The effect of transgenic cucumbers expressing thaumatin on selected immunity parameters in rats *

The effect of a diet containing the fruits of genetically modified cucumber plants expressing thaumatin on the non-specific resistance parameters of the consumer was determined. For five weeks 24 laboratory rats allocated to 3 groups were fed freeze-dried isoproteinous diets containing 15% transgenic or non-transgenic cucumber ffuits of the same variety, or a standard mixture. The phagocytic activity of neutrophils and monocytes was determined by flow cytometry (Phagotest and Bursttest). Feeding transgenic cucumber caused a statistically significant increase in neutrophilic granulocytes that activated oxidation processes after stimulation of PMA and fMLP. The fluorescence intensity of neutrophils was higher when E. coli was used as the chemotactic agent.


INTRODUCTION
Introduction of the gene controlling the synthesis of a sweet protein, thaumatin (Szwacka et al., 2000), may considerably affect the diversification and feed use of plants.Synthesis of new protein may reveal allergenic or immunogenic activity and have unknown biological activities.
An unpredictable risk results from possible changes in the concentration of secondary metabolites and from interactions between the components of the diet (Conner and Jacobs, 1999).In the reported studies, the response of the immune system of rats receiving a diet containing transgenic cucumbers was assessed on the basis of non-specific resistance parameters.

MATERIAŁ AND METHODS
The cucumber fruits of variety Borszczagowski, containing the gene coding the synthesis of thaumatin (Szwacka et al., 2000) were culti vated at the Department of Plant Genetics, Breeding and Biotechnology of Warsaw Agricultural University.The fruits of transgenic and non-transgenic plants were collected in similar growth and maturity phases, cut frozen (-30°C) and freeze dried.The transgenic cucumbers contained more crude protein and less crude fibrę and somewhat less minerał compounds as compared with the non-transgenic fruits (Kosieradzka et al., 2001).The nutritional experiment lasted 5 weeks.Twenty-four małe rats from the outbred IF 2 Jaz herd with a mean body weight of about 150 g were divided into 3 groups and kept in individual cages.The rats were fed a semisynthetic isoproteinous diet balanced according to NRC (1976).Freeze-dried transgenic and non-transgenic cucumbers constituted 15% of dry matter of the experimental diets.The control group received a diet without cucumbers.The diets were supplemented with a vitamin mixture (AOAC,1975) and a minerał mixture (NRC, 1976), in diets with the cucumbers, the minerał supplement had the following composition: CaHP0 4 *2H 2 0, 139.44; maize starch, 12.15; K,S0 4 10.20; NaCl, 14.50; CaC0 3 , 3.15; Na,HP0 4 , 3.21; MgO, 3.75; mixture of tracę elements, 2.70.After the completion of the nutritional experiment, and after 12 h of fasting, the rats were anaesthetized with ketamine; blood samples were collected from the heart into heparin-protected test tubes and cooled to a temperaturę of 4°C.The phagocytic activity of neutrophils and monocytes was determined by flow cytometry (Bohmer et al., 1992), using Phagotests and Bursttests (Orpegen Pharma, D-69115 Heildelberg, Germany).The results were analysed using the Statgraphics Plus 2.1 for Windows program.The significance of differences between the groups was compared by single-factor analysis of variance and Duncan's test.

RESULTS AND DISCUSSION
Feeding transgenic cucumbers did not affect the number of phagocytising neu trophils and monocytes and fluorescence intensity related to the level of phagocytosis by neutrophils and monocytes (Table 1).
Higher activity of neutrophils, as stimulated by E. coli, and higher fluorescence intensity was found in the rats fed on transgenic cucumbers.A highly statistically significant difference was found between the control group and groups receiving non-transgenic and GM cucumbers in the percentage of neutrophils capable of killing with oxygen-dependent mechanisms after fMLP and PMA stimulation (Table 2).The increase of non-specific immunity of the rats due to feeding the GM cucumber-containing diet may be a conseąuence of a change in the chemical composition of transgenic fruits inducing the immunological response of monocytes and neutrophils (Chandra and Sarchielli, 1993;Ferrante et al., 1996).However, earlier studies did not reveal any response of the experimental animals to the presence of thaumatin-producing cucumbers in the diet (Kosieradzka et al., 2003).

CONCLUSIONS
The 15% addition of freeze-dried fruits of transgenic cucumbers overexpressing sweet protein to the rat diet caused an increase in the values of selected parameters of non-specific immunity, an increase in the number of neutrophils that activated oxidation processes after PMA and fMLP stimulation, and an increase in neutrophil activity, i.e. higher neutrophil fluorescence intensity when E. coli was used as the chemotactic factor.