Toxicological Evaluation of Brassica napus Extract Containing Vanadium,Nutricultured in Jeju Water

Abstract

This study evaluated the mutagenicity and acute toxicity of the juice extract of nutricultured Brassica napus containing vanadium (BECV). The BECV was prepared by nutriculture for 7 days in Jeju water containing vanadium. The mutagenic effects of BECV were investigated using the bacterial reverse mutation test, chromosome aberration test, and micronucleus test. Based on the results of the mutagenicity test, we propose that BECV is not a mutagenicity-inducing agent. In the acute oral toxicity study, male and female Sprague-Dawley rats were administered a single limiting dose of 0.014, 0.14, or 1.4 μg BECV/kg body weight; the rats were then observed for 7 days. No acute lethal effect was observed at the maximal dose of 1.4 μg BECV/kg body weight. In the subacute study, male and female rats were administered once daily, by oral gavage, a dose of 0.028, 0.14, and 0.7 μg/kg body weight of BECV for 28 days. No significant toxicity was observed not only hematological, biochemical, and pathological parameters but also the body and organ weights when compared to controls. The level of BECV with no observed adverse effects in male and female rats was 0.7 μg/kg body weight (concentration of vanadium in BECV) in the subacute toxicity study.

Introduction

Vanadium is a trace mineral present in some foods, most commonly in vegetables such as mushrooms, dill seed, black pepper, and parsley and foods such as cereals, fruits, and shellfish.^1,2 It is essential for normal bone growth, although it is required in very small amounts. Studies have indicated some unique beneficial effects of vanadium, particularly its anticarcinogenic potential at very low doses without any adverse toxicity.^3,4 Vanadium is also known to exert insulin-like effects in in vitro as well as in vivo systems.^5,6 Continuous oral administration of bis(maltolato)oxovanadium, an organic vanadium compound, reduced the food intake and body weight in Zucker diabetic fatty rats.⁷ An organic vanadium compound, such as sodium orthovanadate (SOV), improves the glucose homeostasis in three different genetic animal models of obesity and insulin resistance, namely the obese fatty (fa/fa) Zucker rat,⁸ the genetically diabetic (db/db) mouse,⁹ as well as the genetically obese and diabetic (ob/ob) mouse.¹⁰ In these studies, the doses of SOV varied from 0.25 to 0.6 mg/mL in drinking water, and the duration of treatment was from 13 days to 7 weeks. Treatment resulted in marked improvement of glucose homeostasis, as assessed by reduced hyperglycemia and hyperinsulinemia with increased glucose utilization.^8

–11 The most common toxic effects of vanadium compounds are diarrhea, decreased fluid and food uptake, dehydration, and reduction in body weight gain^12

–16; these can be corrected by adding NaCl to the drinking water, adjusting the pH of the solution to neutrality, and by gradually increasing the dose of vanadium. A recent comparative study demonstrated that organic vanadium compounds are much safer as antidiabetic agents than inorganic vanadium salts.¹⁷

However, Kitani et al. ¹⁸ reported the allergic and inflammatory potential at high concentrations (>1 mM) of V₂O₅ and orthovanadate (Na₃VO₄), based on the release of histamine from human basophils, rat mast cells, and rat basophilic leukemia (2H3) cells in the presence of hydrogen peroxide.

Jeju Island, the largest island in Korea, is a volcanic island located about 140 km south of the Korean peninsula. The geological feature of Jeju Island is composed predominantly of permeable basalts into which rain and stream waters easily percolate. In general, since basalts contain the highest concentrations of vanadium, Jeju water contains vanadium dissolved from basalt and is present in an oxidized state at a concentration of 0.01 mg/L vanadium. Recent reports have presented the beneficial effects of Jeju water containing vanadium for the treatment of diabetes, through the activation of AMP-activated protein kinase.¹⁹ Also, Kim et al. ²⁰ reported the antioxidant property of Jeju water via scavenging of the superoxide anion induced by xanthine/xanthine oxidase and of hydroxyl radicals induced by the Fenton reaction (H₂O₂ + FeSO₄).

Brassica napus was nutricultured in Jeju water, from which they bioaccumulated vanadium. In the present study, we focused on the toxicological evaluation of B. napus extracts containing vanadium to investigate their potential use as a source of functional food ingredients.

Materials and Methods

Extraction of B. napus containing vanadium

B. napus was nutricultured with vanadium concentrates (0.06 ± 0.03 mg/L) from Jeju water for 7 days (20°C ± 3°C, 55% ± 5.0% relative humidity). After 7 days, extraction of the nutricultured B. napus was performed by using juicers (HD-2233; Hyundai Household Appliances Co., Ltd., Incheon, Korea). The vanadium content of the B. napus extract containing vanadium (BECV) was measured as follows: 5 g of BECV was precisely taken into a microwave Teflon vessel and 10 mL of 65% nitric acid (Suprapur; Merck, Darmstadt, Germany) was added. The digestion vessel was placed in a microwave unit (START D; Milestone, Sorisole, Italy), and temperature was raised to 180°C for 20 min using a microwave digestion system, followed by holding for 15 min. After digestion was completed, decomposed BECV was cooled and filtered through a 0.45-μm syringe filter (PTFE; Whatman, Cardiff, Wales, United Kingdom), placed in a 25-mL volumetric flask, and diluted with ultrapure water. Standard solutions were prepared by diluting the quality control standard (Perkin Elmer, Shelton) with 2% nitric acid (concentrate of standard solution 0.01–1 mg/L). Samples and standard solutions were analyzed by ICP-OES (Optima 7300DV; Perkin Elmer, Shelton). The content of BECV contained 0.083 mg/L vanadium. This extract was used for mutagenicity test and toxicity test.

Mutagenicity test

Mutagenicity was tested by the following standard methods: bacterial reverse mutation test (Ames test), chromosome aberration test, and micronucleus test (standard plate incorporation assay).^21
–23 S9 rat liver (Sprague-Dawley) was purchased from Molecular Toxicology, Inc. (Boone, NC). All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Chungbuk National University (CBNUA-305-11-01).

Bacterial reverse mutation test (Ames test)

Mixture A, containing 0.1 mL of BECV (at final concentrations of vanadium 0.42, 0.83, 2.08, 4.15, 6.23, and 8.3 ng/plate), 0.5 mL of S9 mix or phosphate buffer, and 0.1 mL of fresh culture of the tester strain (Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA; cell density of ∼10⁸ cells/mL), was added to a tube containing 2 mL of Top agar (supplemented with 0.75% agar and 0.5% NaCl). The tube was then gently vortexed, and the mixture was overlaid onto glucose minimal agar plate containing 1.5% agar, 0.02% MgSO₄·7H₂O, 0.2% citric acid, 1% K₂HPO₄, 0.35% NaHNH₄PO₄·4H₂O, and 2% glucose. The extracts were tested with and without the S9 mix, and triplicate plates were poured for each dose of mutagen. Diagnostic mutagens including 2-nitrofluorene, sodium azide, 9-aminoacridine, 2-(2-furyl-3-(5-nitro-2-furyl) acrylamide, and 2-aminoanthracene were used as the positive controls. After incubation at 37°C for 48 h, the number of revertant colonies was scored. A compound was considered a mutagen if there was a twofold increase in the number of revertants compared with the spontaneous revertants (negative control), or there was a dose-related increase in the number of revertants in one or more strains.

Chromosome aberration test

Chromosome aberration test was performed using the Chinese hamster ovary (CHO) both in the presence and absence of S9 metabolic activation. All examination substance and positive control (mitomycin C [MMC] for the treatments without S9 and cyclophosphamide for the treatments with S9) concentrations were prepared using ultrapure water as a vehicle. Before the definitive chromosome aberration test, a dose range–finding study was conducted and the effects of BECV on cell growth were then compared with the effects of the negative control. Based on the results, 0.9, 1.8, and 3.6 ng/mL BECV were selected for the chromosome aberration test. The definitive test was assessed via two different procedures: a 6 h exposure followed by an 18 h recovery (with or without metabolic activation) and a 24 h continuous exposure.

Micronucleus test

To evaluate the genotoxicity of BECV, a micronucleus test was performed using the bone marrow cells of specific pathogen-free male Institute of Cancer Research mice. On the basis of dose range–finding test, there was no observed difference of sensitivity between female and male rats, and dead rats were not found. So the highest dose of the main test was determined to be 0.7 μg/kg BECV. After 1 week acclimation of 6-week-old male mice, the test substance was orally administered once a day interval at a dose of 0 (sterilized deionized distilled water, negative control), 0.125, 0.35, and 0.7 μg/kg BECV, and five rats per each group were used. The positive control (MMC) was administered intraperitoneally once at a dose of 2000 μg/kg BECV. After 24 h, the mice were sacrificed and the femur bones were removed. The bone marrow was collected by perfusion of 0.5 mL fetal bovine serum. The suspension of bone marrow cells was centrifuged at 168 g for 5 min, using a model 5810R centrifuge (Eppendorf AG, Hamburg, Germany), to discard the supernatant, and the bone marrow cells were floated on the slide glass, dried, and fixed with methanol. After stained with 3% Giemsa buffer before microscopic examination, slides were rinsed with 0.004% citric acid solution and air-dried. The observation was performed with blind method. The slides were observed under the fluorescent microscope at the magnification of >1000 × . Micronucleated polychromatic erythrocytes were counted in 2000 polychromatic erythrocytes per rats. As an index of inhibition of bone marrow cell proliferation, the ratio (%) of polyglutamic erythrocytes to 500 total erythrocytes per individual was calculated.

Toxicity test

For single-dose toxicity test, 6-week-old Sprague-Dawley rats (male and female) were purchased from DH Biolink (Chungbuk, Korea) and acclimatized for 7 days before commencement of the toxicity test. Three rats were housed per cage, under controlled environmental conditions (22°C ± 3°C, 50% ± 10% relative humidity, 200–300 lux). The rats were divided into four groups (n = 10 in each group; n = 5 per gender). The control group was administered distilled water. Three experimental groups were administered one dose of BECV by oral gavage. The vanadium concentrations in BECV administered to the three experimental groups were 0.014, 0.14, and 1.4 μg/kg body weight. All rats were carefully observed for signs of morbidity, clinical symptoms, and mortality immediately after dosing and for the subsequent 7 days. The Functional Observational Battery test with included various functions were performed daily during the experiment period. After 7 days, the rats were sacrificed by blood exhaustion via the abdominal aorta (performed under ether anesthesia); an autopsy was performed for all rats, and all organs were subjected to gross examination.

Repeated-dose toxicity of BECV was performed following the same protocol used for single-dose toxicity tests. The vanadium concentrations in BECV evaluated in the three experimental groups were 0.028, 0.14, and 0.7 μg/kg body weight, once daily for 28 days by oral gavage. Clinical signs and mortality were observed twice daily. At the end of the 28 days, all rats were fasted overnight and sacrificed under anesthesia. Whole blood sample was collected from the abdominal aorta in ethylenediaminetetraacetic acid containing tubes (BD Vacutainer; BD, Franklin Lake, NJ) for hematological analysis, and serum was obtained by centrifuging whole blood at 13,475 g for 10 min at 4°C using a model Smart R17 centrifuge (Hanil Science Industrial, Daejeon, Republic of Korea).

Histological examination

All animals subjected to the repeated-dose toxicity test underwent a complete necropsy, after which we evaluated the organ weights and histological examinations. Tissues (liver, lung, spleen, kidney, heart, thymus, adrenal gland, brain, testis, and ovary) were fixed in 10% buffered formaldehyde solution and embedded in paraffin. The tissue-embedded paraffin blocks were cut into 4 μm sections, stained with hematoxylin and eosin, and examined under a light microscope.

Hematological examination

Whole blood sample was examined by automated hematology analyzer (Siemens Medical ADVIA 2120; Bayer, Leverkusen, Germany). The criteria evaluated included white blood cell count (WBC), neutrophil count (NEUT), lymphocyte count (LYMP), monocyte count (MONO), eosinophil count (EOS), basophil count (BASO), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), and platelets (PLT).

Blood biochemical examinations

The serum sample was used to determine the total protein, albumin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, creatinine, blood urine nitrogen, total cholesterol, glucose, and triglycerides by enzymatic methods using an automatic analyzer (Hitachi 7080 Clinical Analyzer; Hitachi, Tokyo, Japan).

Statistical analysis

All data were analyzed with the SPSS 12.0 statistical software (SPSS, Chicago, IL). Data are expressed as mean ± standard deviation. Statistical differences were examined independently using the Student's t-test and Pearson's correlation test. A P-value of <.05 is considered significant.

Results

Bacterial reverse mutation test (Ames test)

Within the tested dose range of vanadium (0.42, 0.83, 2.08, 4.15, 6.23, and 8.3 ng/plate), no growth inhibition was observed in the test strains (S. typhimurium TA98, TA100, TA1535, and TA1537, and E. coli WP2uvrA) with or without the presence of S9 mix (data not shown). Since the test strains showed no toxicity, the same range of BECV concentrations was applied for the test.

The Ames test results are shown in Table 1; the revertant numbers induced by extracts (0.42, 0.83, 2.08, 4.15, 6.23, and 8.3 ng/plate) for all test strains were similar to that of the negative control (spontaneous revertant, without extract) and much lower than that of positive controls (with diagnostic mutagens). Based on the Ames test results, BECV showed no mutagenicity under the tested dose range.

Table 1.

Ames Test of Juice Extract of Nutricultured Brassica napus Containing Vanadium Toward Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA

		Revertant colonies (CFU/plate) ^b
		Base pair substitution type			Frame-shift type
S9 mix ^a	Groups	TA100	TA1535	WP2uvrA	TA98	TA1537
−	Control^c	226 ± 4.2	55 ± 10.5	348 ± 26.7	23 ± 3.8	5 ± 2.3
	Positive control^d	736 ± 34.5	1197 ± 101.6	2885 ± 442.0	2636 ± 268	2305 ± 861.8
	BECV 0.42 μg/plate	182 ± 19.7	59 ± 10.1	370 ± 20.8	18 ± 2.5	4 ± 2.1
	BECV 0.83 μg/plate	182 ± 25.2	56 ± 8.7	375 ± 19.5	22 ± 4.5	5 ± 2.6
	BECV 2.08 μg/plate	175 ± 26.0	58 ± 7.2	406 ± 20.4	17 ± 1.2	3 ± 1.5
	BECV 4.15 μg/plate	211 ± 14.2	67 ± 4.6	401 ± 31.6	25 ± 4.2	6 ± 0.6
	BECV 8.3 μg/plate	265 ± 18.9	79 ± 5.6	419 ± 28.4	26 ± 2.1	6 ± 1.2
+	Control^c	307 ± 28.6	104 ± 10.8	311 ± 23.3	29 ± 3.6	9 ± 1.5
	Positive control^d	3068 ± 49.3	204 ± 17.0	1249 ± 45.0	2656 ± 49.3	1478 ± 141.8
	BECV 0.42 μg/plate	299 ± 21.5	111 ± 10.5	308 ± 20.5	25 ± 3.8	9 ± 1.0
	BECV 0.83 μg/plate	298 ± 6.0	107 ± 9.8	299 ± 10.7	28 ± 3.1	8 ± 1.5
	BECV 2.08 μg/plate	300 ± 40.0	126 ± 7.8	282 ± 22.9	24 ± 6.5	7 ± 0.6
	BECV 4.15 μg/plate	300 ± 29.2	117 ± 14.3	315 ± 9.6	32 ± 5.5	9 ± 1.7
	BECV 8.3 μg/plate	381 ± 51.1	150 ± 5.5	331 ± 6.6	39 ± 2.1	9 ± 2.1

S9 is a metabolic activation system consisting of the postmitochondrial fraction of the livers of rats.

Values are expressed in mean ± SD of triplicates.

Negative control: without extract was treated with aseptic water, spontaneous revertants/plate.

Positive control: TA98, TA100, TA1535, TA1537, and WP2uvrA with S9: 2-AA (1 μg/plate), 2-AA (1 μg/plate), 2-AA (2 μg/plate), 2-AA (2 μg/plate), and 2-AA (2 μg plate), respectively. TA98, TA100, TA1535, TA1537, and WP2uvrA without S9: 2-NF (5 μg/plate), SA (1.5 μg/plate), SA (1.5 μg/plate), 9-AA (80 μg/plate), and AF2 (0.005 μg/plate), respectively.

2-NF, 2-nitrofluorene; 2-AA, 2-aminoanthracene; 9-AA, 9-aminoacridine; AF2, 2-(2-furyl-3-(5-nitro-2-furyl) acrylamide; BECV, Juice extract of nutricultured Brassica napus containing vanadium; CFU, colony-forming unit; SA, sodium azide; SD, standard deviation.

Chromosome aberration test

Based on the results of the concentration range–finding study, BECV was tested at concentrations varying from 0.9, 1.8, and 3.6 ng/mL. Dose-related changes in cytotoxicity were noted at every concentration tested. The results of the definitive chromosome aberration test with CHO for BECV are presented in Table 2.

Table 2.

Results of Chromosome Aberration Test in the Chinese Hamster Ovary Cells Exposed to Juice Extract of Nutricultured Brassica napus Containing Vanadium

					No. of cells with structure aberrations						No. of cells with numerical aberrations
Test substance	Dose (ng/mL)	S9 mix	Time of exposure (h)	No. of cells analyzed	ctb	cte	csb	cse	Total (%)	Gap (%)	end	dip	pol	Total (%)
Negative control	0	−	6–18	100	2	0	0	1	3 (1.5)	6 (3.0)	0	0	0
Negative control	0	−	6–18	100	0	0	0	0	3 (1.5)	6 (3.0)	0	0	0
BECV	3.6	−	6–18	100	1	0	3	1	7 (3.5)	5 (2.5)	0	0	0
	3.6	−	6–18	100	0	0	2	0	7 (3.5)	5 (2.5)	0	0	0
	1.8	−	6–18	100	0	0	0	2	2 (1.0)	5 (2.5)	0	0	0
	1.8	−	6–18	100	0	0	0	0	2 (1.0)	5 (2.5)	0	0	0
	0.9	−	6–18	100	0	0	0	0	0 (0.0)	3 (1.5)	0	0	0
	0.9	−	6–18	100	0	0	0	0	0 (0.0)	3 (1.5)	0	0	0
MMC	50	−	6–18	100	4	7	10	9	53 (26.5)	32 (16.0)	0	0	2	2 (1)
MMC	50	−	6–18	100	3	6	7	7	53 (26.5)	32 (16.0)	0	0	0	2 (1)
Negative control	0	+	6–18	100	1	0	0	0	2 (1.0)	3 (1.5)	0	0	0
Negative control	0	+	6–18	100	1	0	0	0	2 (1.0)	3 (1.5)	0	0	0
BECV	3.6	+	6–18	100	1	1	0	2	7 (3.5)	6 (3.0)	0	0	0
	3.6	+	6–18	100	2	0	1	0	7 (3.5)	6 (3.0)	0	0	0
	1.8	+	6–18	100	1	0	4	0	6 (3.0)	8 (4.0)	0	0	0
	1.8	+	6–18	100	1	0	0	0	6 (3.0)	8 (4.0)	0	0	0
	0.9	+	6–18	100	2	0	1	0	3 (1.5)	7 (3.5)	0	0	0
	0.9	+	6–18	100	0	0	0	0	3 (1.5)	7 (3.5)	0	0	0
B[a]P	20,000	+	6–18	100	6	10	12	12	52 (26.0)	42 (21.0)	0	0	2	2 (1)
B[a]P	20,000	+	6–18	100	7	0	6	0	52 (26.0)	42 (21.0)	0	0	0	2 (1)
Negative control	0	−	24–0	100	0	0	0	0	1 (0.5)	4 (2.0)	0	0	0
Negative control	0	−	24–0	100	1	0	0	0	1 (0.5)	4 (2.0)	0	0	0
BECV	3.6	−	24–0	100	0	0	0	0	0 (0.0)	4 (2.0)	0	0	0
	3.6	−	24–0	100	0	0	0	0	0 (0.0)	4 (2.0)	0	0	0
	1.8	−	24–0	100	1	0	0	1	3 (1.5)	5 (2.5)	0	0	0
	1.8	−	24–0	100	0	0	1	0	3 (1.5)	5 (2.5)	0	0	0
	0.9	−	24–0	100	0	0	0	0	1 (0.5)	8 (4.0)	0	0	0
	0.9	−	24–0	100	0	0	1	0	1 (0.5)	8 (4.0)	0	0	0
MMC	50	−	24–0	100	1	8	12	8	65 (32.5)	39 (18.5)	0	0	0	1 (0.5)
MMC	50	−	24–0	100	4	21	9	2	65 (32.5)	39 (18.5)	0	0	1

Gap, chromosome and chromatid gap; ctb, chromatid break; cte, chromatid exchange; csb, chromosome break; cse, chromosome exchange; end, endoploid; dip, diploid; pol, polyploidy; B[a]P, benzo[a]pyrene; MMC, mitomycin C.

Micronucleus test

The results of the bone marrow micronucleus test in mice for BECV are shown in Table 3. No deaths occurred as result of the administration of the test substance, and the general symptoms were not observed. Under the conditions used in this test, BECV did not show any evidence of genotoxic potential.

Table 3.

Results of Micronucleus Test in Mice Administered Juice Extract of Nutricultured Brassica napus Containing Vanadium

Test substance	Dose (μg/kg)	Route	Time (h)	Animal no.	PCE	NCE	PCE/(PCE + NCE)	MNPCE/PCE
Negative control	0	PO	24	SN1	280	226	0.553	17/2000
				SN2	289	211	0.578	14/2000
				SN3	284	216	0.568	11/2000
				SN4	313	187	0.626	13/2000
				SN5	293	207	0.586	11/2000
				Total			—	66/10000
				Mean ± SD			0.582 ± 0.027	13.2 ± 2.5
BECV	0.7	PO	24	SN6	316	184	0.632	16/2000
				SN7	318	182	0.636	11/2000
				SN8	342	158	0.684	17/2000
				SN9	268	232	0.536	6/2000
				SN10	275	225	0.550	12/2000
				Total			—	62/10000
				Mean ± SD			0.608 ± 0.063	12.4 ± 4.4
	0.35	PO	24	SN11	274	226	0.548	6/2000
				SN12	291	209	0.582	11/2000
				SN13	288	212	0.576	8/2000
				SN14	260	240	0.520	11/2000
				SN15	270	230	0.540	6/2000
				Total			—	42/10000
				Mean ± SD			0.553 ± 0.026	8.4 ± 2.5
	0.125	PO	24	SN16	269	231	0.538	9/2000
				SN17	284	216	0.568	11/2000
				SN18	270	230	0.540	9/2000
				SN19	271	229	0.542	9/2000
				SN20	269	231	0.538	10/2000
				Total			—	48/10000
				Mean ± SD			0.545 ± 0.013	9.6 ± 0.9^**
Positive control (MMC)	2000	IP	24	SN21	265	235	0.530	235/2000
				SN22	367	133	0.734	247/2000
				SN23	272	228	0.544	216/2000
				SN24	251	249	0.502	237/2000
				SN25	281	219	0.562	204/2000
				Total			—	1139/10000
				Mean ± SD			0.574 ± 0.092	227.8 ± 17.4

Significant difference from control by Dunnet's t-test: ^** P < .01.

IP, intraperitoneal; MNPCE, micronucleated polychromatic erythrocytes; PCE, polychromatic erythrocytes; PO, per os; NCE, normochromatic erythrocyte.

Single- and repeated-dose toxicity study

At the single- and repeated-dose toxicity study, all the treated rats appeared normal during the experimental period, and all the rats survived during the experimental period. Also, histological examination showed no significant findings in the control groups and experimental groups (data not shown). There were no significant clinical symptoms or death of rats that could be attributed to the treatment. Therefore, the results indicate that vanadium in BECV, even at the upper-limit doses, does not induce any clinical signs and toxic symptoms.

In the single-dose toxicity study, there were no significant differences in the body weight gain of male and female rats treated with BECV (data not shown). Figure 1 shows the body weight change of rats subjected to the repeated-dose toxicity study. Compared with the control group, the administration of BECV for 28 days, at a dose of 0.028, 0.14, and 0.7 μg/kg (as vanadium concentration in BECV) of body weight, did not result in any difference in the body weight of male and female experimental rats. After 4 weeks of treatment, the weights of the internal organs were evaluated. The liver, spleen, thymus, lung, kidney, heart, brain, adrenal glands, and reproductive organs (testis and ovary) from the male and female experimental rats showed no difference when compared with the control group (Tables 4 and 5).

FIG. 1.

Changes in body weights of (A) male and (B) female Sprague-Dawley rats orally administered BECV for 28 days. Data points are expressed in mean ± SD (n = 10 in each group; n = 5 per gender). Control: without extract was treated with aseptic water. BECV, juice extract of nutricultured Brassica napus containing vanadium.

Table 4.

Organ Weight Changes in Male Rats Administered Juice Extract of Nutricultured Brassica napus Containing Vanadium Daily by Oral Gavage for 28 Days

Groups	Liver (g)	Lung (g)	Spleen (g)	Kidney (g)	Heart (g)	Testis (g)	Adrenal gland (g)	Thymus (g)	Brain (g)
Control^a	11.91 ± 0.65	1.61 ± 0.11	0.84 ± 0.11	1.35 ± 0.11	1.18 ± 0.11	1.70 ± 0.15	0.03 ± 0.01	0.75 ± 0.13	2.03 ± 0.23
BECV 0.028 μg/kg	12.64 ± 0.80	1.54 ± 0.19	0.90 ± 0.16	1.34 ± 0.14	1.26 ± 0.11	1.67 ± 0.07	0.02 ± 0.00	0.77 ± 0.34	1.99 ± 0.08
BECV 0.14 μg/kg	10.60 ± 2.01	1.45 ± 0.17	0.90 ± 0.09	1.27 ± 0.09	1.24 ± 0.12	1.67 ± 0.11	0.03 ± 0.01	0.58 ± 0.16	2.04 ± 0.10
BECV 0.7 μg/kg	11.22 ± 0.77	1.66 ± 0.22	0.87 ± 0.23	1.40 ± 0.15	1.19 ± 0.14	1.70 ± 0.10	0.03 ± 0.01	0.68 ± 0.04	1.95 ± 0.22

Data are expressed in mean ± SD (n = 5).

Negative control: without extract was treated with aseptic water.

Table 5.

Organ Weight Changes in Female Rats Administered Juice Extract of Nutricultured Brassica napus Containing Vanadium Daily by Oral Gavage for 28 Days

Groups	Liver (g)	Lung (g)	Spleen (g)	Kidney (g)	Heart (g)	Ovary (g)	Adrenal gland (g)	Thymus (g)	Brain (g)
Control^a	6.50 ± 0.45	1.28 ± 0.06	0.67 ± 0.08	0.74 ± 0.06	0.82 ± 0.09	0.06 ± 0.01	0.04 ± 0.00	0.48 ± 0.08	1.87 ± 0.12
BECV 0.028 μg/kg	6.54 ± 0.22	1.14 ± 0.12	0.63 ± 0.06	0.86 ± 0.03	0.86 ± 0.06	0.05 ± 0.01	0.04 ± 0.00	0.46 ± 0.07	1.81 ± 0.12
BECV 0.14 μg/kg	6.03 ± 0.60	1.18 ± 0.07	0.62 ± 0.03	0.84 ± 0.09	0.81 ± 0.07	0.05 ± 0.01	0.04 ± 0.01	0.45 ± 0.03	1.85 ± 0.10
BECV 0.7 μg/kg	6.16 ± 0.42	1.13 ± 0.13	0.58 ± 0.05	0.83 ± 0.06	0.85 ± 0.03	0.05 ± 0.01	0.03 ± 0.01	0.43 ± 0.12	1.83 ± 0.06

Data are expressed in mean ± SD (n = 5).

Negative control: without extract was treated with aseptic water.

Hematological and blood biochemical analysis

In the repeated-dose toxicity study, whole blood was collected at 28 days after treatment. Hematological parameters were measured, including WBC, NEUT, LYMP, MONO, EOS, BASO, RBC, HGB, HCT, MCV, MCH, MCHC, and PLT. Their proportions (%) remained unchanged after BECV administration in both male (Table 6) and female rats (data not shown).

Table 6.

Changes of Hematological Parameters in Male Rats Administered Juice Extract of Nutricultured Brassica napus Containing Vanadium Daily by Oral Gavage for 28 Days

	Groups
Hematological parameter	Control^a	BECV 0.028 μg/kg	BECV 0.14 μg/kg	BECV 0.7 μg/kg
RBC (1 × 10⁶cells/μL)	8.70 ± 0.27	8.40 ± 0.20	8.60 ± 0.32	8.80 ± 0.40
HGB (g/dL)	20.60 ± 0.76	19.70 ± 0.56	20.50 ± 0.62	20.10 ± 1.06
HCT (%)	53.00 ± 2.56	51.80 ± 1.96	53.50 ± 1.81	52.00 ± 3.57
MCV (fL)	60.70 ± 1.51	61.40 ± 0.94	62.30 ± 0.85	59.00 ± 1.40
MCH (pg)	23.60 ± 0.36	23.30 ± 0.15	23.90 ± 0.48	22.80 ± 0.67
MCHC (g/dL)	38.90 ± 0.49	38.00 ± 0.40	38.40 ± 0.51	38.70 ± 1.19
PLT (1 × 10³cells/μL)	1301.40 ± 184.41	1345.20 ± 227.82	1245.40 ± 189.25	1393.00 ± 128.12
WBC (1 × 10³cells/μL)	9.70 ± 1.38	9.10 ± 1.56	6.00 ± 1.98	10.10 ± 2.26
NEUT (%)	5.78 ± 1.46	7.40 ± 2.01	7.60 ± 2.05	6.10 ± 0.51
LYMP (%)	88.00 ± 2.57	87.80 ± 1.42	87.60 ± 4.59	90.80 ± 1.17
MONO (%)	2.06 ± 0.40	1.50 ± 0.52	1.20 ± 0.16	1.80 ± 0.90
EOS (%)	3.70 ± 2.46	2.60 ± 1.12	3.20 ± 2.95	0.80 ± 0.41
BASO (%)	0.20 ± 0.07	0.20 ± 0.04	0.20 ± 0.08	0.20 ± 0.08

Data are expressed in mean ± SD (n = 5).

Negative control: without extract was treated with aseptic water.

BASO, basophil count; EOS, eosinophil count; HCT, hematocrit; HGB, hemoglobin; LYMP, lymphocyte count; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; MCV, mean cell volume; MONO, monocyte count; NEUT, neutrophil count; PLT, platelet; RBC, red blood cell count; WBC, white blood cell count.

There was no significant difference between the parameters of blood biochemistry in the control and experimental groups (Tables 7 and 8) for either rat genders. Throughout, no toxicity was observed in these studies, based on the results of hematological and serum biochemical parameters.

Table 7.

Blood Biochemistry Changes in Male Rats Administered Juice Extract of Nutricultured Brassica napus Containing Vanadium Daily by Oral Gavage for 28 Days

	Groups
Biochemistry parameter	Control ^a	BECV 0.028 μg/kg	BECV 0.14 μg/kg	BECV 0.7 μg/kg
ALB (g/dL)	1.48 ± 0.22	1.14 ± 0.25	1.18 ± 0.36	1.26 ± 0.64
ALP (U/L)	264.18 ± 21.36	232.00 ± 32.85	192.05 ± 48.07	236.75 ± 49.61
CRE (mg/dL)	0.48 ± 0.04	0.44 ± 0.11	0.36 ± 0.11	0.48 ± 0.13
GLU (mg/dL)	133.66 ± 14.57	116.20 ± 16.24	120.84 ± 19.81	132.33 ± 17.43
AST (U/L)	85.63 ± 12.75	89.63 ± 28.65	114.47 ± 21.86	93.57 ± 2.32
ALT (U/L)	36.13 ± 3.01	31.64 ± 6.38	29.00 ± 6.54	28.28 ± 6.37
TC (mg/dL)	56.65 ± 1.50	52.63 ± 5.64	57.95 ± 24.31	50.72 ± 16.96
TP (g/dL)	4.54 ± 0.56	4.30 ± 0.75	3.95 ± 0.90	4.10 ± 1.30
TG (mg/dL)	74.28 ± 7.44	88.88 ± 21.35	65.62 ± 24.17	69.60 ± 15.40
BUN (mg/dL)	19.92 ± 2.71	19.54 ± 5.17	19.30 ± 6.06	19.98 ± 0.56

Data are expressed in mean ± SD (n = 5).

Negative control: without extract was treated with aseptic water.

ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urine nitrogen; CRE, creatinine; GLU, glucose; TC, total cholesterol; TG, triglycerides; TP, total protein.

Table 8.

Blood Biochemistry Changes in Female Rats Administered Juice Extract of Nutricultured Brassica napus Containing Vanadium Daily by Oral Gavage for 28 Days

	Groups
Biochemistry parameter	Control ^a	BECV 0.028 μg/kg	BECV 0.14 μg/kg	BECV 0.7 μg/kg
ALB (g/dL)	2.50 ± 0.16	2.32 ± 0.22	2.43 ± 0.25	2.44 ± 0.15
ALP (U/L)	202.88 ± 61.40	224.65 ± 35.23	202.70 ± 50.31	191.63 ± 15.61
CRE (mg/dL)	0.60 ± 0.00	0.60 ± 0.07	0.60 ± 0.10	0.60 ± 0.10
GLU (mg/dL)	137.32 ± 15.27	136.04 ± 27.84	132.98 ± 23.22	130.80 ± 1.82
AST (U/L)	111.17 ± 6.09	93.40 ± 11.01	108.13 ± 21.77	94.22 ± 15.41
ALT (U/L)	30.28 ± 6.25	28.66 ± 4.09	28.88 ± 3.53	26.15 ± 1.05
TC (mg/dL)	104.38 ± 14.03	103.47 ± 18.94	83.34 ± 13.64	93.70 ± 16.46
TP (g/dL)	6.25 ± 0.19	6.00 ± 0.24	6.00 ± 0.18	5.86 ± 0.27
TG (mg/dL)	25.17 ± 12.57	24.43 ± 7.05	19.30 ± 2.56	24.62 ± 7.26
BUN (mg/dL)	19.74 ± 2.58	19.66 ± 2.52	20.78 ± 4.79	21.74 ± 2.82

Data are expressed in mean ± SD (n = 5).

Negative control: without extract was treated with aseptic water.

Discussion

In the bacterial reverse mutation test (Ames test), no reproducible and dose-dependent increase in revertants to prototrophy was obtained with any of the tester strains (S. typhimurium TA98, TA100, TA1535, and TA1537, and E. coli WP2uvrA) exposed to BECV, either in the presence or absence of S9 mix. In the chromosome aberration test, BECV did not induce chromosomal aberrations. In the micronucleus test, BECV did not affect the micronucleus induction of bone marrow cells. Therefore, based on the results of the mutagenicity testing, we propose that BECV is not a mutagenicity-inducing agent.

In the present study, morbidity, clinical signs, and mortality rates were examined after treatment with indicated doses of BECV (as mentioned in the Materials and Methods section). None of the doses administered resulted in any untoward symptoms or abnormal clinical signs, and no deaths were observed during the experimental period (data not shown). In the repeated-dose toxicity test, we observed similar results: there were no instances of any abnormal symptoms or deaths (data not shown). Histological examination of the organs of all rats treated with BECV for 4 weeks (liver, lung, spleen, kidney, heart, thymus, adrenal gland, brain, testis, and ovary) were determined using hematoxylin–eosin staining. All concentrations of BECV failed to induce any histological lesions in the organs.

We further investigated any possible toxicities of BECV in rats after a single oral administration (0.04, 0.14, and 1.4 μg/kg body weight, as vanadium concentration in BECV) or once daily for 28 days (0.028, 0.14, and 0.7 μg/kg body weight, as vanadium concentration in BECV). In hematological and blood biochemical analysis, none of the parameters observed was affected by the BECV. This suggests that BECV had no adverse effect on the hematopoiesis of blood cells and caused no injury to the liver and kidney. Hence, we conclude that no adverse effects of BECV were observed in either single- or repeated-dose toxicity tests.

This study demonstrates that BECV (B. napus extract containing vanadium) lacks genotoxicity under the bacterial reverse mutation test. The results also show no acute lethal effects at the maximal tested dose of 1.4 μg/kg body weight (as vanadium concentration in BECV) in rats. Histopathological examination in the subacute toxicity study revealed that BECV did not cause any significant toxic effect in rats. The subacute toxicity study also confirmed the safety of the BECV after repeated oral administration of 0.7 μg/kg body weight (as vanadium concentration in BECV) for 28 days. We thereby conclude that BECV is safe in both male and female rats at the limited dose level, and our results indicate no significant adverse effects of BECV in rats. We therefore suggest that a safe dose of BECV has the potential to be beneficial as a source of functional food ingredients.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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