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Forming a chromium-based interstrand DNA crosslink: Implications for carcinogenicity
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The mutagenic and carcinogenic properties of chromium(VI) complexes have been ascribed to the formation of ternary Cr(III)-small molecule-DNA complexes. As part of these laboratories’ efforts to establish the structure and properties of discrete binary and ternary adducts of Cr(III) and DNA at a molecular level, the properties of Cr(III)-cysteine-DNA, Cr(III)-ascorbate-DNA, and Cr(III)-glutathione-DNA complexes formed from Cr(III) were examined. These studies determined the composition of previously described “pre-reacted” chromium cysteinate and chromium glutathione. Neither of these complexes nor “chromium ascorbate” form ternary complexes with DNA as previously proposed. In fact, these Cr(III) compounds do not measurably bind to DNA and cannot be responsible for the mutagenic and carcinogenic properties ascribed to ternary Cr(III)-cysteine-DNA and Cr(III)-ascorbate-DNA adducts. The results of biological studies where “ternary adducts” of Cr(III), cysteine, glutathione, or ascorbate and DNA were made from “pre-reacted” chromium cysteinate or chromium glutathione or from “chromium ascorbate” must, therefore, be interpreted with caution.
The mutagenic and carcinogenic properties of chromium(VI) complexes have been ascribed to the formation of ternary Cr(III)-small molecule-DNA complexes. As part of these laboratories efforts to establish the structure and properties of discrete binary and ternary adducts of Cr(III) and DNA at a molecular level, the properties of Cr(III)-histidine-DNA complexes formed from Cr(III) were examined. These studies determined the composition of previously described “prereacted” chromium histidinate and reveal the reaction of “prereacted” chromium histidinate with DNA does not form ternary complexes as previously proposed. The products instead are chromium histidinate complexes weakly bound, probably in the minor groove, to DNA. These weakly bound adducts cannot be responsible for the mutagenic and carcinogenic properties ascribed to ternary Cr(III)-histidine-DNA adducts. The results of biological studies where “ternary adducts” of Cr(III), histidine, and DNA were made from “prereacted” chromium histidinate must, therefore, be interpreted with caution.
Chromium(VI) is a carcinogen and mutagen, and its mechanisms of action are proposed to involve binding of its reduction product, chromium(III), to DNA. The manner in which chromium(III) binds DNA has not been established, particularly at a molecular level. Analysis of oligonucleotide duplex DNAs by NMR, EPR, and IR spectroscopies in the presence of chromium(III) allows the elucidation of the Cr binding site. The metal centers were found to interact exclusively with guanine N7 positions. No evidence of chromium interactions with other bases or backbone phosphates nor of Cr forming intra‐strand crosslinks between neighboring guanine residues was observed.
Hexavalent chromium is a human carcinogen activated primarily by direct reduction with cellular ascorbate and to a lesser extent, by glutathione. Cr(III), the final product of Cr(VI) reduction, forms six bonds allowing intermolecular crosslinking. In this work, we investigated the ability of Cr(VI) to cause interstrand DNA crosslinks (ICLs) whose formation mechanisms and the presence in human cells are currently uncertain. We found that in vitro reduction of Cr(VI) with glutathione showed a sublinear production of ICLs, yield of which was less than 1% of total Cr-DNA adducts at the optimal conditions. Formation of ICLs in fast ascorbate-Cr(VI) reactions occurred during a short reduction interval and displayed a linear dose-dependence with the average yield of 1.3% of total adducts. In vitro production of ICLs was strongly suppressed by increasing buffer molarity, indicating inhibitory effects of ligand-Cr(III) binding on the formation of crosslinking species. The presence of ICLs in human cells was assessed from the impact of ICL repair deficiencies on Cr(VI) responses. We found that ascorbate-restored FANCD2-null and isogenic FANCD2-complemented cells showed similar cell cycle inhibition and toxicity by Cr(VI). XPA-null cells are defective in repair of Cr-DNA monoadducts but stable knockdowns of ERCC1 or XPF in these cells with extended time for the completion of crosslinking reactions did not produce any sensitization to Cr(VI). Our results together with chemical and steric considerations of Cr(III) reactivity suggest that ICL generation by chromate is probably an in vitro phenomenon occurring at conditions permitting formation of Cr(III) polymers.
Cisplatin is one of the most effective anticancer agents widely used in the treatment of solid tumors. It is generally considered as a cytotoxic drug which kills cancer cells by damaging DNA and inhibiting DNA synthesis. How cells respond to cisplatin-induced DNA damage plays a critical role in deciding cisplatin sensitivity. Cisplatin-induced DNA damage activates various signaling pathways to prevent or promote cell death. This paper summarizes our current understandings regarding the mechanisms by which cisplatin induces cell death and the bases of cisplatin resistance. We have discussed various steps, including the entry of cisplatin inside cells, DNA repair, drug detoxification, DNA damage response, and regulation of cisplatin-induced apoptosis by protein kinases. An understanding of how various signaling pathways regulate cisplatin-induced cell death should aid in the development of more effective therapeutic strategies for the treatment of cancer.
DNA cross-links were found in nuclei isolated from the liver and kidney of rats treated with chromate. A dose-dependent relationship between chromate exposure and total DNA cross-links was determined using the alkaline elution technique. DNA cross-links in kidney were mainly DNA-protein in nature. Chromate also induced a small amount of interstrand DNA cross-links in kidney. Liver nuclei contained protein-associated DNA single strand breaks in addition to DNA-protein cross-links. The organotropic DNA damage induced by chromate is discussed in relation to the carcinogenicity and toxicity of chromium(VI) compounds.
Chromium(III) complexes currently being sold as dietary supplements were tested for their ability to cause chromosomal aberrations in Chinese hamster ovary cells. Complexes were tested in soluble and particulate forms. Chromium picolinate was found to produce chromosome damage 3-fold to 18-fold above control levels for soluble doses of 0.050, 0.10, 0.50, and 1.0 mM after 24 h treatment. Particulate chromium picolinate doses of 8.0 micrograms/cm2 (corresponding to a 0.10 mM solublized dose) and 40 micrograms/cm2 (0.50 mM) produced aberrations 4-fold and 16-fold above control levels, respectively. Toxicity was measured as a decrease in plating efficiency relative to controls. The above treatments produced > or = 86% survival for all doses except 1.0 mM chromium picolinate, which produced 69 +/- 10% survival. Chromium nicotinate, nicotinic acid, and chromium(III) chloride hexahydrate did not produce chromosome damage at equivalent nontoxic doses. Damage was inferred to be caused by the picolinate ligand because picolinic acid in the absence of chromium was clastogenic. Data are evaluated in terms of their relevance to human exposure based on pharmacokinetic modeling of tissue accumulation and are discussed in terms of literature reporting toxic effects of picolinic acid.
The effects of 8 wk of daily chromium supplementation (3.3-3.5 mumol as chromium chloride or chromium picolinate) or placebo (0.1 mumol Cr) and weight training were examined in 36 men in a double-blind design. Strength, mesomorphy, fat-free mass, and muscle mass increased with resistance training independently of chromium supplementation (P < 0.0001). Protein, magnesium, zinc, copper, and iron intakes equalled or exceeded the recommended dietary allowance (RDA) or estimated safe and adequate daily dietary intake (ESADDI) during training and did not change significantly from pretraining intakes (P > 0.05). Chromium supplementation increased the serum chromium concentration and urinary chromium excretion without a difference as a result of the chemical form of chromium (P < 0.05). Resistance training was associated with a significant decrease (P < 0.05) in serum ferritin, total-iron-binding capacity, transferrin saturation, the ratio of enzymatic to immunoreactive ceruloplasmin, and plasma copper, independently of chromium supplementation. However, transferrin saturation was decreased more with chromium picolinate supplementation (24%) than with chromium chloride or placebo (10-13%). Compared with pretraining values, urinary magnesium excretion increased (P < 0.05) and urinary zinc output tended to decrease during the first 4 wk of resistance training and then returned to baseline values for the final 4 wk, which suggests an adaptation in mineral excretion in response to weight training. These findings suggest that routine chromium supplementation has no beneficial effects on body- composition change or strength gain in men. Whether chromium supplementation of individuals with diminished chromium nutriture facilitates propitious changes in body structure and function remains to be determined.
If chromium is an essential metal it must have a specific role in an enzyme or cofactor, and a deficiency should produce a disease or impairment of function. To date, no chromium-containing glucose tolerance factor has been characterized, the purpose of the low-molecular-weight chromium-binding protein is questionable, and no direct interaction between chromium and insulin has been found. Furthermore, chromium(3+) is treated like the toxic metals arsenic, cadmium, lead and mercury in animals. Chromium(3+) may be involved in chromium(6+)-induced cancers because chromium(6+) is converted to chromium(3+) in vivo, and chromium(3+) is genotoxic and mutagenic. Although there is no direct evidence of chromium deficiencies in humans, dietary supplements exist to provide supraphysiological doses of absorbable chromium(3+). Chromium(3+) may act clinically by interfering with iron absorption, decreasing the high iron stores that are linked to diabetes and heart disease. If so, this would make chromium(3+) a pharmacological agent, not an essential metal.
A protocol is presented for the synthesis of chromium(III) complexes of the type cis-[Cr(diimine)2(1-methylimidazole)2](3+). These compounds exhibit large excited-state oxidizing powers and strong luminescence in solution. Emission is quenched by added guanine, yielding rate constants that track the driving force for guanine oxidation. The cis-[Cr(TMP)(DPPZ)(1-MeImid)2](3+) species binds strongly to duplex DNA with a preference for AT base sites in the minor groove and may serve as a precursor for photoactivated DNA covalent adduct formation.
Polynuclear complexes with hydroxide (or oxide) as bridging ligands constitute an important class of complexes. They are formed by hydrolysis of mononuclear aqua complexes of most metal ions and, therefore, constitute an important aspect of the hydrolytic chemistry of metal ions. They display a chemistry which is interesting and is also relevant in relation to applied chemistry and to biochemistry. This chapter reviews the chemistry of hydroxo-bridged chromium(III), cobalt(III) complexes, and includes studies of the related rhodium(III) and iridium(III)systems. The review focuses the chemical properties of these complexes, and special emphasis has been made on reactions that involve the cleavage or formation of a hydroxo bridge. Reactions of bridging groups other than hydroxide and reactions of nonbridging ligands are also discussed in the chapter. A large number of X-ray crystal-structure determinations have been made during the past two decades. The hydroxo-bridged chromium(III) oligomers constitute a very interesting class of complexes from a spectroscopic and magnetochemical point of view. The most important methods for obtaining structural information about solutions are probably potentiometric pH measurements and visible/ultraviolet spectroscopy, but many other techniques have been applied, including bridge-cleavage experiments, magnetic measurements, and electron spin resonance (ESR) spectroscopy.
In the 1950s, Mertz and Schwarz published a series of experiments providing evidence that chromium (Cr) is an essential nutrient forming the active component of the “glucose tolerance factor.” This factor enhances the binding of insulin to its receptors, potentiating the action of insulin on carbohydrate metabolism. Cr deficiency in patients on total parenteral nutrition (TPN) is associated with the development of glucose intolerance, glycosuria, weight loss, peripheral neuropathy, ataxia, and encephalopathy. In these patients, the infusion of trivalent Cr has resulted in a resolution of neurological changes and re-establishment of glucose tolerance. Recently, others have cautioned against the administration of Cr in parenteral nutrition, because it is a contaminant of parenteral nutrition solutions and also because circulating Cr levels have been found to be elevated in patients on parenteral nutrition. However, no relationship was noted between the response to Cr in these patients and their plasma levels. This observation was recently confirmed in another patient with severe neuropathy and glucose intolerance who responded to Cr infusion despite high plasma levels of Cr. Plasma levels may not be appropriate for the assessment of Cr status in patients receiving TPN. The development of unexpected glucose intolerance and/or neuropathy would suggest the presence of Cr deficiency. In such patients, the diagnosis of deficiency by infusing Cr daily for 2 weeks to correct an abnormal glucose tolerance will confirm the diagnosis of Cr deficiency. The subsequent improvement in neuropathy, when present, following the administration of Cr would support this diagnosis. J. Trace Elem. Exp. Med. 12: 85–89, 1999. © 1999 Wiley-Liss, Inc.
The binding of Schiff base complexes of chromium(III) of the type [Cr(salen)(H2O)2]+ and [Cr(salprn)(H2O)2]+, where salen denotes 1,2-bis(salicylideneamino)ethane and salprn denotes 1,3-bis(salicylideneamino)propane to calf thymus DNA has been investigated by absorption, emission, circular dichroism, melting temperature and viscosity measurements. These chromium(III) complexes showed absorption hyperchromicity accompanied by red shift in charge transfer band, fluorescence enhancement, increase in melting temperature, some structural changes in CD spectra and changes in specific viscosity when bound to calf thymus DNA. The binding constant Kb has been determined from absorption measurements for both the complexes and found to be (2.5±0.4)×103 M−1 for [Cr(salen)(H2O)2]+ and (1.7±0.3)×104 M−1 for [Cr(salprn)(H2O)2]+. From the binding stoichiometry of DNA-[Cr(salprn)(H2O)2]+, the number of binding site size has been determined and found to be ten base pairs per bound complex molecule. The chromium(III) complexes also bring about single strand cleavage in plasmid DNA. The experimental results show that the chromium(III) complexes bind to DNA by non-intercalative mode. Major groove binding is the preferred mode of interaction for these Schiff base complexes of chromium(III).
Three complexes of chromium(III) and histidine were investigated by variable temperature magnetic susceptibility and ESR. Two monomeric stereosoimers of Cr(L-histidine)2(NO3) were studied; the trans(imidazole) and trans(carboxylate)isomers exhibited Curie-Weiss behaviour in the 6–300 K temperature region. The magnetic susceptibility of the related binuclear complex, [Cr(L-histidine)2(OH)]2, showed ferromagnetic behaviour at low temperature (J/k = +0.15 K) lending strong support to the proposed hydroxo-bridged structure.
For over 100 years it has been known that, during the synthesis of basic, dark green chromium carboxylates, violet compounds are also formed. The first such species has now been characterized. The complex cation 1 was obtained upon reduction of chromium(VI) oxide with formic acid and isolated as its p-toluenesulfonate. Complex 1 is the first Cr complex containing one μ-hydroxo and two μ-carboxylato ligands.
The nutritional supplement chromium picolinate, [Cr(pic)3], has been proposed to damage DNA, presumably through the catalytic formation of reactive oxygen species. For this mechanism to be important in vivo, the compound needs to bind to or be in close proximity of DNA. Recent studies have shown that [Cr(pic)3] does not accumulate in the nucleus and is not stable for prolonged periods of time in cells; this work also demonstrates that the supplement has little if any propensity to bind to DNA. However, as the supplement enters cells intact and is potentially capable of generating oxidative damage where it does occur, experiments were performed to probe for several forms of oxidative damage in rats. [Cr(pic)3] was found to raise urinary and cellular 8-OHdG levels in a time dependent fashion, and the compound was found to significantly increase lipid peroxidation in vivo. Thus, oxidative DNA damage (and lipid damage) from [Cr(pic)3] in whole animals has been observed for the first time.
A new program, PHI, with the ability to calculate the magnetic properties of large spin systems and complex orbitally degenerate systems, such as clusters of d-block and f-block ions, is presented. The program can intuitively fit experimental data from multiple sources, such as magnetic and spectroscopic data, simultaneously. PHI is extensively parallelized and can operate under the symmetric multiprocessing, single process multiple data, or GPU paradigms using a threaded, MPI or GPU model, respectively. For a given problem PHI is been shown to be almost 12 times faster than the well-known program MAGPACK, limited only by available hardware. © 2013 Wiley Periodicals, Inc.
The synthetic biomimetic triaqua-μ-oxohexapropionatotrichromium(III) nitrate when given intravenously has been shown previously to lower fasting blood plasma triglycerides and cholesterol concentrations in rats; thus, the cation has the potential to serve as a therapeutic agent. Its ability to function in vivo presumably is dependent on its ability to mimic the action of the natural, bioactive, chromium-binding oligopeptide chromodulin in stimulating insulin receptor kinase activity. For this to happen, the cation presumably should be incorporated into insulin-sensitive cells intact. Examination of the distribution of 51Cr and 14C from 51Cr- and [1-14C]-propionate labeled trinuclear cation in rats after injection with the trinuclear complex for 2 weeks suggests that the cation enters cells intact; however, the cation appears to degrade within 24 h after injection. Chromium from the complex is excreted in the urine as chromodulin.
The biologically-active form of chromium, low-molecular-weight chromium-binding substance (LMWCr), activates the insulin-dependent tyrosine protein kinase activity of insulin receptor (IR). The site of activation was shown to be on the active site fragment of the β subunit of IR. As LMWCr previously has been proposed to contain a multinuclear chromic assembly, the ability of multinuclear chromium assemblies to activate IR kinase activity has been probed. The trinuclear cation [Cr3O(O2CCH2CH3)6(H2O)3]+ (1) has been found to activate IR activity in a fashion almost identical to that of LMWCr using rat adipocytic membrane fragments and an active site fragment of IR, while a variety of other chromic complexes have in contrast been found to be ineffective or to inhibit kinase activity. The activation of IR kinase activity by complex 1, its stability in aqueous and strongly acidic solution, and its low molecular weight suggest that it potentially could be used in a treatment for adult-onset diabetes.
IntroductionHydroxo-Bridged DimersSubstituted Hydroxo-Bridged DimersHalogen-Bridged DimersDimers with Mixed Bridges
Despite forty years of research on the potential role of chromium in carbohydrate and lipid metabolism, significant progress has only recently been made regarding the mode of action of chromium at a molecular level. The oligopeptide low-molecular-weight chromium-binding substance (LMWCr) may function as part of a novel insulin-signaling autoamplification mechanism. The proposed mechanism of action also sheds some light on the potential of chromium-containing compounds as nutritional supplements or in the treatment of adult-onset diabetes and other conditions. The potential relationship between the results of recent studies on diabetic patients and the proposed mode of action of LMWCr are discussed.
The biochemistry of chromium(III) has been a poorly understood field of endeavor. Despite four decades of investigation, only recently has a somewhat clear picture of the role of Cr been refined. Chromium(III) is required for proper carbohydrate and lipid metabolism in mammals, although chromium deficiency is difficult to achieve. Conditions that increase circulating glucose and insulin concentrations increase urinary chromium output. Chromium is excreted after an insulin challenge in the form of the oligopeptide chromodulin. Chromodulin may be the key to understanding the role of chromium at a molecular level as the molecule has been found to bind to activated insulin receptor, stimulating its kinase activity. An examination of the history of studies of chromium picolinate and glucose tolerance factor illustrates the difficulties and problems associated with biochemical studies dealing with chromium(III).
The magnetic exchange behaviour for μ-hydroxo and μ-acetato double-bridged chromium(III) dimer is investigated based on calculations of density functional theory combined with the broken-symmetry approach. It is demonstrated that there is a magnetic exchange cooperative effect of the two bridging ligands in a double-bridged dimer systems with approximate equal coupling intensity. Meanwhile, the calculated results reveal that the deprotonation of the μ-hydroxo ligand causes a sharp increase of the magnetic exchange interaction between the chromium centers. Replacing either the μ-hydroxo bridging ligand by one water bridging ligand or the μ-acetato bridging ligands by two terminal water ligands produces a relatively reasonable model to examine the contribution on the magnetic exchange interaction of another individual bridging ligand.
Chromium(III) tris(picolinate), Cr(pic)3, is currently a very popular nutritional supplement; however, at physiologically-relevant concentrations, it has recently been demonstrated to cleave DNA [J.K. Speetjens, R.A. Collins, J.B. Vincent, S.A. Woski, Chem. Res. Toxicol. 12 (1999) 483]. A number of other chromium-containing compounds have been proposed as substitutes for Cr(pic)3. Of particular interest are low-molecular-weight chromium-binding substance (LMWCr) and [Cr3O(O2CCH2CH3)6(H2O)3]+1. The former compound has recently been identified as the biologically active form of chromium in mammals, activating the kinase activity of insulin receptor in the presence of insulin. Complex 1 is a functional biomimetic for LMWCr. Both compounds have been proposed as possible nutritional supplements and therapeutics for adult-onset diabetes. This work demonstrates that these complexes, unlike Cr(pic)3, are poor DNA-cleaving agents and may represent safer materials for human consumption.
Drinking water supplies in many geographic areas contain chromium in the +3 and +6 oxidation states. Public health concerns are centered on the presence of hexavalent Cr that is classified as a known human carcinogen via inhalation. Cr(VI) has high environmental mobility and can originate from anthropogenic and natural sources. Acidic environments with high organic content promote the reduction of Cr(VI) to nontoxic Cr(III). The opposite process of Cr(VI) formation from Cr(III) also occurs, particularly in the presence of common minerals containing Mn(IV) oxides. Limited epidemiological evidence for Cr(VI) ingestion is suggestive of elevated risks for stomach cancers. Exposure of animals to Cr(VI) in drinking water induced tumors in the alimentary tract, with linear and supralinear responses in the mouse small intestine. Chromate, the predominant form of Cr(VI) at neutral pH, is taken up by all cells through sulfate channels and is activated nonenzymatically by ubiquitously present ascorbate and small thiols. The most abundant form of DNA damage induced by Cr(VI) is Cr-DNA adducts, which cause mutations and chromosomal breaks. Emerging evidence points to two-way interactions between DNA damage and epigenetic changes that collectively determine the spectrum of genomic rearrangements and profiles of gene expression in tumors. Extensive formation of DNA adducts, clear positivity in genotoxicity assays with high predictive values for carcinogenicity, the shape of tumor-dose responses in mice, and a biological signature of mutagenic carcinogens (multispecies, multisite, and trans-sex tumorigenic potency) strongly support the importance of the DNA-reactive mutagenic mechanisms in carcinogenic effects of Cr(VI). Bioavailability results and kinetic considerations suggest that 10-20% of ingested low-dose Cr(VI) escapes human gastric inactivation. The directly mutagenic mode of action and the incompleteness of gastric detoxification argue against a threshold in low-dose extrapolation of cancer risk for ingested Cr(VI).
The mechanism of DNA damage induced by Cr(III) complexes is currently unknown even though it is considered to be the ultimate biologically active oxidation state of chromium. In this study, we have employed the Salmonella reversion assay to identify mutagenic Cr(III) complexes. Cyclic voltammetry was used to differentiate the redox kinetics between mutagenic and selected nonmutagenic Cr(III) species. Plasmid relaxation of supercoiled DNA was employed to show in vitro interactions with plasmid DNA and correlate the interactions with the electrochemical behavior and biological activity. The results of this study demonstrate that the mutagenic Cr(III) complexes identified in the Salmonella reversion assay display characteristics of reversibility and positive shifts of the Cr(III)/Cr(II) redox couple consistent with the ability of these Cr(III) complexes to serve as cyclical electron donors in a Fenton-like reaction. These same mutagenic complexes display an ability to relax supercoiled DNA in vitro, presumably by the induction of single-strand breaks. Nonmutagenic complexes were selected to test different ligands to determine how the ligand directs the activity of Cr(III) complexes. All nonmutagenic complexes tested thus far have shown classical irreversibility, more negative reduction potentials, and an inability to relax supercoiled plasmid DNA. These results suggest that the mechanism by which chromium complexes potentiate mutagenesis involves an oxygen radical as an active intermediate. These data also demonstrate the effect of associated ligands with regard to the ability of a metal to generate an active redox center.
Eleven male and nine female adult subjects were given one of the following five carbohydrate-drink combinations (per kg body wt) on five mornings separated by greater than or equal to 2 wk: 1) 1.0 g glucose, 2) 0.9 g uncooked cornstarch, 3) 1.0 g glucose followed 20 min later by 1.75 g fructose, 4) 0.9 g uncooked cornstarch followed 20 min later by 1.75 g fructose, and 5) water followed 20 min later by 1.75 g fructose. Glucose plus fructose was the most insulinogenic followed by glucose alone, starch plus fructose, starch alone, and water plus fructose. The urinary losses of chromium followed a similar pattern. Subjects with the highest concentrations of circulating insulin displayed decreased ability to mobilize chromium on the basis of urinary chromium excretion. Therefore, urinary chromium losses are related to the insulinogenic properties of carbohydrates.
The selective paramagnetic relaxation of oligonucleotide protons of d(GTGCAC)_2 by Δ- and Λ-Ni(phen)_3^(3+) and Δ- and Λ-Cr(phen)_3^(3+) has been examined to obtain some structural insight into the noncovalent binding of tris(phenanthroline) metal complexes to DNA. The experiments demonstrate that the relative rate of relaxation of different oligonucleotide protons by the paramagnetic metal complex varies with the chirality of the metal complex and, to a lesser extent, the metal charge. The proton most efficiently relaxed in all cases is the adenosine AH2, which is situated in the minor groove of the oligonucleotide helix. For both Λ-Ni(phen)_3^(2+) and Λ-Cr(phen)_3^(3+), the order of relaxation rates varies as AH2 » AH8 > G3H8 = TMe = C4H5. For Δ-Ni(phen)_3^(2+) it varies as AH2 > G3H8 > AH8 > TMe = C4H5 and for Δ-Cr(phen)_3^(3+) as AH2 > TMe = G3H8 = AH8 > C4H5. Distances between the metal center and oligonucleotide
protons were calculated on the basis of the relaxation data, and these distances were used to generate a set of models to describe the interactions of the rigid metal complex with the helix. For A-isomers, the data are consistent with a predominant surface-bound association in the minor groove of the DNA helix. The results for A-isomers correlate better with models that incorporate also a major groove intercalative mode. Despite the absence of hydrogen-bonding groups in the metal complex, the surface-bound model of the phenanthroline complex in the minor groove of DNA resembles the noncovalent association seen with other DNA groove binding molecules.
A biologically active, low-molecular-weight, chromium-binding substance present in milk (M-LMCr) was isolated from bovine colostrum and purified more than 2000 times by means of ethanol precipitation and successive ion-exchange and Sephadex gel chromatographies. The purified M-LMCr appeared to be an anionic organic Cr compound with a molecular weight of 1500, as determined by gel permeation chromatography. It contained aspartic acid, glutamic acid, glycine and cysteine in a ratio of 5:4:2:1 and no detectable carbohydrate. Although we were unable to detect nicotinic acid, some ultraviolet-absorbing (lambda max 260 nm) chemical structure was shown to be a constituent. Purified M-LMCr stimulated the rates of both [U-14C]glucose oxidation and [3-3H]glucose conversion into lipid in rat epididymal adipocytes at Cr concentrations greater than 1.5 ng/mL in relation to insulin action. This substance appears to have properties similar to those of glucose tolerance factor in yeast and the low-molecular-weight, chromium-binding substance present in mammalian liver. The role of M-LMCr in Cr nutrition and detoxication is discussed.
A low-molecular-mass chromium-binding substance (LMCr), which is recognized as a detoxification ligand of chromium, was isolated from the livers of rabbits injected intravenously with K2Cr2O7 (200 μmol Cr/kg body wt) as a biologically active form. LMCr appears as an anionic, organic Cr compound with a relative molecular mass of 1500. It is composed of glutamic acid or glutamine, glycine, cysteine and aspartic acid or asparagine with a Cr/amino-terminal residue ratio of 4:1.
The purified LMCr (10–300 ng Cr/ml) shows in vitro activities comparable to those of glucose tolerance factor in relation to insulin action. In the presence of insulin it enhances [U-14C]glucose conversion to 14CO (23–30% up) in rat epididymal adipocytes above the value obtained with insulin alone. LMCr also stimulates the rate of [3-3H]glucose incorporation into lipid by 30–40% with insulin or by 15–23% without insulin, as compared with the basic value obtained with insulin alone or without insulin. These findings suggest that LMCr plays essential roles in both glucose metabolism and detoxification of invaded Cr in the body.
Uptake of chromium (VI) and subsequent induction of DNA damage was examined in liver and blood cells of 14-day chick embryos after injection of sodium dichromate onto the inner shell membrane. Maximal loss of chromium from the inner shell membrane and distribution of chromium in liver, lung and blood was observed 2 h after injection. DNA strand breaks, interstrand cross-links and DNA--protein cross-links were measured using the alkaline elution technique. In chick embryo liver, chromium (VI) induced DNA cross-links in the absence of strand breaks. Maximal DNA cross-linking was detected in the liver 8 h after injection. Little or no DNA damage remained in the liver 10-24 h after injection. In contrast, chromium (VI) induced DNA strand breaks in the absence of cross-links in chick embryo blood cells. Maximal DNA strand breakage was observed in blood cells 8 h after injection. High levels of DNA strand breaks were present in blood cells even 24 h after treatment. These intra-embryonic tissue differences in chromium (VI)-induced DNA damage may be a result of the differences in glutathione, cytochrome P-450, other pathways of chromium (VI) metabolism or chromatin organization which exist in liver and blood cells.
The role of glutathione and cytochrome P-450 in the production of DNA damage by chromium(VI) was examined in chicken embryo hepatocytes by the alkaline elution technique. Cellular levels of glutathione and cytochrome P-450 were altered by treating the hepatocytes with N-acetyl-L-cysteine, buthionine sulfoximine, isopentanol, or beta-naphthoflavone. A dramatic increase in chromium(VI)-induced DNA strand breaks was observed after increasing glutathione levels in the cells. Chromium(VI)-induced DNA strand breaks were even more numerous when the level of cytochrome P-450 was also increased. Upon depletion of glutathione levels and induction of cytochrome P-450 or cytochrome P-448, little or no DNA strand breaks or DNA interstrand cross-links were observed after chromium(VI) treatment. Chromium(VI)-induced DNA-protein cross-links generally decreased after either increases or decreases in cellular levels of glutathione or cytochrome P-450 or P-448. These results suggest that glutathione enhances chromium(VI)-induced DNA damage through metabolic activation of chromium(VI). The possible production of reactive chromium species upon metabolism by glutathione and cytochrome P-450 or P-448 and their involvement in DNA damage is discussed.
Chromium (Cr) content of the self-selected diets of 10 adult males and 22 females was determined. Each subject collected duplicate food and beverage samples on a daily basis for seven consecutive days. The 7-day average intake for males was 33 +/- 3 micrograms (mean +/- SEM), range 22-48 micrograms, and intake for females was 25 +/- 1, range 13-36. Mean Cr intake per 1000 cal was approximately 15 micrograms. Approximately 90% of the diets analyzed were below the minimum suggested safe and adequate daily intake for Cr of 50 micrograms. Chromium absorption was inversely related to dietary intake; absorption at a dietary Cr intake of 10 micrograms was approximately 2% and, with increasing intake to 40 micrograms, Cr absorption decreased to 0.5%. These data demonstrate that the average daily intake of chromium from self-selected diets is well below the minimum suggested safe and adequate intake and that Cr absorption, at levels found in typical US diets, is inversely related to dietary intake.
The distribution of low-molecular-weight, chromium-binding substance (LMWCr) and high-molecular-weight, chromium-binding substance (HMWCr) in the organ cytosol were analyzed by means of Sephadex G-25 gel filtration, after a single i.p. injection of K2Cr2O7 (280 mumol, Cr/Kg) to mice (male dd, 23 +/- 2 g). The amount of Cr in LMWCr per mouse was highest in the liver (83 micrograms), followed by those in the kidney (10 micrograms) and other organs (3-1 micrograms), with lesser amounts of Cr in HMWCr in all the organs. In these organs LMWCr was found to bind 3-28 times the amount of Cr to that in the in vivo binding after the in vitro incubation with K2Cr2O7 at 37 degrees C, showing a high Cr binding capacity of the substance. No inductive formation of LMWCr was observed in the liver even after daily repetitive administration of Cr (150 mumol/Kg, 4 days). Time course studies on the liver and the kidney of mice injected with K2Cr2O7 showed no difference in the accumulation of Cr in LMWCr and in the ratio of Cr in LMWCr to that in HMWCr between the organs at intervals of from 5 min to 24 hr after the injection. The comparative affinity of Cr(III) for LMWCr and for the serum proteins decreases in the order LMWCr, transferrin, albumin. The transfer of Cr from LMWCr to albumin and vice versa was almost negligible. However, significant amounts of the metal transfer was found from LMWCr to transferrin and vice versa, and from albumin to transferrin. These findings suggest that LMWCr is distributed widely in the body and it quickly binds invaded Cr in stable form at an organ site, especially in the liver, with participation of albumin or/then transferrin. This supports the hypothesis that LMWCr plays a large role in Cr detoxification.
From liver of dogs injected iv with potassium dichromate (38 mg/kg body wt), a low-molecular-weight chromium-binding substance (LMCr) was purified into two subfractions, LMCr I and LMCr II, which differ in physical and chemical properties. LMCr I was identified to be an anionic, organic chromium compound with a molecular weight of 1500. It contained glutamic acid, glycine, and cysteine as the predominant amino acids and firmly bound chromium in a ratio of one chromium(III) to one molecule of LMCr I. LMCr II was isolated in crystalline form and demonstrated to be a water-soluble, inorganic chromium(III) complex consisting of Na2HPO4 . 7H2O and Na2HPO4 . 2H2O. Although its crystallization reduced the chromium content, it had a maximum chromium-binding capacity as much as one chromium per one phosphorus in water. The mixture of LMCr I and LMCr II as approximated to be the natural composition showed a lower acute toxicity as measured by lethality in mice and had higher rates of urinary excretion and renal clearance in rabbits, accompanied by lower rates of renal tubular reabsorption and retention in kidney and liver than potassium dichromate(VI) and chromium(III) chloride. Pretreatment with chromium-free LMCr II remarkably reduced the mortality rates of mice acutely poisoned with chromium chloride. These results indicate that LMCr plays an important role in the detoxification and excretion of chromium in mammals.
DNA lesions were detected in rat organ nuclei following an i.p. injection of sodium dichromate. Kidney, liver, and lung nuclei were examined for DNA interstrand cross-links, strand breaks, and DNA-protein cross-links using the alkaline elution technique. The time course for formation of cross-links in kidney nuclei revealed the presence of DNA interstrand and DNA-protein cross-links 1 hr after injection of sodium dichromate. By 40 hr in kidney, DNA interstrand cross-links had been repaired, but DNA-protein cross-links persisted. In liver nuclei, the time course for formation of cross-links after injection of dichromate showed a maximum in DNA-protein cross-linking at 4 hr and a maximum in DNA interstrand cross-linking at 2 hr. By 36 hr, in the liver, both types of lesions had been repaired. In lung nuclei, both DNA interstrand and DNA-protein cross-links were observed 1 hr after dichromate injection; however, by 36 hr, only DNA-protein cross-links persisted. No DNA lesions were detectable in kidney 1 hr after an i.p. injection of chromium(III) chloride. Chromium distribution in rat kidney, liver, and lung was measured and is discussed with respect to the observed DNA lesions. The lung and kidney may be more sensitive than liver to chromium-induced DNA damage, an observation which correlates with the reported toxicity and carcinogenicity data for chromium(VI) in both animals and humans.
DNA damage by chromate in chick embryo hepatocytes has been correlated with the effect of chromate on inducible cell functions. Treatment of chick embryo hepatocytes with chromium(VI) in the form of sodium chromate resulted in the rapid uptake of chromate and the induction of DNA lesions in a time- and concentration-dependent manner. DNA interstrand cross-links, strand breaks and DNA-protein crosslinks, as measured by the alkaline elution technique, were observed after treatment of the hepatocytes with chromate concentrations (2.5- 0 microM) which did not affect cell viability. The effect of chromate on inducible cell functions was measured by assaying propylisopropylacetamide-induced accumulation of porphyrin and glucuronidation of phenol red by intact cells. Chromate inhibited propylisopropylacetamide-induction of porphyrin accumulation and phenol red glucuronidation in a time- and concentration-dependent manner which paralleled DNA damage. DNA damage was removed and inducibility of porphyrin accumulation by propylisopropylacetamide plus deferoxamine methanesulfonate was restored 21 h following a 2 h pretreatment with chromate. Chromium(III) in the form of chromic nitrate at concentrations up to 25 times those used with chromate had no effect on DNA damage or the induction of porphyrin accumulation and phenol red glucuronidation by propylisopropylacetamide in the cultured chick hepatocytes.
The effects of chromium (Cr) supplementation on insulin secretion and glucose clearance (KG) during intravenous glucose tolerance tests (IVGTTS) were assessed in rats with impaired glucose tolerance due to dietary Cr deficiency. Male Wistar rats were maintained after weaning on a basal low-Cr diet containing 55% sucrose, 15% lard, 25% casein. American Institute of Nutrition (AIN)-recommended levels of vitamins, no added Cr, and an altered mineral content as required to produce Cr deficiency and impaired glucose tolerance. The Cr-supplemented group ([+Cr] n = 6) were provided with 5 ppm Cr as CrCl3 in the drinking water, and the Cr-deficient group ([-Cr]n = 5) received purified drinking water. At 12 weeks on the diet, both groups of rats were hyperinsulinemic (+Cr, 103 +/- 13; -Cr, 59 +/- 12 microU/mL) and normoglycemic (+Cr, 127 +/- 7; -Cr, 130 +/- 4 mg/dL), indicating insulin resistance. After 24 weeks, insulin levels were normal (+Cr, 19 +/- 5; -Cr, 21 +/- 3 microU/mL) and all rats remained normoglycemic (+Cr, 124 +/- 8; -Cr, 131 +/- 6 mg/dL). KG values during IVGTTS were lower in -Cr rats (KG = 3.58%/min) than in +Cr rats (KG = 5.29%/min), correlating with significantly greater 40-minute glucose areas in the -Cr group (P < .01). Comparisons of 40-minute insulin areas indicated marked insulin hyperresponsiveness in the -Cr group, with insulin-secretory responses increased nearly twofold in -Cr animals (P < .05). Chromium deficiency also led to significant decreases in cyclic adenosine monophosphate (cAMP)-dependent phosphodiesterase (PDE) activity in spleen and testis (P < .01). In these studies, Cr deficiency was characterized by both beta-cell hypersecretion of insulin and tissue insulin resistance that were associated with decreased tissue levels of cAMP PDE activity.
We have previously shown that trivalent chromium can bind to purified DNA and form lesions capable of obstructing DNA replication in vitro. Trivalent chromium is not, however, carcinogenic to humans. Rather, it is the end product of the intracellular reduction of hexavalent chromium, which is carcinogenic. The process of chromium reduction yields several reactive intermediates which may also interact with DNA, perhaps producing different lesions than those generated when trivalent chromium binds DNA. The present study was undertaken to determine whether the treatment of DNA with hexavalent chromium in the presence of ascorbate (the intracellular reductant responsible for most in vivo chromium reduction), would also generate DNA lesions capable of obstructing replication. Using increasing chromium concentrations and a constant ascorbate:chromium ratio of 0.5:1 to generate biologically relevant adduct levels, a DNA polymerase arrest assay revealed that polymerase arresting lesions were formed and were indistinguishable from those generated by trivalent chromium, in that the most prominent arrests sites were one base upstream of guanine residues on the template strand. Measurement of the amount of chromium bound to template DNA in relation to the number of arrests demonstrated that only a subset (18.5%) of the chromium adducts were capable of causing polymerase arrest. Arrest assays performed with increasing ratios of ascorbate to chromium showed that high ratios (> or = 5:1) resulted in decreased polymerase arrests. DNA interstrand crosslinks in the arrest assay template were detected by renaturing agarose gel electrophoresis, and were shown to decrease markedly with increasing ascorbate to chromium ratios, whereas chromium binding levels remained unchanged. These results strongly implicate DNA interstrand crosslinks as the polymerase arresting lesion. The present study confirms and extends our previous study with trivalent chromium, and suggests that while the initial chemical nature of the DNA lesions formed by either trivalent chromium or reductive intermediates of hexavalent chromium may differ, their effect on DNA replication is the same.
Carcinogenic chromium (Cr6+) enters cells via the sulfate transport system and undergoes intracellular reduction to trivalent chromium, which strongly adducts to DNA. In this study, the effect of adducted trivalent chromium on in vitro DNA synthesis was analyzed with a polymerase-arrest assay in which prematurely terminated replication products were separated on a DNA sequencing gel. A synthetic DNA replication template was treated with increasing concentrations of chromium(III) chloride. The two lowest chromium doses used resulted in biologically relevant adduct levels (6 and 21 adducts per 1,000 DNA nucleotides) comparable with those measured in nuclear matrix DNA from cells treated with a 50% cytotoxic dose of sodium chromate in vivo. In vitro replication of the chromium-treated template DNA using the Sequenase version 2.0 T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH) resulted in dose-dependent polymerase arrest beginning at the lowest adduct levels analyzed. The pattern of polymerase arrest remained consistent as chromium adduct levels increased, with the most intense arrest sites occurring 1 base upstream of guanine residues on the template strand. Replication by the DNA polymerase I large (Klenow) fragment as well as by unmodified T7 DNA polymerase also resulted in similar chromium-induced polymerase arrest. Interstrand cross-linking between complementary strands was detected in template DNA containing 62, 111, and 223 chromium adducts per 1,000 DNA nucleotides but not in template containing 6 or 21 adducts per 1,000 DNA nucleotides, in which arrest nevertheless did occur. Low-level, dose-dependent interstrand cross-linking between primer and template DNA, however, was detectable even at the lowest chromium dose analyzed. Since only 9% of chromium adducts resulted in polymerase arrest in this system, we hypothesized that arrest occurred when the enzyme encountered chromium-mediated interstrand DNA-DNA cross-links between either the template and a separate DNA molecule or the template and its complementary strand in the same molecule. These results suggest that the obstruction of DNA replication by chromium-mediated DNA-DNA cross-links is a potential mechanism of chromium-induced genotoxicity in vivo.
Evidence in the literature suggests that the trace element chromium may have a role in glucose homeostasis through the regulation of insulin action. We have previously reported a significant reduction in plasma chromium levels in healthy individuals, following a 75 g oral glucose load, and after meals and glucose-dependent uptake of chromium in insulindependent tissues in vitro . However, in vivo it is unclear whether the changes in plasma chromium relate to changes in plasma glucose or insulin.
The present study describes a series of euglycaemic hyperinsulinaemic clamps designed to attempt to define the initiator of changes in plasma chromium levels in ten healthy individuals.
The data showed a significant ( P <0·01) reduction in fasting plasma chromium levels following glucose infusion and an initial bolus of insulin. Significant ( P <0·02) increases in post-clamp urinary chromium excretion were insufficient to explain the decrease in plasma levels. During the recovery phase of an extended two-phase clamp protocol we found plasma insulin levels decreased by 70% within 10 min, associated with an increase in plasma chromium levels of 30% and no significant change in plasma glucose level.
These data indicate that alterations in plasma glucose are unlikely to be directly related to changes in plasma chromium, whilst supporting the hypothesis that plasma insulin may influence plasma levels of this trace element. In contrast, plasma zinc was unaffected throughout these clamp studies.
Journal of Endocrinology (1993) 139, 339–345
The purpose of this study was to determine if diet or various metabolites alter chromium (Cr) uptake and distribution in rats. Radioactively labeled Cr was detected within 15 min of oral administration to rats, and the total amount retained remained relatively constant from 1 to 24 h. Dietary Cr intake did not alter Cr retention or distribution. The majority of the Cr was retained in the carcass. However, when the amount of labeled Cr was expressed per gram of tissue, the highest amounts of Cr were found in the kidneys, spleen, and pancreas. Pharmacological doses of insulin, epinephrine, glucagon, and dibutyryladenosine-3'-5'cyclic monophosphate, prostaglandins A1, A2, B1, B2, E1, E2, F1 alpha, and F2 alpha did not significantly influence Cr retention. Glucose, sucrose, nicotinic acid, glutathione, and other metabolites administered orally in conjunction with labeled Cr also did not significantly alter Cr retention. These data indicate that most nutrients and metabolites do not alter Cr retention and distribution. The regulation of Cr homeostasis appears to be at the level of excretion.
Chromium is essential for proper carbohydrate and lipid metabolism in mammals, although the mechanism of this action has previously proved elusive. Low-molecular-weight chromium-binding protein (LMWCr), a biologically active form of chromium in mammals, potentiates the effect of insulin on the conversion of glucose into lipid and into carbon dioxide in isolated adipocytes. Kinetics studies indicate that LMWCr isolated from bovine liver activates phosphotyrosine phosphatase (PTP) activity in adipocyte membranes while having no intrinsic phosphatase activity. This activation is directly proportional to the amount of added LMWCr. The pattern of inhibition of this activity in the presence of a number of known phosphatase inhibitors suggests the involvement of a membrane phosphotyrosine phosphatase similar to PTP1A' or PTP1B. We propose that chromium plays a biological role in the activation of a membrane phosphotyrosine phosphatase.