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 ProfessorEnvironmental and Occupational HealthGraduate School of Public HealthUniversity of Pittsburgh

Essay Reader:Hasan Guclu, PhD ______________________________________Assistant ProfessorHealth Policy and ManagementGraduate School of Public HealthUniversity of Pittsburgh

ii

Copyright © by Qingqing He2015

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

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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

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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 FOOD SAFETY ASSESSMENT..........................................................6

2.1.1 Toxicity study...............................................................................7

2.1.2 Allergenicity test..........................................................................7

2.2 ECOLOGICAL SAFETY EVALUATION..............................................8

2.2.1 Parasitoids....................................................................................9

2.2.2 Predators....................................................................................10

2.2.3 Other non-target organisms......................................................11

2.3 POTENTIAL ADVERSE IMPACT OF BT RICE.................................12

3.0 DISCUSSION.............................................................................................14

BIBLIOGRAPHY......................................................................................................18

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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).

3

Since then, various Bt rice lines expressing insecticidal genes including Cry1Aa,

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

4

midgut cell receptors and inserts into cell membranes to form pores or ion channels.

These 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

5

of food poisoning associate with genetically modified rice (35).

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).

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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

7

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 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

8

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).

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

9

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,

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

10

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.

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

11

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.

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

12

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

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.

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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).

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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 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

15

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

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

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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 control rice insects if the remaining concerns can be addressed to qualify it for

commercial planting.

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