Nutritional supplement chromium picolinate generates chromosomal aberrations and impedes progeny development in Drosophila melanogaster

Chromium picolinate, [Cr(pic)(3)], is a popular nutritional supplement found in a variety of consumer products. Despite its popularity, safety concerns over its use have arisen. The supplement has been shown to generate clastogenic damage, mitochondrial damage, oxidative damage, and mutagenic effects in cultured cells and oxidative DNA damage and lipid peroxidation in rats. Recently [Cr(pic)(3)] has been demonstrated to generate heritable genetic change and delays in progeny development in Drosophila melanogaster. Based on the damage to chromosomes of cultured cells and of animal models, similar chromosome damage appeared to be a likely source of the mutagenic effects of the supplement in Drosophila. The current three-part study examines the effects of several chromium-containing supplements and their components on hatching and eclosion rates and success of development of first generation progeny of adult Drosophila fed food containing these compounds. It further examines the effects of the compounds on longevity of virgin male and female adults. Finally, the chromosomes in the salivary glands of Drosophila late in the third instar larval stage, which were the progeny of Drosophila whose diets were supplemented with nutritional levels of [Cr(pic)(3)], are shown to contain on average over one chromosomal aberration per two identifiable chromosomal arms. No aberrations were observed in chromosomes of progeny of untreated flies. The results suggest that human consumption of the supplement should be a matter of concern and continued investigation to provide insight into the requirements of chromium-containing supplements to give rise to genotoxic effects.     

Potential of Chromium(III) Picolinate for Reproductive or Developmental Toxicity Following Exposure of Male CD-1 Mice Prior to Mating

Chromium(III) picolinate, [Cr(pic)₃], is a commonly used nutritional supplement in humans, which has also been approved for use in animals. Health concerns have arisen over the use of [Cr(pic)₃]. At high [Cr(pic)₃] doses, developmental toxicity tests in female mice have shown a higher litter incidence of split cervical arch in exposed fetuses, but this was not consistently reproducible. In the current study, male CD-1 mice were used to further assess the potential for reproductive or developmental toxicity. Four weeks prior to mating, the males were fed a diet providing 200 mg/kg/day [Cr(pic)₃] for comparison with untreated controls. Females were not treated. Each male was mated with two females, which were sacrificed on gestation day 17, and their litters were examined for adverse effects. Mating and fertility indices were not significantly altered by treatment. Male exposure to [Cr(pic)₃] also had no effect on prenatal mortality, fetal weight, or gross or skeletal morphology. These results suggest that paternal dietary exposure to chromium(III) picolinate has little potential for adverse reproductive effects, even at exposure levels considerably higher than expected human exposures from nutritional supplements (1 mg of Cr per day or less).     

Comparison of the potential for developmental toxicity of prenatal exposure to two dietary chromium supplements, chromium picolinate and [Cr3O(O2CCH2CH3)(6(H2O)3]+, in mice

BACKGROUND: Chromium(III) is generally thought to be an essential trace element that allows for proper glucose metabolism. However, chromium(III) picolinate, Cr(pic)₃, a popular dietary supplement form of chromium, has been shown to be capable of generating hydroxyl radicals and oxidative DNA damage in rats. The cation [Cr₃O(O₂CCH₂CH₃)₆(H₂O)₃]⁺, Cr3, has been studied as an alternative supplemental source of chromium. It has been shown to increase insulin sensitivity and lower glycated hemoglobin levels in rats, making it attractive as a potential therapeutic treatment for gestational diabetes. To date, no studies have been published regarding the safety of Cr3 supplementation to a developing fetus. METHODS: From gestation days (GD) 6–17, mated CD‐1 female mice were fed diets delivering either 25 mg Cr/kg/day as Cr(pic)₃, 3.3 or 26 mg Cr/kg/day as Cr3, or the diet only to determine if Cr3 could cause developmental toxicity. Dams were sacrificed on GD 17, and their litters were examined for adverse effects. RESULTS: No signs of maternal toxicity were observed. No decrease in fetal weight or significantly increased incidence of skeletal defects was observed in the Cr3 or Cr(pic)₃ exposed fetuses compared to the controls. CONCLUSION: Maternal exposure to either Cr(pic)₃ or Cr3 at the dosages employed did not appear to cause deleterious effects to the developing offspring in mice. Birth Defects Res (Part B), 80:1–5, 2007. © 2007 Wiley‐Liss, Inc.     

High-dose chromium(III) supplementation has no effects on body mass and composition while altering plasma hormone and triglycerides concentrations

Chromium is generally believed to be an essential element and is often claimed to have value as a weight loss or muscle building agent. Recent studies in humans and rats have failed to demonstrate effects on body composition, although recent studies with pharmacological doses of the cation [Cr(III)₃O(O₂CCH₂CH₃)6(H₂O)₃]⁺ (or Cr3) (≤1 mg Cr/kg body mass) in rats have noted a trend toward body mass loss and fat mass loss. Thus, the effects of large gavage doses of Cr3 (1–10 mg Cr/kg) on body mass, organ mass, food intake, and blood plasma variables (insulin, glucose, leptin, cholesterol, and triglycerides) were examined over a 10-wk period using male Sprague-Dawley rats. No effects on body composition were noted, although Cr3 administration lowered (p<0.05) plasma insulin, leptin, and triglycerides concentrations. As Cr3 is absorbed greater than 10-fold better than commercially available nutritional supplements, the lack of an effect of the Cr(III) compound at these levels of administration clearly indicates that Cr(III) supplements do not have an effect on body composition at any reasonable dosage.     

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

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 (P1-P4) 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 microM 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 microM 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.     

The biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ decreases plasma cholesterol and triglycerides in rats: towards chromium-containing therapeutics

3 O(O₂CCH₂CH₃)₆ (H₂O)₃]⁺ 1 and a naturally occurring, biologically active form of chromium, low-molecular-weight chromium-binding substance (LMWCr), to rats are described. Given that the complexes are proposed to function by interacting with insulin receptor, trapping it in its active conformation, in contrast to current chromium-containing nutrition supplements, which only serve as sources of absorbable chromium, changes in lipid and carbohydrate metabolism would be expected. After 12 weeks administration (20 μg/kg body mass), compound 1 results in 40% lower levels of blood plasma LDL cholesterol, 33% lower levels of total cholesterol, and significantly lower HDL cholesterol and triglyceride; these results are in stark contrast to those of administration of other forms of Cr(III) to rats, which have no effect on these parameters. LMWCr, in contrast to 1, has no effect as it probably is degraded in vivoor excreted. These results are interpreted in terms of the mechanism of chromium action in response to insulin and the activation of insulin receptor, and the potential for the rational design of chromium-containing therapeutics is discussed. Received: 27 May 1999 / Accepted: 4 October 1999     

Comparison of acute absorption of commercially available chromium supplements

Chromium (Cr) supplements are available as picolinate, nicotinate or chloride (the latter primarily in multivitamin-mineral supplements). The picolinate form has been reported to be the best absorbed and most efficacious, but some reports question which form has superior absorption. The present study examined acute Cr absorption, based on 24h urinary Cr values, for picolinate, two types of nicotinate, and chloride in young adult, non-overweight females. College-aged women were given 200 microg of Cr as each of the four supplement types in random order accompanied by a small standardized meal, separated by at least a week washout. Cr picolinate produced significantly higher 24h urinary Cr than either of two nicotinate supplements or Cr chloride given in a multivitamin-mineral supplement. This difference was seen for absolute values of the urinary Cr and for percent increases. In conclusion, based on an indirect measure of acute absorption, Cr picolinate was superior to three other Cr complexes commonly sold as supplements.     

Effects of Pre- and Postnatal Exposure to Chromium Picolinate or Picolinic Acid on Neurological Development in CD-1 Mice

Chromium picolinate, Cr(pic)3, a popular dietary supplement marketed as an aid in fat loss and lean muscle gain, has also been suggested as a therapy for women with gestational diabetes. The current study investigated the effects of maternal exposure to Cr(pic)3 and picolinic acid during gestation and lactation on neurological development of the offspring. Mated female CD-1 mice were fed diets from implantation through weaning that were either untreated or that contained Cr(pic)3 (200 mg kg(-1) day(-1)) or picolinic acid (174 mg kg(-1) day(-1)). A comprehensive battery of postnatal tests was administered, including a modified Fox battery, straight-channel swim, open-field activity, and odor-discrimination tests. Pups exposed to picolinic acid tended to weigh less than either control or Cr(pic)3-exposed pups, although the differences were not significant. Offspring of picolinic acid-treated dams also appeared to display impaired learning ability, diminished olfactory orientation ability, and decreased forelimb grip strength, although the differences among the treatment groups were not significant. The results indicate that there were no significant effects on the offspring with regard to neurological development from supplementation of the dams with either Cr(pic)3 or picolinic acid.     

Conservatism of sites of tRNA loci among the linkage groups of severalDrosophila species

Summary The sites of seven tRNA genes (Arg-2, Lys-2, Ser-2b, Ser-7, Thr-3, Thr-4, Val-3b) were studied by in situ hybridization.¹²⁵I-labeled tRNA probes fromDrosophila melanogaster were hybridized to spreads of polytene chromosomes prepared from fourDrosophila species representing different evolutionary lineages (D. melanogaster, Drosophila hydei, Drosophila pseudoobscura, andDrosophila virilis). Most tRNA loci occurred on homologous chromosomal elements of all four species. In some cases the number of hybridization sites within an element varied and sites on nonhomologous elements were found. It was observed that both tRNA ₂ ^Arg and tRNA ₂ ^Lys hybridized to the same site on homologous elements in several species. These data suggest a limited amount of exchange among different linkage groups during the evolution ofDrosophila species.     

Tissue and subcellular distribution of chromium picolinate with time after entering the bloodstream

Chromium picolinate, [Cr(pic)(3)], is a popular nutritional supplement; however, the fate of the complex in vivo has not previously been established. Consequently, rats were administered [51Cr(pic)(3)] intravenously and the fate of the radiolabel in the urine, blood plasma, tissues, and subcellular components of hepatocytes was followed for the first 24 h after injection. The supplement leaves the blood stream rapidly appearing in the urine and entering tissue cells intact. Kidney, muscle, and liver possess most of the absorbed radiolabel. In hepatocytes, the radiolabel appears most rapidly in the nucleus and mitochondria, then in the cytosol, and finally in the lysosomes and microsomes. Thus, while the lifetime of the supplement in vivo is brief, it enters cells rapidly intact. The significance of the lifetime and distribution of [Cr(pic)(3)] in relationship to recent reported potential DNA damage from the supplement is discussed.     

Tests for parental imprinting in the nematode Caenorhabditis elegans

Summary The mutation him-6(e1423) leads to generalized chromosomal nondisjunction during meiosis in oogenesis and spermatogenesis of C. elegans. As a result, gametes nullisomic or disomic for each of the six chromosomes occur at appreciable frequency. Crosses utilizing marked him-6 strains were used to generate and identify exceptional euploid progeny which had received both homologues of a marked autosome either from the male parent or from the female parent. Examples of all ten possible exceptions were identified and found to be viable and fertile. These results (together with previous data for the X chromosome) indicate that major chromosomal imprinting effects do not occur during gametogenesis in this organism.     

Chromium (D-phenylalanine)3 improves obesity-induced cardiac contractile defect in ob/ob mice

Objective: Low‐molecular weight chromium compounds, such as chromium picolinate [Cr(pic)₃], improve insulin sensitivity, although toxicity is a concern. We synthesized a novel chromium complex, chromium (d ‐phenylalanine)₃ [Cr(d ‐phe)₃], in an attempt to improve insulin sensitivity with reduced toxicity. The aim of this study was to compare the two chromium compounds on cardiac contractile function in ob/ob obese mice. Research Methods and Procedures: C57BL lean and ob/ob obese mice were randomly divided into three groups: H₂O, Cr(d ‐phe)₃, or Cr(pic)₃ (45 µg/kg per day orally for 6 months). Results: The glucose tolerance test displayed improved glucose clearance by Cr(d ‐phe)₃ but not Cr(pic)₃. Myocytes from ob/ob mice exhibited depressed peak shortening (PS) and maximal velocity of shortening/relengthening (±dL/dt), prolonged time‐to‐PS and time‐to‐90% relengthening (TR90), reduced electrically stimulated rise in intracellular Ca²⁺ (Δfura‐2 fluorescence intensity), and slowed intracellular Ca²⁺ decay. Although a 3‐month Cr(d ‐phe)₃ treatment for a separate group of ob/ob and lean 2‐month‐old mice only rectified reduced ±dL/dt in ob/ob mice, all mechanical and intracellular Ca²⁺ abnormalities were significantly attenuated or ablated by 6 months of Cr(d ‐phe)₃ but not Cr(pic)₃ treatment (except TR90). Sarco(endo)plasmic reticulum Ca²⁺ ATPase activity and Na⁺‐Ca²⁺ exchanger expression were depressed in ob/ob mice, which were reversed by both Cr(d ‐phe)₃ and Cr(pic)₃, with a more pronounced effect from Cr(d ‐phe)₃. Cr(d ‐phe)₃ corrected reduced insulin‐stimulated glucose uptake and improved basal phosphorylation of Akt and insulin receptor, as well as insulin‐stimulated phosphorylation of Akt and insulin receptor in ob/ob myocytes. Heart homogenates from ob/ob mice had enhanced oxidative stress and protein carbonyl formation compared with the lean group, which were attenuated by both Cr(d ‐phe)₃ and Cr(pic)₃. Discussion: Our data suggest that the new Cr(d ‐phe)₃ compound possesses better cardio‐protective and insulin‐sensitizing properties against obesity.     

Protective effects of β-glucan extracted from Agaricus brasiliensis against chemically induced DNA damage in human lymphocytes

β-Glucans (BGs) are polysaccharides that are found in the cell walls of organisms such as bacteria, fungi, and some cereals. The objective of the present study was to investigate the genotoxic and antigenotoxic effects of BG extracted from the mushroom Agaricus brasiliensis (= Agaricus blazei Murrill ss. Heinemann). The mutagenic activity of BG was tested in single-cell gel electrophoresis assays with human peripheral lymphocytes. In addition, the protective effects against the cooked food mutagen 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and (+/−)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), which is the main metabolite of B[a]P, and against ROS (H₂O₂)-induced DNA damage, were studied. The results showed that the compound itself was devoid of mutagenic activity, and that a significant dose-dependent protective effect against damage induced by hydrogen peroxide and Trp-P-2 occurred in the dose range 20–80 μg/ml. To investigate the prevention of Trp-P-2-induced DNA damage, a binding assay was carried out to determine whether BG inactivates the amine via direct binding. Since no such interactions were observed, it is likely that BG interacts with enzymes involved in the metabolism of the amine.     

Influence of Parental Age on Endoreduplication of Giant Chromosomes and Some Quantitative Traits in Drosophila melanogaster Descendants

We studied the influence of parental age on the degree of polyteny of giant chromosomes and expressivity of mutation eyeless in Drosophila melanogaster descendants. The parental age equal to six days exerted an adverse effect on the function of endoreduplication of giant chromosomes in straineyeless. The highest degree of polyteny was observed in descendants of four- and ten-day imago. The maximum reduction of eye facets was observed in descendants of four-day imago, while in the progeny of older parents, the mutation expressivity was sharply reduced. Relations between the changes in chromosome polyteny, expressivity of mutation eyeless, and earlier studied components of adaptation were statistically analyzed in descendants of aging parents of this Drosophila strain.     

Differential mutagenic response in selected lines of Drosophila melanogaster

Summary The mutagenic efficiency of ionizing radiations has been tested on different lines of Drosophila melanogaster. It has been shown that differential lethal effects are obtained when irradiated females from different lines are mated to flies carrying heterozygous lethal genes. The results seem not to be attributable to differential expression of the lethality in the various crosses performed with the irradiated flies. This might suggest that gene activity is involved in the expression of the mutagenic effects of radiations.     

Ascorbate acts as a highly potent inducer of chromate mutagenesis and clastogenesis: linkage to DNA breaks in G2 phase by mismatch repair

Here we examined the role of cellular vitamin C in genotoxicity of carcinogenic chromium(VI) that requires reduction to induce DNA damage. In the presence of ascorbate (Asc), low 0.2–2 μM doses of Cr(VI) caused 10–15 times more chromosomal breakage in primary human bronchial epithelial cells or lung fibroblasts. DNA double-strand breaks (DSB) were preferentially generated in G₂ phase as detected by colocalization of γH2AX and 53BP1 foci in cyclin B1-expressing cells. Asc dramatically increased the formation of centromere-negative micronuclei, demonstrating that induced DSB were inefficiently repaired. DSB in G₂ cells were caused by aberrant mismatch repair of Cr damage in replicated DNA, as DNA polymerase inhibitor aphidicolin and silencing of MSH2 or MLH1 by shRNA suppressed induction of γH2AX and micronuclei. Cr(VI) was also up to 10 times more mutagenic in cells containing Asc. Increasing Asc concentrations generated progressively more mutations and DSB, revealing the genotoxic potential of otherwise nontoxic Cr(VI) doses. Asc amplified genotoxicity of Cr(VI) by altering the spectrum of DNA damage, as total Cr-DNA binding was unchanged and post-Cr loading of Asc exhibited no effects. Collectively, these studies demonstrated that Asc-dependent metabolism is the main source of genotoxic and mutagenic damage in Cr(VI)-exposed cells.     

Evolutionary changes in non-histone chromosomal proteins within the Drosophila melanogaster group revealed by monoclonal antibodies

Monoclonal antibodies directed against nonhistone chromosomal proteins of D. melanogaster were tested for crossreactivity with the homologous antigens of various Drosophila species. — By indirect immunofluorescence it could be shown that three antibodies react only with polytene chromosomes of species of the D. melanogaster subgroup, and only much less with chromosomes of other species of Drosophila. — With chromosomes of various other species of the Sophophora or Drosophila radiations only a reaction at background level could be observed. — The results suggest that the three antibodies react with different antigenic determinants of a single protein whose conformation changed rather fast during evolution of the Drosophilidae.     

Insulin Receptors and Downstream Substrates Associate with Membrane Microdomains after Treatment with Insulin or Chromium(III) Picolinate

We have examined the association of insulin receptors (IR) and downstream signaling molecules with membrane microdomains in rat basophilic leukemia (RBL-2H3) cells following treatment with insulin or tris(2-pyridinecarbxylato)chromium(III) (Cr(pic)₃). Single-particle tracking demonstrated that individual IR on these cells exhibited reduced lateral diffusion and increased confinement within 100 nm-scale membrane compartments after treatment with either 200 nM insulin or 10 μM Cr(pic)₃. These treatments also increased the association of native IR, phosphorylated insulin receptor substrate 1 and phosphorylated AKT with detergent-resistant membrane microdomains of characteristically high buoyancy. Confocal fluorescence microscopic imaging of Di-4-ANEPPDHQ labeled RBL-2H3 cells also showed that plasma membrane lipid order decreased following treatment with Cr(pic)₃ but was not altered by insulin treatment. Fluorescence correlation spectroscopy demonstrated that Cr(pic)₃ did not affect IR cell-surface density or compete with insulin for available binding sites. Finally, Fourier transform infrared spectroscopy indicated that Cr(pic)₃ likely associates with the lipid interface in reverse-micelle model membranes. Taken together, these results suggest that activation of IR signaling in a cellular model system by both insulin and Cr(pic)₃ involves retention of IR in specialized nanometer-scale membrane microdomains but that the insulin-like effects of Cr(pic)₃ are due to changes in membrane lipid order rather than to direct interactions with IR.     

Chromium uptake,retention and reduction in photosynthetic <Emphasis Type="Italic">Euglena gracilis</Emphasis>

Photosynthetic Euglena gracilis grown with different K₂CrO₄ concentrations was analyzed for its ability to take up, retain and reduce Cr(VI). For comparison, cells were also exposed to CrCl₃. Cellular Cr(VI) uptake at pH 7.2 showed a hyperbolic saturation pattern with K _m of 1.1 mM, V _m of 16 nmol (h × 10⁷ cells)⁻¹, and K _i sulfate of 0.4 mM. Kinetic parameters for sulfate uptake were similar, K _m = 0.83 mM, V _m = 15.9 nmol (h × 10⁷cells)⁻¹ and K _i chromate = 0.3 mM. The capacity to accumulate chromium depended on the ionic species, external concentration and pH of the incubation medium. Cr(VI) or Cr(III) accumulation was negligible in the acidic (pH 3.5) culture medium, in which Cr(VI) was abiotically reduced to Cr(III). At pH 7.2 Cr(VI) was fully stable and high accumulation (>170 nmol/1 × 10⁷ cells at 1 mM K₂CrO₄) was achieved; surprisingly, Cr(III) accumulation was also significant (>35 nmol/1 × 10⁷ cells at 1 mM CrCl₃). Cr(VI) was reduced by cells at pH 7.2, suggesting the presence of an external reductive activity. Cr(VI) induced an increased cysteine and glutathione content, but not in phytochelatins suggesting that chromium accumulation was mediated by monothiol compounds.     

Transformed roots of Crepis capillaries — a sensitive system for the evaluation of the clastogenicity of abiotic agents

The presence of a large number of pollutants, including mutagenic agents in the environment is a problem of a major concern. Rapid progress in plant biotechnology, especially in the development of cell transformation methods, including the production of transformed roots – ‘hairy roots’ – has opened new possibilities to use transformed root cultures in plant bioassays for the evaluation mutagenic effects of different agents. We have used Crepis capillaris hairy roots for evaluation of cytogenetic effects of mutagenic treatment. Effects of maleic acid hydrazide (MH) and X-ray treatment were analysed in chromosomal aberration, sister chromatid exchange (SCE) and TUNEL tests. Comparison of cytogenetic effects in hairy roots and roots of seedlings showed a much higher sensitivity of hairy roots, which makes them convenient material for monitoring DNA damage after mutagenic treatment.