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ISIS Report 18/07/07

GM Safflower with Human Pro-Insulin

Regulators show cavalier disregard for the safety of threatened species as well as human beings in proposed release of the GM pharm crop. Prof. Joe Cummins

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Proposed release of transgenic safflower shrouded in secrecy

USDA-APHIS conducted an Environmental Assessment (EA) [1] in response to an application (06-363-103r), received from SemBioSys, Inc to field test a transgenic safflower (Carthamus tinctorius) line 4438-5A that produces human pro-insulin. The transgenic safflower was engineered to express an oleosin-human pro-insulin protein exclusively in its seed. The field site (<1 acre) is located on private property in Lincoln County, WA, and will be surrounded on all sides by a 50 ft fallow strip. The exact location of the site is withheld from the public; but the application and risk assessment are open for public comment at http://www.regulations.gov/fdmspublic/component/main until 23 July 2007.

Pro-insulin is the precursor to insulin, normally made in the beta cell of the islets of Langerhans of the human pancreas. The protein is synthesized in the endoplasmic reticulum (membrane stacks within the cell), where it is folded and two sulphydryl (-SH) groups are oxidized into a disulphide bond (-S-S-). It is then transported to the Golgi apparatus (a special organelle) where it is packaged into secretory vesicles, and processed by a series of proteases into mature insulin. Mature insulin has 39 less amino acids; 4 are removed altogether, and the remaining 35 amino acids - the C-peptide - are cut out from the middle of the pro-insulin molecule; the two ends segments - the B chain and A chain - remain connected by the disulphide bond formed earlier [2, 3].

A patent application [4] describes the genetic modifications for high expression of human insulin in plants, including shortening the C-peptide by four amino acids.  The APHIS report [1] notes further that the human pro-insulin has two amino acids removed for stability in plants plus 11 C terminal amino acids added to ensure retention of the protein in the endoplasmic reticulum of the plant seed cell. The pro-insulin sequence was fused to the Arabidopsis oleosin gene, to be exclusively expressed in seeds. Expression of the fused gene was controlled by the phaseolin promoter and terminator sequences from common bean. The bean promoter drives seed-specific transcription of the synthetic pro-insulin.  A selectable marker is regulated by the parsley ubquitin promoter and terminator, and was deemed confidential business information even though it is said to be the most commonly used selectable marker in plants, and had been used in many previous field trials [1]. Animal feeding tests evaluating the toxicity of the neither the synthetic pro-insulin nor the marker gene and its proteins were  included with the EA.

Site of release in area with threatened species

The area selected for the transgenic safflower field test releases - “sagebrush steppe” - is dry and dominated by sagebrush. Resident animals include the sage grouse, sage sparrows, loggerhead shrikes, and even the once ubiquitous black-tailed hare or “jackrabbit”. According to USAD/APHIS [1], the threatened species in the test area also include bald eagle, pygmy rabbits, Columbian white tailed deer and grey wolf, and the plant species Spalding’s catchfly and Ladies’ tresses. Pygmy rabbits are the most threatened species, the Columbia pygmy rabbit feeds mainly on sagebrush and its number may be as low as 30 or less. There has been limited success in breeding the rabbits in captivity [5, 6]. The pygmy rabbit is likely to feed on the transgenic safflower seeds with potentially detrimental (even fatal) consequences. The USDA/APHIS report claims there will be no toxicity from ingesting seeds from the transgenic safflower, from contact or from inhaling dust and debris [1]. Even if that were true - and there is evidence ignored by APHIS suggesting that the ingested pro-insulin from transgenic safflower is active (see below) - the disruption of the habitat of the pygmy rabbit by human activities and transportation is likely to drive the threatened animals to extinction. APHIS displays a cavalier disregard for the threatened species, ignoring studies that do not support their conclusions.

Evidence of potential harm to threatened species ignored

There is at least one report showing that transgenic pro-insulin can effectively reduce blood glucose in rats. Feeding a bracken fungus, Ganoderma lucium, modified with a gene for human pro-insulin to diabetic rats reduced their blood glucose [7]; presumably the modified fungus cell wall and endoplasmic reticulum prevent rapid degradation of pro-insulin, allowing the transgenic organism to deliver insulin to the diabetic animal. Cholera toxin pro-insulin fusion proteins were produced in lettuce and tobacco plants; and when powdered transgenic plant preparations were fed to diabetic mice, oral tolerance to insulin was produced, preventing the autoimmune degradation of insulin-producing beta cells in the pancreas [8]. Human insulin produced in Arabidopsis seeds was activated by exposure to the common digestive enzyme trypsin [9]. The APHIS report presumes that human pro-insulin will be degraded too rapidly for it to become activated when ingested by animals, but the studies cited show that may not the case. Furthermore, functional argentine peptides were found to enhance intestinal absorption of insulin such peptides may be encountered commonly in anti-microbial peptides [10]. Seed debris may produce dust that contains human pro-insulin, and it is worth noting that inhaled insulin is an available option for human therapy [11]. The APHIS report dismisses the possibility that inhaled debris and dust from the transgenic safflower could be active, but provides no experimental evidence to support that conclusion.

APHIS implies that wild animals would not be affected by human insulin [1], but rabbits were among the animals first used in the discovery of insulin, and continue to be used as experimental animals in current studies on insulin action [12]. Furthermore, birds [13] and snakes [14] also respond to human insulin; and it is probably safe to say that all of the threatened species, and human beings are potential victims of the release of food crops modified to produce human insulin. The APHIS report notes that grain crops surrounding the transgenic safflower plot will provide a more attractive “free lunch” for birds and mammals than the transgenic safflower; that is a fallacious and dangerous assumption because the ‘free lunch’ will attract both foragers and predators to the test site. Furthermore, the fallow strip around the test plot is unlikely to discourage browsers such as rabbits that feed at night to avoid predators.

Safe haven for pharm crops but deadly for humans and wild life

Eastern Washington State is rapidly being transformed into a haven for transgenic crops modified to produce pharmaceuticals. Along with previous safflower field test releases, large plantings of humanized barley are being tested. The exact locations of such tests are not disclosed and people living near the test sites are unaware of the potential hazards to their health.   The impact of such developments on threatened species is also ignored and dismissed by APHIS.  The APHIS report reads more like a public relations document for the company rather than an independent critical evaluation of the company proposal. This is potentially deadly for humans and wildlife, and the agency should be held to public account.

References

  1. USDA-APHIS Environmental Assessment In response to permit application (06-363-103r), received from SemBioSys, Inc. for a field-test to produce human proinsulin (line 4438-5A) in genetically engineered safflower (Carthamus tinctorius) seeds U.S. Department of Agriculture Animal and Plant Health Inspection Service Biotechnology Regulatory Services 06_363103r 06/22/2007  http://www.regulations.gov/fdmspublic/component/main
  2. Wikipedia Proinsulin 2007 http://en.wikipedia.org/wiki/Proinsulin
  3. Davidson, H. Proinsulin processing. Cell Biochemistry and Biophysics 2004 Supplement, 143-57.
  4. Molony M, Boothe J, Keone R, Nykiforuk C and Van Rooijen.  Method for production of insulin in plants,  2005 US Patent 2005/0039235A1
  5. Washington Department of Fish and Wildlife Pygmy Rabbit 1995
  6. Hays D. Washington Department of Fish and Wildlife Washington Pygmy Rabbit 2003 Recovery Plan Update addendum to 1995 above
  7. Ni T, Hu Y, Sun L, Chen X, Zhong J, Ma H and Lin Z.  Oral route of mini-proinsulin-expressing Ganoderma lucidum decreases blood glucose level in streptozocin-induced diabetic rats. Int J Mol Med. 2007, 20(1), 45-51.
  8. Ruhlman T, Ahangari R, Devine A, Samsam M and Daniell H.         Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts--oral administration protects against development of insulitis in non-obese diabetic mice.  Plant Biotechnol J. 2007, 5(4), 495-510.
  9. Nykiforuk CL, Boothe JG, Murray EW, Keon RG, Goren HJ, Markley NA and Moloney MM. Transgenic expression and recovery of biologically active recombinant human insulin from Arabidopsis thaliana seeds. Plant Biotechnol J. 2006,:77-85.

  10. Morishita M, Kamei N, Ehara J, Isowa K and Takayama K. A novel approach using functional peptides for efficient intestinal absorption of insulin. J Control Release 2007, 118(2), 177-84.
  11. Guevara CA. Inhaled insulin for diabetes mellitus. N Engl J Med. 2007, 356(20):2106-7.
  12. Barillas R, Friehs I, Cao-Danh H, Martinez JF, del Nido PJ. Inhibition of glycogen synthase kinase-3beta improves tolerance to ischemia in hypertrophied hearts. Ann Thorac Surg. 2007, 84(1), 126-33.
  13. Remage-Healey L and Romero LM. Corticosterone and insulin interact to regulate glucose and triglyceride levels during stress in a bird. Am J Physiol Regul Integr Comp Physiol. 2001, 281(3), R994-1003.
  14. Sidorkiewicz E and  Skoczylas R. Effect of insulin on the blood sugar level in the grass snake (Natrix natrix L.). Comp Biochem Physiol A. 1974, 48(3), 457-64.
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