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Subchronic toxicity study of GH transgenic carp
Food and Chemical Toxicology 50 (2012) 3920–3926
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Subchronic toxicity study of GH transgenic carp Ling Yong a, Yu-Mei Liu b, Xu-Dong Jia a, Ning Li a, Wen-Zhong Zhang a,⇑ a b
National Institute for Nutrition and Food Safety, China CDC, Beijing 100021, China College of Applied Arts and Sciences, Beijing Union University, Beijing 100191, China
a r t i c l e
i n f o
Article history: Received 18 April 2012 Accepted 27 July 2012 Available online 4 August 2012 Keywords: Growth hormone transgenic carp Subchronic toxicity Sprague–Dawley rat Endocrine disruption
a b s t r a c t A subchronic toxicity study of GH (growth hormone) transgenic carp was carried out with 60 SD rats aged 4 weeks, weight 115125 g. Ten male and 10 female rats were allotted into each group. Animals of the three groups (transgenic carp group (GH–TC), parental carp group (PC) and control group) were fed soyand alfalfa-free diet (SAFD) with 10% GH transgenic carp powder, 10% parental carp powder or 10% common carp powder for 90 consecutive days, respectively. In the end of study, animals were killed by exsanguination via the carotid artery under diethyl ether anesthesia, then weights of heart, liver, kidneys, spleen, thymus, brain, ovaries and uterus/testis were measured. Pathological examination of organs was determined. Endocrine hormones of triiodothyronine (T3), thyroid hormone (T4), follicle-stimulating hormone (FSH), 17b-estradiol (E2), progesterone (P) and testosterone (T) levels were detected by specific ELISA kit. Parameters of blood routine and blood biochemical were measured. The weights of the body and organs of the rats, food intake, blood routine, blood biochemical test and serum hormones showed no significant differences among the GH transgenic carp-treated, parental carp-treated and control groups (P > 0.05). Thus, it was concluded that at the dose level of this study, GH transgenic carp showed no subchronic toxicity and endocrine disruption to SD rats. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The application of growth hormone (GH) treatment and altered GH gene expression in gene modified fishes have greatly increased aquaculture production and efficiency (McLean and Donaldson, 1993; Zhu et al., 1985; Gross et al., 1992; Devlin et al., 1994). It has been clearly demonstrated by studies that all-fish GH transgenic carp could grow faster, and the transgenic gene could inherit from generation to generation (Zhong et al., 2009a). A one-year study indicated that F(4) all-fish GH transgenic carp grew significantly fast with higher and constant serum GH levels in comparison with the control (Zhong et al., 2009b). Due to the shortened breeding period as well as decreased fishing cost in fast-growing transgenic fish, aquaculture production and economic efficiency could be significantly promoted (Ye et al., 2011). Fast-growing fishes have been considered as the best candidates of marketing transgenic animals for human consumption (Zbikowska, 2003). Abbreviations: GH, growth hormone; SD rats, Sprague–Dawley rats; SAFD, soyand alfalfa-free diet; T3, triiodothyronine; T4, thyroid hormone; FSH, folliclestimulating hormone; E2, 17b-estradiol; P, progesterone; T, testosterone; GMOs, genetically modified organisms; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TP, total protein; ALB, albumin; ALP, alkaline phosphatase; BUN, urea nitrogen; CRE, creatinine; CHO, total cholesterol; TG, triglyceride. ⇑ Corresponding author. Tel.: +86 010 67776535. E-mail address: [email protected] (W.Z. Zhang). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.07.064
The fast-growing genotypes of P0 all-fish GH transgenic carp were initially produced by microinjection of the pCAgcGHc into the fertilized eggs of carp (Yellow River variety). In all-fish GH transgenic fish, all constructs are derived from fish including the promoter, enhancer and coding sequence. The all-fish gene construct pCAgcGHc was a recombinant construct of grass carp Ctenopharyngodon idella (Valenciennes) growth hormone cDNA (gcGHc) of which the expression was driven by the b-actin gene promoter of carp (pCA) (Wang et al., 2001). The F1, F2 and F3 generation were, respectively, 1.6 times (Wang et al., 2001), 1.8–2.5 times and 1.4–1.9 times (Li et al., 2007) the body mass of non-transgenic counterparts under hatchery-reared conditions, which indicated that growth enhancement could be stably sustained through generations. The F5 generation transgenic fish was produced from crosses between a wildtype female and an F4 hemizygous transgenic male of a fast-growing transgenic strain on 25 April 2007 (Duan et al., 2009). However, the food safety of GMOs (Genetically Modified Organisms) still needs further assessment before commercial production and subsequent human consumption. For example, 90-day studies with SD rats reported three GM corn varieties (NK 603, MON 810 and MON 863) were probably of sex- and dosedependent effects on kidney and liver (Séralini et al., 2009; Vendômois et al., 2009). The harmful effects inevitably aroused suspicion of GMOs’ consumption among people and further debates in scientific community. It would be better for the acceptance of
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biotechnologies by the public, to close the scientific debate by longer, more detailed, and transparent toxicological tests on GMOs, especially when the GMOs were still experimental (Vendômois et al., 2010). Now this situation must invoke more comprehensive studies about the GH transgenic carp. So far, the food safety of GH transgenic carp has hardly been evaluated either in vivo or in vitro. Conventional toxicological tests (Chen et al., 2002; Zhang et al., 2000) were carried out by animal studies feeding with transgenic fish, but current data did not show significant difference in hematological index, morphology and tissue pictures of the main organs between treatment and the control. Since the emerging of GMOs, the subchronic toxicity study has been recommended for the safety assessment prior to commercialization of the production of biotechnology. In this study, a 90-day subchronic toxicity study to evaluate GH transgenic carp was carried out. The study aimed to explore whether the F5 generation GH transgenic carp was associated with adverse effects on mammals after oral intake, with putative relevance to humans by the 90-day rat study. Our study will provide relevant scientific evidence for food safety evaluation of GH transgenic carp. 2. Materials and methods 2.1. Materials The F5 generation GH transgenic carps and its parental carps were provided by Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). Common carps used as control were bought from a market in Beijing. The T3(triiodothyronine), T4(thyroid hormone), FSH(follicle-stimulating hormone), E2(17b-estradiol), P(progesterone) and T(testosterone) ELISA kits were bought from Cayman and BIO-LAB INC (USA). Low temperature high-speed centrifuge was from Beckman (Allegra X-22R, USA), auto chemistry analyzer was from Hitachi (7080, Japan), hematology analyzer was from Beckman Coulter (Ac. Tdiff2™, USA), microplate reader and plate washer was from Gene (Synergy 4, Hong Kong, China). GH transgenic carps, parental carps and common carps were vacuum freezedried and grinded into power, then mixed into the soy- and alfalfa-free diet, which was supplied by institute of laboratory animal sciences (license numbers: [SCXK(JING)2009-0008]). The formula of the feeds was made according to ‘‘GB 14924.3-2010: Laboratory animals—Nutrients for formula feeds’’. Diets containing of GH transgenic carp, parental carp and common carp were analyzed for nutrient composition as well as anti-nutrient and contaminant levels to verify suitability for use in animal diets. Composition analysis (nutrients: proximates, crude fiber, individual amino acids, minerals [Ca, P, Mg, K, Na, Zn, Mn, Cu, Fe], individual fatty acids, vitamins [B1/thiamine, B2/riboflavin, B6/pyridoxine, niacin, folic acid, vitamin E, and b-carotene], and anti-nutrients [trypsin inhibitor
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activity, phytic acid]. Additional vitamins (pantothenic acid) and minerals (Se) were quantified. Results of nutrition analysis see Table 1. The composition of diets all meet the requirements of Chinese national standard: Laboratory animals— Nutrients for formula feeds. 2.2. Animals and housing environment Immature Sprague–Dawley (SD) rats, 4 weeks old, were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) [license numbers: SCXK (JING) 2006–0009]. The rats were housed in an animal room maintained at 24 ± 1 °C and 50 ± 10% relative humidity with the altering 12:12-h light–dark cycle. The rats were housed in stainless steel, wire-mesh cages with one per cage. Food and distilled water were provided ad libitum. The treatment was conducted in accordance with the ‘‘Guiding Principles in the Use of Animals in Toxicology’’ by SOT. 2.3. Subchronic toxicity study The subchronic toxicity study was performed in accordance with ‘‘General Guidelines for Designing and Conducting Toxicity Studies’’ (FDA, 2000). All animals were checked by a physical examination for clinical signs of ill health and were observed for 7 days following their arrival. Then the rats were weighed, then weightranked, and were randomly divided into 3 groups, including GH transgenic carp group (GH–TC), parental carp group (PC) and control group. Each group was treated with soy- and alfalfa-free diet containing 10% GH transgenic carp powder, 10% parental carp powder or 10% common carp powder by weight in place of the commodity fish meal typically used, for 90 consecutive days, respectively. The rats were weighted and killed at the end of the study. Blood samples were collected in plastic tubes and were centrifuged for 10 min at 3000 rpm at 4 °C to obtain sera. Sera were separated and stored at 20 °C until assay for T3, T4, FSH, E2, P and T. Heart, liver, kidneys, spleen, thymus, brain, ovaries and uterus/testis were excised, carefully eliminated of excess adhering connective tissues and fat, weighed immediately and then immersion fixed in formalin. These tissues were embedded in paraffin after fixation, and were cut into 4 lm sections. Hematoxylin–eosin staining was used and the slides were examined by light microscopy. 2.4. Statistical analyses Numerical data are presented as means ± standard deviation (SD), and multiple comparison tests for the different dose groups were conducted. Homogeneity of variance was examined using the Levene test. If the Levene test indicated no significant deviations from homogeneity in the variance, the data were analyzed by oneway analysis of variance followed by Dunnett’s multiple comparison to determine the group comparisons that were significantly different. In the case of significant deviations from variance with the Levene test, the Dunnett’s T3 test was conducted to identify the significant group comparison pairs. Statistical analyses were conducted using SPSS for Windows release 12.0 K, (SPSS Inc., Chicago, IL, USA), and P-values <0.05 were considered significantly different.
3. Results 3.1. Body weight, food consumption, food utility, organ weights and organ pathology
Table 1 The nutritional analysis of soy- and alfalfa-free diet (g/g, %). Control Composition Moisture Crude protein Carbohydrate Crude fat Ash Crude fiber Calcium Total phosphorus Calcium / total phosphorus Amino acids Arg His Ile Leu Lys Met + Cys Phe + Tyr Thr Trp Val
8.86 23.8 65.6 5.03 5.62 4.79 1.25 0.91 1.37 1.43 0.654 0.956 1.80 1.38 0.89 1.48 1.02 0.265 1.08
PC 8.67 23.3 65.9 5.74 5.16 4.85 1.26 0.92 1.37 1.36 0.667 0.952 1.89 1.42 0.75 1.50 1.01 0.297 1.12
GH–TC 9.10 23.6 65.0 5.37 6.01 4.67 1.24 0.88 1.41 1.41 0.642 0.918 1.91 1.47 0.80 1.53 0.985 0.283 1.07
No changes in behavior, posture or gait was observed in either sex. Body weight and weight gains are similar between the three groups (see Fig. 1). No significant difference was found on food
Fig. 1. The animal growth curves of the subchronic toxicity.
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Table 2 Effects on week’s food-intake of rats (±SD, g) (n = 10). Group
GH–TC
Sex
Female
1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 week 9 week 10 week 11 week 12 week 13 week
96.31 ± 8.54 109.62 ± 11.54 129.41 ± 13.24 150.64 ± 14.21 164.85 ± 12.58 169.86 ± 10.59 173.59 ± 16.34 176.34 ± 12.05 176.89 ± 9.87 179.54 ± 10.54 180.54 ± 11.89 181.96 ± 13.56 181.68 ± 10.54
PC
Control
GH–TC
95.81 ± 9.85 110.85 ± 11.55 131.58 ± 10.88 152.58 ± 13.54 168.85 ± 14.25 170.52 ± 9.54 171.29 ± 10.11 177.25 ± 12.30 179.54 ± 10.25 179.52 ± 11.26 181.35 ± 13.52 180.89 ± 13.22 181.25 ± 11.98
96.81 ± 10.54 108.55 ± 9.67 128.81 ± 13.61 149.67 ± 14.59 167.89 ± 12.79 171.25 ± 12.99 172.54 ± 14.66 176.55 ± 15.55 178.61 ± 13.49 181.25 ± 13.02 180.22 ± 10.08 181.25 ± 13.84 182.16 ± 13.80
PC
Control
99.25 ± 6.35 138.25 ± 10.22 169.44 ± 10.23 179.08 ± 12.51 187.23 ± 13.05 192.43 ± 10.24 193.45 ± 8.12 194.20 ± 12.05 196.08 ± 9.15 197.54 ± 10.54 200.16 ± 11.59 201.22 ± 13.20 203.85 ± 11.54
97.51 ± 11.02 140.66 ± 9.43 171.45 ± 7.11 182.25 ± 13.54 190.22 ± 10.55 193.58 ± 10.53 194.12 ± 13.02 195.45 ± 11.06 196.59 ± 12.05 198.45 ± 10.25 198.69 ± 12.84 201.55 ± 11.02 202.11 ± 12.05
Male 98.51 ± 10.26 142.56 ± 6.84 176.89 ± 11.54 180.88 ± 13.49 188.25 ± 6.84 190.84 ± 9.25 194.26 ± 10.85 188.85 ± 11.45 195.82 ± 9.34 196.58 ± 11.54 199.84 ± 13.65 205.54 ± 12.85 205.15 ± 11.85
Table 3 Effects on week’s food utilization of rats (±SD, %) (n = 10). Group
GH–TC
Sex
Female
1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 week 9 week 10 week 11 week 12 week 13 week
25.39 ± 3.15 26.54 ± 5.44 12.75 ± 3.45 15.52 ± 4.51 11.16 ± 4.61 14.55 ± 3.25 3.64 ± 1.25 4.93 ± 2.53 3.65 ± 1.05 6.23 ± 2.54 6.84 ± 2.45 2.41 ± 0.24 1.36 ± 0.85
PC
Control
GH–TC
PC
Control
33.20 ± 7.01 47.08 ± 6.33 22.95 ± 4.55 30.04 ± 6.21 17.02 ± 3.05 15.87 ± 6.05 11.68 ± 5.05 9.35 ± 2.86 11.82 ± 2.59 12.15 ± 3.06 13.84 ± 5.52 8.96 ± 2.56 3.93 ± 1.53
34.40 ± 8.52 41.62 ± 6.88 24.31 ± 5.46 22.92 ± 6.15 17.18 ± 5.44 13.45 ± 3.59 12.55 ± 5.77 8.46 ± 3.44 11.31 ± 2.98 9.77 ± 3.05 11.70 ± 5.11 6.17 ± 3.15 3.25 ± 1.25
Male 24.96 ± 5.66 27.82 ± 3.65 15.54 ± 3.89 19.10 ± 6.54 8.76 ± 2.22 9.34 ± 3.15 5.65 ± 1.89 5.66 ± 2.56 5.20 ± 1.62 9.00 ± 3.61 5.16 ± 2.54 2.35 ± 1.02 2.64 ± 1.22
22.46 ± 4.66 23.87 ± 5.26 18.53 ± 3.85 13.05 ± 5.48 9.42 ± 3.55 14.33 ± 6.88 6.68 ± 3.62 8.05 ± 2.55 6.84 ± 3.21 6.33 ± 2.41 4.25 ± 2.01 3.95 ± 1.52 1.78 ± 0.59
consumption and food utilization between controls and treatment groups (see Tables 2 and 3). Organ weights and organ weight/body weight of GH–TC group showed no significant difference comparing with both PC group and control group (see Tables 4 and 5). Bw means body weight. Means with a common superscript letter are significantly different (P < 0.05). Of the organs examined, there were no histological changes observed in the heart, liver, kidneys, spleen, thymus, brain, ovaries and uterus/testis (see Fig. 2). 3.2. T3, T4, FSH, E2, P and T measurement The serum concentrations of T3, T4, FSH, E2, P and T were measured with specific Enzyme-Linked Immuno Sorbent Assay (ELISA) kit. Microplate reader was used to read OD 450 nm. No significant difference was observed (see Table 6). 3.3. The effect of GH–TC on blood routine and blood biochemical indicators of rats Auto chemistry analyzer and hematology analyzer were used to detect the blood routine and blood biochemical parameters of rats. The GH–TC rats showed no significant differences with those in PC and control group in blood routine and blood biochemical indicators (see Tables 7 and 8). The indicators of blood routine includes white blood cell (WBC), HGB (hemoglobin), PLT (Platelets), lymphocyte% (LYM%), intermediate cell% (MID%) and neutrophils% (GRN%). The indexes of blood biochemical are alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP),
35.61 ± 5.85 42.76 ± 9.85 22.46 ± 5.44 29.51 ± 5.38 14.36 ± 2.06 17.36 ± 5.06 12.36 ± 4.03 10.02 ± 3.52 7.65 ± 2.55 11.05 ± 3.56 11.60 ± 4.01 6.01 ± 2.54 2.72 ± 1.05
albumin (ALB), alkaline phosphatase (ALP), urea nitrogen (BUN), creatinine (CRE), total cholesterol (CHO) and triglyceride (TG). 4. Discussion Fishes is considered as the best candidate for the first marketable transgenic animal for human consumption. Since the first transgenic fish was successfully produced in the mid-1980s (Zhu et al., 1985), transgenetic studies have been conducted in over 35 fish species, half of which are important for aquaculture (e.g. carps, tilapia, catfish and salmonids). Transgenesis in fish has been extensively reviewed in many aspects during the last decade (Fletcher and Davies, 1991; Houdebine and Chourrout, 1991; Gong and Hew, 1995; Chen et al., 1996; Iyengar et al., 1996; Devlin, 1997; Maclean, 1998; Hackett et al., 1999; Hackett and Alvarez, 2000; Zbikowska, 2003). To date, competitive conditions and potential ecological risk of transgenic fish have been fully studied (Duan et al., 2009; Muir and Howard, 1999, 2001; Sundström et al., 2005; Devlin et al., 2006; Hu et al., 2007). On the other hand, studies for food safety assessment are less conducted. Guillén et al. carried out a food safety evaluation of transgenic tilapia in comparison to wild tilapia by 22 human volunteers, which is by far the only transgenic fish study performed on human. No effects were detected in human healthy volunteers after the consumption of tiGH-transgenic tilapia as an alternative feeding source (Guillén et al., 1999). Sun et al. fed cats with GH transgenic carp for three months and concluded that there is no difference of the physiological indicator and hematological index between cats fed with GH transgenic carp
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L. Yong et al. / Food and Chemical Toxicology 50 (2012) 3920–3926 Table 4 Effects on organ weights of rats (±SD, g) (n = 10). Sex
Group
Heart
Liver
Kidneys
Spleen
Thymus
Brain
Testis
Ovaries
Uterus
Male
GH–TC PC Control GH–TC
1.66 ± 0.26 1.62 ± 0.18 1.69 ± 0.19 1.17 ± 0.06
12.09 ± 1.07 11.63 ± 1.76 11.80 ± 1.37 6.79 ± 0.67
3.09 ± 0.62 3.11 ± 0.66 3.07 ± 0.67 2.28 ± 0.29
0.85 ± 0.14 0.83 ± 0.17 0.80 ± 0.14 0.70 ± 0.28
0.46 ± 0.11 0.52 ± 0.14 0.52 ± 0.12 0.33 ± 0.06
2.11 ± 0.27 2.04 ± 0.25 2.10 ± 0.14 1.88 ± 0.23
3.32 ± 0.65 2.93 ± 0.53 3.53 ± 0.42 –
– – – 0.16 ± 0.06
– – – 0.55 ± 0.21
Female
PC Control
1.13 ± 0.11 1.11 ± 0.11
7.61 ± 0.93 7.05 ± 0.69
2.23 ± 0.25 2.19 ± 0.24
0.63 ± 0.10 0.56 ± 0.11
0.35 ± 0.09 0.37 ± 0.06
1.89 ± 0.18 1.82 ± 0.31
– –
0.16 ± 0.04 0.15 ± 0.03
0.63 ± 0.11 0.52 ± 0.13
Table 5 Effects on body composition of rats (±SD, %) (n = 10). Sex
Group
Heart weight/bw, %
Liver weight/bw, %
Kidneys weight/bw, %
Spleen weight/bw, %
Thymus weight/bw, %
Brain weight/bw, %
Testis weight/bw, %
Ovaries weight/bw, %
Uterus weight/bw, %
Male
GH–TC PC Control GH–TC
0.34 ± 0.05 0.32 ± 0.03 0.36 ± 0.02 0.42 ± 0.05
2.44 ± 0.13 2.27 ± 0.22 2.48 ± 0.24 2.44 ± 0.15
0.68 ± 0.09 0.66 ± 0.05 0.68 ± 0.06 0.69 ± 0.15
0.17 ± 0.03 0.16 ± 0.03 0.17 ± 0.03 0.25 ± 0.09
0.09 ± 0.02 0.10 ± 0.03 0.11 ± 0.03 0.12 ± 0.02
0.43 ± 0.05 0.40 ± 0.03 0.44 ± 0.02 0.68 ± 0.10
0.67 ± 0.14 0.58 ± 0.11 0.75 ± 0.09 –
– – – 0.06 ± 0.02
– – – 0.20 ± 0.08
Female
PC Control
0.39 ± 0.05 0.38 ± 0.04
2.62 ± 0.38 2.44 ± 0.27
0.65 ± 0.38 0.66 ± 0.27
0.22 ± 0.04 0.19 ± 0.03
0.12 ± 0.04 0.13 ± 0.02
0.66 ± 0.13 0.63 ± 0.11
– –
0.06 ± 0.01 0.05 ± 0.01
0.22 ± 0.06 0.18 ± 0.05
Fig. 2. Photomicrographs of rat organs fixed in 10% neutral-buffered formalin and stained with hematoxylin and eosin (H&E). A–C: caudate lobe, kidney, spleen from a male control rat; D–F: caudate lobe, kidney, spleen from a male rat treated with GH transgenic carp; G-I: brain, testis from a male control rat and ovary from a female control rat; JL: brain, testis from a male rat treated with GH transgenic carp and ovary from a female rat treated with GH transgenic carp. The section shows no histological change in main organs in photomicrographs.
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Fig. 2. (continued)
Table 6 Effects on rats’ hormone levels (±SD) (n = 10). Sex
Group
T3 (ng/ml)
T4 (lg/ml)
FSH (IU/ml)
E2 (pg/ml)
P (lg/ml)
T (pg/ml)
Female
GH–TC PC Control GH–TC
5.95 ± 0.47 6.19 ± 1.26 4.50 ± 1.11 5.32 ± 1.25
2.47 ± 0.44 2.70 ± 1.69 2.48 ± 2.51 1.28 ± 0.67
4.22 ± 1.26 4.10 ± 1.29 4.30 ± 0.41 0.73 ± 0.13
14.14 ± 9.44 10.04 ± 5.31 11.19 ± 5.56 –
2.91 ± 0.14 2.87 ± 0.15 2.93 ± 0.31 –
– – – 382.47 ± 25.64
Male
PC Control
4.62 ± 1.02 4.89 ± 1.16
1.37 ± 1.00 1.23 ± 0.16
0.64 ± 0.12 0.70 ± 0.26
– –
– –
365.80 ± 24.70 408.84 ± 12.27
Table 7 Effects on blood routine of rats (±SD) (n = 10). Sex
Group
WBC
HGB
PLT
LYM%
MID%
GRN%
Female
GH–TC PC Control GH–TC
13.08 ± 4.74 11.61 ± 3.11 10.19 ± 1.65 14.7 ± 1.93
190.00 ± 19.83 195.8 ± 12.32 195.90 ± 15.72 220.30 ± 17.54
192.20 ± 49.26 196.80 ± 33.70 227.7 ± 53.11 496.7 ± 82.60
80.67 ± 5.42 82.18 ± 3.44 78.35 ± 5.71 76.10 ± 6.71
6.90 ± 1.61 6.44 ± 1.41 8.28 ± 2.45 8.36 ± 1.25
12.434.15 11.38 ± 3.49 13.37 ± 4.12 15.54 ± 6.35
Male
PC Control
15.08 ± 2.92 14.46 ± 6.07
211.10 ± 17.63 188.60 ± 66.62
508.50 ± 84.28 420.70 ± 58.43
76.74 ± 6.68 78.58 ± 3.36
7.56 ± 1.18 7.89 ± 1.44
15.70 ± 6.08 13.53 ± 2.77
and control. However, the animal they chose is not proper to extrapolate to human, and the number of animals is limited (Sun et al., 1998).
According to Pharmacology and Toxicology Study Specification of National Ministry of Health, Zhang et al. (Zhang et al., 2000) fed mice with all-fish gene transgenic carps and the results
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L. Yong et al. / Food and Chemical Toxicology 50 (2012) 3920–3926 Table 8 Effects on blood biochemical indicators of rats (±SD) (n = 10). Sex
Group
ALT
AST
TP
ALB
ALP
BUN
CRE
CHO
TG
Female
GH–TC PC Control GH–TC
35.10 ± 9.70 29.50 ± 6.75 30.33 ± 12.39 39.40 ± 16.30
211.00 ± 17.09 217.90 ± 25.35 241.17 ± 47.49 294.60 ± 51.27
83.83 ± 7.52 80.98 ± 4.87 79.35 ± 15.70 72.94 ± 5.65
48.38 ± 3.37 47.02 ± 1.94 41.53 ± 10.48 41.41 ± 2.72
59.25 ± 5.57 66.10 ± 13.41 69.17 ± 20.20 139.90 ± 33.58
8.73 ± 2.11 9.29 ± 1.66 8.58 ± 0.83 7.38 ± 0.93
72.23 ± 6.38 72.65 ± 6.97 61.30 ± 13.71 60.02 ± 8.09
1.77 ± 0.27 1.83 ± 0.13 1.69 ± 0.37 1.46 ± 0.23
0.58 ± 0.08 0.48 ± 0.08 0.52 ± 0.12 1.08 ± 0.45
Male
PC Control
48.40 ± 17.50 32.40 ± 9.41
272.90 ± 30.36 276.10 ± 29.61
74.43 ± 7.94 73.75 ± 4.44
41.87 ± 3.38 41.93 ± 1.56
133.40 ± 30.66 104.20 ± 15.18
7.15 ± 1.00 6.21 ± 1.32
58.17 ± 9.27 60.91 ± 6.75
1.55 ± 0.39 1.38 ± 0.23
1.06 ± 0.30 0.89 ± 0.27
revealed that they were substantially equivalent to normal carp on the aspect of physiology and pathology. But pharmaceuticals are different in composition, concentration, intake and many other aspects with foods, which make this study not appropriate for food safety assessment. Chen et al. (2002) used grass carp with Hu-a-IFN gene to feed rats, and no significant difference in hematological index, morphology and histopathological examination of the main organs between the treatment group and control group was observed. Liu et al. (2011) evaluated the androgenic and anti-androgenic effects of GH transgenic carp (the same carp that we used in this study) in male rats and concluded that GH transgenic carp does not have any androgenic or anti-androgenic properties in vivo screening tests. Our subchronic toxicity study was conducted to test for possible adverse effects of using GH transgenic carp for food before its marketization. In our study, GH transgenic carp, parental carp and common carp were used to replace the commodity fish meal typically used. All diets were analyzed quantitatively for nutrient composition and contaminants. This method has been used in many studies of food safety assessment of GMOs (He et al., 2009; Malley et al., 2007; Appenzeller et al., 2009a,b). In this feeding study, no significant difference was found on average body weights, organ weights, body compositions, food consumption and food utility of the male and female rats administered the GH transgenic carp, parental carp or common carp. Histopathological examination showed no pathological changes that need concern. In subchronic toxicity study, change of organ weight coefficient directly shows developing condition of animal organs and can figure out adverse effect from external environment (containing food) at an early date. The organ coefficients in our experiment showed no significant difference among GH–TC, PC and control group. In addition, serum T3, T4, FSH, E2, P and T levels are useful indicators for endocrine disruption. In this study, the serum concentrations of them showed no significant changes in treatment with GH transgenic carp, as compared to the PC and control group. These results indicate that GH transgenic carp has no endocrine disruption effects. The enzyme activity in blood is an important indicator for animal state of health. For example, AST and ALT mainly exist in histiocyte and are little in blood. When infection or injure happens in hepatic tissue, the content of AST and ALT in blood would be higher, which makes the two enzymes important indicators of liver health. In our study, the blood routine and blood biochemical parameters indicate that rats fed with GH transgenic carp showed no significant difference (P > 0.05) compared with those in other groups. In conclusion, this study demonstrates that at this dose level (10% contained in diet), GH transgenic carp shows no subchronic toxicity and endocrine disruption in SD rats. Although a classical 90-day subchronic toxicity study may not achieve adequate sensitivity to detect toxic components at extremely low level and their effects, many countries and organizations still take it as an important procedure of food safety assessment. In China, a national
standard, ‘‘GB_15193.1-2003: Procedures for toxicological assessment of food’’ takes 90-day study a necessary procedure of food safety assessment, which is in accordance with international guidelines. With respect to the safety testing of GM food and feed, the Guidance Document of the GMO Panel states (EFSA, 2006a, Section 3 7.8.4): ‘‘If the composition of the GM plant is modified substantially, or if there are any indications for the potential occurrence of unintended effects, based on the preceding molecular, compositional, phenotypic or agronomic analysis, not only new constituents, but also the whole GM food and feed should be tested’’. In these cases, the testing programme should include at least a 90-day toxicity study in rodents. However, the study needs to be repeated and deepened. It is recommended that GMOs need more serious standardized tests on at least three mammalian species for at least three months employing larger sample sizes, and up to one and two years before commercialization (Séralini et al., 2009). They also called for the criteria for testing adverse health effects overall for GM food or feed products. Here we call for the same treat of GM carp before we put it into the market. Conflict of Interest The authors declare no conflicts of interest. References Appenzeller, M.L., Malley, A.L., MacKenzie, A.S., Hoban, D., Delaney, B., 2009a. Subchronic feeding study with genetically modified stacked trait lepidopteran and coleopteran resistant (DAS-Ø15Ø7-1xDAS-59122-7) maize grain in Sprague–Dawley rats. Food Chem. Toxicol. 47, 1512–1520. Appenzeller, M.L., Munley, M.S., Hoban, D., Sykes, P.G., Malley, A.L., Delaney, B., 2009b. Subchronic feeding study of grain from herbicide-tolerant maize DPØ9814Ø-6 in Sprague–Dawley rats. Food Chem. Toxicol. 47, 2269–2280. Chen, K.J., Zhang, H.Y., Zhang, X.W., Xiao, T.Y., Chen, L.X., Su, J.M., Wang, D.G., 2002. A study on the safety of feeding transgenic grass carps to rat. J. Hunan Agric. Univ. (Nat. Sci.) 28, 147–149 (In Chinese). Chen, T.T., Vrolijk, N.H., Lu, J.K., Lin, C.M., Reimschuessel, R., Dunham, R.A., 1996. Transgenic fish and its application in basic and applied research. Biotechnol. Annu. Rev. 2, 205–236. Devlin, R.H., 1997. Transgenic salmonids. In: Houdebine, L.M. (Ed.), Transgenic Animals: Generation and Use. 105–117. Devlin, R.H., Yesaki, T.Y., Biagi, C.A., Donaldson, E.M., 1994. Extraordinary salmon growth. Nature 371, 209–210. Devlin, R.H., Sundström, L.F., Muir, W.M., 2006. Interface of biotechnology and ecology for environmental risk assessments of transgenic fish. Trends Biotechnol. 24, 89–97. Duan, M., Zhang, T., Hu, W., Sundström, L.F., Wang, Y., Li, Z., Zhu, Z., 2009. Elevated ability to compete for limited food resources by ‘all-fish’ growth hormone transgenic common carp Cyprinus carpio. J. Fish Biol. 75 (6), 1459–1472. FDA Redbook 2000: IV.B.1 General Guidelines for Designing and Conducting Toxicity Studies November 2003. Fletcher, G.L., Davies, P.L., 1991. Transgenic fish for aquaculture. Genet. Eng. 13, 331–370. He, X.Y., Tang, M.Z., Luo, Y.B., Li, X., Cao, S.S., Yu, J.J., Delaney, B., Huang, K.L., 2009. A 90-day toxicology study of transgenic lysine-rich maize grain (Y642) in Sprague–Dawley rats. Food Chem. Toxicol. 47, 425–432. Houdebine, L.M., Chourrout, D., 1991. Transgenesis in fish. Experientia 47, 891–897. Gong, Z., Hew, C.L., 1995. Transgenic fish in aquaculture and developmental biology. Curr. Top. Dev. Biol. 30, 177–214. Gross, M.L., Schneider, J.F., Moav, N., Moav, B., Alvarez, C., Myster, S.H., Liu, Z., Hallerman, E.M., Hackett, P.B., Guise, K.S., Faras, A.J., Kapuscinski, A.R., 1992. Molecular analysis and growth evaluation of northern pike (Esox lucius) microinjected with growth hormone genes. Aquaculture 103, 253–273.
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Guillén, I., Berlanga, J., Valenzuela, C.M., Morales, A., Toledo, J., Estrada, M.P., Puentes, P., Hayes, O., Fuente, J., 1999. Safety evaluation of transgenic tilapia with accelerated growth. Mar. Biotechnol. 1, 2–14. Hackett, P.B, Alvarez, M.C., 2000. The molecular genetics of transgenic Fish. In: Fingerman, M., Nagabhushanam, R. (Eds), Recent Advances in Marine Biotechnology, In: Aquaculture, vol. 4, Science Publishers, Inc., Enfield, NH, USA, pp. 77–145. Hackett, P.B., Izsvak, Z., Ivics, Z., Caldovic, L., 1999. Development of genetic tools for transgenic animals. In: Murray, J.D., Anderson, G.B., Oberbauer, A.M., McGloughlin, M.M. (Eds.), Transgenic Animals in Agriculture. CAB International Wallingford, UK, pp. 19–35. Hu, W., Wang, Y.P., Zhu, Z.Y., 2007. Progress in the evaluation of transgenic fish for possible ecological risk and its containment strategies. Sci. China, Ser. C Life Sci. 50, 573–579. Iyengar, A., Muller, F., Maclean, N., 1996. Regulation and expression of transgenes in fish – a review. Transgenic Res. 5, 147–166. Li, D., Fu, C., Hu, W., Zhong, S., Wang, Y., Zhu, Z., 2007. Rapid growth cost in ‘‘all-fish’’ growth hormone gene transgenic carp: reduced critical swimming speed. Chin. Sci. Bull. 52, 1501–1506. Liu, Y., Zhang, W., Yong, L., Zhao, X.H., Jia, X.D., Li, N., 2011. An assessment of androgenic/anti-androgenic effects of GH transgenic carp by Hershberger assay. Biomed. Environ. Sci. 24 (4), 445–449. Maclean, N., 1998. Regulation and exploitation of transgenes in fish. Mutat. Res. 399, 255–266. Malley, A.L., Everds, E.N., Reynolds, J., Mann, C.P., Lamb, I., Rood, T., Schmidt, J., Layton, J.R., Prochaska, M.L., Hinds, M., Locke, M., Chui, C.F., Claussen, F., Mattsson, L.J., Delaney, B., 2007. Subchronic feeding study of DAS59122-7 maize grain in Sprague–Dawley rats. Food Chem. Toxicol. 45, 1277–1292. McLean, E., Donaldson, E.M., 1993. The role of growth hormone in the growth of poikilotherms. In: Schreibman, M.P., Scanes, C.G., Pang, P.K.T. (Eds.), The Endocrinology of Growth, Development, and Metabolism in Vertebrates. Academic Press, San Diego, CA, pp. 43–71. Muir, W.M., Howard, R.D., 1999. Possible ecological risks of transgenic organism release when transgenes affect mating success: sexual selection and the Trojan gene hypothesis. Proc. Nat. Acad. Sci. USA 96, 13853–13856.
Muir, W.M., Howard, R.D., 2001. Fitness components and ecological risks of transgenic release: a model using Japanese medaka (Oryzias latipes). Am. Nat. 158, 1–16. Séralini, G.E., Spiroux de Vendômois, J.S., Cellier, D., Sultan, C., Buiatti, M., Gallagher, L., Antoniou, M., Dronamraju, K.R., 2009. How subchronic and chronic health effects can be neglected for GMOs, pesticides or chemicals. Int. J. Biol. Sci. 5 (5), 438–443. Sundström, L.F., Lõhmus, M., Devlin, R.H., 2005. Selection on increased intrinsic growth rates in coho salmon Oncorhynchus kisutch. Evolution 59, 1560–1569. Sun, X.W., Yan, X.C., Liang, L.Q., Cao, D.C., Zhang, L.M., 1998. Research on transgenic fish as food. Chin. High Technol. Lett. 3, 45–49 (In Chinese). Vendômois, J.S., Roullier, F., Cellier, D., Séralini, G.E., 2009. A Comparison of the effects of three GM corn varieties on mammalian health. Int. J. Biol. Sci. 55 (7), 706–726. Vendômois, J.S., Cellier, D., Vélot, C., Clair, E., Mesnage, R., Séralini, G.E., 2010. Debate on GMOs health risks after statistical findings in regulatory tests. Int. J. Biol. Sci. 6, 590–598. Wang, Y., Hu, W., Wu, G., Sun, Y., Chen, S., Zhang, F., Zhu, Z., Feng, J., Zhang, X., 2001. Genetic analysis of ‘‘all-fish’’ growth hormone gene transferred carp (Cyprinus carpio L.) and its F1 generation. Chin. Sci. Bull. 46, 1174–1177. Ye, X., Tian, Y.Y., Gao, F.Y., 2011. Progress in transgenic fish techniques and application. Yi Chuan 33 (5), 494–503 (In Chinese). Zbikowska, H.M., 2003. Fish can be first–advances in fish transgenesis for commercial applications. Transgenic Res. 12, 379–389. Zhang, F.Y., Wang, Y.P., Hu, W., Cui, Z.B., Zhu, Z.Y., Yang, J., Peng, R.X., 2000. Physiological and pathological analysis of the mice fed with ‘‘all fish’’ gene transferred yellow river carp. Chin. High Technol. Lett. 7, 17–19 (In Chinese). Zhong, S., Luo, D.J., Wu, G., Xu, J., Wang, Y.P., Zhu, Z.Y., 2009a. The growth promoting activity of two different kinds of reconstructed terminators in transgenic common carp. Yi Chuan 31 (8), 831–836 (In Chinese). Zhong, S., Wang, Y.P., Pei, D.S., Luo, D.J., Liao, L.J., Zhu, Z.Y., 2009b. A one-year investigation of the relationship between serum GH levels and the growth of F(4) transgenic and non-transgenic common carp Cyprinus carpio. J. Fish Biol. 75 (5), 1092–1100. Zhu, Z., Li, G., He, L., Chen, S., 1985. Novel gene transfer into the fertilized eggs of goldfish (Carassius auratus L. 1758). Zeitschrift fur Angewandte Ichthyologie 1, 31–34.
Contents lists available at SciVerse ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Subchronic toxicity study of GH transgenic carp Ling Yong a, Yu-Mei Liu b, Xu-Dong Jia a, Ning Li a, Wen-Zhong Zhang a,⇑ a b
National Institute for Nutrition and Food Safety, China CDC, Beijing 100021, China College of Applied Arts and Sciences, Beijing Union University, Beijing 100191, China
a r t i c l e
i n f o
Article history: Received 18 April 2012 Accepted 27 July 2012 Available online 4 August 2012 Keywords: Growth hormone transgenic carp Subchronic toxicity Sprague–Dawley rat Endocrine disruption
a b s t r a c t A subchronic toxicity study of GH (growth hormone) transgenic carp was carried out with 60 SD rats aged 4 weeks, weight 115125 g. Ten male and 10 female rats were allotted into each group. Animals of the three groups (transgenic carp group (GH–TC), parental carp group (PC) and control group) were fed soyand alfalfa-free diet (SAFD) with 10% GH transgenic carp powder, 10% parental carp powder or 10% common carp powder for 90 consecutive days, respectively. In the end of study, animals were killed by exsanguination via the carotid artery under diethyl ether anesthesia, then weights of heart, liver, kidneys, spleen, thymus, brain, ovaries and uterus/testis were measured. Pathological examination of organs was determined. Endocrine hormones of triiodothyronine (T3), thyroid hormone (T4), follicle-stimulating hormone (FSH), 17b-estradiol (E2), progesterone (P) and testosterone (T) levels were detected by specific ELISA kit. Parameters of blood routine and blood biochemical were measured. The weights of the body and organs of the rats, food intake, blood routine, blood biochemical test and serum hormones showed no significant differences among the GH transgenic carp-treated, parental carp-treated and control groups (P > 0.05). Thus, it was concluded that at the dose level of this study, GH transgenic carp showed no subchronic toxicity and endocrine disruption to SD rats. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The application of growth hormone (GH) treatment and altered GH gene expression in gene modified fishes have greatly increased aquaculture production and efficiency (McLean and Donaldson, 1993; Zhu et al., 1985; Gross et al., 1992; Devlin et al., 1994). It has been clearly demonstrated by studies that all-fish GH transgenic carp could grow faster, and the transgenic gene could inherit from generation to generation (Zhong et al., 2009a). A one-year study indicated that F(4) all-fish GH transgenic carp grew significantly fast with higher and constant serum GH levels in comparison with the control (Zhong et al., 2009b). Due to the shortened breeding period as well as decreased fishing cost in fast-growing transgenic fish, aquaculture production and economic efficiency could be significantly promoted (Ye et al., 2011). Fast-growing fishes have been considered as the best candidates of marketing transgenic animals for human consumption (Zbikowska, 2003). Abbreviations: GH, growth hormone; SD rats, Sprague–Dawley rats; SAFD, soyand alfalfa-free diet; T3, triiodothyronine; T4, thyroid hormone; FSH, folliclestimulating hormone; E2, 17b-estradiol; P, progesterone; T, testosterone; GMOs, genetically modified organisms; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TP, total protein; ALB, albumin; ALP, alkaline phosphatase; BUN, urea nitrogen; CRE, creatinine; CHO, total cholesterol; TG, triglyceride. ⇑ Corresponding author. Tel.: +86 010 67776535. E-mail address: [email protected] (W.Z. Zhang). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.07.064
The fast-growing genotypes of P0 all-fish GH transgenic carp were initially produced by microinjection of the pCAgcGHc into the fertilized eggs of carp (Yellow River variety). In all-fish GH transgenic fish, all constructs are derived from fish including the promoter, enhancer and coding sequence. The all-fish gene construct pCAgcGHc was a recombinant construct of grass carp Ctenopharyngodon idella (Valenciennes) growth hormone cDNA (gcGHc) of which the expression was driven by the b-actin gene promoter of carp (pCA) (Wang et al., 2001). The F1, F2 and F3 generation were, respectively, 1.6 times (Wang et al., 2001), 1.8–2.5 times and 1.4–1.9 times (Li et al., 2007) the body mass of non-transgenic counterparts under hatchery-reared conditions, which indicated that growth enhancement could be stably sustained through generations. The F5 generation transgenic fish was produced from crosses between a wildtype female and an F4 hemizygous transgenic male of a fast-growing transgenic strain on 25 April 2007 (Duan et al., 2009). However, the food safety of GMOs (Genetically Modified Organisms) still needs further assessment before commercial production and subsequent human consumption. For example, 90-day studies with SD rats reported three GM corn varieties (NK 603, MON 810 and MON 863) were probably of sex- and dosedependent effects on kidney and liver (Séralini et al., 2009; Vendômois et al., 2009). The harmful effects inevitably aroused suspicion of GMOs’ consumption among people and further debates in scientific community. It would be better for the acceptance of
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biotechnologies by the public, to close the scientific debate by longer, more detailed, and transparent toxicological tests on GMOs, especially when the GMOs were still experimental (Vendômois et al., 2010). Now this situation must invoke more comprehensive studies about the GH transgenic carp. So far, the food safety of GH transgenic carp has hardly been evaluated either in vivo or in vitro. Conventional toxicological tests (Chen et al., 2002; Zhang et al., 2000) were carried out by animal studies feeding with transgenic fish, but current data did not show significant difference in hematological index, morphology and tissue pictures of the main organs between treatment and the control. Since the emerging of GMOs, the subchronic toxicity study has been recommended for the safety assessment prior to commercialization of the production of biotechnology. In this study, a 90-day subchronic toxicity study to evaluate GH transgenic carp was carried out. The study aimed to explore whether the F5 generation GH transgenic carp was associated with adverse effects on mammals after oral intake, with putative relevance to humans by the 90-day rat study. Our study will provide relevant scientific evidence for food safety evaluation of GH transgenic carp. 2. Materials and methods 2.1. Materials The F5 generation GH transgenic carps and its parental carps were provided by Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). Common carps used as control were bought from a market in Beijing. The T3(triiodothyronine), T4(thyroid hormone), FSH(follicle-stimulating hormone), E2(17b-estradiol), P(progesterone) and T(testosterone) ELISA kits were bought from Cayman and BIO-LAB INC (USA). Low temperature high-speed centrifuge was from Beckman (Allegra X-22R, USA), auto chemistry analyzer was from Hitachi (7080, Japan), hematology analyzer was from Beckman Coulter (Ac. Tdiff2™, USA), microplate reader and plate washer was from Gene (Synergy 4, Hong Kong, China). GH transgenic carps, parental carps and common carps were vacuum freezedried and grinded into power, then mixed into the soy- and alfalfa-free diet, which was supplied by institute of laboratory animal sciences (license numbers: [SCXK(JING)2009-0008]). The formula of the feeds was made according to ‘‘GB 14924.3-2010: Laboratory animals—Nutrients for formula feeds’’. Diets containing of GH transgenic carp, parental carp and common carp were analyzed for nutrient composition as well as anti-nutrient and contaminant levels to verify suitability for use in animal diets. Composition analysis (nutrients: proximates, crude fiber, individual amino acids, minerals [Ca, P, Mg, K, Na, Zn, Mn, Cu, Fe], individual fatty acids, vitamins [B1/thiamine, B2/riboflavin, B6/pyridoxine, niacin, folic acid, vitamin E, and b-carotene], and anti-nutrients [trypsin inhibitor
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activity, phytic acid]. Additional vitamins (pantothenic acid) and minerals (Se) were quantified. Results of nutrition analysis see Table 1. The composition of diets all meet the requirements of Chinese national standard: Laboratory animals— Nutrients for formula feeds. 2.2. Animals and housing environment Immature Sprague–Dawley (SD) rats, 4 weeks old, were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) [license numbers: SCXK (JING) 2006–0009]. The rats were housed in an animal room maintained at 24 ± 1 °C and 50 ± 10% relative humidity with the altering 12:12-h light–dark cycle. The rats were housed in stainless steel, wire-mesh cages with one per cage. Food and distilled water were provided ad libitum. The treatment was conducted in accordance with the ‘‘Guiding Principles in the Use of Animals in Toxicology’’ by SOT. 2.3. Subchronic toxicity study The subchronic toxicity study was performed in accordance with ‘‘General Guidelines for Designing and Conducting Toxicity Studies’’ (FDA, 2000). All animals were checked by a physical examination for clinical signs of ill health and were observed for 7 days following their arrival. Then the rats were weighed, then weightranked, and were randomly divided into 3 groups, including GH transgenic carp group (GH–TC), parental carp group (PC) and control group. Each group was treated with soy- and alfalfa-free diet containing 10% GH transgenic carp powder, 10% parental carp powder or 10% common carp powder by weight in place of the commodity fish meal typically used, for 90 consecutive days, respectively. The rats were weighted and killed at the end of the study. Blood samples were collected in plastic tubes and were centrifuged for 10 min at 3000 rpm at 4 °C to obtain sera. Sera were separated and stored at 20 °C until assay for T3, T4, FSH, E2, P and T. Heart, liver, kidneys, spleen, thymus, brain, ovaries and uterus/testis were excised, carefully eliminated of excess adhering connective tissues and fat, weighed immediately and then immersion fixed in formalin. These tissues were embedded in paraffin after fixation, and were cut into 4 lm sections. Hematoxylin–eosin staining was used and the slides were examined by light microscopy. 2.4. Statistical analyses Numerical data are presented as means ± standard deviation (SD), and multiple comparison tests for the different dose groups were conducted. Homogeneity of variance was examined using the Levene test. If the Levene test indicated no significant deviations from homogeneity in the variance, the data were analyzed by oneway analysis of variance followed by Dunnett’s multiple comparison to determine the group comparisons that were significantly different. In the case of significant deviations from variance with the Levene test, the Dunnett’s T3 test was conducted to identify the significant group comparison pairs. Statistical analyses were conducted using SPSS for Windows release 12.0 K, (SPSS Inc., Chicago, IL, USA), and P-values <0.05 were considered significantly different.
3. Results 3.1. Body weight, food consumption, food utility, organ weights and organ pathology
Table 1 The nutritional analysis of soy- and alfalfa-free diet (g/g, %). Control Composition Moisture Crude protein Carbohydrate Crude fat Ash Crude fiber Calcium Total phosphorus Calcium / total phosphorus Amino acids Arg His Ile Leu Lys Met + Cys Phe + Tyr Thr Trp Val
8.86 23.8 65.6 5.03 5.62 4.79 1.25 0.91 1.37 1.43 0.654 0.956 1.80 1.38 0.89 1.48 1.02 0.265 1.08
PC 8.67 23.3 65.9 5.74 5.16 4.85 1.26 0.92 1.37 1.36 0.667 0.952 1.89 1.42 0.75 1.50 1.01 0.297 1.12
GH–TC 9.10 23.6 65.0 5.37 6.01 4.67 1.24 0.88 1.41 1.41 0.642 0.918 1.91 1.47 0.80 1.53 0.985 0.283 1.07
No changes in behavior, posture or gait was observed in either sex. Body weight and weight gains are similar between the three groups (see Fig. 1). No significant difference was found on food
Fig. 1. The animal growth curves of the subchronic toxicity.
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Table 2 Effects on week’s food-intake of rats (±SD, g) (n = 10). Group
GH–TC
Sex
Female
1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 week 9 week 10 week 11 week 12 week 13 week
96.31 ± 8.54 109.62 ± 11.54 129.41 ± 13.24 150.64 ± 14.21 164.85 ± 12.58 169.86 ± 10.59 173.59 ± 16.34 176.34 ± 12.05 176.89 ± 9.87 179.54 ± 10.54 180.54 ± 11.89 181.96 ± 13.56 181.68 ± 10.54
PC
Control
GH–TC
95.81 ± 9.85 110.85 ± 11.55 131.58 ± 10.88 152.58 ± 13.54 168.85 ± 14.25 170.52 ± 9.54 171.29 ± 10.11 177.25 ± 12.30 179.54 ± 10.25 179.52 ± 11.26 181.35 ± 13.52 180.89 ± 13.22 181.25 ± 11.98
96.81 ± 10.54 108.55 ± 9.67 128.81 ± 13.61 149.67 ± 14.59 167.89 ± 12.79 171.25 ± 12.99 172.54 ± 14.66 176.55 ± 15.55 178.61 ± 13.49 181.25 ± 13.02 180.22 ± 10.08 181.25 ± 13.84 182.16 ± 13.80
PC
Control
99.25 ± 6.35 138.25 ± 10.22 169.44 ± 10.23 179.08 ± 12.51 187.23 ± 13.05 192.43 ± 10.24 193.45 ± 8.12 194.20 ± 12.05 196.08 ± 9.15 197.54 ± 10.54 200.16 ± 11.59 201.22 ± 13.20 203.85 ± 11.54
97.51 ± 11.02 140.66 ± 9.43 171.45 ± 7.11 182.25 ± 13.54 190.22 ± 10.55 193.58 ± 10.53 194.12 ± 13.02 195.45 ± 11.06 196.59 ± 12.05 198.45 ± 10.25 198.69 ± 12.84 201.55 ± 11.02 202.11 ± 12.05
Male 98.51 ± 10.26 142.56 ± 6.84 176.89 ± 11.54 180.88 ± 13.49 188.25 ± 6.84 190.84 ± 9.25 194.26 ± 10.85 188.85 ± 11.45 195.82 ± 9.34 196.58 ± 11.54 199.84 ± 13.65 205.54 ± 12.85 205.15 ± 11.85
Table 3 Effects on week’s food utilization of rats (±SD, %) (n = 10). Group
GH–TC
Sex
Female
1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 week 9 week 10 week 11 week 12 week 13 week
25.39 ± 3.15 26.54 ± 5.44 12.75 ± 3.45 15.52 ± 4.51 11.16 ± 4.61 14.55 ± 3.25 3.64 ± 1.25 4.93 ± 2.53 3.65 ± 1.05 6.23 ± 2.54 6.84 ± 2.45 2.41 ± 0.24 1.36 ± 0.85
PC
Control
GH–TC
PC
Control
33.20 ± 7.01 47.08 ± 6.33 22.95 ± 4.55 30.04 ± 6.21 17.02 ± 3.05 15.87 ± 6.05 11.68 ± 5.05 9.35 ± 2.86 11.82 ± 2.59 12.15 ± 3.06 13.84 ± 5.52 8.96 ± 2.56 3.93 ± 1.53
34.40 ± 8.52 41.62 ± 6.88 24.31 ± 5.46 22.92 ± 6.15 17.18 ± 5.44 13.45 ± 3.59 12.55 ± 5.77 8.46 ± 3.44 11.31 ± 2.98 9.77 ± 3.05 11.70 ± 5.11 6.17 ± 3.15 3.25 ± 1.25
Male 24.96 ± 5.66 27.82 ± 3.65 15.54 ± 3.89 19.10 ± 6.54 8.76 ± 2.22 9.34 ± 3.15 5.65 ± 1.89 5.66 ± 2.56 5.20 ± 1.62 9.00 ± 3.61 5.16 ± 2.54 2.35 ± 1.02 2.64 ± 1.22
22.46 ± 4.66 23.87 ± 5.26 18.53 ± 3.85 13.05 ± 5.48 9.42 ± 3.55 14.33 ± 6.88 6.68 ± 3.62 8.05 ± 2.55 6.84 ± 3.21 6.33 ± 2.41 4.25 ± 2.01 3.95 ± 1.52 1.78 ± 0.59
consumption and food utilization between controls and treatment groups (see Tables 2 and 3). Organ weights and organ weight/body weight of GH–TC group showed no significant difference comparing with both PC group and control group (see Tables 4 and 5). Bw means body weight. Means with a common superscript letter are significantly different (P < 0.05). Of the organs examined, there were no histological changes observed in the heart, liver, kidneys, spleen, thymus, brain, ovaries and uterus/testis (see Fig. 2). 3.2. T3, T4, FSH, E2, P and T measurement The serum concentrations of T3, T4, FSH, E2, P and T were measured with specific Enzyme-Linked Immuno Sorbent Assay (ELISA) kit. Microplate reader was used to read OD 450 nm. No significant difference was observed (see Table 6). 3.3. The effect of GH–TC on blood routine and blood biochemical indicators of rats Auto chemistry analyzer and hematology analyzer were used to detect the blood routine and blood biochemical parameters of rats. The GH–TC rats showed no significant differences with those in PC and control group in blood routine and blood biochemical indicators (see Tables 7 and 8). The indicators of blood routine includes white blood cell (WBC), HGB (hemoglobin), PLT (Platelets), lymphocyte% (LYM%), intermediate cell% (MID%) and neutrophils% (GRN%). The indexes of blood biochemical are alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP),
35.61 ± 5.85 42.76 ± 9.85 22.46 ± 5.44 29.51 ± 5.38 14.36 ± 2.06 17.36 ± 5.06 12.36 ± 4.03 10.02 ± 3.52 7.65 ± 2.55 11.05 ± 3.56 11.60 ± 4.01 6.01 ± 2.54 2.72 ± 1.05
albumin (ALB), alkaline phosphatase (ALP), urea nitrogen (BUN), creatinine (CRE), total cholesterol (CHO) and triglyceride (TG). 4. Discussion Fishes is considered as the best candidate for the first marketable transgenic animal for human consumption. Since the first transgenic fish was successfully produced in the mid-1980s (Zhu et al., 1985), transgenetic studies have been conducted in over 35 fish species, half of which are important for aquaculture (e.g. carps, tilapia, catfish and salmonids). Transgenesis in fish has been extensively reviewed in many aspects during the last decade (Fletcher and Davies, 1991; Houdebine and Chourrout, 1991; Gong and Hew, 1995; Chen et al., 1996; Iyengar et al., 1996; Devlin, 1997; Maclean, 1998; Hackett et al., 1999; Hackett and Alvarez, 2000; Zbikowska, 2003). To date, competitive conditions and potential ecological risk of transgenic fish have been fully studied (Duan et al., 2009; Muir and Howard, 1999, 2001; Sundström et al., 2005; Devlin et al., 2006; Hu et al., 2007). On the other hand, studies for food safety assessment are less conducted. Guillén et al. carried out a food safety evaluation of transgenic tilapia in comparison to wild tilapia by 22 human volunteers, which is by far the only transgenic fish study performed on human. No effects were detected in human healthy volunteers after the consumption of tiGH-transgenic tilapia as an alternative feeding source (Guillén et al., 1999). Sun et al. fed cats with GH transgenic carp for three months and concluded that there is no difference of the physiological indicator and hematological index between cats fed with GH transgenic carp
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L. Yong et al. / Food and Chemical Toxicology 50 (2012) 3920–3926 Table 4 Effects on organ weights of rats (±SD, g) (n = 10). Sex
Group
Heart
Liver
Kidneys
Spleen
Thymus
Brain
Testis
Ovaries
Uterus
Male
GH–TC PC Control GH–TC
1.66 ± 0.26 1.62 ± 0.18 1.69 ± 0.19 1.17 ± 0.06
12.09 ± 1.07 11.63 ± 1.76 11.80 ± 1.37 6.79 ± 0.67
3.09 ± 0.62 3.11 ± 0.66 3.07 ± 0.67 2.28 ± 0.29
0.85 ± 0.14 0.83 ± 0.17 0.80 ± 0.14 0.70 ± 0.28
0.46 ± 0.11 0.52 ± 0.14 0.52 ± 0.12 0.33 ± 0.06
2.11 ± 0.27 2.04 ± 0.25 2.10 ± 0.14 1.88 ± 0.23
3.32 ± 0.65 2.93 ± 0.53 3.53 ± 0.42 –
– – – 0.16 ± 0.06
– – – 0.55 ± 0.21
Female
PC Control
1.13 ± 0.11 1.11 ± 0.11
7.61 ± 0.93 7.05 ± 0.69
2.23 ± 0.25 2.19 ± 0.24
0.63 ± 0.10 0.56 ± 0.11
0.35 ± 0.09 0.37 ± 0.06
1.89 ± 0.18 1.82 ± 0.31
– –
0.16 ± 0.04 0.15 ± 0.03
0.63 ± 0.11 0.52 ± 0.13
Table 5 Effects on body composition of rats (±SD, %) (n = 10). Sex
Group
Heart weight/bw, %
Liver weight/bw, %
Kidneys weight/bw, %
Spleen weight/bw, %
Thymus weight/bw, %
Brain weight/bw, %
Testis weight/bw, %
Ovaries weight/bw, %
Uterus weight/bw, %
Male
GH–TC PC Control GH–TC
0.34 ± 0.05 0.32 ± 0.03 0.36 ± 0.02 0.42 ± 0.05
2.44 ± 0.13 2.27 ± 0.22 2.48 ± 0.24 2.44 ± 0.15
0.68 ± 0.09 0.66 ± 0.05 0.68 ± 0.06 0.69 ± 0.15
0.17 ± 0.03 0.16 ± 0.03 0.17 ± 0.03 0.25 ± 0.09
0.09 ± 0.02 0.10 ± 0.03 0.11 ± 0.03 0.12 ± 0.02
0.43 ± 0.05 0.40 ± 0.03 0.44 ± 0.02 0.68 ± 0.10
0.67 ± 0.14 0.58 ± 0.11 0.75 ± 0.09 –
– – – 0.06 ± 0.02
– – – 0.20 ± 0.08
Female
PC Control
0.39 ± 0.05 0.38 ± 0.04
2.62 ± 0.38 2.44 ± 0.27
0.65 ± 0.38 0.66 ± 0.27
0.22 ± 0.04 0.19 ± 0.03
0.12 ± 0.04 0.13 ± 0.02
0.66 ± 0.13 0.63 ± 0.11
– –
0.06 ± 0.01 0.05 ± 0.01
0.22 ± 0.06 0.18 ± 0.05
Fig. 2. Photomicrographs of rat organs fixed in 10% neutral-buffered formalin and stained with hematoxylin and eosin (H&E). A–C: caudate lobe, kidney, spleen from a male control rat; D–F: caudate lobe, kidney, spleen from a male rat treated with GH transgenic carp; G-I: brain, testis from a male control rat and ovary from a female control rat; JL: brain, testis from a male rat treated with GH transgenic carp and ovary from a female rat treated with GH transgenic carp. The section shows no histological change in main organs in photomicrographs.
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Fig. 2. (continued)
Table 6 Effects on rats’ hormone levels (±SD) (n = 10). Sex
Group
T3 (ng/ml)
T4 (lg/ml)
FSH (IU/ml)
E2 (pg/ml)
P (lg/ml)
T (pg/ml)
Female
GH–TC PC Control GH–TC
5.95 ± 0.47 6.19 ± 1.26 4.50 ± 1.11 5.32 ± 1.25
2.47 ± 0.44 2.70 ± 1.69 2.48 ± 2.51 1.28 ± 0.67
4.22 ± 1.26 4.10 ± 1.29 4.30 ± 0.41 0.73 ± 0.13
14.14 ± 9.44 10.04 ± 5.31 11.19 ± 5.56 –
2.91 ± 0.14 2.87 ± 0.15 2.93 ± 0.31 –
– – – 382.47 ± 25.64
Male
PC Control
4.62 ± 1.02 4.89 ± 1.16
1.37 ± 1.00 1.23 ± 0.16
0.64 ± 0.12 0.70 ± 0.26
– –
– –
365.80 ± 24.70 408.84 ± 12.27
Table 7 Effects on blood routine of rats (±SD) (n = 10). Sex
Group
WBC
HGB
PLT
LYM%
MID%
GRN%
Female
GH–TC PC Control GH–TC
13.08 ± 4.74 11.61 ± 3.11 10.19 ± 1.65 14.7 ± 1.93
190.00 ± 19.83 195.8 ± 12.32 195.90 ± 15.72 220.30 ± 17.54
192.20 ± 49.26 196.80 ± 33.70 227.7 ± 53.11 496.7 ± 82.60
80.67 ± 5.42 82.18 ± 3.44 78.35 ± 5.71 76.10 ± 6.71
6.90 ± 1.61 6.44 ± 1.41 8.28 ± 2.45 8.36 ± 1.25
12.434.15 11.38 ± 3.49 13.37 ± 4.12 15.54 ± 6.35
Male
PC Control
15.08 ± 2.92 14.46 ± 6.07
211.10 ± 17.63 188.60 ± 66.62
508.50 ± 84.28 420.70 ± 58.43
76.74 ± 6.68 78.58 ± 3.36
7.56 ± 1.18 7.89 ± 1.44
15.70 ± 6.08 13.53 ± 2.77
and control. However, the animal they chose is not proper to extrapolate to human, and the number of animals is limited (Sun et al., 1998).
According to Pharmacology and Toxicology Study Specification of National Ministry of Health, Zhang et al. (Zhang et al., 2000) fed mice with all-fish gene transgenic carps and the results
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L. Yong et al. / Food and Chemical Toxicology 50 (2012) 3920–3926 Table 8 Effects on blood biochemical indicators of rats (±SD) (n = 10). Sex
Group
ALT
AST
TP
ALB
ALP
BUN
CRE
CHO
TG
Female
GH–TC PC Control GH–TC
35.10 ± 9.70 29.50 ± 6.75 30.33 ± 12.39 39.40 ± 16.30
211.00 ± 17.09 217.90 ± 25.35 241.17 ± 47.49 294.60 ± 51.27
83.83 ± 7.52 80.98 ± 4.87 79.35 ± 15.70 72.94 ± 5.65
48.38 ± 3.37 47.02 ± 1.94 41.53 ± 10.48 41.41 ± 2.72
59.25 ± 5.57 66.10 ± 13.41 69.17 ± 20.20 139.90 ± 33.58
8.73 ± 2.11 9.29 ± 1.66 8.58 ± 0.83 7.38 ± 0.93
72.23 ± 6.38 72.65 ± 6.97 61.30 ± 13.71 60.02 ± 8.09
1.77 ± 0.27 1.83 ± 0.13 1.69 ± 0.37 1.46 ± 0.23
0.58 ± 0.08 0.48 ± 0.08 0.52 ± 0.12 1.08 ± 0.45
Male
PC Control
48.40 ± 17.50 32.40 ± 9.41
272.90 ± 30.36 276.10 ± 29.61
74.43 ± 7.94 73.75 ± 4.44
41.87 ± 3.38 41.93 ± 1.56
133.40 ± 30.66 104.20 ± 15.18
7.15 ± 1.00 6.21 ± 1.32
58.17 ± 9.27 60.91 ± 6.75
1.55 ± 0.39 1.38 ± 0.23
1.06 ± 0.30 0.89 ± 0.27
revealed that they were substantially equivalent to normal carp on the aspect of physiology and pathology. But pharmaceuticals are different in composition, concentration, intake and many other aspects with foods, which make this study not appropriate for food safety assessment. Chen et al. (2002) used grass carp with Hu-a-IFN gene to feed rats, and no significant difference in hematological index, morphology and histopathological examination of the main organs between the treatment group and control group was observed. Liu et al. (2011) evaluated the androgenic and anti-androgenic effects of GH transgenic carp (the same carp that we used in this study) in male rats and concluded that GH transgenic carp does not have any androgenic or anti-androgenic properties in vivo screening tests. Our subchronic toxicity study was conducted to test for possible adverse effects of using GH transgenic carp for food before its marketization. In our study, GH transgenic carp, parental carp and common carp were used to replace the commodity fish meal typically used. All diets were analyzed quantitatively for nutrient composition and contaminants. This method has been used in many studies of food safety assessment of GMOs (He et al., 2009; Malley et al., 2007; Appenzeller et al., 2009a,b). In this feeding study, no significant difference was found on average body weights, organ weights, body compositions, food consumption and food utility of the male and female rats administered the GH transgenic carp, parental carp or common carp. Histopathological examination showed no pathological changes that need concern. In subchronic toxicity study, change of organ weight coefficient directly shows developing condition of animal organs and can figure out adverse effect from external environment (containing food) at an early date. The organ coefficients in our experiment showed no significant difference among GH–TC, PC and control group. In addition, serum T3, T4, FSH, E2, P and T levels are useful indicators for endocrine disruption. In this study, the serum concentrations of them showed no significant changes in treatment with GH transgenic carp, as compared to the PC and control group. These results indicate that GH transgenic carp has no endocrine disruption effects. The enzyme activity in blood is an important indicator for animal state of health. For example, AST and ALT mainly exist in histiocyte and are little in blood. When infection or injure happens in hepatic tissue, the content of AST and ALT in blood would be higher, which makes the two enzymes important indicators of liver health. In our study, the blood routine and blood biochemical parameters indicate that rats fed with GH transgenic carp showed no significant difference (P > 0.05) compared with those in other groups. In conclusion, this study demonstrates that at this dose level (10% contained in diet), GH transgenic carp shows no subchronic toxicity and endocrine disruption in SD rats. Although a classical 90-day subchronic toxicity study may not achieve adequate sensitivity to detect toxic components at extremely low level and their effects, many countries and organizations still take it as an important procedure of food safety assessment. In China, a national
standard, ‘‘GB_15193.1-2003: Procedures for toxicological assessment of food’’ takes 90-day study a necessary procedure of food safety assessment, which is in accordance with international guidelines. With respect to the safety testing of GM food and feed, the Guidance Document of the GMO Panel states (EFSA, 2006a, Section 3 7.8.4): ‘‘If the composition of the GM plant is modified substantially, or if there are any indications for the potential occurrence of unintended effects, based on the preceding molecular, compositional, phenotypic or agronomic analysis, not only new constituents, but also the whole GM food and feed should be tested’’. In these cases, the testing programme should include at least a 90-day toxicity study in rodents. However, the study needs to be repeated and deepened. It is recommended that GMOs need more serious standardized tests on at least three mammalian species for at least three months employing larger sample sizes, and up to one and two years before commercialization (Séralini et al., 2009). They also called for the criteria for testing adverse health effects overall for GM food or feed products. Here we call for the same treat of GM carp before we put it into the market. Conflict of Interest The authors declare no conflicts of interest. References Appenzeller, M.L., Malley, A.L., MacKenzie, A.S., Hoban, D., Delaney, B., 2009a. Subchronic feeding study with genetically modified stacked trait lepidopteran and coleopteran resistant (DAS-Ø15Ø7-1xDAS-59122-7) maize grain in Sprague–Dawley rats. Food Chem. Toxicol. 47, 1512–1520. Appenzeller, M.L., Munley, M.S., Hoban, D., Sykes, P.G., Malley, A.L., Delaney, B., 2009b. Subchronic feeding study of grain from herbicide-tolerant maize DPØ9814Ø-6 in Sprague–Dawley rats. Food Chem. Toxicol. 47, 2269–2280. Chen, K.J., Zhang, H.Y., Zhang, X.W., Xiao, T.Y., Chen, L.X., Su, J.M., Wang, D.G., 2002. A study on the safety of feeding transgenic grass carps to rat. J. Hunan Agric. Univ. (Nat. Sci.) 28, 147–149 (In Chinese). Chen, T.T., Vrolijk, N.H., Lu, J.K., Lin, C.M., Reimschuessel, R., Dunham, R.A., 1996. Transgenic fish and its application in basic and applied research. Biotechnol. Annu. Rev. 2, 205–236. Devlin, R.H., 1997. Transgenic salmonids. In: Houdebine, L.M. (Ed.), Transgenic Animals: Generation and Use. 105–117. Devlin, R.H., Yesaki, T.Y., Biagi, C.A., Donaldson, E.M., 1994. Extraordinary salmon growth. Nature 371, 209–210. Devlin, R.H., Sundström, L.F., Muir, W.M., 2006. Interface of biotechnology and ecology for environmental risk assessments of transgenic fish. Trends Biotechnol. 24, 89–97. Duan, M., Zhang, T., Hu, W., Sundström, L.F., Wang, Y., Li, Z., Zhu, Z., 2009. Elevated ability to compete for limited food resources by ‘all-fish’ growth hormone transgenic common carp Cyprinus carpio. J. Fish Biol. 75 (6), 1459–1472. FDA Redbook 2000: IV.B.1 General Guidelines for Designing and Conducting Toxicity Studies November 2003. Fletcher, G.L., Davies, P.L., 1991. Transgenic fish for aquaculture. Genet. Eng. 13, 331–370. He, X.Y., Tang, M.Z., Luo, Y.B., Li, X., Cao, S.S., Yu, J.J., Delaney, B., Huang, K.L., 2009. A 90-day toxicology study of transgenic lysine-rich maize grain (Y642) in Sprague–Dawley rats. Food Chem. Toxicol. 47, 425–432. Houdebine, L.M., Chourrout, D., 1991. Transgenesis in fish. Experientia 47, 891–897. Gong, Z., Hew, C.L., 1995. Transgenic fish in aquaculture and developmental biology. Curr. Top. Dev. Biol. 30, 177–214. Gross, M.L., Schneider, J.F., Moav, N., Moav, B., Alvarez, C., Myster, S.H., Liu, Z., Hallerman, E.M., Hackett, P.B., Guise, K.S., Faras, A.J., Kapuscinski, A.R., 1992. Molecular analysis and growth evaluation of northern pike (Esox lucius) microinjected with growth hormone genes. Aquaculture 103, 253–273.
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