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[SANET-MG] pharm crops being produced and marketed in USA
April 12, 2004
Prof. Joe Cummins
?Genetically modified biopharmaceutical products are being produced and
marketed in the United States of America?
There has been a great deal of discussion about the testing of rice genetically
modified to produce the human gene products lysozyme and lactoferrin in the
United States (1) so far those tests in a stalemate. Nevertheless,
Sigma-Aldrich chemical company of the United States has been marketing the
biopharmaceutical products trypsin, avidin, and beta-glucuronidase (GUS)
processed from transgenic maize for at least two years (2) while Prodigene
Corporation markets aprotinin (AproliZean)(3) as does Sigma-Aldrich but from a
non-food or feed member of the tobacco family(4) For example,. Trypsin is a
digestive enzyme used extensively in research , to treat disease and in food
processing, the product TrypZean is marketed as an animal free product (to
avoid contamination with animal viruses or prions) it is produced jointly by
Sigma-Aldrich and Prodigene biotechnology company (Prodigene is the company
that faced problems after contaminating food crops with biopharmaceuticals in
the United States (5)). The production of genetically modified (GM) food crops
follows a strict process in the United States, first controlled field tests
are undertaken for a number of seasons then the proponent applies for
deregulation of the GM crop following reviews by the Animal Plant Health
Service (APHIS) of the United States Department of Agriculture (SDA), the Food
and Drug Administration (FDA) and the Environmental Protection Agency (EPA)
when the GM crop includes a plant incorporated bio-pesticide. Upon completion
of the process the GM crop is deemed to be de-regulated and can be grown
without monitoring. None of the GM crops producing the products listed above
appear to have been deregulated. The production of those biopharmaceutical and
their marketing seems to have been accomplished without the benefit of final
regulatory approval.
Production and marketing of the biopharmaceutical crops listed above has done
with apparently full cooperation of the FDA and USDA (the agriculture
department has proprietary interest in some of the biopharmaceuticals).
Approval of the biopharmaceuticals and their production appears to have been
done using a tricky backdoor procedure based on a loophole in the regulation
of field test releases. According to Pew initiative on food and
biotechnology ?current APHIS regulations do allow the commercialization of a
GE crop without a prior affirmative approval by the agency and without public
notice. Developers are not required to file a petition for nonregulated status
before they produce a plant commercially. It is possible for developers to grow
plants at a commercial scale under notification or field trial
permits, even if the plants might pose some identifiable environmental or human
health risk(6).? Crop production facilities are permitted as ?field tests?,
locations of such facilities are designated confidential business information
and is not disclosed to inhabitants even though the genes and products of
such sites can easily contaminate both crops, ground water and surface water.
There seems to be no direct way to find production facilities to any but
producers and government regulators. The United States government seems
committed to going ahead with a procedure that bypasses public input and
scrutiny and which if ,when finally revealed, threatens the marketability of
US food exports. In contrast, the Canadian Food Inspection Service maintains
that ?plant products of test sites cannot be marketed? (7) even though
numerous plant biopharmaceutical products have been tested.
Production of the commercial biopharmaceuticals was , for the most part,
achieved using maize. Maize is a crop of fundamental importance and should not
have been messed with in the way it has to produce biopharmaceuticals. An
approach of that type might be excused on the basis that the products are
benign. However, the products are not benign. Exposure to such products will
result in characteristic impact on humans and animals. Each of the products
will be briefly reviewed below. But first the regulation of plant derived
biopharmaceuticals was reviewed in 2000 (8). And in 2004 by the Pew Initiative
(6). Only the Pew report came to grips with the practice of marketing
virtually untested products commercialized with out public input. A review of
the Prodigene products was published in 2002 (9). As indicated earlier test
plot permits for crops producing biopharmaceutical proteins are usually
designated confidential business information so that the nature of the product
is hidden from the public , the exact location of test sites is not declared,
but APHIS does record the crop and the state in which the modified crop is
tested. During 2003 and 2004 ProdiGene had test plots in Nebraska, Texas, Iowa
and Missouri (10), so residents of other states can feel relieved about
their neighbors corn.
Trypsin is an enzyme produced in the pancreas and it breaks down proteins in
digestion. The enzyme is used in a number of laboratory applications, it is
used in wound treatment and to treat diabetes. The enzyme is used in food
processing and often put into infant formulations to aid in digestion. The
plant produced product is desirable because it is free of prions and animal
viruses.(11). The method for modifying crop plants and preparation of the gene
for trypsin is described in US patent 6,087,558 (12). The gene for trypsin is
isolated from bovine pancreas, the gene is actually to produce trypsinigen
which has six additional amino acids at the start of the enzyme (these amino
acids are cleaved to make active trypsin).The DNA inserted into the corn plans
comprises the ubiquitin promoter, including the first exon and intron; the
barley alpha amylase export signal sequence; a trypsinogen encoding sequence;
pinII terminator(potato protease inhibitor II terminator); 35S promoter and
terminator with the moPAT (maize optimized PAT) selectable marker. The PAT gene
is for glufosinate resistance the maize is herbicide tolerant but the gene is
mainly for selecting transformed cells.
According to material data safety sheets provided by trypsin manufacturers the
product is capable of causing allergy is a skin, eye and respiratory irritant
and may be a mutagen (13,14). Known allergens should not be produced in food
crops.
Avidin is a protein found in bird eggs, it functions to bind the vitamin
biotin which is required for many insect pests. The pests are inactivated by
the absence of the necessary vitamin. Maize transgenic for avidin production
is resistant to storage insect pests (16) Friends of the Earth did an
excellent case study of avidin corn providing substantial evidence that the
protein caused dangerous biotin deficiency in humans and animals leading to
immune deficiency and growth retardation (17). Even marginal biotin deficiency
is teratogenic in mice (18)and implicated in human birth defects (19). Biotin
is used a great deal but mainly as a diagnostic too. Commercial production
using transgenic maize has been described in a publication (20) and in
patents (21,22).The genes used in transgenic corn are similar to those
described above for trypsin. A chicken egg white avidin gene was modified in
genetic code to make a maize optimum gene, The ubiquitin promoter along with
first exon and intron were joined to a barley signal sequence for localizing
avidin in the grain, after the avidin sequence the potato protease inhibitor
II terminator sequence. A long with the avidin related a sequence with the
selectable Bar gene (glufosinate resistance ) driven by a double and a CaMV
promoter, tobacco mosaic virus leader sequence and maize alcohol dehydrogenase
intron, the transcription terminator was from the potato protease as above.
The avidin produced in maize has similar properties to avidin produced in egg
white but it is clearly a unique gene product that requires independent safety
assessment.
Aprotinin is a protease inhibitor normally prepared from bovine pancreas and
lung. Recombinant apoprotinin produced in plants is currently marketed as
incidated above. Bill Freese of friends of the earth provided a very fine
review of the product and the problem of allergy and pancreatic disease from
ingestion of the biopharmaceutical (23). Some findings additional to those
covered by Bill Freese are discussed as follows. Aprotinin is listed as a
reproductive hazard (24). There is serious danger to those exposed to
aprotinin after having had a previous exposure. Fore example a two year old
child suffered severe anaphylactic shock after a a test dose of aprotinin
(25). Fatal anaphylaxis followed aprotinin exposure in aa local application
of fibrin glue (26 or a similar application led to an immediate skin reaction
following reexposure to fibrin sealent (27). Secret field testing of plant
based recombinant aproptinin could result in severe or fatal anaphylaxis
either in a brief exposure in the maize field of one previously treated during
surgery or exposure of one exposed to the maize field then treated during
surgery.
Recombinant aprotinin derived from the bovine gene was produced in maize
(28,29). The genetic construction was similar to those described above for
maize based biopharmaceuticals. The ubiquitin promoter along with the ubiquitin
intron was linked to a signal sequence from barley amylase, the aprotinin
sequence was followed by the transcription terminator for potato protease II.
A selectable marker, bar (glufosinate tolerance) was added along with the
double CaMV promoter , tobacco mosaic virus omega leader and intron from maize
alcohol dehydrogensase along with the potato protease II terminator . The
recombinant aprotinin does not appear to have been extensively tested using
animals even though its glycosylation pattern may differ from the animal
product and effect its allergenicity and stability.
The final commercial recombinant product produced in maize is
beta-Glucuronidiase (GUS). The gene is used in a wide array of experimental
situations but does not appear to have therapeutic importance. It has been
observed that formula milk for infants had a low content of GUS while mother?s
milk had elevated GUS. Elevated GUS has been implicated in bilirubinaemia
(jaundice) of breast fed infants (30) and breast fed infants of diabetic
mothers (31). GUS is used extensively as a marker believed to have liitle
effect on the phenotype of the test organism, however, GUS was found to
enhance the feeding activity of the peach aphid (32) suggesting that the marker
may not be entirely without effect on the phenotype of organisms.
Commercial production of recombinant GUS was reported in 1998 (33). The
recombinant GUS gene was isolated from E. coli. The maize construction was
similar to those described previously for the other recombinant proteins
inclusing the promoters, introns etc. and selectable marker described above.
Codon alterations to accommodate protein synthesis in plants, a surprising
omission because bacterial genes do not normally perform well in higher plants
unless the gene codons are adjusted for the plant cell.
In conclusion, the secretive production of truly dangerous pharmaceuticals in
food crops is a truly disturbing development. Production of products such as
aprotinin may have fatal consequences to the unknowingly exposed The sale of
such products without transparent public approval is a threat to the
residents, not from the products themselves, but from the knowing exposure of
the public through the food crop and concealment of that information by
authorities. In a democratic society we should insure that elected local
officials take care to insure that the secret production is not permitted in
the area. If the elected officials are uninterested or willing to connive with
the bureaucrats they should be replaced at election or by recall.
References
1.Cummins,J. ?Pharm crops near you? 2004 http://www.i-sis.org.uk/ pp1-5
2.Horn,M,Woodward,S. and Howard,J. ?Plant molecular farming:systems and
products? 2004 Plant Cell Reports ? 2004 in press DOI:
10.1007/s00299-004-0767-1
3.Prodigene ?Aprolizean recombinant aprotinin from maize?2004
http://www.prodigene.com/pdf/AproliZean(tm)%20Backgrounder.pdf
4.Yahoo Finance ?LSBC and Sigma-Aldrich announce an agreement to manufacture and
distribute plant-produced recombinant aprotinin? 2004
http://biz.yahoo.com/bw/040308/85333_1.html
5.USDA NewsRelease ?USDA announces actions regarding plant protection act
violations involving prodigene,inc? 2002
http://www.usda.gov/news/releases/2002/12/0498.htm
6.Pew Initiative on food and biotechnology ?Issues in the regulation of
genetically engineered plants and animals? 2004 pp1-174
7.Perron,F. personal communication CFIA 2004
8.Graham,S. ?Plant derived biologics meeting ? 2000 pp1-145
http://www.fda.gov/cber/minutes/plnt1040500.pdf
9.Hood,E. ?From green plants to industrial enzymes? 2002 Enzyme and Microbial
Technology? 30,279-83
10.APHIS ?Prodigene permit activity? 2004 APHIS-USDA 2004
http://www.aphis.usda.gov/cgi-bin/parse-company.pl pp1-2
11.Prodigene ?TrypZean recombinant trypsin from maize? 2004 pp1-3
http://www.prodigene.com/pdf/TrypZean(tm)%20Backgrounder.pdf
12.Howard,J. and Hood,E. ?Commercial production of proteases in plants? United
States Patent 6,087,558 pp1-18
13. Safety data ?Safety (MSDS) data for trypsin from bovine pancreas? 2003 pp1-2
http;//physchem.ox.ac.uk/MSDS/TR/trypsin.html
14.Specialty Media ?MSDS EDTA,MSDS and trypsin MSDS?2004 pp1
http://www.specialitymedia.com/05Resources/MSDS%20SM-2002-C.htm4/
15. McGraw,L. ?Avidin an egg-citing insecticide protein in corn? 2000
Agricultural Research http://www.ars.usda.gov/is/AR/archive/aug00/egg0800.htm
16. Kramer,K, Morgan,T, Throne,J, Dowell,F, Bailey,M. and Howard,J.
?Transgenic avidin maize is resistant to storage insect pests? 2000 Nature
Biotech 18,670-5
17. Freese,B. ?Biopharming:case study of avidin corn? 2002 Friends of the Earth
http://www.foe.org/biopharm/csavidin.pdf
18.Mock,D,Mock,N,Stewart,C,LaBorde,J. and Hansen,D. ?Marginal biotin deficiency
is teratogenic in ICR mice? 2003 J.Nutr.133,2519-25
19. Zemleni,J. and Mock,D. ?Marginal biotin is teratogenic (in human)? 2000
Proc.Soc. Exp.Biol.Med.223,14-21
20. Hood,E, Witcher,D,Maddock,S, Meyer,T, Baszczynski,C, Bailey,M, Flynn,P,
Register.J, Marshall,L, Bond,D, Kulisek,E, Kusnad,A , Evangelista,R,
Nikolov,Z,
Wooge,C, Mehigh,R, Hernan,R, Kappel,W, Ritland,D, Ping ,C, Howard,L and
Howard,J. ?Commercial production of avidin from transgenic maize:
characterization of transformant, production, processing, extraction and
purification? 1997 Molecular Breeding 3,291-306
21. Bazynski,C,Hood,E,Maddox,S,Myer,T,Register,J,Witcher,D. and Howard,J.
?Commercial production of avidin in plants? 1998 United States patent 5,767,379
pp1-15
22.Albertson,M,Howard,J. and Maddox,S. ?Induction of male sterility in plants by
expression of high levels of avidin? 1999United States patent 5,962,769
23. Freese,B. ?Biopharming:case study of corn producing aprotinin? 2002 Friends
of the Earth pp1-5 http://www.foe.org/biopharm/csaprotinin.pdf
24.Research Safety ?Appendix B:Reproductive Hazard? 2002 pp1-22
http;//www.northwestern.edu/research-safety/pdf
25. Ryckwaert,Y,Barthlet,Y,Bonnet-Boyer,M,Rochette,A,Capdevila,X. and d?Athis,F.
?Anaphylactic shock after a test dose of aprotinin in pediatric orthopedic
surgery? 1999 AnnFrAnesthReanim 18,904-8
26.Oswald,A,Joly,L,Gury,C,Disdet,M,Leduc,V. and Kanny,G. ?Fatal intraoperative
anaphylaxis related to aprotinin after local application of fibrin glue? 2003
Anasthesiology 99,762-3
27.Beierlein,W,Scheule,A,Antoniadis,G,Braun,C. and Schlosser,R. ?An immediate
allergic skin reaction to aprotinin after exposure to fibrin sealant? 2000
Transfusion 40,302-5
28. Zhong,G, Peterson,D, Delaney,D, Bailey,M, Witcher,D, Register ,J, Bond,D,
Li,C, Marshall,L, Kulisek,E, Ritland,D, Meyer,T, Hood,E. and Howard,J.
?Commercial production of aprotinin in transgenic maize seeds? 1999 Molecular
Breeding 5,345-56
29. Baszczynski ,C, Czapla,T, Hood,E, Meyer,T, Peterson,D,Rao,G, Register,J,
Witcher,D. and Howard,J. ?Commercial production of aprotinin in plants? 1998
United States patent 5,824,870 pp1-15
30.Gourley,G. and Arend,R. ?beta-Glucuronidase and hyperbilirubinaemia in breast
fed and formula fed babies? 1986 Lancet 22, 644-6
31. Sirota,L, Ferrera,M,lerer,N. and Dulitzky,F. ?Betagucuronidase and
hyerbiirubinaemia in breast fed infants of diabetic mothers? 1992
Arch.Dis.Child 67,120-1
32. Cherqui A, Alla S, Saguez J, Doury G, Sangwan-Norreel B. and Giordanengo P.
?Probiotic effects of beta-glucuronidase on the peach-potato aphid Myzus
persicae (Aphididae)? 2003 J. Insect. Physiol. 49,1199-209
33. Witcher,D, Hood,E, Peterson,D, Bailey,M, Bond,D, Kusnadi, A,
Evangelista,R, Nikolov,Z, Wooge,C, Mehigh,R, Kappel, W, Register,J. and Howard
,J ? Commercial production of â-glucuronidase (GUS): a model system for the
production of proteins in plants? 1998 Molecular Breeding 4,301-12
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