Thursday, September 30, 2010

Some Issues in the Bt brinjal

I must stress that I have not made and cannot make coherently brief comments on the very important Bt brinjal problem that will give a terse pertinent insight. This is not expected. It is required nevertheless at least from an academic point of view from an interested academic.

The new point I will try to stress on is the necessity for tasting the brinjal using knowledge from the gene-science of tasting that is recently available. The second is to try to examine the notion that Bt technology is safe because the toxin protein crystals are digested only in the alkaline medium of the insect and this should not be important in our acidic digestive medium.

There are too many ifs and buts and on-the-one-hand and on-the-other-hand aspects that prevent such a presentation. I have tried to make pertinent and relevant remarks that requires some (penance-like in a sense) study. Some of these points I have highlighted in bold, and some others which should be important to an interested expert (which I am afraid to highlight). I have made as serious a study that my background allows in the one week time that I gave myself, sensing an urgency, because of commercial pressures on the government.

I continue writing this blog from the previous blog (the numbering of figures commence from the previous one) more to inform myself about the Bt problem; hopefully, it is also to inform possible readers about the issues involved. I do not vouchsafe for the authenticity of my sources but I have cited most and some more of the literature available on the net readily available for reference.

Before we go into the whys and why-nots and the ifs and if-nots one requires knowing what the Bt about the brinjal bush is all about.

On pushing further than what I did in the previous blog one finds (see for instance, Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensisOne Species on the Basis of Genetic Evidence, by Erlendur Helgason et al, Appl Environ Microbiol. 2000 June; 66, 2627, cited more than 450 times) that other bacterial strain that belongs to the Bt or Bc family that show wide phenotypes (the visible characteristics of an organism resulting from the interaction between its genetic makeup and the environment) and pathological effects (processes of a disease, observable either with the naked eye or by microscopy, or, at a molecular level, as inferred from biochemical tests) is Bacillus anthracis, which causes the acute fatal disease anthrax (a highly infectious, often fatal, bacterial disease of mammals, especially cattle and sheep, that is transmissible to humans and causes skin ulcers cutaneous anthrax or a form of pneumonia when inhaled pulmonary anthrax) and is a potential biological weapon due to its high toxicity (causing evacuation of the US Senate building when traces of anthrax was found in the mail in the amerithrax episode a week after 9/11).

Bacillus cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. Despite the recognition of Bacillus cereus as a food-borne pathogen over 50 years ago and the identification of several enterotoxin (any toxin produced by bacteria that causes the vomiting and diarrhea associated with food poisoning) genes, its virulence mechanisms have still not been fully elucidated. In contrast to the differences in phenotypes, Helgason et al show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group (that includes Bt!!).

This is no surprise to the academic kinds since it was known from at least 1994 (Carlson et al in Appl. Environ. Microbiology, 60, 1719; that Bacillus cereus and Bacillus thuringiensis should be regarded as one species. It has been known for some time, for instance, that enzyme systems for monounsaturated fatty acid synthesis in Bacillus thuringiensis and Bacillus anthracis prefer normal fatty acids instead of branched-chain fatty acids as substrates for binding that eventually cause the damage.

Most Bacillus thuringiensis strains were typeable like Bc thereby providing further evidence of the close-relatedness of Bacillus cereus and Bacillus thuringiensis. Recent phage (a virus that infects bacteria and may integrate into the genetic material of its host cell; bacteriophages are used as vectors in gene cloning and have other biotechnological uses) typing studies on finding the origin of food poisoning studies on Bc showed new morphotype (Phage 11) species with an isometric head and a very long contractile tail with long wavy tail fibers and was one of the largest viruses known. This need not be a disconnected point since it shows that changes are happening

There are further problems.

When one talks about Bt there are dozens and dozens of them multiplying and mutating all the time.

Bacillus thuringiensis and its strains have presently been classified into 84 serovars (a group of bacteria that share a characteristic set of antigens) making it the most diverse among the Bacillus cereus species. The taxonomic (the science of classifying plants, animals, and microorganisms into increasingly broader categories based on shared features. Traditionally, organisms were grouped by physical resemblances, but in recent times other criteria such as genetic matching have also been used) value of these serovars is currently not settled. As early as 1994, Nakamura had written “… Bacillus thuringiensis serovars canadensis and kenyae consisted of two DNA relatedness groups, each of which exhibited levels of intragroup relatedness of 80% or higher and levels of intergroup relatedness of 60 to 70%.

Bt serovars are not identical. Genetic engineering with this variation can hardly be expected to be controlled.

There are four major d-endotoxins (toxins produced within some bacteria that are released only when the bacteria disintegrate) crystal protein classes classified as Cry-I, CryII, CryIII and CryIV (what else?). CryI are for moths and butterflies (lepidopetra, red border box in Fig 3), CryIII are for beetles ((coleoptera, green border box in Fig 3), CryIV are for flies (diptera, blue border box in Fig 3, the figure with a yellow border is a high resolution picture of crystals on butterfly wings) and CryII for lepidoptera and diptera. There are subclasses of these d-endotoxins grouped as A, B, C, … and a, b, c, … (as simple as that).

The Bt brinjal end-game is to use mainly lepidopetra (moths and butterflies) crystal proteins Cry1ab and Cry1ac.

There are some obvious issues which we can briefly address and highlight.
Why Bt Brinjal?
What Bt Brinjal? With what taste?
What Environmental Effects?
Whose Liability?

Why Bt Brinjal?

Why indeed? This question comes to everybody’s mind when they first hear of Bt brinjal. Couldn’t they find something else? From a strategy point of view this could be the most important question that requires the most time to deliberate upon by denying permission several times.

There has been no evaluation of whether there is actually any ground level requirement for Bt brinjal at all given the Indian mind-set and given the known protests and resistances of several farmers’ groups.

Brinjal is one of our more traditional vegetables. However, it does not contribute in any significant way to our food requirements

It is not related to solving problems associated with a food shortage, impending or not. “Hunger in the future will probably be due to production limitations,… inappropriate solutions that are not sustainable … would not address the major causes of shortage..” (N. P. Stoner, Global Science Policy leader , Biotechnology, Dow Agrosciences, Indianapolis). Do we require an external expert to point out what seems to be obvious?

Bt crops do not show considerable increase in yield as compared to other competitive practices. One should seriously examine the statement Only about 3 to 4 percent of the 28 percent increase in corn yield over the past 13 years comes from the Bt trait. Purported benefits of genetically engineered crops are determined against a limited set of current agricultural practices that do not adequately represent the range of available technologies. … organic farmers typically produce yields comparable to …Bt corn crops produce yields comparable to best industrial methods. (D. Gurian Sherman, Union of concerned scientists, non-profit science-based organization started in 1969 at MIT)

Speaking with a purely devious mind (which happens to be the first requirement in anything that is not straight) one may imagine that Bt brinjal is a test-case as well as an opportunity to gauge the Indian mind set, especially the regulatory ones, as well as to test the pliability of scientists and bureaucrats to bend to multinational wishes.

The Bt brinjal has all the important unsolved questions that is plaguing the industry in terms of permissions. Monsanto and their Indian clones would like India to join Paraguay and Argentina as the only nation to have more than half their cultivated land in the GM-fold. India, with its lax rules, chalta hai attitudes, elastic legal system, malleable leadership, deficient in national identity is looked upon as a country of opportunity for those who have exhausted their opportunities elsewhere. This is the time, to make hay when Bt "sun” of biotechnology is rising in India? Once the humble brinjal is pushed through by not framing rules there would be other bio-technologies that would be slipped in.

As recently as 22nd sept 2010 Mahyco announced that it is undertaking partnerships with public institutions and private companies to introduce more than 200 products in the crop market, including wheat, rice, cotton, sorghum, pearl millet, mustard and several vegetables such as chilli, okra, brinjal, tomato, cauliflower, bottle gourd, bitter gourd.

It hurts to ask the obvious. Why did they invest in so much if they were not sure about the policy on GM food?

What is particularly worrying is that “important” proponents of Bt brinjal such as C. Kameshwara Rao (executive secretary of a Bangalore-based Foundation for Biotechnology Awareness and Education whose source of funding is not known to me) writes to the minster “Should we not recognize the toil of our own outstanding Agriculture Universities and a private partner who is equally committed? … Why should we ever think that they will compromise on the environmental and health safety of the nation? There is no reason for the government to delay the release of Bt brinjal. In a couple of years one would know its success or otherwise in the field and farmers would provide the answer.” The mind-boiling fallacy is that if the farmers are to find the answer in one or two years what is the “scientist” doing that they cannot wait for a year or two. More importantly, why don’t the scientists themselves request Rao not to support them?

It is the same Kameswara Rao who warns “… that even if Bt brinjal cannot be grown legally, farmers may start cultivating it anyway, as has happened with Bt cotton …

What Bt Brinjal? What taste?

We are quite happy with our brinjal. The Bt brinjal targets the shoot and borer insects. There are some of us who prefer buying brinjal with borer holes as we are satisfied that the particular brinjal does not have pesticide. They are genuinely organic. Except for the fact that we would be eating a living being if we accidentally eat the insect in some form, we usually just cut the brinjal, preferably without hurting the borer, throw away the small infected part and eat the rest of the brinjal. I personally don’t know of the known scientifically verified effects (good or bad) in such accidental brinjal insect consumption.

At the present time, the number of brinjals with borer holes are virtually absent except those of the small farmer. It is mainly our present plastic or in-organic western-oriented populace (WOPs) who don’t like the sight of creepy crawleys and would like to HIT or ZAP them with insecticides that prefer the borer-free vegetables. They like their brinjals zapped and hibred and virtually tasteless.

It is known that in earlier agricultural practice various vegetable crops are rotated so that the insect community which prefers a particular vegetable is not stabilized. Further, it seems that the various varieties of brinjal are best adapted to the particular soil. Local varieties of brinjal (with their own toxins) should actually be preferred so as to minimize the use of pesticides rather than market driven varieties which are easy meat for the insects.

A recent article on the brinjal by Bhat and Vasanthi (Asian Agri-History, 12, 169 ( 2008)) ( which I found on the net does a fairly competent job on the antiquity, cultivation and use of Brinjal in India. They stress the point that natural brinjal itself has been regarded as poisonous. The varieties of brinjal available in India seems to be due to several factors which could have been influenced by the cooking quality and the ingredients added for the preferences defined by the local cuisine. Brinjal has also been associated with poisonous substances and the selection and breeding of brinjals could have been to reduce such poisons just as the selection of various spices for seasoning in the final stages, or other additives (say, turmeric or yogurt) in the medium of cooking could have been as a result of selection by generation of breeding and feeding.

It is important to note the word of caution from M S Swaminathan when he writes that one should “… collect, catalogue and conserve the existing genetic variability in brinjal. Such a collection must be carefully preserved, before we permit the extinction of the gifts of thousands of years of natural evolution and human selection.” More importantly, he suggested that we “… undertake a careful study of the chronic effects of Bt brinjal on health. This is analogous to the studies carried out on the impact of tobacco smoking on the incidence of lung cancer …”. I could not agree with him more.

The unstated implications of Swaminathan’s comments are that he is not sure whether the Bt lobby is not hiding something the way the tobacco lobby did.

There is no doubt that brinjal as medicinal properties especially with respect to type 2 diabetes. I have benefited from it personally. The uncooked wilder varieties (paneerful for vegetable cheese rennet or sundakkai for more kozhumbu) seems to be the more efficient.

In the minister’s report, Dr. G. Sivaraman, of the National Siddha Pharmacopia Committee, of the Government of India gave actual figures from the Minister|s report on the variation in Bt brinjal that could affect the way the ayurvedic practitioner would approach plant synergy. For instance, the test analysis showed nearly 40% changes between wild an Bt brinjal in methanol extracts of solanine (increases in Bt brinjal) and solamargine (decreases in Bt brinjal). Such changes are brought about by stress in the plant, they say, and which I believe. The ratio of solasonine to solamargine which is nearly unity in the wild brinjals is nearly doubled in the Bt brinjal. These alkaloids show effectiveness in anticancer treatments as per a Korean study. They act synergetically so that a change in their ratio could have large changes in their effectiveness, say in medicine or in the ayurvedic formulations as well.

This is a perfectly valid and important point. It is strange that the latest EEC-II report recorded such changes but did not comment on it.

As far as my food is concerned, the very important testing of changes due to different cooking procedures were not carried out. Dr. Sivaraman draws attention, for instance, to the seasoning methods that are traditionally used for nullifying toxic effects during processing or cooking.

Changes in taste of the brinjal to a given individual are an indicator of changes in chemistry to which the individual is sensitive.

The Indian food research institute, CFTRI, does not seem to be in a position to perform taste panel tests of brinjal. The EEC-II has dismissed this requirement as they claim it is not based on food safety but consumer choice. This is surprising as it is through the taste that the safe varieties of brinjal have evolved through selective breeding.

I suppose one should start a brinjal taster programme just as one does with coffee tasting, and wine tasting and tea tasting. This is vital since we are dealing with crucial tests that are pertinent to the safe health of a nation that cannot be allowed to be judged by Western standards for the Indian nation.

The science of tasting has evolved considerably ever since Japanese scientist wished to automate cooking procedure by examining taste sensors. The basic premise in these studies is that “… the ability to taste was important to ensure acquisition of nutrients and to avoid toxic substances.

Quite contrary to the EEC-II opinion tasting is not just a matter of consumer choice but consumer safety.

The science of tasting involves G-proteins (guanine nucleotide binding proteins) which are involved in sending chemical signals outside and also in responding to signals that require changes inside the cell. The trans-membrane receptors involving G-proteins are the G-protein-coupled receptors (GPCRs). A good article to read is in Physiol Behav. 2006, 88, 215 on Diverse tastes: Genetics of sweet and bitter perception, by Reed Tanaka and McDaniel. They note that “… taste receptors have naturally occurring alleles that can be evaluated in a cell-based assay system (i.e., heterologous expression systems), and these alleles can also be used to predict how the receptor function will change using computer modeling, and then the DNA sequence can be compared across species to determine the origins in taste differences.” They then go on to state that “Both bitter and sweet compounds bind to G-protein coupled receptors GPCRs. However, in the case of sweet and umami (savoury) receptors the family is small, with only three known genes but in the case of bitter receptors the number is large, with perhaps as many as 30 to 50 genes.

Why do our bitter taste receptors have such a large variety of genes? It seems that subtle changes in bitterness could indicate major changes in toxicity. Breeding by tasting is the most sensible thing to do and should be compulsory for any GMO food crops. In the last few years there has been extensive progress in finding the variety of such receptor genes. Associating this variety with changes in the environment and thereby studying the suitability of GM crops should be a priority for MEF.

What Environmental Concerns?

One of the well known major concerns in biotechnology is the question of gene flow which is what it implies… flow of genes from one population to another. This point is important when one worries whether such a gene flow would affect some of the other varieties of brinjal including those that grows wildly in India. India is a center of domestication or genetic diversity for brinjal and there should be concern regarding gene flow. Suman Sahai writes to the minister on this valid point:- Farmers have been working for thousands of years to domesticate wild plants like the Solanacae family, to make them safe for eating. Much of this exercise involved breeding out the toxins…Genetic engineering …produce a toxin … This seems to be a process working to reverse several thousand year of efforts.. There are apprehensions that not only new toxins could develop but that old toxin that were removed by the technology would also re-appear.

Doug Gurian-Sherman, of the Union for concerned scientists and author of “Real Scoop” which gives regular analysis on environmental issues in general writes on the possible environmental impact of Bt brinjal in India on the environment, the following;-
Several wild relatives of brinjal are found in India and have been shown to be sexually compatible with brinjal. And it appears that at least one wild relative grows in or near brinjal fields. Further, methods to prevent gene flow from crops to wild relatives currently do not exist (there are some methods that can slow this process in some crops). Gene flow from Bt brinjal to wild relatives, would therefore be certain. Whether Bt gene becomes a permanent part of the environment in India would then depend on the properties of the gene in the wild plant – something that cannot be predicted qithout performing tests. No such tests have been performed according to the tests available to me. He further adds that because of a wild weedy relative sugar beets in California, seed production of GE sugar beets is not allowed in California, which is not a center of domestication or genetic diversity for any crop.

The issue of toxicity is usually quantified by the event of death and not by the quality of living. That should be ridiculous but it is not.
One of the problems about examining the toxicity of various bio-products is the lethal concentration (LC). How much will you require to achieve 50 % death (LC50). Mixed protein fractions from various subspecies of Bt sometimes gave greater activity than expected from the individual fractions. Effects of such synergistic interactions are difficult to reproduce because of several factors such as large variation in toxicity probably due to lack of proper control experiments; there is also the dependence on the bio-assay conditions of the host; e.g., solubility of the crystals, larval age, diet, natural variations in insect populations.

The problem of reproducibility is dismissed as an inherent and therefore intractable problem. It is not treated as variety in synergicity.

Thus Dr. J. Gowrishankar, Director Centre for DNA Finger Printing and Diagnostics, Nampally, Hyderabad, writes “It is inappropriate to quote insignificant observations out of context as many such observations seen at one time point do not persist at the next point or not observed in the other sex.”

In this context Jack Heinemann of the Centre for Integrated Research in Biosafety writes somewhat scathingly, I thought, “All scientific studies that form part of a safety evaluation must involve a comparator. The comparator must be appropriate and used consistently. The purpose of the comparator is to provide the standard baseline for all measurements, and be the single common element in all experiments using material grown in multiple locations and years. It is impossible to determine if either of these rules were followed in the dossier for Bt brinjal.

This is echoed by N. P. Stoner, Global Science Policy leader , Biotechnology, Dow Agrosciences, Indianapolis, who writes that “there is a fundamental flaw in not showing the use of an appropriate comparator.

Some of the more serious-looking protests are from ordinary non-professionals. Unfortunately it is not clear whether the anti Bt lobby may not be as manipulative as the pro Bt lobby. The pro Bt lobby includes the popular fiction writer, Jeffrey M. Smith, who is probably making his own commercial killing feeding public discomfort by propagating anti Bt stories. These stories include those of mass death of sheep by feeding on dry Bt-cotton crop by one (preliminary) report by one Ashish Gupta and others (which has been removed from the net). Another by one Carrie Swadener in the Journal of Pesticide Reform v.14, n.3 of 1994 which is cited by many from the anti Bt lobby. The report seems to have some substance. When I searched for Carrie Swadener I got directed to several porn sites including a friendly invitation from someone named Carrie “right in Pune” where I live.

There is little mention on the effect of domestic animals who graze on Bt crops. Reports such as “At least 1 820 sheep were reported dead after grazing on post-harvest Bt cotton crops; the symptoms and post-mortem findings strongly suggest they died from severe toxicity” are dismissed as being motivated or unsubstantiated. Details of such reports which has not yet been removed from the net is one,, by a Dr. Mae-Wan Ho titled The Bt trail of dead sheep, ill workers and dead villagers over three years. In this report human death in Phillipines due to exposure to Bt maize crops has been reported.

In western countries commercial concerns and public health efforts are present to increase fruit and vegetable intake by reducing the bitter tastes of the foods which are “healthy”. This is what they call consumer choice. 25% of American food production is geared towards junk food. I don’t know how many billion dollars waste is there. The Indian IT-dominated spending class must eat junk food to meet their work pressure or their pleasure wastes. Junk food removal should contribute greatly to pesticide reduction because it is the junk food lobby that require pesticides. Junk food-grains are not naturally evolved and are therefore pest-prone.

A little bitterness in healthy natural brinjals can only do them good. Diversity in bitter tastes is required to suit individual variations when taste dominates nutrition which in turn dominates health. It saves on the insecticide investment and eliminated Bt requirement.

Another point that requires being de-stressed and re-examined is the high alkalinity (high pH or high basicity) in the gut of insects where Bt crystal proteins are processed to provide the insecticidal effect. Human gut is acidic and so it is deemed safe towards such toxins.

This need not necessarily be so. It has to be remembered that the toxin binding takes place at membrane walls. Membrane walls have lipid molecule which have a long tail and a head-group which could be charged depending on the head-group (acid or base) and the basicity or acidity of the gut. In the un-damaged state the molecules are packed close together as in (a)of Scheme 1 when the inter-chain (tail-tail) attraction prevails. The head-group may be charged depending on the pH. The repulsion between like-charges (for instance) effectively increases the size of the head-group when there will be crowding, as shown in Scheme 1(b). This crowding or repulsion weakens the membrane wall as in Scheme 1(c). If the membrane wall is strong (tail-tail attractive interaction is strong) the head-group size will have to reduce by becoming uncharged irrespective of the nature (pH) of the medium. If the uncharged state is alkaline (say a basic amino group) the interface will be alkaline even when the bulk is acidic.

In bilayer membrane systems as in Scheme 1(d) and (e), the effect of charging and crowding, as in (d), may be accommodated by flip-over of some of the molecules. This, in turn will change an optimal or required ion-balance.

I don’t think this aspect has been examined in anz detail. In one my publications in Langmuir (published by the American Chemical Society) I have shown that this may be the case. For instance, titanyl oxalate is stable in acidic solution and is hydrolyzed in alkaline medium to titanium dioxide. When the surface of an acidic solution of titanyl oxalte is covered with a single molecular layer of long-chain hydrocarbon molecule with an amino head-group, titanium oxide is formed at the interface of the amine monolayer and the bulk acid.


A serious issue is the liability laws. One important point made by Suman Sahai in his letter to the minister is concerning liability. There is no liability law in the country … In case of adverse health impacts or adverse impacts on other non-Bt farms who would judge the damage and who would be liable to pay compensation … what would be the quantum of damage. Indeed, where is the government regulations in place?

One of the suggestions put forward to the minister, which I whole heartedly support, is that liability should be placed on Bt Brinjal patent owners … , Research and scientific bodies involved in the Biosafety studies of Bt brinjal calling it safe; The committees that give clearance – alleging safety. Policy makers permitting potentially hazardous products… I suppose Indian industry has already protected itself from such liabilities… It will nevertheless be important to test it in a court of law.

Germany is among the first European country to fix liability for possible damage on farmers who use GM seed. It requires the setting up of a public location register to record areas used for GMO cultivation. This law puts an onus on the farmers. In India farmers are coaxed, bluffed, bribed into buying

Dr. J. Gowrishankar, Director Centre for DNA Finger Printing and Diagnostics, Nampally, Hyderabad writes instead that EC II report was unnecessary entailing waste of time of its expert members and the burden of proof with regard to GMOs should be seen as innocent until proven guilty rather than the other way around. He also endorsed the view that it is “inappropriate to quote insignificant observations out of context as many such observations seen at one time point do not persist at the next point or not observed in the other sex.

That is what one means by saying throwing caution to the genes?.

It is imperative that there should be compulsory labelling of a Bt crop field or a Bt product. One of the signs that could be used after suitable modification is given below.

This blog does not claim to make an extensive analysis. As mentioned earlier it is meant first of all to give some additional insights to the ordinary reader on what the problem means. It is intended finally to add a little more to the kind of problems the Indian mind should concern itself with --- which it must.

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