QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Atkinson, H. J. Articles by Robbins, M. Search for Related Content PubMed PubMed Citation Articles by Atkinson, H. J. Articles by Robbins, M.

---

Nutrient Interactions and Toxicity
Research Communication

Prima Facie Evidence That a Phytocystatin for Transgenic Plant Resistance to Nematodes Is Not a Toxic Risk in the Human Diet¹

Centre for Plant Sciences, University of Leeds, Leeds, LS2 9JT, UK and ^* BIBRA International, Carshalton, Surrey, SM5 4DS, UK

²To whom correspondence should be addressed. E-mail: h.j.atkinson{at}leeds.ac.uk.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
A protein-engineered rice cystatin (OcID86) provides transgenic, partial crop resistance to plant nematodes. This study determined whether its oral uptake has adverse effects on male Sprague-Dawley rats when they are administered by oral gavage 0.1–10 mg OcID86/kg body weight daily for 28 d. Body weight and water and food intakes were unaltered for most of the study. The only significant changes in fresh weight of nine organs were for the liver (4% decrease; P < 0.05) and the empty cecum (14% increase; P < 0.05) at the two lowest doses and the highest dose of OcID86, respectively. No abnormalities in either organ were detected by histochemistry. There were no changes in the urine or in hematological variables measured, and blood serum revealed no dose-dependent responses for any of 17 variables measured. OcID86 was degraded by boiling with a 50% loss of its inhibition of papain after 9.2 ± 8.0 min. It also showed >95% loss of such inhibition after 15 s in simulated gastric fluid. The results suggest that the no effect level (NOEL) for OcID86 is >10 mg/(kg · d). This provides a range of dietary exposure >200–2000 fold depending upon the promoter used to control its expression in potato.

KEY WORDS: • toxicity • cystatin • OcID86 • anti-nematode • biopesticide

Several transgenic proteins have potential for partial or full control of particular crop pests. For instance, the proteinaceous -endotoxin (Bt)³ of Bacillus thuringiensis is a biopesticide that already protects crops such as corn from some insects (1). The U.S. Environmental Protection Agency has suggested three general criteria for defining the food safety of such orally ingested biopesticides. The protein should be rapidly broken down in digestive fluids when present in the diet. It should be structurally unrelated to a known food allergen or toxin and should not display oral toxicity when administered at high doses (2). Bt meets these criteria when expressed in plants. There is a wide margin of exposure (MOE) between the maximum levels likely in the diet and the no effect level (NOEL) for this protein (3). The current work represents the first study of food safety for a distinct transgenically expressed protein that may soon be used as an antifeedant for pest control.

Transgenic resistance for plant parasitic nematodes could reduce the $100 billion of damage these pests cause to world agriculture each year. It can also help reduce the need for nematicides that risk environmental harm and raise health-related concerns such as groundwater contamination in the United States (4). Transgenic expression of a cysteine proteinase inhibitor (cystatin) imposes nutritional stress on nematodes, suppressing their growth and fecundity. A rice cystatin (OcI) has been protein-engineered to enhance its inhibition constant (K_i) for this purpose. The improved variant, OcID86, lacks its 86th amino acid (5). This and other cystatins provided both partial control of a nematode in a field trial with potato (6) and full control when stacked with natural, partial resistance (7). Constitutively expressed OcID86 does not harm nontarget arthropods that consume potato crops (8) or perturb soil microorganism communities (9). Furthermore, expression of antinematode proteins can be limited to the root systems where nematodes feed (10), thus reducing the expression of the novel cystatin in potato tubers.

This work examines the toxicity of OcID86 to a mammal. Cystatins are not novel to the human diet. They occur naturally in seeds such as those of rice and maize (11) and they are present in potato tubers (12). Cystatins also occur at high levels in egg-white (13); they are present in human saliva and are thus swallowed continually (14). There has been no previous work on the toxicity of cystatins used as biopesticides. Previous work suggested that cowpea trypsin inhibitor was not toxic to rats although a 30% increase in pancreas size occurred (15).

Most previous toxicological studies of proteins that may be used in transgenic crops (16) have relied on rodents, and that work helped direct this study. This work measures variables in all categories used before (16) plus urine analysis and a variety of clinical chemistry measurements. Our goal was to provide an initial study of toxicity after oral uptake of OcID86 in rats. We chose to use isolated cystatin and not the protein expressed in plants in this first work to avoid the issues of experimental design with diets involving raw or cooked potato.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Heat stability of OcID86.

OcID86 was expressed in Escherichia coli and collected and stored at -70°C as before (5) until use. Aliquots were made up to g protein in 50 mmol/L Tris/HCl buffer at pH 7.4 with 1 mmol/L NaEDTA and one protease inhibitor tablet/50 mL (Complete, Roche Diagnostics, Mannheim, Germany) or in the same buffer in which soluble potato tuber proteins had been added to 1.0 g/L. The samples were immediately placed in a boiling water bath and samples removed at various times and plunged into liquid nitrogen before storage at -70°C until assay. The level of OcID86 in all the samples was then determined by Sandwich ELISA using standard methods (17) OcID86 at 0.01 g/L in Tris buffer without protease inhibitors (see above) was boiled for 0–50 min and assayed for inhibition of papain using four replicates as detailed below.

Digestion of OcID86 and its function as a cystatin in simulated gastric fluid (SGF).

The SGF was made with 3.2 g pepsin/L (Sigma-Aldrich, Poole, UK, 14,500 kU/L) in HCl at pH 1.2 as previously described (18). Cystatin was incubated at 37°C over a time course of 0–10 min in SGF. Digestion was ended by adding 0.2 mol/L Na₂CO₃ and the OcID86 remaining in the sample was measured in three subsamples. Two subsamples were assayed on a SDS/PAGE gel with controls. One gel was stained for total protein using coomassie stain; the second was Western blotted using a monoclonal antibody raised against OcID86 by The Central Science laboratory, York, UK. Aliquots of 100 ng Papain in 50 µL of buffer (0.15 mol/L MES/OH at pH 5.8 with 4 mmol/L of both NaEDTA and DTT) were added to the third subsample and preincubated for 30 min before its activity was measured from a linear increase in OD at 415 nm over 15 min for a synthetic substrate (pGlu-Phe-Leu-p-NA; Sigma-Aldrich, Poole, UK). This assay was conducted with four replicates in both the presence and absence of potato proteins.

Dosing of rats with OcID86.

Male Sprague-Dawley rats, aged 4–5 wk, from a barrier maintained colony (Harlan UK, Bicester, UK) were housed in groups of 5 for 14 d before the experiment. They were sole occupants of the room with 12 h light/24 h and 15 air changes/h. They were fed a nutritionally adequate maintenance diet (RM1[E] SQC.FG, Special Diets Services, Witham, UK) and had free access to food and tap water in bottles throughout the study. Rats whose body weights were within 2 SD of the mean body weight were selected before the experiment and allocated by random numbers to the treatment groups.

Conduct of the experiment with rats.

OcID86 was expressed in E. coli (see above) and frozen at -70°C in 25-mg aliquots. One or two aliquots were thawed per day and diluted to 1.0, 0.1 or 0.01 g/L with dH₂0. The prepared dosing solutions were administered by oral gavage at a dose volume of 10 mL/kg body weight to give daily administered doses of 0 (control), 0.1, 1.0 or 10.0 mg/(k · d) for 28 consecutive days.

Throughout the study, the rats were weighed and observed daily for change in clinical condition. Food and water consumption for each cage of rats was recorded twice a week over the periods between body weight recordings. Urine was collected from all rats for 4 h on the day before killing, during which time they were housed singly in cages designed for such collection. Food, but not water was removed from the rats overnight before necropsy.

Biochemical and physiological analysis.

The urine collected on the day before necropsy was examined for volume, refractive index and pH. Glucose, protein, ketones, bilirubin, urobilinogen and blood pigments were measured semiquantitatively (Integra 400, Roche Diagnostics UK, Lewes, UK). A microscopic examination of the urine sediment was also made.

At necropsy, the rats were killed by exsanguination under a barbiturate overdose; blood collected into tubes containing EDTA anticoagulant was analyzed, using a Technicon H1 hematology analyzer for total erythrocytes, total and differential leukocytes, hemoglobin concentration, mean cell volume and platelet count. Additionally, hematocrit, mean cell hemoglobin and mean cell hemoglobin concentration were determined by calculation. A sample of blood was also used to determine the number of reticulocytes using flow cytometry (Beckman Coulter UK, High Wycombe, UK) after incubation with Coriphosphine. Another blood sample collected into citrate anticoagulant was used to measure prothrombin time, activated partial thromboplastin time and fibrinogen levels. Serum separated from the blood was analyzed with a chemical analyzer (Roche Integra 400) for glucose, urea, total protein, albumin, creatinine, calcium, phosphate, chloride, total bilirubin plus sodium and potassium concentrations. In addition, activities of alkaline phosphatase, alanine aminotransferase, lactate dehydrogenase, aspartate aminotransferase and -glutamyltransferase were determined. All of these assays were carried out at BIBRA International using a clinical chemical analyser (Integra 400, Roche Diagnostics UK).

During the necropsy procedures, a macroscopic examination of the internal viscera and cavities was made, and the heart, kidneys, liver, spleen, testes and thymus were removed and weights recorded. Additionally, mesenteric lymph node and pancreas weights were obtained after fixation. A comprehensive range of organs and tissues was retained in buffered 40g/L formaldehyde to allow follow-up of any indications of toxic effects associated with particular organs. The cecum, heart, kidneys, liver, pancreas, spleen, sternum, and thymus of each rat were processed, embedded in wax and sections stained with hematoxylin and eosin before examination under a microscope.

Statistical methods.

Results are expressed as means ± SEM. Differences were considered significant at P < 0.05. Procedures are described in a standard text (19) The data from the experiments with rats were tested for normality by Kolmogorov-Smirnov test and for homogeneity of variance by Bartlett’s test. Both tests were nonsignificant and treatments were compared by one-way ANOVA followed by the least significance difference (LSD) test. Regression analysis was used to determine whether any variables that changed significantly showed a dose-responsive effect. Multiple analysis of variance was used to compare the effect of the presence or absence of potato proteins with time in the boiling experiment. Linear calibration after regression analysis provided estimates of the time for 50% loss (with 95% CI) of OcID86 in the ELISA and papain inhibition assays.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
A reduction of 50% in the ability of OcID86 to inhibit papain and to be recognized by an antibody in ELISA occurred after boiling for 9.2 ± 8.0 and 24.1 ± 11.6 min, respectively (Fig. 1). The presence of potato proteins did not affect the latter value. OcID86 was rapidly digested in SGF. Both total protein and Western blotting suggested that 50% of OcID86 was lost in <5 s (Fig. 2). OcID86 in SGF progressively failed to inhibit papain with 97.7 ± 2.10% of this ability lost after 15 s in the presence of 1 g potato proteins/L of buffer.

View larger version (21K): [in this window] [in a new window]   FIGURE 1 Reduction of OcID86 levels with duration of boiling an aqueous sample measured by the inhibition of papain or by ELISA. Data points are means, n = 4. Values in italics are estimated times for 50% reduction ± 95% CI.

View larger version (63K): [in this window] [in a new window]   FIGURE 2 Digestion of OcID86 (II) by pepsin (I) in simulated gastric fluid (SGF). Controls are all in SGF. They are C1, OcID86 without pepsin and C2 and C3, pepsin without OcID86 at 0 and 10 min. III is a Western blot showing loss of detection of OcID86 by a monoclonal antibody over 0–2 min in SGF; no other bands were detected in this blot. Protein markers of different Mr values are shown.

Only a few of the variables measured in the rats were affected by the administration of any of the doses of OcID86 (see Table 1). Food intakes did not differ on six of the 4-d periods. At both d 3–7 and 7–10 of dosing, rats administered doses of 1 mg OcID86/d had slightly higher daily feed intakes (P < 0.05) than controls (6–7%).

View this table: [in this window] [in a new window]   TABLE 1 Summary of results from the toxicological study of male Sprague-Dawley rats administered the cystatin OcID861

Of the nine organs weighed, the only effects detected were a slight increase in empty cecum weight at the highest dose and a decrease in liver fresh weight at the two lowest doses of OcID86 (P < 0.05). In neither case did histopathological examination reveal any abnormalities.

The hematological variables measured did not differ among the groups. Analysis of the blood serum revealed minor changes in 4 of the 17 variables examined, including slightly higher potassium (P < 0.05) and creatinine levels (P < 0.01) at the highest dose compared with controls (Table 1).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
OcID86 was less heat stable than rice cystatin (OcI), which is fully active after 30 min of boiling in phosphate buffer (20). It has a heat stability similar to that of lima bean cystatin, which retains 15% of its activity after boiling for 15 min (21) but is more stable than some proteins from potato tubers such as peroxidases (22). OcID86 did not withstand conditions that simulated those in the stomach, which contributes to its safety after oral ingestion. Its lack of stability is similar to that of several Bt proteins that were no longer detectable after 30–60 s in simulated gastric fluid (3). One indicator that a protein is unlikely to be an allergen is its rapid partial digestion by pepsin in simulated gastric fluid (18), providing their fragments do not retain allergenicity (23). There was no evidence that OcID86 was digested into stable fragments larger than the size of the smallest protein marker used (Fig. 2). This finding is consistent with other work suggesting that OcID86 is not a hazardous allergen (Hilary Atkinson, BIBRA International, personal communication).

There were very few changes in rats detected in the very wide range of measurements made. Only one change, that of a reduced liver weight occurred at the two concentrations of 0.1 and 1.0 mg OcID86/kg body weight; however, this represented only a 4% reduction in weight and was not dose responsive. The change in creatinine levels in the serum was the sole change that was progressive with dose, but this trend was not significant. We conclude that daily administration by oral gavage of OcID86 at levels of up to 10 mg/kg body weight for 28 consecutive days has no significant toxic effects on male Sprague-Dawley rats under the defined conditions of this experiment. Therefore the NOEL is >10 mg OcID86/(kg · d).

Previous rat feeding studies with high levels of cowpea trypsin inhibitor administered for 28 d caused a 20% reduction in live weight relative to controls and a 30% increase in pancreas weight (15). Consumption of OcID86 at the levels used in this work did not have such effects. The fresh body weight and relative pancreas weight were 256.5 ± 8.80 g and 0.51 ± 0.056 g/100 g fresh weight, respectively, for controls and 258.8 ± 12.25 g and 0.53 ± 0.053/100 g fresh weight, respectively, for the rats dosed daily with 10 mg/kg OcID86. The absence of a digestive role for cysteine proteinases in the mammal intestine and lack of effects in the current work suggest that OcID86 used for nematode control would not be toxic in food.

We can estimate the maximum level of OcID86 that a human might consume from potatoes expressing it transgenically to protect potato roots from nematodes. A level of 0.1 g/100 g soluble protein as OcID86 was detected in potato tubers expressing the cystatin under control of a constitutive promoter (CaMV35S). This represents 10 mg cystatin/kg potato fresh weight after cooking based on tubers having 20 g protein/kg fresh weight. The highest annual consumption of potatoes is 136 kg/person in Poland, i.e., 0.37 kg/d (24). At this rate of consumption, an adult of 80 kg would ingest 0.047 mg cystatin/(kg · d) from cooked transgenic potatoes expressing OcID86. This extreme example provides a dietary MOE of 215-fold if 10 mg OcID86/(kg · d) is assumed to be the NOEL. The root-specific promoters used to provide nematode resistance such as that from a root-specific tubulin (10) reduce the levels of OcID86 below our detection limit of 0.01 g/100 g total protein as cystatin (data not shown). Therefore, the minimum dietary MOE using these plants would be at least 2000-fold. The dietary exposure margin for OcID86 may not require further definition given that acute dietary MOE of >100 are not a cause of concern in the diet (25). This seems an appropriate level of definition in this case because cystatins are a normal part of the human diet. Other sources of cystatin exceed the OcID86 levels in transgenic tubers even in the highly dependent potato diet given above. A meal including two chicken eggs provides 3.72 mg cystatin/100 g fresh weight of egg (13). Human saliva also provides 13 mg of swallowed cystatin/d (14).

Overall, we conclude that a prima facie case is established for the lack of a risk of toxicity to humans arising from expression of OcID86 to control nematodes in roots. A larger dietary exposure margin has been defined for Bt than the minimum defined in this work for OcID86, but Bt is not similar to ingredients that occur naturally in saliva and several human foods.

	FOOTNOTES

 
¹ Supported by The Scottish Executive of Rural Affairs and the Department for International Development of the UK government.

³ Abbreviations used: Bt, proteinaceous -endotoxin of Bacillus thuringiensis; MOE, margin of exposure; NOEL, no effect level; OcI, rice cystatin; SGF, simulated gastric fluid.

Manuscript received 31 July 2003. Initial review completed 11 September 2003. Revision accepted 17 October 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Environmental Protection Agency (2000) Bt Plant-Pesticides Registration Action Document 2000 http://www.epa.gov/scipoly/sap/2000/october/brad5_benefits_corn.pdf (accessed 10/8/03).

2. Environmental Protection Agency (2000) Biopesticides Registration Action Document Preliminary Risks and Benefits Sections. Bacillus thuringiensis Plant-Pesticides: U. S. Environmental Protection Agency 2000 http://www.epa.gov/scipoly/sap/2000/october/brad1_execsum_overview.pdf (accessed 10/8/03).

3. Betz, F. S., Hammond, B. G. & Fuchs, R. L. (2000) Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests. Regul. Toxicol. Pharmacol. 32:156-173.[Medline]

4. Gustafson, D. I. (1993) Pesticides in Drinking Water 1993 Van Nostrand Reinhold New York, NY.

5. Urwin, P. E., Atkinson, H. J., Waller, D. A. & McPherson, M. J. (1995) Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida. Plant J. 8:121-131.[Medline]

6. Urwin, P. E., Troth, K. M., Zubko, E. I. & Atkinson, H. J. (2001) Effective transgenic resistance to Globodera pallida in potato field trials. Mol. Breed. 8:95-101.

7. Urwin, P. E., Jayne Green, J. & Atkinson, H. J. (2003) Resistance to Globodera spp conferred by a plant cystatin alone and enhancement by a cystatin pyramided with natural resistance. Mol. Breed. 12:263-269.

8. Cowgill, S. E., Wright, C. & Atkinson, H. J. (2002a) Transgenic potatoes with enhanced levels of nematode resistance do not have altered susceptibility to nontarget aphids. Mol. Ecol. 11:821-827.[Medline]

9. Cowgill, S. E., Bardgett, R. D., Kiezebrink, D. T. & Atkinson, H. J. (2002b) The effect of transgenic nematode resistance on non-target organisms in the potato rhizosphere. J. Appl. Ecol. 39:915-923.

10. Lilley, C. J., Urwin, P. E., Johnston, K. A. & Atkinson, H. J. (2003) Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne and Globodera spp. Plant Biotechnol. J. (in press).

11. Richardson, M. (1991) Seed storage proteins: the enzyme inhibitors. Methods Plant Biochem. 5:259-305.

12. Rodis, P. & Hoff, J. E. (1984) Naturally occurring protein crystals in the potato. Plant Physiol. 74:907-911.[Abstract/Free Full Text]

13. Awade, A. C. (1996) On hen egg fractionation: applications of liquid chromatography to the isolation and the purification of hen egg white and egg yolk proteins. Z. Lebensm.-Unters -Forsch. 202:1-14.

14. Veerman, E.C.I., vandenKeybus, P.A.M., Vissink, A. & Amerongen, A.V.N. (1996) Human glandular salivas: their separate collection and analysis. Eur. J. Oral Sci. 104:346-352.[Medline]

15. Pusztai, A., Grant, G., Brown, D. J., Stewart, C. & Bardocz, S. (1992) Nutritional evaluation of the trypsin (EC 3.4.21.4) inhibitor from cowpea (Vigna unguiculata Walp). Br. J. Nutr. 68:783-791.[Medline]

16. Kuiper, H. A., Kleter, G. A., Noteborn, H.P.J.M. & Kok, E. J. (2001) Assessment of the food safety issues related to genetically modified foods. Plant J. 27:503-528.[Medline]

17. Harlow, E. & Lane, D (1988) Antibodies: A Laboratory Manual 1988 Cold Spring Harbor Laboratory Cold Spring Harbor, NY.

18. Astwood, J. D., Leach, J. N. & Fuchs, R. L. (1996) Stability of food allergens to digestion in vitro. Nat. Biotechnol. 14:1269-1273.[Medline]

19. Snedecor, G. W. & Cochran, W. G. (1989) Statistical Methods 8th ed. 1989 Iowa State University Press Ames, Iowa.

20. Abe, K., Kondo, H. & Arai, S. (1987) Purification and characterization of a rice cysteine proteinase inhibitor. Agric. Biol. Chem. 51:2763-2768.

21. Lawrence, J. C. & Nielsen, S. S. (2001) Partial isolation and characterization of a cysteine proteinase inhibitor from lima bean (Phaseolus lunatus). J. Agric. Food Chem. 49:1020-1025.[Medline]

22. Boucoiran, C. F. S, Kijne, J. W. & Recourt, K. (2000) Isolation and partial characterization of thermostable isoperoxidases from potato (Solanum tuberosum L.) tuber sprouts. Agric. Biol. Chem. 48:701-707.

23. Fu, T.-J. (2002) Digestion stability as a criterion for protein allergenicity assessment. Ann. N.Y. Acad. Sci. 964:99-110.[Abstract/Free Full Text]

24. Madonado, L (1998) CIP Potato Facts, Potato Indicators 1998 http://www.cipotato.org/market/potatofacts/potind.htm (accessed 10/8/03).

25. Environmental Protection Agency (1998) Dietary Exposure Analysis for Methidathion in Support of the Reregistration Eligibility Decision 1998 http://www.epa.gov/pesticides/op/methidathion/methidathion_14.pdf (accessed 10/8/03).

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Atkinson, H. J. Articles by Robbins, M. Search for Related Content PubMed PubMed Citation Articles by Atkinson, H. J. Articles by Robbins, M.