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Possible Human Health Hazards of Genetically Engineered Bt Crops

Comments on the human health and product characterization sections of EPA's
Bt Plant-Pesticides Biopesticides Registration Action Document
By Michael Hansen, Ph.D. Consumer Policy Institute/Consumers Union

Presented to the EPA Science Advisory Panel Arlington, VA
October 20, 2000

Thank you for the opportunity to present the comments of the Consumer
Policy Institute/Consumers Union on a subset of the Environmental
Protection Agency's Bt Plant-Pesticides Biopesticides Registration Action
Document. We would like to comment on two questions: human health
questions and product characterization. We believe that the human health
data that EPA presently requires are inadequate and that more data need to
be taken. Below, we propose some of the additional data that should be
required before any of these crops can be reregistered.

Please provide comment on whether the human health data is an adequate
evaluation of the risk from the Bt proteins. What, if any, additional data
is necessary to assess the risk from the Bt-expressing plant-pesticide
products?

We would particularly like to address the issues of allergenicity,
antibiotic resistance marker genes and acute toxicity testing.

Allergenicity

We do not think that the human health data that EPA currently has are
adequate. In particular, EPA seems to have ignored a crucial study that
suggests that the Bt delta endotoxin is an inhalant allergen, which could
present risks, in an occupational sense, to farmworkers and millworkers
that are exposed to dust from the processing of Bt crops.

EPA maintains that "After decades of widespread use of Bacillus
thuringiensis as a pesticide (it has been registered since 1961), there
have been no confirmed reports of immediate or delayed allergic reactions
to the delta-endotoxin itself despite significant oral, dermal and
inhalation exposure to the microbial product" (EPA, 2000: pg. IIB6). We
believe that this statement is at best misleading. Last year, an
EPA-funded study was published in Environmental Health Perspectives titled
"Immune responses in farm workers after exposure to Bacillus thuringiensis
pesticides." The article pointed out that more work needs to be done to
evaluate the allergenic potential of Bt sprays, as there have been 3
studies that are suggestive of the Bt sprays having an effect. The authors
are concerned because, as they point out, "approximately 75 percent of
asthma cases are triggered by allergens and mordibity and mortality due to
asthma have increased considerably over the past 20 years" (Bernstein et
al, 1999: 570).

The study consisted of a surveillance program of farm workers
before and after exposure to Bt pesticides. This study does not show a
definitive link between exposure to Bt sprays and occupationally related
respiratory symptoms, but did find that a number of the workers exhibited skin
sensitization and presence of IgE and IgG antibodies with those responses
being more numerous in those workers with higher levels of exposure. Both
skin sensitization and IgE antibodies are components of an allergic response.

As part of the study, the scientists were able to show that 2 of the farm
workers studied had a positive skin-prick test to the Btk spore extract
containing the pro-delta-endotoxin active component, as well IgE
antibodies. This means that there are now skin and serologic agents that
could be used to test the potential allergenicity of the various
delta-endotoxins that have been engineered into Bt crops. Although the
authors say that their results should allay some of the concerns about the
allergenicity of transgenic foods from Bt crops, they clearly say that they
now have the skin and serologic agents to do such tests: "Because
reactivity to the Btk pro-delta-endotoxin was only encountered in 2 of 123
workers sensitized by the respiratory route, it is unlikely that consumers
would develop allergic sensitivity after oral exposure to transgenic foods
(e.g., tomatoes, potatoes) that currently contain the gene encoding this
protein. However, future clinical assessment of this possibility is now
feasible because of the availability of reliable Bt skin and serologic
reagents developed during the course of this investigation" italics added
(Bernstein et al., 1999: 581).

Given that such Btk skin and serologic agents exist, we feel that all the
Bt crops should be retested using these skin and serologic reagents.
Although the reaction was to the pro-delta-endotoxin, separate genetic
studies (gel electrophoresis and hybridized blot analysis) demonstrated the
presence of genes for the Cry 1Ab and Cry1Ac delta endotoxins in both spray
formulations (Javelin and Agree) to which the workers had been exposed.
Truncated version of the Cry1Ab and Cry1Ac are present in the Bt corn and
Bt cotton events, respectively. Unless the allergenic epitopes are all
found in the part of the delta endotoxin that is removed during truncation,
one could reasonably expect that the Bt corn and cotton crops would contain
an allergenic epitope. Indeed, Furthermore, use of these reagents would be
superior to the current criteria presently used to evaluate the
allergenicity of these crops: amino acid sequence homology to known
allergens; resistance to acid and gastric digestion; heat stability/heat
resistance; and molecular size. None of these criteria are exact as the
state of science in the field of allergenicity is still in its infant
stages. (SAP, 2000: 7; www.epa.gov/scipoly/sap/2000/february/foodal.pdf).

Clearly, if skin and serologic reagents from humans exist for a given
protein, then any allergenicity testing must use such reagents. If the
reagents become available after the crops have been approved, the companies
should be required to retest those crops because the human reagents are far
more accurate than the four criteria presently being used.

Since one of the co-authors of the paper, Dr. Donald Doerfler, is an EPA
scientist, we wonder why the EPA hasn't already moved to conduct these
tests. With the use of these skin and serologic reagents, the testing of
Bt crops would not take that long and would be relatively inexpensive. So
why hasn't such a test been carried out?

EPA has argued that there occupational exposure to the Cry9C protein (or
the other Cry proteins inserted into corn, cotton) is negligible, or
presents no risk because the Cry proteins are not toxic to people
(Biopesticide Fact Sheet, 1999). Yet the EPA presented no study to
substantiate the claim of negligible exposure.

In fact, there is scientific evidence that occupational exposure to grain
dust can lead to allergic symptoms, with the classic case being bakers'
asthma (Baur, 1998). Recent studies have also implicated corn dust in
respiratory dsyfunctions including acute respiratory inflamation (Park et
al., 1998; Wohlford-Lenane et al., 1999) and in glove-lubricant-powder
derived allergy (Crippa et al., 1997). Thus, corn dust can clearly convey
allergens, and the pro-delta-endotoxin is potentially allergenic, so there
is ample evidence to be concerned about occupational exposure to grain
dusts, especially corn.

Interestingly, while the authors of that study found that farm workers had
skin reactions and IgE antibodies to Bt spray, they could not link any
respiratory symptoms to the occupational exposure. However, this could be
a result of the fairly low levels of Bt that the farm workers were exposed
to. The concentration of delta-endotoxin in the Bt crops, particularly
corn, is between one to two orders of magnitude higher compared to Bt
sprays. That's why the insect resistance management strategy is called the
"high dose" strategy. Furthermore, the concentration of the Cry9C protein
in the seed is one to two orders of magnitude higher than the concentration
of Cry1Ab or Cry1Ac in corn and cotton, respectively-18.6 µg/gm (kernal)
for Cry9C vs. 1.4 µg/gm (kernel), 0.19-0.39 µg/g (grain), and 1.62 µg/g for
Cry1Ab-Bt11, Cry1Ab-MON810, and Cry1Ac, respectively (EPA, 2000: pg.
IIC17). So, the concentration of Cry9C in corn dust could conceivably be 2
to 3 orders of magnitude higher than they level of endotoxin found in
foliar Bt sprays.

So, the Bt crops have far higher levels of endotoxin in the grain and
leaves than do the foliar Bt sprays. Furthermore, while farm workers are
exposed to the foliar Bt sprays, workers in mills or other areas where
grains are being processed would be exposed to grain dust and so could
conceivably be exposed to far higher quantities of the Bt endotoxin than a
farm worker would.

Antibiotic resistance marker genes

In 1991-1992, when FDA was developing its policy of GE plants, the
conventional wisdom in the scientific community was that DNA was a very
fragile molecule that would be readily broken down in the environment and
would not survive digestion in the gut. We now know that both assumptions
may not always be valid (Traavik, 1998). Even though DNases (molecules
that break down DNA) are widely distributed in the environment, free DNA
has been found in all ecosystems (marine, fresh water, sediments) studied
(Lorenz and Wackernagel, 1994). Indeed, pooled data suggest that free DNA
is present in significant amounts in the environment. Larger amounts of
DNA are extracted from soil than can be extracted from the cells in the
soil (Steffan et al., 1988). Further studies have shown that this free DNA
in the soil comes from microorganisms that no longer occur in that habitat
(Spring et al., 1992) thus demonstrating that DNA can out-survive the
organism it came from and still be capable of being taken up and expressed
by microorganisms. Finally, yet other studies have found that pollution
(i.e. xenobiotics) can affect the survivability of DNA and the possibility
of its transfer to other organisms (Traavik, 1998).

These data lead to serious concerns about the antibiotic resistance marker
genes that are present in virtually all engineered plants presently on the
market. These genes code for proteins that confer resistance to a given
antibiotic. The possibility therefore exists that these genes for
antibiotic resistance could be taken up by bacteria, thus exacerbating the
already very serious problem of antibiotic resistance in disease causing
organisms.

In mammalian system, the question is whether foreign DNA can survive
digestion, be taken up through the epithelial surfaces of the
gastrointestinal or respiratory tract or not, or be excreted in feces.
Studies in the 1970s (Maturin and Curtiss, 1977) and 1980s (McAllan, 1982)
in rats and ruminants, respectively, suggested that nucleic acids (e.g. DNA
and RNA) failed to find evidence that DNA survived digestion.
Consequently, many scientists assumed that DNA was readily digested.
However, the methods used to detect DNA were not very sensitive. In the
mid-1990s, researchers in Germany, re-investigated the issue, using far
more sensitive methods (Schubbert et al., 1994). Mice were fed DNA from
the M13 bacteriophage either by pipette or by adding it to the feed
pellets. Using sensitive hybridization methods and PCR (polymerase chain
reaction) the authors found 2-4% of the M13 DNA in feces and 0.01-0.1% in
the blood-both in serum and cell fraction. Sizeable DNA fragments (almost
a quarter of the M13 genome) could be found up to 7 hours after uptake.

If free DNA is not immediately digested in the gastrointestinal tract, the
possibility also exists that it can be transferred to bacteria that live
there. A recent study utilizing a simulated human gut demonstrated that
naked DNA had a half-life of 6 minutes, more than enough time for such DNA
to transform bacteria (ref to come).

In another experiment, a genetically engineered plasmid was found to
survive (6 to 25%) up to an hour of exposure to human saliva (Mercer et
al., 1999). Partially degraded plasmid DNA also successfully transformed
Streptococcus gordonii, a bacteria that normally lives in the human mouth
and pharynx although the frequency of transformation dropped exponentially
with time. Transformation occurred with either filter-sterilized human
saliva or unfiltered saliva. The study also found that human saliva
contains factors that increase the ability of resident bacteria to become
transformed by "naked" DNA. Since transgenic DNA from food is highly
unlike to be completely broken down in the mouth, it may be able to
transform resident bacteria. Of particular concern would be the uptake of
transgenic DNA containing antibiotic resistance marker genes, which are
found the majority of GE crops presently on the market. It should be
pointed out that the antibiotic marker gene present in Novartis' Bt corn,
which codes for resistance to ampicillin, is under the control of a
bacterial promoter rather than a plant promoter which would further
increase the possibility of expression of the ampicillin resistance gene if
it were taken up by bacteria.

In September, 1998, the British Royal Society put out a report on genetic
engineering that called for ending the use of antibiotic resistance marker
genes in engineered food products (Anonymous, 1998). In May, 1999, the
British Medical Association, which represents some 85% of the doctors in
Britain, released a report calling, in part, for a prohibition on the use
of antibiotic resistance mark genes in genetically engineered plants: "The
BMA believes that the use of antibiotic resistance marker genes in GM
foodstuffs is a completely unacceptable risk, however slight, to human
health. . . Recommendations . . . 6. There should be a ban on the use of
antibiotc resistance marker genes in GM food" (BMA, 1999).

In the European Union Directive 90/220/EEC deals with the deliberate
release of GEFs into the environment. The European Commission is in the
process of coming with a revised version of the Directive. This revised
version contains a provision which would phase out the use of antibiotic
resistance marker genes by 2005 (European Commission Services, 2000)

We therefore urge EPA to prohibit use of antibiotic resistance marker genes
as there is no consumer benefit for the presence of such genes in
engineered foods and a potential risk.

Acute toxicity testing

At present, EPA requires only an acute toxicity feeding test.
Furthermore, the delta-endotoxin that is used in these feeding tests come
from a genetically engineered bacteria rather than from the transgenic
plant itself. The EPA assumes that there are no real difference between a
delta-endotoxin produced by an engineered bacteria and one produced in a
plant.

We believe this ignores the phenomenon of post-translational processing,
which consists of the modification of a protein after it has been
translated from the genetic message. And such post-translational
processing can have a significant impact on the structure and function of a
gene. Furthermore, post-translational processing can differ between
organisms, so that the same gene expressed in different genetic backgrounds
may have the same amino acid sequence but may differ in structure and
function. Examples of such processing includes glycosylation and
acetylation.

Glycosylation consists of the addition of sugar groups (usually
oligosaccharides) and can dramatically affect the three-dimensional
structure and thus, function of a protein. Indeed, glycosylation is
thought to be connected to allergenic and immunogenic responses (Benjuoad
et al., 1992). The data presented to the EPA suggest that the
delta-endotoxins are not glycosylated in the plants.

Acetylation of proteins consists of the addition of acetyl groups to
certain amino acids, thereby modifying their behavior. Although
incompletely understood, acetylation of the amino acid lysine has been most
studied in certain groups of proteins that bind with DNA-histones and
high-mobility group proteins-and such acetylation appears to be involved
with the regulation of interaction of these proteins with negatively
charged DNA molecules (Csordas, 1990). However, it has been discovered
that some the lysine residues in rbGH are acetylated, to form
epsilon-N-acetyllysine when it is produced in E. coli . Harbour et al.
(1992) found this to occur at lysine residues 157, 167, 171 and 180 or
rbGH, while Violand et al. (1994) found it at residues 144, 157, and 167.
The creation of this mutant amino acid may be overlooked because "(T)he
identification of this amino acid cannot be determined by simple amino acid
analysis because the acetyl group is labile to the acidic or basic
conditions normally used for hydrolysis" (Violand et al, 1994: 1089). The
effect this has on the safety, structure and function of rbGH is not known
as it hasn't been actively studied.

The differences in glycosylation and acetylation that can happen when
transgenes are expressed in plants or bacteria can possibly affect toxicity
and therefore lend further support to the need for toxicity testing using
the whole engineered food. Even if there are no differences in
glycosylation (as appears to be the case for the delta-endotoxins),
acetylation of lysine residue(s) could cause differences. The presence of
such mutant lysine residues could easily be missed as routine amino acid
analysis will remove the acetyl group; to find if there are mutant lysine
residues, one must specifically look produce the transgene of interest
(gene for herbicide tolerance or Bt endotoxin, for example). Thus,
whenever possible, EPA should require the companies to use material derived
from the transgenic plants themselves in toxicity studies rather than
bacterially-derived proteins.

Product Characterization

Please provide comment on the quality and thoroughness of the product
characterization review. What additional data, if any, should be evaluated
in order to adequately characterize the Bt-expressing plant-pesticide
products?

Information has appeared in the scientific literature related to the safety
of foods derived from genetically engineered (GE) plants which collectively
suggests that the EPA's present regulatory approach is insufficient to
ensure that foods from Bt crops not pose health risks to those who consume
it. This information relates to unexpected and unpredicted effects of gene
insertions, and instability of the genetic characteristics that are
introduced. This information leads to the view that EPA must scrutinize
genetically engineered foods more closely than it has so far, and in
particular should require long-terms (one to two year) animal feeding
studies of the whole engineered food. Requiring a more detailed molecular
characterization for each genetic transformation event will also help EPA
evaluate the potential for risk and may provide a means for EPA to decide
how much additional testing is needed. At present the level of molecular
characterization data required by the EPA is very inadequate.

The studies which lead to greater concern about unexpected effects can be
put into two categories: unpredictability of the location and expression
of transgenic DNA inserts; and differences resulting from
post-translational processing (e.g. proteins from the same gene are not
identical in differing organisms).

Unpredictability of the location and expression of transgenic DNA
underlines need for long-term toxicity tests of engineered food.

The process of insertion of genetic material via GE is unpredictable with
regard to a number of parameters, including: the number of inserts of
transgenic DNA, their location (chromosome, chloroplast, mitochondria),
their precise position (i.e. where and on which chromosome), their
structure, and their functional and structural stability. While all of
these parameters can have consequences, perhaps the most important is the
random or semi-random nature of the physical location of the genetic
insert. The inability to control where the insertion happens is of key
importance. This means that each transformation event is unique and cannot
be replicated because the precise location of the insertion of genetic
material always will be different.

The variable insertion site can have a number of unpredictable, and
potentially negative, consequences (Doerfler et al., 1997). The insertion
site can affect expression of the inserted transgene itself as well as the
expression of host genes (i.e. genes in the recipient organisms). The
former is known as the "position effect". A classic example involved
attempting to suppress the color of tobacco and petunia flowers via the
transfer of a synthetically created gene designed to turn off (via
anti-sense technology) a host pigment gene (van der Krol et al., 1988).
The expected outcome was that all the transformed plants would have the
same color flowers. However, the transformed plants varied in terms of the
amount of color (or pigmentation) in their flowers as well as the pattern
of color in the individual flowers. Not only that, but as the season
changed (i.e. in different environments), some the flowers also changed
their color or color pattern. The factors contributing to the position
effect are not fully understood.

The expression of host genes can be influenced by the location of the
genetic insertion as well. If the material inserts itself into "the middle"
of an important gene, that gene would functionally be turned off. In one
experiment, insertion of viral genetic material into a mouse chromosome
lead to disruption of a gene which resulted in the death of the mouse
embryos (Schnieke et al., 1983). If the "turned off" gene happened to
code for a regulatory protein which prevented the expression of some toxin,
the net result of the insertion would be to increase the level of that toxin.

The genetic background of the host plant can also affect the level of
expression of the transferred gene, which explains the common observation
that varieties of the same plant species varied widely in the ease with
which they can be genetically engineered (Doerfler et al., 1997; Traavik,
1998). In some varieties, the trait can be expressed at high enough levels
to have the desired impact. In others, the expression level is too low to
have the desired impact. In general though, scientists do not really
understand why some plant varieties yield more successful results in GE
than other varieties.

To get around the common problem of an insufficient level of expression of
a desired gene product, powerful regulatory elements-particularly
promoters/enhancers-are inserted along with the desired transgene and used
to maximize gene expression. The promoter has numerous elements that
enable it to respond to signals from other genes and from the environment
which tell it when and where to switch on, by how much and for how long.
When inserted into another organism as part of a "genetic construct," it
may also change the gene expression patterns in the recipient chromosome(s)
over long distances up- and downstream from the insertion site. If the
promoter (plus associated transgenes) is inserted at very different places
on a given chromosome or on different chromosomes, the effects may be very
different; it will depend on the nature of the genes that are near the
insertion site. This uncertainty of insertion site, along with the
promoter means that for all transgenic plants, there will be a fundamental
unpredictability with regard to: expression level of the inserted foreign
gene(s); expression of a vast number of the recipient organism's own genes;
influence of geographical, climate, chemical (i.e. xenobiotics) and
ecological changes in the environment; and transfer of foreign genetic
sequences within the chromosomes of the host organism, and vertical and/pr
horizontal gene transfer to other organisms. Such unpredictability
explains the common observations that different insertion events in the
same variety can vary greatly in terms of the level of expression of the
desired transgene and that the majority of transformation events do not
yield useful results (i.e. the transgenic plant is defective in one way or
another).

The unpredictable influence of the environment may explain what went wrong
in Missouri and Texas with thousands of acres of Monsanto's glyphosate
tolerant cotton and Bt cotton, respectively. In Missouri, in the first
year of approval, almost 20,000 acres of this cotton in malfunctioned. In
some cases the plants dropped their cotton bolls, in others the tolerance
genes were not properly expressed, so that the GE plants were killed by the
herbicide (Fox, 1997). Monsanto maintained that the malfunctioning was due
to "extreme climatic conditions." A number of farmers sued and Monsanto
ended up paying millions of dollars in out-of-court settlements. In Texas,
a number of farmers had problems with the Bt cotton in the first year of
planting. In up to 50% of the acreage, the Bt cotton failed to provide
complete control (a so-called "high dose") to the cotton bollworm
(Helicoverpa zea). In addition, numerous farmers had problems with
germination, uneven growth, lower yield and other problems. The problems
were widespread enough that the farmers filed a class action against
Monsanto. Last fall, Monsanto settled the case out of court, again by
paying the farmers a significant sum (Schanks [plaintiffs attorney],
personal communication). If there could be this unexpected effect on the
growing characteristics of the cotton, it is theoretically possible that
their could be changes in the plant itself which affect the nutritional or
safety characteristics of the plant (used as cattle feed) or the seed (the
oil from which is used in a number of food products). This raises the
question of whether EPA should establish procedures for assuring safety in
the long term.

The unpredictability associated with the process of genetic engineering
itself could lead to unexpected effects such as the production of a toxin
that doesn't normally occur in a plant or the increase in a level of a
naturally occurring toxin. An example of the former occurred in an
experiment with tobacco plants engineered to produce gamma-linolenic acid.
Although the plants did produce this compound, another metabolic pathway
ended up producing higher quantities of a toxic compound, octadecatetraenic
acid, which does not exist in non-engineered plants (Reddy and Thomas,
1996).

An example of the latter occurred in an experiment involving yeast where
genes from the yeast were duplicated and then reintroduced via genetic
engineering (Inose and Murata, 1995). The scientists found that a
three-fold increase in an enzyme in the glycolytic pathway,
phoshofructokinase, resulted in a 40-fold to 200-fold increase of
methylglyoxal (MG), a toxic substance which is know to be mutagenic (i.e.
tests positive in an Ames test). This unexpected effect occurred even
though the inserted genetic material came from the yeast itself. As the
scientists themselves concluded, "Although, except for the case of
microbes, we have no information as to the toxic effect of MG in foods on
human beings, the results presented here indicate that, in genetically
engineered yeast cells, the metabolism is significantly disturbed by the
introduced genes or their gene products and the disturbance brings about
the accumulation of the unwanted toxic compound MG in cells. Such
accumulation of highly reactive MG may cause a damage in DNA, thus
suggesting that the scientific concept of "substantially equivalent" for
the safety assessment of genetically engineered food is not always applied
to genetically engineered microbes, at least in the case of recombinant
yeast cells. . . . Thus, the results presented may raise some questions
regarding the safety and acceptability of genetically engineered food, and
give some credence to the many consumers who are not yet prepared to accept
food produced using gene engineering techniques" (Inose and Murata, 1995: ).

Another study published in Lancet in late 1999 used potatoes that were
genetically engineered to contain a chemical from the snow drop plant (a
lectin, Galanthus nivalis agglutinin [GNA]) to increase resistance to
insects and nematodes. Feeding experiments with rats demonstrated a number
of potentially negative effects (Ewen and Pusztai, 1999). The study found
variable effects on the gastrointestinal tract, including proliferation of
the gastric mucosa. Interestingly, the potent proliferative effect on the
jejunum was seen only in the rats fed GE potatoes with contained the GNA
gene but not in rats fed non-transgenic potatoes to which GNA had been
added. Indeed, a previous feeding study utilizing GNA with a 1,000-fold
higher concentration than the level expressed in the GE potatoes had found
no proliferative effect (Pusztai et al., 1990). The authors proposed "that
the unexpected proliferative effect was caused by either the expression of
other genes of the construct or by some form of positioning effect in the
potato genome caused by GNA gene insertion" (Ewen and Pusztai, 1999:
1354). Such a fine-grained feeding study, which involved utilizing young
rats which were still growing and involved weighing various organs and
looking very carefully for effects on various organ systems and the immune
system is far more detailed than the general feeding studies done utilizing
GE plants. While many criticisms have been leveled at this study, we
believe it raises important questions that merit further research.

Because of the unexpected effects that are theoretically possible and which
have been seen in various experiments, we feel EPA should require long-term
animal feeding studies using the whole food product. Such testing should
be done on growing animals, so that effects on various organ systems can be
readily observed. In addition, fairly extensive data should be taken on
the weights of various organs and on histopathology and immunology. In
addition, there should be follow-up feeding studies if any data from the
lab or field demonstrates that the genetic insert is unstable. FDA should
propose its procedures for public comment so that it can get further input
from the scientific community and others.

The most commonly used promoter in plant genetic engineering is one from
the cauliflower mosaic virus (CaMV); all GE crops on the market contain it.
A promoter has numerous elements that enable it to respond to signals from
other genes and from the environment which tell it when and where to switch
on, by how much and for how long. A CaMV promoter is used for a number of
reasons: because it is a very powerful promoter, because it is active in
all plants-monocots, dicots, algae-and inE. coli and because it is not
greatly influenced by environmental conditions or tissue types. CaMV has
two promoter, 19S and 35S, but the 35S is the one most frequently used
because it is the most powerful. The powerful nature of the CaMV 35S
promoter means that it is not readily controlled by the host genes that
surround it and often yields a high expression level of the transgene next
to it. This is not unexpected as CaMV is a virus that is designed to
hijack a plant cell's genetic machinery and make many copies of itself.
This also means that it is designed to overcome a plant cell's defensive
devices to prevent foreign DNA from being expressed. In the case of
transgenic crops, however, the CaMV promoter is used to put the transgenes
outside the normal regulatory circuits of the host organism and have them
expressed a very high levels. Being placed outside of normal regulatory
circuits may be one of the reasons why GEFs are known to be so unstable
(Finnegan and McElroy, 1994). The questions raised by the extensive use of
the CaMV 35S promoter in engineered crops should be investigated with
further research (Ho et al., 1999)

Post-translational processing

Another area of study that raises serious questions about the safety of
transgenic traits is the phenomenon of post-translational processing, which
consists of the modification of a protein after it has been translated from
the genetic message. And such post-translational processing can have a
significant impact on the structure and function of a gene. Furthermore,
post-translational processing can differ between organisms, so that the
same gene expressed in different genetic backgrounds may have the same
amino acid sequence but may differ in structure and function. Examples of
such processing includes glycosylation, acetylation, and methylation.

Glycosylation and acetylation were covered in the previous section on
acute toxicity testing.

Methylation is the process of putting methyl groups on a molecule.
Methylation of DNA, which occurs with the nucleotide bases cytosine and
adenosine, is important as this appears to prevent that piece of DNA from
being expressed (or "turned on"). Methylation is one of the mechanisms
behind the phenomenon of "gene silencing," whereby a cell "turns off" a
gene. Transgenic work has found that if you try to insert multiple copies
of a gene into a plant, the plant will frequently turn off all, or all but
one, of the copies of the transgene (Finnegan and McElroy, 1994). Indeed,
some scientists now think that gene silencing is an important defense
mechanism that plants use to prevent foreign DNA from being expressed
(other mechanisms exist to try to degrade the foreign DNA before it can
enter the nucleus of the cell) (Traavik, 1998; Ho, 1998). This should be
combined with the recent finding that tobacco plants may contain large
numbers of copies of paratetroviral-like sequences, in some cases reaching
copy numbers of about 10,000 (Jakowitsch et al. 1999). This study is quite
striking as it was previously thought that plant viruses rarely integrate,
if at all, into host genomes. Furthermore, such integrated viral genetic
material is normally silenced via methylation, so that there could be a lot
of dormant viral sequences in plants. Interestingly, the cauliflower
mosaic virus promoter (CaMV 35) used in virtually all transgenic plants on
the market is a pararetrovirus-derived sequence (i.e. CaMV is a
pararetrovirus).

With methylation, the danger exists that the CaMV 35S promoter, being a
very powerful "on switch" that can have effects thousands of base pairs
upstream and downstream from an insertion point, could inadvertently "turn
on" a foreign gene that has previously been silent. Given the studies in
the last couple of years that suggest that horizontal gene transfer may be
more common than previously thought and that most such foreign DNA, if it
survives and is able to incorporate itself in the host genome, is
frequently "silenced" via methylation, there's a potential risk that some
nasty dormant genetic material is inadvertently turned on due to the
presence of the CaMV promoter. Thus, it becomes important to know the
exact insertion site of any and all genetic construct as well as knowing
what the genetic sequence is for thousands of bases pairs upstream and
downstream from the insertions site, and do long term toxicity tests with
the whole engineered food.

What molecular characterization data EPA should require

Because of all the reasons stated above and because of the random nature
of the genetic transformation process each random insertion of transgenic
DNA will differ in location and in structure from all other inserts. It
will be accompanied by a different pattern of unintended positional and
pleiotropic effects due, respectively, to the location of the insert and
the functional interaction of the insert with host genes. Thus, each
transgenic line resulting from the same process, despite using the same
vector system and plant materials under the same conditions will be
distinct, and must be treated as such. Consequently, we think EPA should
require the companies to submit data for each separate transgenic line.
For every line, EPA should require a complete molecular characterization of
each line with respect to the identity, stability and unintended positional
and pleiotropic effects. And based on the results of such characterization,
the agency could decide on how much toxicity data to require.

The components of a complete molecular characterization for molecular
identity would include, for each transgenic or transformed line:
. Total number of inserts of transgenic DNA
. Location of each insert (organelle [chloroplast, mitochondria, etc.] or
chromosomal)
. Exact chromosomal position of each insert
. Structure of each insert (whether duplicated, deleted, rearranged, etc.)
. Complete genetic map of each insert including all elements (coding
region, noncoding regions, marker gene, promoters, enhancers, introns,
leader sequences, terminators, T-DNA borders, plasmid sequences, linkers,
etc. including any truncated, incomplete sequences)
. Complete (nucleotide) base sequence of each insert
. (Nucleotide) base sequence of at least 10kbp (10,000 base pairs) of
flanking host genome DNA on either side of the insert, including changes in
methylation patterns

To determine stability, the EPA needs data on both functional stability
(level of expression remains constant over time and over successive
generations) and structural stability (location in the genome and
structural arrangement of the insert). For functional stability, EPA would
need data on the level of expression of the transgene over time-throughout
the lifetime of the plant as well as over a number of generations (say 3 to
5 generations). For structural stability, the EPA would need data on the
physical location of the insert in the genome as well as the structure of
the insert-throughout the lifetime of the plant as well as over successive
generations (say 3 to 5). In addition, the EPA should require appropriate
molecular probes for each insert with flanking host genome (organelle
sequence) sequences in order to monitor the structural stability of the
insert.

To test for unintended positional effects, the EPA could look carefully at
the methylation patterns of the genes in the flanking host genome DNA (data
we suggest be required under molecular identity characterization). To look
for pleiotropic (as well as positional effects), each transformed line must
be identified in terms of total protein profile and metabolic profiles.
The total protein profiles would help to monitor for unintended changes in
the pattern of gene expression while the metabolic profile would help to
monitor for unintended changes in metabolism. The use of mRNA
fingerprinting and protein fingerprinting as part of the protein profiles
would represent a better, finer screen for detecting novel biochemical,
immunological or toxicological hazards. Some such tests have been
suggested by a Dutch government team and should be more carefully
considered by the FDA (Kuiper et al., 1998). If any of these tests found
differences, there would be more reasons to ask for more comprehensive
toxicity testing.

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