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Witness Statement
January 27, 2001 Prof. Joe Cummins Professor Emeritus of Genetics
University of Western Ontario jcummins@uwo.ca
"Three issues in the safety of genetically modified
crops"
It is a privilege and an honor to participate in this important review
of the safety of genetically modified crops. I thank the commission for
considering the matters that I have submitted below.
The issues that I will comment on include first: Genetically Modified
(GM) Baculovirus vectors used in crop insect control and mammalian gene
therapy. The second issue : Bacillus thuringiensis (Bt) toxins reportedly
toxic to mammals. The third issue: The impact of bacterial genes in GM
crops on the immune system (the CpG effect).
I will comment on each issue below and separately provide copies of key
references in pdf format.
GM Baculovirus vectors: Baculovirus are virus that infect
insects, they are very stable and may remain dormant in the environment
for years before infecting insects. The virus can be purified and produced
in quantity to be used in insect control. Since the virus multiplies and
persists, its use in pest control seems promising. The virus alone has a
relatively low killing power and slow action. When a gene for a potent
toxin such as scorpion toxin or a gene affecting a juvenile hormone is
added to the virus, it kills faster and fewer insects survive infection.
Numerous field tests of modified virus sprayed on crops have been
undertaken, often accompanied by loud expressions of concern from the
public. Soon after GM virus were developed for insect control, it was
found that baculovirus were capable of infecting human liver cells and
produced relatively little toxicity to the infected cells. For that reason
baculovirus vectors were developed to treat liver disease. Interestingly,
the fact that baculovirus can infect human liver cells seems to have been
ignored by those developing the virus for commercial pest control. The
following discussion will deal with the use of baculovirus vectors and
their safety. I understand that there has been a great deal of pressure
tohasten approval of the GM baculovirus for pest control.
Ecological considerations for the impact of recombinant baculovirus
insecticides have been studied extensively (Richards et al 1998). The
study focussed on baculovirus containing scorpion toxin because that
construction has been most widely studied. Impact on non-target insects is
extrapolated from insects of related phylogeny, a practice difficult to
defend. The recombinant baculovirus was very persistent and capable of
reshaping an ecosystem. Modification of baculovirus host range specificity
has been achieved by inserting or deleting genes (Theim 1997).
Baculovirus is a circular DNA duplex, it replicates in the insect cell
nucleus and replication is prone to the generation of defective genomes by
deletion (Wu et al 1999). The mode of virus replication seems to make the
recombinant virus highly unpredictable and prone to generating potentially
undesirable variants. This important finding has not yet been taken into
account in risk analysis of recombinant baculovirus insecticides and gene
therapy vectors.
The scorpion toxins used with recombinant baculovirus have been
selected to avoid human neurotoxicity, and as much as possible toxicity to
non-target animals. However, the allergenicity of toxins and their
behavior, as for example, in triggering autoimmunity in human liver
infection, has not yet been studied. In insect control the depressant
toxin was more effective than the excitatory toxin in recombinant
baculovirus (Gershburg et al 1998).
Recombinant baculovirus containing Bacillus thuringiensis toxin have
not proven successful in controlling insect pests (Martens et al 1995).
However, recombinant baculovirus modifying juvenile hormone proved
effective in insect control (Bonning et al 1999). Recombinant baculovirus
containing an antisense fragment to the c-myc oncogene proved effective in
target insect control (Lee et al 1997). The behavior of the myc oncogene
recombinant vector bears careful study regarding non-target animals and
its impact during human liver infection.
Baculovirus vectors efficiently transfer genes into human liver cells
(Hofmann et al 1995; Boyce and Bucher 1996). The vectors transferred into
human liver tissues most effectively in perfused liver tissue because
serum components hampered virus transfer (Sandig et al 1996). Human
conditions associated with defects in complement should allow liver
transfer of recombinant baculovirus. Inhibitors of complement facilitate
baculovirus gene transfer (Hofmann and Strauss 1998). Hybrid
baculovirus-adeno virus vectors have been used to deliver genes to human
cells (Palombo et al 1998). Baculovirus vectors have beeen used to deliver
hepatitis B to human liver efficiently to allow study of hepatitis B drug
therapy (Delaney et al 1999). Baculovirus vectors proved very effective in
delivering genes to human brain cells (Sarkis et al 2000). Crops treated
with baculovirus may be promoted as "brain food".
In conclusion baculovirus vectors are being used to control insect
pests because they are effective and persist for a long time in the
environment. Baculovirus vectors are also being used in gene therapy of
human liver. These areas of research seem to exist as two solitudes and
the risks of one are not evaluated in the context of the other. The most
disconcerting finding is the one showing that replication of the
baculovirus is inherently unpredictable. However, there may be some who
believe that we should all have unlabelled liver gene therapy with our
salad.
References
1.Bonning,B,Possee,R and Hammock,B "Insecticidal efficacy of a
recombinant baculovirus expressing JHE-KK, a modified juvenile hormone
esterase"1999 J Invertebr Pathol 73,234-6
2.Boyce,F and Bucher,N Baculovirus-mediated gene transfer into mammalian
cells" 1996Proc. Natnl Acad Sci USA 93,2348-52
3.Delaney,W,Miller,T, and Isom,H "Use of the hepatitis B virus
recombinant baculovirus-Hep G2 system to study the effects of beta 2’,3’
dideoxy 3’thiaceydine on replication of hepatitis B virus and
accumulation of covalently closed circular DNA"1999 Antimicrob Agents
Chemother 43,2017-26
4.Gershburg,E,Stockholm,D,Froy,O,Rashi,S,Gurevitz,M and Chejanovsky,N "Baculovirus
mediated expression of a scorpion depressant toxin improves the
insecicidal efficacy achieved with excitatory toxins" 1998 FEBS Lett
422,132-6
5.Hofmann,C,Sandig,V,Jennings,G,Rudolph, Schlag,P and Strauss,M "Efficient
gene transfer into human hepatocytes by baculovirus vectors" 1995
Proc. Nantl Acad Sci USA 92,10099-103
6.Hofmann,C and Strauss,M "Baculovirus mediated gene therapy in the
presence of human serum or blood facilitated by inhibition of the
complement system" 1998 Gene Ther 5,531-6
7.Lee,S,Qu,X,Chen,W,Poloumieko,A,MacAfee,N,Morin,B,Lucarotti,C and
Krause,M "Insecticidal activity of a recombinant baculovirus
containing an antisense c-myc fragment" 1997 J Gen Virol 78,273-81
8.Martens,J,Knoester,M,Weijts,F,Groffen,S,Hu,Z,Bosch,D and Vlack,J "Characterization
of baculovirus insecticides expressing tailored Bacillus thuringiensis
Cry1A9b) crystal proteins" 1995 J Invertebr Pathol 66,249-57
9.Palombro,F,Mociotti,A,Recchia,A,Cortese,R,Ciliberto,G and LaMonica,N "Site
specific integration in mammalian cells mediated by a new hybrid
baculovirus-adeno-associated virus vector" 1998 J Virol 72,5025-34
10.Richards,A,Matthews,M and Christain,P "Ecological considerations
for the environmental impact evaluation of recombinant baculovirus
insecticides" 1998Ann Rev. Entomol 43,493-517
11.Sandig,V,Hofmann,C,Steinert,S,Jennings,Gschlagg,P and Strauss,M "Gene
transfer into hepatocytes and human liver tissue by baculovirus vectors"
1996 Human Gene Ther 20,1937-45
12. Sarkis,C.,Seugera,C,Petres,S,Buchet,D,Riet,J,Edelman,L and Mallet,J.
"Efficient transduction of neural cells in vitro and in vivo by a
bculovirus derived vector" 2000 Proc Natnl Acad USA 97,14638-43
13.Thiem,S "Prospects for altering host range for baculovirus
bioinsecticides" 1997 Curr Opin Biotechnol 8,317-22
14.Wu,Y and Lui,G and Carstens,E "Replication, integration, and
packaging of plasmid DNA cotransformation with baculovirus viral DNA"
1999 J Virol 73,5473-80
Bacillus thuringiensis toxins reportedly toxic to mammals: Bacillus
thuringiensis (Bt) is used both as an insecticidal spray and as a source
of toxin genes for incorporation into GM crops. The spray is used
extensively in organic agriculture and in that capacity it is safe so long
as the food products are washed to remove the bacterium and its spores.
The GM crops have toxin in each and every cell and cannot be cleaned to
remove the toxin. Bt is also used as an aerial spray to control moths
damaging forest resources but in that capacity the spray is used in urban
centers and parks and schoolyards have been sprayed while children were
playing. Teschke et al ( 2001) found that Bt spray drifted for over a
kilometer from the target area of the spray well beyond the area
predicted.
Aerial spraying with Bacillus thuringiensis (Bt) has been allowed in
urban settings even though there is evidence that the spray causes gastric
problems and illness within the people directly contacting the spray. New
evidence shows that those exposed to the spray may develop a distinct
allergic response. Bernstein et al. (1999) found that field workers
exposed to Bt spray experienced allergic skin sensitization and induction
of IgE and IgG antibodies to the spray. There was a significant in
positive skin tests 1 and 4 months after exposure to Bt spray and there
was evidence that number of positive responses increased with increasing
exposure of the workers. Vazquez-Padron et al (1999) found that the Bt
toxin Cry 1 Ac was a potent immunogen in mice when administered by
intraperitoneal injection or intragastric administration. The exposure
caused greater antibody production than did cholera toxin. People prone to
allergy should avoid the districts slated for spraying and pray for rain
to wash away the residues.
Bt has been implicated in human illness and distress in the past but
current evidence suggests that people with weak immune systems (older
people, very young people, people treated for cancer or people with AIDS)
may face toxic shock and death from breathing the spray. Hernandez et al
(1999) took a Bt strain they had isolated from severe human necrosis and
found to induce necrosis in the skin of immunosuppressed mice and
intranasally infected immunocompetent mice. The mice died within 8 hours
in a clinical toxic-shock syndrome. It has also been found that Bt may
have Bacillus cereus like toxins (Azam et al 2000).
Recently there has been considerable international concern about the
contamination of the human food chain with StarLink corn containing
Bacillus thuringiensis (Bt) toxin Cry 9. Bt Cry 9 toxin had evident
allergenicity in test animals and had been approved for use in animal feed
alone but was found to have contaminated corn and corn products destined
for human consumption. Bt toxins are the products of a number of genes and
genes that differ between Bt varieties.
The EPA review of the Bt toxin Cry 9 is found at:
http://www.epa.gov/oppbppd1/biopesticides/cry9c/cry9c-peer_review.htm
"The results of intraperitoneal injection of corn powder extracts
into BN rats indicate that both the control and transgenic corn powders
are able to induce IgE or reagininc [regain is the allergist’s
term for IgE antibody] antibody responses by the PCA assay [PCA is
the passive cutaneous anaphylaxis assay ] . The use of corn powder
immunogen decreases the rate of the immune response to the Cry9C protein
compared to the bacterial preparation. However, the lowest responding dose
for Cry9C was similar for the two preparations (between 0.1 and 0.4 µg
Cry9C). The control challenge test with the heterologous antigen of
control corn powder or transgenic corn powder in the day 42 sera samples
indicated that there was significant reactivity from the corn portion of
the extracts themselves in the PCA assay. It is unclear, given this
background reactivity, how conclusions can be made about the reactivity of
the Cry9C protein alone. The PCA results from oral sensitization with
ovalbumin II, control corn extract, bacterial Cry9C and transgenic corn
(apparently supplemented with bacterial Cry9C) indicated that an IgE or
reagin antibody response was elicited in naïve Sprague-Dawley rats.
Ovalbumin sensitized serum produced a low frequency of responders and a
weak dose response between the 5.0 and 50.0 mg/kg dose levels on days 28
through 42. The control corn also produced a positive oral sensitization
response but this was only examined at the 50 mg/kg dose. Oral dosing with
bacterial Cry9C have a positive PCA response as did the Cry9C amended
transgenic corn extract. The frequency of response to bacterial Cry9C
began to diminish in day 42 sera. The Cry9C amended transgenic corn had a
higher frequency of responders and the frequency remained high on day 42
PCA response. Western blot analysis indicated that Cry9C protein bands
could be recognized in the rat sera from both exposure routes."
The quote above from the EPA provides clear evidence Cry 9 in corn is
allergenic in mammals. The fact that corn powder alone is allergenic has
been known by those milling corn. The Cry 9 allergin was recognized in the
corn powder. Consequently, Cry 9 GM corn is significantly enhanced in its
ability to cause allergy in mammals. Tests such as the passive cutaneous
anaphylaxis (PCA) and laboratory examinations such as Western blots which
isolate and identify particular antibodies (the test is similar to the DNA
fingerprint used in crime detection) provide strong evidence that Cry9 is
an allergin. The repeated claim that GM corn modified with BtCry9 is "suspected"
of causing allergy seems a peculiar use of language by those claiming to
be superior scientists.
These results show that an allergic (IgE) response was associated with
Cry9 in corn powder. Cry 9 containing corn was fed to millions of farm
animals, and probably as many humans eating corn products contaminated
with corn designated only for farm animal use. Thus, any evidence
indicating IgE response to Cry 9 corn should be taken very seriously and
not be allowed to be buried by bureaucrats.
Another study (Fares and El-Sayed 1999) shows that Bt Cry 1 toxin which
is used extensively in corn and cotton products (oil and seed meal) used
in human and animal food damages the mammalian ileum. Damage to the ileum
can produce chronic illness such as fecal incontinence and/or flu like
upsets of the digestive system.
Ileum: Final and longest segment of the small intestine. It is
specifically responsible for the absorption of vitamin B12 and the
reabsorption of conjugated bile salts . The ileum is about 4 m (13 feet)
in length and extends from the jejunum (the middle section of the small
intestine) to the ileocecal valve, which empties into the colon (large
intestine). The ileum is suspended from the abdominal wall by the
mesentery. The smooth muscle of the ileum's walls is thinner than the
walls of other parts of the intestines, and its peristaltic contractions
are slower. The ileum's lining is also less permeable than that of the
upper small intestine. Small collections of lymphatic tissue (Peyer's
patches)are embedded in the ileal wall, and specific receptors for bile
salts and vitamin B12 are contained exclusively in its lining; about 90
percent of the conjugated bile salts in the intestinal contents is
absorbed by the ileum.
In conclusion, Bt in bacterial spores is probably safe for food because
the toxin can be washed off. GM Bt toxins, however, cannot be washed off.
Bt toxins have been found to be allergenic or to create damage to the
digestive system of mammals. Bt spray to control forest moths is unsound
when large urban centers are sprayed and drift is poorly controlled and
accounted for.
References
- Bernstein,I,Bernstein,J,Miller,M,Tiewzieva,S,Bernstein,D,Lummus,Z,
Selgrade,M,Doerfler,D and Seligy,V "Immune responses in farm
workers after exposure to Bacillus thuringiensis pesticides" 1999
Environ Health Perspect 107,575-82
- Fares ,N and El-Sayed,A "Fine Structural Changes in the Ileum of
Mice Fed on -Endotoxin-Treated Potatoes and Transgenic Potatoes"
1999 : Natural Toxins Volume 6, 219-233
- Hernandez,E,Ramisse,F,Cruel,T,leVagueresse,R andCavallo,J "Bacillus
thuringiensis serotype H34 isolated from human and insecticidal strains
3a3b and H14 can lead to death of immunocompetent mice after pulmonary
infection"1999 FEMS immunol Med Microbiol 24,43-7
- Tayabali,A and Seligi,V. "Human Cell
Exposure Assays of Bacillus thuringiensis Commercial Insecticides:
Production of Bacillus cereus-Like Cytolytic Effects from Outgrowth of
Spores"2000 Environmental Health Perspectives 108,919-30
- Teschke, K, Chow,Y. Bartlett,K, Ross,A and van Netten ,C"Spatial
and Temporal Distribution of Airborne Bacillus thuringiensis var.
kurstaki during an Aerial Spray Program for Gypsy Moth Eradication"
2001 Environ Health Perspect 109:47–54
- 6.Vazquez-Padron,R,Moreno-Fierros,L,Neri-Bazan,L,de la Riva,G, and
Lopez-Revilla,R "Intragastric and intraperitoneal administration of
Cry 1Ac prototoxin from Bacillus thuringiensis induces systemic and
mucosal antibody response in mice" Life Sci 64,1897-99
The impact of bacterial DNA on the immune system (the CpG effect):
Essentially all of the GM crops marketed or being field tested presently
contain bacterial sequences as a part of the plasmids used for delivering
genes and many of the primary crop protection genes are of bacterial
origin. Such genes include Bt and most herbicide tolerance genes. DNA
vaccines have generated a huge literature and clinical applications
showing the activity and cellular incorporation of DNA administered by
oral, inhalation, injection, vaginal or dermal application (Molling
1997,Donnoley et al 1997 and Gurunathan et al 2000). Ingestion of bacteria
does not appear to be an effective means of delivering DNA because the
bacterial cell walls effectively contain the nucleic acid (for example, in
yogurt the milk products are digested but the bacteria of the culture are
passed intact). Lysis genes have been found necessary and effective in
triggering release of DNA for mucosal vaccine delivery (Jani and Mekalanos
200). In contrast , the crops eaten by animals release oligonucleotides
and DNA peptide complexes during digestion and such molecules circulate to
a significant degree.
The bacterial genes used in constructing GM crops have a property that
impacts on the immune system over and above the ability to produce
antibodies. Eukaryote DNA has relatively low frequencies of the
dinuleotide motif CpG and that motif is methylated and plays a role in
gene regulation while bacteria and their viruses have a high frequency of
the CpG motif that is usually unmethylated. Apparently the CpG motif in
DNA molecules and oligonucleotides provides a signal that the immune
system recognizes and initiates a primary sequence of reactions leading to
activation of the immune system leading to inflammation (Manders and
Thomas 2000 and Gurunathan et al 2000). Oligonucleotides rich in the CpG
motif are used to enhance immunization. Inflammation is an essential part
of the immune response but it adversely affects existing conditions such
as autoimmune disease. Furthermore, it has been found that CpG
oligonucleotides rescue B cell lymphoma cells from anti-IgM mediated
growth inhibition (Han et al 1999). The oligonucleotide acts as a promoter
of lymphoma.
Finally, Gorecki and Simons (1999) pointed out a danger to the fetus in
DNA vaccination of the mother. That danger was the creation of tolerance
in the fetus leading to individuals more susceptible to infection and/or
they may become carriers. The introduction of genes with bacterial CpG
motif to the fetus is likely to have untoward consequences.
In conclusion, the bacterial genes used in GM crops have been found to
have significant impacts on the individuals ingesting GM crops. The
impacts include inflammation and lymphoma promotion. The consequence of GM
food genes being incorporated into the chromosomes of somatic cells of
those consuming GM food and their unborn has been ignored by those charged
with evaluating the hazards of GM crops.
References
1.Donnelly,J,Ulmer,U,Shiver,J and Lui,M. "DNA Vaccines"1997
Annu Rev Immunol 15,617-48
2.Gorecki,D and Simons,J "The dangers of DNA vaccination" 1999
Nature Medicine 5,126
3.Guunathan,S,Klinman,D and Seder,R. "DNA Vaccines" 2000 Annu
Rev. Immunol 18,927-74
4.Hsu,S,Chung,S,Robertson,D,Ralph,L,Chelvarajan,R and Bondada,S 1999 "CpG
oligodeoxynucleotides rescue BKS-2 immature B cell lymphoma from
anti-Ig-M-mediated growth inhibition by up-regulating of egr-1"
International Immunology 6,871-9
5.Jain,V and Mekalanos,J "Use of lambda phage S and R gene products
in an inducible lysis system from Vibrio cholerae and Salmonella enterica
servovar Typhimurium-Based vaccine delivery systems" 2000 Infection
and Immunity 68,986-9
6.Manders,P and Thomas,R "Immunology of DNA vaccines: CpG motifs
and antigen presentation" Inflamm Res 49,199-205
7.Molling,K "Naked DNA for vaccine or therapy" 1997 JMolMed
75,242-6
8.Schubbert,R,Renz,D,Schmitz,B and Doerfler,W "Foreign (M13) DNA
ingested by mice reaches peripheral leucocytes, spleen,and liver via the
intestinal wall mucosa and can be covalently linked to mouse DNA"
1997 Proc. Natnl Acad Sci USA 94,961-6
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