Infection followed by disease will depend on the microorganisms ability to multiply in the host
and on the host's ability to resist or control the infection. It has proved useful to categories all
microorganisms into 4 groups which define their pathogenicity to humans; only the first group are
non-pathogens. This categorisation applies only to the infectivity towards humans, and is of
significance only, therefore, for the contained use of organisms:
|
Hazard Group 1 |
Organisms that are most unlikely to cause human disease |
|
Hazard Group 2 |
Organisms capable of causing human disease and which may be a hazard to
laboratory workers, but are unlikely to spread to the community. Laboratory
exposure rarely produces infection and effective prophylaxis or effective
treatment is usually available |
|
Hazard Group 3 |
Organisms that may cause severe human disease and present a serious
hazard to laboratory workers. They may present a risk of spread to the
community, but there is usually effective prophylaxis or treatment available |
|
Hazard Group 4 |
Organisms that cause severe human disease and are a serious hazard to
laboratory workers. They may present a high risk of spread to the
community, and there is usually no effective prophylaxis or treatment
|
The intention of this categorisation, which applies to non-modified organisms as well, is to
identify appropriate containment which would be required to protect those working with the
organisms. The higher the hazard group, the greater the containment required to control the
organism and ensure that it does not infect those working with it.
Pathogenicity is not a simple characteristic. Many genes must interact appropriately for a microbe
to cause disease. the pathogen must possess and express characteristics such as recognition
factors, adhesion ability, toxigenicity and resistance to host defence systems. Single gene
modifications of organisms with no pathogenic potential or history, or even the introduction of
multiple genes unlikely to confer pathogenicity are unlikely to result in unanticipated
pathogenicity. For example, E. coli K12 has been disabled to remove some of the factors that
might be associated with pathogenicity (wild type E. coli is a Hazard Group 2 pathogen). The factors
which have been lost include the cell-surface K antigen, part of the LPS side chain, the adherence
factor (fimbriae) that enable adherence to epithelial cells of human gut, resistance to lysis by
complement and some resistance to phagocytosis. This variant of E. coli is a common host
organism for genetic modifications within the laboratory.
The starting point for the risk assessment is, therefore, an assumption that the level of risk
associated with the modified organism is at least as great as that of the host organism (until
proved otherwise, either by direct observation, or by argument where the factors which are likely
to enhance or decrease pathogenicity are considered as in the case of K12 above). Whether in the
laboratory or in industry the capacity to choose a host means that in all but a few cases the host
organism will have been chosen to be in Hazard Group 1. It is assumed that the modified
organism will be used under the same containment as the host wild-type organism unless the
modification inserts information which would alter the pathogenicity.
The vector has also to be considered, both for its own potential for pathogenicity and for its
ability to transfer the insert to other than the intended organism -- horizontal transfer of the
information. Most vectors used for E. coli contain no sequences which might result in pathogenic
behaviour. The presence of genes coding for antibiotic resistance might be of concern. For most
of these the antibiotic resistance is already common in the environment.
Most common E. coli vectors are transfer deficient, but the ability to transfer information either
directly or with the assistance of other plasmids and the host range of the vector must be taken
into account when considering the safety of the mechanism of insertion of the required genes into
the host organism.
The properties of the insert are again of importance in considering the risk assessment for the
modified organism. Clearly if the information encodes a toxic gene product, or one which is
known to be likely to modify the pathogenicity of the organism into which it is inserted, the
greater the risk. If the gene product is non-toxic and is not one which may pose a risk to the
people working with the organism in containment, the risk management will largely be based on
the pathogenicity of the host organism.
In most instances the characteristics of the donor organisms are of less relevance to the risk
assessment than those of the host. If the donor organism is merely used as a source of well
characterised DNA for a selectable phenotype or a promoter or other control sequence, the
characteristics of the donor are unimportant to the risk assessment. If however, the insert
contains genes which are biologically active, producing toxins or virulence factors, then
information from the donor organism is of consequence. The construction of cDNA or genomic
libraries make it essential to consider all the possible hazards associated with the donor organism,
and in this instance, the hazard group may well have to be the higher of the two within which the
host and donor fall.
It is now possible to examine the modified organism and consider the likely risk. During the
1970's Dr. Sidney Brenner and others in the United Kingdom attempted to systematise the
approach by considering three factors -- Access, Damage, and Expression. The approach was
incorporated in the United Kingdom's approach to risk assessment for contained use of bacteria,
and is discussed in detail in a document produced by the Advisory Committee on Genetic
Modification in the United Kingdom. The latest version of the guidance was published in 1999
and provides clear guidance as to the risk assessment for the contained use of genetically
modified microorganisms (including any cells in culture). The guidance note is free and may be
obtained from the Health & Safety Executive in Britain. More information is available by looking
at the newsletters published by the ACGM which are available on the Internet on
http://www.shef.ac.uk/~doe.
Access is a measure of the probability that a modified micro-organism, or the DNA contained
within it, will be able to enter the human body and survive there. It is a function of both host and
vector. Depending on the organism being used, there are a number of routes of entry which allow
access. The properties of the vector, particularly mobilisation functions need to be taken into
account. In general if the organism is capable of colonising humans then access is high, whereas if
the host is disabled so as to require the addition of specific nutrients not available in humans or
outside of the culture media and is also sensitive to physical conditions or chemical agents present
in humans, then the access factor is likely to be low.
Expression and Damage are usually associated with the insert and the gene product.
Expression is a measure of the anticipated or known level of expression of the inserted DNA; if
the 'gene' inserted is intended to be expressed at a high level, for example, by deliberate in-frame
insertion down-stream of a strong promoter, expression is likely to be high. If the insert is simply
there to allow probes to detect the DNA, and is non-expressible DNA, i.e. with no foreseeable
biological effect or gene containing introns which the host is incapable of processing, then the
expression factor will be low. Examination of the final product, the modified organism itself, will
determine the actual expression, which may be higher or lower than expected.
Damage is a measure of the likelihood of harm being caused to a person by exposure to the
GEM, and is independent of either expression or access. It is associated with the known or
suspected biological activity of the DNA or of the gene product. The activity of the organism
which results in any toxic, allergenic or pathogenic effect need be taken into account within this
parameter. It may be that the biological activity of a protein is dependent on the host cell system
in which it is expressed. An oncogene expressed in a bacterium will have no discernible effect,
when present in a human cell, problems may arise. The full biological function of many gene
products require post-translational modification which will not occur within a bacterial cell
normally. The potential biological activity of the gene product should be considered in the context
of where an how it has been expressed and the effect on its structure and activity of the mode of
manufacture. The 'damage' might be from
- a toxic substance or pathogenic determinant that is likely to have a significant biological effect
- damage is high
- a biologically active substance which might have a deleterious effect if delivered to a target
tissue
- a biologically active substance which is very unlikely to have a deleterious effect or where it
could not approach the normal body level. When cloning in E. coli the 'worst case' would be if
all the E. coli in a person were replaced by the modified organism expressing a foreign
polypeptide in an active form at a high rate. If all of these are absorbed in an active form and
arrive at a site where they might have their maximum effect, what would be the damage?
- a gene sequence where any biological effect is unlikely because of known properties of the
protein or because of the high levels encountered in nature.
Once an estimate of each of these parameters has been made (in the United Kingdom this is
numerical in steps of 10-3), they may be combined. The result provides a qualitative measure of
the risk, and allows a containment level to be assigned for the use of the organism in order to
protect those working with the GEM.
Unfortunately, this Brenner scheme is only easily applicable to a small class of experimental uses
of modified micro-organisms, but the number of experiments in research laboratory environments
which fit the requirements for the application of this scheme make its retention useful.
Modified organisms may be used in containment in laboratories (or pilot plants) or may be used in
an industrial setting. It may be that the primary distinction here is not the size of plant or type of
organism, but rather the skill and training of those working in the facility.
It is likely that a research or development laboratory will be working with organisms which pose
a greater threat to either the individuals working therein or to the environment than do those
organisms developed for large scale factory use. The great majority of organisms used in
industrial production are well-characterised, 'familiar' organisms capable of being used under
conditions of 'Good Industrial Large Scale Practice' or GILSP. Given that it is usually possible to
'choose' the parental organism into which a gene is inserted for a particular 'industrial' purpose,
there would be no good reason to choose an organism likely to pose problems to either those
working in the facility, or to the environment in the event of an escape.
The same logic would apply to the development stage where 'industrial' use of the modified
organisms is being planned. There is a possible extra hazard in that it is at this stage that the
modified genes may be inserted into the organism, and the unpredictability of insertion site may,
arguably, require slightly greater care than that taken at the production facility.
In the research laboratory, organisms may be pathogenic to humans and/or to the environment, as
it is here that fundamental research would be conducted. Experiments will involve organisms and
/or inserts which may be injurious to the health of the workers or to those who are incidentally on
site in the laboratory.
How are GMO's handled?
When the technology was first used, it involved the modification of organisms within a laboratory
under very controlled conditions. The risks were perceived to be only to those working in the
laboratory, and containment conditions were devised to attempt to ensure that the organism
would not escape into the environment, or, if it should, it would have been designed so as not to
survive in the open. This resulted in the assessment of risk only being associated with human
health.
The technology has changed, and for the last ten years or more modified organisms have been
used as biological factories within industrial environments. The volume of material may be
considerably greater in the industrial or commercial environment than in the laboratory, and the
individuals working with the organism may be less knowledgeable or competent at handling the
organism. This implies that there is the possibility of accidental escape in a volume great enough
for the modified organism to survive and persist in the open environment. There is also a risk of
incidental release where waste from the industrial plant is not as carefully monitored or
controlled as it would be in the laboratory. Hence in assessing the risks associated with industrial
use of modified organism we have to take into account both the impact on human health (both for
those working in the 'plant' and for those living close to it) and the possible environmental effects
which may occur.
Modified organisms may be deliberately released into the environment. They may be crop plants
which have been modified to change their characteristics or micro-organisms which are used, for
example, for bioremediation on heavily polluted land. In both instances, the risk assessment which
might be required would have to take into account the impact on the environment, which would
include the health and safety of those humans living and working near the site of release.
Risk assessment is not an exact science. When a new characteristic is introduced into an
organism, we are not absolutely certain of the site of introduction, and therefore of unrelated
effects which may modify the organism in ways which we may not be looking for. The new gene
or its products may interact in unexpected ways within our organism, or significantly alter the
manner in which the organism interacts with its environment. As the risk when working with
organisms in containment is largely restricted to considering the effect on human health and
safety, the procedure may more readily be tabulated than when it is the environment that is
considered the primary concern.
The first steps in risk assessment are to examine the host organisms, donor organisms, vector
used for transfer of the gene, and the expected gene products.
- Where an organism has been used in containment for a very long time, and its
characteristics have been described in detail, we are familiar with the organism. E. coli
or Saccharomyces cerevisae are organisms about which a great deal is known. We
know, for example, that no pathogenic strains of bakers' or brewers' yeast have ever
been observed. These organisms are familiar. This familiarity allows some confidence
in attempting to identify risks associated with their modification.
- The first presumption we are likely to make is that the modified organism is at least as
hazardous as the host. For example, work with modified haemolytic streptococci will
proceed in the laboratory in a similar way as with other streptoccoci of this type and
known pathogenicity. However, more precautions are normally required for modified
organisms as introduced external DNA might increase the hazard usually attached to
these haemolytic streptococci. Formally such potential increase of the hazard is
expressed by classification of the manipulated strain in higher risk category. The
formulation "might increase" is important since it reflects the lack of our familiarity with
the new strain. In some cases we shall observe the opposite - the new strain will be less
invasive, the haemolysis less expressed, in short - the strain will represent lower hazard
to human health. Nevertheless, since we cannot depend for sure on this in advance we
are obliged to initially treat the new strain as more dangerous.
From this example we see that it is the absence of familiarity which brings the necessity of
precautions when handling genetically modified organisms. This is also why we are asked to
document our experiments and observations in more detail than working with common
organisms. If any unexpected effect is observed in the later stages of an experiment careful
documentation will make it possible to trace the experiment back and eventually come to the
sources of the observed effect. On the other hand our detailed documentation will contribute to
the building up of familiarity which in the future may result in amendment of the risk assessment
and assignment of a lower level of containment than that initially assigned.
What is the essence of precautions required by regulations for contained use? The use of the
GMO should be "contained". The containment could be physical, where there are real barriers to
prevent escape, or biological, where the organism is designed not to be able to survive in any
environment other than that of the laboratory. Physical containment means that we are asked to
keep the GMO within barriers which prevent its escape from the designed space. In this way the
GMO will be under control. Such barriers are usually represented by walls, fences, boxes, filters
and other mechanical constructions safely preventing the GMO from escaping
Barriers may be also of a chemical nature. Solution of phenol or hydrochloric acid will prevent
bacteria from invading the environment. Also heat is used in many systems, e.g. fermentors.
Certain principles can help to improve containment but single factors are not sufficient to fulfil the
conditions of containment. Laminar flow, negative air pressure and in many cases also so called
biological barriers fall in this category.
"Biological barriers" need careful consideration. Let us have a strain of bacteria representing a
risk to the environment which is not able to synthesise lysine and folic acid. Consequently it will
grow only in media supplemented with these two growth factors. Can we consider this deficiency
as a "biological barrier" which will prevent survival and spreading of this strain when it escapes
from containment? In practice, are we allowed to pour a culture of such strain in the drain? The
answer is no. Sewage water contains great selection of organic compounds many of them can be
considered as "growth factors" for auxotrophic bacteria. Let us have another example. Can we
open the door of a stable and let the transgenic cow go free for a pasture on the meadow?
Certainly we can. There is no possibility that the gene introduced in the genome of the cow will
escape from the animal and will contaminate the environment. (but only if it is the female!)
These examples bring us to releases of modified organisms. In general we can call "release" any
removal of the GMO from physical barriers which limited its presence within a closed space. This
could be accidental 'escape' or intentional action. When we start using GMO's we must think
about the possibility that such accident may occur, think of the likely consequences of the escape,
and if necessary, prepare steps which have to be taken.
When considering deliberate release it is clear that the risks will differ depending on the organism
released. Modifying an animal virus so that is capable of binding to human receptors is likely to
pose significant risk to the human population, and a risk assessment would normally indicate that
this should not be allowed. We have already indicated that the release of a cow into a field
constitutes (effectively) no risk. Ten years ago a company in the USA was in serious trouble
when their researchers placed pots with several seedlings of a transgenic tree on the flat roof of
the building without the permission of the authorities. To-day such an experiment would be
classified as of little risk, particularly if the plants were not allowed to produce pollen. Only a
burglar stealing the pots or a wind of an tornado strength might spread the modified genome of
these trees in the environment. It is only recently that permission for field application of
transgenic microorganisms introduced into soil has been given. At the current stage of knowledge
we are not able to predict nor to monitor the transmission of a gene in the community of native
soil microorganisms. Therefore nobody has been willing to step in the dark. Again we see how
important is our familiarity with traits and behaviour of parent organisms and extend that to
those which result from gene engineering techniques.
Plants require special handling. As soon as they form pollen their genome can be transferred to
other plants of the same or close species. Since this transfer may occur through different vectors -
wind, insects, man, water - measures ought to be taken to eliminate the possibility. Examples
include the use of "male-sterile" varieties which does not form pollen. Alternatively, flowers may
removed before the pollen is formed, or the flowers could be 'bagged' to ensure that pollen cannot
escape. This is not normally possible in large-scale field experiments. We may attempt to ensure
that sexually compatible plants are a considerable distance away from those genetically modified
so that the transfer of pollen is unlikely. In such cases the "safe zone" around the field should be
kept free of relatives which could be pollinated by the transgenic cultivar. The size of this zone
could be assessed from the largest possible radius the pollinating insects (e.g. honey bees) can
travel. In the case of wind pollination this distance could be very large, unless the pollen is viable
for a relatively short time. Many experiments have been performed to attempt to identify 'isolation
zones' for particular crop species.
In general the handling of GMOs is dominated by two precautions: to protect the health and
safety of people who have a direct interaction with the GMO (laboratory workers, factory
workers, cleaners in the laboratory) and to protect the environment which will include people,
water, earth, air.
