Article pubs.acs.org/JAFC
Subchronic Immunotoxicity Assessment of Genetically Modified Virus-Resistant Papaya in Rats Hsin-Tang Lin,†,‡ Wei-Cheng Lee,§ Yi-Ting Tsai,§ Jhaol-Huei Wu,§ Gow-Chin Yen,⊥ Shyi-Dong Yeh,∥ Ying-Huey Cheng,# Shih-Chieh Chang,⊗ and Jiunn-Wang Liao*,§ †
Food and Drug Administration, Ministry of Health and Welfare, Taipei City115, Taiwan, Republic of China College of Bioresources, National I-Lan University, I-Lan 260, Taiwan 260, Republic of China § Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung 402, Taiwan, Republic of China ⊥ Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 402, Taiwan, Republic of China # National Plant Genetic Resources Center, Taiwan Agricultural Research Institute, Taichung 413, Taiwan, Republic of China ∥ Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan, Republic of China ⊗ Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan, Republic of China ‡
ABSTRACT: Papaya is an important fruit that provides a variety of vitamins with nutritional value and also holds some pharmacological properties, including immunomodulation. Genetically modified (GM) papaya plants resistant to Papaya ringspot virus (PRSV) infection have been generated by cloning the coat protein gene of the PRSV which can be used as a valuable strategy to fight PRSV infection and to increase papaya production. In order to assess the safety of GM papaya as a food, this subchronic study was conducted to assess the immunomodulatory responses of the GM papaya line 823-2210, when compared with its parent plant of non-GM papaya, Tainung-2 (TN-2), in Sprague−Dawley (SD) rats. Both non-GM and GM 823-2210 papaya fruits at low (1 g/kg bw) and high (2 g/kg bw) dosages were administered via daily oral gavage to male and female rats consecutively for 90 days. Immunophenotyping, mitogen-induced splenic cell proliferation, antigen-specific antibody response, and histopathology of the spleen and thymus were evaluated at the end of the experiment. Results of immunotoxicity assays revealed no consistent difference between rats fed for 90 days with GM 823-2210 papaya fruits, as opposed to those fed non-GM TN-2 papaya fruits, suggesting that with regard to immunomodulatory responses, GM 823-2210 papaya fruits maintain substantial equivalence to fruits of their non-GM TN-2 parent. KEYWORDS: immunotoxicity, subchronic, PRSV-GM papaya fruit, rats
■
INTRODUTION Immunotoxicity refers to the adverse effects of a substance on the structure or function of the immune system, or on other systems, as a result of immune system dysfunction. An immunomodulatory effect is considered adverse or immunotoxic if it suppresses humoral or cellular immunity of a host against infectious or neoplastic disease or it causes unnecessary tissue damage mediated by autoimmunity, hypersensitivity, or inflammation.1 Testing for direct immunotoxicity includes assessment of a number of nonfunctional parameters of the immune system, such as routine hematology, and the weight and histology of lymphoid organs and tissues. Otherwise, functional assays can confirm effects on the capacity of natural killer cells (NK) cytotoxicity and lymphocyte proliferation in response to mitogen stimulation.2 Although genetic engineering (GE), genetically modified (GM) crops have been experimentally used to produce a variety of proteins, blood components, coagulation factors, various interferons, and other therapeutic entities.3 But no GM crops producing therapeutic proteins have received regulatory approval yet. Commercially cultivated GM crops include soybeans, maize, cotton, canola, potatoes, and tomatoes. At present, the most widely grown GM crops contain new genes that confer herbicide tolerance or insect resistance.4 GM crops © 2016 American Chemical Society
may contain newly expressed proteins that are described as “intractable”.5 Safety assessments of these newly produced proteins may require acute and 28-day repeated dose studies in rats or mice. A randomized design is recommended for 90-day toxicity studies when testing whole food/feed.6 In addition, the developmental immunotoxicity (DIT) study for chemical pesticides under Regulation 1107/2009 may be considered as an alternative approach to the multigeneration reproductive toxicity study in rats for GM whole food/feed.7 In regards to allergic potency and immunomodulatory responses specifically, for determination of the allergic potency of GM soybeans, which contain 2S albumin from the Brazil nut, skin-prick testing in humans was conducted. The results indicated that three subjects had positive reactions to extracts of Brazil nuts and transgenic soybeans and negative reactions to soybean extract.8 Feeding GM maize to weaning and aged mice for 30 and 90 days has shown immune alterations induced by the percentage of T and B cells and of CD4+, CD8+, γδT, and αβT subpopulations.9 The immunmodulating effect of Cry1Ab Received: Revised: Accepted: Published: 5935
May 18, 2016 July 9, 2016 July 10, 2016 July 11, 2016 DOI: 10.1021/acs.jafc.6b02242 J. Agric. Food Chem. 2016, 64, 5935−5940
Article
Journal of Agricultural and Food Chemistry protein from Bacillus thuringiensis (Bt) and PHA-E lectin from kidney beans (Phaseolus vulgaris erythroagglutinin) has been examined in 90-day feeding studies in Wistar rats. Both PHA-E lectin and Cry1Ab protein were capable of inducing an antigenspecific antibody response.10 Furthermore, the Cry1Ab protein or other compositional differences in GM Bt-maize might cause minor alterations in intestinal responses in juvenile salmon, but without affecting overall survival, growth performance, development or health.11 However, no adverse immunotoxicological effects of GM corn with the Bt Cry1Ah gene were found when feeding mice for 30 days,12 or in a safety study of rats fed with GM corn with Cry1Ab for 90 days.13 Papaya is an important fruit that provides a variety of vitamins with nutritional value and also holds some pharmacological properties, including immunomodulation.14 However, Papaya ringspot virus (PRSV) is a destructive virus that affects the production of papaya. A GM papaya plant has been developed, which can be used as a valuable strategy to fight PRSV infection and to increase papaya production. Molecular characterization of transgenic papaya has demonstrated that the coat protein gene (CP gene) of Taiwan strain PRSV YK has been successfully inserted into the genome of Tainung No. 2 (TN-2) papaya by liquid-phase wound infection with Agrobacterium tumefaciens, which contained the CP gene of PRSV. Molecular analysis of nine selected transgenic papaya lines that exhibited different levels of resistance revealed that the expression level of the transgene is negatively correlated with the degree of resistance, suggesting that the resistance is manifested by a RNA-mediated mechanism.15,16 Four transgenic papaya lines (16-0-1, 17-0-1, 17-0-5, and 18-2-4) expressing the CP gene of PRSV were evaluated under field conditions for their reaction to PRSV infection and fruit production in 1996 to 1999. None of the transgenic lines showed severe symptoms of PRSV, whereas control nontransgenic plants were 100% severely infected 3 to 5 months after planting. In the first and second experiments, transgenic lines yielded 10.8 to 11.6 and 54.3 to 56.7 times more marketable fruit, respectively, than controls. All transgenic plants produced fruit of marketable quality with no ringspots or distortion.15 The transgenic papaya lines provide broadspectrum resistance against various PRSV strains under greenhouse conditions and show potential for controlling PRSV infection in papaya.15,16 In our previous study, the 90-day feeding toxicity of GM papaya fruit did not reveal adverse effects in rats and indicates that GM papaya fruits may be substantially equivalent to their non-GM parent plants.17,18 However, the immunomodulatory properties of GM papaya remain unclear. The aim of this study was to evaluate the effects of GM papaya fruit (823-2210) on the immune responses of rats and compare it to its conventional counterpart (TN-2).
control in this study was Tainung No. 2 (TN-2). They were cultivated in a screen house in a certified isolated test field at the Taiwan Agricultural Research Institute. Fresh papaya fruit pulp was harvested and lyophilized (Gold Chia Fong Biotech Co. Wufeng, Taiwan). After lyophilization, the weight of lyophilized papaya fruit was approximately one-ninth of fresh papaya fruit. The lyophilized papaya fruits were ground into powder and stored at −20 °C before use. The molecular markers for the characterization of these two GM papaya lines (which were resistant to PRSV) and non-GM TN-2, were confirmed in our previous reports.17,19 Animals. The 5-week-old male and female (nulliparous and nonpregnant) Sprague−Dawley albino rats were obtained from Biolasco Taiwan Co., Ltd. (I-Lan, Taiwan). The housing facility was maintained under appropriate environmental conditions at 21 ± 2 °C with 50−70% humidity under a 12-h light/12-h dark cycle. Autoclaved Rat Chow (Purina 5010, MO, USA) and reverse-osmosis water were available ad libitum. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of National Chung Hsing University (IACUC: 10104). Experimental Designs. Rats were divided into ten groups, with five male and five female rats designated to each group. The control animals were given reverse osmosis water with a basal diet. Test samples were prepared by dissolving non-GM TN-2 and GM 823-2210 lyophilized papaya fruit in reverse osmosis water, and the consistencies were based on the daily dose of 1 g/kg body weight (low dose group) and 2 g/kg body weight (high dose group). Rats were administered the test samples (non-GM TN-2 and GM 823-2210 papaya fruit) for 90 days by oral gavage. However, one female rat was mismatched at day 0 during grouping to make 4 male and 6 female rats in the GM 823-2210 papaya fruit 2 g/kg body weight group. The weekly body weight and feed consumption were recorded. At the end of treatment (day 91), all animals were fasted and sacrificed under 2% isoflurane (Halocarbon Laboratories, USA) anesthesia in an inhalation chamber (MSS 003, Benchtop Small Animal Anesthesia Unit, U.K.). Flow Cytometry Analysis for Immunophenotyping. Blood was collected from the abdominal aorta into collection tubes (K3 EDTA syringes) (Vacutainer, NJ, USA). White blood cell counts and phenotyping of peripheral blood mononuclear cells (PBMCs) were conducted. Briefly, PBMCs from blood were obtained using RBC lysis buffer (0.899% ammonium chloride, 0.1% sodium bicarbonate, and 0.0037% disodium EDTA per liter). The following antibodies were used for surface marker staining and were obtained from BD Biosciences (CA, USA): FITC mouse IgG1, κ isotype control (clone MOPC-31C), and FITC labeled mouse anti-rat CD3+ monoclonal antibody (clone G4.18) for total T cell staining. The FITC labeled mouse anti-rat CD4+ monoclonal antibody (clone OX-35) was used for staining T helper cells, FITC labeled mouse anti-rat CD8b+ monoclonal antibody (clone 341) for T cytotoxic cells, FITC labeled mouse anti-rat CD45RA+ monoclonal antibody (clone OX-33) for B cells, and FITC mouse anti-rat CD161a+ monoclonal antibody (clone 10/78) for NK cells, respectively. PBMCs were stained with the above antibodies for 30 min on ice in the dark. Flow cytometry analysis was performed using a FACScan (Becton Dickinson Immunocytometry system) and data were analyzed using CELLQuest software (BD Biosciences, CA, USA). The relative percentages of lymphocytic subpopulations were gated on isotype control.
■
MATERIALS AND METHODS Test Samples. The coat protein (CP) gene of Papaya ringspot virus (PRSV) was transferred to Agrobacterium tumefaciens. Transformation of embryogenic papaya was conducted by liquid-phase wounding with Agrobacterium tumefaciens, which contained the CP gene of PRSV19. The hybrid line (823-2210) generated by crossing 2210 with 823 was used in this study. Line 2210 was obtained by backcrossing the monoresistant transgenic papaya line 18-2-4 with the nonGM Sunrise line. When 18-2-4 was backcrossed with the nonGM Thailand variety, line 823 was obtained. The non-GM 5936
DOI: 10.1021/acs.jafc.6b02242 J. Agric. Food Chem. 2016, 64, 5935−5940
Article
Journal of Agricultural and Food Chemistry
Statistical Analysis. Data are expressed as mean ± standard deviation. The comparisons were designed to determine whether differences were attributable to the nonGM TN-2 and GM 823-2210 treated groups at the same dosage. All the student t-tests statistical analyses were conducted using Excel software (MicroSoft, Washington, USA) and statistical significance levels (p < 0.05) were determined by 2-tailed tests with paired comparison in 1 g/ kg groups (n = 5), and unpaired comparison in 2 g/kg groups for the misidentification of gender (n = 4 and 6), respectively.
Spleen Cell Proliferation Assay. The spleen cells of rats were harvested and washed from the spleen by RPMI-1640 with 10% FBS (Gibco BRL, Life Technologies, Inc., NY, USA) and isolated by Ficoll-Paque density gradient centrifugation (Pharmacia Biotech Limited, NY, USA). The spleen cells were resuspended in RPMI-1640 with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco BRL), and the spleen cell concentration was adjusted to 1 × 106/mL. The spleen cells (1 mL per well) were cultured in the absence (10 μL RPMI-1640 as blank control) or presence of specific mitogen. T cells were stimulated with 10 μL (1 mg/mL) ConA (Sigma, MO, USA), and B cells were stimulated with 10 μL (1 mg/mL) LPS (Sigma, MO, USA). The plates were incubated at 37 °C for 72 h in a humidified incubator with 5% (v/v) CO2 and 95% (v/v) air. Seventy-two hours later, the cultures were centrifuged at 450g for 5 min. Then, 1 mL (2 mg/mL) of MTT solution (thiazolyl blue tetrazolium bromide, Sigma, MO. USA) was added to each well. Plates were incubated at 37 °C with 5% CO2 for 30 min. Then, the cultures were centrifuged at 450g for 5 min. Finally, 100 μL DMSO (dimethyl sulfoxide, Merck Millipore, Germany) was added to the wells to dissolve the formazan particles and then the absorbance at 570 nm of each well was measured with a microplate ELISA reader (Dynex MRX II, USA). ConA-induced T cell proliferation was expressed by the absorbance differences between the ConAstimulated well and the control well (no-ConA-stimulated). LPS-induced B cell proliferation was expressed by the absorbance differences between the LPS-stimulated well and the control well (no-LPS-stimulated). The activity was expressed as % of blank control. Immunoglobulin Assays. To evaluate serum total IgG, IgM, and IgE antibody levels, serum samples were collected from all animals in different test groups and diluted to 1:50000 for IgG, 1:2000 for IgM, and 1:10 for IgE. One hundred microliters of rat IgG, IgM, and IgE standards (Bethyl Laboratories, Montgomery, USA) were diluted serially in diluent and the prediluted serum samples were added to the wells. The plates were then incubated for 60 min at room temperature. After the plates had been washed four times with washing buffer by a microplate washer (ELx50 Microplate Strip Washer, BioTek, USA), 100 μL of anti-rat IgG, IgM, and IgE detection antibody (Bethyl Laboratories) were added to the wells for 60 min. After washing four times, 100 μL of HRP solution was added to the plates and incubated for about 30 min. After washing four times, 100 μL of TMB substrate solution was added to each well and incubated for about 30 min in the dark. The reaction was stopped by adding 100 μL of stop solution and the absorbance at 450 nm was measured with an ELISA reader (Dynex MRX II, USA). The concentration of immunoglobulin was expressed as IgG and IgM (mg/mL) and IgE (ng/mL), respectively. Histopathological Analysis. Necropsies were performed in which the spleen and thymus were collected and weighed after dissection, examined grossly, fixed in 10% buffered formalin for 1 week, routinely processed, and then embedded in paraffin wax. Two-micrometer sections of spleen and thymus were stained with hematoxylin and eosin (H&E) for histopathological examination. For semiquantitative grading, lesion severity was graded using the criteria described by Shackelford et al.20 Lesion severity was graded as follows: 1 = minimal (
