BioSafety Journal Pontificia Universidad Católica de Valparaíso ISSN: 1366 0233 Vol. 3, Num. 1, 1997

Safety evaluation of transgenic potatoes transformed with synthetic antibacterial peptide gene - toxicological test

WANG Ying^1, QIU Jinsong^1, SUN Yibin^1, QU Xianming^2, TANG Yixiong^3, JIA Shirong^3*

^1 Institute of Science and Technology, National Committee of Family Planning, Beijing 100081;
^2 Shanghai Biotechnology Research Center, Chinese Academy of Sciences, Shanghai 200233;
^3 Biotechnology Research Center, Chinese Academy of Agricultural Sciences, Beijing 100081
* Author to whom correspondence should be addressed.

Received: April 6th 1997
Accepted: May 10th 1997
Published: May 21st 1997

Code Number: BY97006
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SUMMARY

Cecropin B is an antibacterial peptide commonly existing in the insect immune system. During the past decade, we have designed and synthesized various novel antibacterial peptides and corresponding genes, such as modified Cecropin B, Shiva A and Shiva 2A (an analog of Cecropin B with 40% of amino acid sequence homology) and WHD. 3 clones with significant enhanced resistance to bacterial wilt (caused by Pseudomonas solanacearum) have been obtained. As part of the biosafety assessment of the transgenic potato, here we report the results of toxicological tests obtained using the synthetic peptide Shiva 2A. The acute toxicity study used the Ames, micronucleus and chromosome aberration tests together with accumulative toxicity to mice.

Keywords: release GMO

INTRODUCTION

Modern biotechnology based on recombinant DNA technology has made it possible to introduce new traits into plants, with great potential for crop improvement applicable to agriculture. Potato is one of the most important crop plants used in genetic engineering, because of the importance of the potato crop throughout the world, the relative ease to regenerate and transform it in vitro and genetic limitations associated with traditional potato breeding. Numerous transgenic potatoes transformed with specific genes have been reported and some are already commercialized (Dale, 1995). For commercial release of transgenic potato, it is important to accumulate solid scientific data for safe environmental release and food consumption. In this respect, safety evaluation based on pollen dispersal, the possibility of transgenic potato crossing with related wild species, toxicity (solanine), allergenicity, major nutritional components and anti-nutritional factors, as well as agronomic characteristics has been carried out and reported (Conner, 1994, 1995; Dale et al., 1992, 1994, 1995; Eijlander & Stiekema, 1994; Fuchs et al., 1995; Heeres et al., 1994; Tynan et al.) However, to the best of our knowledge, the safety assessment of transgenic potato containing an anti- bacterial peptide gene has not yet been reported.

Cecropin B is an antibacterial peptide commonly existing in the insect immune system. Because of the wide-spectrum antibacterial activity to both Gram positive and negative bacteria of Cecropin B, the gene is expected to be useful for plant genetic engineering to provide enhanced resistance to bacterial pathogens. During the past decade, we have designed and synthesized various novel antibacterial peptides and corresponding genes such as modified Cecropin B, Shiva A and Shiva 2A (an analog to Cecropin B with 40 % of amino acid sequence homology), and WHD. The synthetic anti- bacterial peptide genes were introduced into seven Chinese potato cultivars via Agrobacterium-mediated gene transfer. Through repeated disease resistance evaluation of the transgenic potato lines both in greenhouse and in field nurseries, 3 clones with significant enhanced resistance to bacterial wilt (caused by Pseudomonas solanacearum) have been obtained. The level of the expressed antibacterial peptide is about 60 ug per kilogram of fresh tuber tissue. As a part of the biosafety assessment of the transgenic potato, here we report the results of toxicological tests carried out.

Since it is difficult to isolate large amount of expressed antibacterial peptide from the transgenic potatoes, we used the synthetic peptide Shiva 2A in the experiments. The acute toxicity study included evaluation of the synthetic peptide with regard to: genotoxic potential to bacteria (Ames test), effect on genetic damage (micronucleus test), effect on spermatocytes of mice testes (chromosome aberration test) and accumulative toxicity to rodents.

Evaluation of the genotoxic potential of the synthetic peptide to the bacterium Salmonella typhimurium

The genotoxic potential of the antibacterial peptide to Salmonella typhimurium was studied by a standard Ames test. The effects of the synthetic peptide Shiva 2A on back mutation of Salmonella typhimurium strains TA97, TA98, TA100 and TA102 were investigated with or without the addition of the metabolic activator S9 mix (the 900g supernatant of rat liver homogenate). For comparison, effects of 2,4,7-trinitro-9-fluorenone, methyl methanesulfonate (MMS), aminofluorene (2-AF), 2-hydroxyanthraquinone and daunomycin on back mutation of different bacterial strains were also investigated, as positive controls. No significant difference in back mutation frequency was detected in the tests with addition of 0.1, 1.0 or 2.5 ug Shiva 2A per petri dish as compared to the negative control (physiological saline), whether S9 mix was added or not, while significant back mutations were observed in the positive controls. The growth of the bacteria was inhibited when 5.0 ug Shiva 2A was added per petri dish (Tables 1, 2). This showed that antibacterial peptide Shiva 2A indicated negative in the standard Ames test.

Table 1 Ames tests of the positive controls

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            PS*        TNF*     Daunomycin     MMS*     2-AF*   HAQ
------------------------------------------------------------------------
Bacterial              Dose (ug/petri dish) and number of back mutations
Strains                -------------------------------------------------
                         0.2        10         3         50       80
------------------------------------------------------------------------
TA 97
-S9       168+/- 24.3   >1000 
+S9       182+/- 15.1                                  >1000

TA98
-S9        32+/-4.2              385+/-30.1 
+S9        39+/-3.5                                    >1000

TA100 
-S9       188+/-17.0                          >1000
+S9       190+/-14.1                                   >1000

TA102
-S9       294+/-40.1    >1000
+S9       257+/-15.1                                           840+/-46.5 
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Table 2 Ames tests of anti bacterial peptide Shiva 2A

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                 Dose (mg/petri dish) and number of back mutations
-----------------------------------------------------------------
Bacterial        0         0.1          1.0         2.5       5.0*
strains                                                     
-----------------------------------------------------------------
TA97 
-S9         168+/-24.3  141+/-20.1   155+/-33.2  166+/-11.5    NA 
+S9         182+/-15.1  212+/-25.0   214+/-31.1  185+/-22.0    NA

TA98
-S9          32+/-4.2    34+/-9.7     43+/-10.4  31+/-3.6      NA 
+S9          39+/-3.5    31+/-5.1     30+/-4.2   34+/-9.2      NA

TA100
-S9         188+/-17.0  195+/-19.4   197+/-29.7  182+/-14.1    NA 
+S9         190+/-14.1  180+/-11.3   176+/-7.0   192+/-5.7     NA

TA102 
-S9         294+/-40.1  290+/-36.1   247+/-28.3  297+/-20.5    NA 
+S9         257+/-15.1  284+/-17.4   310+/-46.0  293+/-16.7    NA 
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Evaluation of genetic damage to mice

Micronucleus occurring in myelocytes is usually a marker for determining the degree of genetic damage. In the present experiment, the effects of Shiva 2A on the frequency of micronucleus formation in mouse myelocytes were studied to see if the antibacterial peptide would lead to such nuclear damage in rodents.

Kunming mice (n=10/dose group, half male and half female) were orally administered twice with Shiva 2A at a dose level of 20, 40 and 100 ug/kg, respectively, at an interval of 24 hours. Control mice were orally treated with physiological saline (negative control), or with cyclophosphamide at 40000 ug/kg via intraperitoneal injection (positive control). The mice were killed 6 hours after the second administration, and the polychromatic erythrocytes with micronucleus in the mouse sternum marrow were counted. The result showed that the average frequency of micronucleus formation of the test groups fed with Shiva 2A ranged from 0.11% to 0.19%, while those of the negative and positive control groups were 0.21% and 3.11%, respectively (Table 3). This suggests that the synthetic peptide will not lead to genetic damage in mice at the dose levels used in this study.

Table 3 Micronucleus tests of Shiva 2A

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Test groups   Dose (ug/kg)   Detected cells   Frequency of    P Values      
                             (No. of animals) micronucleus
                                              formation (%)
                                                _
                                               (X+/-SD)   
----------------------------------------------------------------------
Physiological                  10,000 (10)      0.21+/-0.06    >0.05 
saline
Cyclophosphamide  40,000       10,000 (10)      3.11+/-0.041   <0.01

Shiva 2A              20       10,000 (10)      0.17+/-0.015   >0.05

Shiva 2A              40       10,000 (10)      0.11+/-0.06    >0.05

Shiva  2A            100       10,000 (10)      0.19+/-0.015   >0.05
---------------------------------------------------------------------

Chromosome aberration in spermatocytes of testes

Adult male Kunming mice (n=5/dose group) were orally administered with synthetic Shiva 2A at daily doses of 100, 200 and 300 ug/kg, respectively, for successive five days. Control mice were treated orally with physiological saline (negative control) or with cyclophosphamide (40000 ug/kg) via intraperitoneal injection (positive control). Two weeks after the last dose, the mice were subjected to colchicine treatment (4 mg/kg) via intraperitoneal injection and then killed after another two hours. The testes of the mice were taken out and observation of chromosome aberration in spermatocytes was conducted. No significant difference in the frequency of chromosome aberration was found between the test groups administered with Shiva 2A and the negative control group, while in the positive control group, the average chromosome aberration frequency was 30-fold higher than that of the negative control group (Table 4). This result showed that the antibacterial peptide Shiva 2A did not induce genetic abnormality in the male sexual cells.

Table 4 Effects of Shiva 2A on mouse chromosome aberration

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Test      Dosages   Detected cells Cells with Abberation   P 
groups    (ug/kg)    (animals)    chromosome frequency  Values              
                                  aberration         (%)
----------------------------------------------------------------
Physio-
logical                250 (5)          1         0.4
saline
Shiva 2A    100        250 (5)          3         1.2     >0.05

Shiva  2A   200        250 (5)          3         1.2     >0.05

Shiva 2A    300        250 (5)          4         1.6     >0.05

Cyclo-      40000      250 (5)         29        11.6     <0.01
phosphamide 
------------------------------------------------------------------

Accumulative toxicity

Shiva 2A was administered orally to 40 Kunming mice (male and female half each) during a period of 28 days. The daily dose for the first 4 days was 8.78 ug/kg, corresponding to an approximate human daily consumption of 9kg transgenic antibacterial peptide-potato. The daily dose levels for the following periods were increased by 50% every four days, and that for the last four days was 100 ug/kg, corresponding to an approximate human daily consumption of 100 kg transgenic potato. No death of the mice nor difference in body weight gain was observed between the test group administered with Shiva 2A and the control group administered with physiological saline (Table 5, Figure 1). No pathological abnormality has been observed in the organs, including brain, liver, cardiac muscle, kidney, adrenal, stomach, small intestine, spleen, testes, ovary and uterus, of the mice fed with Shiva 2A. The coefficient of accumulation was 11.3, K>5, suggesting that the synthetic peptide had no accumulative toxicity to mice at a total dose of 1130 ug/kg during 28 days.

Figure 1 Profile of the weight gain of mice during the feeding period with Shiva 2A

--------------------------------------------------------------------
Dose/       1-4    5-8    9-12   13-16   17-20   21-24   25-28
Periods
(days)
Daily  
doses 
(ug/kg)     8.78  13.17   19.75   29.63   44.44   66.67   100
Total Doses 
in 4 days 
(ug/kg)    35.12  52.68  79.00   118.52  177.76  266.68   400 
Accumul-
ative total 
doses 
(ug/kg)    35.12  87.80  166.80  285.32  463.08  729.76  1129.76
Number of 
mice died

Male         0       0       0       0       0       0       0

Female       0       0       0       0       0       0       0
--------------------------------------------------------------------

CONCLUSION

Antibacterial peptide Shiva 2A gave negative results in a standard Ames test. Results of the micronucleus test and histochemical observation of chromosome aberration in spermatocytes of male mice suggested that the peptide will not act as an agent causing nuclear and chromosome damage either in somatic and sexual cells. No accumulative toxicity was observed for the mice administered with Shiva 2A at a total dose of ca. 1130 ug/kg during 28 days. All these results have indicated that the transgenic potatoes transformed with antibacterial peptide genes will not show toxicity to rodents. For commercialization of the transgenic potatoes, the food safety assessment aiming at establishment of substantial equivalence to the commercially existed potato cultivars is to be carried out.

REFERENCES

Conner, A.J., (1995). Case study: food safety evaluation of transgenic potato. In: Report of a WHO Workshop Application of the principles of substantial equivalence to the safety evaluation of foods or food components from plants derived by modern biotechnology. WHO, 1995, pp. 23-35.

Conner, A.J., (1994). Biosafety assessment of transgenic potatoes: environmental monitoring and food safety evaluation. In: Jones, D.D., (Ed.) Proceedings of the 3rd International Symposium on the Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms. The Univ. of California, Oakland, California, pp. 245-265.

Dale, P.J. & McPartland, H.C., (1992). Field performance of transgenic potato plants compared with controls regenerated from tuber discs and shoot cuttings. Theor. Appl. Genet. 84:585-591.

Dale, P.J., (1995). R & D regulation and field trialling of transgenic crops. TIBTECH 13:1-11.

Dale, P.J., McPartland, H.C., Parkinson R., MacKay G.R., Scheffler J.A., (1992). Gene dispersal from transgenic crop by pollen. In: Casper, R. & Landsmann, J., (Eds.) Proceedings of the 2nd Symposium on the Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms. Biologische Bundesanstalt fur Land- und Forstwirtschaft, Braunschweig, Germany. pp. 73-78.

Eijlander, R., Stiekema, W.J., (1994). Biological containment of potato (Solanum tuberosum)-outcrossing to its related wild species black nightshade (Solanum nigrum) and bittersweet (Solanum dulcamara). Ser. Plant Report 7: 29-40.

Fuchs, R.L., Rogan, G.J., Keck, P.J., Love, S.L., Lavrik, P.B. (1995). Safety evaluation of Colorado potato beetle-protected potatoes. In: Report of a WHO Workshop "Application of the principles of substantial equivalence to the safety evaluation of foods or food components from plants derived by modern biotechnology. WHO, 1995, pp. 63-78.

Heeres, P., Swaaj, A.C., Bruinenberg, P.M., Kuipers, A.G.J., Visser, RGF., Jacobsen, E. (1994). Biosafety aspects of field testing with transgenic amylose-free potatoes. In: Jones DD (Ed.) Proceedings of the 3rd International Symposium on the Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms. Univ. of California, Oakland, California, pp. 271-289.

McPartlan, H.C., Dale, P.J., (1994). An assessment of gene transfer by pollen from field-grown transgenic potatoes to non-transgenic potatoes and related species. Transgenic Res. 3: 216-225.

Tynan, J.L., Williams M.K., Conner, A.J., (1990). Low frequency of pollen dispersal from a field trial of transgenic potatoes. J. Genet. Breed 44: 303- 306.

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