Chemical properties and biotoxicity of several chromium picolinate derivatives

JIB-10078; No of Pages 9 Journal of Inorganic Biochemistry xxx (2016) xxx–xxx

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Chemical properties and biotoxicity of several chromium picolinate derivatives Bin Liu a,⁎, Yanfei Liu a, Jie Chai a, Xiangquan Hu a, Duoming Wu b, Binsheng Yang a,⁎ a b

Institute of Molecular Science, Key Laboratory of Chemical Biology of Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, China The First Hospital of Lanzhou University, Lanzhou, China

a r t i c l e

i n f o

Article history: Received 19 July 2016 Received in revised form 31 August 2016 Accepted 13 September 2016 Available online xxxx Keywords: Chromium picolinate Toxicity Crystal structure Cyclic voltammetry Ovotransferrin

a b s t r a c t As a man-made additive, chromium picolinate Cr(pic)3 has become a popular dietary supplement worldwide. In this paper Cr(pic)3 and its new derivatives Cr(6-CH3-pic)3 (1), [Cr(6-NH2-pic)2(H2O)2]NO3 (2) and Cr(3-NH2pic)3 (3) were synthesized, and complexes 1 and 2 were characterized by X-ray crystal structure (where pic = 2-carboxypyridine). The relationship between the chemical properties and biotoxicity of these complexes was fully discussed: (1) The dynamics stability of chromium picolinate complexes mainly depends on the Cr\\N bonds length. (2) There is a positive correlation between the dynamics stability, electrochemical potentials and generation of reactive oxygen species through Fenton-like reaction. (3) However, no biological toxicity was observed through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and sub-chronic oral toxicity study for these chromium picolinate compounds. Together, our findings establish a framework for understanding the structure-property-toxicity relationships of the chromium picolinate complexes. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Chromium(III) was first proposed to be an essential trace element for mammals about fifty years ago, which may increase sensitivity to insulin and thus participate in carbohydrate and lipid metabolism [1–5]. The status of chromium has recently been challenged, and the status is a matter of current debate [6–8]. Part of the confusion with the status of Cr arises from pharmacological effects of Cr3 + at supranutritional doses. At certain dosages Cr(III) can improve impaired glucose and lipid homeostasis in animals with stresses on the glucose and lipid metabolism systems of type 2 diabetes [9]. Nevertheless, supplemental trivalent chromium appears to be a useful tool in the world's fight against epidemic-like appearances of various manifestations of the metabolic syndrome, especially obesity and diabetes [10]. The pharmacological effects of supplementary Cr(III), administered mostly in the form of chromium picolinate, Cr(pic)3, have been studied extensively [11–12]. Given the popularity of Cr(pic)3 as a dietary supplement and food supplement worldwide, investigation of any potential health risk caused by this compound is necessary. In the past decade, conflicting results have appeared on Cr(pic)3 toxicity [13–18]. In a study commissioned by the National Institutes of Health (USA), it showed that Cr(pic)3 fed up to 5% of the diet for two years to male and female rats and mice did not induce biologically toxicity [19].

⁎ Corresponding authors. E-mail addresses: [email protected] (B. Liu), [email protected] (B. Yang).

Recent reports suggest that the coordinated ligands play an important role in the toxic behavior of chromium(III) compounds [20–21]. In recent in vitro investigations, physiologically relevant concentrations of Cr(pic)3 and biological reductants, resulted in catalytic production of hydroxyl radicals, which cleaved DNA [22]. This ability stems from the combination of chromium(III) and picolinate; the picolinate ligand shifts the redox potential of the chromic center such that it is susceptible to reduction. The reduced Cr(II) species interacts with dioxygen to produce reduced oxygen species including hydroxyl radical. The question whether the physicochemical property affect the redox potential to generate hydroxyl radical and cytotoxicity has not been clearly addressed to date. The relevance of structure to biologically active chromium and its safety used as a dietary supplement have not been established. It is of interest to note the role of ligands on the cytotoxicity of Cr(pic)3 complex. It is interesting that 2-carboxypyridine as one of many ligands that can be used to study the effect of structure on chromium metabolism and the cytotoxicity, if any, to which this activity can be controlled by choice of ligands. Prior to the work described herein, only scattered information exist on the affect of derivatives of chromium picolinate complex on toxicity. The aim of this study was to clarify a variety of related physicochemical properties of chromium picolinate complexes from a chemical point of view, such as the structure, stability, redox potential, generation of hydroxyl radical and biotoxicity, and the relationship between these factors were discussed in this paper. In this work, picolinate ligand and its derivatives with different substituent groups were employed and three Cr(III) complexes Cr(6-CH3-pic)3 (1), [Cr(6-NH2-

http://dx.doi.org/10.1016/j.jinorgbio.2016.09.006 0162-0134/© 2016 Elsevier Inc. All rights reserved.

Please cite this article as: B. Liu, et al., Chemical properties and biotoxicity of several chromium picolinate derivatives, J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2016.09.006

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Scheme 1. Physicochemical properties of Cr(pic)3 derivatives and toxicity.

pic)2(H2O)2]NO3 (2), Cr(3-NH2-pic)3 (3) and Cr(pic)3 were synthesized and characterized (pic = 2-carboxypyridine). The concerned content was shown in Scheme 1. 2. Experimental section 2.1. Materials and instruments Unless otherwise stated all chemicals were obtained from Aladdin and used without further purification. All manipulations were performed under aerobic conditions. MCF-7 cancer cells were provided by the Gene Engineering Center of Shanxi University. Dulbecco Minimum Essential Media (DMEM) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Thermo Fisher Biological and Chemical Product (Beijing, China). The heatinactivated fetal bovine serum (FBS) was from GIBCO (Grand Island, NY). Apoovotransferrin was purchased from Sigma. ESI-MS spectra were recorded on an Agilent 6520 Accurate-Mass QTOF LC/MS mass spectrometer. UV–visible (UV–Vis) spectra were measured with a Varian 50 BIO spectrophotometer. Elemental analyses were measured on a Vario EL III analyzer. IR spectra were recorded with a Bruker TENSOR 21 FT-IR spectrophotometer. Water-jacketed CO2 cell incubator was from Shanghai Lishen Scientific Instruments Co., Ltd. 2.2. Synthesis of Cr(6-CH3-pic)3 (1) An aqueous solution of 6-methylpyridine-2-carboxylic acid (6-CH3pic, 1.92 mmol, 263 mg) and triethylamine (1.92 mmol, 266 μL) was added dropwise to an aqueous solution of Cr(NO)3·9H2O (0.640 mmol, 256 mg) under stirring at 100 °C for 1 h. Purple block crystals were obtained by slow evaporation of the reaction mixture at room temperature (yield 53%). Elemental analysis: Calcd. For C21H18N3CrO6 (%): C 54.79, H 3.94, N 9.13. Found (%): C 54.72, H 4.06, N 9.21. Selected FT-IR data (KBr pellet, cm−1): 3456 (br), 3089 (w), 1678 (s), 1667 (s), 1134 (s), 1118 (m), 1020 (m), 480 (m), 563 (w) (Fig. S1). ESI+ mass spectra (m/z): 461.06 for [Cr(6-CH3-pic)3] (Fig. S2).

2.3. Synthesis of [Cr(6-NH2-pic)2(H2O)2]NO3·H2O (2) An aqueous solution of Cr(NO)3·9H2O (0.640 mmol, 256 mg), 6aminopyridine-2-carboxylic acid (6-NH2-pic, 1.28 mmol, 177 mg) and 450 μL HNO3 was stirred for 60 min at 100 °C. The solution color changed from dark blue to light yellow in approximately 20 min. The solution was filtered and allowed to stand at room temperature for 2 weeks. Large yellow green needlelike crystals were obtained for an X-ray diffraction study (yield 42%). Elemental analysis: Calcd. For C12H16CrN5O10 (%): C 32.59, H 3.65, N 15.83. Found (%): C 32.52, H 3.79, N 15.72. Selected FT-IR data (KBr pellet, cm−1): 3410 (s), 3221 (s), 1664 (s), 1305 (s), 827 (m), 769 (m), 484 (m) (Fig. S1). 2.4. Synthesis of Cr(3-NH2-pic)3 (3) An aqueous solution of 3-aminopyridine-2-carboxylic acid (3-NH2pic, 1.92 mmol, 265 mg) and triethylamine (1.92 mmol, 266 μL) was added dropwise to an aqueous solution of Cr(NO)3·9H2O (0.640 mmol, 256 mg) under stirring at 100 °C for 1 h. The solution was filtered and brown block crystals were obtained after 2 weeks (yield 47%). Elemental analysis: Calcd. For C18H15CrN6O6 (%): C 46.66, H 3.26, N 18.14. Found (%): C 46.42, H 3.06, N 18.23. Selected FT-IR data (KBr pellet, cm−1): 3294 (br), 1643 (s), 1328 (s), 1240 (m), 1132 (m), 879 (m), 694 (m), 497(m) (Fig. S1). ESI+ mass spectra (m/z): 464.05 for [Cr(3-NH2-pic)3] (Fig. S3). The structure models for these complexes are shown in Fig. 1. Cr(pic)3 was synthesized and crystallized according the literature [23]. Selected IR data (cm− 1): 3526 (w), 3454 (w), 1681 (s), 1608 (m), 1567 (w), 1472 (w), 1351 (m), 1326 (s), 1289 (s), 1261 (w), 1239 (w), 1153 (m), 1051 (m), 863 (m), 767 (m), 715 (m), 659 (w), 475 (m) (Fig. S1). 2.5. X-ray structure determination The crystal data were collected with a Bruker SMART diffractometer with Mo-Ka (0.71073 Å) radiation at 293 K. The structure was solved

Fig. 1. Structural representation for the chromium(III) complexes.

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Table 1 Crystal data and refinement details for complexes 1 and 2.

Empirical formula Formula weight System, space group a/Å b/Å c/Å α/° β/° γ/° θ h, k, l Tot. Uniq. Data R(int) Nreflections, Nparameter Volume/Å3 Z, calculated density/Mg/m3 F(000) Final R indices [I N 2sigma(I)] R indices (all data) CCDC reference numbers:

Cr(6-CH3-pic)3 (1)

Cr(6-NH2-pic)2(H2O)2 (2)

C21H18N3CrO6 460.38 Monoclinic, C2/c 31.1910(11) 8.2907(3) 15.4109(6) 90 91.8960(10) 90 2.88 to 28.35 −41 b h b 35, −10 b k b 11, −20 b l b 20 19,222/4963 [R(int) =

C12H16N5CrO10 442.30 Triclinic, P1 7.9692(13) 8.3091(12) 14.0182(19) 102.572(5) 92.885(5) 103.311(5) 2.99 to 27.64 −10 b h b 9, −10 b k b 10, −18 b l b 17 12,617/4015 [R(int) =

0.0342] 4963/283 3983.0(3) 8, 1.535

0.0444] 4015/287 876.7(2) 2, 1.675

1896 R1 = 0.0394, wR2 = 0.1002

454 R1 = 0.0460, wR2 = 0.0903

R1 = 0.0566, wR2 = 0.1090 1,472,540

R1 = 0.0901, wR2 = 0.1058 1,472,553

Fig. 2. ORTEP diagram of [Cr(6-CH3-pic)3] (1).

and refined with SHELXTL-97 program package. Selected crystallographic data and selected angles and bond distances are shown in Tables 1 and 2, respectively. The structure was solved by direct methods SHELX97 [24] and subsequent differences Fourier map and then refined on F2 by a full-matrix least-squares procedure using anisotropic displacement parameters [25]. Atomic scattering factors are from International Tables for X-ray Crystallography [26] and molecular graphics from SHELXTL [27]. CCDC reference numbers: 1472540, 1472553.

extinction coefficient of 91,200 M−1 cm−1. Difference UV spectra for the reaction of 10 μM complexes 1 and 2 with 10 μM apoOTf at different times in 5 mM HCO− 3 were monitored [29–30]. In addition, to study the stability of Cr(III) complexes, 100 equiv. of EDTA was added to Cr(III) complexes in 0.01 M Hepes, pH 7.4 at 37 °C. The UV spectra were monitored as a function of time.

2.6. Cyclic voltammetry

2.8. Hydroxyl radical experiment

Cyclic voltammetric data were obtained using a Forth-based voltammetry program. Tetrabutyl ammonium perchlorate (TBAP, 0.1 M) was used as supporting electrolyte. Solutions (1.0 mM) of all complexes in DMSO in glassy carbon electrolyte were scanned at 100 mV/s sweep rates with a platinum electrode as auxiliary electrode and saturated calomel (SCE) as reference electrode [28].

First, the potential of chromium compounds to generate hydroxyl radicals in vitro was assessed by the traditional Fenton-like reaction method [31]. Briefly, a reaction mixture containing either Cr(pic)3 or complexes 1–3 (100 μM), 2-deoxyribose (4 mM) and ascorbic acid (Vc, 100 μM) in potassium phosphate buffer (pH 7.4, 10 mM) was incubated at 37 °C for 30 min, then hydrogen peroxide (H2O2, 100 μM) was added and cultured for 3 h. An aliquot of the mixture was treated with 2.8% (w/v) thiobarbituric acid and 1% (w/v) trichloroacetic acid and heated at 90 °C for 30 min, rapidly cooled and the amount of chromogen formed in the sample was measured by its absorption at 532 nm. FerricEDTA (100 μM) was used as a positive control.

2.7. The transfer of Cr(III) to apoovotransferrin (apoOTf) Apoovotransferrin was used to study the transfer of Cr(III)·The concentration was determined from the absorbance at 278 nm using an

Table 2 Bond distances for complexes 1, 2 and Cr(pic)3 (Å). Complex 1 Cr\ \N1 Cr\ \N2 Cr\ \N3 Cr\ \O1 Cr\ \O2 Cr\ \O3 N(1)\ \Cr(1)\ \N(2) N(3)\ \Cr(1)\ \N(1) N(3)\ \Cr(1)\ \N(2) O(5)\ \Cr(1)\ \N(1) O(5)\ \Cr(1)\ \O(3) O(5)\ \Cr(1)\ \O(1) O(5)\ \Cr(1)\ \O(3) O(3)\ \Cr(1)\ \N(1) O(3)\ \Cr(1)\ \N(2)

Complex 2 2.1025(16) 2.1489(17) 2.1003(15) 1.9272(14) 1.9555(13) 1.9319(15) 90.43(6) 162.86(6) 104.29(6) 86.57(7) 165.81(6) 92.47(7) 87.84(6) 106.23(6) 79.68(6)

Cr\ \N1 Cr\ \N2 Cr\ \O1 Cr\ \O2 Cr\ \O3 Cr\ \O5 N(1)\ \Cr(1)\ \N(2) O(1)\ \Cr(1)\ \N(1) O(1)\ \Cr(1)\ \N(2) O(2)\ \Cr(1)\ \N(1) O(2)\ \Cr(1)\ \N(2) O(5)\ \Cr(1)\ \N(1) O(5)\ \Cr(1)\ \N(2) O(1)\ \Cr(1)\ \O(2) O(5)\ \Cr(1)\ \O(3)

Cr(pic)3 2.074(2) 2.065(2) 1.990(2) 1.985(2) 1.9553(18) 1.9432(18) 178.15(10) 89.33(9) 88.95(9) 91.16(9) 90.57(9) 98.44(8) 80.89(8) 179.45(10) 178.79(8)

Cr\ \N1 Cr\ \N2 Cr\ \N3 Cr\ \O2 Cr\ \O3 Cr\ \O5 N1\ \Cr\ \N2 N1\ \Cr\ \N3 N2\ \Cr\ \N3 O1\ \Cr\ \O2 O2\ \Cr\ \N3 O2\ \Cr\ \O3 O1\ \Cr\ \O3 N2\ \Cr\ \O2 N2\ \Cr\ \O3

2.047 (2) 2.053 (2) 2.058 (2) 1.957 (2) 1.949 (2) 1.950 (2) 92.24 168.49 97.24 175.51 88.73 90.16 94.3 80.75 170.83

Note: Distances and angles data for Cr(Pic)3 were from ref. [23].

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with MTT was removed and the formazan product formed was solubilized with 200 μL DMSO. The plates were shaken for 10 min. The optical density of each well was determined at 490 nm. As a control experiment, 1% (V:V) DMSO in DMEM medium was used. 2.10. Oral toxicity study Healthy male and female C57 mice weighing between 20 and 30 g were used in this study. Animals were purchased from the animal house, First Hospital of Shanxi Medical University. The animals were fed commercial pellet feed (purchased from Nanjing Jiancheng Bioengineering Institute). Food and water were provided ad libitum during acclimation and throughout the study. The animals were maintained at 22 °C (±3 °C) and at relative humidity of approximately 50–60%. 12 h light and 12 h dark cycle was maintained throughout the experimental period. The mice were acclimatized to laboratory conditions for one week and divided into three groups of six rats each. Group 1: Control treated with vehicle (normal saline) for 90 days. Group 2: Treated with 500 mg/kg b.w. of Cr(pic)3 for 90 days. Group 3: Treated with 450 mg/kg b.w. of 1 for 90 days. At the end of the experimental period, all the animals were killed and their pancreas, liver and kidney tissues were quickly excised and washed with saline. The tissue samples were then subjected to histopathological examination. Fig. 3. ORTEP diagram of [Cr(6-NH2-pic)2(H2O)2]NO3·H2O (2).

3. Results and discussion Second, in order to explore the production mechanism of hydroxyl radical, Cr(pic)3 or complexes 1–3 (100 μM) and hydrogen peroxide (100 μM) was incubated in potassium phosphate buffer (PBS, pH 7.4, 10 mM) at 37 °C for 4 h. Then 0.01 g/mL diphenylcarbazide and 0.1 M H2SO4 was added into the solution, the UV–visible absorption spectra were measured. 2.9. MTT assay Effects of all complexes on viability of MCF-7 cancer cells were determined by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test [32]. Cells were diluted with DMEM medium to 2.5 × 104 cells/mL and aliquots (4.5 × 103 cells/180 μL) were placed in individual wells in 96-multiplates to incubate for 12 h. Then 20 μL Cr(III) complex (1.0 × 10−3 M) solution was added into the cultured cells for 24 h, subsequently, cells were incubated with 20 μL MTT (5 mg/mL, PBS) in DMEM medium for another 4 h at 37 °C. The medium

3.1. Structural analysis The crystal structure and labeling scheme for complex 1 are shown in Fig. 2 and Fig. S4. Selected interatomic distances are summarized in Table 2. The Cr(III) center is a distorted octahedron (3 + 3) with three N atoms and three O atoms surrounding by three 6-CH3-pic ligands. It is of interest to note that complex 1 is the meridional isomer with purple color, while Cr(pic)3 is the meridional isomer with red color [23]. This is consistent with assumptions in the literature that meridional isomers of CrL3 (where L = a bidentate amino carboxylate ligand) are purple and that only facial isomers are red [33], but is disproved by characterization of Cr(pic)3. Through careful analysis the angles in Table 2, such as N1\\Cr\\N3 (162.86°), N2\\Cr\\O5 (165.8°), O1\\Cr1\\O3 (172.45°), N2\\Cr\\N3 (104.30°) and O3\\Cr\\N3 (85.33°), it was found that Cr(III) center was in a seriously distorted octahedron configuration than that of Cr(pic)3. Meanwhile, the Cr\\N bond lengths for 1 range from 2.1003

Fig. 4. Comparative cyclic voltammogram of (a) complexes 1–3, Cr(pic)3 and Cr(NO3)3; (b) ligands in DMSO at scan rate = 100 mV/s.

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Fig. 5. Difference UV spectra for the reaction of 10 μM complexes 1 and 2 with 10 μM apoOTf at different times in 5 mM HCO− 3 : (a) 1, (b) 2. 37 °C, 0.01 M Hepes, pH 7.4. Inset: a plot of (A∞ − A0) / [a(A∞ − At)] vs. time, a = 10 μM.

(13) to 2.1489 (17) Å, the Cr\\O bond lengths range from 1.9272(14) to 1.9555(13) Å. The average Cr\\N and Cr\\O bond length for 1 is 2.1172 and 1.9382 Å, and the average Cr\\N and Cr\\O bond length for Cr(pic)3 is 2.053 and 1.952 Å, respectively. The Cr\\N bond distances of 1 are more longer than that of Cr(pic)3 (Δ = 0.0642 Å). Obviously, it is the presence of N ortho-position substitutive group 6-CH3 that causes the seriously distorted octahedron, which further weakens the bond strength of Cr\\N. Therefore, the stereo-hindrance effect of 6-CH3 group may play a dominant role on the octahedron distortion and Cr\\N bond weakness. Single crystal X-ray diffraction of 2 is presented in Fig. 3 and Fig. S5. The symmetric unit of 2 consists of one Cr(III), two 6-NH2-pic ligands and two H2O molecules. The Cr center is six-coordinated by N and O atoms of two 6-amino-pyridine-2-carboxylate anions and two O atoms of water molecule displaying an octahedral configuration. The bond angles N(1)\\Cr(1)\\N(2) (178.15°) and O(5)\\Cr(1)\\O(3) (178.79°) nearly approach to 180°. The angles related to the central Cr(III) are 98.44(8)° for O(5)\\Cr(1)\\N(1), 80.40(8)° for O(3)\\Cr(1)\\N(1), 100.26(8)° for N(2)\\Cr(1)\\O(3) and 80.89(8)° for N(2)\\Cr(1)\\O(5) with the sum of 359.99° closes to 360°, which shows that N(1), N(2), O(3) and O(5) together with Cr(1) are nearly coplanar [34]. Bond angles O(1)\\Cr(1)\\O(2) (179.45°), O(2)\\Cr(1)\\N(2) (90.579°) and O(1)\\Cr(1)\\N(2) (88.95°) indicate that two water molecules perpendicular to the plane. The average bond length Cr\\N and Cr\\O with 6-NH2-pic ligand are 2.0695 Å, 1.949 Å, respectively. It is comparable to the bond length reported for Cr(pic)3 (2.053 Å for Cr\\N, 1.952 Å for Cr\\O bond). The bond lengths analysis of Cr(1)\\O(1) (1.985 Å), Cr(1)\\O(2) (1.990 Å) Cr(1)\\O(3) (1.9553 Å) and Cr(1)\\O(5) (1.9432 Å) suggest that oxygen atoms from water molecules have weaker coordination ability than that of carboxylic oxygen atoms of ligand. Indeed, it seems quite remarkable that the stereo-hindrance effect of 6-NH2 group on Cr\\N bond herein can be ignored. 3.2. Electrochemical studies In the current study, cyclic voltammetry (CV) is used to determine if the accompanying pic ligand direct the biotoxicity of Cr(III) complexes

by shifting the reduction potentials and conferring reversibility of the redox couples. Complexes 1–3, Cr(pic)3 and Cr(NO3)3 in DMSO were investigated by cyclic voltammetry, respectively. All experiments were conducted in anodic and cathodic scan modes at same scan rate (100 mV/s), the summarized data are presented in Fig. 4(a) and (b). As demonstrated in Fig. 4(a), neither reduction wave nor oxidation wave are observed for the CV curve of Cr(NO3)3 in this condition. The voltammogram of 1 shows a cathodic (−1.124 V vs. SCE) and an anodic (−1.04 V vs. SCE) wave. The separation between the two peak potentials ΔEp = 84 mV (ΔEp = Epc − Epa) in a reversible electron-transfer process should be close to 58/n mV (where n is the number of electrons transferred), even will be N58/n [35–36]. It is attributed to the quasi-reversible Cr3+/Cr2+ redox process. Such a quasi-reversible behavior is typical for many Cr(III)/Cr(II) couple, because of Jahn–Teller distortion expected in the case of Cr(II) ion. The E1/2 values of 1 are −1.042/−0.929 V for Cr(III)/Cr(II) couple wave. Another redox couples appeared at Ere = −1.860 and Eox = −1.735 V, which is attributed to the quasi-reversible Cr2 +/Cr+ redox process. The E1/2 values of 1 are −1.749/−1.552 V for Cr(II)/Cr(I) couple wave. The CV of the complex 2 indicates reduction of Cr(III) to Cr(II) at a cathodic peak potential, Epc of −1.234 V versus SCE in DMSO solution. Reoxidation of the Cr(II) species occurred at − 1.160 V upon reversal of scan, ΔEp = 74 mV. The E1/2 values of 2 for Cr(III)/Cr(II) appear at − 1.095/− 1.069 V. The second cathodic peak appeared at Ere = −1.54 V (Er1/2 value = −1.460 V), but anodic wave was not observed. Just like Cr(NO3)3, the presence of two coordinated H2O molecules in complex 2 affected the anodic peak potential to a large extent. The CV of 3 reveals −1.250/−1.305 V for Cr(III)/Cr(II) couple wave. The E1/2 values for Cr(III)/Cr(II) are −1.095/−1.069 V. For Cr(II)/Cr(I), the E1/2 values are − 1.810/− 1.72 V. Similarly, the redox couples of Cr(pic)3 occurred at −1.04/−1.124 V. Comparing the metal-centered reduction potential values of the three species discussed above, − 1.124 (1) ≈ − 1.126 (Cr(pic)3) N − 1.234 (2) N − 1.305 V (3) vs. SCE respectively, an apparent cathodic shift (more negative) is observed for complexes 2 and 3 than 1 and Cr(pic)3. In addition, the oxidation potential values follow the sequence of − 1.04 (1) N − 1.07 (Cr(pic)3) N − 1.16 (2) N − 1.25 (3). In a word, it shows a trend with

Table 3 The Cr\ \N bonds, N\ \Cr\ \N′ angles, redox potentials, cell viability, transfer rate constant and generation of hydroxyl radicals for Cr(III) complexes.

1 Cr(pic)3 2 3

Bond length Cr\ \N (Å)

Bond angles N\ \Cr\ \Naxis

Er1/2/V

Eo1/2/V

A (•OH)

Cell viability

k'EDTA h−1

k″apoOTf M−1 h−1

2.117 2.053 2.0695 No data

162.86 168.49 178.15 No data

−1.042 −1.052 −1.095 −1.22

−0.929 −0.973 −1.069 −1.16

0.0815 0.459 0.574 0.732

90% 82% 72% 85%

0.2312 0.001 0.0417 0.00001

1.21 × 104 No data 3.18 × 103 No data

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the pic and methyl derivative having no difference and a more negative shift is observed as more electron withdrawing substitutes are added. 3.3. Dynamics stability 3.3.1. Transfer of Cr(III) to apoOTf The iron-transport protein transferrin, the second most abundant protein in blood serum, has been proposed to serve as the major chromium transport agent, although this opinion has just been challenged [8]. In this paper, in order to compare the Cr(III) transfer efficiency of these complexes, apoovotransferrin (apoOTf) was utilized in place of serum transferrin because of its ready availability in quantity and its cost; the binding properties of apoOTf are nearly identical to serum transferrin [30]. The addition of Cr(III) to apoOTf results in enhancement of the intensity of the UV absorption bands at ca. 240 and 291 nm (arising in part from tyrosine residues which serve as metal ligands) as a result of Cr(III) binding. Cr(pic)3 does not release its chromium efficiently to biological chromium-binding species including apoOTf [37]. Fig. 5 shows the difference UV spectra of the mixture of 10 μM 1 or 2 with 1.0 equiv. of apoOTf at 37 °C blanked as protein solution. With time going, it reveals a significant enhancement in the absorbance at 291 and 240 nm. The increase is attributed to the binding of Cr(III) to residues of the protein. The second-order rate constant k″ at 37 °C is calculated to be (1.21 ± 0.05) × 104 M−1 h− 1 and (3.18 ± 0.08) × 103 M−1 h− 1 for 1 and 2, respectively. However, no changes are observed for 3 and Cr(pic)3 throughout this competition reaction for 100 h, which is consistent to the literature [37]. It indicates that 3 is very stable and the Cr(III) cannot be transferred to apoOTf.

Fig. 6. Generation of hydroxyl radicals by complexes 1–3 and Cr(pic)3. Control group contained Fe-EDTA (100 μM). Sample containing 4 mM desoxyribose sugar, 100 μM Vc, 100 μM H2O2 in 10 mM phosphate buffer (pH 7.4). Results are mean ± SD of three independent experiments.

3.4. Generation of hydroxyl radicals

generate more active redox center. This redox active center has the potential to generate more oxygen radicals. It is generally accepted that the generation of hydroxyl radicals by Ferric-EDTA was due to the Fenton reaction [38], and the process includes two step: 1) Fe3+ must be related to the metal centered reduction Fe3 + + Vc → Fe2 +; 2) Oxidation Fe2 + + H2O2 → Fe3 + + •OH. However, it is reported that Cr(III) can be oxidized to high valent Cr by H2O2 directly at pH 7.4 condition. Therefore, it is reasonable to suggest that oxidation of Cr(III) by H2O2 directly with the formation of oxo(hydroxo)Cr(IV) or Cr(V) complexes may be the actual mechanism [14]. To identify this deduction, Cr(pic)3 or complexes 1–3 (100 μM) was incubated with hydrogen peroxide (100 μM) in potassium phosphate buffer (pH 7.4, 10 mM) at 37 °C for 4 h. The production of high valent Cr species was measured using diphenylcarbazide though UV–visible absorption spectra. As shown in Fig. 7, the characteristic absorption peak at 540 nm for high valent Cr species (Cr(VI), Cr(V) and Cr(IV)) appeared which indicated that these Cr(III) complexes could be oxidized to high valent Cr directly by hydrogen peroxide at pH 7.4. Among these complexes, the amount of Cr(VI) species of Cr(pic)3 is consistent with literature [14], the maximum amount high valent Cr was produced by 1. Herein, thermodynamic stability or other factors of Cr(III) complex is thought to be the determining factor. On the other hand, there has

The chromium picolinate complex, Cr(pic)3, is known as a bioavailable source of chromium(III), where the picolinate acts as a Cr(III) transporter. Recent reports have indicated that Cr(pic)3 can be oxidized to Cr(VI) by H2O2 at pH 7.4 resulting in the catalytic generation of the potent DNA-damaging hydroxyl radical [5,7]. In order to compare with Cr(pic)3, the potential of chromium compounds to generate hydroxyl radicals in vitro was assessed by the traditional method (Fenton-like reaction). As a control experiment, generation of hydroxyl radicals by the free ligands of these complexes was shown in Fig. S7. As shown in Fig. 6, Ferric-EDTA complex in the presence of ascorbate generated hydroxyl radicals via the Fenton reaction caused significant damage to 2-deoxyribose. The model complex Cr(pic)3 induced an obvious increase in hydroxyl radical production [22]. Interestingly, the amount of chromogen produced by 1 was lower than that of blank, while 2 and 3 induced an obvious increase in hydroxyl radical production than Cr(pic)3. The UV values follow the sequence of (1) b Cr(pic)3 b (2) b (3). This sequence is just in contrast to that of oxidation potential values. Obviously, the more negative potential values it behaves, the more hydroxyl radicals it produces. Less hydroxyl radicals was generated by 1 under the conditions tested. While 3-position amino group on 3 alter the electrochemical behavior of chromium picolinate to

Fig. 7. The UV–visible absorption spectra for the reaction of Cr(V,VI) with diphenylcarbazide (0.01 g/mL). Cr(V,VI) was produced by complex 1–3 or Cr(pic)3 (100 μM) + H2O2 (100 μM) in 10 mM phosphate buffer (pH 7.4). Inset: the absorbance at 540 nm of these complexes. Results are mean ± SD of three independent experiments.

3.3.2. Transfer of Cr(III) to EDTA In addition, EDTA as a simple competitive ligand was added to study the stability of these Cr(III) complexes. To examine the transfer of Cr(III), 100 equiv. of EDTA was added to 5.0 mM complex 1–3 in 0.01 M Hepes at pH 7.4, and then the mixture was stored at 37 °C. The UV–vis spectra were recorded until the spectra became constant as time (Fig. S6). The first-order rate constant k′ was obtained from a general approach, as shown in Table 3. Significant differences were found between these rates constants: 0.2312 (1) N 0.0417 (2) N 0.001 (Cr(pic)3) N 0.00001 h−1 (3), among which complex 1 is the fastest one for the competitive process. This sequence is accordance to that of apoOTf.

Please cite this article as: B. Liu, et al., Chemical properties and biotoxicity of several chromium picolinate derivatives, J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2016.09.006

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3.5. Cytotoxic tests

Fig. 8. Cytotoxic effect of complexes 1–3 on cancer cell lines. The cytotoxicity of 1–3 and Cr(pic)3 (1.0 × 10−4 M) were evaluated on MCF-7 cancer cell lines (2.5 × 104 cells/mL) by the MTT assay for 24 h. 1% DMSO (V:V) was used as a control experiment. Results are mean ± SD of six independent experiments.

been much interest generated in the reaction of Cr(VI) with Vc since the observation that ascorbate is one of the major reductants of Cr(VI) in rodents and humans [39]. It is known that the hydroxyl radicals can be produced easily by the reaction of CrO2− and Vc [40]. So far, it can be 4 safely concluded that production of hydroxyl radicals in Fig. 5 is closely related to the following steps: 1) Cr(III) + H2O2 → Cr(VI, V, IV) + H2O; 2) Cr(VI, V, IV) + Vc and/or H2O2 → Cr(III) + •OH. In a word, chromium picolinate and it derivatives can be oxidized to toxic high valent Cr species by hydrogen peroxide in neutral aqueous media. However, it is needed to note that the 100 μM hydrogen peroxide used in this paper is not a biologically relevant condition.

To further evaluate the practical toxicity of these Cr(III) complexes, cytotoxicity was determined on MCF-7 cancer cells using MTT assay for 24 h, results are mean ± SD of six independent experiments. DMSO was used as a control experiment. The results were shown in Fig. 8 and Fig. S8. Fig. 8 shows the viability of cells treated with Cr(pic)3 or complexes 1–3 as monitored by MTT assay. Overall, the difference is not statistically significant. Cell ability is 81% for Cr(pic)3, up to 90% for 1, about 70% for 2 and 81% for 3, respectively. Complex 1 has low toxicity for tumor cells than other species, which is consistent with the result of hydroxyl radicals experiment. The cell ability for 3 is higher than 2 and Cr(pic)3, which is beyond our expectation. It indicates that cell toxicity of Cr(III) complex does not absolutely depend from the ability of hydroxyl radicals generation in vitro. The cell toxicity of Cr(III) complex in cells is a complex process [7]. On the other hand, it should be pointed out that the cells are exposed to intact Cr(pic)3 in vitro, which would not occur for cells in the human body. 3.6. Sub-chronic oral toxicity study Histopathological examination for kidney, liver and pancreas histology sections of C57 rats with Cr(pic)3 and 1 are shown in Fig. 9. Kidney histology sections of C57 rats treated orally with different complexes showed normal structures. Control group showed thin glomeruli with normal vascularity. Many collecting tubules are normal. Both of Cr(pic)3 and 1 group showed distal collecting tubules and the interstitial tissues with normal architecture. Histological features of the liver of control rats showed normal structures. No adverse effect was found on the histoarchitecture of hepatocytes of mice administered acutely with 500 mg/kg dose of Cr(pic)3 and 450 mg/kg b.w. dose of 1. The central vein, and the hepatocytes are arranged in a regular manner for Cr(pic)3 and 1. Specially, mild

Fig. 9. Oral toxicity study for 90 days: kidney, liver and pancreas histology sections of C57 rats with Cr(pic)3 and 1. Magnifications of all the stained sections were 400×. Control: normal architecture. Cr(pic)3 (500 mg/kg b.w.) and 1 (450 mg/kg b.w.).

Please cite this article as: B. Liu, et al., Chemical properties and biotoxicity of several chromium picolinate derivatives, J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2016.09.006

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B. Liu et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx

mononuclear in filtration and mild loss of hepatocyte architecture were not observed for these complexes. Similarly, non-toxicity was found through pancreas histology section analysis. As shown in extension reviews, the toxic effects of Cr(pic)3 arises only in situations when the contact molecule is present (cell culture, injection into mammals, animals with simple digestive systems such as fruit flies); in mammals, the compound degrades in the stomach and is non-toxic [5]. 3.7. Relationship between physicochemical properties The structure and different substituent groups have apparent effect on the Cr(III) complexes electrochemical behavior, Cr(III) transfer rate, ability of generation of hydroxyl radicals as well as cell cytotoxicity. The results were listed in Table 3 and the relationships between these factors were shown in Fig. 10. In general, the stability of complexes largely depends upon the coordination bond strength of Cr\\N or Cr\\O [41], and the induced effect of substituent on electron density of pyridine N atoms should be bigger than that of carboxylic O atom. For Cr(pic)3 or its derivatives, the difference of Cr\\O bonds have not distinct differences, while the Cr\\N bonds exist significant differences. To be specific, stereo-hindrance effect of 6-position group causes the octahedron distortion and seriously weakens the Cr\\N bond in 1:3 type complexes. The relationship of Cr\\N bond with Cr(III) transfer rate are shown in Fig. 10(a). It shows that Cr\\N bonds have important effect on the stability of Cr(pic)3 or its derivatives: the shorter the Cr\\N bonds, the slower the transfer of

Cr(III). Herein, stereo-hindrance effect of 6-CH3 group maybe play a dominant role on the octahedron distortion and Cr\\N bond weakness than the induced effect. The production of hydroxyl radicals through Fenton-like reaction can be reflected by its electrochemical behavior. Negative shifts of the redox couples could allow the complex to serve as a good reductant in the oxidation reaction with H2O2. The more negative oxidation potential values it behaves, the more hydroxyl radicals it will produce. This deduction was identified by Fig. 10(b) and (c): the sequence of Er1/2/V or Eo1/2/V (1 N Cr(pic)3 N 2 N 3) is just well accordant to that of generation of hydroxyl radicals (1 b Cr(pic)3 b 2 b 3). Among these complexes, 1 behaves more positive redox couples, and it generate the least hydroxyl radicals through Fenton-like reaction. That is, there is a closely correlation between the potential values and potential to produce oxygen species through Fenton-like reaction. However, the sequence of cytotoxicity for these complexes, 1 b 3 b Cr(pic) 3 b 2, is not well consistent with that of hydroxyl radicals (Fig. 10(d)). Among these complexes, 1 has the lowest toxicity while 2 behaves the highest toxicity. Different from other complexes, 2 poses two-coordinated H2O molecular and + 1 positive charge. It suggested that cytotoxicity is not only related to the ability to generation hydroxyl radicals, but also involve other factors, possibly such as the hydrophilic or lipophilic characteristic, charge, DNA damage mode and so on. Moreover, 100 μM hydrogen peroxide used in this paper is not a biologically relevant condition. In the presence of normal biological reductants such as glutathione, dithiothreitol and vitamin C, generation hydroxyl radicals can be ignored.

Fig. 10. The relation between reduction potentials, Cr\ \N bond, generation of hydroxyl radicals and rate constant for Cr(III) complexes 1, 2, 3 and Cr(pic)3.

Please cite this article as: B. Liu, et al., Chemical properties and biotoxicity of several chromium picolinate derivatives, J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2016.09.006

B. Liu et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx

Work is in progress to examine how the ligands affect the uptake and biological activity of chromium. 4. Conclusions Taken together, The evidences suggest that Cr(pic)3 and its derivatives have different physicochemical property, such as structure, electrochemical behavior, stability, potential to generate oxygen radicals and cytotoxicity. There was a positive correlation between the stability and potential to produce oxygen species in vitro. Additionally, the generation of oxygen radicals is mainly depended on the electrochemical potentials: the more negative oxidation potential values it behaves, the more hydroxyl radicals it produces through Fenton-like reaction. However, the difference on cytotoxicity is not statistically significant and these chromium picolinate complexes are non-toxic through subchronic oral toxicity study. In sum, this work provides a framework for integrating the structure-property-toxicity into a mechanistic understanding of the chromium picolinate complexes. Abbreviations pic 2-carboxypyridine 6-CH3-pic 6-methylpyridine-2-carboxylic acid 6-NH2-pic 6-aminopyridine-2-carboxylic acid 3-NH2-pic 3-aminopyridine-2-carboxylic acid apoOTf apoovotransferrin TBAP Tetrabutyl ammonium perchlorate CV cyclic voltammetry MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide k′ first-order rate constant k″ second-order rate constant

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Acknowledgements We are very grateful for financial support from the National Natural Science Foundation of China (No. 21271122, 21571117) and Research Project Supported by Shanxi Scholarship Council of China (2013-018, 2015-021). Supplementary data CCDC 1472540 (for 1) and 1472553 (for 2) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: (+ 44) 1223-336-033; or [email protected]). Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jinorgbio.2016.09.006.

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Please cite this article as: B. Liu, et al., Chemical properties and biotoxicity of several chromium picolinate derivatives, J. Inorg. Biochem. (2016), http://dx.doi.org/10.1016/j.jinorgbio.2016.09.006