SAFETY ASSESSMENT OF THE BACILLUS THURINGIENSIS
TRANSGENIC RICE EXPRESSING CRY1C PROTEIN IN CHINA
by
Qingqing He
BMed, Central South University, XiangYa School of Medicine, China, 2012
Submitted to the Graduate Faculty of
Environmental and Occupational Health
Graduate School of Public Health in partial fulfillment
of the requirements for the degree of
Master of Public Health
University of Pittsburgh
2015
UNIVERSITY OF PITTSBURGH
GRADUATE SCHOOL OF PUBLIC HEALTH
This essay is submitted
by
Qingqing He
on
April 6, 2015
And approved by
Essay Advisor:
James Peterson, PhD
______________________________________
Associate Professor
Environmental and Occupational Health
Graduate School of Public Health
University of Pittsburgh
Essay Reader:
Hasan Guclu, PhD
______________________________________
Assistant Professor
Health Policy and Management
Graduate School of Public Health
University of Pittsburgh
ii
Copyright © by Qingqing He
2015
iii
James Peterson, PhD
SAFETY ASSESSMENT OF THE BACILLUS THURINGIENSIS
TRANSGENIC RICE EXPRESSING CRY1C PROTEIN IN CHINA
Qingqing He, MPH
University of Pittsburgh, 2015
Abstract
Bacillus thuringiensis (Bt) is a bacterium that has been used as a biological pest
control for a long time. The Cry1C protein expressed by Bt genes are toxic to specific
species of rice insects. Although Bt transgenic crops have been widely planted, the
potential health risk to human beings still remains a concern. The rice line expressing
Cry1C protein is currently undergoing risk assessment. Based on the literature
reviewed, it cannot be concluded the Cry1C protein in the Bt transgenic rice is safe. In
addition to the environmental issues, it is important to consider the public health
relevance, such as food allergies, toxicology and genetic mutation. Further research
is needed before this rice line gets biosafety certification.
Keywords: Bt transgenic rice; Cry1C protein; food safety; ecological impact
iv
TABLE OF CONTENTS
1.0
INTRODUCTION ......................................................................................... 1
1.1
HISTORY OF GENETICALLY MODIFIED RICE IN CHINA ................ 3
1.2
ADVANTAGES OF BT TRANSGENIC RICE ...................................... 4
2.0
ANALYSIS .................................................................................................. 6
2.1
2.2
2.3
3.0
FOOD SAFETY ASSESSMENT.......................................................... 6
2.1.1
Toxicity study............................................................................... 7
2.1.2
Allergenicity test .......................................................................... 7
ECOLOGICAL SAFETY EVALUATION ............................................. 8
2.2.1
Parasitoids ................................................................................... 9
2.2.2
Predators .................................................................................... 10
2.2.3
Other non-target organisms ..................................................... 11
POTENTIAL ADVERSE IMPACT OF BT RICE ................................ 12
DISCUSSION ............................................................................................ 14
BIBLIOGRAPHY ...................................................................................................... 18
v
1.0
INTRODUCTION
Rice is the major food crop in China. The history of rice cultivation in China dates
back over 7000 years (1). China is one of the most important rice producing and
exporting countries in the world, 20% of agricultural land in China is used to plant rice,
and 18% is for corn in 2008 (2). In order to feed its 1.3 billion people, China has tried
several ways to control insect pests of rice and increase yields. More than 200
species of rice insects are found across the six rice-growing areas in China (3).
Among them, lepidoptera is the largest order of rice insects. Rice stem borers, one of
the important subgroups of lepidopteran insects, cause losses of 1.69 billion US
dollars every year (1).
Genetically modified technology has been used to control crop pests all over the
world for many years. This strategy has been considered as effective and
environmentally friendly. Bacillus thruingiensis (Bt) is a bacterium used in biological
pest control. Genes from Bt are introduced into crops to express the crystal proteins
(4). Crystal proteins are a type of toxins highly toxic to the specific species of insects.
Six different genes are found among the 20 Cry1 sequences, Cry1Aa, Cry1Ab,
Cry1Ac, Cry1B, Cry1C and Cry1D (37). The Cry1Aa, Cry1Ab and Cry1Ac genes
show more than 80% amino acid identity, while the other three genes are quite
1
different from each other (37). The Cry1C protein, encoded by the cry1c gene, shows
resistance to a variety of lepidopteran insects (15). Cry1C protein binds with different
site in the midgut of pests than Cry1Aa, Cry1Ab, and Cry1Ac proteins (16). So Cry1C
can be considered as an alternative choice to Cry1A toxins. It also can be combined
with Cry1A gene and hence increase the toxicity to rice insects (7, 15). This group of
proteins is effective against several orders of insects, including Lepidoptera,
Colepotera and Diptera (5) and their biotechnology developed very quickly. In 2009,
about 20.7 million acres area were planted with Bt transgenic crops all over the world
(6). However, only a few Bt genes are used to control lepidopteran insects. Cry1Ab,
Cry1Ac and Cry1Ac/Cry1Ab are the most common Bt genes encoded into genetically
modified plants (7). Cry1C protein is not a common toxin so that researchers are now
assessing its safety. There are studies shows the plants encoded Cry1C genes can
against pests which resistant to Cry1A proteins (39, 40) and the combination of Cry1A
and Cry1C genes results in the ability to kill more of the targeted pests (41). On
October 2009, the Chinese Ministry of Agriculture issued two Bt rice lines, both
expressing Cry1Ab/Ac protein, with biosafety certificates for commercial production
(1). Meanwhile, other transgenic rice lines are under investigation.
This review will consider the history of genetically modified rice development in
China. As a new member of Bacillus thurigiensis toxin family, Cry1C protein must be
tested and proven to be safe not only to animals but also the environment before it is
widely used in genetically modified rice. Public health concerns and the ecological
2
impact of planting Bt transgenic rice expressing Cry1C protein will be reviewed by
examining the relevant published literatures.
1.1
THE HISTORY OF GENETICALLY MODIFIED RICE IN CHINA
Rice insects have caused serious yield loss in past years. According to a report by
Sheng et al. (17), there was a 3.1% yield loss caused by rice stem borers in China in
2003, with an estimated cost of 965 million dollars. In addition to this, China also
spent approximately 735 million dollars on insecticides and other insect control
measures. Many control technologies for rice insects have been developed during the
long history of rice growing in China (1). Among these strategies, famers mostly rely
on the chemical insecticides mostly. However, this chemical control strategy has
several disadvantages, such as being harmful to the environment, a possible source
of food poisoning etc. By 2005, the usage of insecticides for crops in China had been
doubled since 1990 (18). So, a safer and more effective strategy needs to be
developed.
With the successful development of Bt transgenic crops all over the world,
Chinese government and agricultural researchers began to study the Bt transgenic
rice to control rice insects. In 1989, scientists from the Chinese Academy of
Agricultural Sciences (CAAS) generated the earliest Bt transgenic rice in China (19).
Since then, various Bt rice lines expressing insecticidal genes including Cry1Aa,
3
Cry1Ab, Cry1C and Cry2A have been developed and are being tested based on the
biosafety regulations and policies for genetically modified rice in China. In 1993, the
first biosafety regulation for genetically modified organisms was created and issued
by the Chinese Ministry of Science and Technology (1). The latest regulation was
issued in 2001 by the State Council (1).
1.2
ADVANTAGES OF BT TRANSGENIC RICE
Stemborers and leaffolders are the two most damaging kinds of rice insect worldwide
(7). They belong to the lepidopteran order and cause severe yield loss in rice. For a
very long time, farmers mostly relied on insecticides to control the pests. However,
the widespread use of insecticides resulted in an increased range of pests resistant to
the insecticides. Furthermore, the larger amount of pesticides applied to the
rice-growing areas have increased the cost of rice production and raised
environmental concerns.
The insecticidal mechanism of crystal proteins is not completely understood, but
the basic steps of toxicity have been studied. It is thought that after the insects absorb
the toxins, they enter the digestive tracts and become soluble. Then the proteases in
the midgut of insects cleave the toxins in several stages to produce the active
protease-resistant toxin core protein. After that, the protein both binds to midgut cell
receptors and inserts into cell membranes to form pores or ion channels. These
4
processes involve both reversible and irreversible binding steps. Irreversible binding
is thought to be associated with the insertion of the toxin into the cell membrane after
which it is resistant to proteases. The combined action between toxins and
membranes eventually kills the cells in the midgut, so that the insects cannot absorb
food and die (37). Cry1C protein combines different sites from Cry1Ab, Cry1Ac
proteins. So if two different crystal protein genes are encoded together into a
genetically modified crop, it should become more toxic to the target insects.
Tobacco is the first plant that was transgenically modified with Bt genes in 1987
(31). Since then, Bt genes have been studied and developed very quickly worldwide
(7). By 2004, the global area under commercial cultivation with Bt transgenic plants
has reached 22.5 million hectares (7, 32). The development of genetically modified
crops began in the late 1980s in China.
In 1999, a field trial was conducted in Hubei Province (33). There were two fields,
Bt transgenic rice and non-Bt rice. The results showed that without insecticides, Bt
rice gave 29% more yield than the control. This field study indicated the benefit of the
rice encoded with the Bt genes. More reports or surveys revealed that when planting
Bt transgenic rice, less money was spent on insecticides. Farmers growing Bt
transgenic rice only spend 4.56 dollars per hectare per season on insecticides,
compared to 35.74 dollars spent on insecticides by non-Bt rice producers (1, 34).
Moreover, genetically modified rice is environmentally friendly and there is no report
of food poisoning associate with genetically modified rice (35).
5
2.0
ANALYSIS
It is important to make both a safety and an environmental assessment of the
genetically modified food before release them to market. Thousands of tests have
been done and not all of the results showed negative effects.
2.1
FOOD SAFETY ASSESSMENT
Individual types of crystal proteins show specific toxicity to certain pests. The toxin will
combine with specific receptors in the gut and cause subsequent death of cell of
insects (8). To date, there is no report of pathologic damage in mammals caused by
crystal proteins. The reason has been considered to be the lack of crystal protein
binding sites in mammals (9, 10). However, the advantage or potential damage of
transgenic rice expressing Cry1C protein in human is still uncertain (11). Several
laboratory studies have been conducted to assess the food safety in small animals,
like mice and the results were negative (11-15).
6
2.1.1
Toxicity study
In the past 40 years, many toxicity studies on pesticide crystal proteins showed no
significant adverse effects (11). The rodents fed with genetically modified (GM) rice
expressing different types of crystal proteins did not show significant differences in
body weight, blood samples analysis, or organ weight compared with the ones fed
with non-GM rice (11-14).
Tang et al. (11) reported that T1C-1 genetically modified rice expressing Cry1C
protein had no adverse effects on Sprague Dawley rats’ behavior or weight gain after
a 90-day study. The results of hematology analysis and fecal samples between the
T1C-1 group and controls showed no significant difference. The results are similar to
those of another study by Cao et al. (12) who conducted an acute toxicity experiment
of Cry1C protein in ICR (Institute of Cancer Research) mice. After giving 5 g/kg body
weight of Cry1C protein, no mortality or visible signs of toxic effects were observed in
these mice for 14 days.
2.1.2
Allergenicity test
As a new and novel food, potential allergenicity associated with the Cry1C protein is
an important consideration. Food allergies can affect people’s health and even can
cause death. Cao et al (12) had conducted an amino acid sequence homology search
and did not find any similarity between the Cry1C protein and any other known toxic
7
or allergenic proteins. This was a good start to investigating the possibility of an
allergic reaction. The allergenicity evaluation of Cry1C protein was also carried out on
animals. A laboratory study in Brown Norway rats was undertaken to assess the
potential allergenicity of Cry1C toxin (15). The results showed Cry1C protein did not
result in production of antigen-specific immunoglobulin (IgG2a) or increased
histamine levels compared to peanut agglutinin, potato acid phosphatase and
ovalbumin. Based on the results of these two studies, the conclusion can be drawn
that the Cry1C protein has very low probability of having any significant allergenicity.
2.2
ECOLOGICAL SAFETY EVALUATION
According to the field survey, there are about 200 species of rice insects in China.
However, in just one rice-planting region in South China, there are more than 228
species of rice insect predator (1, 20). In order to control the rice insects, farmers use
large amounts of insecticides and subsequently, cause resistance to emerge in pests.
Compared to this chemical control strategy, transgenic insect-resistant rice is
generally thought to be much more friendly toward the environment. However, the
impact on the ecology is still an open question. Most of the current limited studies
were focused on the adverse effects of the parasitoids and predators (21).
8
2.2.1
Parasitoids
Parasitoids are a kind of organism that attach to the host and complete their
development there. Ultimately, they will kill or eat the host. If the host feeds on the Bt
transgenic plants, then the parasitoids will also be exposed to the Cry toxins, so it is
important to assess the potential impact on them. Parasitoids are natural enemies of
insects and play an indispensable role in the ecological chain. They have been used
as a biological control strategy for years. Some studies have found adverse effects on
the parasitoids when their hosts consumed Bt transgenic plant tissues (22-24)
including longer development time, lower population, and lower weight. As the results
were considered to be indirect (host mediated), the direct impact still needs to be
assessed.
Chen et al. (25) conducted a study to assess both the direct and indirect effect of
the Cry1C protein on Diademga insulare, an important endoparasitoid of Plutella
xylostella. They found that the Cry1c protein had no deleterious impacts on Diademga
insulare compared to common insecticides. A similar result was observed in another
study in which Schuler et al. (26) tested the impact of Cry1Ac protein on Cotesia
plutellae, another endoparasitoid of Plutella xylostella, and found that there was no
difference in production quantity or behavior between the Bt-plant diet group and
controls. Even though the final outcomes showed no significant negative impact,
there were adverse effects detected during the development period. Consequently,
9
the impact of Cry protein on parasitoids still remains a concern requiring further
studies.
2.2.2
Predators
Predators are a major group of non-target organism exposured to transgenic
insecticide-resistance plants. Before commercialization, it is important to clarify the
possible impact of Bt transgenic rice on non-target organisms.
A 2-year field study conducted by Xu et al. (27) revealed a negative effect on the
arthropod predators of Cnaphalocrocis medinalis of three transgenic rice lines
(expressing Cry1Ab/Cry1Ac, Cry1C and Cry2A). Cnaphalocrocis medinalis is one of
the lepidopteran pests which can heavily damage the rice leaves and reduce the
production level. Their results show that the density of Cnaphalocrocis medinalis was
much lower in the transgenic rice fields than in the non-transgenic filed, however, the
population of the arthropod predators was not significantly affected differently
compared to the filed planting non-transgenic rice. Another non-target predator,
Propylea japonica, has been assessed regarding the potential effect of crystal protein
too. Propylea japonica is not only a predator of young larvae of Lepidoptera, but also
a pollen feeder (28). Thus, they are exposed to Cry protein in both direct and indirect
ways in the field of transgenic rice. Li et al. (28) found that Propylea japonica was not
sensitive to Cry1C protein when exposed to 10-times higher concentration than that
detected in pollen from Bt transgenic rice.
10
To date, no adverse effect has been identified in predators exposed to Cry1C
protein. However, only limited predators were subjected to testing and, so more
representative species of predators need to be evaluated.
2.2.3
Other non-target organisms
Leafhoppers have been identified as a significant group of non-target herbivores in
China (1). Lu et al. (29) conducted a study under laboratory and field conditions to
assess the potential impact of Cry1C on Nephotettix cincticeps. The laboratory results
indicated that no significant difference in biological parameters was observed
between Bt and non-Bt rice diet group. However, in the field study, a shorter
preoviposition period (a period between the emergence of a female insect and the
beginning of laying egg) was found in the Bt rice diet group, expressing Cry1C protein,
compared to the non-Bt rice diet group, although the seasonal density and population
dynamics were similar between the Bt and non-Bt rice fields.
The green lacewing is helpful to human beings in that it can eat mites and aphids.
It may be exposed to the Bt plants also. The potential impact of Cry1C protein on
Chrysoperia sinica, one kind of green lacewing, has been evaluated in laboratory
bioassays (30). The results demonstrated that neither larvae nor adults of
Chrysoperia sinica were sensitive to Cry1C toxin. The authors concluded that the
growing Bt rice expressing Cry1C protein would not cause a risk to Chrysoperia
sinica.
11
2.3
POTENTIAL ADVERSE IMPACT OF BT RICE
Although some of the Cry1C toxin safety studies found negative impacts, more
laboratory and field research need to be carried out. To date, there have been limited
studies with transgenic rice expressing Cry1C protein and many more studies are
required to assess its combined effects with other crystal proteins such as Cry1Ab
and Cry1Ac. So the safety of Bt rice expressing Cry1C protein remains a concern.
Moreover, if Bt transgenic crops were to be planted widely, the possible ecology
impact might become a significant issue. The Bt genes expressing crystal toxins can
spread to wild plant species and as a result, pest resistance to cry proteins will
increase (36). The fact that the biochemical insecticides containing Bt crystal toxins
have the issue of pest resistance may become a concern for the Bt transgenic plants.
There is a report about resistance to Cry1C protein expressed by Bt broccoli. Zhao et
al. (42) found a strain of diamorback moth which is resistant to Cry1C toxin that can
even complete their entire life cycle on the transgenic broccoli expressing high level
of Cry1C protein rather than being killed at neonate instars. Even though the
mechanism of crystal protein is basically understood, the model of pest resistance to
these toxins is still unclear (43).
Another concern is the potential health impact by consuming Bt transgenic rice.
To date, all the evaluation of the Bt rice is based on animals. There is no long time
critical trial to assess the potential effect on human beings. Rice is the staple food in
China, especially in the south, where people eat rice as part of every meal. The
12
amount of rice people consume is much more than other Bt transgenic crops, such as
corn, broccoli etc. So the crystal proteins people obtain from rice represents the
largest portion. There are some remaining issues. First, unpredicted mutations may
happen in the transgenic genes and result in production of new toxins or human
allergens. Second, the nutritional value of the genetically modified rice may be
reduced compared to the traditional rice. Third, the cry proteins produced by Bt genes
may not be digested completely in human beings. Also there is the potential that the
Bt gene could be transmitted to fetus if a pregnant woman were to consume Bt
transgenic rice frequently. It is not known if the toxin could be harmful to the
development of the fetus. Finally, the durations of all the reported tests of the crystal
proteins are quite short, no longer than 90 days. The potential negative impact of Bt
transgenic rice on human beings may only be detected after several generations,
posing a potential health risk to offspring several decades in the future.
13
3.0
DISCUSSION
A tremendous number of studies regarding food safety and environmental impact of
Bt transgenic rice have been conducted in China. Compared to the amount of
research focused on Cry1Ab or Cry1Ac proteins, limited work has been done on
Cry1C protein. The two rice lines that have been biosafety certified are expressing
Cry1Ab/Cry1Ac. If the rice line expressing Cry1C protein is to be certified, more risk
assessment is needed. There is no doubt that this technology brings obvious benefit
to farmers. The yield of rice can be increased and the cost of planting reduced.
However, there are still many concerns remaining. Rice is the predominant food in
China of a 1.3 billion population. In order to meet the food demand for this huge
population, further studies need to be carried out to get a better understanding of
crystal proteins and to determine any potential negative effects on both animals and
ecology (1).
There are six main rice-growing areas in China. However, due to rice insects, the
production levels are reduced and in the future it may not meet the food supply
requirement. Chemical pesticides represent the main strategy to control pest damage,
but this causes several problems: pest resistance to pesticides, environmental
contamination, food poisoning, increased cost of planting etc. Thus, a genetic
engineering strategy has been sought. Bacillus thuringiensis is a gram-positive
bacterium that has been used as a biological pesticide. Further research has shown
14
more effective killing of pests when the relevant Bt genes are encoded into the crops.
Bt genes and the crystal proteins they produce have been widely studied and applied
to agriculture. The rice line expressing Cry1C toxin is relatively new compared to the
one expressing Cry1Ab and Cry1Ac toxins. Although several laboratory and field
studies have been conducted in relation to Cry1C toxin, and most of the results
showed negative impact, Cry1C protein cannot yet be certified as safe for the
following reasons.
First of all, the toxicity research is limited and only based on rats. The safety
assessment of Cry1C conducted by Cao et al. (12) is a good research in which the
researchers have considered several aspects of toxicity. The results indicated that
Cry1C protein is neither a potential toxin nor allergen. However, the Cry1C protein in
this study was obtained from Escherichia coli not from Bt rice. So, it cannot be
concluded that the Cry1C protein in the Bt rice is safe. Furthermore, while drug safety
or effect assessment can initially be tested on rats, before final approval and release
to market, critical primate testing and human clinical trials need to be carried out to
better predict the possible impact on human beings. Genetically modified rice is not a
drug, but people will consume it everyday in large quantities. The possible
accumulation of crystal protein in the body has not been studied yet. Therefore,
long-term exposure to Cry1C protein remains a concern.
Second, environmental impact is not only based on the effect of non-target
organisms, other aspects should be considered. Emergence of Insect resistance to
crystal protein is the main issue. If Bt rice is widely planted, some pests will become
15
resistant to the crystal protein. After several years or decades, the amount of pests
resistant to crystal proteins will have increased and, thus, the benefit of Bt rice will be
reduced. Another potential result of resistance to crystal proteins is the occurrence of
super weeds. Since congeneric weeds get damaged by the pest that has gained
resistance to either Bt biological pesticides, or Bt genes, the growth of super weeds
will be promoted. Subsequently, the super weeds can spread and break the
ecological balance. Similar results can be caused by gene flow. The Bt genes in the
pollen of rice can be transferred to nearby plants. This will disturb biological diversity.
Lastly, any organism living in the soil or water should be considered a non-target
species that is subject to exposure to Cry1C protein. While, the degradation rate of
crystal protein in the soil has been found to be rapid (36), it is still worthwhile to
evaluate this aspect of the potential environmental impact of Cry1C protein.
In conclusion, based on the current information, the genetically modified rice
expressing Cry1C protein is considered to be of low or negative risk to humans and
the environment, but further laboratory and field studies are urgently needed and
ongoing to achieve final safety certification. There are some other considerations
need to take into account. Since the combination of two or more types of crystal
protein should be more effectively toxic to insects, toxicity assessment of Cry1C
combined with other crystal proteins needs to be conducted. Additionally, social
impacts need to be assessed. China is a rice exporting country. The release of
commercial genetically modified rice may affect the trading relationship between
China and other countries (1). Genetically modified rice is seemingly a good choice to
16
control rice insects if the remaining concerns can be addressed to qualify it for
commercial planting.
17
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