Food and Chemical Toxicology 100 (2017) 34e41
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Food and Chemical Toxicology
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Safety assessment of genetically modified milk containing human
beta-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
b
c
College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China
Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China
Medical College, Yan'an University, Yan'an 716000, Shaanxi, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 September 2016
Received in revised form
8 December 2016
Accepted 10 December 2016
Available online 12 December 2016
In recent years, transgenic technology has been widely applied in many fields. There is concern about the
safety of genetically modified (GM) products with the increased prevalence of GM products. In order to
prevent 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 milk
derived from these transgenic cattle thus contained HBD3. The objective of the present study was to
analyze the nutritional composition of HBD3 milk and conduct a 90-day feeding study on rats. Rats were
divided into 5 groups which consumed either an AIN93G diet (growth purified diet for rodents recommended by the American Institute of Nutrition) with the addition of 10% or 30% HBD3 milk, an
AIN93G diet with the addition of 10% or 30% conventional milk, or an AIN93G diet alone. The results
showed 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 histopathology were normal in those rats that consumed diets containing HBD3. No adverse effects were
observed 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/).
Keywords:
Genetically modified milk
HBD3
Safety assessment
90-Day feeding study
Abbreviations:
AIN93G diet
Growth purified diet for rodents
recommended by the American Institute of
Nutrition
ALB
albumin
ALP
alkaline phosphatase
ALT
alanine aminotransferase
AST
aspartate aminotransferase
BUN
urea nitrogen
CAC
Codex Alimentarius Commission
FAO
Food and Agriculture Organization
GLO
globulin
GM
genetically modified
HBD3
human beta-defensin-3
HE
hematoxylin-eosin
* Corresponding author. College of Veterinary Medicine, Northwest A&F University, 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.2016.12.012
0278-6915/© 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/).
X. Chen et al. / Food and Chemical Toxicology 100 (2017) 34e41
35
OECD
Organization for Economic Co-operation
and Development
TBIL
total bilirubin
T-CHOL
total cholesterol
TG
triglyceride
TP
total protein
WHO
World Health Organization
w/w
(weight/weight)
1. Introduction
Human beta-defensin-3 (HBD3) is a small, cationic, host defense
peptide comprised of 45 amino acid residues, which was first isolated from human lesional psoriatic scales and cloned from keratinocytes by Harder et al. (2001). HBD3 possesses six conserved
cysteine residues that facilitate both broad antimicrobial activity
against many pathogenic microbes and diverse innate immune
functions (Van Hemert et al., 2012). Previous studies have shown
that HBD3 is effective against the majority of Gram-positive and
Gram-negative bacteria, including a number of multiple antibiotic
resistant strains (Maisetta et al., 2006; Zasloff, 2002). The antimicrobial effects of HBD3 differ for different strains of bacteria. In
an in vitro study of oral cavity bacteria, aerobes were found to be
more sensitive to HBD3 than anaerobes (Joly et al., 2004). HBD3
also has inhibitory effects against fungi and viruses (Dhople et al.,
2006). Quinones-Mateu et al. (2003) reported that HBD3 can
inhibit replication of the human immunodeficiency virus. In addition, HBD3 has chemotaxis effects on immature dendritic cells and
memory T cells (Scudiero et al., 2010). Therefore, HBD3 plays an
important role in the regulation of immunity. In the immune system, HBD3 acts as a bridge linking innate immunity and acquired
immunity.
Mastitis is the most common disease related to milk production
in dairy cows. Mastitis causes great economic losses due to a
decrease in the quality and quantity of milk production and the
increased cost of disease treatment (Kerro Dego et al., 2002; Sinha
et al., 2014). Mastitis is an inflammatory response to pathogenic
microorganisms entering through the teat canal and multiplying in
the mammary gland (Oviedo-Boyso et al., 2007). Many different
bacteria can cause mastitis, including contagious and environmental bacteria such as Staphylococcus aureus, Escherichia coli, and
Streptococcus dysgalactiae. Antibiotics are the most common
treatment for mastitis. However, antibiotics are not an ideal treatment as there are various different pathogenic bacteria species that
could cause infection and the overuse of antibiotics also causes
problems such as drug-resistant bacterial strains and milk containing antibiotic residues which is unfit for consumption.
Transgenic animals are animals where one or more genes from
one organism has been transferred to another by using genetic
engineering technologies (Hino, 2002). With the continued development of transgenic technology, genetically modified (GM)
products are becoming more prevalent in daily life. When new GM
products are developed, testing must be performed to determine
whether the new trait will affect the nutritional value of the
product or consumer health. The safety assessment of GM products
focuses primarily on potential allergenic compounds in the food,
the nutritional content of the food, possible expression of antibiotic
selection 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 GM
products, which are accepted by most nations and organizations,
support the concept of substantial equivalence and the steps
involved for the scientific evaluation of each GM product are
formed in a case by case basis (Codex, 2008; OECD, 1993). Feeding
studies are often used to assess the safety of food products,
including GM products. Traditionally, a feeding study is conducted
within 30 days or 90 days for a general health assessment. If the GM
product is for a special population such as infants or the elderly,
special parameters in addition to general health may need to be
evaluated, and thus a feeding study may be conducted using animals 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 and
El-Sayed, 1998), maize grain (He et al., 2008, 2009), and animal
products such as meat and milk (S. Liu et al., 2013; Yamaguchi et al.,
2007; Zhou et al., 2011).
In order to prevent mastitis in dairy cows, our group
exploited the broad-spectrum antimicrobial activity of HBD3
and produced transgenic cattle expressing HBD3 specifically in
their mammary glands to prevent colonization in this area by
the bacteria that cause mastitis. Lactation in the transgenic
cattle was comparable to healthy conventional cattle, and the
milk from transgenic cows repressed the growth of both
S. aureus and E. coli. (Yu et al., 2013).
A previous in vitro study showed the GM milk containing HBD3
was easy to digest, and did not cause any adverse effects on the
general and gastrointestinal health of the mice in the study (Chen
et al., 2016). Rat and mouse models are the commonly-used
model animals in feeding studies. Here, we analyzed the composition of HBD3 GM milk and the general health of rats following
consumption of HBD3 GM milk, which included analysis of body
weight, food consumption, blood biochemistry, relative organ
weight, and pathology. A 90-day feeding study was conducted in
accordance with the Chinese Toxicology Assessment Procedures and
Methods for Food Safety (Chinese standard GB 15193.13e2003).
2. Materials and methods
2.1. Test sample
The GM milk containing HBD3 was produced by transgenic
cattle with HBD3 inserted into a “safe harbor” in the bovine
genome by phiC31 integrase. The concentration of HBD3 in the
milk was measured during the lactation period using ELISA, and it
ranged from 3.9 to 10.4 mg/ml (Yu et al., 2013). We selected the
milk with the highest HBD3 concentration for use in this study.
36
X. Chen et al. / Food and Chemical Toxicology 100 (2017) 34e41
The conventional milk was acquired from Yangling Keyuan Cloning Co., Ltd., China.
2.2. Nutritional composition analysis of milk
Analyses of protein, fat, lactose, and solids of GM and non-GM
milk were performed in accordance with standard methods (Chinese Standard GB 5009.5e2010, GB 5413.3e2010, GB 5413.5e2010,
and GB 5413.39e2010, respectively). Amino acid, mineral, and
vitamin composition of GM and non-GM milk were also measured
in accordance with standard methods (Chinese Standard GB/T
5009.124e2003, GB 5413.21e2010, and GB 5413.9e2010,
respectively).
2.3. Experimental animals and diets
Sixty male and 60 female pathogen-free Sprague Dawley rats
were obtained from the Laboratory Animal Center of the Fourth
Military Medical University (Xi'an, Shaanxi, China). All rats were four
weeks old at the start of treatment and were housed in a polypropylene plastic cage with ad libitum access to water and food. Room
temperature was maintained 22 C ± 2 C, with a 50% ± 10% relative
humidity, five air changes per hour, and a 12-h light/dark cycle. Rats
were acclimatized for five days and fed with the AIN93G diet (growth
purified diet for rodents recommended by the American Institute of
Nutrition) before they were randomly divided into five groups with
12 rats/sex/group. Experimental groups were fed diets supplemented with 10% or 30% (w/w) GM milk according to the AIN93G
diet, and control groups were fed diets supplemented with the corresponding concentration of conventional milk. A further group that
served as the negative control was fed with only the AIN93G diet. In
order to simplify the reference to each group, those animals fed the
AIN93G diet alone, the AIN93G diet 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 with the addition of 30% conventional milk were
designated as the C, 10G, 30G, 10N, and 30N group, respectively.
To meet the nutritional requirements of the AIN93G diet, the
composition 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 phosphate, and 0.6% salt. The 30G diet, 10G diet, 30N diet and 10N diet
were the AIN93G diet with the addition of the corresponding
concentrations of milk. All feed ingredients were combined and
thoroughly mixed to ensure homogeneity. The dough was cut into
appropriately sized pellets and oven-dried. During pellet production, drying temperatures were set to 55 C in order to maintain
protein activity. All diets were divided into aliquots sufficient for a
single-day feeding and vacuum-packed to prevent potential
decomposition of the fodder due to long-term air exposure. The
bioactivity of HBD3 in pellet was examined by analyzing its resistant capacity to Staphylococcus aureus (Fig. S1).
The methodology of the study was approved by the Animal Care
Commission of the College of Veterinary Medicine, Northwest A&F
University. Each animal received humane care. The study was
performed 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 mortality
and signs of morbidity or clinical signs of toxicity. We also observed
their appearance (piloerection, kyphosis, disheveled fur, and secreta), their behavior (altered grooming or nesting), and activity
(altered exploring) each day. Body weight and food consumption of
each rat were recorded weekly.
2.5. Organ weight, gross necropsy, and pathology
At the end of the feeding study, rats were anesthetized and
killed by cervical dislocation. A thorough necropsy of major organs
was performed and the heart, liver, spleen, lungs, and kidneys
(paired) were excised, examined, and weighed. Tissue samples
were fixed in 4% buffered formaldehyde for more than 24 h before
histological processing, then embedded in paraffin in order to
create 4e6-mm thick sections which were stained with
hematoxylin-eosin (HE) for light microscopy (OLYMPUS, BX-50,
Tokyo, Japan). For histopathological analysis, we observed the
pericardium and myocardial fibers of the heart; the capsule, hepatic
artery, and hepatic vein of the liver; the capsule, red pulp, and
white pulp of the spleen; the pleurae, alveoli, alveolar space,
bronchia, and blood vessels of the lungs; and the capsule, renal
cortex and medulla, glomerulus, and renal tubular of the kidneys.
2.6. Blood biochemistry
Before collecting blood samples, rats were subject to fasting
overnight with water provided ad libitum. The rats were anesthetized and blood samples were collected from the orbital sinus
vein. The serum chemistry parameters alanine aminotransferase
(ALT), aspartate aminotransferase (AST), alkaline phosphatase
(ALP), total protein (TP), albumin (ALB), globulin (GLO), total bilirubin (TBIL), urea nitrogen (BUN), total cholesterol (T-CHOL), and
triglyceride (TG) were measured with an auto-analyzer (Hitachi
7180, Tokyo, Japan).
2.7. Statistical analysis
Statistical comparisons were designed to determine whether
significant differences between values were attributable to the
consumption of HBD3. Data obtained from groups fed the 10G and
30G diets were first compared with data from the group fed the 10N
and 30N diets, respectively, and then compared with that from the
group fed the AIN93G diet. All data were analyzed using SPSS 20.0
statistical software (IBM Corporation, Somers, NY, USA). Data were
tested by one-way ANOVA and least-significant difference tests.
Data is presented as the mean ± SD, with p < 0.05 considered as
significantly different.
3. Results
3.1. Milk nutritional composition analysis
The nutritional compositions of the GM and conventional milk
used in the feeding trial were subjected to comprehensive analysis.
The results showed that the gross composition of GM and conventional milk was not statistically different (Table 1). All values
were within the normal range according to the Chinese Standard.
Amino acid, mineral, and vitamin composition of GM milk were
also not significantly different when compared with those of conventional milk (Table 2, Table 3, and Table 4, respectively). These
results also conformed to the requirements of the Chinese national
food safety standard.
Table 1
Gross composition of non-GM and GM milk (Mean ± SD, n ¼ 3).
Non-GM milk
Fat (g/100 g)
Protein (g/100 g)
Lactose (g/100 g)
pH
Solids (g/100 g)
3.57
2.99
4.87
6.69
8.13
±
±
±
±
±
0.25
0.18
0.04
0.02
0.05
GM milk
3.44
3.24
4.83
6.73
8.27
±
±
±
±
±
0.21
0.13
0.11
0.02
0.11
X. Chen et al. / Food and Chemical Toxicology 100 (2017) 34e41
Table 2
Amino acid composition of non-GM and GM milk (Mean ± SD, n ¼ 3).
Amino acid (mg/g)
Non-GM milk
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Cys
Val
Met
Lle
Leu
Tyr
Phe
Lys
His
Arg
1.70
0.93
1.20
3.67
4.33
0.43
0.73
0.00
1.33
0.40
1.07
2.23
1.13
1.17
1.63
1.13
0.83
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
GM milk
0.10
0.06
0.10
1.80
0.25
0.06
0.06
0.00
0.06
0.10
0.06
0.21
0.06
0.15
0.12
0.06
0.06
1.73
0.87
1.20
4.73
4.47
0.47
0.80
0.00
1.37
0.43
1.03
2.33
1.17
1.20
1.70
1.13
0.77
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.06
0.06
0.10
0.21
0.23
0.06
0.10
0.00
0.06
0.12
0.06
0.15
0.06
0.17
0.10
0.06
0.06
Table 3
Mineral composition of non-GM and GM milk (Mean ± SD, n ¼ 3).
Ca (mg/kg)
Mg (mg/kg)
P (mg/kg)
K (mg/kg)
Na (mg/kg)
Fe (mg/kg)
Zn (mg/kg)
Se (mg/kg)
Non-GM milk
GM milk
750.19 ± 32.26
127.24 ± 3.80
1896.14 ± 15.14
2601.17 ± 53.11
490.51 ± 79.25
4.33 ± 0.71
14.58 ± 0.37
0.02 ± 0.00
807.55 ± 139.10
127.20 ± 5.07
1895.63 ± 31.62
2609.21 ± 51.53
490.25 ± 77.25
4.46 ± 1.05
14.51 ± 0.42
0.02 ± 0.00
Table 4
Vitamin composition of non-GM and GM milk (Mean ± SD, n ¼ 3).
Vitamin
Non-GM milk
A(mg/100 g)
E(mg/100 g)
B1 (mg/100 g)
B2 (mg/100 g)
0.03
0.07
0.07
0.35
±
±
±
±
0.01
0.02
0.01
0.01
GM milk
0.03
0.07
0.07
0.47
±
±
±
±
0.02
0.01
0.01
0.14
3.2. Clinical signs, body weights, and food consumption
Over the course of the feeding trial, we observed the rats for
clinical signs of toxicity or morbidity each day. The body weight and
food consumption of each rat were measured weekly. All rats survived the duration of the feeding trial. No clinical signs of toxicity
were observed in any of the groups. The appearance, behavior, and
activity of all rats were normal. There were no significant differences in body weight or food consumption between rats fed
different diets. Body weight in 30G and 30N groups were slightly
higher than that in other groups in male and female rats (Fig. 1).
According to the mean daily food consumption, we calculated the
total consumption of HBD3. The total consumption of HBD3 of 30G
and 10G groups in male rats were approximately 5876.405 mg and
1883.735 mg, respectively. The total consumption of HBD3 of 30G
and 10G groups in female rats were approximately 4393.494 mg and
1462.374 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 the
measured parameters. The levels of TG and TBIL of males in the 30G
group were slightly lower than those in the C group. However, there
37
was no difference between 30G and 30N groups regarding TG and
TBIL levels. In female rats, ALT in 30G and 10N groups were lower
than that in the C group, and ALT in the 10N group was significant
lower than that in the 10G group. Although there were several
differences observed concerning blood biochemistry results, all the
parameters were within the normal range.
3.4. Gross necropsy and relative organ weight
A complete necropsy was conducted on all rats. No treatmentrelated adverse gross lesions were observed during the entire
necropsy process. No statistical difference was observed in relative
organ weight between experimental treatment groups and the
control group (Table 7).
3.5. Histopathology
Following histopathological observation, sporadic microscopic
changes were found in all groups. No severe histopathological lesions were observed in any organ tissue sections (Fig. 2). For the
heart, the pericardium was intact without exudate. No degeneration, necrosis, atrophy, hypertrophy, or inflammatory cell infiltration in myocardial fibers was observed. No obvious differences in
the heart were observed between each group. For the liver, the
capsule of the liver was intact in each group. There was no obvious
connective tissue hyperplasia. The central hepatic vein, the small
branches of hepatic artery and the hepatic vein were normal in each
group. For the spleen, the capsule of the spleen of each group was
complete. The structure of red pulp and white pulp were clear.
There was no significant difference in each group. For the lungs, the
pleurae of the lung were smooth without exudate in all groups. No
collapsed alveoli or bullous alveoli were observed. There was no
exudate in the alveolar space. The structure of the bronchia and
blood vessels of the lung appeared normal. For the kidneys, the
capsule of the kidney was intact with a clear cortico-medullary
junction in each group. Glomerular and renal tubular epithelial
cell size were within the normal range in each group.
4. Discussion
Since the development of advanced genetic technologies, it is
possible to transfer a gene from one organism to another without
sexual reproduction. This process allows desirable alterations to be
made to animal genomes (Gaj et al., 2013). Transgenic technology is
widely used in medicine, agriculture, research, and industry. In
animal science, a large number of these GM products are treated as
potential food. The concern about the safety of GM products arises
with the increased prevalence of GM products. Many international
organizations have designed various guidelines to assess the safety
of GM products, such as the Food and Agriculture Organization
(FAO) of the United Nations, the World Health Organization (WHO),
the Organization for Economic Co-operation and Development
(OECD), and the Codex Alimentarius Commission (CAC). Regardless
of country-of-origin or governing organization, GM products
should undergo a strict evaluation before they are allowed to enter
the market. A 30- or 90-day feeding study is traditionally used to
assess the safety of a GM product. For GM products with varying
final purposes, different animals will be used for the feeding
studies. For example, cattle and sheep are consistently selected in
feeding trials to study the effects of GM products on the composition and yield of milk (Calsamiglia et al., 2007). Once animal models
consume GM products, informative data can be generated by
analyzing and comparing multiple health related parameters.
Mastitis is a highly prevalent disease in dairy industries
worldwide and results in great economic losses. Traditional
38
X. Chen et al. / Food and Chemical Toxicology 100 (2017) 34e41
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 of
male 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 AIN93G
diet 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 with
the addition of 30% conventional milk, respectively.
Table 5
Blood biochemistry of male rats following 90 days. (Mean values ± SD, n ¼ 12).
ALT(U/L)
AST(U/L)
ALP(U/L)
TP(g/L)
ALB(g/L)
GLO(g/L)
TG(mmol/L)
TBIL(mmol/L)
T-CHOL(mmol/L)
BUN(mmol/L)
30G
30N
10G
10N
C
41.6 ± 5.3
122.9 ± 24.1
92.8 ± 20.3
53.1 ± 3.3
22.9 ± 1.5
30.2 ± 1.9
1.02 ± 0.31*
0.95 ± 0.11*
1.86 ± 0.24
6.94 ± 0.83
42.4 ± 3.7
131.5 ± 17.8
89.4 ± 21.7
52.9 ± 2.7
23.1 ± 1.9
29.8 ± 2.0
1.13 ± 0.25
1.06 ± 0.21
1.90 ± 0.19
7.12 ± 1.13
40.9 ± 6.2
126.1 ± 22.5
91.9 ± 17.3
53.9 ± 2.5
21.8 ± 1.7
32.1 ± 2.2
1.24 ± 0.19
1.12 ± 0.13
1.79 ± 0.18
7.25 ± 0.92
42.7 ± 5.5
119.5 ± 29.2
88.5 ± 21.2
53.1 ± 3.0
22.0 ± 0.9
31.1 ± 1.7
1.29 ± 0.33
1.09 ± 0.18
1.75 ± 0.33
7.17 ± 0.72
40.9 ± 4.4
124.6 ± 18.6
93.4 ± 27.1
52.1 ± 1.8
22.3 ± 1.4
29.8 ± 2.4
1.32 ± 0.36
1.12 ± 0.15
1.79 ± 0.23
7.59 ± 1.29
ALT: alanine aminotransferase, AST: aspartate aminotransferase, ALP: alkaline phosphatase, TP: total protein, ALB: albumin, GLO: globulin, TG: triglyceride, TBIL: total bilirubin, 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 the
addition 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.
treatment of the disease involves the administration of antibiotics.
However, this practice promotes the development of antibioticresistant bacteria strains and results in the disposal of milk that
contains antibiotics residues. Previous studies showed that transgenic dairy animals that produce anti-bacterial proteins in their
milk have less mastitis (J. Liu et al., 2013; Wall et al., 2005; Yang
X. Chen et al. / Food and Chemical Toxicology 100 (2017) 34e41
39
Table 6
Blood biochemistry of female rats following 90 days. (Mean values ± SD, n ¼ 12).
ALT(U/L)
AST(U/L)
ALP(U/L)
TP(g/L)
ALB(g/L)
GLB(g/L)
TG(mmol/L)
TBIL(mmol/L)
T-CHOL(mmol/L)
BUN(mmol/L)
30G
30N
10G
10N
C
32.9 ± 6.4*
109.7 ± 16.8
49.8 ± 7.1
56.2 ± 2.9
30.7 ± 2.1
25.5 ± 2.0
0.69 ± 0.10
0.92 ± 0.09
1.61 ± 0.16
5.62 ± 0.74
35.7 ± 6.6
107.9 ± 21.3
47.0 ± 12.9
56.7 ± 3.5
29.8 ± 2.5
26.9 ± 2.4
0.71 ± 0.15
0.94 ± 0.11
1.59 ± 0.20
5.47 ± 0.82
40.3 ± 4.8
111.7 ± 19.2
50.2 ± 8.6
57.1 ± 3.7
31.3 ± 1.6
25.8 ± 1.9
0.75 ± 0.11
0.89 ± 0.13
1.56 ± 0.09
5.54 ± 1.01
33.6 ± 5.2*△
108.2 ± 13.2
51.5 ± 10.1
56.6 ± 3.9
30.5 ± 2.3
26.1 ± 1.7
0.73 ± 0.21
0.91 ± 0.08
1.55 ± 0.12
5.27 ± 0.63
41.6 ± 5.5
116.1 ± 19.8
53.3 ± 10.7
56.2 ± 4.1
31.0 ± 1.7
25.2 ± 2.3
0.76 ± 0.26
0.96 ± 0.17
1.58 ± 0.22
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 7
Organ/body weight in male and female rats following 90 days. (Mean values ± SD, n ¼ 12).
30G
Male
heart
liver
spleen
lungs
kidneys (paired)
Female
heart
liver
spleen
lungs
kidneys (paired)
30N
10G
10N
C
0.31
2.86
0.15
0.31
0.61
±
±
±
±
±
0.02
0.28
0.05
0.07
0.08
0.30
2.72
0.17
0.31
0.65
±
±
±
±
±
0.04
0.33
0.02
0.04
0.06
0.30
2.52
0.17
0.28
0.64
±
±
±
±
±
0.03
0.37
0.03
0.06
0.04
0.35
2.63
0.18
0.30
0.64
±
±
±
±
±
0.04
0.24
0.03
0.08
0.05
0.29
2.74
0.19
0.27
0.63
±
±
±
±
±
0.06
0.40
0.04
0.11
0.06
0.33
2.70
0.19
0.38
0.68
±
±
±
±
±
0.04
0.26
0.03
0.04
0.05
0.35
2.75
0.20
0.40
0.66
±
±
±
±
±
0.04
0.17
0.02
0.06
0.11
0.30
2.82
0.20
0.41
0.69
±
±
±
±
±
0.05
0.22
0.03
0.06
0.07
0.32
2.72
0.18
0.37
0.68
±
±
±
±
±
0.04
0.24
0.04
0.04
0.05
0.33
2.63
0.21
0.41
0.68
±
±
±
±
±
0.02
0.27
0.05
0.03
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.
et al., 2011). HBD3 is an ideal candidate protein to protect the body
from bacterial invasion (Roosen et al., 2004). Our group used
phiC31 integrase to insert HBD3 into the cattle genome. Mastitis
was less prevalent amongst the resulting transgenic cattle and the
milk containing HBD3 also repressed the growth of bacteria (Yu
et al., 2013). In addition, the HBD3 transgenic cattle were healthy.
The sequence of the vector in transgenic cattle was integrated (Yu
et al., 2013). The lactation was comparable between the transgenic cattle and the healthy conventional cattle. Compared with
antibiotics, HBD3 shows antibacterial activity at very low concentration. HBD3 also expressed at low concentration in the transgenic
cattle. It is not easy for pathogens to develop resistance to HBD3.
Therefore, it is a better way to prevent dairy cow mastitis by producing the HBD3 transgenic cattle.
A recent study assessed the effects of GM milk containing HBD3
on the gastrointestinal health of mice. The study was conducted to
evaluate multiple health parameters including general physical
examination, gastric emptying function, intestinal permeability,
mouse intestinal microflora composition, the possibility of horizontal gene transfer, and in vitro digestion of HBD3 milk. No adverse
effects were found on gastrointestinal health resulting from consumption of GM milk containing HBD3. The HBD3 milk was easily
digested in a simulated gastric fluid in vitro. Although a slight difference was observed in the mouse intestinal microflora composition, the dominant bacteria species were similar between those
animals fed with GM diets and those fed with non-GM diets (Chen
et al., 2016). Other feeding studies of transgenic milk containing
antimicrobial proteins also resulted in the modulation of intestinal
microflora (Hu et al., 2012; Maga et al., 2006).
Given the differences in physiological function and structure
between rats and mice, different reactions to the same product are
possible. Previous studies showed that rats displayed no pathological changes following feeding trials with GM glyphosate-
tolerant soybean (Sakamoto et al., 2007). However, other studies
showed that some modifications occurred in hepatocyte nuclei of
mice fed diets containing GM glyphosate-tolerant soybean
(Malatesta et al., 2002, 2005). Therefore, in this study, we conducted a feeding study to evaluate the presence of any unintended
adverse effects on rats associated with the consumption of diets
containing GM milk at different doses. The results showed there
was no difference in the composition of GM and conventional milk.
The body weight, food consumption, relative organ weight, and
histopathology of each group were normal and no statistical differences were observed between groups. Significant differences
were observed between groups for some blood biochemistry parameters, e.g., ALT activity. The ALT levels in female rats of the 30G
and 10N groups were slightly lower than those of the C group. Many
studies have shown that ALT activity undergoes significant diurnal
variation, and ALT activity in the afternoon can be up to 45% higher
than its activity in the morning (Cordoba et al., 1998). In this study,
the difference observed in ALT level may be the result of differences
in the timing of blood collection. In addition, ALT level typically
declines as liver fibrosis progresses (Kim et al., 2008). However, the
liver histopathological results of female rats in 30G and 10N groups
revealed that the tissue did not contain lesions. In general, ALT
levels less than fivefold the upper limit of the normal range should
be verified (Kim et al., 2008). All the analyzed parameters were
within the normal range for rats of this age. Thus, we consider that
the differences in ALT level in female rats of the 30G and 10N
groups are not biologically significant. The TG level in male rats of
the 30G group was slightly lower than that in the C group. In terms
of pathological condition, a declining level of the TG value is
accompanied by decreased body weight (Gavino et al., 2000).
However, the body weights of male rats of the 30G group were not
decreased during the study. In addition, the TG level in male rats of
the 30G group was within the normal range. High-concentration
40
X. Chen et al. / Food and Chemical Toxicology 100 (2017) 34e41
We used somatic cell count meter (KangDaZhiXin KD-400,
China) to count the somatic cells in the transgenic milk. There
were 232,000 cells per milliliter milk on the average, which was
within the normal range. Somatic cells in the transgenic milk could
express EGFP. However, the amount of EGFP in the transgenic milk
was few to detect using western bloting. Therefore, we did not
consider the effect from EGFP.
In this study, no rats showed pathologic changes after they
consumed approximately maximum 5876.405 mg HBD3. As no
feeding study reports the total consumption of HBD3 by rats, this
result may be a guide for the future feeding studies of HBD3.
In summary, based on the principles of the GM product safety
assessment and other previous studies, we conducted a study to
assess the composition of GM milk and the general health of rats
fed GM milk in their diet. The results showed that GM milk
composition was normal and did not significantly differ to the
composition of conventional milk. In addition, the results showed
that all rats were healthy during the feeding trial. Thus, GM milk
containing HBD3 did not have any adverse effects on rats.
Competing financial interests
The authors declare that there are no competing financial
interests.
Author contributions
Conceived and designed the experiments: XC, MG, and YZ.
Performed the experiments: XC, MG, DL, SY, KY, and YZ. Analyzed
the data: XC and MG. Wrote the paper: XC.
Acknowledgements
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.
This work was supported by the National Major Project for
Production of Transgenic Breeding (No. 2013ZX08007-004).
Appendix A. Supplementary data
TBIL serves as a marker of hepatobiliary disorders. Serum concentrations of TBIL within the physiological range were shown to be
inversely associated with metabolic syndrome (Choi et al., 2013). In
metabolic syndrome, a lower value for TBIL is accompanied by
higher values for ALT, TG, and CHOL (Wu et al., 2011). However, ALT,
TG, and CHOL concentrations in male rats of the 30G group were
lower than those in the control group. Therefore, the differences
observed in TG and TBIL levels were not an evidence that HBD3
milk is unsafe. No rats exhibited clinical symptoms during the
feeding trial. In previous studies, differences were also observed in
the blood biochemistry parameters of different feeding groups
(Chen et al., 2016; Yuan et al., 2013; Zou et al., 2016).
The transgenic milk is mainly applied to change milk ingredients
more closely to the human milk, to produce pharmaceutical proteins as mammary gland bioreactor, and to improve the resistance
of mastitis (X. Liu et al., 2014; Wang et al., 2008; Yang et al., 2011).
Although there may be several physiological indexes different with
those of the corresponding control group, feeding studies of
different transgenic milk did not show a detrimental effect on the
consumer health. All experimental animals survived the duration of
the 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 many
areas including immune responses and reproduction (Ansar et al.,
1985). Therefore, they may have different responses to the same
stimulus. In this study, no differences were observed between male
and female rats for any of the analyzed parameters.
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 found
online at http://dx.doi.org/10.1016/j.fct.2016.12.012.
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ASSIGNMENT - SWOT Analysis in the Life Sciences (100 points/10%)
Addresses Course Outcomes 1, 2, 3 and 4:
Interpret and critically analyze primary scientific literature to assess the validity and reliability of scientific results
and evaluate the conclusions drawn from these data
Demonstrate proficiency in scientific principles, techniques and applications in the life sciences to evaluate
experimental design and determine compliance with standards of protocol and ethical practice
Effectively communicate scientific principles, concepts, methods, and research findings based on critical analysis
of primary literature, industry reports, and other life sciences resources
Pose vital and relevant scientific questions to identify problems, challenges, and opportunities for the
development of innovative products and services in the life sciences
SWOT, which represents Strengths, Weaknesses, Opportunities and Threats, is an analysis that is often used during strategic
business planning. It can serve as a precursor to any sort of company action, such as exploring new initiatives or identifying
possible areas for change. SWOT's primary objective is to help organizations develop a full awareness of all the factors
involved making in a decision. Although at first glance the SWOT analysis seems more fitting for a business course, its
application in biotechnology is very valuable. Many start-up companies or new products and services in the life sciences
are initiated by researchers who recognize challenges and opportunities to investigate complex phenomena and, in the
process, make new discoveries, encounter seemingly insurmountable technical problems, or recognize an opportunity to
serve other industries. The SWOT analysis enables these researchers to identify both internal influences (strengths and
weaknesses) and external influences (opportunities and threats) that will have an impact on their identified opportunity. This
process is often based on the observations gleaned from primary scientific literature – the first step in the scientific method.
Recall from last week’s critical analysis assignment that assessing the authenticity of the research findings by careful critical
analysis serves to broaden the understanding of the subject matter, and provide a background for conducting further study.
This week you will apply your ability to read and critically analyze primary scientific literature to identifying future
research challenges and/or opportunities for which you will employ a SWOT analysis.
For this assignment, you will read primary article B which is based on a specific area of biotechnology. You will also read
a few resources that will help you think creatively and innovatively before you complete the SWOT analysis. You will then
analyze and critique the article for its innovative potential in one of four areas:
1) development of a new research method
2) development of a new research project
3) development of a new product
4) development of a new service
THE ASSIGNMENT
Imagine that you are the CEO of a biotechnology company looking to invest in a new research method, a new
research project, a new product, or a new service. You have just read an interesting primary research article
(primary article B, which will be provided) and you are evaluating its potential for innovation. After reading primary
article B, as well as the SWOT analysis resources, please address each of the following items.
I. Paper Format (10 points)
A. Title page: Name, Course Name, Instructor Name, Paper Title, Due Date (2.5)
B. Sections of paper numbered and labelled properly (2.5)
C. Type double-spaced, 11-point, Times New Roman font paper with 1-inch margins (2)
D. 3-4 pages (not including title page and reference page) (3)
II. Paper Content (70 points) SWOT Analysis - Label each section of your paper according to the labels A-B below.
A. Introduction (20 points)
1. Summary (5) - summarize primary article B including the observations, question, hypothesis, experiment,
results, and conclusion of the article.
2. Eureka! moment (5) - In 4-5 sentences, describe a Eureka! moment - an aspect of the research
(introduction, materials, methods, results, discussion, conclusion) that stood out as you read primary article B
that you believe could lead to an innovative research method, project, product, or service.
3. Innovation (5) – Read all of the required resources and watch the required videos listed below on the topics
of innovation and creativity. Make notes about key take-away messages (you need not submit these notes.)
Apply what you have learned from the videos and slides to fully and specifically explain your innovative
method, service, product, or research based on the Eureka! moment you have identified.
4. Creativity (5) - explain the ways in which your innovative method, research, product, or service is creative
and can contribute to the biotechnology field.
B. SWOT Analysis (50 points) – Based on your proposed innovative method, research, product, or service,
fill in the SWOT Analysis Template provided below and embed it into your paper. Use the provided SWOT
Analysis Questions to guide you as you fill in the template.
1. Strengths (10) Explain any internal resources that would make the innovation possible
2. Weaknesses (10) Explain any internal factors that could hinder innovation
3. Opportunities (10) Explain any external factors that could contribute to the success of your innovation
4. Threats (10) Explain any external threats that could hinder the success of your innovation
5. SWOT Analysis summary (10) - Present a similar or related method, research, product, or service
to your chosen innovation from at least two additional recent primary journal articles. Based on the recent
primary literature, what do you regard as the most promising aspect of your proposed innovation and what do
you regard as the biggest threat to the future of your proposed innovation? (Be sure to cite the resources)
III. Paper References (15 points) If no references are cited, a grade of zero for this assignment will be awarded.
A. Use at least 10 references (5) All references should be cited throughout the paper: DO NOT USE WIKIPEDIA
B. In-text references in APA format (5)
C. End references in APA-format (5)
IV. Paper Grammar (5 points)
Spelling, punctuation, capitalization, sentence construction, and paragraph construction will be considered in the
grading of this assignment.
Please submit your paper as a Word document to the appropriate assignment folder. Please label your paper with
your first name, last name and SWOT Analysis. For example: Sharon Brown – SWOT Analysis.
Read
primary article B
SWOT ANALYSIS RESOURCES
Innovation in 5 Minutes
http://www.slideshare.net/Brokenbulbs/understand-innovation-in-5-minutes?related=2
Short Practical Steps to Developing an Innovator’s DNA (Slide Presentation):
http://www.slideshare.net/sivapriya28/the-innovators-dna?related=2
What is Creativity and Innovation?
http://www.slideshare.net/ingosigge/creativity-innovation-18790832?related=3
Use the resources listed below to help you choose a specific area of interest for the opportunities section of
the assignment:
Career Trends
http://www.nhscareers.nhs.uk/explore-by-career/healthcare-science/careers-in-healthcarescience/careers-in-life-sciences/
http://www.nhscareers.nhs.uk/explore-by-who-you-are/undergraduates-and-recent-graduates/
http://www.nature.com/naturejobs/science/articles/10.1038/nj7393-277a
Service Trends
http://www.lifesciencepatents.nl/en/
http://lifesciencesadvisory.com/services
http://www.indeed.com/q-Technology-Transfer-jobs.html
http://www.federallabs.org/employment/
Technology Trends
www.bio.org
http://www.biotech-now.org/
https://www.youtube.com/watch?v=ptqPJGTsIoM Future Technologies That Will Change the
World
http://www.marketsandmarkets.com/Market-Reports/life-science-chemical-biotechinstrumentation-market-38.html
http://aami-bit.org/loi/bmit OR https://www.mhealthevidence.org/journal-title/biomedicalinstrumentation-technology-association-advancement-medical-instrumentation
http://www.unboundmedicine.com/medline/journal/Biomedical_instrumentation technology
http://www.biotechmedia.com/y2001-ed-bit.html
SWOT ANALYSIS TEMPLATE – FILL IN AND EMBED IN YOUR PAPER
Internal
Strengths
1.
Weaknesses
1.
External
Opportunities
1.
Threats
1.
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