Herediras 91: 1-3 (1979)

Mutagenicity of Bacillus thuringiensis exotoxin 111. Sister chromatid exchange in rats in vivo MARKETTA KAHKONEN, ULLA GRIPENBERG, GUNNEL CARLBERG,’ TYTTI MERETOJA+ and MARJA SORSA Department of Genetics and Department of Microbiology,’ University of Helsinki, Finland

K A H K O N E N , M., GRIPENBERG, U., CARLBERG, G . , MERETOJA. T. and S O R S A . M. 1979. Mutagenicity of Bacillus fhuringiensis exotoxin. 111. Sister chromatid exchange in rats in vivo. Herediras 91: 1-3. Lund, Sweden. ISSN 0018-0661. Received January 22. 1979 Sublethal doses of the autoclaved supernatant of exotoxin-producing Bacillus lhuringiensis serotype I (“AS I*’)were given intrapentoneally to rats, which were killed 12 h later. The sister chromatid exchange (SCE) frequencies in the bone marrow cells of exotoxin-treated animals were within normal ranges (3.1-33 per cell); no differences between exotoxin-treated and control animals were observed. Signs of C-mitotic influences were, however, found in cells of the test animals. Previous long-term feeding experiments with exotoxin have been repeated and extended to comprise 6 months. In the bone marrow cells of treated rats, no clastogenic effects were observed. The results of the tests performed, thus, revealed no mutagenic or clastogenic potency of B. rhuringiensis exotoxin. Risks of cytogenetic damage caused by exotoxin in amounts normally used to control pest insects are thus considered negligible. UUa Gripenberg. Deparfmenr of Genetics, P . Rautatickaru 13, SF-00100 Helsinki 10, Finland

Exotoxin produced by certain serotypes of BacilMeanwhile, the long-term feeding experiments lus thuringiensis has proved to be highly effective of rats, whose drinking water was substituted by to control pest insects, especially flies (CARLBERG,the autoclaved supernatant of B . thuringiensis unpublished). Various mammalian and submam- (“AS I”), were extended from a period of three malian tests for mutagenicity have previously months up to six months. We want to include the been carried out with these products (MERETOJA et additional results to the previously reported findal. 1977; LINNAINMAA et al. 1977; SHARMA and SAHU ings regarding the mutagenic effects of B . thuring1977). In some experiments toxic or lethal con- iensis exotoxin. centrations of the agent caused clastogenic effects in human and rat cells. In submammalian tests sublethal concentrations induced a slight Material and methods mutagenic activity in two of four test types. The sister chromatid exchange method (SCE) For the SCE tests, young female rats of the strain has in many cases proved more sensitive and Sprague-Dowley were treated with 5-bromodeoxreliable than previously used techniques for yuridine (BrdU) in vivo. Intraperitoneal injecchromosome aberrations (CARRANO et al. 1978). tions, in concentration 50 mg/kg bw, were given The test is considered an indicator of chromosome hourly to the animals during 9 h. The last injection damage and repair and obviously reflects a differ- included a sublethal dose of exotoxin ( 2 . 5 ml “AS ent type of genetic lesion than chromosome aber- I ” in glutaminate medium, 10 times concentrated; 1977). For these reasons and be- for further details see MERETOJA rations (EVANS et al. 1977). The cause previous tests left the question of the safety control animals received pure glutaminate of B. thuringiensis preparations to some extent medium. The rats were killed 12 h later. Bone unsettled, we decided to investigate the possible Deceased December lS”‘, 1977 SCE effects of exotoxin in rats. +

1

2

M K A H K O N E N IT A L .

Hrrrrlirus Y I ( I Y 7 Y )

Tlihh I. The occurrence of sister chromatid exchanges (SCE's) in bone marrow cells of rats injected with sublethal doses of exotoxin("AS I") Animal examined. no.

Number of mrtaphases examined

Number of aneuploid cells

Number of SCE's per cell k SE

101

17

3.S? 0.2

64 68

IS 23

3 . 2 + 0.1 3. I f 0.2

26 I04

7 33

3 . 2 ? 0.4 3 . 4 2 0.2

Ti,.\! I 2 3 ('oil f r o /

4 5

marrow tissue was sampled from the femur. Preparations were made according to previously described methods. Differential staining of the sister chromatids was performed with the FPG technique (PIIRRY and WOI.FF1974). 100 adequately stained metaphases of good quality were examined in each series; in some series, however, the intended number of good metaphases could not be achieved. The number of SCE's per cell as well a s the number of aneuploid cells were recorded. In the repeated long-term feeding experiments the male rats used were divided into three groups, each group consisting of five animals. For rats in the test group the drinking water was substituted with autoclaved supernatant of the glucose-containing nutrient medium of the exotoxin-producing serotype 1 of B. thuritigic~nsis ("AS I"). The concentration used in these series was IOO%~-v/v. Rats in control group I received tap water for their fluid intake. Control group I 1 was given glucosecontaining nutrient medium. The feeding experiments lasted h months. Bone marrow preparations were examined for the occurrence of chromatid and chromosome gaps and breaks.

Results Table 1 shows no increase in the number of aberrations in the bone marrow cells of rats treated in vivo with sublethal doses of exotoxin. The mean number of SCE's varied between 3.13.5 per cell in three treated animals. In two control rats the number of SCE's was 3.2, respectively 3.4. No significant differences could be observed. A few metaphases with a very high degree of

polyploidy, contracted chromosomes, pulverization and pycnosis were observed in the present test series. The controls failed to express corresponding cytogenetic abnormalities. Table 2 shows the occurrence of chromosome changes in bone marrow cells of rats fed with exotoxin during a 6 months period. The aberration frequency varied between 0.0-7.4 in the test group. In the control groups the aberration frequencies varied between 0.0-1 I .8 (control I ) and 0.0-3.3 (control 11). N o significant differences could be demonstrated. The test group expressed, however, an increased mitotic response and a higher quality of chromosome preparations than the controls.

Discussion and conclusion The treatment with the autoclaved supernatant of exotoxin-producing B . /hitringic~~r.sis( " A S 1 . ' ) failed to demonstrate any increase in the S C E frequency in the bone marrow cells of rats treated in vivo. C-mitotic properties and binucleation have earlier been reported in plant material ( L I N N A I N M A A et al. 1977; S H A K M A et a]. 1978) after exotoxin treatment. I n the mammalian tissue examined, no binucleate cells have been observed. Signs of C-mitotic influences were found also in rats: highly polyploid cells were seen in the bone marrow preparations. Simultaneous occurrence of C-mitotic effects in cells with normal SCE frequencies supports the view, that exotoxin scarcely causes chromosomal lesions i n treated animals. In previously reported in vitro studies (MEREIOJA et al. 1977) toxic concentrations of exotoxin caused clastogenic effects in human blood cultures. A slight increase in the number of breaks in blood cultures given exotoxin-free supernatant of the growth medium of B. t/iitri/r,yic.ri.si.s serotype 3 ( " A S 3"). non-producer of exotoxin, indicated influences of other non-specified factors. Lethal doses of exotoxin also resulted in clastogenic effects in rats: the control receiving pure nutrient medium. however, also showed increased breakage rates. The mitogenic stimulation observed in the present animal tests is in good agreement with previously reported increased mitogenicity caused by cell-free supernatants of several bacteria ( M I : R ~ . Iand ~ J ACARmt..w 1977). In reevaluating the clastogenic effects of exo-

MUTAGENICITY OF EXOTOXIN. 111

Heredifas 91 (1979)

3

Table 2. The occurrence of chromosome aberrations in bone marrow cells of rats fed with exotoxin (“AS I ” ) during six

months Animal examined. no.

Number of metaphases examined

Number of aneuploid cells

Percentage of cells with chromosome aberrations Gaps

Tesf 6 7 8 9

100 100 100 56

10

27

13 3 14 8 3

Breaks

Total

I .o

3.0

4.0

4.0

-

-

-

-

3.1

7.4

Confrol I

II

I2 13 14 15 Conrrol I 1 16

34 41 33

2

100

8

5.9 4.9

7

-

11.8 4.9

IS

I .o

I .o

-

too few mitoses too few mitoses

17 18

50

12

61

13

19

27

7

20

36

27*

* 22 metaphases revealed only 41 chromosomes

toxin, the following points should be stressed. When increased breakage rates have appeared, the exotoxin concentration used in the experiments has been extremely high and many times exceeding the concentration used for control of flies in piggeries ( C A R L B t x 1978). Furthermore, the breakage rate seemed to vary considerably, also in some of the controls. In the repeated experiments the different series gave more uniform breakage frequencies and, thus, the results appear more reliable. In conclusion, the reported findings indicate that the exotoxin-containing preparations of B. thrtririgicvisis, when used in proper concentrations for the control of pest insects, most probably do not cause cytogenetic damage to other living organisms. A ~ k f l o i ~ ~ l u d g n i e f l-f . ~We . are most indebted to Miss Tarja

Valimaki for technical assistance. The Academy of Finland and Kemira Oy have financially supported this study.

Literature cited CARLBERG, G. 1978. Karpasten mikrobiologinen lorjunta antanut lupaavia tuloksia. - Kiiyfiinnon maamies 5 : 78-79

CARRANO. A. V., THOMPSON. L. H . . L I N I ) ~ .P. . A . and M I N K L E R . J . L. 1978. Sister chromatid exchange a s an indicator of mulagenesis. - Narure 271: 551-553 EVANS. H. J . 1977. What are sister chromatid exchanges. Chromosomcs Toduy 6: 7 1-72 LINNAINMAA. K.. SORSA, M . . CARLBERG, G . . GRIPENBERG. U. and MERETOJA,T. 1977. Mutagenicity of Bacillus fhuringiensis exotoxin. II. Submammalian tests. - Heredifus 8.5: 113-122 M I ; R k . I O J A . T. and CARl.BIRG. G. 1977. The effect ofBacillus fhuringiensis and of cell-free supernatants of some other bacteria on the mitotic acitivity of human lymphocytes. FEMS Microhiol. Leffers2: 109-1 I I M E R E T O J A , T., CARLBERG. G . . GRIPENBIRG. U.. LINN A I N M A A . K. and SORSA. M. 1977. Mutagenicity of Bacillus fhuririgiensis exotoxin. 1. Mammalian tests. - Heredifus 85: 105-1 I 2 PI..RRY. P. E. and WOLFF, S . 1974. New Giemsa method for the differential staining of sister chromatids. - Nature 251: 156158

SHARMA, C . B. S. R. and S A H U ,R. K. 1977. Cytogenetic hazards from agricultural chemicals. I. A preliminary study o n the responses of root meristems to exotoxin from Bucillus fhuringiensis. a constituent of a microbial insecticide. thuricide. - Mufur. Res. 46: IY-26 SHARMA. C. B. S . R . . SAHU. R. K. and PANIGRAHI. S. 197R. Inhibition of cytokinesis by a bacterial toxin - thuringiensinA. - Curyolugia 31: 8%93

Mutagenicity of Bacillus thuringiensis exotoxin III. Sister chromatid exchange in rats in vivo.

Herediras 91: 1-3 (1979) Mutagenicity of Bacillus thuringiensis exotoxin 111. Sister chromatid exchange in rats in vivo MARKETTA KAHKONEN, ULLA GRIPE...
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