Unusual reactivity in a commercial chromium supplement compared to baseline DNA cleavage with synthetic chromium complexes

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Abstract

Commercially available chromium supplements were tested for their DNA cleavage ability compared with synthetic chromium(III) complexes, including chromium(III) tris-picolinate [Cr(pic)3], basic chromium acetate [Cr3O(OAc)6]+, model complexes, and recently patented Cr-complexes for use in supplements or therapy. Four different supplements (P1P4) were tested for their DNA cleaving activity in the presence and the absence of H2O2, dithiothreitol (DTT) or ascorbate. One supplement, P1, showed nicking of DNA in the absence of oxidant or reductant at 120 μM metal concentration. Different lot numbers of P1 were also tested for DNA cleavage activity with similar results. Commercial supplements containing Cr(pic)3 nicked DNA at ⩾120 μM metal concentrations in the presence of 5 mM ascorbate or with excess hydrogen peroxide, analogous to reactions with synthetic Cr(pic)3 reported elsewhere. Another chromium (non-Cr(pic)3) supplement, P2, behaves in a comparable manner to simple Cr(III) salts in the DNA nicking assay. Chromium(III) malonate [Cr(mal)2] and chromium(III) acetate [Cr(OAc)] can nick DNA in the presence of ascorbate or hydrogen peroxide, respectively, only at higher metal concentrations. The Cr(III) complexes of histidine, succinate or N-acetyl-l-glutamate do not nick DNA to a significant degree.

Introduction

DNA cleavage assays in the presence of various metals are a simple, rapid and sensitive probe of metal mediated processes, such as hydrolysis or metal–DNA redox reactions, that may have biological significance [1], [2]. These assays may use plasmid, single-stranded DNA or double-stranded DNA fragments or other nucleic acids to determine initial modes of reactivity between metal ions and these biological materials. Plasmid DNA cleavage reactions giving rise to uncut (supercoiled), nicked (relaxed supercoil) or cut (linear) forms of the plasmid may be used for concentration and time dependent assays of redox, hydrolytic or other reactivity with the nucleic acids.

While much is known about the toxicology of chromium (especially the oxidation states 4, 5, and 6) [3], [4], [5], [6], literature on the necessity of Cr(III) as an essential trace element and its biochemical speciation, even with extensive reviews [7], [8], [9], [10], [11], is not definitive [11], [12], [13]. There are good examples of work exploring amino acid–Cr(III) chemistry [14], [15], [16], [17], [18], [19], [20], especially redox processes related to biological systems [21], but a definitive answer as to the nature of a Cr(III) “glucose tolerance factor”, the biological form of chromium(III) and its mechanism of action has not been determined [11], [22], [23].

The work of John Vincent and coworkers in characterizing a “low molecular weight” chromium binding peptide (LMWCr) has sought to clarify the role and mechanism of chromium usage in the body [23]; this complex may turn out to be the elusive “glucose tolerance factor”. LMWCr has recently been shown to possibly play a role in the insulin-dependent glucose uptake pathway. The apparent action of the chromium complex is to stimulate and prolong glucose metabolism inside insulin sensitive cells after external binding of insulin. The role of Cr(III) and of LMWCr are of interest to researchers in the areas of adult-onset diabetes, carbohydrate and lipid metabolism, toxicology, and bioinorganic chemistry.

Chromium(III) picolinate (Cr(pic)3) (Fig. 1) has some advantage over simple chromium salts in dietary uptake [11] and has become a popular nutritional supplement over the past decade. Cr(pic)3 is available over the counter in the form of tablets, sports drinks, etc., however, there is concern regarding deleterious effects of Cr(pic)3 in the recent literature [2], [24], [25]. In addition to supplements for human consumption, chromium supplementation of animal feeds is practiced [26], and there are recent patents for the use of other Cr(III) complexes as supplements of both types [27], [28].

Here, we are using DNA reactivity as a sensitive probe of interaction of Cr(III) complexes with DNA. The present study focuses on the testing of store brand chromium supplements, Cr(pic)3 and various models of the “low molecular weight chromium” substance for their DNA cleavage activity and making general comparisons among the compounds tested. We tested reactivity of four store brands of nutritional chromium supplements with pUC 19 DNA. Also, we repeated previously reported DNA reactions with synthetic Cr(pic)3 [2] and with ‘basic’ chromium acetate (Cr(OAc)) (Fig. 1) [29]. The purpose for repeating those reactions is to confirm the expected results in our hands and to compare these with supplements and other Cr(III) compounds. Also completed was the testing of various new model complexes of low molecular weight chromium binding peptide. The models synthesized and tested were trans-chromium malonate (Cr(mal)2), a chromium bis-histidine complex, and chromium diacid complexes: chromium succinate and chromium(N-acetyl-l-glutamate) (Fig. 1).

Section snippets

Caution

Ethidium bromide is a suspected mutagen. Chromium in the oxidation states, II, IV, V, and VI, are known or potentially hazardous species. Chromium(III) may also present hazard under certain conditions. Care should be taken while handling these materials.

General

Reagents were used as supplied unless otherwise noted. pUC 19 DNA was obtained from New England BioLabs Inc. and used as received. l-Ascorbic acid was obtained from Sigma–Aldrich Chemical, Inc. Cr(pic)3 and trans-chromium(III) malonate [Cr(mal)2

Results

Reactions of pUC 19 DNA with chromium(III) complexes were analyzed by gel electrophoresis. The reactions were evaluated by comparing the relative amounts of nicked (relaxed supercoil) or cut (linear) forms of the plasmid to the amount of uncut (supercoiled) plasmid present in control reactions. The reactivity of DNA was compared with commercial chromium supplements, Cr(pic)3 and other synthetic chromium(III) complexes.

Discussion

In this project, commercially available Cr supplements were tested for their ability to nick DNA. By coincidence, the first Cr(pic)3 containing supplement tested, P1a, exhibited unusual ability to cleave DNA compared to other manufacturers’ chromium supplements containing various forms of Cr. Two other supplements containing Cr(pic)3 (P3, P4) showed lower levels of DNA nicking activity consistent with Cr(pic)3 studied by Vincent and coworkers [2]. A third Cr supplement containing another form

Abbreviations

    DTT

    D,L-1,4-Dithiothreitol

    Cr(pic)3]

    Chromium(III) trispicolinate

    [Cr(mal)2]

    trans-chromium(III) malonate

    ESR

    electron paramagnetic resonance

    [Cr(His)2]

    trans-imidazole-chromium(III) histidine

    [Cr(OAc)]

    ‘basic’ chromium(III) acetate, [Cr3O(OAc)6]+

    LMWCr

    low molecular weight chromium-binding peptide(s)

    TBE

    Tris/boric acid/EDTA buffer

Acknowledgement

We thank Dr. Dipesh Ghosh (UMKC) for samples of chromium succinate and chromium N-acetyl-l-glutamate. This work was supported by an award from the American Heart Association, and by funds from the University of Missouri Research Board. M.M.R. was supported by an award from the UMKC SEARCH undergraduate research program.

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