Biomimetic oxidation of chromium(III): Does the antidiabetic activity of chromium(III) involve carcinogenic chromium(VI)?

Article abstract of DOI:10.1002/anie.200460113

The insulin-enhancing activities of some Cr^III complexes, such as [Cr₃O(OCOEt)₆-(OH₂)₃]⁺ (1), previously attributed to specific interactions of Cr^III ions with cellular insulin receptors, are more likely to be caused by the formation of [CrO₄]^2- (2). Oxidation of 1 to 2 by biologically relevant oxidants, including enzymes (see scheme), and inhibition of an isolated protein tyrosine phosphatase by a Cr^VI complex are reported.

Full text of DOI:10.1002/anie.200460113

Communications
Bioinorganic Chemistry
Biomimetic Oxidation of Chromium(iii): Does the
Antidiabetic Activity of Chromium(iii) Involve
Carcinogenic Chromium(vi)?**
Irma Mulyani, Aviva Levina, and Peter A. Lay*
The proposed role of CrIII ions as an essential human
micronutrient, responsible for insulin activation in glucose
and fat metabolism,[1] is currently under dispute.[2,3] The most
recent mechanistic hypothesis on the biological activity of the
CrIII ion assumes the existence of a specific biomolecule that
binds CrIII ions, chromodulin, which enhances interactions of
insulin with its cellular receptors.[4] No definitive structural
information on chromodulin is available as yet,[4] and its
isolation procedure is controversial.[3,5] A well-characterized
trinuclear
oxo–carboxylato
complex,
[CrIII3O(OCO-
Et)6(OH2)3]+ (1)[6] has been proposed as a structural and
[*] I. Mulyani, Dr. A. Levina, Prof. P. A. Lay
Centre for Heavy Metals Research
School of Chemistry
University of Sydney
Sydney, NSW 2006 (Australia)
Fax: (+61)2-9351-3329
E-mail: p.lay@chem.usyd.edu.au
[**] Financial support for this work was provided by an Australian
Research Council Discovery grant, including an Australian Profes-
sional Fellowship (to P.A.L.). I.M. is grateful to Chemistry Depart-
ment, Bandung Institute of Technology, Indonesia, for partial
funding of her postgraduate studies at the University of Sydney. We
thank Dr. Michael Davies (Heart Research Institute, Sydney) for
helpful discussions.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200460113
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Table 1: Typical conditions and results of CrIII oxidations.
functional mimetic of chromodulin[7] and a safer potential
therapeutic agent for Type II diabetes and related disorders[8]
than the controversial nutritional supplement, [CrIII(pic)3] (2,
pic = picolinato(1À) = pyridine-2-carboxylato(1À)).[9]
Substrate[a]
Oxidant[b]
Buffer[c]
t [h][d]
[CrVI] [mm][e]
1
1
1
1
1
1
H2O2 (1.0)
H2O2 (1.0)
H2O2 (0.10)
H2O2 (1.0)
H2O2 (1.0)
H2O2 (1.0)
H2O2 (1.0)
H2O2 (1.0)
ClOÀ (0.25)
GO[i]
A
A
A
B
C
D
A
1.0
6.0
6.0
6.0
6.0
1.0[f]
6.0
6.0
6.0
6.0
6.0
12Æ2
33Æ6
4.0Æ0.7
10Æ1
20Æ1
8Æ2
Although biological oxidations of CrIII species to geno-
toxic and carcinogenic CrVI or CrV species[3] were previously
considered impossible,[10] evidence is mounting for the
feasibility of such oxidations under physiologically relevant
conditions.[3,11] Herein we propose, for the first time, that the
reported insulin-enhancing activities of some CrIII com-
pounds,[1,8] previously attributed to specific interactions of
CrIII ions with cellular insulin receptors,[4] are caused by intra-
or extracellular oxidations of CrIII to CrV and/or CrVI
compounds, which act as protein tyrosine phosphatase
(PTP) inhibitors by mechanisms similar to those for a well-
known insulin mimetic VV species (isoelectronic to CrVI).[12]
Strong oxidants, such as H2O2 and ClOÀ, are produced
naturally by cells, particularly those involved in the immune
response, through the reactions of oxidase enzymes with
reductants and O2.[13] While oxidation of CrIII to CrVI ions by
H2O2 in strongly alkaline media is well-known and is widely
used in analytical chemistry,[14] only scattered reports exist on
the oxidation of some CrIII complexes (with nonbiological
ligands) by H2O2 or ClOÀ in neutral or weakly basic aqueous
media (pH 7–9).[15] We chose complex 1 as a main substrate
for the systematic studies of the oxidation of CrIII species
under physiologically relevant conditions owing to its known
biological activity[8] and proposed structural similarity to a
purported natural factor that contains CrIII ions.[4,7] Complex 1
can also serve as a representative example of numerous
polynuclear species formed on hydrolysis of CrIII complexes
in neutral aqueous solutions.[16] It is relatively stable at pH 7.4
and 378C, although partial hydrolysis (leading to di- and
mononuclear species) occurs on the timescale of hours, as
revealed by electrospray mass spectrometry (see the Support-
ing Information).
[g]
CrCl3
10Æ1
1.0Æ0.1
23Æ2
14Æ3
9Æ1
2
1
1
1
A
B[h]
A
XO[j]
A
[a] In all reactions, [CrIII]0 =0.10 mm. [b] Concentrations (mM) are given
in parentheses. [c] Designations: A is 4-(2-hydroxyethyl)-1-piperazine-
ethanesulfonate (HEPES, 0.10m, pH 7.4); B is phosphate (0.10m,
pH 7.4); C is tris(hydroxymethyl)aminomethane (Tris, 50 mm, pH 7.0),
containing NaCl (0.10m), ethylenediamine-N,N,N’,N’-tetraacetate
(EDTA, 0.20 mm), bovine serum albumin (BSA, 1.0 mgmLÀ1), and Brij
35 (0.001%); and D is fetal bovine serum (undiluted). [d] All the
reactions were carried out at 378C. [e] Average results and standard
deviations of three independent experimental series. [f] Longer reaction
times led to partial reduction of the formed CrVI. [g] Hydrolysis products
of CrCl3. [h] No CrVI formation was observed in buffer A, probably
because of ClOÀ reduction by the buffer. [i] Glucose oxidase
(0.10 UmLÀ1), glucose (5.0 mm), and ambient air O2. [j] Xanthine
oxidase (0.10 UmLÀ1), xanthine (saturated, ~1 mm), and ambient air
O2.
EPR spectroscopy)[3,11] were formed under the studied
conditions (Table 1).
The abilities of a CrVI complex, as well as of a model CrV
complex, [CrVO(ehba)2]À (ehba = 2-ethyl-2-hydroxybutan-
oato(2À)),[20] to inhibit a well-characterized[21] microbial
PTP[22] in vitro were comparable to those of VV under the
same conditions (Figure 1). The PTP used contained a
Cys(X5)Arg catalytic domain (X is any amino acid), which
is common for all the known microbial and mammalian
PTPs.[21] The buffer used in the phosphatase reactions
(corresponding to C in Table 1) contained small amounts of
Oxidations of 1 by H2O2 or ClOÀ at pH 7.4 led to the
formation of [CrVIO4]2À, detected from its characteristic
absorbance in electronic spectra (lmax = 372 nm, emax ~ 4.7 ”
103 mÀ1 cmÀ1, see the Supporting Information).[14a] Quantita-
tive determination of CrVI species was performed by the
diphenylcarbazide method[17] after the removal of uncon-
verted H2O2 by catalase (see the Supporting Information for
experimental details), typical results are presented in Table 1.
Micromolar concentrations of CrVI ions were produced in
the reactions of 1 (Cr-ion concentration, 0.10 mm) with sub-
mm concentrations of H2O2 or ClOÀ, as well as with two well-
known H2O2-producing enzymatic systems, based on glucose
oxidase[18] or xanthine oxidase,[19] on the hours timescale at
pH 7.0–7.4 and 378C (Table 1). This is the first evidence for
oxidation of CrIII ions by enzymatic systems. Kinetic studies of
the reaction of 1 with the glucose oxidase system showed that
the formation of CrVI species is preceded by the formation of
H2O2 (see the Supporting Information). Notably, oxidation of
1 by H2O2 was efficient in a protein-rich environment and
Figure 1. Inhibition of a recombinant Yersinia enterocolitica PTP (from
Sigma) by CrVI ions (Na2CrO4), CrV ions (Na[CrO(ehba)2]), or VV ions
(Na3VO4) in buffer C (see note [c] to Table 1), at 378C (reaction time
30 min), by using p-nitrophenylphosphate (10 mm) as a substrate.
Averaged results and standard deviations of three independent experi-
mental series are shown; experimental details are given in the Support-
ing Information.
even in undiluted blood serum (buffers C and D in Table 1).
VI
Relatively lowbut detectable levels of Cr
species were
formed upon the oxidation of 2 or hydrolysed CrCl3 by H2O2
(Table 1).[16] No detectable levels of CrV species (ꢀ 1 mm, by
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Communications
thiols ([RSH] ꢁ 7 mm, see the Supporting Information for
more easily, Table 1) is efficient in healthy animals.[8] A stable
chelate complex, 2, is relatively nonreactive in vitro (Table 1);
indeed, its stability in gastrointestinal media is one of the
main reasons for its use as a nutritional supplement.[1]
However, 2 is known to be metabolized by hepatic enzymes
with the release of more reactive CrIII species.[28] Rapid
hydrolysis of CrCl3 in neutral aqueous media with the
formation of insoluble products[16] is a likely reason for its
relatively inefficient oxidation to CrVI (Table 1), as well as for
the controversies regarding its biological activity.[1]
In conclusion, the ease of oxidation of 1 to carcinogenic
CrVI under biologically relevant conditions (Table 1) warrants
further research into the safety of using 1 (or any other CrIII
compound) as a nutritional supplement or therapeutic
agent.[8]
details), which helped to maintain the active reduced state of
the Cys residue in the catalytic domain of the enzyme.[21] To
ensure that the inhibitory effects of CrVI and CrV specues were
not due to the depletion of the antioxidant thiols in the buffer,
the abilities of CrVI and CrV complexes to reduce the PTP
activity in the presence of excess thiol (dithiothreitol,
[RSH] = 1.0 mm) were also demonstrated (see the Supporting
Information). Furthermore, the efficiency of PTP inhibition
by CrVI species (0.10 mm) was not significantly decreased in
the presence of the most abundant biological reductant,
glutathione (GSH) at a high physiological concentration
(10 mm, Figure 1).[22] These data suggest that CrVI and CrV
species are likely to react with PTPs even in the thiol-rich
intracellular environment. To our knowledge, this is the first
example of inhibition of an isolated PTP by CrVI or CrV
compounds. No significant (ꢀ 20%) effects on the PTP
activity were observed for CrIII compounds (up to 0.50 mm Experimental Section
of 1, 2, or CrCl3).[23]
Full details of the used reagents, preparation of reaction solutions and
instrumental techniques are given in the Supporting Information.
Reactions of [CrO4]2À ions with PTPs probably involve
reversible formation of relatively stable five-coordinate
thiolato complexes with Cys residues in the catalytic domains
of the enzymes (Figure 2),[24] consistent with the generally
Received: March 25, 2004 [Z460113]
Keywords: bioinorganic chemistry · biological activity ·
.
chromium · enzyme inhibitors · oxidation
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