SUNS 4506 Monday 13 September 1999

Geneva, 7 Sep (Chakravarthi Raghavan) -- The current security measures and regulations for research and production of genetically modified organisms (GMOs) and GM-products are based on some "unsound" earlier assumptions about survival and transfer abilities of micro-organismsand concepts of biological containment.

As such, the security measures and regulations need changes and updating, three biologists and micro-biologists at the Institute for Applied Ecology, Frieburg (Germany) conclude.

The authors of the study say that any biosafety protocol being considered must cover transfer of GMOs intended for contained use and these should be included in the Advanced Informed Agreement procedures.

In the just published study, "Survival, Persistence, Transfer: An Update on current knowledge of GMOs and the fate of their recombinant DNA," Doctors Beatrix Tappeser, Manuela J"ger and Claudia Eckelkamp present data on survival of GMOs in different environments, persistence of recombinant DNA, and what is
currently known of different gene transfer mechanisms involved. The data and evidence, they say, show that:

* GMOs can survive or transfer their transgenes to indigenous organisms;

* DNA is more stable than has been hitherto imagined;

* the DNA taken up with the food is not completely degraded in the gastro-intestinal tract, but has rather been found to enter white blood cells and spleen and liver cells; and

* DNA can even be transferred to the cells of foetuses, as has been shown in new-born mice - where transfer probably took place via the placenta.

The existing state of knowledge, the need for considerable further research and possible adverse effects, underscore the need for a "precautionary" approach in regulating the contained use and deliberate release of GMOs, the three scientists say in the study.

Also, the difficulties of extrapolating results of study from one region or environment to another differing environment makes it imperative that the scope of the biosafety protocol should cover the transfer of GMOs intended for contained use, and include this in the Advanced Informed Agreement procedures, they add.

The study, published by the Third World Network, is the third in its "TWN Biotechnology & Biosafety Series". The publication comes on the eve of resumption of international efforts to agree on a biosafety protocol to the UN Convention on Biological Diversity (CBD).

Informal consultations are to be held in Vienna next week (15-19 September) to prepare for the resumed session of the Extra- ordinary session of the Conference of Parties (ExCOP) for the adoption of the Protocol on Biosafety to the UN CBD.

Genetically modified products, and the right and ability of countries to regulate these products and trade, including imports, are also looming as a major trade issue -- with the United States backing its powerful corporations, and trying to use the WTO rules to force acceptance of these products, using scientifically questionable concepts like 'substantial equivalence', and asking those objecting to prove they are
unsafe.

Tappeser is a biologist and coordinator of the Department of Risk Assessment in Genetic Engineering at the Institute of Applied Ecology, Freiburg, a non-profit, citizen-funded research institution in Germany. J"ger studied biology, micro-biology, molecular genetics and virology and works at the institute focusing on genetically engineered and novel foods. Echelkamp is a biologist and scientific collaborator at the institute working on risk assessment and evaluation of genetic engineering in different areas of application, particularly risk assessment of GMOs, environmental risks of cultivation of GEd plants in agriculture and evaluation of safety of GEd foodstuffs.

In an introduction to the study, the three authors explain that during the last few years the release, whether tolerated or permitted, of GMOs and their nucleic acids into various environments has increased worldwide.

Legislation governing containment of GMOs has been deregulated and safety measures have been relaxed throughout the industrial world, as genetic engineering has not occasioned any obvious accident or visible negative impact during the two decades of its rapid development and constantly increasing use.

According to the WHO Report 1996, over the last 10-15 years, there has been an increase in frequency of outbreaks of new and re-emerging infectious diseases. Current pathogen strains are often resistant to known treatments, some even to nearly all commonly used anti-biotics.

Horizontal gene transfer is now recognized to be the main avenue of exchange of genetic material in the microbial world, and hence also of the exchange and spread of antibiotic resistance genes.

These developments, the three scientists say, give rise to two questions: does extensive use of antibiotic resistance genes in GE contribute to the increase in frequency of antibiotic resistance in bacterial pathogens? and what will be the outcome of a spread of recombinant genes analogous to the spread of antibiotic resistance genes?

Are risk assessment procedures available for monitoring the fate of GMOs and their recombinant DNA? Are we capable of detecting ecological impacts or health impacts in early warning systems?

"The answer to these questions has to be no...

"We are only beginning to understand microbial ecology. There is a lack of basic knowledge by which to judge possible impacts of a given GMO or recombinant DNA on different environments... We do not know enough of the special conditions under which gene transfer takes place. What are the selective conditions which facilitate the transfer of specific genes and may, for example, promote transfer of recombinant gene constructs?

"A pre-requisite for the possibility of GMOs and recombinant DNA contributing to the spread of newly cloned genes is the viability and/or persistence of GMOs and their recombinant DNA in certain environments. Another point requiring analysis is the extent to which artificial vectors facilitate and/or enhance the probability of horizontal gene transfer."

The authors in the study present what they say is the latest data on survival of GMOs in different environments, persistence of recombinant DNA, and what is currently known of different gene transfer mechanisms involved.

Citing various studies, they note that digestive systems of vertebrates and invertebrates alike offer possibilities for bacteria ingested with food to get in close contact with each other and with micro-flora of the animal, and there can be prolonged bacterial survival compared with other environments, and due to high density of micro-organisms increase the possibility of gene transfer.

The bacteria can be disseminated by new host animals, e.g. earthworms, with some bacteria even changing metabolic activities and capacity for survival during passage through the digestive tract. Earthworm activities may also influence the rate of transfer of conjugative plasmids from Pseudomonos fluorescens to autochthonous soil micro-organisms; it is also possible to transfer mechanisms other than conjugation - thus contributing to the dissemination of DNA from GMOs to endogenous microflora of animals.

Studies of digestive systems of rats, different strains of Lactococcus lactis colonising the rat intenstine and transfer of plasmids between these bacteria, and of the same bacteria in human intestines yielded similar results - digestive tract of germfree animals are rapidly colonisable by all kinds of bacteria, but succeeded only temporarily in non-germfree controls.

These studies conclude that even those micro-organisms, only temporarily detectable in the digestive tract, are able to transfer their conjugative plasmids to endogenous microflora, and the rate of such events depend on structure of the plasmids themselves.

Studies on presence and survival of GMOs in wastewater and sludge, suggest that survival has to do with competitors, and easonal fluctuation of predators. One study on survival and gene transfer in percolating filter beds, which are layered with living cells similar to river epilithon, showed two inoculated Pseudomonas strains survived whole investigation time of 145 days, and there was transfer of conjugative plasmid harboured by one of these strains to another. While GMOs do not seem to persist in settlement tanks, two yeasts tested in aeration basin remained detectable for more than 25 days.

Similar data have been found on aquatic ecosystems - with survival for e.g. higher for E.coli and Campylobacter jejuni increasing in filtered water, sterile tapwater etc where predators are eliminated. Bacteria taken from natural environment, transformed into a plasmid and released again as GMOs have very high chance of establishing themselves in the original ecosystems. Bacteria not adapted to environmental conditions, like E.coli ETEC, are not only able to survive within their hosts at body temperatures, but persist in a cultivable stage for weeks in aquatic environments at temperatures between 0 and 16 degrees celsius.

Soil environments are especially complex and heterogenous and it is difficult to predict chance of survival for newly introduced GMOs; they depend on various circumstances, including presence of predators.

Any analysis of survival of GMOs in soils has to include plants growing there: a recombinant strain of bacillus amyloliquefaciens, was no longer detectable in the soil of the planted microcosms 6 days after inoculation, but persisted on the grass for at least 70 days. Genetically modified strains of phytopathogenic bacteria Xanthomonas campstris, inoculated on cabbage host plants, survived for six months and were distributed to other cruciferous plants and even unrelated plants. The persistence of bacteria inoculated in plants may also depend on plant species used - two strains of Pseuomonas fluorescens remained at constant level on leaf surfaces of capsicum and eggplants during a 30-day glass-house experiment, but decreased on leaves of strawberry and tomato.

Risk assessment, the study says, cannot be satisfied by assumption or proof that a given GMO will not survive - but need to extend to the fate of the DNA, which may be stably integrated, eventually expressed and may provide advantages for indigenous micro-organisms.

Other studies show that isolated DNA does not only originate from dead and lysed cells (lyses is process of disintegration of bacterial or other cells), but can also be actively secreted.

Analysing data from studies of persistence of naked DNA in waste- water treatment plants (water/sludge), in aquatic systems and in soils, the three authors of the study say that if isolated DNA can persist in different environmental media, it is quite possible for digestive systems of animals and humans to take up these nucleic acids with the food or drinking water.

Gene transfer to date has been found by means of specially designed plasmids, but the spread of anti-biotic resistance markers throughout bacterial communities shows that gene transfer is likely to happen not only in more or less artificial settings but also under natural conditions. Some plasmids originating from 'gram-positive' bacteria have also been isolated from 'gram- negative,' stressing the possibility of a wide distribution of genetic information. But it has not been possible to ascertain whether this has been through conjugation (direct contact), transduction (gene transfer process mediated by bacteriophages, which require living cells) or transformation (which does not depend on living cells as donors of nucleic acids).

Establishment of plasmids does not depend on homology, but only on suitable origins of replication.

On the one hand special plasmids are being designed to reinforce constraints of biological containment (safety vectors without transfer genes), while on the other, progress in cloning relies on shuttle vectors capable of transgressing existing borders between bacterial classes and even kingdoms.

These various studies, and the conclusions outlined at the beginning about survival and transfer abilities of micro- organisms, suggest that there are now nucleic acid constructs containing sequences which contribute not only to effective replication in different cellular backgrounds but also to stability and integration via recombination, transfer and extraordinary expression. The essence of the current state of art is that these nucleic acid constructs are especially designed to fulfil these jobs.

Neither laboratory nor in situ studies on survival, DNA persistence and gene transfer produce a realistic measure of what can happen in complex terrestrial, aquatic and gut environment.

Crude as they may be, validated microcosm and mesocosm studies imitating natural conditions as accurately as possible are nevertheless an indispensable means of testing and prognosticating the influence of some of the biotic and abiotic factors operating on GMOs in natural environment.

What have been neglected so far, in designing such studies, are the selective forces that may act on the spread of recombinant genes -- especially in disturbed or polluted environments; contaminants like heavy metals or high salt concentrations that may facilitate gene transfer have been neglected.

The authors cite an invited speaker at a recent seminar organized by the Norwegian Biotechnology Advisory Board on Antibiotic resistance, Marker Genes and Transgenic Plants as saying:

"The extent and consequences of horizontal gene transfer are apparent in the evolution of antibiotic resistant micro- organisms. Evidence suggests that horizontal gene transfer may be equally frequent among multi-cellular eukaryotic organisms. But the actual and potential frequencies of gene transfer are poor indicators of risk: very common genes are not maintained in nature if unselected and rare genes become common extremely quickly if they are the subject of selection.

"What remains essential to assessing risk is identifying all potential selective pressures a recombinant gene might be suited to neutralise. New evidence suggests that current knowledge of evolutionary theory is inadequate to predict the fate of recombinant organisms or recombinant genes."
Tappeser,J"ger and Eckelkamp conclude from this and other studies cited and discussed by them, that "until there is sufficient evidence that a given GMO or its recombinant genes will not pose any environmental stress or health impact, we should abide by the precautionary approach in regulating the contained use and
deliberate release of GMOs."

"This implies," they add, "putting a stop to deregulation in favour of contained use and to tolerated releases from production plants where environmental impacts are not routinely assessed. We should bear in mind soil ecology has much to do with mineralisation and nutrient flow, which again is dependant on enzymatic properties of the organismal network.

"Genes currently perceived as harmless like many enzyme-coding genes (or organisms containing them) may alter soil chemistry and so create selective pressure on conditions favourable to survival of GMOs."

Also, they further stress, there is no way to extrapolate (the results of controlled studies and experiments) from one region or environment to another, differing environment. This is specially true when GMOs are transferred to ecosystems and climates which differ greatly from those in which they were first developed and
used.

While this is evident and acknowledged for deliberate release, it also holds true for contained use in production plants with multiple pathways for escape.

"Therefore the scope of a biosafety protocol should cover transfer GMOs intended for contained use and have these included in the Advanced Informed Agreement procedures."