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Nutritional supplement chromium picolinate generates chromosomal aberrations and impedes progeny development in Drosophila melanogaster
Mutation Research 610 (2006) 101–113
Nutritional supplement chromium picolinate generates chromosomal aberrations and impedes progeny development in Drosophila melanogaster Dontarie M. Stallings a,c , Dion D.D. Hepburn a,c , Meredith Hannah a,c , John B. Vincent a,c,∗ , Janis O’Donnell b,c a
Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487-0336, USA Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487-0344, USA Coalition for Biomolecular Products, The University of Alabama, Tuscaloosa, AL 35487-0336, USA
b c
Accepted 5 June 2006 Available online 2 August 2006
Abstract 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Chromium picolinate; Chromium nicotinate; Cr3; Drosophila; Chromium toxicity
1. Introduction As dietary trivalent chromium is not readily absorbed and chromium picolinate, [Cr(pic)3 ], is more readily
∗ Corresponding author. Tel.: +1 205 348 9203; fax: +1 205 348 9104. E-mail address: [email protected] (J.B. Vincent).
1383-5718/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2006.06.019
absorbed [1], [Cr(pic)3 ] has become popular as a nutritional supplement and is often touted to assist in body mass loss and changes in body composition. It currently can be widely found in an extensive variety of products including sports drinks, “health” shakes, and chewing gums. Previously, the compound has generally been considered safe since no acute toxic effects have been observed in numerous clinic trials [2]. In addition, rats fed diets of 100 mg of Cr as [Cr(pic)3 ] per
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kg of food during a 24-week period exhibited no evidence of acute toxicity [3]. [Cr(pic)3 ] has even been investigated as a potentially useful agent for alleviating the symptoms of atypical depression and reportedly has some beneficial effects [4]. Since its introduction as a supplement, however, sporadic reports of deleterious effects associated with [Cr(pic)3 ] supplementation have appeared. These include anemia, liver dysfunction, and renal failure [5,6] and cognitive, perceptual, and motor alterations [7]. Additionally, recent reviews and metaanalyses of studies of healthy individuals consuming [Cr(pic)3 ] (including individuals in weight training programs) indicate that the supplement has no significant effects on body mass or body composition or on blood serum insulin and glucose levels [2,8–11]. Evidence of deleterious cellular and genetic effects of [Cr(pic)3 ] have been accumulating. Stearns et al. [12] reported that [Cr(pic)3 ] induced clastogenic damage in Chinese hamster ovary cells. Mitochondrial damage [13] and mutagenic effects [14] were subsequently reported in these cells as well. [Cr(pic)3 ]-induced chromosomal and oxidative damage have also been reported in a murine macrophage cell line [15,16]. Additionally, [Cr(pic)3 ] has been found to induce mutagenic responses in mouse lymphoma cells with and without the addition of S9 [17]. Further evidence comes from animal studies. Increases in urinary and tissue 8-hydroxydeoxyguanosine [18] and lipid peroxidation [18,19] have been observed in rats given the supplement. Recently, these laboratories have demonstrated that doses of [Cr(pic)3 ] equivalent to or lower than those of humans consuming commercial [Cr(pic)3 ] supplements generate heritable genetic changes and delays in progeny development in Drosophila melanogaster [20]. Not all Cr(III) complexes produce these mutagenic effects. These effects seem to arise from either reactive oxygen species and/or high valent Cr oxo/hydroxo species generated from the intact complex in the presence of biological reducing agents or from the picolinate ligand itself after Cr is released [12,13,21,22]. For example, CrCl3 at comparable doses does not give rise to any genetic changes or developmental delays in Drosophila [20], appears to be nonmutagenic in cell cultures [14,23], and does not generate reactive oxygen species [21]. Thus, the mutagenic effects and other damage do not appear to arise from Cr3+ . As [Cr(pic)3 ] damages DNA in vitro [21] and chromosomes in Chinese hamster ovary cells [12] and human macrophages [15,16] and generates oxidative DNA damage in rats [18], similar chromosome damage seems to be a likely source of the mutagenic effects of [Cr(pic)3 ] in Drosophila.
Effects on health and development of Drosophila have been studied from egg deposition to the death of the adult. These developmental studies have been extended to other chromium(III) complexes proposed or currently sold as nutritional supplements, the known chromium(III)-containing mutagen [Cr(2,2 bipyridine)3 ]3+ , [Cr(bpy)3 ]3+ , and their organic components. Additionally, the effects of [Cr(pic)3 ] prepared in the laboratory were compared with those of the supplement prepared by its commercial supplier Nutrition 21. The studies found small or no effects on Drosophila from nutritionally relevant amounts of Cr as “chromium nicotinate”, [Cr3 O(propionate)6 (H2 O)3 ]+ , nicotinic acid, or propionic acid. [Cr(bpy)3 ]3+ and 2,2 bipyridine were found to be toxic at these levels, while [Cr(pic)3 ] was found to delay development of progeny regardless of source. Additionally, salivary gland chromosomes from third instar larval progeny of Drosophila whose diets were supplemented with [Cr(pic)3 ] were examined to determined whether heritable chromosomal damage could be detected. [Cr(pic)3 ] was found to induce over one chromosomal aberration per two identifiable chromosome arms. 2. Materials and methods 2.1. Chemicals and reagents Compounds were used as received unless otherwise indicated. [Cr(pic)3 ]·H2 O was prepared by the method of Press et al. [24] or a gift of Nutrition 21. The identity and purity of the samples was confirmed by elemental analysis (C, H, N by Gallbraith Laboratories, Knoxville, TN). [Cr(bpy)3 ]3+ [25], “chromium nicotinate”, Cr(nic)2 [26], and [Cr3 O(propionate)6 (H2 O)3 ]+ , Cr3 [27], were prepared by literature methods. Doubly deionized water was used throughout. All operations were performed in plastic-ware whenever possible. 2.2. Strains and standard media D. melanogaster was used in all studies. An isogenized line of the wild-type strain Canton S was used for cytogenetic analysis. All other studies used an isogenized line of the wild-type strain Oregon R. Both stocks and experimental cultures were maintained in a controlled environment incubator at 25 ◦ C with 65% relative humidity. All studies were conducted on standard D. melanogaster medium (agar, Brewers yeast, cornmeal, sucrose, and 0.12% methylparaben) [20] unless otherwise noted. Some lifespan and F1 progeny studies were performed using instant Drosophila media formula 4-24 (Carolina Biological), rather than standard medium which is autoclaved. Instant media were prepared by mixing an equal volume of media and appropriate aqueous solution just prior to use.
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Chromium solutions or solutions of their organic components, at the appropriate concentration, were substituted for water in tests for toxicity. Cr concentrations were 260 g Cr/kg media. Toxicity tests performed with the organic components contained a concentration equivalent to the ligand concentration of their Cr complexes at 260 g Cr/kg media. Electronic spectra of aqueous solutions or suspensions of chromium picolinate, Cr3, and Cr(nic)2 , are identical before and after autoclaving, suggesting that the complexes are stable against autoclaving. 2.3. Egg deposition and hatching The effect of supplementation on egg deposition and hatching was determined by comparing the amount of eggs deposited/hatched on control media versus those deposited/hatched on supplemented media. Five virgin females and five virgin males, 24 h after eclosion, were placed on a 1% agar egg laying medium prepared with either water, Cr complexes at 260 g Cr/kg media, or organic compounds corresponding to the ligand concentration of their Cr complexes at 260 g Cr/kg media. The number of eggs deposited per day for three consecutive days and the percentage of eggs that hatched were determined. 2.4. Effects on viability and development Adult Drosophila that had been reared on standard, nonsupplemented Drosophila media were collected 6–12 h posteclosion. Five virgin females and five virgin males were placed in vials containing either standard medium, media which contains Cr complexes at 260 g Cr/kg medium, or media which contains organic compounds corresponding to the ligand concentration of their Cr complexes at 260 g Cr/kg media and placed in a 25 ◦ C incubator 65% relative humidity. Each supplement set contained 25 vials. After 3 days the adult Drosophila were removed from the vials, and the developmental stage of the resulting progeny was assessed every 24 h. The number of pupal cases and the number of progeny adults were scored to assess supplemental effects on progeny. Chromium tris(bipyridine) and 2,2 -bipyridine were found to be toxic to the flies at a concentration of 260 g Cr/kg medium or the corresponding ligand concentration. All flies on media containing these two compounds died within 3 days, and all eggs laid during this time failed to hatch (data not shown). Medium containing 2,2 -bipyridine developed a distinct orange tint upon autoclaving. This color is characteristic of iron-bipyridine complexes. Thus, the toxic effects observed with the bipyridine-containing media probably reflect those of its iron complexes. 2.5. Longevity Adult Drosophila that had been reared on standard, nonsupplemented Drosophila medium were collected 6–12 h posteclosion. Cultures were established at a density of 15 males and 15 females; parents were removed from the bottles after
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3 days. Cultures were maintained in a 25 ◦ C incubator 65% relative humidity. First generation progeny from these bottles were collected at 6–12 h post-eclosion and placed in an 18 ◦ C incubator for 24 h to mature. After incubation separated males and females were placed into vials containing either standard Drosophila media, media which contained Cr complexes at 260 g Cr/kg media, or media which contained organic compounds corresponding to the ligand concentration of their Cr complexes at 260 g Cr/kg media. Each supplement set contained 25 vials each of 10 virgin males or 10 virgin females. The vials were placed in a 25 ◦ C incubator at 65% relative humidity and scored for viability every other day. Survivors were transferred to fresh media every 7 days. 2.6. Chromosome spreads Adult wild-type flies were allowed to lay on standard media and media supplemented with 260 g Cr as [Cr(pic)3 ] per kg medium (five males and five females per vial; 10 vials in each group). Their progeny were allowed to develop to the adult stage on this medium. From both groups, single male progeny were collected and mated to untreated wild-type females, and their progeny were allowed to develop to the late third instar larval stage. Chromosome spreads were prepared from the salivary glands of late third instar larvae using standard methods [28]. Chromosomes were examined using phase optics and either a 60× or 100× oil immersion lens on a Nikon Eclipse E600 microscope. Chromosome arms were identified using Drosophila chromosome maps from Lindsley and Zimm [29]. 2.7. Statistics All results are presented as means ± S.D. Significance of differences of means were evaluated using the pooled Student’s t-test; means were considered statistically different if P < 0.05.
3. Results and discussion 3.1. Effects on viability and development In order to determine whether previously observed effects on viability and developmental rate of the progeny were specific to chromium picolinate, untreated adults derived from standard culture were placed on medium supplemented with [Cr(pic)3 ], [Cr(bpy)3 ]3+ , “chromium nicotinate”, and [Cr3 O(propionate)6 (H2 O)3 ]+ , 260 g Cr as the complexes per kg medium, for a single generation. [Cr(pic)3 ] provided by Nutrition 21 and prepared by the method of Ref. [24] was examined as well. In addition, picolinic acid, nicotinic acid, propionic acid, and bipyridine at concentrations equivalent to the maximum ligand concentrations if the Cr(III) complexes were completely dissociated when tested were examined. The developmental stage of the
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resulting progeny was assessed every 24 h. The number of pupal cases and the number of adult progeny were scored to assess fertility effects on progeny reared on each Cr compound. [Cr(pic)3 ] is a popular nutritional supplement that has previously been shown to give rise to mutagenic effects in Drosophila [20]. [Cr(bpy)3 ]3+ is a known mutagen in the Salmonella reversion assay [30]. “Chromium nicotinate” is also a popular commercial chromium supplement but has been poorly characterized [31]. The insoluble complex is probably a polymer and possesses the approximate formula Cr(nicotinate)2 (H2 O)3 (OH) [32]. [Cr3 O(propionate)6 (H2 O)3 ]+ , also called Cr3, has been shown increase insulin sensitivity and lower fasting blood plasma triglycerides and total and LDL cholesterol levels in healthy rats and rat models of type 2 diabetes [33–35]; consequently, the complex has been proposed as a potential therapeutic agent. Drosophila in this study were fed on media containing 260 g Cr/kg media. In comparison, rat diets containing less than 100 g Cr/kg diet are considered Cr deficient [36]. Anderson and Kozlovsky have determined that the Cr content of a composite human diet sample was approximately 100 g/kg diet; mean Cr intake per 1000 calories was ∼15 g such that adult males intake ∼33 g per day and females intake 28 g per day [37]. Most Cr dietary supplements provide 200–600 g Cr per day if the suggested dosage is followed. Consequently, the fruit flies are feeding on media with a concentration of Cr well below that of a supplemented human diet. It has been shown previously that ingestion of this quantity has no acute effects of adult Drosophila; however, juvenile stages are significantly more susceptible to deleterious effects from [Cr(pic)3 ] at this concentration [20]. These deleterious effects on juvenile stages were also observed in the current work. The numbers of progeny reaching pupation and eclosion were observed to be diminished when [Cr(pic)3 ] from either source was tested (Fig. 1A and B). Moreover, the number of uneclosed pupae increased with either source of [Cr(pic)3 ] (Fig. 1C). Treatment with picolinic acid also resulted in an increase of developmental arrest during pupariation, although the effects were smaller in magnitude than after [Cr(pic)3 ] treatment (Fig. 1). In the cases of [Cr(bpy)3 ]3+ and 2,2 -bipyridine, no eggs hatched. In fact, the presence of the compounds in the food had adverse effects on the adult flies, particularly the female flies. In consequence, additional female flies were added to these vials to maintain their numbers during the 3day laying period; thus, the compounds were actually toxic to the flies in addition to preventing development of the eggs. In contrast, “chromium nicotinate”, nico-
tinic acid, Cr3, and propionic acid did not result in any observable delays in development (Fig. 2A and B). Additionally, these compounds did not increase the percentage of uneclosed pupae (Fig. 2C). However, some effects were noted for two of these compounds. The average total number of pupae appearing per vial was significantly (P < 0.05) larger for Cr3 compared to the control (54.1 versus 47.6) and significantly smaller for “chromium nicotinate” (44.4) compared to the control. Similar effects were noted for the average total number of adults appearing per vial ([Cr3 O(propionate)6 (H2 O)3 ]+ , 52.0; “chromium nicotinate”, 42.2; control 45.6). Three possible explanations exist for the results with chromium picolinate and picolinic acid: (1) the adults delayed mating on the medium; (2) [Cr(pic)3 ] or picolinic acid could adversely affect the general state of adult health resulting in fewer fertilized eggs or abnormal embryonic development; (3) the progeny developing on these supplements could be directly affected. It appears that neither the first nor the second explanation applies to these results. Adults have been monitored after exposure to [Cr(pic)3 ], and no significant differences from the control adults in either adult survival or behavior during the 3-day interval in which parents were maintained on test media were found (vide infra). Therefore, the adults seem to be in reasonable health and exhibiting no adverse effects on viability in this time frame (vide infra). To address the possibility of delays in egg deposition or diminution of egg numbers, adults at 24 h post-eclosion were placed on a hard agar egg laying medium prepared with either water or the Cr compounds or their components (except [Cr(bpy)3 ]3+ or 2,2 -bipyridine which were already shown to be toxic), and the number of eggs deposited each day for 3 days was determined. Previously, [Cr(pic)3 ] and picolinate have been shown to not have any observable effect on egg laying [20]. The results summarized in Fig. 3 show that the number of eggs laid per day per female on any of the supplemented media is not significantly different from the number laid on control medium. In addition, no evidence for delays in egg deposition was observed. Similarly, no effects were observed on the hatching of the eggs (data not shown). Therefore, the most likely explanation for the adverse effects of [Cr(pic)3 ] and picolinic acid is that they are acting directly upon the immature progeny, resulting in progeny lethality and slowed development of survivors. In fact, this level of [Cr(pic)3 ] in the diet of adult males flies results in over one chromosomal aberrations per two arms of polytene chromosomes of salivary gland cells of third instar stage larvae (vide infra). Genotoxic effects from the nutritional supplement have been observed in other studies; Stearns and cowork-
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Fig. 1. Effect of [Cr(pic)3 ] and picolinic acid on development. (A) The average number of pupae formed per vial per day in cultures containing media supplemented with chromium picolinate, Cr(pic)3 , produced by the method of Ref. [24] or provided by Nutrition 21 or picolinic acid, Hpic, or standard medium as a function of time. (B) Average eclosion of adult progeny per vial per day. (C) Percentage of unhatched pupae. * P < 0.05. For [Cr(pic)3 ]-supplemented media, the Cr concentration of the media was 260 g/kg food. For Hpic, the picolinate concentration was equivalent to that of the ligand for the [Cr(pic)3 ]-supplemented media. Progeny were produced by five males and five females per vial allowed to lay eggs for 72 h at 25 ◦ C.
ers in 2002 reported that [Cr(pic)3 ] is mutagenic at the hypoxanthine phosphoribosyltransferase locus in CHO cells over the concentration range of 0.2–1 mM [14], while Whittaker and coworkers found [Cr(pic)3 ] induced mutagenic responses in mouse lymphoma cells [17].
The effects of the various Cr complexes can be rationalized based on their ligand composition. Earlier studies have shown that mutagenic forms of Cr(III) possessed chelating ligands containing imine nitrogens coordinated to the metal. DNA damage by the complexes
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Fig. 2. Effect of chromium compounds and ligands on development. (A) The average number of pupae formed per vial per day in cultures containing media supplemented with [Cr3 O(propionate)6 (H2 O)3 ]+ , Cr3; propionic acid, Hprop; “chromium nicotinate”, Cr(nic)2 ; or nicotinic acid, Hnic, or standard media as a function of time. (B) Average eclosion of adult progeny per vial per day. (C) Percentage of unhatched pupae from cultures. For Cr compound-supplemented media, the Cr concentration of the media was 260 g Cr/kg food. For Hprop and Hnic, the propionic acid and nicotinic acid concentrations were equivalent to that of the ligand for the chromium compound-supplemented media. Progeny were produced by five males and five females per vial allowed to lay eggs for 72 h at 25 ◦ C.
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Fig. 3. Number of eggs per female laid on standard media and media supplemented with chromium picolinate, Cr(pic)3 ; picolinic acid, Hpic; “chromium nicotinate”, Cr(nic)2 ; nicotinic acid, Hnic; [Cr3 O(propionate)6 (H2 O)3 ]+ , Cr3; or propionic acid, Hprop. For Cr compound-supplemented media, the Cr concentration of the media was 260 g Cr/kg food. For Hpic, Hprop and Hnic, the picolinic acid, propionic acid, and nicotinic acid concentrations were equivalent to that of the ligand for the chromium compound-supplemented media.
required the presence of dioxygen [30]. This suggests that Cr complexes able to be involved with redox processes could be mutagenic and that imine ligands could shift the redox potential of Cr(III) appropriately for such chemistry to occur. [Cr(bpy)3 ]3+ (with six imine ligands bound to the Cr(III) center) was previously shown to be a mutagen [30], and in the current study had such drastic effects as to be toxic to adult flies and lethal to developing eggs. Of the other Cr(III) complexes, only [Cr(pic)3 ] (with three imine ligands bound per Cr) displayed dramatic effects, resulting in decreased success of hatching and eclosion and developmental delays. Cr3, “chromium nicotinate”, and CrCl3 [20], all lacking imine ligands bound to chromium, resulted in no discernable developmental delays, and only chromium nicotinate resulted in any decrease in progeny success. (Note that while nicotinic acid, 3-carboxypyridine, contains an imine nitrogen, the nitrogen is not bound to Cr [31].) Lay and coworkers have proposed that toxicological effects of chromium nutritional supplements could result from in vivo oxidation to chromate, [Cr(VI)O4 ]2− [38]; [Cr(pic)3 ] and [Cr3 O(O2 CCH2 CH3 )6 (H2 O)3 ]+ were shown to react with 1 mM H2 O2 to generate Cr(V) and possibly Cr(IV) species, which could disproportionate to form Cr(VI). Both compounds appeared to have similar abilities to generate chromate under these conditions. The contrasting effects of [Cr(pic)3 ] and the trinuclear cation in Drosophila in the current study are in contradiction to this proposal. In addition to [Cr(pic)3 ] and [Cr(bpy)3 ]3+ , two of the ligands, picolinic acid and 2,2-bipyridine resulted in observable effects. These can readily be explained. Autoclaving food containing 2,2-bipyridine results in
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the appearance of a distinct orange tint in the food; this color is characteristic of [Fe(bpy)3 ]3+ ; thus, the heated solution of food with the ligands can result in the production of metal complexes. [Fe(bpy)3 ]3+ is known to be a potent generator of reactive oxygen species in the presence of reductants and air or hydrogen peroxide. Similarly, Fe(III) picolinate complexes have been shown to generate reactive oxygen species [39]. Enhanced lipid peroxidation in mitochondria [40] and inhibition of mitosis [41] and, at high concentrations (3 mM), bacteriocidal effects [42] by picolinic acid has been proposed to arise from its ability to form iron complexes. However, [Cr(pic)3 ] and [Cr(bpy)3 ]3+ are stable against autoclaving. [Cr(pic)3 ] is likely to be broken down in the body by enzymatic degradation of the ligands and not by ligand release [43], and [Cr(pic)3 ] at physiologically relevant concentrations has been shown to generate reactive oxygen species in the presence of reductants and dioxygen or hydrogen peroxide [21]. Thus, the similar, but not identical, effects of [Cr(pic)3 ] compared to picolinic acid probably result from the similar ability of the nutritional supplement compared to Fe picolinates to generate reactive oxygen species and genotoxic effects. The effects of picolinic acid have been examined with other genera of flies than Drosophila. Unfortunately, the concentrations of picolinic acid utilized were much higher [44–46], making comparisons difficult. For example, Levinson and Bergmann [44] found for Musca vicina that picolinic acid affected larvae survival, weight, and success of pupation and also ultimately the success of larvae developing into adults. However, the lowest concentration of picolinic acid used was 2.19 g/kg diet (compared to 1.83 mg/kg in the current study, more than a 1000-fold less), and some effects required significantly higher concentrations to be observed. One difference in the current study and this laboratory’s previous study on the effects of [Cr(pic)3 ] and picolinic acid on Drosophila is that in the current study the effects from picolinic acid are less than those of the nutritional supplement, where as the deleterious effects were essentially equivalent in the previous study [20]. While the effects of picolinic acid have been found to be concentration dependent (D. D. D. Hepburn, J. O’Donnell, and J. B. Vincent, unpublished results) in a similar fashion to [Cr(pic)3 ] [20], the magnitude of the effects from picolinic acid have been found to vary from one batch of food to another whereas effects from [Cr(pic)3 ] are uniform. This is consistent with picolinic acid forming metal complexes with metal ions in the media components; use of different lots of components in the food, probably with minor alterations in metal ion content, could lead to the subtle differences in behavior.
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Fig. 4. Effect of chromium compounds and ligands on development of Drosophila maintained on instant media. (A) The average number of pupae formed per vial per day in cultures containing instant media supplemented with [Cr(pic)3 ]; picolinic acid, Hpic; Cr3; or propionic acid, Hprop, or instant media as a function of time. (B) Average eclosion of adult progeny per vial per day. (C) Percentage of unhatched pupae from cultures. P < 0.05. For Cr compound-supplemented media, the Cr concentration of the media was 260 g Cr/kg food. For propionic and nicotinic acids, the propionic acid and nicotinic acid concentrations were equivalent to that of the ligand for the chromium compound-supplemented media. Progeny were produced by five males and five females per vial allowed to lay eggs for 72 h at 25 ◦ C.
To establish whether autoclaving of the media was affecting the chromium complexes, the media was prepared in an alternative manner. Water or aqueous solutions of the chromium compounds or ligands were mixed with instant media and used immediately. The effects of the Cr compounds (and ligands) were essentially identical to those using autoclaved media (Fig. 4). Chromium picolinate-treated media still resulted in a 3-day delay in and decrease of success in hatching and eclosion (Fig. 4A), while a smaller delay and increased success for hatching and eclosion was observed for picolinic acid-treated media (Fig. 4B). Additionally, picolinic acid and [Cr(pic)3 ] resulted in an increase in the percentage of uneclosed pupae (Fig. 4C). In contrast, no effect was observed from Cr3-treated or propionic acid-treated media. These results are not surprising as aqueous solutions or suspensions of the Cr compounds appear to be stable to autoclaving. Thus, the effects of the Cr com-
pounds are readily reproducible and independent of the manner of media preparation. 3.2. Effects on lifespan To better address the effects of [Cr(pic)3 ] and picolinic acid on the health of the adult flies, an examination of the effects of these compounds on the lifespan of both virgin male and virgin female Drosophila was undertaken using both standard autoclaved and instant media. Within 4 days, males on [Cr(pic)3 ]-treated instant medium (Fig. 5A) began deviating from controls and by 8 days were exhibiting statistically significant reductions in viability. This result continued and resulted in significant reduced lifespan in the treated population. The survival of the picolinic acid-treated males was statistically indistinguishable from controls until day 42, when the number of surviving flies becomes
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Fig. 5. Lifespan of (A) virgin male and (B) virgin female adult flies maintained on instant medium and instant media supplemented with chromium picolinate, [Cr(pic)3 ], or picolinic acid, Hpic. The Cr concentration of the [Cr(pic)3 ]-treated media was 260 g/kg food. The picolinate concentration of the Hpic-treated media was equivalent to that of the ligand for the [Cr(pic)3 ]-supplemented media.
lower than that of the control. Longevity of female Drosophila (Fig. 5B) is similarly adversely affected by [Cr(pic)3 ] ingestion; however, picolinic acid appears to affect females more strongly than males as viability is significantly lower after only 16 days. In contrast, Cr3, chromium nicotinate, and their ligands had no observable effects on longevity (Fig. 6). However, both males and females exhibit marginally increased in the Cr3supplemented group compared to the control. Results were essentially identical when autoclaved standard media was utilized (results not shown), again indicating the choice of medium was not crucial to the outcome. 3.3. Effects of [Cr(pic)3 ]·H2 O on polytene chromosomes Previously, lethal mutations created in parental germline cells by dietary supplementation with chromium
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Fig. 6. Lifespan of (A) virgin male and (B) virgin female adult flies maintained on instant medium and instant media supplemented with Cr3, propionic acid, chromium nicotinate, or nicotinic acid. The Cr concentration of the Cr compound-treated media was 260 g/kg food. The ligand concentrations of the ligand-treated media were equivalent to those of the ligand for the Cr compound-treated media.
picolinate were shown were shown to be heritable by progeny two generations removed from exposure [20]. However, the results of these X-linked mutagenesis studies, while demonstrating the mutagenic effects of the supplement, do not provide information on the nature of the mutations. Previous observations of clastogenic damage by this compound in cultured mammalian cells suggested the possibility that lethal mutations might be associated with chromosomal breakage. In the current study, polytene chromosomes of nuclei from the salivary glands of third instar larvae were examined to assess the nature and frequency of damage inherited by progeny of treated Drosophila parents. In the larval stage of Drosophila development, most cells do not divide but go through repeated cycles of DNA replication. Homologous chromosomes remain precisely aligned and paired, except where a chromosomal aberration in one
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Fig. 7. Representative chromosomal aberrations in nuclei of larval progeny of [Cr(pic)3 ]-treated fathers. (A) Inversion (Inv.) in the 3L chromosomal arm. (B) Translocation (Tran.) of the X and 3L chromosomal arms. (C) Deletion (Del.) in the 2R chromosomal arm.
homolog disrupts pairing. These sites of nonpairing are easily visible in cytological preparations of these polytene chromosomes, which in the case of salivary glands, are composed of over 1000 aligned DNA molecules. The size of these chromosomes allows for analysis of the nature of chromosomal aberrations, the identification of the chromosomal arm on which they occur, and a quite precise mapping of the exact position of these lesions. Changes to the chromosomal arms from nuclei of heterozygous progeny from [Cr(pic)3 ]-treated males and untreated females were scored for the number and types of aberrations compared to those of untreated wild-type
nuclei. Referral to published maps of Drosophila chromosomes allowed an analysis of mutations among chromosome arms and a more precise placement of chromosome breakpoints assuring that a single aberration from multiple nuclei of a single individual were not repeatedly scored as independent events [29]. Chromosome aberrations of all classes were observed in chromosome arms of treated flies, including inversions, translocations, and deficiencies (Fig. 7). An incidence of at least one aberration per every two chromosome arms was observed for the [Cr(pic)3 ]-treated group, while none was observed in control samples. While these results were convincing,
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every chromosome arm could not clearly be identified in every squash. Thus, only aberrations confirmed in at least two nuclei from the same individual and only those found on chromosome arms that could be assigned were counted. A total of 43 chromosome arms were examined in the control group; there were no observable aberrations. In the treated group, 47 arms were examined; a total of 25 arms (53%) were positively identified as containing one or more aberrations. In some cases, the aberrations were complex, and combinations of two or more classes of aberrations occurred close to or at the same point. Chromosome aberrations were fairly randomly distributed between the chromosome arms; approximately equal numbers of incidences of deficiencies, inversions, and translocations were present. 4. Discussion Cr picolinate, previously shown to be mutagenic in Drosophila [20], was found to lead to developmental delays and decreases in success rates of hatching and eclosion. This was found independent of whether the compound was prepared in this laboratory or by Nutrition 21. In contrast, [Cr3 O(propionate)6 (H2 O)3 ]+ , “chromium nicotinate” [Cr(nic)2 (OH)(H2 O)3 ]n , nicotinic acid, and propionic acid at equivalent levels had little or no effect on development; thus, nicotinic acid, propionic acid, and their chromium complexes are not anticipated to be mutagenic in Drosophila. Similarly, chromium chloride (CrCl3 ·6H2 O) had no deleterious effects on Drosophila development in a previous study [20]. The results parallel those of Sugden et al., who analyzed the effects of a series of chromium compounds in the Salmonella reversion assay [18]; only chromium compounds with imine ligands were found to be mutagenic. The effects of the imine ligands were postulated to arise from shifts in the redox potential of the chromic centers [18]. Of the complexes examined in this study, only [Cr(pic)3 ] and [Cr(bpy)3 ]3+ possess imine ligands coordinated to their Cr(III) centers. Both chromium picolinate and picolinic acid (or metal picolinate complexes generated during autoclaving), but not Cr3 nor chromium nicotinate (nor their ligands), have detrimental effects on longevity of male and female Drosophila at nutrition supplement levels in addition to affecting embryonic and juvenile development. Cr(III) tris(2,2 bipyridine) and 2,2 -bipyridine were toxic to fruit flies at nutrition supplement levels. The effects of bipyridine are believed to arise from the formation of metal bipyridine complexes during autoclaving. The results suggest that the effects of picolinic acid on development [20] may arise from the formation of metal complexes
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during autoclaving. Thus, [Cr(pic)3 ] and picolinic acid have detrimental effects of all stages of the life cycle of Drosophila. Given the deleterious effects of [Cr(pic)3 ] but not Cr3 nor chromium nicotinate in these experiments, the ability of [Cr(pic)3 ] to generate chromosomal aberrations was examined. In the [Cr(pic)3]-treated group, 53% of the identified chromosomal arms were positively identified as containing one or more aberrations, while no aberrations were observed for the identified chromosomal arms of the control group. The current studies show unequivocally for the first time that [Cr(pic)3 ] supplementation at levels equivalent to human dietary supplementation causes deleterious effects at a molecular level in a whole animal that is widely accepted as a model for the study of human disease. The results report here report long term consequences for adults consuming [Cr(pic)3 ] at supplement levels. Moreover, a possibility exists for heritable deleterious effects, which could be of concern for adults in their reproductive years. Acknowledgments Funding for this research was provided by NIH (DK 62094 (J.B.V.) and GM 62879 (J.O.)) and the American Diabetes Association (J.B.V.). M.H. was supported by an NSF REU grant (to Lowell Kispert and J.B.V.). D.M.S. was supported by a Future Faculty Fellowship. Nutrition 21 provided a sample of chromium picolinate. J.B.V. is the inventor or co-inventor of 5 patents on the use of chromium-containing oligopeptides or complexes (including [Cr3 O(propionate)6 (H2 O)3 ]+ ) as nutritional supplements or therapeutic agents. References [1] R.A. Anderson, N.A. Bryden, M.M. Polansky, K. Gautschi, Dietary chromium effects on tissue chromium concentrations and chromium absorption in rats, J. Trace Elements Exp. Med. 9 (1996) 11–25. [2] J.B. Vincent, The potential value and toxicity of chromium picolinate as a nutritional supplement, weight loss agent, and muscle development agent, Sports Med. 33 (2003) 213–230. [3] R.A. Anderson, N.A. Bryden, M.M. Polansky, Lack of toxicity of chromium chloride and chromium picolinate in rats, J. Am. Coll. Nutr. 16 (1997) 273–279. [4] J.R.T. Davidson, K. Abraham, K.M. Connor, M.N. McLeod, Effectiveness of chromium in atypical depression: a placebocontrolled trial, Biol. Psychiatr. 53 (2003) 261–264. [5] J. Cerulli, D.W. Grabbe, I. Gauthier, M. Malone, M.D. McGoldrick, Chromium picolinate toxicity, Ann. Pharmacother. 32 (1998) 428–431. [6] W.G. Wasser, V.D. D’Agati, Chronic renal failure after ingestion of over-the-counter chromium picolinate, Ann. Intern. Med. 126 (1997) 410.
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Nutritional supplement chromium picolinate generates chromosomal aberrations and impedes progeny development in Drosophila melanogaster Dontarie M. Stallings a,c , Dion D.D. Hepburn a,c , Meredith Hannah a,c , John B. Vincent a,c,∗ , Janis O’Donnell b,c a
Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487-0336, USA Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487-0344, USA Coalition for Biomolecular Products, The University of Alabama, Tuscaloosa, AL 35487-0336, USA
b c
Accepted 5 June 2006 Available online 2 August 2006
Abstract 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Chromium picolinate; Chromium nicotinate; Cr3; Drosophila; Chromium toxicity
1. Introduction As dietary trivalent chromium is not readily absorbed and chromium picolinate, [Cr(pic)3 ], is more readily
∗ Corresponding author. Tel.: +1 205 348 9203; fax: +1 205 348 9104. E-mail address: [email protected] (J.B. Vincent).
1383-5718/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2006.06.019
absorbed [1], [Cr(pic)3 ] has become popular as a nutritional supplement and is often touted to assist in body mass loss and changes in body composition. It currently can be widely found in an extensive variety of products including sports drinks, “health” shakes, and chewing gums. Previously, the compound has generally been considered safe since no acute toxic effects have been observed in numerous clinic trials [2]. In addition, rats fed diets of 100 mg of Cr as [Cr(pic)3 ] per
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kg of food during a 24-week period exhibited no evidence of acute toxicity [3]. [Cr(pic)3 ] has even been investigated as a potentially useful agent for alleviating the symptoms of atypical depression and reportedly has some beneficial effects [4]. Since its introduction as a supplement, however, sporadic reports of deleterious effects associated with [Cr(pic)3 ] supplementation have appeared. These include anemia, liver dysfunction, and renal failure [5,6] and cognitive, perceptual, and motor alterations [7]. Additionally, recent reviews and metaanalyses of studies of healthy individuals consuming [Cr(pic)3 ] (including individuals in weight training programs) indicate that the supplement has no significant effects on body mass or body composition or on blood serum insulin and glucose levels [2,8–11]. Evidence of deleterious cellular and genetic effects of [Cr(pic)3 ] have been accumulating. Stearns et al. [12] reported that [Cr(pic)3 ] induced clastogenic damage in Chinese hamster ovary cells. Mitochondrial damage [13] and mutagenic effects [14] were subsequently reported in these cells as well. [Cr(pic)3 ]-induced chromosomal and oxidative damage have also been reported in a murine macrophage cell line [15,16]. Additionally, [Cr(pic)3 ] has been found to induce mutagenic responses in mouse lymphoma cells with and without the addition of S9 [17]. Further evidence comes from animal studies. Increases in urinary and tissue 8-hydroxydeoxyguanosine [18] and lipid peroxidation [18,19] have been observed in rats given the supplement. Recently, these laboratories have demonstrated that doses of [Cr(pic)3 ] equivalent to or lower than those of humans consuming commercial [Cr(pic)3 ] supplements generate heritable genetic changes and delays in progeny development in Drosophila melanogaster [20]. Not all Cr(III) complexes produce these mutagenic effects. These effects seem to arise from either reactive oxygen species and/or high valent Cr oxo/hydroxo species generated from the intact complex in the presence of biological reducing agents or from the picolinate ligand itself after Cr is released [12,13,21,22]. For example, CrCl3 at comparable doses does not give rise to any genetic changes or developmental delays in Drosophila [20], appears to be nonmutagenic in cell cultures [14,23], and does not generate reactive oxygen species [21]. Thus, the mutagenic effects and other damage do not appear to arise from Cr3+ . As [Cr(pic)3 ] damages DNA in vitro [21] and chromosomes in Chinese hamster ovary cells [12] and human macrophages [15,16] and generates oxidative DNA damage in rats [18], similar chromosome damage seems to be a likely source of the mutagenic effects of [Cr(pic)3 ] in Drosophila.
Effects on health and development of Drosophila have been studied from egg deposition to the death of the adult. These developmental studies have been extended to other chromium(III) complexes proposed or currently sold as nutritional supplements, the known chromium(III)-containing mutagen [Cr(2,2 bipyridine)3 ]3+ , [Cr(bpy)3 ]3+ , and their organic components. Additionally, the effects of [Cr(pic)3 ] prepared in the laboratory were compared with those of the supplement prepared by its commercial supplier Nutrition 21. The studies found small or no effects on Drosophila from nutritionally relevant amounts of Cr as “chromium nicotinate”, [Cr3 O(propionate)6 (H2 O)3 ]+ , nicotinic acid, or propionic acid. [Cr(bpy)3 ]3+ and 2,2 bipyridine were found to be toxic at these levels, while [Cr(pic)3 ] was found to delay development of progeny regardless of source. Additionally, salivary gland chromosomes from third instar larval progeny of Drosophila whose diets were supplemented with [Cr(pic)3 ] were examined to determined whether heritable chromosomal damage could be detected. [Cr(pic)3 ] was found to induce over one chromosomal aberration per two identifiable chromosome arms. 2. Materials and methods 2.1. Chemicals and reagents Compounds were used as received unless otherwise indicated. [Cr(pic)3 ]·H2 O was prepared by the method of Press et al. [24] or a gift of Nutrition 21. The identity and purity of the samples was confirmed by elemental analysis (C, H, N by Gallbraith Laboratories, Knoxville, TN). [Cr(bpy)3 ]3+ [25], “chromium nicotinate”, Cr(nic)2 [26], and [Cr3 O(propionate)6 (H2 O)3 ]+ , Cr3 [27], were prepared by literature methods. Doubly deionized water was used throughout. All operations were performed in plastic-ware whenever possible. 2.2. Strains and standard media D. melanogaster was used in all studies. An isogenized line of the wild-type strain Canton S was used for cytogenetic analysis. All other studies used an isogenized line of the wild-type strain Oregon R. Both stocks and experimental cultures were maintained in a controlled environment incubator at 25 ◦ C with 65% relative humidity. All studies were conducted on standard D. melanogaster medium (agar, Brewers yeast, cornmeal, sucrose, and 0.12% methylparaben) [20] unless otherwise noted. Some lifespan and F1 progeny studies were performed using instant Drosophila media formula 4-24 (Carolina Biological), rather than standard medium which is autoclaved. Instant media were prepared by mixing an equal volume of media and appropriate aqueous solution just prior to use.
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Chromium solutions or solutions of their organic components, at the appropriate concentration, were substituted for water in tests for toxicity. Cr concentrations were 260 g Cr/kg media. Toxicity tests performed with the organic components contained a concentration equivalent to the ligand concentration of their Cr complexes at 260 g Cr/kg media. Electronic spectra of aqueous solutions or suspensions of chromium picolinate, Cr3, and Cr(nic)2 , are identical before and after autoclaving, suggesting that the complexes are stable against autoclaving. 2.3. Egg deposition and hatching The effect of supplementation on egg deposition and hatching was determined by comparing the amount of eggs deposited/hatched on control media versus those deposited/hatched on supplemented media. Five virgin females and five virgin males, 24 h after eclosion, were placed on a 1% agar egg laying medium prepared with either water, Cr complexes at 260 g Cr/kg media, or organic compounds corresponding to the ligand concentration of their Cr complexes at 260 g Cr/kg media. The number of eggs deposited per day for three consecutive days and the percentage of eggs that hatched were determined. 2.4. Effects on viability and development Adult Drosophila that had been reared on standard, nonsupplemented Drosophila media were collected 6–12 h posteclosion. Five virgin females and five virgin males were placed in vials containing either standard medium, media which contains Cr complexes at 260 g Cr/kg medium, or media which contains organic compounds corresponding to the ligand concentration of their Cr complexes at 260 g Cr/kg media and placed in a 25 ◦ C incubator 65% relative humidity. Each supplement set contained 25 vials. After 3 days the adult Drosophila were removed from the vials, and the developmental stage of the resulting progeny was assessed every 24 h. The number of pupal cases and the number of progeny adults were scored to assess supplemental effects on progeny. Chromium tris(bipyridine) and 2,2 -bipyridine were found to be toxic to the flies at a concentration of 260 g Cr/kg medium or the corresponding ligand concentration. All flies on media containing these two compounds died within 3 days, and all eggs laid during this time failed to hatch (data not shown). Medium containing 2,2 -bipyridine developed a distinct orange tint upon autoclaving. This color is characteristic of iron-bipyridine complexes. Thus, the toxic effects observed with the bipyridine-containing media probably reflect those of its iron complexes. 2.5. Longevity Adult Drosophila that had been reared on standard, nonsupplemented Drosophila medium were collected 6–12 h posteclosion. Cultures were established at a density of 15 males and 15 females; parents were removed from the bottles after
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3 days. Cultures were maintained in a 25 ◦ C incubator 65% relative humidity. First generation progeny from these bottles were collected at 6–12 h post-eclosion and placed in an 18 ◦ C incubator for 24 h to mature. After incubation separated males and females were placed into vials containing either standard Drosophila media, media which contained Cr complexes at 260 g Cr/kg media, or media which contained organic compounds corresponding to the ligand concentration of their Cr complexes at 260 g Cr/kg media. Each supplement set contained 25 vials each of 10 virgin males or 10 virgin females. The vials were placed in a 25 ◦ C incubator at 65% relative humidity and scored for viability every other day. Survivors were transferred to fresh media every 7 days. 2.6. Chromosome spreads Adult wild-type flies were allowed to lay on standard media and media supplemented with 260 g Cr as [Cr(pic)3 ] per kg medium (five males and five females per vial; 10 vials in each group). Their progeny were allowed to develop to the adult stage on this medium. From both groups, single male progeny were collected and mated to untreated wild-type females, and their progeny were allowed to develop to the late third instar larval stage. Chromosome spreads were prepared from the salivary glands of late third instar larvae using standard methods [28]. Chromosomes were examined using phase optics and either a 60× or 100× oil immersion lens on a Nikon Eclipse E600 microscope. Chromosome arms were identified using Drosophila chromosome maps from Lindsley and Zimm [29]. 2.7. Statistics All results are presented as means ± S.D. Significance of differences of means were evaluated using the pooled Student’s t-test; means were considered statistically different if P < 0.05.
3. Results and discussion 3.1. Effects on viability and development In order to determine whether previously observed effects on viability and developmental rate of the progeny were specific to chromium picolinate, untreated adults derived from standard culture were placed on medium supplemented with [Cr(pic)3 ], [Cr(bpy)3 ]3+ , “chromium nicotinate”, and [Cr3 O(propionate)6 (H2 O)3 ]+ , 260 g Cr as the complexes per kg medium, for a single generation. [Cr(pic)3 ] provided by Nutrition 21 and prepared by the method of Ref. [24] was examined as well. In addition, picolinic acid, nicotinic acid, propionic acid, and bipyridine at concentrations equivalent to the maximum ligand concentrations if the Cr(III) complexes were completely dissociated when tested were examined. The developmental stage of the
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resulting progeny was assessed every 24 h. The number of pupal cases and the number of adult progeny were scored to assess fertility effects on progeny reared on each Cr compound. [Cr(pic)3 ] is a popular nutritional supplement that has previously been shown to give rise to mutagenic effects in Drosophila [20]. [Cr(bpy)3 ]3+ is a known mutagen in the Salmonella reversion assay [30]. “Chromium nicotinate” is also a popular commercial chromium supplement but has been poorly characterized [31]. The insoluble complex is probably a polymer and possesses the approximate formula Cr(nicotinate)2 (H2 O)3 (OH) [32]. [Cr3 O(propionate)6 (H2 O)3 ]+ , also called Cr3, has been shown increase insulin sensitivity and lower fasting blood plasma triglycerides and total and LDL cholesterol levels in healthy rats and rat models of type 2 diabetes [33–35]; consequently, the complex has been proposed as a potential therapeutic agent. Drosophila in this study were fed on media containing 260 g Cr/kg media. In comparison, rat diets containing less than 100 g Cr/kg diet are considered Cr deficient [36]. Anderson and Kozlovsky have determined that the Cr content of a composite human diet sample was approximately 100 g/kg diet; mean Cr intake per 1000 calories was ∼15 g such that adult males intake ∼33 g per day and females intake 28 g per day [37]. Most Cr dietary supplements provide 200–600 g Cr per day if the suggested dosage is followed. Consequently, the fruit flies are feeding on media with a concentration of Cr well below that of a supplemented human diet. It has been shown previously that ingestion of this quantity has no acute effects of adult Drosophila; however, juvenile stages are significantly more susceptible to deleterious effects from [Cr(pic)3 ] at this concentration [20]. These deleterious effects on juvenile stages were also observed in the current work. The numbers of progeny reaching pupation and eclosion were observed to be diminished when [Cr(pic)3 ] from either source was tested (Fig. 1A and B). Moreover, the number of uneclosed pupae increased with either source of [Cr(pic)3 ] (Fig. 1C). Treatment with picolinic acid also resulted in an increase of developmental arrest during pupariation, although the effects were smaller in magnitude than after [Cr(pic)3 ] treatment (Fig. 1). In the cases of [Cr(bpy)3 ]3+ and 2,2 -bipyridine, no eggs hatched. In fact, the presence of the compounds in the food had adverse effects on the adult flies, particularly the female flies. In consequence, additional female flies were added to these vials to maintain their numbers during the 3day laying period; thus, the compounds were actually toxic to the flies in addition to preventing development of the eggs. In contrast, “chromium nicotinate”, nico-
tinic acid, Cr3, and propionic acid did not result in any observable delays in development (Fig. 2A and B). Additionally, these compounds did not increase the percentage of uneclosed pupae (Fig. 2C). However, some effects were noted for two of these compounds. The average total number of pupae appearing per vial was significantly (P < 0.05) larger for Cr3 compared to the control (54.1 versus 47.6) and significantly smaller for “chromium nicotinate” (44.4) compared to the control. Similar effects were noted for the average total number of adults appearing per vial ([Cr3 O(propionate)6 (H2 O)3 ]+ , 52.0; “chromium nicotinate”, 42.2; control 45.6). Three possible explanations exist for the results with chromium picolinate and picolinic acid: (1) the adults delayed mating on the medium; (2) [Cr(pic)3 ] or picolinic acid could adversely affect the general state of adult health resulting in fewer fertilized eggs or abnormal embryonic development; (3) the progeny developing on these supplements could be directly affected. It appears that neither the first nor the second explanation applies to these results. Adults have been monitored after exposure to [Cr(pic)3 ], and no significant differences from the control adults in either adult survival or behavior during the 3-day interval in which parents were maintained on test media were found (vide infra). Therefore, the adults seem to be in reasonable health and exhibiting no adverse effects on viability in this time frame (vide infra). To address the possibility of delays in egg deposition or diminution of egg numbers, adults at 24 h post-eclosion were placed on a hard agar egg laying medium prepared with either water or the Cr compounds or their components (except [Cr(bpy)3 ]3+ or 2,2 -bipyridine which were already shown to be toxic), and the number of eggs deposited each day for 3 days was determined. Previously, [Cr(pic)3 ] and picolinate have been shown to not have any observable effect on egg laying [20]. The results summarized in Fig. 3 show that the number of eggs laid per day per female on any of the supplemented media is not significantly different from the number laid on control medium. In addition, no evidence for delays in egg deposition was observed. Similarly, no effects were observed on the hatching of the eggs (data not shown). Therefore, the most likely explanation for the adverse effects of [Cr(pic)3 ] and picolinic acid is that they are acting directly upon the immature progeny, resulting in progeny lethality and slowed development of survivors. In fact, this level of [Cr(pic)3 ] in the diet of adult males flies results in over one chromosomal aberrations per two arms of polytene chromosomes of salivary gland cells of third instar stage larvae (vide infra). Genotoxic effects from the nutritional supplement have been observed in other studies; Stearns and cowork-
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Fig. 1. Effect of [Cr(pic)3 ] and picolinic acid on development. (A) The average number of pupae formed per vial per day in cultures containing media supplemented with chromium picolinate, Cr(pic)3 , produced by the method of Ref. [24] or provided by Nutrition 21 or picolinic acid, Hpic, or standard medium as a function of time. (B) Average eclosion of adult progeny per vial per day. (C) Percentage of unhatched pupae. * P < 0.05. For [Cr(pic)3 ]-supplemented media, the Cr concentration of the media was 260 g/kg food. For Hpic, the picolinate concentration was equivalent to that of the ligand for the [Cr(pic)3 ]-supplemented media. Progeny were produced by five males and five females per vial allowed to lay eggs for 72 h at 25 ◦ C.
ers in 2002 reported that [Cr(pic)3 ] is mutagenic at the hypoxanthine phosphoribosyltransferase locus in CHO cells over the concentration range of 0.2–1 mM [14], while Whittaker and coworkers found [Cr(pic)3 ] induced mutagenic responses in mouse lymphoma cells [17].
The effects of the various Cr complexes can be rationalized based on their ligand composition. Earlier studies have shown that mutagenic forms of Cr(III) possessed chelating ligands containing imine nitrogens coordinated to the metal. DNA damage by the complexes
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Fig. 2. Effect of chromium compounds and ligands on development. (A) The average number of pupae formed per vial per day in cultures containing media supplemented with [Cr3 O(propionate)6 (H2 O)3 ]+ , Cr3; propionic acid, Hprop; “chromium nicotinate”, Cr(nic)2 ; or nicotinic acid, Hnic, or standard media as a function of time. (B) Average eclosion of adult progeny per vial per day. (C) Percentage of unhatched pupae from cultures. For Cr compound-supplemented media, the Cr concentration of the media was 260 g Cr/kg food. For Hprop and Hnic, the propionic acid and nicotinic acid concentrations were equivalent to that of the ligand for the chromium compound-supplemented media. Progeny were produced by five males and five females per vial allowed to lay eggs for 72 h at 25 ◦ C.
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Fig. 3. Number of eggs per female laid on standard media and media supplemented with chromium picolinate, Cr(pic)3 ; picolinic acid, Hpic; “chromium nicotinate”, Cr(nic)2 ; nicotinic acid, Hnic; [Cr3 O(propionate)6 (H2 O)3 ]+ , Cr3; or propionic acid, Hprop. For Cr compound-supplemented media, the Cr concentration of the media was 260 g Cr/kg food. For Hpic, Hprop and Hnic, the picolinic acid, propionic acid, and nicotinic acid concentrations were equivalent to that of the ligand for the chromium compound-supplemented media.
required the presence of dioxygen [30]. This suggests that Cr complexes able to be involved with redox processes could be mutagenic and that imine ligands could shift the redox potential of Cr(III) appropriately for such chemistry to occur. [Cr(bpy)3 ]3+ (with six imine ligands bound to the Cr(III) center) was previously shown to be a mutagen [30], and in the current study had such drastic effects as to be toxic to adult flies and lethal to developing eggs. Of the other Cr(III) complexes, only [Cr(pic)3 ] (with three imine ligands bound per Cr) displayed dramatic effects, resulting in decreased success of hatching and eclosion and developmental delays. Cr3, “chromium nicotinate”, and CrCl3 [20], all lacking imine ligands bound to chromium, resulted in no discernable developmental delays, and only chromium nicotinate resulted in any decrease in progeny success. (Note that while nicotinic acid, 3-carboxypyridine, contains an imine nitrogen, the nitrogen is not bound to Cr [31].) Lay and coworkers have proposed that toxicological effects of chromium nutritional supplements could result from in vivo oxidation to chromate, [Cr(VI)O4 ]2− [38]; [Cr(pic)3 ] and [Cr3 O(O2 CCH2 CH3 )6 (H2 O)3 ]+ were shown to react with 1 mM H2 O2 to generate Cr(V) and possibly Cr(IV) species, which could disproportionate to form Cr(VI). Both compounds appeared to have similar abilities to generate chromate under these conditions. The contrasting effects of [Cr(pic)3 ] and the trinuclear cation in Drosophila in the current study are in contradiction to this proposal. In addition to [Cr(pic)3 ] and [Cr(bpy)3 ]3+ , two of the ligands, picolinic acid and 2,2-bipyridine resulted in observable effects. These can readily be explained. Autoclaving food containing 2,2-bipyridine results in
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the appearance of a distinct orange tint in the food; this color is characteristic of [Fe(bpy)3 ]3+ ; thus, the heated solution of food with the ligands can result in the production of metal complexes. [Fe(bpy)3 ]3+ is known to be a potent generator of reactive oxygen species in the presence of reductants and air or hydrogen peroxide. Similarly, Fe(III) picolinate complexes have been shown to generate reactive oxygen species [39]. Enhanced lipid peroxidation in mitochondria [40] and inhibition of mitosis [41] and, at high concentrations (3 mM), bacteriocidal effects [42] by picolinic acid has been proposed to arise from its ability to form iron complexes. However, [Cr(pic)3 ] and [Cr(bpy)3 ]3+ are stable against autoclaving. [Cr(pic)3 ] is likely to be broken down in the body by enzymatic degradation of the ligands and not by ligand release [43], and [Cr(pic)3 ] at physiologically relevant concentrations has been shown to generate reactive oxygen species in the presence of reductants and dioxygen or hydrogen peroxide [21]. Thus, the similar, but not identical, effects of [Cr(pic)3 ] compared to picolinic acid probably result from the similar ability of the nutritional supplement compared to Fe picolinates to generate reactive oxygen species and genotoxic effects. The effects of picolinic acid have been examined with other genera of flies than Drosophila. Unfortunately, the concentrations of picolinic acid utilized were much higher [44–46], making comparisons difficult. For example, Levinson and Bergmann [44] found for Musca vicina that picolinic acid affected larvae survival, weight, and success of pupation and also ultimately the success of larvae developing into adults. However, the lowest concentration of picolinic acid used was 2.19 g/kg diet (compared to 1.83 mg/kg in the current study, more than a 1000-fold less), and some effects required significantly higher concentrations to be observed. One difference in the current study and this laboratory’s previous study on the effects of [Cr(pic)3 ] and picolinic acid on Drosophila is that in the current study the effects from picolinic acid are less than those of the nutritional supplement, where as the deleterious effects were essentially equivalent in the previous study [20]. While the effects of picolinic acid have been found to be concentration dependent (D. D. D. Hepburn, J. O’Donnell, and J. B. Vincent, unpublished results) in a similar fashion to [Cr(pic)3 ] [20], the magnitude of the effects from picolinic acid have been found to vary from one batch of food to another whereas effects from [Cr(pic)3 ] are uniform. This is consistent with picolinic acid forming metal complexes with metal ions in the media components; use of different lots of components in the food, probably with minor alterations in metal ion content, could lead to the subtle differences in behavior.
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Fig. 4. Effect of chromium compounds and ligands on development of Drosophila maintained on instant media. (A) The average number of pupae formed per vial per day in cultures containing instant media supplemented with [Cr(pic)3 ]; picolinic acid, Hpic; Cr3; or propionic acid, Hprop, or instant media as a function of time. (B) Average eclosion of adult progeny per vial per day. (C) Percentage of unhatched pupae from cultures. P < 0.05. For Cr compound-supplemented media, the Cr concentration of the media was 260 g Cr/kg food. For propionic and nicotinic acids, the propionic acid and nicotinic acid concentrations were equivalent to that of the ligand for the chromium compound-supplemented media. Progeny were produced by five males and five females per vial allowed to lay eggs for 72 h at 25 ◦ C.
To establish whether autoclaving of the media was affecting the chromium complexes, the media was prepared in an alternative manner. Water or aqueous solutions of the chromium compounds or ligands were mixed with instant media and used immediately. The effects of the Cr compounds (and ligands) were essentially identical to those using autoclaved media (Fig. 4). Chromium picolinate-treated media still resulted in a 3-day delay in and decrease of success in hatching and eclosion (Fig. 4A), while a smaller delay and increased success for hatching and eclosion was observed for picolinic acid-treated media (Fig. 4B). Additionally, picolinic acid and [Cr(pic)3 ] resulted in an increase in the percentage of uneclosed pupae (Fig. 4C). In contrast, no effect was observed from Cr3-treated or propionic acid-treated media. These results are not surprising as aqueous solutions or suspensions of the Cr compounds appear to be stable to autoclaving. Thus, the effects of the Cr com-
pounds are readily reproducible and independent of the manner of media preparation. 3.2. Effects on lifespan To better address the effects of [Cr(pic)3 ] and picolinic acid on the health of the adult flies, an examination of the effects of these compounds on the lifespan of both virgin male and virgin female Drosophila was undertaken using both standard autoclaved and instant media. Within 4 days, males on [Cr(pic)3 ]-treated instant medium (Fig. 5A) began deviating from controls and by 8 days were exhibiting statistically significant reductions in viability. This result continued and resulted in significant reduced lifespan in the treated population. The survival of the picolinic acid-treated males was statistically indistinguishable from controls until day 42, when the number of surviving flies becomes
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Fig. 5. Lifespan of (A) virgin male and (B) virgin female adult flies maintained on instant medium and instant media supplemented with chromium picolinate, [Cr(pic)3 ], or picolinic acid, Hpic. The Cr concentration of the [Cr(pic)3 ]-treated media was 260 g/kg food. The picolinate concentration of the Hpic-treated media was equivalent to that of the ligand for the [Cr(pic)3 ]-supplemented media.
lower than that of the control. Longevity of female Drosophila (Fig. 5B) is similarly adversely affected by [Cr(pic)3 ] ingestion; however, picolinic acid appears to affect females more strongly than males as viability is significantly lower after only 16 days. In contrast, Cr3, chromium nicotinate, and their ligands had no observable effects on longevity (Fig. 6). However, both males and females exhibit marginally increased in the Cr3supplemented group compared to the control. Results were essentially identical when autoclaved standard media was utilized (results not shown), again indicating the choice of medium was not crucial to the outcome. 3.3. Effects of [Cr(pic)3 ]·H2 O on polytene chromosomes Previously, lethal mutations created in parental germline cells by dietary supplementation with chromium
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Fig. 6. Lifespan of (A) virgin male and (B) virgin female adult flies maintained on instant medium and instant media supplemented with Cr3, propionic acid, chromium nicotinate, or nicotinic acid. The Cr concentration of the Cr compound-treated media was 260 g/kg food. The ligand concentrations of the ligand-treated media were equivalent to those of the ligand for the Cr compound-treated media.
picolinate were shown were shown to be heritable by progeny two generations removed from exposure [20]. However, the results of these X-linked mutagenesis studies, while demonstrating the mutagenic effects of the supplement, do not provide information on the nature of the mutations. Previous observations of clastogenic damage by this compound in cultured mammalian cells suggested the possibility that lethal mutations might be associated with chromosomal breakage. In the current study, polytene chromosomes of nuclei from the salivary glands of third instar larvae were examined to assess the nature and frequency of damage inherited by progeny of treated Drosophila parents. In the larval stage of Drosophila development, most cells do not divide but go through repeated cycles of DNA replication. Homologous chromosomes remain precisely aligned and paired, except where a chromosomal aberration in one
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Fig. 7. Representative chromosomal aberrations in nuclei of larval progeny of [Cr(pic)3 ]-treated fathers. (A) Inversion (Inv.) in the 3L chromosomal arm. (B) Translocation (Tran.) of the X and 3L chromosomal arms. (C) Deletion (Del.) in the 2R chromosomal arm.
homolog disrupts pairing. These sites of nonpairing are easily visible in cytological preparations of these polytene chromosomes, which in the case of salivary glands, are composed of over 1000 aligned DNA molecules. The size of these chromosomes allows for analysis of the nature of chromosomal aberrations, the identification of the chromosomal arm on which they occur, and a quite precise mapping of the exact position of these lesions. Changes to the chromosomal arms from nuclei of heterozygous progeny from [Cr(pic)3 ]-treated males and untreated females were scored for the number and types of aberrations compared to those of untreated wild-type
nuclei. Referral to published maps of Drosophila chromosomes allowed an analysis of mutations among chromosome arms and a more precise placement of chromosome breakpoints assuring that a single aberration from multiple nuclei of a single individual were not repeatedly scored as independent events [29]. Chromosome aberrations of all classes were observed in chromosome arms of treated flies, including inversions, translocations, and deficiencies (Fig. 7). An incidence of at least one aberration per every two chromosome arms was observed for the [Cr(pic)3 ]-treated group, while none was observed in control samples. While these results were convincing,
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every chromosome arm could not clearly be identified in every squash. Thus, only aberrations confirmed in at least two nuclei from the same individual and only those found on chromosome arms that could be assigned were counted. A total of 43 chromosome arms were examined in the control group; there were no observable aberrations. In the treated group, 47 arms were examined; a total of 25 arms (53%) were positively identified as containing one or more aberrations. In some cases, the aberrations were complex, and combinations of two or more classes of aberrations occurred close to or at the same point. Chromosome aberrations were fairly randomly distributed between the chromosome arms; approximately equal numbers of incidences of deficiencies, inversions, and translocations were present. 4. Discussion Cr picolinate, previously shown to be mutagenic in Drosophila [20], was found to lead to developmental delays and decreases in success rates of hatching and eclosion. This was found independent of whether the compound was prepared in this laboratory or by Nutrition 21. In contrast, [Cr3 O(propionate)6 (H2 O)3 ]+ , “chromium nicotinate” [Cr(nic)2 (OH)(H2 O)3 ]n , nicotinic acid, and propionic acid at equivalent levels had little or no effect on development; thus, nicotinic acid, propionic acid, and their chromium complexes are not anticipated to be mutagenic in Drosophila. Similarly, chromium chloride (CrCl3 ·6H2 O) had no deleterious effects on Drosophila development in a previous study [20]. The results parallel those of Sugden et al., who analyzed the effects of a series of chromium compounds in the Salmonella reversion assay [18]; only chromium compounds with imine ligands were found to be mutagenic. The effects of the imine ligands were postulated to arise from shifts in the redox potential of the chromic centers [18]. Of the complexes examined in this study, only [Cr(pic)3 ] and [Cr(bpy)3 ]3+ possess imine ligands coordinated to their Cr(III) centers. Both chromium picolinate and picolinic acid (or metal picolinate complexes generated during autoclaving), but not Cr3 nor chromium nicotinate (nor their ligands), have detrimental effects on longevity of male and female Drosophila at nutrition supplement levels in addition to affecting embryonic and juvenile development. Cr(III) tris(2,2 bipyridine) and 2,2 -bipyridine were toxic to fruit flies at nutrition supplement levels. The effects of bipyridine are believed to arise from the formation of metal bipyridine complexes during autoclaving. The results suggest that the effects of picolinic acid on development [20] may arise from the formation of metal complexes
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during autoclaving. Thus, [Cr(pic)3 ] and picolinic acid have detrimental effects of all stages of the life cycle of Drosophila. Given the deleterious effects of [Cr(pic)3 ] but not Cr3 nor chromium nicotinate in these experiments, the ability of [Cr(pic)3 ] to generate chromosomal aberrations was examined. In the [Cr(pic)3]-treated group, 53% of the identified chromosomal arms were positively identified as containing one or more aberrations, while no aberrations were observed for the identified chromosomal arms of the control group. The current studies show unequivocally for the first time that [Cr(pic)3 ] supplementation at levels equivalent to human dietary supplementation causes deleterious effects at a molecular level in a whole animal that is widely accepted as a model for the study of human disease. The results report here report long term consequences for adults consuming [Cr(pic)3 ] at supplement levels. Moreover, a possibility exists for heritable deleterious effects, which could be of concern for adults in their reproductive years. Acknowledgments Funding for this research was provided by NIH (DK 62094 (J.B.V.) and GM 62879 (J.O.)) and the American Diabetes Association (J.B.V.). M.H. was supported by an NSF REU grant (to Lowell Kispert and J.B.V.). D.M.S. was supported by a Future Faculty Fellowship. Nutrition 21 provided a sample of chromium picolinate. J.B.V. is the inventor or co-inventor of 5 patents on the use of chromium-containing oligopeptides or complexes (including [Cr3 O(propionate)6 (H2 O)3 ]+ ) as nutritional supplements or therapeutic agents. References [1] R.A. Anderson, N.A. Bryden, M.M. Polansky, K. Gautschi, Dietary chromium effects on tissue chromium concentrations and chromium absorption in rats, J. Trace Elements Exp. Med. 9 (1996) 11–25. [2] J.B. Vincent, The potential value and toxicity of chromium picolinate as a nutritional supplement, weight loss agent, and muscle development agent, Sports Med. 33 (2003) 213–230. [3] R.A. Anderson, N.A. Bryden, M.M. Polansky, Lack of toxicity of chromium chloride and chromium picolinate in rats, J. Am. Coll. Nutr. 16 (1997) 273–279. [4] J.R.T. Davidson, K. Abraham, K.M. Connor, M.N. McLeod, Effectiveness of chromium in atypical depression: a placebocontrolled trial, Biol. Psychiatr. 53 (2003) 261–264. [5] J. Cerulli, D.W. Grabbe, I. Gauthier, M. Malone, M.D. McGoldrick, Chromium picolinate toxicity, Ann. Pharmacother. 32 (1998) 428–431. [6] W.G. Wasser, V.D. D’Agati, Chronic renal failure after ingestion of over-the-counter chromium picolinate, Ann. Intern. Med. 126 (1997) 410.
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