Evaluation of the Potential Effect of Cry1Ab Expressing Straw on ICR Mice

Cry1Ab toxin has been effectively integrated into crops such as rice and cotton for pest control, and the safety evaluation of transgenic rice has attracted widespread attention. Nevertheless, the effects of transgenic rice straw on animal model are still unclear. Hence, the present study conducted an integrated analysis to evaluate the unintended effects of transgenic rice straw expressing Cry1Ab protein on the Institute of Cancer Research (ICR) mice under 90-day treatment. The results indicated that Cry1Ab rice straw had no signicant effects on the behavior and body weight of mice. In addition, physiological indicators, including hemogram, blood biochemistry, apoptosis rate, and calcium ion concentration of the blood lymphocytes, displayed no alterations under Cry1Ab protein stress. Similarly, Cry1Ab rice straw had no adverse effects on several antioxidase activities (i.e., catalase, superoxide dismutase, peroxidase, glutathione peroxidase, and acetylcholine esterase). Moreover, we recorded that Cry1Ab stress did not adversely impact the sperm quality and follicular development of male and female ICR mice. Collectively, this integrated analysis indicates that Cry1Ab rice straw has no adverse or toxic effects on ICR mice after 90-day treatment and provides multi-level perspectives to assess the safety of genetically modied crops on non-target mammals.


Introduction
Rice (Oryza sativa L.) is the most important food crop in China, and more than half of the world's population relying on rice as the primary food intake (Li et al., 2018;Liu et al., 2020). Therefore, increasing rice yield and improving rice quality has become a crucial strategic target of sustainable development. Nonetheless, pests such as Sesamia inferens, Tryporyza incertulas, and Nilaparvata lugens have become disastrous factors restricting rice yield (Liu et (Savary et al., 2019). Hence, preventing and solving the adverse effects of rice pests on economic crops (e.g., rice, cotton and corn) has become a hot issue in the current social development.
Bacillus thuringiensis (Bt) is a gram-positive bacterium that can secrete multiple parasporal crystals (Melo et al., 2016). The parasporal crystals have speci c insecticidal activity against Coleoptera, Homoptera, Lepidoptera and other speci c insects (Bel et al., 2020;Melo et al., 2016). Hitherto, more than 100 insecticidal crystal proteins have been isolated, and the Cry1Ab/Ac protein is recognized as one of the most widely used Bt protein. With the gradual maturity of transgenic technology, transgenic Bt crops, including cotton, corn, and rice, have been primarily utilized in different areas (Li et al., 2019;Riaz Marral et al., 2020;Song et al., 2020). Nonetheless, the promotion of genetically modi ed crops (GMCs) is restricted to some extent. Some organizations believe that GMCs, as "new species", occupy the new environment and may also cause genetic environment pollution or adverse effects on human health.
Previous studies have indicated that broilers fed VIP3A transgenic maize and Bt transgenic maize have no signi cant difference in performance compared with the control (Raybould and Vlachos, 2011).
Similarly, there were no signi cant alterations in growth and development indexes of pigs fed with Bt maize (Liu et al., 2018). In addition, many scholars have carried out a large number of studies on the drug and toxicology of transgenic Bt rice on rats (Wang et al., 2014;Wang et al., 2013a). Feeding Bt protein powder caused intestinal disorder in mice, which was manifested by changes in the number of bi dobacteria (Schrøder et al., 2007). However, growing studies have shown that under long-term and chronic Bt stress, the body weight, feed intake, and organ index of mice fed with transgenic rice have little difference compared with that of the control, and various indexes of blood (blood phase, blood biochemistry, etc.) were unchanged, and organ biopsy results showed no disease and toxic effects Compared with non-transgenic rice, there were no signi cant differences in organ indexes, physiological behavior, blood indexes, and immune capacity of experimental animals under Bt stress. Nevertheless, rice is one of the dominant food crops in China, and rice straw is an important feed source for cattle (especially in southern China) to survive the winter. Therefore, it is still of great signi cance to evaluate GMCs' biological impact on organisms' derivatives. In this study, Institute of Cancer Research (ICR) mice were fed with Bt rice straw powder for 90 days, then the growth indexes, physiological indicators and antioxidants of mice were detected, and it was found that Bt rice straw powder did not have adverse effects on mice.

Ethics statement
This study was approved by Hunan Normal University ethics committee. All experiments were carried out in compliance with the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations.

Rice straw sample preparation
The rice straw of the transgenic Shanyou 63 rice expressing Cry1Ab protein and its non-transgenic parental wild type Shanyou 63 rice were obtained from the Life Science College, Hunan Science and Technology University. The rice straw was kept in a ventilated, clean, and dry place under 23 ± 2℃.

Feed
Control (non-Bt rice straw) feed: B-grade mice feed was purchased from Hunan Slack Scene of Laboratory Animal Co., LTD. The ordinary feed contains the following main nutrients: protein > 20%, crude fat > 4%, crude ber < 5%, crude ash < 8%, moisture < 10%, calcium 1.0-1.8%, phosphorus 0.6-1.2%. The control feed was prepared by uniformly mixing the non-Bt rice straw powder (ultra ne grinding) with the ordinary feed at the ratio of 1:3. Feed was kept in a ventilated, clean, and dry place under 23 ± 2℃.
Bt rice straw feed: For the feeding test group of mice, the ordinary feed was uniformly mixed with Bt rice straw powder (ultra ne grinding) and ordinary feed ingredient at the ratio of 1:3. Feed was kept in a ventilated, clean, and dry place under 23 ± 2℃.Cry1Ab content in the transgenic rice straw was quanti ed using a Bt Cry1Ab/Ac ELISA reagent box (American Enviro Logix Corporation). The nal Bt rice straw feed contained 92.21 ng of Cry1Abper gram of feed and the control feed had no detectable Cry1Ab protein.

Animals preparation
Sixty mature and pathogen-free (SPF) (ICR) mice (30 males and 30 females) were purchased from the Hunan Slack Scene of Laboratory Animal Co., LTD. All animals were housed in groups (5 females or males in each group). Each group was housed in a stainless steel wire cage (0.5 m 3 per cage) under 23 ± 2℃and 40-60% relative humidity at the Hunan SJA Laboratory Animal Co., Ltd. The test groups (15 females and 15 males) were fed with the Bt rice straw feed daily for 90 d, and those in the control group (15 females and 15 males) were fed with control feed for 90 d. Male mice are grouped separately from female mice. After the 90 d feeding trial, all mice were euthanized, and tissue samples were collected. ICR mice were weighted individually before and after dissection.
All procedures were carried out according to the approved guidelines of the Hunan Community Rules of Animal Care of Hunan Agricultural University Veterinary Services (China). All experimental protocols were approved by the Veterinary Inspection Department of Hunan Agricultural University (China). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

Blood and tissue sample preparation
Individual blood samples were collected from eyeball veins and stored in heparin anticoagulation tubes for hemogram, blood biochemistry, and lymphocyte tests. Liver, brain, spleen, kidney, marrow, heart, ovary and testis were removed respectively for viscera index and enzyme activity assays. The blood and organ samples were immediately processed after dissection. Sperm quality and ovarian follicle growth and development were tested according to the method of Smith et al. (1995).
The apoptosis rate and calcium ion level in the blood lymphocytes were analyzed using the FCM FP1000 Flow Cytometry System (Beckman Corporation). The blood biochemistry indicators were tested using the OLYMPUS AU400 automatic biochemical analyzer. After dissection, organs, including liver, brain, spleen, kidney, marrow, heart, ovary, and testis, were individually weighed. Tissues (0.2 g per sample) from each of these organs were collected and used to assay activities of various enzymes, including catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), glutathione peroxidase (GSH-Px and acetylcholine esterase (AchE). Chinese Nanjing Jiancheng Bioengineering Institute manufactured all reagents.

Data analysis
The Signi cant difference (p < 0.05) was examined by the Wilcoxon signed-rank test using the SPSS 19.0.

Effects of Cry1Ab rice straw on hematological indicators
No adverse effects were found in a clinical appearance on animal behavior, including the diet intake, drinking and daily behavior (activity, posture, gait, external appearance) of ICR mice (data not shown). A total of 18 categories of hematological indicators were measured, and all the results are presented in Table 1. All data showed that the measured values were in the normal range. No signi cant difference was detected between the test and control groups.  Table 2 presents all measurements for nine biochemical blood factors. Three of them, including ALB, ALT, and ALT/AST, were slightly higher in the treatment group than in the control group, while LDH was lower in the treatment group than in the control group. Nevertheless, none of these differences were statistically signi cant. Effect of Cry1Ab rice straw on the apoptosis rate of blood lymphocytes Although the number of apoptosis cells were 0.1% and 0.3% in the control group and test group of female respectively, while the number of apoptosis cells was 0.2% in the male control group and 0.3% in the male test group. Notably, the differences between the test and control had not attained the signi cant level statistically (Fig. 1).
Effect of Cry1Ab rice straw on the calcium ion content of blood lymphocytes As shown in Fig. 2, there were no signi cant differences in calcium ion content in the blood lymphocytes among groups during the whole study period, indicating no obvious effect on the blood lymphocytes.
Effect of Cry1Ab rice straw on the enzyme activities of serum and organs There was no signi cant difference in CAT, GSH-Px, POD, SOD, and AchE activities in the serum between the test and control mice (Fig. 3). In addition, as shown in Fig. 4, there were no signi cant differences in organ weight between the test and control groups in the 90d studies. This result indicated that Cry1Ab protein did not affect organ development in mice. The activities of CAT, GSH-Px, POD, SOD and AchE in various organs of 90 d mice groups are shown in Fig. 5. For the female and male mice, the changes of these enzyme activities between the test group and the control group had no signi cant differences.
Effect of Cry1Ab rice straw on the reproductive ability of the male and female mice As shown in Fig. 6 and Table 3, there were no signi cant differences in sperm grading, sperm count, sperm vitality, sperm motility rate, sperm abnormality rate, and sperm acrosome intact rate of male mice between the test group and control group. In addition, no signi cant differences in the number of follicles were detected between the test and control groups (Fig. 7). As shown in Fig. 8, under 40× magni cation, all levels of follicle and corpus luteum were visible in the control and test groups. The number of follicles at all developmental stages is quite abundant; under 100× magni cation, the structures of medulla and cortex and the follicular cavity were visible clearly, with clear texture; under 400× magni cation, the morphology of the follicles was clear and complete, the contour rules and basement membrane were complete, the oocyte was surrounded by multi-layer granulosa cells around. No pathological ndings during necropsy and no group-related histopathologic observations were found (Fig. 8).

Discussion
The basic safety assessment of genetically modi ed (GM) crops is based on the principle of "substantial equivalence" concept, but the "substantial equivalence" principle is a starting point rather than an endpoint (König et al., 2004;Muccilli et al., 2020). Giving the randomly inserted trait and the potential synergism with extrinsic factors of action mode of Bt protein, the expression level and recombination mode of Bt protein in different parts of the plant is not the same . In any safety assessment of GM crops case, we all need novel and comprehensive methods, concepts and ideas to probe into the intended and/or unintended effects of GM technology and crops on target and/or non-target organisms, for the GM technology and crops need to be based on a scienti c foundation. Thus, it has also become crucial to apply the case-by-case approach to assess the toxicity of GM plants, especially Bt rice, which plays an signi cant role in human health. Considering that the straw is the winter livestock feed for cattle in southern China, the unintended effects of Bt rice straw on mammalian especially on its reproductive system should also be valued despite much of the previous literature involving the safety assessment of Bt rice on mice,.
When compared with the control mice, the dietary intake, drinking and daily behavior of ICR mice in the Cry1Ab rice straw fed group were normal and no toxicity or mortality was recorded during the 90-d experimental period. In a similar study, no adverse effects of Cry1Ab protein (Bt rice) on animal behavior or weight increasing on Wistar rats were found (Schrøder et al., 2007). The physiological changes of the blood can directly re ect the health condition of animals. Apoptosis is an important indicator of animal toxicology and pathology (Zeng et al., 2015). Calcium ion has an extremely close relationship with apoptosis and participates in signal transduction regulation of apoptosis pathway (Clapham, 2007). This study demonstrated that no advert effects of Cry1Ab protein exist on the hemogram (Table 1), blood biochemistry (Table 2), apoptosis rate and calcium ion concentration of the blood lymphocytes ( Fig. 1-2), and CAT, GSH-Px, POD, SOD, AchE activities of serum (Fig. 3). The results showed that the Bt rice straw had no signi cant in uence on the blood biochemistry of mice. These results are consistent with the previous research in a 90-d study of Sprague Dawley rats fed with transgenic T1C-1 rice expressing Cry1C protein (Tang et al., 2012).
Organ index indicates the ratio of the organ weight to animal weight, which can directly re ect the status of animal disease or organ suffered from toxic effect. In the 90 d Bt rice feeding study, we found that the organ index of ICR mice has no signi cant differences between the test and control groups (Fig. 4). These results are consistent with previous studies, which showed Cry1Ab does not affect weight gain of Sprague-Dawley rat and Wistar rat (Schrøder et al., 2007;Wang et al., 2002). The antioxidants, including CAT, GSH-Px, POD and SOD that present widely in aerobic organisms can eliminate the free radicals (Jones, 2008;Sharma et al., 2018). In addition, AchE is closely related to neuronal development and regeneration (Fossati et al., 2015). In our study, we detected some enzyme activities related with reproduction in ICR mice organs in the test group, which showed that CAT, GSH-Px, POD, SOD, and AchE enzyme activities had no signi cant differences compared with those in the control group (Fig. 5). This phenomenon suggested that the growth and development of ICR mice organs didn't be in uenced by taking in Bt rice straw powder for a long period (90 d). And Wang et al. (2013b) showed that there were no signi cant differences in rat organ weight and enzyme activities between the Cry1Ab protein in test group and the control group in a 90 d study.
The sperm density is the most basic parameter in the reproductive safety assessment, and sperm motility is the main indicator of sperm quality. Sperm abnormalities have long been associated with male infertility and sterility, and sperm density and quality play a substantial role in fertilization and pregnancy outcomes (Chenoweth, 2005). As shown in Fig. 6 and Table 3, the sperm density was in the normal range and was not signi cantly different between the test and control groups. This nding also showed that Cry1Ab protein exerted no detectable adverse effects on mice sperm vitality, sperm motility rate, sperm abnormalities and sperm acrosome intact rate, which are concordant with several previous studies (Ning et al., 2000;Wang et al., 2002). The safety assessment of Cry1C protein from genetically modi ed rice also showed that the Cry1C protein had no signi cant effect on the incidence of sperm abnormality in mice (Cao et al., 2010). In addition, it was reported that there was no negative effect of Bt rice on testis of Sprague-Dawley rats (Wang et al., 2004). Another research also pointed out that Bt corn had neither longterm effects nor multi-generational dominant toxic effects on the development of germ cells and testes in the mice reproductive system, yet the Cry 1Ab have no association with the testes development of rats (Brake et al., 2004;Betz et al., 2000). The differences in ovarian organ index and follicle number at different levels were insigni cant (Fig. 7). The granule cells were complete, and the corpus was visible in the test and control groups of female mice. The ovarian slices texture and structure were clear, and the morphology of follicles was no signi cant differences between the test and control groups (Fig. 8). The results showed that no adverse effects of Bt rice straw on the ovary and follicle of mice. The results were consistent with no signi cant difference in CAT, GSH-Px, POD, SOD and AchE in testes and ovary and corroborated each other. Thus, all results showed no adverse effects of Bt rice straw on the reproductive safety of female and male mice in the 90-d study. Our ndings are supported by a study on three generations of mice fed with transgenic rice, which reported no potential adverse effect on the male reproductive axis, copulation index, fertility index, gestation length, live-birth rate and litter number (Zhou et al., 2012).
Cry proteins have high species-speci c toxicity against certain insects. The mode of action in the target insect is through speci c receptors in the gut, which is highly alkaline, with binding of the toxin resulting in pore-formation, osmotic imbalance, cell lysis and subsequent death of the insect (Palma et al., 2014). Due to species-speci city, the Cry proteins are regarded harmless or non-toxic to mammals, including humans. Hitherto, numerous data from toxicity studies have showed that no signi cant adverse effects of Cry proteins on body weight and clinical observations. The Bt insecticidal toxin-encoding gene has a long history of safe use in biotechnology (Mendelsohn et al., 2003). Animal feeding trials is an important assessment method for potential adverse effects of GM crops, and a 90 d rodent feeding study with GM food is suitable and valid (Poulsen et al., 2007). Nevertheless, animal's complex physiological structure and function sometimes could bring unforeseen di culties in the experiment process and results (Zhou et al., 2012). We need to evaluate possible potential effects from transgenic crops through a large number of comprehensive analysis assessments.

Conclusion
Rice is the main food crop of humanity, the further complex and long-term safety assessment study of Bt rice is required and necessary. Currently, there are few reports on the safety assessment of Bt rice straw. This study is focused on assessing potential unintended effects of Bt rice straw rather than determining qualitative and quantitative intrinsic toxicity of Bt rice constituents. Summing up the obtained results, no adverse effect was observed in ICR mice feeding Bt rice straw feed compared with the conventional nontransgenic rice straw feed in a 90 d study.

Declarations
Statements and Declarations

Data Availability
The data used to support the ndings of this study are available from the corresponding author upon request.
coordinate means the percent of necrosis cells and the late apoptosis cells. The B2 coordinate means the percent of dead cells of mechanical damage. The B3 coordinate means the percent of survived cells. The B4 coordinate means the percent of the early apoptosis cells. The total apoptosis rate was the sum of B2 and B4 Figure 2 Effects of transgenic Bt rice straw on calcium currents of lymphocytes of mice blood. D1: the female control group. D2: the male control group. D3: the female test group. D4: the male test group Page 18/18 The grading of sperm in the treated and control mice Pathological sections of follicles between the test and control mice Note: A, C, and E are sliced sections of control groups in 40×,100× and 400× magni cation respectively; B, D, and F are sliced sections of test groups of 40×, 100× and 400× magni cation, respectively.