Dr. Purnananda Guptasarma, Ph.D (Professor & Head, Dept. of Biological Sciences @ IISER Mohali) speaks in a Public Debate on January 24, 2014 at MICROCON on "Why Bt is Not Safe?" during a Public Debate on GMO's. You need to understand this to understand What's Wrong with Bt. GMO's.

1. Purnananda Guptasarma, Ph.D
Professor & Head, Dept. of Biological Sciences @ IISER Mohali
&
Director, MHRD Centre of Excellence @ IISER Mohali
(Center for Protein Science, Design and Engineering)
Genetically-modified organisms
Public Debate Panel - Jan 24, 2015 – MICROCON 2015, Panjab University

2. Bacillus thuringiensis (Bt)
is a bacterium
Bt produces toxic
proteins that can easily
form crystals
The molecular structures of these
toxic (Cry) proteins have been
determined in atomic detail.
The proteins (Bt toxins) are toxic to insects,
and to their larval (worm-like) forms.
Thus they act as insecticides.
Earlier, Bt toxins were sprayed. Now plants are made to produce them, through gene introduction.
SOME BACKGROUND FACTS ABOUT Bt

3. With INSECTS, therefore,
eating Bt Crop
= eating Bt toxin
= death
The Bt toxin thus
kills the insects…
that eat the plants…
that make the Bt toxin…
which is a recombinant protein…
produced in the plant’s tissues…
through expression of a bacterial gene…
inserted into the plant’s genome…
through genetic engineering. Some people think that this is a really
smart way of keeping insects
from destroying crop.
Actually, it’s a really smart implementation of a smart idea
Death for the insect, and life for the crop !
SOME MORE BACKGROUND FACTS ABOUT Bt

4. However, would we want to eat such a plant ?
Because, if we eat the plants, we are going to eat
the toxin too……
Can we be sure that ingesting the toxin is safe ?
Of course, some scientists and companies are
telling us that no harm will come to humans…..
I’m just not persuaded yet that they fully
understand what they are talking about ……

5. The Bt toxin is a protein…….
I work with proteins. I engineer proteins. I engineer
organisms to produce proteins.
I think that many GMO-derived proteins are safe.
I am not persuaded that the Bt toxin is safe for
ingestion by humans.

6. Let us see examine how the Bt toxin kills insects…..
…….And how insect-like we are (or aren’t).

7. QUESTION :
Does mankind yet have a mature understanding of
Bt toxin’s mechanism of action ?

8. Cell Membrane
Membrane Pore Formation by Bacterial Pore-Forming Toxins
(PFTs)
Consequences
 Free diffusion of ions and small molecules
 Disruption of permeability barrier function of membrane
PFT
Extracellular Space
Cytosol

9. One thing that Bt toxin does is to penetrate and rupture intestinal microvilli, and
lyze epithelial cells…..it paralyses and dissolves the intestines and allows gut
bacteria to infect the whole body. But that is just what we know currently…

10. The scientific literature suggests that man does not yet know, or
understand
• the exact manner in which Bt toxin proves to be toxic to insects …..
- Some believe that toxicity and death result from intestinal
cell rupture, mediated with/without binding of toxin to cell
surface receptors
- Others believe that death owes to binding to
cadherins, intracellular signaling and cell death

11. In 2010, there was
no unanimity
about how the toxin
acts..there is still no
unanimity!

12. !

13. I would prefer that companies and scientists
postpone declarations of safety to a point of
time that comes, at least technically,…
…after they have understood how the Bt toxin
actually acts to kill insect gut cells and
insects ….
i.e., whether through pore-formation, or
through signal transduction, or both!

14. QUESTION :
Does mankind yet have a mature understanding of
receptors involved in Bt toxin action ?

15. The scientific literature suggests that man does
not yet know, or understand
• all the cell membrane components with which
the Bt toxin interacts,
• whether a cell-surface Bt toxin-binding
receptor is required under all conditions, or
whether the toxin can insert without a receptor,
as is now proving to be the case with many
bacterial alpha helical and beta sheet-based
PFTs.

16. In a paper published in 2005 in the Journal of Cell Science, here’s what the
authors had to say, in a ‘matter-of-fact’ way, in the paper’s introduction :
“These observations indicated that APN and CLPs are not the only components
involved in mediating Cry protein toxicity. Consequently, the presence and
correct assembly of other membrane components has recently been proposed as
a prerequisite for Cry1A toxicity.”
There’s much more to ‘Cry’ than meets the eye :

17. The highlighted text : From the paper’s introduction

18. Even in 2008, scientists
remained perplexed by the
diversity of discovered
receptors (cadherins,
aminopeptidases,
alkaline phosphatases) of the
Bt toxin in cells from different
insects, since this suggests
that Bt toxin interacts with
many diverse proteins.
So, the mode of action is still
being studied !
i.e., man does not yet
understand all that the Bt
toxin can do inside living
systems !

19. And in 2010 the list of likely receptors of the Bt toxin has become larger, and now
Includes glycolipids, glycoconjugates
and various other molecules

20. I would prefer that companies and scientists
postpone declarations of safety to a point of
time that comes, at least technically,…
…after they have understood the entire
gamut of likely receptors - for pore-formation,
involving the pore-forming domain - or for signal
transduction - involving any one of Bt toxin’s three
structural domains…
In fact, I would like to insist that this be done before I am
asked, or forced, to eat any Bt-based food.
Without seeing this data, how can I agree that a
protein that can bind to such a diverse set of insect
proteins will not also manage to bind to some
proteins on human insect gut cells, for instance to cadherin
(CAD) or cadherin-like proteins (CLPs) present on every cell?

21. QUESTION :
What about man’s understanding of Bt toxin activation ?

22. Scientists assure us that activation only happens
at alkaline pH, and not at acidic pH, and involves
proteolysis (although they have not yet
identified the specific proteases that truncate
the Bt toxin).
Admittedly, it is true that insect guts are mostly
alkaline, whereas the human gut is mildly acidic-
to-neutral and only sometimes very mildly
alkaline.
But are the guts of all insects alkaline? Also,
are ‘non-alkaline gut’ insects immune to toxin?

24. There is confusion.
Insects with mildly acidic or neutral guts (like human)
are susceptible, if the toxin is pre-solubilized in
mildly alkaline solution before entering the gut!
So, can a little bit of Digene or Gelusil (or other base)
taken after a meal of Bt-food solubilize the toxin ?
How alkaline? How acidic ? Do we know ?

25. On the other hand, can not taking Digene or Gelusil
cause the protein to lose some solubility and
aggregate into precipitates that might elicit some
other reaction, involving mucosal-associated
lymphoid tissue and T-cell-independent B-cell
activation ?
Do we know ?
And if any major gut protease in insects can cleave
the toxin, how do we know that human guts lack
such proteases ?

26. And do serine proteases do the job of activation by
cleaving the protein, or do they degrade the toxin
by over-cleaving the protein ? Why is there
‘double-speak’ on this ? Because all insects are
not the same. Nor are all humans.
Do serine protease inhibitors truly enhance activity
of the toxin in some instances, as claimed ?
Then, why have some companies produced Bt-
soyabean which is a rich source of the well-known
serine protease inhibitor known as soyabean
trypsin inhibitor?
And who will reckon for the minority population of
toxin that could be activated?

27. Protein behavioral characteristics, such as stability, activation, folding, aggregation etc
follow certain patterns such as sigmoidal, bell-shaped, exponential curves etc
Nothing is 100 percent proof! Even if one says that the toxin only acts at mildly
alkaline pH, there is bound to be some activity at neutral or mildly acidic pH
where ‘negligible’ activity can ‘accumulate’ over time to have serious
consequences!
100 percent
0 percent
0.1 percent

28. Nothing is 100 percent proof! Even if one says that the toxin is only activated
optimally under certain conditions, there is bound to be some activation
outside the ‘bounds’ identified by in vitro or ex vivo assays. These ‘negligible’
activations can ‘accumulate’ over time to have serious consequences!
Protein behavioral characteristics, such as stability, activation, folding, aggregation etc
follow certain patterns such as sigmoidal, bell-shaped, exponential curves etc

29. I would prefer that companies and scientists
postpone declarations of safety to a point of
time that, at least technically, comes …
…after they have demonstrated beyond reasonable
doubt that no toxin activation can, or does, occur, in
humans
In fact, I would like to insist that this be done before I am
asked, or forced, to eat any Bt-based food.
Without seeing this data, how can I agree that – over time –
as I eat kilogram after kilogram of vegetable matter laden
with Bt toxin in every cell – there won’t be any damage?

30. QUESTION :
What about man’s understanding of after-effects ?

31. The scientific literature suggests that there is
enough reason to doubt Bt toxin’s safety

35. Look at research data from 2007/2008, and this carefully-worded abstract of a paper
showing significant histopathological and biochemical changes in three generations of rats
fed on Bt corn. Rats have short lives, and still these changes showed up.

36. Then, there is the business of
how these toxins (both alpha-
helical and beta-sheet based
PFTs) form SDS-resistant
oligomers which are also
protease-resistant

37. Protease-resistant oligomers will
not be digested in the stomach
or intestine
They may be transported to organs, like the rogue
prion protein (responsible for CJD and mad cow
disease) – which is similarly resistant to
proteolytic digestion – gets transported to (and
accumulates in) different organs including the
appendix, by lymphocytes in mucosal associated
lyphoid tissue

38. Cry proteins form crystals at suitably
high concentrations
Do we want Cry protein crystals
forming in those organs in our body known to
accumulate ‘stuff’ and undergo stone formation
or calcification ?
….such as the appendix, kidney or pineal gland ?
Do we want allergies ? Do we want damaged
intestinal ileums ? Do we want persistent diarhea ?
Do we want more irritable bowel syndrome ?
Is all this necessary ? Is there a guarantee ?

39. Then why should we be in a
hurry to expose humans to this
toxin?

40. Are insects (and insect cells) so different from
humans (and human cells) that there is no need to
investigate the effects on Bt toxins on humans over
long years, and many trials,
as is commonly done for drugs?
At least drugs save lives.
What will Bt-fods like Bt-brinjal save ?

41. Here’s another issue that vexes me !
The agri-biotech scientific community emphasizes that the
cells of insects are so very different from human cells that a
toxin killing insect cells will have no effect on human cells!
The pharma-biotech community emphasizes that the cells
of insects so very similar to human cells that we can make
therapeutic proteins and antibodies using these cells as
factories, and elicit almost human-like glycosylation!

42. Let us take the well-known insect larval (worm) stage
of the insect pest known as the ‘fall armyworm’,
also known as - Spodoptera frugiperda
and ask two simple questions
1) Are the cells of Spodoptera frugiperda (Sf) killed by the Bt toxin ?
2) Are the cells of Spodoptera frugiperda sufficiently like human cells
to be considered to be ‘models’ of human cells?

43. 1) Bt toxin kills Spodoptera frugiperda (Sf) cells
(this is so well-known that only a representative FDA document is presented as evidence)

44. Sf cell-derived H1N1 flu vaccines are
already being administered to
humans – as injectibles - in Europe
and Australia for the last month or
more, and more such developments
are taking place every month !
Sf cell-derived human proteins have
been approved for research use – as
human equivalents - for the last 15
years, or more !
FDA is on the verge of allowing Sf-
derived therapeutic proteins and
vaccines.
That is how human-like Sf cells are
held to be, by scientists !
2) Spodoptera frugiperda (Sf) cells are human-like

45. In fact, Spodoptera frugiperda (Sf) cells are believed
to be so ‘human-like’ that – after Chinese Hamster
Ovary (CHO) cells – Sf cells, infected by genetically
engineered baculovirus vectors, are current held to be
the second-best option for production of active
glycosylated human antibodies and other human (and
non-human) proteins for potential use in humans.
So, Sf cells are human-like.
And they are susceptible to the Bt toxin.
What does that say about the likely susceptibility of
human cells, given the right conditions ?

46. What is meant by “the right conditions “ ?
• Pre-solubilization of the toxin in mildly alkaline conditions
• trypsin-like proteases to activate the protoxin
• cadherins, aminopeptidases, alkaline phosphatases and
any number of as-yet-undiscovered potential receptor proteins,
transcription factors etc., which could trigger gene-expression
cascades that we may have no control over, in a population-wide
manner.
all of which could help the toxin to oligomerize and puncture
holes in cells, inside an unknown number of humans, manifesting in
different ways, or trigger cell-death by other signalling means!
Such a possibility would pull the plug on Bt crops even if they
were as life-saving as a drug ! And we know that with drugs, it
makes sense to administer them even with known side-effects,
when lives need to be saved !

47. I am not sure that I understand which lives need to be
saved, by feeding people with Bt crops……
Especially when problems with policy, stocking, hoarding
and distribution create more artificial famines than
any real lack of food…..
And given that a profit-making company developing a Bt
crop is anyway unlikely to give away the seeds
charitably to the poor and the hungry.
Gandhi said that there is enough for everyone’s need but
not enough for everyone’s greed.
I am not sure that we should eat Bt crops simply to
rationalize the research expenditures made by
scientists, or companies, in fulfilling commitments
made by such companies to investors and stake-holders

48. MOOT QUESTION
Should a non-life-saving GM crop enter the market in a matter of
months, or years, with
• insufficient independent safety-testing ,
• blanket denials regarding side-effects,
• clear evidence of immaturity of understanding of mode of action,
• clear evidence of profit motive
• clear evidence of being anti-poor (refer to ‘terminator’ seeds)
• known evidence of being life-threatening to other organisms,
• and no possibility of ever being labeled in our sabzi-mandi
?

49. MOOT QUESTION - cont’d
When even a new medicine takes
• a decade or more of safety-testing,
• declaration of side-effects,
• administration through doctors aware
of these side-effects
• life-saving potential
?
In my humble opinion, as a protein
scientist and protein engineer …….no.