Pathol. 2 418-432 BURGERJON, A. 1974. Effects physiologiques et mutagenes sur les Insects de la toxine thermostable de Bacillus thuringiensis Berliner. Ann. Parasitol. Hum. Comp. 48: 835-844 BURGEMON, A,, BIACHE, G. and CALS,PH. 1969. Teratology of the Colorado potato beetle, Leptinotarsa decemlineara, as provoked by larval administration of the thermostable toxin of Bacillus thuringiensis. - J. Inverreh. Pathol. 14: 274-278 CANTWELL, G . E., HEIMPEL, A. M. and THOMPSON, M. J. 1964. The production of an exotoxin by various crystal-forming bacteria related to Bacillus rhuringiensis var. thuringiensis Berliner. - J . Insect Pathol. 6 : 466-480 CARLBERG, G. 1973. Biological effects of the termostable p-exotoxin produced by different serotypes of Bacillus thuringiensis. - Rep. Dept. Microhiol. Univ. Helsinki 6 CONNER, K. M. and HANSEN,P. A. 1967. Effects of valine, leucine and isoleucine on the growth of Bacillus rhuringiensis and related bacteria. - J . Inverreh. Pathol. 9 12-18 DUNN,P. H. 1960. Control of houseflies in bovine feces by a feed additive containing Bacillus thuringiensis var. thuringiensis Berliner. - J . Insect Pathol. 2 13-16 J., SEBESTA,K., HORSKA, K., SAMEK, 2.. DOLEJA,L. FARKAS, and SORM.F. 1969. The structure of exotoxin of Bacillus thuringiensis var. gelechiae. - Collection Czech. Chetn. Commun.34: 1118-1120 DE
~
GRAHAME-SMITH, D. G., ISAAC,P., HEAL,D. J. and BOND, R. P. M. 1975. Inhibition of adenyl cyclase by an exotoxin of Bacillus thuringimsis. - Nature 253: 58-60 HARNDEN, D. G. 1964. Cytogenetic studies on patients with virus infections and subjects vaccinated against yellow fever. - Am. J. Hum. Genet. 16: 204-213 B. G. and IGNOFFO, C. M. 1972. The KIM,Y . T., GREGORY. p-exotoxin of B. rhuringiensis. - J . Inverreh. Pathol. 20: 46-50 LAM,A. B. Q.and WEBSTER, J. M. 1972. Effect the DD-136 Nematode and of a p-exotoxin preparation of B. rhuringiensis var. thuringiensis on Leatherjachets, Tipuh paludosa larvae. - J . Inverreb. Parhol. 20: 141-149 MACKEDONSKI, V. V. 1975. Effect of the exotoxin of Bacillus rhuringiensis on the biosynthesis and maturation of mouse liver nuclear RNA. - Biochim. Biophys. Actu 390: 319-326 V. V., NIKOLAEV. N., SEBESTA,K., and MACKEDONSKI, HADJIOLOV, A. A. 1972. Inhibition of ribonucleic acid biosynthesis in mice liver by the exotoxin of 8. thuringiensis. Biochim. Biophys. Acia 2 7 2 56-66 MIKHAILOVA, G . R. and GORSHUNOVA. L. 1975. Investigation of chromosomes in bone marrow cells of mice inoculated subsequently with various vaccines. - Tsitol. Genet. 9: 460-461 NICHOLS, W. W.1963. Relationships of viruses, chromosomes Hereditas 50: 53-80 and carcinogenesis. NICHOLS,W. W., LEVAN, A,, HALL,B. and OSTERGREN, G. 1962. Measles-associated chromosome breakage. - Hereditas 48: 367-370 SCHERMER, S. 1967. The white rat. - In The Blood Morphology of Laboratory Animals, 3 ed. (Ed. S . SCHERMER), Davis. Philadelphia, p. 41 -60 K. 1973. The fate of exotoxin from SEBESTA.K. and HORSKA, Bacillus thuringiensis in mice. .- Collection Czech. Chem. Commun. 3 8 2533-2537 SEBESTA, K.,HORSKA.K. and VANKOVA, J. 1967. Isolation, purification and toxicity of a thermostable exotoxin from the strain of Bacillus gelechiae. - In Insect Pathology und Microbial Control(Ed. P. A. VANDERLAAN), Norrh-Holland, Amsterdam, p. 238-242 K.,HORSKA,K. and VANKOVA, J. 1969a. Isolation SEBESTA, and properties of the insecticidal exotoxin of B. ~huringietuis var. gelechiae. - Collection Czech. Commun. 34: 891 -900 J. 1969b. Inhibition SEBESTA, K., HORSKA, K. and VANKOVA, of de novo KNA synthesis by the insecticidal exotoxin of B. thuringiensis var. gelechiae. - Collection Czech. Chrm. Commun. 34: 1789- I79 I SMUCKLER, E. A. and HADJIOLOV, A. A. 1972. Inhibition of hepatic DNA-dependent RNA polymerases by the exotoxin of B. rhuringiensis in comparison with the effects of rr-amanitin and cordycepin. rBiochem. J. 12Y. 153-166 SOMERVILLE, H. J. and SWAIN,H. M. 1975. Temperaturedependent inhibition of polyphenylalanine formation by the exotoxin of Bucillus thuringiensis. - FEBS Letters 54: 330-333 H:T.and WOLFENBERGER. D. A., GUERRA, A. A., DULMAGE, CAREIA,R. D. 1972. Properties of the p-exotoxin 01' B. thuringiensis IMC 10,001 against the tobacco budworm. J. Econ. Entomol. 65: 1245- I248 ~
Mutagenicity of Bacillus thuringiensis exotoxin I. Mammalian tests TYTTI MERETOJA, GUNNEL CARLBERG,' ULLA GRIPENBERG, KAIJA LINNAINMAA AND MARJA SORSA Department of Genetics and Department of Microbiology,' University of Helsinki, Finland
MERETOIA,T., CARLBERG, G., GRIPENBERG, U.,LINNAINMAA, K.and SORSA,M. 1977. Mutagenicity of Bacillus thuringiensis exotoxin 1. Mammalian tests. - Hereditas 85: 105-1 12. Lund, Sweden. ISSN 0018-0661. Received December 12, 1976
The mutagenicity of B. thuringiensis preparations was tested in different mammalian systems. Human blood cultures were used for studying cytological effects on the cells after in vitro treatment with autoclaved supernatant on B. fhuringiensis serotype I (producer of exotoxin), serotype 3 (non-producer) and purified exotoxin. Bone-marrow cells and cultured blood cells of rats were studied after long-term feeding with varying concentrations of supernatant of serotype I . Cytological effects of acute exposure to lethal concentrations of exotoxin were studied in the same cell systems. In human blood cultures toxic concentrations of exotoxin induced a significant increase in the incidence of chromosomal aberrations. Also the treatment with supernatant of serotype 3 revealed a slight clastogenic effect, although no mitotic arrest could be observed. In in vivo experiments with rats no clastogenic effects could be demonstrated in bone-marrow cells. Low concentrations of serotype 1 supernatant did not affect blood metaphases of the treated animals either. Clastogenic effects appeared when the drinking water was substituted by 50 or 100% of the agent during three months. Similarly, clastogenic effects were observed in blood cells of rats given lethal doses of serotype 1 supernatant. Marja Sorsa, Department of Genetics. P . Rautatiekatu 13, SF-00100 Helsinki 10, Finland
The two main insecticidal toxines synthetized by Bacillus thuringiensis are: the proteinaceous endotoxin widely used in the control of Lepidopterous pest insects, and the exotoxin, a nucleotide. The latter is nevertheless produced only by certain serotypes. During the last decade commercial exotoxin containing B. thuringiensis preparations have been used for microbial control of flies. The exotoxin is structurally related to ATP, and inhibits the RNA synthesis in vitro (DEBARJAC and DEWNDER1965, 1968; BONDet al. 1969; SEBFSTAet al. 1967, 1969a; FARKAS et al. 1969; BEEBEEet al. 1972; SMUCKLER and HADJIOLOV 1972; MACKEDONSKI et al. 1972; MACKEWNSKI 1975). In addition, inhibition of DNA synthesis has been demonstrated (KIMet al. 1972; CARLBERG 1973). It is also toxic for mammals by injection (DEBARJACand RIOU1969; SEBESTAet al. 1969b), and therefore the use of exotoxin is no longer recommended. Recently exotoxin has been shown to be a competitive inhibitor of adenyl cyclase (GRAHAME-SMITR et al. 1975) and at high concentrations it inhibits cellfree protein synthesis (SOMERVILLE and SWAIN19f5). Exotoxin is able to cross cell and nuclear membranes
in mammalian systems (SMUCKLER and HADJIOLOV 1972; BEEBEE et al. 1972; MACKEWNSKI et al. 1972). It is known to cause severe teratological effects in susceptible insects (e.g. DUNN1960; BRIGGS1960; CANTWELL et al. 1964, BURGERJON et al. 1969; WOLFENBERGER et al. 1972; LAMand WEBSTER 1972). The only evidence so far, concerning mutagenesis of exotoxin, is based on the observations of inherited deformations in Leptinotarsa (BURGERJON 1974). Thus an effective search on the possible mutagenic properties of exotoxin is well motivated. The object of this paper is to study the effects of B. thuringiensis preparations on rats in vivo after long-term feeding and acute exposure, and on human blood cells in vitro.
Material and methods 1. Source of exotoxin Previous experiments have shown that purified exotoxin has a biological effect different from the crude autoclaved culture supernatant (CARLBERG 1973).
106
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Hereditas 85 (1977)
AL.
Because the crude culture is the preparation generally used in the microbial control of pest insects, both broth culture of B. thuringiensis and pure exotoxin were used in the experiments. B. thuringiensis serotype 1, known as a constant exotoxin producer, was grown on nutrient broth containing 1.O% glucose, and on a modified Conner and Hansen’s medium (CONNER and HANSEN1967) containing glutaminate instead of casamino acids. Growth was continued at 28°C in shake flasks (Gyrotory shaker, 200 rpm) for one week. The growth medium was centrifuged and the supernatant autoclaved for 20 min at 120°C. B. thuringiensis serotype 3, which does not produce exotoxin, was grown and treated as serotype 1. The purified exotoxin was kindly supplied by Dr. R. Bond, Shell Research Ltd., Borden Microbiological Laboratory, Sittingbourne, Kent, UK. In the present work the concepts “ASI” and “AS3” are used for autoclaved supernatants, and “ET Bond” for the purified substance. The media per se, used as controls, are called nutrient medium and glutaminate medium.
injected with an equal volume of pure nutrient medium. In control group 2, the animals were untreated. From previous studies (CARLBERG 1973) exotoxin-treated mice were known to die on the third day after injection. In these experiments the rats were sacrificed on the second day. Bone-marrow tissue of the femur as well as heart-blood were sampled for chromosome studies. Bone-marrow cells were suspended in Hanks’ solution, incubated one hour with Velbe (Lilly)0.02 m1/3ml, followed by hyptr tonic treatment with 0.075 M KCl solution, and fixed with methanol-glacial acetic acid (3:1). The slides were air-dried and stained with 10% solution of Giemsa. The blood of rats was cultured according to the microculture technique used for human cells. In addition, the animals were submitted to pathological and bacteriological examinations. The following organs were included in the histological screening: heart, liver, kidneys (Scharlach Rot, haematoxylin-eosin, PAS), lungs, spleen, adrenals, thymus, thyroid gland, pancreas, abdomen, intestine, bladder, ovary, womb, cerebrum, cerebellum arid parotis (haematoxylin-eosin, PAS).
2. Human blood cultures
Whole blood microcultures, established according to conventional methods, were used to test the effects of exotoxin on human cells in vitro. To minimize the known individual variation in cell growth and reactivity, blood from the same healthy female donor was used throughout the experiments, thus allowing a strict comparison of results in successive test systems. Separate cultures which were to be compared were always established from the same blood sample and cultured at the same time. Each test included several parallel cultures and was repeated two or more times. The agents to be tested were added to the cell suspensions for the entire culture period. Air-dried slides were stained with 10% solution of Giemsa stain. Inhibition of mitotic activity was used as a measure of cytotoxicity. To estimate the mitotic activity or mitotic index, the number of mitoses per 10,OOO cells was counted in each culture. Metaphase plates were selected for detailed chromosome analysis. The number of aneuploid metaphases and the occurrence of structural chromosome and chromatid aberrations including breaks and fragments were recorded. 3. Acute exposure experiments with rats
Female rats of the same age were injected intraperitoneally with a lethal dose of “ASI” (0.02 ml/g, concentrated ten times). In control group 1 rats were
4. Long-term feeding experiments with rats Different concentrations of “ASI” grown in glutaminate medium were given to female rats of the same age for three months in drinking water. “ASI” grown in glutaminate medium instead of nutrient medium was chosen because of its greater toxicity to house flies (CARLBERC, unpubl.) in order to elicit the most striking effects of the agent. Pure medium was given to the control animals. The animals were thereafter sacrificed, the bone-marrow cells and blood sampled, and pathological examinations performed as above.
Results 1. Human lymphocyteculture tests
Cytotoxic effects of exotoxin. -The effect of varying concentrations of “ASl” grown in nutrient medium, affecting the cells during the entire culture time, was tested. In preliminary cultures polymorphonuclear leukocytes were shown to be affected so that in higher concentrations (over 10%) no polymorphs could be seen on the slides, while they were frequently present on the controls. The same effect was noted in cultures containing supernatant of B. thuringiensis serotype 3, which does not produce exotoxin. However, purified exotoxin seemed to have
MUTAGENICITY OF EXOTOXIN I
Hereditas 85 (1977)
107
\ I-
.1
1
2
I
4
I
8
10
15
2 0 %(v/v)
CONCENTRATION OF EXOTOXIN
Fig. I . The effect of "ASI" on the mitotic activity of human lymphocytes. The mitotic index ratio gives the ratio of mitotic indices in "AS1" and control cultures.
Table 1. The Occurrence of aneuploidy and chromosomal aberrations in metaphase cells from cultures containing toxic concentration of exotoxin In each case 100 metaphases examined Conc.
Aneuploid cells
Cells with chromosomal aberrations Gaps alone
AS1 nutrient medium
Breaks
Total
a
20%-v/v
3
4
26
30
Control nutrient medium
20%-v/v
1
6
3
9
AS1 glutaminate medium
20%-v/v
3
2
16
18
Control glutaminate medium
20%-v/v
1
2
3
5
Control AS3 nutrient medium
20%-v/v
2
1
7
8
'ET-Bond'
20~g/ml
1
-
10
10
1
-
2
2
Control redest. water
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Table 2. The occurrence of chromosomal aberrations in bone-marrow cells of rats injected with lethal doses of exotoxin Metaphases examined
Cells with chromosomal aberrations Gaps alone
Test group animal I animal 2 animal 3 animal 4
I00 100 100
Control group 1 animal 5 animal 6
100
Control group 2 animal I
2 1 1 2
I00
100
Total
4 9
6 10 12 10
II
8 9 4(=7/100)
9
2(=3/100) 1
I
2
~
60
Breaks
6(=10/100)
Table 3. The occurrence of aneuploidy and chromosomal aberrations in cultured rat lymphocytes after injection with lethal doses of exotoxin In each case 100 metaphases examined Aneuploid cells
Cells with chromosomal aberrations Gaps alone
Breaks
Total
2
3 6
24 25
21
5
Control group I animal 5
1
I
10
11
Control group 2 animal 7
3
7
14
21
Test group animal I animal 3
31
no effect of this kind. When considering the toxicity however, significant. Autoclaved supernatant of of B. thuringiensis preparations, the comparison of the , “ASI” shows more pronounced effects than does the effects of the three agents is inevitable. On the other purified exotoxin. The slight increase in the frehand, the quantity of polymorphs is known to vary, quency of breaks in “AS3” cultures indicates a thus complicating the interpretation of the results in possible influence of metabolic factors other than successive sets of experiments. Therefore, in order to exotoxin in the medium. Even though 4% v/v “ASI” remove polymorphonuclear leukocytes, the blood in the cultures had no effect on cell growth, it seemed was allowed to sediment overnight in a glass bottle to induce chromosome breakage. At this concentrabefore establishing cultures. tion the frequency of metaphases with aberrations Fig. 1 shows the relation between the mitotic index was 17% (cells with gaps alone 3/100, cells with and the concentration of exotoxin. Total inhibition breaks 14/100). In control cultures the aberration of cell growth could not be demonstrated. frequency was 6% (all breaks). Clastogenic effects of exotoxin. - The distinctly toxic concentration of 20% v/v was selected when comparing the effects of different B. thuringirnsis 2. Acute exposure experiments with rats preparations. Table I gives the results of a detailed A test group of four female rats was given a lethal chromosome analysis of the metaphase plates. The dose of “ASl” as an i.p. injection. Two rats injected occurrence of aneuploidy is not statistically signifi- with the same volume of pure nutrient medium and cant. The incidence of chromosomal aberrations is, one rat with no treatment served as controls. The
MUTAGENICITYOF EXOTOXINI
Hereditas 85 (1977)
109
Table 4. The occurrence of chromosomal aberrations in bone-marrow cells of rats fed with exotoxin during three months In each case 100 metaphases examined Conc. of AS1
Cells with chromosomal aberrations Gaps alone
Breaks
Total
Control animal 1 animal 2
6 4
7 4
Test animal 3 animal 4 animal 5
2 9 9
3 I1
5
6
3
3
2
3
-
4
5 4
animal 6 animal 7 animal 8 animal 9
animals were sacrificed on the second day after treatment and examined for pathologic changes. Macroscopically the animals were normal. In histological screening the only changes observed in the test group animals were liver changes, including fatty liver and haemorrhagia, and calcification of kidneys. Bacteriological findings were negative. The results of the chromosome analysis of bonemarrow cell metaphases are listed in Table 2. As shown, “ASl” had no effect on induction of chromosomal aberrations. Table 3 gives the results of a detailed analysis of rat lymphocyte cultures. In the occurrence of aneuploidy no difference could be demonstrated between test and control animals. Instead, the incidence of chromosomal aberrations is significantly higher in treated animals. 3. Long-term feediig experiments with rats
Three test groups of young female rats were given in drinking water l000/,, 50% and 5% concentrations of “ASI” grown in glutaminate medium. After 3 months of treatment all animals were in good condition. Veterinary examination revealed no pathological changes except calcification in tubuli renales observed both in test and control animals. Bacteriologic findings were negative. Altogether 100 bone-marrow metaphases were analysed in detail for the occurrence of chromosomal aberrations. The results are presented in Table 4. According to the results exotoxin had no effect on the incidence of aneuploidy or chromosomal aberrations
5% 5%
10
in bone-marrow cells. However, observation of bonemarrow cells revealed a lack of megakaryocytes in animals of the 100% group with none or very few megakaryocytes on the slides. The blood picture of the rats was not examined prior to culturing, thus permitting only limited conclusions on the Occurrence of different cell types. Lack of polymorphonuclear leukocytes in animals of the 100% group was, however, obvious. In addition, disintegration of cells and a very low number of mitoses could be seen on the slides. In blood cultures of the 50% group, cell debris was obvious, as well. In the 5 % group, however, no effect of the treatment could be observed. In the control group, the substitution of drinking water totally by glutaminate medium induced a strong inhibition of mitotic activity in cultured blood cells. Thus only a few mitoses were available for the analysis. No disintegration of cells could be observed. The experiment was repeated later and identical results were obtained. The results of the chromosome analysis of rat blood cells are presented in Table 5 . In the 5 % group no effect on the incidence of chromosomal aberrations could be demonstrated. However, when taking into account the relatively low frequency of chromosomal aberrations in the control group, the chromosomal damage (cf. Fig. 2) and cell debris observed in the 50% and 100% groups can largely be attributed to the “ASI” feeding, although the possibility of some effect of the glutaminate medium itself cannot be excluded.
110
T. MERETOJA ET AL.
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.-E
Herediras 8.5 (1977)
MUTAGENICITY OF EXOTOXIN I
11 1
Table 5. The occurrence of chromosomal aberrations in cultured lymphocytes of rats fed with exotoxin during three months ~
Conc. of AS1
Metaphases examined
~~
Aneuploid
~
% Cells with chromosomal aberrations
cells Gaps alone
Breaks
Total
Control animal 1 animal 2 animal 3 Test animal 4 animal 5 animal 6
100 5;; 100% 100%
10 24
30 12.5
-
40 66.7
40 66.7
animal 7 animal 8
50 % 50 %
II
9.1 2.9
-
34
2.9
54.5 61.8
54.5 64.7
animal 9 animal 10
5% 5%
5 12
5 13
No mitoses
100
100
Discussion According to the results presented in this paper, the long-term feeding experiments with rats revealed no difference between test and control animals in pathological examinations. Earlier long-term feeding studies with mice were also negative (CARLBERG 1973). SEBFSTAand HORSKA(1972) administered purified exotoxin intraperitoneally to mice and showed it to be rapidly excreted in urine in an unaltered form. In addition, exotoxin was dephosphorylated to a non-toxic form in the tissue. On the other hand, DE BARJAC and LECADET (1975) demonstrated that mice excrete orally given exotoxin rapidly in feces. The urine was totally negative. No accumulation of exotoxin in the tissue could be demonstrated in either of the studies. The substitution ofdrinking water totally by “ASI” did not, however, remain without effects. Lack of megakaryocytes in the bone marrow and polymorphs in the blood cultures could be demonstrated. Many chemicals, radiation and infections are known to induce similar changes. As for chromosomal effects and possible mutagenicity, in in vivo experiments even the total substitution of drinking water by “ASl” for three months had no effect on the incidence of chromosomal aberrations in the bone-marrow cells. The results of the bone-marrow cell analysis of rats given a lethal dose of “ASI” were also negative. Instead, a clear clastogeniceffect was observed in vivo in the blood cells of rats in the 50% and 100% groups of the long-term feeding experiment as well as in the acute exposure test and in human blood cultures
2 2
-
I
in vitro. Hence, the cell debris and the resulting decrease in the number of cells observed in blood cultures of the 50% and 100% groups of rats in the long-term feeding experiment is at least partially explained by the Occurrence of chromosomal aberrations. Because all the animals were sacrificed at the same time we cannot ascertain whether the effects observed were transient, as was the case with toxic changes in pullets induced by B. thuringiensk preparations (BARKER and ANDERSON 1975). While considering the relatively high incidence of chromosomal aberrations in the blood cells of even the control animals, the white blood count in rats should be taken into account. It differs strikingly 1967) with possibly from that of humans (SCHERMER quite different functions. Generally the clastogenic effects, observed only in blood cells, were caused by high concentrations of exotoxin, much above the amounts normally used in the control of pest insects. Chromosomal aberrations have been demonstrated in connection with bacterial and viral products including commonly used vaccines (NICHOLS et al. 1962; NICHOLS 1963; HARNDEN1964; MIKHAIL~VA and GORSHUNOVA 1975). Further studies, taking into account the sensitive reactivity of the immunocompetent cells in blood, are needed for critical consideration of the actual harm of the effects observed, and for drawing final conclusions on the safety of B. thuringiensis preparations. Acknowledgments. -We wish to thank Drs. A . 4 . Andersson and P. Andersson (State Veterinary Medical Institute) for the histological and pathological examination of the rats used in
112
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the experiments. The Academy of Finland and Kemira Oy have financially supported this study.
Literature cited BARJAC,H. and DEDONDER,R. 1965. Isolement d'un nucleotide identifiable d la "toxin thermostable" de Bacillus rhuringiensis var. Berliner. - C . R. Acad. Sci. (Paris) 260: 7050-7053 DE BARJAC, H. and DEWNDER, R. 1968. Purification de la toxine thermostable de Bacillus thuringiensis var. thuringiensis et analyses complementaires. - Bull. Soc. Chim. Biol. 50: 941 -944 DE BARJAC,H. and LECADET, M.-M. 1975. Elimination de I'exotoxine thermostable de B. thuringiensi.9 apres ingestion chez la Souris. - C. R. Acad. Sci. (Paris) 286: 677-679 DE BARJAC, H. and RIOU.J.-Y.1969. Action de la toxine thermostable de Bacillus thuringiensis var. thuringiensis administree a des souris. - Rev. Pathol. Comp. Mid. E.xp. 6 : 367-374 BARKER, R. J. and ANDERSON, W. F. 1975. Evaluation of p-exotoxin of Bacillus thuringiensis Berliner for control of flies in chicken manure. - J . Med. Entomol. 1 2 103-1 10 BEEBEE, T., KORNER,A. and BOND,R. P. M. 1972. Differential inhibition of mammalian ribonucleic acid polymerases by an exotoxin from Bacillus Ihuringiensi.9. -- Biochem. J . 127: 619-624 BOND,R. P. M., BOYCE,C. B. C. and FRENCH, S. J. 1969. A purification and some properties of an insecticidal exotoxin from Bacillus thuringiensis Berliner. - Biorhem. J. 114 477-488 BRIGGS,J . D. 1960. Reduction of adult housefly emergence by the effects of Bacillus spp. on the development of immature forms. - J..Insect Pathol. 2 418-432 BURGERJON, A. 1974. Effects physiologiques et mutagenes sur les Insects de la toxine thermostable de Bacillus thuringiensis Berliner. Ann. Parasitol. Hum. Comp. 48: 835-844 BURGEMON, A,, BIACHE, G. and CALS,PH. 1969. Teratology of the Colorado potato beetle, Leptinotarsa decemlineara, as provoked by larval administration of the thermostable toxin of Bacillus thuringiensis. - J. Inverreh. Pathol. 14: 274-278 CANTWELL, G . E., HEIMPEL, A. M. and THOMPSON, M. J. 1964. The production of an exotoxin by various crystal-forming bacteria related to Bacillus rhuringiensis var. thuringiensis Berliner. - J . Insect Pathol. 6 : 466-480 CARLBERG, G. 1973. Biological effects of the termostable p-exotoxin produced by different serotypes of Bacillus thuringiensis. - Rep. Dept. Microhiol. Univ. Helsinki 6 CONNER, K. M. and HANSEN,P. A. 1967. Effects of valine, leucine and isoleucine on the growth of Bacillus rhuringiensis and related bacteria. - J . Inverreh. Pathol. 9 12-18 DUNN,P. H. 1960. Control of houseflies in bovine feces by a feed additive containing Bacillus thuringiensis var. thuringiensis Berliner. - J . Insect Pathol. 2 13-16 J., SEBESTA,K., HORSKA, K., SAMEK, 2.. DOLEJA,L. FARKAS, and SORM.F. 1969. The structure of exotoxin of Bacillus thuringiensis var. gelechiae. - Collection Czech. Chetn. Commun.34: 1118-1120 DE
~
GRAHAME-SMITH, D. G., ISAAC,P., HEAL,D. J. and BOND, R. P. M. 1975. Inhibition of adenyl cyclase by an exotoxin of Bacillus thuringimsis. - Nature 253: 58-60 HARNDEN, D. G. 1964. Cytogenetic studies on patients with virus infections and subjects vaccinated against yellow fever. - Am. J. Hum. Genet. 16: 204-213 B. G. and IGNOFFO, C. M. 1972. The KIM,Y . T., GREGORY. p-exotoxin of B. rhuringiensis. - J . Inverreh. Pathol. 20: 46-50 LAM,A. B. Q.and WEBSTER, J. M. 1972. Effect the DD-136 Nematode and of a p-exotoxin preparation of B. rhuringiensis var. thuringiensis on Leatherjachets, Tipuh paludosa larvae. - J . Inverreb. Parhol. 20: 141-149 MACKEDONSKI, V. V. 1975. Effect of the exotoxin of Bacillus rhuringiensis on the biosynthesis and maturation of mouse liver nuclear RNA. - Biochim. Biophys. Actu 390: 319-326 V. V., NIKOLAEV. N., SEBESTA,K., and MACKEDONSKI, HADJIOLOV, A. A. 1972. Inhibition of ribonucleic acid biosynthesis in mice liver by the exotoxin of 8. thuringiensis. Biochim. Biophys. Acia 2 7 2 56-66 MIKHAILOVA, G . R. and GORSHUNOVA. L. 1975. Investigation of chromosomes in bone marrow cells of mice inoculated subsequently with various vaccines. - Tsitol. Genet. 9: 460-461 NICHOLS, W. W.1963. Relationships of viruses, chromosomes Hereditas 50: 53-80 and carcinogenesis. NICHOLS,W. W., LEVAN, A,, HALL,B. and OSTERGREN, G. 1962. Measles-associated chromosome breakage. - Hereditas 48: 367-370 SCHERMER, S. 1967. The white rat. - In The Blood Morphology of Laboratory Animals, 3 ed. (Ed. S . SCHERMER), Davis. Philadelphia, p. 41 -60 K. 1973. The fate of exotoxin from SEBESTA.K. and HORSKA, Bacillus thuringiensis in mice. .- Collection Czech. Chem. Commun. 3 8 2533-2537 SEBESTA, K.,HORSKA.K. and VANKOVA, J. 1967. Isolation, purification and toxicity of a thermostable exotoxin from the strain of Bacillus gelechiae. - In Insect Pathology und Microbial Control(Ed. P. A. VANDERLAAN), Norrh-Holland, Amsterdam, p. 238-242 K.,HORSKA,K. and VANKOVA, J. 1969a. Isolation SEBESTA, and properties of the insecticidal exotoxin of B. ~huringietuis var. gelechiae. - Collection Czech. Commun. 34: 891 -900 J. 1969b. Inhibition SEBESTA, K., HORSKA, K. and VANKOVA, of de novo KNA synthesis by the insecticidal exotoxin of B. thuringiensis var. gelechiae. - Collection Czech. Chrm. Commun. 34: 1789- I79 I SMUCKLER, E. A. and HADJIOLOV, A. A. 1972. Inhibition of hepatic DNA-dependent RNA polymerases by the exotoxin of B. rhuringiensis in comparison with the effects of rr-amanitin and cordycepin. rBiochem. J. 12Y. 153-166 SOMERVILLE, H. J. and SWAIN,H. M. 1975. Temperaturedependent inhibition of polyphenylalanine formation by the exotoxin of Bucillus thuringiensis. - FEBS Letters 54: 330-333 H:T.and WOLFENBERGER. D. A., GUERRA, A. A., DULMAGE, CAREIA,R. D. 1972. Properties of the p-exotoxin 01' B. thuringiensis IMC 10,001 against the tobacco budworm. J. Econ. Entomol. 65: 1245- I248 ~
