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Food and Chemical Toxicology 100 (2017) 34e41

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Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate/ foodchemtox

Safety assessment of genetically modified milk containing humanbeta-defensin-3 on rats by a 90-day feeding study

Xin Chen a, 1, Ming-Qing Gao a, b, 1, Dong Liang a, Songna Yin a, c, Kezhen Yao a,Yong Zhang a, b, *

a College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, Chinab Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, Chinac Medical College, Yan'an University, Yan'an 716000, Shaanxi, China

a r t i c l e i n f o

Article history:Received 7 September 2016Received in revised form8 December 2016Accepted 10 December 2016Available online 12 December 2016

Keywords:Genetically modified milkHBD3Safety assessment90-Day feeding study

Abbreviations:AIN93G dietGrowth purified diet for rodentsrecommended by the American Institute ofNutritionALBalbuminALPalkaline phosphataseALTalanine aminotransferaseASTaspartate aminotransferaseBUNurea nitrogenCACCodex Alimentarius CommissionFAOFood and Agriculture OrganizationGLOglobulinGMgenetically modifiedHBD3human beta-defensin-3HEhematoxylin-eosin

* Corresponding author. College of Veterinary Meversity, Taicheng Road 3, Yangling 712100, China.

E-mail address: zhy1956@263.net (Y. Zhang).1 The co-author have the equal contribution.

http://dx.doi.org/10.1016/j.fct.2016.12.0120278-6915/© 2016 The Authors. Published by Elsevier

a b s t r a c t

In recent years, transgenic technology has been widely applied in many fields. There is concern about thesafety of genetically modified (GM) products with the increased prevalence of GM products. In order toprevent mastitis in dairy cows, our group produced transgenic cattle expressing human beta-defensin-3(HBD3) in their mammary glands, which confers resistance to the bacteria that cause mastitis. The milkderived from these transgenic cattle thus contained HBD3. The objective of the present study was toanalyze the nutritional composition of HBD3 milk and conduct a 90-day feeding study on rats. Rats weredivided into 5 groups which consumed either an AIN93G diet (growth purified diet for rodents rec-ommended by the American Institute of Nutrition) with the addition of 10% or 30% HBD3 milk, anAIN93G diet with the addition of 10% or 30% conventional milk, or an AIN93G diet alone. The resultsshowed that there was no difference in the nutritional composition of HBD3 and conventional milk.Furthermore, body weight, food consumption, blood biochemistry, relative organ weight, and histopa-thology were normal in those rats that consumed diets containing HBD3. No adverse effects wereobserved between groups that could be attributed to varying diets or gender.© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

dicine, Northwest A&F Uni-

Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

X. Chen et al. / Food and Chemical Toxicology 100 (2017) 34e41 35

OECDOrganization for Economic Co-operation

and DevelopmentTBILtotal bilirubinT-CHOLtotal cholesterolTGtriglycerideTPtotal proteinWHOWorld Health Organizationw/w(weight/weight)

1. Introduction

Human beta-defensin-3 (HBD3) is a small, cationic, host defensepeptide comprised of 45 amino acid residues, which was first iso-lated from human lesional psoriatic scales and cloned from kera-tinocytes by Harder et al. (2001). HBD3 possesses six conservedcysteine residues that facilitate both broad antimicrobial activityagainst many pathogenic microbes and diverse innate immunefunctions (Van Hemert et al., 2012). Previous studies have shownthat HBD3 is effective against the majority of Gram-positive andGram-negative bacteria, including a number of multiple antibioticresistant strains (Maisetta et al., 2006; Zasloff, 2002). The anti-microbial effects of HBD3 differ for different strains of bacteria. Inan in vitro study of oral cavity bacteria, aerobes were found to bemore sensitive to HBD3 than anaerobes (Joly et al., 2004). HBD3also has inhibitory effects against fungi and viruses (Dhople et al.,2006). Quinones-Mateu et al. (2003) reported that HBD3 caninhibit replication of the human immunodeficiency virus. In addi-tion, HBD3 has chemotaxis effects on immature dendritic cells andmemory T cells (Scudiero et al., 2010). Therefore, HBD3 plays animportant role in the regulation of immunity. In the immune sys-tem, HBD3 acts as a bridge linking innate immunity and acquiredimmunity.

Mastitis is the most common disease related to milk productionin dairy cows. Mastitis causes great economic losses due to adecrease in the quality and quantity of milk production and theincreased cost of disease treatment (Kerro Dego et al., 2002; Sinhaet al., 2014). Mastitis is an inflammatory response to pathogenicmicroorganisms entering through the teat canal and multiplying inthe mammary gland (Oviedo-Boyso et al., 2007). Many differentbacteria can cause mastitis, including contagious and environ-mental bacteria such as Staphylococcus aureus, Escherichia coli, andStreptococcus dysgalactiae. Antibiotics are the most commontreatment for mastitis. However, antibiotics are not an ideal treat-ment as there are various different pathogenic bacteria species thatcould cause infection and the overuse of antibiotics also causesproblems such as drug-resistant bacterial strains and milk con-taining antibiotic residues which is unfit for consumption.

Transgenic animals are animals where one or more genes fromone organism has been transferred to another by using geneticengineering technologies (Hino, 2002). With the continued devel-opment of transgenic technology, genetically modified (GM)products are becoming more prevalent in daily life. When new GMproducts are developed, testing must be performed to determinewhether the new trait will affect the nutritional value of theproduct or consumer health. The safety assessment of GM productsfocuses primarily on potential allergenic compounds in the food,the nutritional content of the food, possible expression of antibioticselection markers, and transgene stability and inheritance

(Domingo, 2016; Domingo and Gine Bordonaba, 2011; Nicolia et al.,2014). The current principles of the safety assessment for GMproducts, which are accepted by most nations and organizations,support the concept of substantial equivalence and the stepsinvolved for the scientific evaluation of each GM product areformed in a case by case basis (Codex, 2008; OECD, 1993). Feedingstudies are often used to assess the safety of food products,including GM products. Traditionally, a feeding study is conductedwithin 30 days or 90 days for a general health assessment. If the GMproduct is for a special population such as infants or the elderly,special parameters in addition to general health may need to beevaluated, and thus a feeding study may be conducted using ani-mals of different ages over varying lengths of time (Malatesta et al.,2008). Many GM products have been the subject of feeding studies,including rice (Schroder et al., 2007; Tang et al., 2012; Yuan et al.,2013), soybeans (Appenzeller et al., 2008), tomatoes (Fares andEl-Sayed, 1998), maize grain (He et al., 2008, 2009), and animalproducts such as meat andmilk (S. Liu et al., 2013; Yamaguchi et al.,2007; Zhou et al., 2011).

In order to prevent mastitis in dairy cows, our groupexploited the broad-spectrum antimicrobial activity of HBD3and produced transgenic cattle expressing HBD3 specifically intheir mammary glands to prevent colonization in this area bythe bacteria that cause mastitis. Lactation in the transgeniccattle was comparable to healthy conventional cattle, and themilk from transgenic cows repressed the growth of bothS. aureus and E. coli. (Yu et al., 2013).

A previous in vitro study showed the GM milk containing HBD3was easy to digest, and did not cause any adverse effects on thegeneral and gastrointestinal health of the mice in the study (Chenet al., 2016). Rat and mouse models are the commonly-usedmodel animals in feeding studies. Here, we analyzed the compo-sition of HBD3 GM milk and the general health of rats followingconsumption of HBD3 GM milk, which included analysis of bodyweight, food consumption, blood biochemistry, relative organweight, and pathology. A 90-day feeding study was conducted inaccordance with the Chinese Toxicology Assessment Procedures andMethods for Food Safety (Chinese standard GB 15193.13e2003).

2. Materials and methods

2.1. Test sample

The GM milk containing HBD3 was produced by transgeniccattle with HBD3 inserted into a “safe harbor” in the bovinegenome by phiC31 integrase. The concentration of HBD3 in themilk was measured during the lactation period using ELISA, and itranged from 3.9 to 10.4 mg/ml (Yu et al., 2013). We selected themilk with the highest HBD3 concentration for use in this study.

Table 1Gross composition of non-GM and GM milk (Mean ± SD, n ¼ 3).

Non-GM milk GM milk

Fat (g/100 g) 3.57 ± 0.25 3.44 ± 0.21Protein (g/100 g) 2.99 ± 0.18 3.24 ± 0.13Lactose (g/100 g) 4.87 ± 0.04 4.83 ± 0.11pH 6.69 ± 0.02 6.73 ± 0.02Solids (g/100 g) 8.13 ± 0.05 8.27 ± 0.11

X. Chen et al. / Food and Chemical Toxicology 100 (2017) 34e4136

The conventional milk was acquired from Yangling Keyuan Clon-ing Co., Ltd., China.

2.2. Nutritional composition analysis of milk

Analyses of protein, fat, lactose, and solids of GM and non-GMmilk were performed in accordance with standard methods (Chi-nese Standard GB 5009.5e2010, GB 5413.3e2010, GB 5413.5e2010,and GB 5413.39e2010, respectively). Amino acid, mineral, andvitamin composition of GM and non-GM milk were also measuredin accordance with standard methods (Chinese Standard GB/T5009.124e2003, GB 5413.21e2010, and GB 5413.9e2010,respectively).

2.3. Experimental animals and diets

Sixty male and 60 female pathogen-free Sprague Dawley ratswere obtained from the Laboratory Animal Center of the FourthMilitaryMedical University (Xi'an, Shaanxi, China). All ratswere fourweeks old at the start of treatment and were housed in a poly-propyleneplastic cagewithad libitumaccess towaterand food.Roomtemperature was maintained 22 �C ± 2 �C, with a 50% ± 10% relativehumidity, five air changes per hour, and a 12-h light/dark cycle. Ratswere acclimatized forfive days and fedwith theAIN93Gdiet (growthpurified diet for rodents recommended by the American Institute ofNutrition) before they were randomly divided into five groups with12 rats/sex/group. Experimental groups were fed diets supple-mented with 10% or 30% (w/w) GM milk according to the AIN93Gdiet, and control groups were fed diets supplemented with the cor-responding concentration of conventionalmilk. A further group thatserved as the negative control was fed with only the AIN93G diet. Inorder to simplify the reference to each group, those animals fed theAIN93G diet alone, the AIN93G diet with the addition of 10% HBD3milk, the AIN93G diet with the addition of 30% HBD3 milk, theAIN93G diet with the addition of 10% conventional milk, and theAIN93G diet with the addition of 30% conventional milk weredesignated as the C, 10G, 30G, 10N, and 30N group, respectively.

To meet the nutritional requirements of the AIN93G diet, thecomposition of animal feed was 24% soybean meal, 30% flour, 5%fish meal, 20% corn, 12% bran, 2% grass meal, 2% yeast power, 1.5%vegetable oil, 1.3% mountain flour, 1.6% Calcium hydrogen phos-phate, and 0.6% salt. The 30G diet, 10G diet, 30N diet and 10N dietwere the AIN93G diet with the addition of the correspondingconcentrations of milk. All feed ingredients were combined andthoroughly mixed to ensure homogeneity. The dough was cut intoappropriately sized pellets and oven-dried. During pellet produc-tion, drying temperatures were set to 55 �C in order to maintainprotein activity. All diets were divided into aliquots sufficient for asingle-day feeding and vacuum-packed to prevent potentialdecomposition of the fodder due to long-term air exposure. Thebioactivity of HBD3 in pellet was examined by analyzing its resis-tant capacity to Staphylococcus aureus (Fig. S1).

The methodology of the study was approved by the Animal CareCommission of the College of Veterinary Medicine, Northwest A&FUniversity. Each animal received humane care. The study wasperformed in accordance with the institution's guidelines.

2.4. Clinical observations, body weight, and food consumption

During the feeding trial, rats were monitored daily for mortalityand signs of morbidity or clinical signs of toxicity. We also observedtheir appearance (piloerection, kyphosis, disheveled fur, and sec-reta), their behavior (altered grooming or nesting), and activity(altered exploring) each day. Body weight and food consumption ofeach rat were recorded weekly.

2.5. Organ weight, gross necropsy, and pathology

At the end of the feeding study, rats were anesthetized andkilled by cervical dislocation. A thorough necropsy of major organswas performed and the heart, liver, spleen, lungs, and kidneys(paired) were excised, examined, and weighed. Tissue sampleswere fixed in 4% buffered formaldehyde for more than 24 h beforehistological processing, then embedded in paraffin in order tocreate 4e6-mm thick sections which were stained withhematoxylin-eosin (HE) for light microscopy (OLYMPUS, BX-50,Tokyo, Japan). For histopathological analysis, we observed thepericardium andmyocardial fibers of the heart; the capsule, hepaticartery, and hepatic vein of the liver; the capsule, red pulp, andwhite pulp of the spleen; the pleurae, alveoli, alveolar space,bronchia, and blood vessels of the lungs; and the capsule, renalcortex and medulla, glomerulus, and renal tubular of the kidneys.

2.6. Blood biochemistry

Before collecting blood samples, rats were subject to fastingovernight with water provided ad libitum. The rats were anes-thetized and blood samples were collected from the orbital sinusvein. The serum chemistry parameters alanine aminotransferase(ALT), aspartate aminotransferase (AST), alkaline phosphatase(ALP), total protein (TP), albumin (ALB), globulin (GLO), total bili-rubin (TBIL), urea nitrogen (BUN), total cholesterol (T-CHOL), andtriglyceride (TG) were measured with an auto-analyzer (Hitachi7180, Tokyo, Japan).

2.7. Statistical analysis

Statistical comparisons were designed to determine whethersignificant differences between values were attributable to theconsumption of HBD3. Data obtained from groups fed the 10G and30G diets were first comparedwith data from the group fed the 10Nand 30N diets, respectively, and then compared with that from thegroup fed the AIN93G diet. All data were analyzed using SPSS 20.0statistical software (IBM Corporation, Somers, NY, USA). Data weretested by one-way ANOVA and least-significant difference tests.Data is presented as the mean ± SD, with p < 0.05 considered assignificantly different.

3. Results

3.1. Milk nutritional composition analysis

The nutritional compositions of the GM and conventional milkused in the feeding trial were subjected to comprehensive analysis.The results showed that the gross composition of GM and con-ventional milk was not statistically different (Table 1). All valueswere within the normal range according to the Chinese Standard.Amino acid, mineral, and vitamin composition of GM milk werealso not significantly different when compared with those of con-ventional milk (Table 2, Table 3, and Table 4, respectively). Theseresults also conformed to the requirements of the Chinese nationalfood safety standard.

Table 2Amino acid composition of non-GM and GM milk (Mean ± SD, n ¼ 3).

Amino acid (mg/g) Non-GM milk GM milk

Asp 1.70 ± 0.10 1.73 ± 0.06Thr 0.93 ± 0.06 0.87 ± 0.06Ser 1.20 ± 0.10 1.20 ± 0.10Glu 3.67 ± 1.80 4.73 ± 0.21Pro 4.33 ± 0.25 4.47 ± 0.23Gly 0.43 ± 0.06 0.47 ± 0.06Ala 0.73 ± 0.06 0.80 ± 0.10Cys 0.00 ± 0.00 0.00 ± 0.00Val 1.33 ± 0.06 1.37 ± 0.06Met 0.40 ± 0.10 0.43 ± 0.12Lle 1.07 ± 0.06 1.03 ± 0.06Leu 2.23 ± 0.21 2.33 ± 0.15Tyr 1.13 ± 0.06 1.17 ± 0.06Phe 1.17 ± 0.15 1.20 ± 0.17Lys 1.63 ± 0.12 1.70 ± 0.10His 1.13 ± 0.06 1.13 ± 0.06Arg 0.83 ± 0.06 0.77 ± 0.06

Table 3Mineral composition of non-GM and GM milk (Mean ± SD, n ¼ 3).

Non-GM milk GM milk

Ca (mg/kg) 750.19 ± 32.26 807.55 ± 139.10Mg (mg/kg) 127.24 ± 3.80 127.20 ± 5.07P (mg/kg) 1896.14 ± 15.14 1895.63 ± 31.62K (mg/kg) 2601.17 ± 53.11 2609.21 ± 51.53Na (mg/kg) 490.51 ± 79.25 490.25 ± 77.25Fe (mg/kg) 4.33 ± 0.71 4.46 ± 1.05Zn (mg/kg) 14.58 ± 0.37 14.51 ± 0.42Se (mg/kg) 0.02 ± 0.00 0.02 ± 0.00

Table 4Vitamin composition of non-GM and GM milk (Mean ± SD, n ¼ 3).

Vitamin Non-GM milk GM milk

A(mg/100 g) 0.03 ± 0.01 0.03 ± 0.02E(mg/100 g) 0.07 ± 0.02 0.07 ± 0.01B1 (mg/100 g) 0.07 ± 0.01 0.07 ± 0.01B2 (mg/100 g) 0.35 ± 0.01 0.47 ± 0.14

X. Chen et al. / Food and Chemical Toxicology 100 (2017) 34e41 37

3.2. Clinical signs, body weights, and food consumption

Over the course of the feeding trial, we observed the rats forclinical signs of toxicity ormorbidity each day. The bodyweight andfood consumption of each rat were measured weekly. All rats sur-vived the duration of the feeding trial. No clinical signs of toxicitywere observed in any of the groups. The appearance, behavior, andactivity of all rats were normal. There were no significant differ-ences in body weight or food consumption between rats feddifferent diets. Body weight in 30G and 30N groups were slightlyhigher than that in other groups in male and female rats (Fig. 1).According to the mean daily food consumption, we calculated thetotal consumption of HBD3. The total consumption of HBD3 of 30Gand 10G groups in male rats were approximately 5876.405 mg and1883.735 mg, respectively. The total consumption of HBD3 of 30Gand 10G groups in female rats were approximately 4393.494 mg and1462.374 mg, respectively (Table S1).

3.3. Blood biochemistry

The blood biochemistry results are shown in Table 5 and Table 6.There were no significant differences in the majority of themeasured parameters. The levels of TG and TBIL of males in the 30Ggroupwere slightly lower than those in the C group. However, there

was no difference between 30G and 30N groups regarding TG andTBIL levels. In female rats, ALT in 30G and 10N groups were lowerthan that in the C group, and ALT in the 10N group was significantlower than that in the 10G group. Although there were severaldifferences observed concerning blood biochemistry results, all theparameters were within the normal range.

3.4. Gross necropsy and relative organ weight

A complete necropsy was conducted on all rats. No treatment-related adverse gross lesions were observed during the entirenecropsy process. No statistical difference was observed in relativeorgan weight between experimental treatment groups and thecontrol group (Table 7).

3.5. Histopathology

Following histopathological observation, sporadic microscopicchanges were found in all groups. No severe histopathological le-sions were observed in any organ tissue sections (Fig. 2). For theheart, the pericardium was intact without exudate. No degenera-tion, necrosis, atrophy, hypertrophy, or inflammatory cell infiltra-tion in myocardial fibers was observed. No obvious differences inthe heart were observed between each group. For the liver, thecapsule of the liver was intact in each group. There was no obviousconnective tissue hyperplasia. The central hepatic vein, the smallbranches of hepatic artery and the hepatic veinwere normal in eachgroup. For the spleen, the capsule of the spleen of each group wascomplete. The structure of red pulp and white pulp were clear.There was no significant difference in each group. For the lungs, thepleurae of the lung were smooth without exudate in all groups. Nocollapsed alveoli or bullous alveoli were observed. There was noexudate in the alveolar space. The structure of the bronchia andblood vessels of the lung appeared normal. For the kidneys, thecapsule of the kidney was intact with a clear cortico-medullaryjunction in each group. Glomerular and renal tubular epithelialcell size were within the normal range in each group.

4. Discussion

Since the development of advanced genetic technologies, it ispossible to transfer a gene from one organism to another withoutsexual reproduction. This process allows desirable alterations to bemade to animal genomes (Gaj et al., 2013). Transgenic technology iswidely used in medicine, agriculture, research, and industry. Inanimal science, a large number of these GM products are treated aspotential food. The concern about the safety of GM products ariseswith the increased prevalence of GM products. Many internationalorganizations have designed various guidelines to assess the safetyof GM products, such as the Food and Agriculture Organization(FAO) of the United Nations, theWorld Health Organization (WHO),the Organization for Economic Co-operation and Development(OECD), and the Codex Alimentarius Commission (CAC). Regardlessof country-of-origin or governing organization, GM productsshould undergo a strict evaluation before they are allowed to enterthe market. A 30- or 90-day feeding study is traditionally used toassess the safety of a GM product. For GM products with varyingfinal purposes, different animals will be used for the feedingstudies. For example, cattle and sheep are consistently selected infeeding trials to study the effects of GM products on the composi-tion and yield of milk (Calsamiglia et al., 2007). Once animalmodelsconsume GM products, informative data can be generated byanalyzing and comparing multiple health related parameters.

Mastitis is a highly prevalent disease in dairy industriesworldwide and results in great economic losses. Traditional

Fig. 1. Body weight and daily food consumption. (A) Mean body weight of male rats (n ¼ 12); (B) Mean body weight of female rats (n ¼ 12); (C) Mean daily food consumption ofmale rats (n ¼ 12); (D) Mean daily food consumption of female rats (n ¼ 12). The C, 10G, 30G, 10N, and 30N group represented those animals fed the AIN93G diet alone, the AIN93Gdiet with the addition of 10% HBD3 milk, the AIN93G diet with the addition of 30% HBD3 milk, the AIN93G diet with the addition of 10% conventional milk, and the AIN93G diet withthe addition of 30% conventional milk, respectively.

Table 5Blood biochemistry of male rats following 90 days. (Mean values ± SD, n ¼ 12).

30G 30N 10G 10N C

ALT(U/L) 41.6 ± 5.3 42.4 ± 3.7 40.9 ± 6.2 42.7 ± 5.5 40.9 ± 4.4AST(U/L) 122.9 ± 24.1 131.5 ± 17.8 126.1 ± 22.5 119.5 ± 29.2 124.6 ± 18.6ALP(U/L) 92.8 ± 20.3 89.4 ± 21.7 91.9 ± 17.3 88.5 ± 21.2 93.4 ± 27.1TP(g/L) 53.1 ± 3.3 52.9 ± 2.7 53.9 ± 2.5 53.1 ± 3.0 52.1 ± 1.8ALB(g/L) 22.9 ± 1.5 23.1 ± 1.9 21.8 ± 1.7 22.0 ± 0.9 22.3 ± 1.4GLO(g/L) 30.2 ± 1.9 29.8 ± 2.0 32.1 ± 2.2 31.1 ± 1.7 29.8 ± 2.4TG(mmol/L) 1.02 ± 0.31* 1.13 ± 0.25 1.24 ± 0.19 1.29 ± 0.33 1.32 ± 0.36TBIL(mmol/L) 0.95 ± 0.11* 1.06 ± 0.21 1.12 ± 0.13 1.09 ± 0.18 1.12 ± 0.15T-CHOL(mmol/L) 1.86 ± 0.24 1.90 ± 0.19 1.79 ± 0.18 1.75 ± 0.33 1.79 ± 0.23BUN(mmol/L) 6.94 ± 0.83 7.12 ± 1.13 7.25 ± 0.92 7.17 ± 0.72 7.59 ± 1.29

ALT: alanine aminotransferase, AST: aspartate aminotransferase, ALP: alkaline phosphatase, TP: total protein, ALB: albumin, GLO: globulin, TG: triglyceride, TBIL: total bili-rubin, T-CHOL: total cholesterol, and BUN: urea nitrogen.*p < 0.05 versus C group.The C, 10G, 30G, 10N, and 30N group represented those animals fed the AIN93G diet alone, the AIN93G diet with the addition of 10% HBD3 milk, the AIN93G diet with theaddition of 30% HBD3 milk, the AIN93G diet with the addition of 10% conventional milk, and the AIN93G diet with the addition of 30% conventional milk, respectively.

X. Chen et al. / Food and Chemical Toxicology 100 (2017) 34e4138

treatment of the disease involves the administration of antibiotics.However, this practice promotes the development of antibiotic-resistant bacteria strains and results in the disposal of milk that

contains antibiotics residues. Previous studies showed that trans-genic dairy animals that produce anti-bacterial proteins in theirmilk have less mastitis (J. Liu et al., 2013; Wall et al., 2005; Yang

Table 6Blood biochemistry of female rats following 90 days. (Mean values ± SD, n ¼ 12).

30G 30N 10G 10N C

ALT(U/L) 32.9 ± 6.4* 35.7 ± 6.6 40.3 ± 4.8 33.6 ± 5.2*△ 41.6 ± 5.5AST(U/L) 109.7 ± 16.8 107.9 ± 21.3 111.7 ± 19.2 108.2 ± 13.2 116.1 ± 19.8ALP(U/L) 49.8 ± 7.1 47.0 ± 12.9 50.2 ± 8.6 51.5 ± 10.1 53.3 ± 10.7TP(g/L) 56.2 ± 2.9 56.7 ± 3.5 57.1 ± 3.7 56.6 ± 3.9 56.2 ± 4.1ALB(g/L) 30.7 ± 2.1 29.8 ± 2.5 31.3 ± 1.6 30.5 ± 2.3 31.0 ± 1.7GLB(g/L) 25.5 ± 2.0 26.9 ± 2.4 25.8 ± 1.9 26.1 ± 1.7 25.2 ± 2.3TG(mmol/L) 0.69 ± 0.10 0.71 ± 0.15 0.75 ± 0.11 0.73 ± 0.21 0.76 ± 0.26TBIL(mmol/L) 0.92 ± 0.09 0.94 ± 0.11 0.89 ± 0.13 0.91 ± 0.08 0.96 ± 0.17T-CHOL(mmol/L) 1.61 ± 0.16 1.59 ± 0.20 1.56 ± 0.09 1.55 ± 0.12 1.58 ± 0.22BUN(mmol/L) 5.62 ± 0.74 5.47 ± 0.82 5.54 ± 1.01 5.27 ± 0.63 5.36 ± 1.07

*p < 0.05 versus C group, △p < 0.05 versus 30N group.30G, 30N, 10G, 10N and C were labeled as in Table 5.

Table 7Organ/body weight in male and female rats following 90 days. (Mean values ± SD, n ¼ 12).

30G 30N 10G 10N C

Maleheart 0.31 ± 0.02 0.30 ± 0.04 0.30 ± 0.03 0.35 ± 0.04 0.29 ± 0.06liver 2.86 ± 0.28 2.72 ± 0.33 2.52 ± 0.37 2.63 ± 0.24 2.74 ± 0.40spleen 0.15 ± 0.05 0.17 ± 0.02 0.17 ± 0.03 0.18 ± 0.03 0.19 ± 0.04lungs 0.31 ± 0.07 0.31 ± 0.04 0.28 ± 0.06 0.30 ± 0.08 0.27 ± 0.11kidneys (paired) 0.61 ± 0.08 0.65 ± 0.06 0.64 ± 0.04 0.64 ± 0.05 0.63 ± 0.06Femaleheart 0.33 ± 0.04 0.35 ± 0.04 0.30 ± 0.05 0.32 ± 0.04 0.33 ± 0.02liver 2.70 ± 0.26 2.75 ± 0.17 2.82 ± 0.22 2.72 ± 0.24 2.63 ± 0.27spleen 0.19 ± 0.03 0.20 ± 0.02 0.20 ± 0.03 0.18 ± 0.04 0.21 ± 0.05lungs 0.38 ± 0.04 0.40 ± 0.06 0.41 ± 0.06 0.37 ± 0.04 0.41 ± 0.03kidneys (paired) 0.68 ± 0.05 0.66 ± 0.11 0.69 ± 0.07 0.68 ± 0.05 0.68 ± 0.06

Organ/body weight expressed as a percentage of organ weight/body weight. 30G, 30N, 10G, 10N and C were labeled as in Table 5.

X. Chen et al. / Food and Chemical Toxicology 100 (2017) 34e41 39

et al., 2011). HBD3 is an ideal candidate protein to protect the bodyfrom bacterial invasion (Roosen et al., 2004). Our group usedphiC31 integrase to insert HBD3 into the cattle genome. Mastitiswas less prevalent amongst the resulting transgenic cattle and themilk containing HBD3 also repressed the growth of bacteria (Yuet al., 2013). In addition, the HBD3 transgenic cattle were healthy.The sequence of the vector in transgenic cattle was integrated (Yuet al., 2013). The lactation was comparable between the trans-genic cattle and the healthy conventional cattle. Compared withantibiotics, HBD3 shows antibacterial activity at very low concen-tration. HBD3 also expressed at low concentration in the transgeniccattle. It is not easy for pathogens to develop resistance to HBD3.Therefore, it is a better way to prevent dairy cow mastitis by pro-ducing the HBD3 transgenic cattle.

A recent study assessed the effects of GMmilk containing HBD3on the gastrointestinal health of mice. The study was conducted toevaluate multiple health parameters including general physicalexamination, gastric emptying function, intestinal permeability,mouse intestinal microflora composition, the possibility of hori-zontal gene transfer, and in vitro digestion of HBD3milk. No adverseeffects were found on gastrointestinal health resulting from con-sumption of GM milk containing HBD3. The HBD3 milk was easilydigested in a simulated gastric fluid in vitro. Although a slight dif-ference was observed in the mouse intestinal microflora composi-tion, the dominant bacteria species were similar between thoseanimals fed with GM diets and those fed with non-GM diets (Chenet al., 2016). Other feeding studies of transgenic milk containingantimicrobial proteins also resulted in the modulation of intestinalmicroflora (Hu et al., 2012; Maga et al., 2006).

Given the differences in physiological function and structurebetween rats and mice, different reactions to the same product arepossible. Previous studies showed that rats displayed no patho-logical changes following feeding trials with GM glyphosate-

tolerant soybean (Sakamoto et al., 2007). However, other studiesshowed that some modifications occurred in hepatocyte nuclei ofmice fed diets containing GM glyphosate-tolerant soybean(Malatesta et al., 2002, 2005). Therefore, in this study, we con-ducted a feeding study to evaluate the presence of any unintendedadverse effects on rats associated with the consumption of dietscontaining GM milk at different doses. The results showed therewas no difference in the composition of GM and conventional milk.The body weight, food consumption, relative organ weight, andhistopathology of each group were normal and no statistical dif-ferences were observed between groups. Significant differenceswere observed between groups for some blood biochemistry pa-rameters, e.g., ALT activity. The ALT levels in female rats of the 30Gand 10N groups were slightly lower than those of the C group.Manystudies have shown that ALT activity undergoes significant diurnalvariation, and ALT activity in the afternoon can be up to 45% higherthan its activity in the morning (Cordoba et al., 1998). In this study,the difference observed in ALT level may be the result of differencesin the timing of blood collection. In addition, ALT level typicallydeclines as liver fibrosis progresses (Kim et al., 2008). However, theliver histopathological results of female rats in 30G and 10N groupsrevealed that the tissue did not contain lesions. In general, ALTlevels less than fivefold the upper limit of the normal range shouldbe verified (Kim et al., 2008). All the analyzed parameters werewithin the normal range for rats of this age. Thus, we consider thatthe differences in ALT level in female rats of the 30G and 10Ngroups are not biologically significant. The TG level in male rats ofthe 30G group was slightly lower than that in the C group. In termsof pathological condition, a declining level of the TG value isaccompanied by decreased body weight (Gavino et al., 2000).However, the body weights of male rats of the 30G group were notdecreased during the study. In addition, the TG level in male rats ofthe 30G group was within the normal range. High-concentration

Fig. 2. Histopathological results of main organs by HE staining. (A) Male rats; (B)Female rats. Scale bar ¼ 100 mm. 30G, 30N, 10G, 10N and C were labeled as in Fig. 1.

X. Chen et al. / Food and Chemical Toxicology 100 (2017) 34e4140

TBIL serves as a marker of hepatobiliary disorders. Serum concen-trations of TBIL within the physiological range were shown to beinversely associated with metabolic syndrome (Choi et al., 2013). Inmetabolic syndrome, a lower value for TBIL is accompanied byhigher values for ALT, TG, and CHOL (Wu et al., 2011). However, ALT,TG, and CHOL concentrations in male rats of the 30G group werelower than those in the control group. Therefore, the differencesobserved in TG and TBIL levels were not an evidence that HBD3milk is unsafe. No rats exhibited clinical symptoms during thefeeding trial. In previous studies, differences were also observed inthe blood biochemistry parameters of different feeding groups(Chen et al., 2016; Yuan et al., 2013; Zou et al., 2016).

The transgenicmilk is mainly applied to changemilk ingredientsmore closely to the human milk, to produce pharmaceutical pro-teins as mammary gland bioreactor, and to improve the resistanceof mastitis (X. Liu et al., 2014; Wang et al., 2008; Yang et al., 2011).Although there may be several physiological indexes different withthose of the corresponding control group, feeding studies ofdifferent transgenic milk did not show a detrimental effect on theconsumer health. All experimental animals survived the duration ofthe feeding trial and no histopathological changes were found(Chen et al., 2016; Hu et al., 2012; Zhou et al., 2011).

Males and females display physiological differences in manyareas including immune responses and reproduction (Ansar et al.,1985). Therefore, they may have different responses to the samestimulus. In this study, no differences were observed between maleand female rats for any of the analyzed parameters.

We used somatic cell count meter (KangDaZhiXin KD-400,China) to count the somatic cells in the transgenic milk. Therewere 232,000 cells per milliliter milk on the average, which waswithin the normal range. Somatic cells in the transgenic milk couldexpress EGFP. However, the amount of EGFP in the transgenic milkwas few to detect using western bloting. Therefore, we did notconsider the effect from EGFP.

In this study, no rats showed pathologic changes after theyconsumed approximately maximum 5876.405 mg HBD3. As nofeeding study reports the total consumption of HBD3 by rats, thisresult may be a guide for the future feeding studies of HBD3.

In summary, based on the principles of the GM product safetyassessment and other previous studies, we conducted a study toassess the composition of GM milk and the general health of ratsfed GM milk in their diet. The results showed that GM milkcomposition was normal and did not significantly differ to thecomposition of conventional milk. In addition, the results showedthat all rats were healthy during the feeding trial. Thus, GM milkcontaining HBD3 did not have any adverse effects on rats.

Competing financial interests

The authors declare that there are no competing financialinterests.

Author contributions

Conceived and designed the experiments: XC, MG, and YZ.Performed the experiments: XC, MG, DL, SY, KY, and YZ. Analyzedthe data: XC and MG. Wrote the paper: XC.

Acknowledgements

This work was supported by the National Major Project forProduction of Transgenic Breeding (No. 2013ZX08007-004).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.fct.2016.12.012.

Transparency document

Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.fct.2016.12.012.

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