Chromium-picolinate induced ocular changes: Protective role of ascorbic acid

Toxicology 226 (2006) 143–151

Chromium-picolinate induced ocular changes: Protective role of ascorbic acid Amany A. Mahmoud a , Sawsan H. Karam b , Mosaad A. Abdel-Wahhab c,∗ a

c

Biochemistry Department, Research Institute of Ophthalmology, Giza, Egypt b Histology Department, Research Institute of Ophthalmology, Giza, Egypt Food Toxicology and Contaminants Department, National Research Center, Dokki, Cairo, Egypt Received 31 May 2006; received in revised form 14 June 2006; accepted 14 June 2006 Available online 30 June 2006

Abstract Chromium-picolinate (Cr-picolinate) is a popular nutritional supplement; however its safety has been questioned with regard to its ability to act as a clastogen. The aim of the present work was to evaluate the biochemical, histological and morphological changes in the cornea and lens following oral administration of Cr-picolinate and the possible protective effect of Vitamin C. Ninety male Sprague-Dawley rats were divided into five groups included the control group, the groups treated with Cr-picolinate (0.8 and 1.5 mg/100 g b.w.) alone or in combination with Vitamin C (0.5 mg/100 g b.w.) for 8 weeks. The results indicated that the high dose of Cr-picolinate induced a significant decrease in SOD, GSH, Na+ -, K+ -ATPase levels, and a significant increase in MDA level. Severe morphological and histological changes in the cornea and lens accompanied with a decrease in the total soluble protein of the lens homogenate and changes in the crystallines fractions in lens. Vitamin C supplementation succeeded to restore these changes to great extent. It could be concluded that consumption of Cr-picolinate for a long time induced several hazards to cornea and lens. Supplementation with extra amounts of Vitamin C may be useful to restrain the Cr-picolinate induced ocular changes. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Chromium-picolinate; Vitamin C; Eye; Cornea; Lens; Oxidative stress; Antioxidant

1. Introduction Chromium is a unique micronutrient essential for normal protein, fat and carbohydrate metabolism (Mertz, 1993). Chromium(III) helps insulin metabolize fat, turn protein into muscle and convert sugar into energy (Anderson, 1998). Deficiency of chromium has been linked to a number of disorder, including symptoms of type 2 diabetes, and can also mimic many signs of cardiovascular diseases (Anderson, 1995).

∗

Corresponding author. Tel.: +20 2 283 1943; fax: +20 2 337 0931. E-mail address: mosaad [email protected] (M.A. Abdel-Wahhab).

Cr-picolinate is commonly used as dietary supplements to provide a bioavailable form of chromium(III). It has been marketed to consumers for use in weight loss, increasing muscle mass and lowering serum cholesterol (Whitteker et al., 2005). However, its safety has recently been questioned, especially with regard to its ability to act as a clastogen (Vincent, 2003). Many studies have suggested that Cr-picolinate supplements may cause renal impairment and cell toxicity when ingested in excess (Cerulli et al., 1998; Speetjens et al., 1999). Nevertheless, no available data were found regarding the effects of Cr-picolinate on the eye. Thus, this study was designed to investigate the long term effects of Crpicolinate intake on the ocular tissues (cornea and lens) with regards to the biochemical and histological view

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and the possible protective effects of Vitamin C supplementation as an antioxidant. 2. Materials and methods 2.1. Chemicals and kits Chromium-picolinate was obtained from Amoun Pharmaceutical Co. (El-Obour City, Cairo, Egypt). Reduced glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) were purchased from Randex Laboratories (San Francisco, CA, USA). Other chemicals were of the highest purity commercially available. 2.2. Animals Ninety male Sprague-Dawley rats (80–100 g b.w.) were obtained from the Animals House Laboratory, Research Institute of Ophthalmology (Giza, Egypt). Rats were maintained on the standard laboratory chow diet (protein: 16.0%; fat: 3.6%; fiber: 4.1%, and metabolic energy: 0.012 MJ) and water ad libitum at the Animal House Laboratory, Research Institute of Ophthalmology (Giza, Egypt). After an acclimation period of 1 week, the animals were distributed into five groups (18 rats/group) and housed individually in stainless steel cages in a temperature-controlled (23 ± 1 ◦ C) and artificially illuminated (12 h dark/light cycle) room free from any source of chemical contamination. All animals were received humane care in compliance with the guidelines of the Animal Care and Use Committee of the Research Institute of Ophthalmology, Egypt. 2.3. Experimental design Animals within different treatment groups were treated for 8 weeks as follows: (1) untreated control group fed on the standard diet; (2) treated orally with Cr-picolinate (0.8 mg/100 g b.w.; low-dose group); (3) treated orally with Cr-picolinate (1.5 mg/100 g b.w.; high-dose group); (4) treated orally with Cr-picolinate at the low dose plus Vitamin C (0.5 mg/100 g b.w.); (5) treated orally with Cr-picolinate at the high dose plus Vitamin C. All animals were examined using slit lamp biomicroscopy (Hagg Shiet-Ger) at weekly intervals starting from the 1st week. At the end of the experimental period, all animals were sacrificed by cervical dislocation (approved by the Local Animal Ethics Committee of the Research Institute of Ophthalmology, Egypt) and the eyes were removed for the following biochemical and histological studies. 2.4. Biochemical analysis 2.4.1. Cornea The corneas from 13 rats within different treatment group were removed from the eye globe by section through the corneo-scleral junction, placed in ice cold distilled water and hand homogenized. The homogenate was centrifuged at 6000 rpm for 30 min at 4 ◦ C. Aliquots from clear supernatant

were utilized for the determination of reduced glutathione (GSH) as described by Tietze (1969), malondialdehyde (MDA) as described by Placer et al. (1966), superoxide dismutase (SOD) as described by Sun et al. (1989) and Na+ -, K+ -ATPase according to Bonting et al. (1961). 2.4.2. Lens The lenses from 10 animals within different treatment group were removed and their membranes were peeled off under microscope, weighted and utilized for the determination of Na+ -, K+ -ATPase, GSH, SOD and MDA as mentioned above. Pools of three pairs of rat lenses were homogenized in distilled water (0.2 g lens/2.5 ml d.w.), and centrifuged under cooling at 8000 rpm for 20 min. The supernatant (soluble lens proteins) was subjected to column chromatographic analysis according to Testa et al. (1965). The column used was 2.6 cm × 100 cm, packed with Sephadex G200 and the mobile phase was Trisbuffer pH 7.5. Fractions of 8 ml/20 min were collected using a fraction collector (universal fraction collector Eldex from USA). The optical density of collected fractions was measured using spectrophotometer (Avian 930 of Kout Ron) at 280 nm. The column was calibrated using standard proteins of molecular weight ranging between 29 and 669 kDa. The soluble lens protein of the lens homogenate was accessed according to Lowry et al. (1951). 2.5. Histological preparation The eyes of the reminder five rats from each group were opened at the corneo-scleral junction, the cornea and the lens were removed and fixed in 4% gluteraldehyde for 0.5 h. The corneas were dissected into small pieces while the lenses were cut into two halves and leaved in 4% gluteraldehyde for 6 h then all the specimens washed in 0.1 molar phosphate buffers overnight at 2 ◦ C. The lenses specimens were soaked in mollifex solution for 10 days to decalcification then dissected into small pieces. All the corneal and lenses specimen were placed in 1.3% osmium tetroxide, dehydrated in ascending grades of alcohol and embedded in araldite CY 212. Semi-thin sections were cut by ultratome, stained with toluidine blue then examined by lympus microscope (Bancroft and Stevens, 1996). 2.6. Statistical analysis All data were statistically analyzed using the general linear models procedure of the statistical analysis system (SAS Institute Inc., 1982). The significance of the differences among treatment groups was determined by Waller–Duncan k-ratio (Waller and Duncan, 1969). All statements of significance were based on probability of P ≤ 0.05.

3. Results 3.1. Ophthalmologic examination The ophthalmologic examination of cornea in the group received the high dose of Cr-picolinate revealed

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Table 1 Effect of Cr-picolinate alone or in combination with Vitamin C on GSH, SOD, MDA and Na+ -, K+ -ATPase in cornea of rats (means ± S.E.) Groups

Parameters GSH (␮g/g wet wt.)

Control Cr-picolinate (low dose) Cr-picolinate (high dose) Cr-picolinate (low dose) + Vitamin C Cr-picolinate (high dose) + Vitamin C

0.09 a 0.087 a 0.06 b 0.079 a 0.083 a

± ± ± ± ±

0.006 0.001 0.005 0.03 0.00

MDA (nmol/g wet wt.) 0.059 a 0.068 a 0.142 b 0.056 a 0.061 a

± ± ± ± ±

0.003 0.02 0.005 0.002 0.002

SOD (U/g protein) 0.55 a 0.45 a 0.28 b 0.49 a 0.52 a

± ± ± ± ±

0.13 0.01 0.1 0.09 0.13

Na+ -, K+ -ATPase (␮mol pi/h/g protein) 0.244 a 0.22 a 0.156 b 0.20 a 0.21 a

± ± ± ± ±

0.08 0.07 0.02 0.07 0.08

Whithin each column, means with different letters are significantly different (P < 0.05).

that severe changes were occurred in the cornea including superficial localized opacity which appeared at the 4th week, superficial diffuse opacity appeared at the 6th week and all layer opacification (diffused or localized) appeared at the 8th week. Whereas, the ophthalmologic examination of the lens revealed central faint cataract (mild opacification) at the 4th week, dense central cataract (severe opacification) at the 6th week and total lens opacity (white cataract) at the 8th week. On the other hand, the animals treated with the low dose of Cr-picolinate were comparable to the controls.

The chromatographic elution pattern of the soluble lens protein fraction of the control rat lens homogenate showed four distinct bands namely α, β1 , β2 and ␥crystallines (Fig. 1a). Whereas, the chromatographic elution pattern of the soluble lens protein of animals treated with Cr-picolinate (1.5 mg/100 g b.w.) showed that β1 and β2 crystalline are diffused together and the ␥-crystalline fraction shifted towards higher molecular weight (Table 3 and Fig. 1b), The total proteins of the soluble fraction of the lens homogenate of rats in this group dropped by 35% with a decrease in the protein contents of the β and ␥ fractions of soluble lens crystalline (Table 4). The daily supplement of Vitamin C to the high dose of Cr-picolinatetreated animals restored the chromatogram of the lens crystalline to more or less of normal position (Fig. 1c).

3.2. Biochemical results The results of biochemical parameters of the corneal homogenate (Table 1) or lens (Table 2) of all animals within different groups revealed that animals treated with Cr-picolinate alone at the low dose level (0.8 mg/100 g b.w.) or those treated with the two tested doses plus Vitamin C were comparable to the controls regarding all the biochemical parameters tested. Whereas, animals treated with Cr-picolinate alone at the high dose level (1.5 mg/100 g b.w.) showed a significant decrease in GSH, SOD and Na+ -, K+ -ATPase levels accompanied with a significant increase in MDA level in corneal homogenate or lens.

3.3. Histological results The cornea of the control group showed normal structure of the five layers and revealed that the epithelium formed of five layers: two layers of superficial flattened epithelial cells, two to three layers of wing polygonal cells and single layer of basal cuboidal cells. The Bowman’s layer modified anterior layer of

Table 2 Effect of Cr-picolinate alone or in combination with Vitamin C on GSH, SOD, MDA and Na+ -, K+ -ATPase in lens of rats (means ± S.E.) Groups

Parameters GSH (␮g/g wet wt.)

Control Cr-picolinate (low dose) Cr-picolinate (high dose) Cr-picolinate (low dose) + Vitamin C Cr-picolinate (high dose) + Vitamin C

4.2 a 3.9 a 1.9 b 4.8 a 4.1 a

± ± ± ± ±

0.3 0.3 0.12 0.001 0.001

MDA (nmol/g wet wt.) 0.27 a 0.31 a 0.51 b 0.30 a 0.32 a

± ± ± ± ±

0.01 0.08 0.02 0.1 0.09

Within each column, means with different letters are significantly different (P < 0.05).

SOD (U/g protein) 85.5 a 79.7 a 53.3 a 82.0 a 81.5 a

± ± ± ± ±

0.9 3.2 1.2 3.1 4.1

Na+ -, K+ -ATPase (␮mol pi/h/g protein) 0.41 a ± 0.37 a ± 0.28 b ± 0.40 a ± 0.36 a ±

0.05 0.1 0.04 0.03 0.08

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Fig. 1. Distribution profile of crystallines in soluble fraction of lens: (a) control group, (b) animal supplemented with 1.5 mg Cr-picolinate alone and (c) animals supplemented with 1.5 mg Cr-picolinate plus 0.5 mg Vitamin C. (Soluble protein was loaded to gel filtration column of Sephadex G200 , Tris-buffer pH 7.5 and proteins peaks were predicted at 280 nm, V0 is the void volume.)

Table 3 Molecular weight (kDa) range of soluble lens proteins in animals treated with the high dose of Cr-picolinate with or without Vitamin C Groups

Control 1.5 mg Cr-picolinate 1.5 mg Cr-picolinate + Vitamin C

Crystallines α

β1

691 691 691

371

β2

γ

154

69 93 69

186 371

154

stroma. The stroma formed of collagenous lamellae running parallel to each other with keratocytes in between. The descemets membrane showed thick basement membrane of the endothelial cells and the endothelium single layer of cells with flattened nucleus (Fig. 2A). The microscopic examination of the cornea in the animals received the low dose level of Cr-picolinate revealed insignificant histological changes. Examination of the cornea in the animals received the high dose of Cr-picolinate showed edema in all layer. Disorganization of the epithelial cells and increased number of superficial layers were the most characteristic feature. The cytoplasm of most epithelial cells contained numerous vacuoles and the nuclei varied in shape. Irregularity of basement membrane was generally noted. Stromal collagen lamellae were irregular and homogenous with vascularization of its anterior portion. In addition, abnormal keratocytes were noticed, where various pathological signs were seen. Some of them became swollen with loss of their long process while others appeared pyknotic. The endothelium revealed vacuolation of the cytoplasm (Fig. 2B). Corneal layers of the animals received the low dose of Crpicolinate plus Vitamin C appeared normal. Whereas, the corneas in the animals received the high dose of Crpicolinate plus Vitamin C appeared more or less normal (Fig. 2C). The microscopic examination of the lens in the control group revealed that it was formed of three layers (Fig. 3a); Lens capsule (homogenous layer); the subcapsular epithelium (layer of cuboidal cells with rounded nuclei) and the cortex (formed of lens fiber parallel to each other). Lenses in the group treated with the low dose level of Cr-picolinate showed nearly normal histological picture. Whereas, animals in the group received the high dose of Cr-picolinate revealed that the lenses appeared highly edematous, lens capsule was thickened and the epithelium appeared swollen, vacuolated with irregularity of the basement membrane. Epithelial nuclei variable in shape with chromatin fragmentation, besides, few nuclei showed signs of pyknosis. In addition, the anterior cortex collagen lamellae were slightly vacuolated and separated (Fig. 3b), while the posterior cortex appeared highly vacuolated (Fig. 3c). The histological examination of the lenses of animals received the high dose of Cr-picolinate plus Vitamin C showed normal appearance. Moreover, the microscopic examination of the lenses in the animals treated with the high dose of Cr-picolinate plus Vitamin C revealed improvement in the histology of the lens to great extent (Fig. 3d).

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Table 4 Total proteins and lens crystallines of animals treated with high dose of Cr-picolinate alone or in combination of Vitamin C Groups

Control Cr-picolinate Cr-picolinate + Vitamin C

Total protein (mg/g wet wt.)

295 189 280

4. Discussion Cr-picolinate is a unique micronutrient essential for normal protein, fat and carbohydrate metabolism. It has been marketed to consumers for weight loss, increasing muscle mass, lowering serum cholesterol and maintains homeostatic control of blood glucose in the body (Whitteker et al., 2005). However, it may cause renal impairment and cell toxicity when ingested in excess (Speetjens et al., 1999). This study was performed to investigate the long term effects of Cr-picolinate intake on the ocular tissues (cornea and lens) and the possible protective effect of Vitamin C supplementation. The selective doses of Cr-picolinate and Vitamin C were literature based (Speetjens et al., 1999; Dey et al., 2001, respectively). The present data indicated that low dose of Crpicolinate (0.8 mg/100 g b.w.) has no biochemical or

Crystallines α

β1

β2

γ

56.7 83.2 70.56

37.2 54.1 42.56

79.65

120.95 49.14 98

68.04

histological effects on the cornea or the lens, whereas, the high dose (1.5 mg /100 g b.w.) caused severe biochemical changes and morphologic characteristics of the cornea and lens, The cornea and lens exhibited a decrease in Na/K ATPase activity accompanied with a decrease in SOD and GSH level and an increase in lipid peroxidation as indicated by increasing MDA level. These biochemical changes were concomitant to the histological alteration in both cornea and lens. Although the mechanistic cytotoxicity of Cr-picolinate is not completely understood, the previous studies supported the conclusion that Cr(III) is relatively nontoxic. Most Cr(III) compound have failed to induce genetic defects in bacteria, yeast or mammalian cell lines (De Flora et al., 1990). Stearns et al. (1995a) stated that organic forms of Cr(III) such as Cr-picolinate and nicotinate have much higher absorption compared to chromium(III) chloride and phosphate salts. However, increased absorption of

Fig. 2. (A) Light micrograph of the control rat cornea showing the normal five layers: (1) epitheliu, (2) Bowman’s layer, (3) stroma, (4) descemet’s membrane and (5) endothelium (toluidine blue 500×), (B) light micrograph of the cornea from the high dose of Cr-picolinate-treated group showing that the pigment epithelium were vacuolated (arrow head) with irregularity of basement membrane (arrow), vascularization of the anterior cortex (v) and swollen of keratocytes (k). Note: focal loss of endothelial nuclei (e) (toluidine blue 1250×), (C) light micrograph of the cornea from the animals treated with the high dose of Cr-picolinate plus Vitamin C showing nearly normal appearance of corneal layers. k, keratocytes; e, endothelium (toluidine blue 1250×).

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Fig. 3. (a) Light micrograph of the control rat lens showing the normal three layers: (1) lens capsule, (2) lens epithelium, (3) lens cortex, (b) light micrograph of the lens specimen in the high dose of Cr-picolinate-treated group showing thickened capsule (c). The epithelium appeared swollen and vacuolated (ep) and its basement membrane showing irregularity. The cortex fibers were vacuolated and separated (f) (toluidine blue 1250×), (c) light micrograph of the lens from the group treated with the high dose of Cr-picolinate plus Vitamin C showing the posterior cortex were highly vacuolated (arrows) (toluidine blue 1250×), (d) light micrograph of the specimen in the animals treated with the high dose of Cr-picolinate plus Vitamin C revealing more or less normal lens (toluidine blue 1250×).

organic-complexed, chromium caused toxicity due to the concentration of this metal in the tissues (Stearns et al., 1995b). Because of its neutral charge and hydrophobic character allow it to readily pass through hydrophobic barriers. Ironically, once the Cr-picolinate is absorbed by cells and because of its ligand composition and the resulting redox potential, the complex can be reduced readily by abundant biological reductants and generate hydroxyl radical via Haber–Weiss and Fenton cycles (Sugden and Rogers, 1992). Moreover, it was previously reported that this reactive oxygen species, may injury the corneal tissues by degrading corneal stromal macromolecules (Cejkova et al., 2004). This may explain the corneal stromal changes reported herein in the high Crpicolinate-treated group as a result of hydroxyl radical and superoxide anions generation. In accordance to our results, multiple studies reported that Cr-picolinate generate hydroxyl radicals and super-

oxide anions radicals leading to lipid peroxidation, mitochondrial and DNA damage (Kareus et al., 2001; Hepburn and Vincent, 2002) thus the Cr-picolinate considered the most toxic form. In addition, Bagchi et al. (2002) conducted a comparative study of Cr-picolinate and niacin-bound chromium(III) (two popular dietary supplement), and reported that Cr-picolinate produced severe oxidative stress and DNA damage and their studies have implicated the toxicity of Cr-picolinate in renal impairment, anemia, tissue edema, neural cell injury enhanced production of hydroxyl radical and depletion of antioxidant enzyme. In an in vitro study, Hepburn et al. (2003) stated that low level of Cr-picolinate can catalytically generates reactive oxygen species in the presence of biological reducing agents. Furthermore, other studies observed that the oxidative damage resulted from the Cr-picolinate supplement in vivo in rats were injected daily for 60

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days, induced a significant increase in urinary-8 hydroxydeoxyguanosine (8-OhdG), a product of oxidative DNA damage in urine and increased the level of lipid peroxidation in tissue. In addition, the oxidative damage may be in turn lead to DNA and chromosome damage (Rindgen et al., 2000; Acharya et al., 2006). In the current study the free radical formation and decreased GSH and the activity of SOD enzyme and increase MDA lead to malfunction of Na/K ATPase pump and epithelial cells degeneration could add to more severe disruption of Na/K ATPase pump causing efflux of Na and water, these events lead to vacuolation and indicative edema reported in the present study. As mentioned previously, the keratocytes appeared as swollen dense mass and some of them contained vacuoles, these results were in accordance with Shrivastava et al. (2005) who studied the effect Cr(III) complex on human dermal fibroblasts and demonstrated surface morphological damage evidenced by cellular blebbing and spike formation accompanied by nuclear damage which reflecting the cell death. These authors suggested that chromium(V) formed as a result of oxidation of chromium(III) by cellular oxidative enzymes in the cytotoxic response. Consequently, chromium(III) complex which is absorbed by the cells and may be oxidized to chromium(V) and must be considered as a potential carcinogen. In the current study, we observed vacuolar degeneration in the corneal endothelium. Free radicals are known to cause cellular damage by damaging plasma membrane (Topaz, 1998) and the presence of these vacuoles indicated the rupture of intracellular organelles membrane (Rubowitz et al., 2003). Margo and Grossin (1991) stated that corneal dehydration is maintained by endothelium and any disorders of endothelium contribute edema resulted in necrosis of keratocytes. Thus, edema and pyknotic signs in keratocytes observed in the present study may be owing to endothelium changes. Concerning to the lens, the decrease level of lens GSH induced lipid peroxidation as indicated by increased level of MDA in the lens homogenate. The scavenger effect of GSH can be diminished directly by consumption or indirectly by a low generation rate due to a progressive free radical generated by Cr-picolinate. In this concern, Reddy et al. (1988) concluded that deficiency of GSH causes marked increase in lens membrane permeability and such lenses are susceptible to oxidative damage resulting in inactivation of Na/K pump leading to impairment of sodium transport and increase Na/water retention which lead to swelling of the lens and increase the oxidation of sulfhydril content of the protein which lead to aggregation of the lens proteins and the occur-

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rence of cataract. This could contribute to the explanation of lens protein aggregation, a drop in the content of soluble part of lens protein and shift the ␥-crystalline fraction to higher molecular weight as reported in the current study in animals treated with the high dose of Cr-picolinate. In our study, we hypothesized that the vacuolization of the lens epithelium resulted from disruption of Na/K ATPase pump as a result of oxidative stress. Such disruption led to entry of water and cause vacuoles as seen in the posterior cortex and lamellar separation of the anterior cortical fibers. On the other hand, few studies reported that, the picolinic acid may contribute to the toxicity resulting from Cr-picolinate administration. Hepburn and Vincent (2002) suggested that, picolinic acid is rapidly cleared from the body, whereas Cr-picolinate passes intact from the blood stream to the kidneys then urine and also passes into cells where it is degraded. Additional support could be found in the study of Manygoats et al. (2002) who studied the effect of Crpicolinate, picolinic acid and chromic chloride in the Chinese hamster ovary and observed swollen of the mitochondria with degrades cristae. Apoptosis was identified by nuclear convolution, fragmentation and cytoplasmic blebbing. They also reported that picolinic acid was much more toxic and concluded that the coordination of Cr(III) by Cr-picolinate ligands may alter the cellular chemistry of Cr(III) to make Cr-picolinate, a toxic from of Cr(III). This may be another explanation to the biochemical and histological changes observed in the current study. So, it can be suggested that the pathogenesis of Cr-picolinate induced corneal and lens injury is multifactorial. Although, some studies have found toxic effects of Cr-picolinate, there are other reports about its safety (Anderson et al., 1997; Lamson and Plaza, 2002; Shinde and Gotal, 2003). These authors stated that chronic treatment with Cr-picolinate at the therapeutic doses that improved glucose tolerance was observed to have no hepatotoxic or nephrotoxic potential. Animals treated with the high dose of Cr-picolinate plus Vitamin C prevented the changes in the cornea and lens. This considered a further indication to the oxidative stress generated in the eye as a result of Cr-picolinate. Kasetsuwan et al. (1999) reported that the eye protect itself from free radical injury by two major protective mechanisms: (1) the endogenous antioxidant enzyme systems such as SOD, catalase and glutathione peroxidase (GPX) and (2) the free radical scavengers such as ascarbate and Vitamin E. Brennan et al. (2000) stated that, Vitamin C is known to be a first-line defense antioxidant mopping up hydroperoxide, the hydroxyl radical

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and singlet oxygen. They added that Vitamin C conferred significant intracellular protection against H2 O2 induced DNA damage. However, administration of Vitamin C plus Crpicolinate led to an improvement in the biochemical parameters and histological picture of the cornea and lens. Previous studies indicated that antioxidants (free radicals scavengers) may ameliorate this risk. Naziroglu et al. (1999) concluded that, Vitamins C and E and selenium can protect the lens against oxidative damage, but the effect of Vitamin C appears to be much greater than that of Vitamin E and selenium, they also stated that MDA level was significantly lower by the administration of Vitamin C. In addition, Kasetsuwan et al. (1999) found that topical ascorbic acid significantly decreases oxygen free radical-induced tissue damage in the cornea. Furthermore, it has been reported that the ability of Vitamins C and E in compensation for GSH depletion to protect against H2 O2 -induced cell death suggests that GSH, Vitamins C and E have common targets in their actions against oxidative damage and supports the preventive or therapeutic use of Vitamins C and E to cobat age and pathology—associated declines in GSH (Shang et al., 2003). In the same concern, Rubowitz et al. (2003) concluded that, ascorbic acid can reduce the amount of endothelial cell loss (after phacoemulsification surgery) and this effect may be due to its free radical scavenging properties, they have also demonstrated that ascorbic acid sharply reduces the amount of intracellular vacuoles in the endothelial cells. Furthermore, Hegde and Varma (2004) found that reactive oxygen species decreased the membrane transport activity as well as the level of adenosine triphosphate (ATP) and glutathione reductase enzymes and using of ascorbate to minimize these toxic effects substantially. More recently, Serbecic and Beutelspacher (2005) studied the effects of Vitamins A, C, and E supplementation on lipid peroxidation and apoptosis in corneal endothelial cells, they showed that supplementation with these antioxidative vitamins significantly prevents the generation of free radical injury, lipid peroxidation, and consequent apoptosis. In conclusion, from the current study, we can conclude that: (1) Cr-picolinate supplementation may cause visual impairment when ingested in excess doses, while low doses can be taken safely, (2) supplementation of Vitamin C afforded protection against damage caused by Cr-picolinate intake and further work is needed to clarify these impressions, and (3) it is advisable to restrict Crpicolinate intake to those persons with proven chromium deficiency biochemically.

Acknowledgement We thank Prof. Kareem Rezk (Eye Clinic, Research Inistitute of Ophthalmology, Giza, Egypt) for his help and encouragement in conducting these experiment. References Acharya, U.R., Mishra, M., Tripathy, R.R., Mishra, I., 2006. Testicular dysfunction and antioxidative defense system of Swiss mice after chromic acid exposure. Reprod. Toxicol. 22 (1), 87–91. Anderson, R.A., 1995. Chromium, glucose tolerance, diabetes and lipid metabolism. J. Adv. Med. 8, 37–49. Anderson, R.A., 1998. Recent advances in the clinical and biochemical manifestation of chromium deficiency in human and animal nutrition. J. Trace Elem. Exp. Med. 11, 241–250. Anderson, R.A., Bryyden, N.A., Polansky, M.M., 1997. Lack of toxicity of chromium chloride and chromium picolinate in rats. J. Am. Coll. Nutr. 16 (3), 273–279. Bagchi, D., Stohs, S.J., Downs, B.W., Bagchi, M., Preuss, H.G., 2002. Cytotoxicity and oxidative mechanisms of different forms of chromium. Toxicology 180 (1), 5–22. Bancroft, J.D., Stevens, A., 1996. Theory and Practice of Histological Techniques, 4th ed. Churchill Livingstone, New York, Edinburgh, London. Bonting, S.L., Simon, K.A., Hawkins, N.M., 1961. Studies on sodiumpotassium activated adenosine triphosphatase: quantitative distribution in several tissues of the cat. Arch. Biochem. Biophys. 95, 416–423. Brennan, L.A., Morris, G.M., Wasson, G.R., Hannigan, B.M., Barnett, Y.A., 2000. The effect of Vitamin C or E supplementation on basal and H2 O2 induced DNA damage in human lymphocytes. Brit. J. Nutr. 84, 159–202. Cejkova, J., Stipek, S., Crkovska, J., Ardan, T., Platenik, J., Cejka, C., Midelfart, A., 2004. UV rays, the prooxidant/antioxidant imbalance in the cornea and oxidative eye damage. Physiol. Res. 53, 1–10. Cerulli, J., Grabe, D.W., Gauthier, I., Malone, M., McGoldrick, M.D., 1998. Chromium picolinate toxicity. Ann. Pharm. 32 (4), 428– 431. De Flora, S., Bagnasco, M., Serra, D., Zanacchi, P., 1990. Genotoxicity of chromium compounds. A review. Mut. Res. 238, 99–172. Dey, S.K., Nayak, P., Roy, S., 2001. Chromium-induced membrane damage: protective role of ascorbic acid. J. Environ. Sci. 13 (3), 272–275. Hegde, K.R., Varma, S.D., 2004. Protective effective effects of ascorbate against oxidative stress in the mouse lens. Biochim. Biophys. Acta 1670 (1), 12–18. Hepburn, D.D., Vincent, J.B., 2002. In vivo distribution of chromium from chromium picolinate in rats and implication for the safety of the dietary supplement. Chem. Res. Toxicol. 15, 93–100. Hepburn, D.D., Xiao, J., Bindom, S., Vincent, J.B., O’Donnell, J., 2003. Nutritional supplement chromium picolinate causes sterility and lethal mutations in Drosophila melanogaster. PNAS 100 (7), 3766–3771. Kareus, S.A., Kelley, C., Walton, H.S., Sinclair, P.R., 2001. Release of chromium III from chromium III picolinate upon metabolic activation. J. Hazard Mater. 84 (2/3), 163–174. Kasetsuwan, N., Francis, M.W., Hsiet, F., Sanchez, D., Peter, J., Mc Donnell, P.J., 1999. The effect of topical ascorbic acid on free

A.A. Mahmoud et al. / Toxicology 226 (2006) 143–151 radical tissue in the cornea after excimer laser corneal surgery. Arch. Ophthalmol. 117, 649. Lamson, D.W., Plaza, S.M., 2002. The safety and efficacy of high-dose chromium. Altern. Med. Rev. 7 (3), 218–235. Lowry, O.H., Rosbough, N.J., Farr, A.L., Randbell, R.T., 1951. Protein measurement with the folin phenal reagent. J. Biol. Chem. 193, 265–275. Manygoats, K.R., Yazzie, M., Stearns, D.M., 2002. Ultrastructural damage in chromium picolinate—treated cells: a TEM study. J. Biol. Inorg. Chem. 7 (7/8), 791–798. Margo, C.E., Grossin, H.E., 1991. Epithelial and stromal edema of the cornea. In: Ocular Histopathology. W.B. Saunders, Philadelphia, London, Toronto, p. 238. Mertz, W., 1993. Chromium in human nutrition: a review. J. Nutr. 123, 626–633. Naziroglu, M., Dilsiz, N., Cay, M., 1999. Protective role of intraperitoneally administrated Vitamins C and E and Selenium on the levels of lipid peroxidation in the lens of rats made diabetic with streptozotocin. Biol. Trace Elem. Res. 70 (3), 223– 232. Placer, Z.A., Cushman, L.L., Johnson, B.C., 1966. Estimation of product of lipid peroxidation (malondialdehyde) in biochemical systems. Anal. Biochem. 16 (2), 359–364. Reddy, V.N., Garadi, R., Chakrapar, U., 1988. Effect of glutathione depletion on cation transport and metabolism in rabbit lens. Ophthalmic Res. 20 (3), 191–199. Rindgen, D., Lee, S.H., Nakajima, M., Blair, I.A., 2000. Formation of a substituted 1, N(6)-etheno-2 -deoxyadenosine adduct by lipid hydroperoxide-mediated generation of 4-oxo-2-nonenal. Chem. Res. Toxicol. 13, 846–852. Rubowitz, A., Assia, E.I., Rosner, M., Topaz, M., 2003. Antioxidant protection against corneal damage by free radicals during phacoemulsification. Invest. Ophth. Vis. Sci. 44 (5), 1866– 1870. SAS Institute, 1982. SAS User’s Guide: Statistics. SAS Institute Inc., Cary, NC. Serbecic, N., Beutelspacher, S.C., 2005. Vitamins inhibit oxidantinduced apoptosis of corneal endothelial cells. Jpn. J. Ophthalmol. 49 (5), 355–362. Shang, f., Lu, M., Dudek, E., Reddan, J., Taylor, A., 2003. Vitamin C and Vitamin E restore the resistance of GSH-depleted lens cells to H2 O2 . Free Rad. Biol. Med. 34 (5), 521–530.

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Shinde, U.A., Gotal, R.K., 2003. Effect of chromium picolinate on histopathological alteration in STZ and neaonatal STZ diabetic rats. J. Cell. Mol. Med. l7 (3), 322–329. Shrivastava, H.Y., Ravikumar, T., Shanmugasundaram, N., Babu, M., Unni-Nair, B., 2005. Cytotoxicity studies of chromium(III) complexes on human dermal fibroblasts. Free Rad. Bio. Med. 38 (1), 58–69. Speetjens, J.K., Collins, R.A., Vincent, J.B., Woski, S.A., 1999. The nutritional supplemented chromium(III) tris(picolinate) cleaves DNA. Chem. Res. Toxicol. 12, 483–487. Stearns, D.M., Wise, J.P., Patierno, S.R., Weterhahn, K.E., 1995a. Chromium III picolinate produces chromosome damage in Chinese hamster ovary cells. Res. commun. 9, 1643–1648. Stearns, D.M., Belbruno, J.J., Wetterhahn, K.E., 1995b. A prediction of chromium(III) accumulation in humans from chromium dietary supplements. FASEB J. 9, 1650–1657. Sugden, K.D., Rogers, S.J., 1992. Oxygen radical-mediated DNA damage by redox-active Cr(III) complexes. Biochemistry 31, 11626–11631. Sun, L., Peterson, T.E., Mc Cormick, M.L., Oberley, L.W., Osbome, J.W., 1989. Improved superoxide dismutase assay for clinical use. Clin. Chem. 35 (6), 1265–1266. Testa, M., Armond, G., Balaz, E., 1965. Separation of the soluble proteins of bovine lenses by column chromatography. Exp. Eye Res. 4, 327–339. Tietze, F., 1969. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: application to mammalian blood and other tissues. Anal. Biochem. 27, 502– 522. Topaz, M., 1998. Possible long term complication in ultrasound— assisted lipoplasty induced by sonoluminescence, sonochemistry, and thermal effect. Long-term possible hazardous effects of ultrasonically assisted lipoplasty. Plast. Reconstr. Surg. 102, 280–281. Vincent, J.B., 2003. The potential value and toxicity of chromium picolinate as a nutritional supplement, weight loss agent and muscle development agent. Support Med. 33 (3), 213–230. Waller, R.A., Duncan, D.B., 1969. A Bayes rule for the symmetric multiple comparison problems. J. Am. Stat. Assoc. 64, 1484–1503. Whitteker, P., San, R.H., Clark, J.J., Sefrid, H.E., Dunkel, V.C., 2005. Mutagenicity of chromium picolinate and its components in Salmonella typhimurium and L5178Y mouse lymphoma cells. Food Chem. Toxicol. 43 (11), 1619–1628.