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

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

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

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 ……

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.

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

QUESTION :

Does mankind yet have a mature understanding of

Bt toxin’s mechanism of action ?

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

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…

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

In 2010, there was

no unanimity

about how the toxin

acts..there is still no

unanimity!

!

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!

QUESTION :

Does mankind yet have a mature understanding of

receptors involved in Bt toxin action ?

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.

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 :

The highlighted text : From the paper’s introduction

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 !

And in 2010 the list of likely receptors of the Bt toxin has become larger, and now

Includes glycolipids, glycoconjugates

and various other molecules

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?

QUESTION :

What about man’s understanding of Bt toxin activation ?

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?

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 ?

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 ?

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?

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

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

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?

QUESTION :

What about man’s understanding of after-effects ?

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

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.

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

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

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 ?

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

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 ?

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!

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?

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

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

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 ?

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 !

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

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

?

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.