Total chromium in the human lens

A simple, quick procedure has been developed for preparation of human lenses for total chromium (Cr) analysis by electrothermal atomic absorption spectrometry (EAAS). This procedure involves wet ashing in a mini-autoclave with HNO₃ and H₂SO₄. Recovery was 101.6%. This procedure is simple to carry out, does not generate corrosive fumes, and minimizes contaminations. Measurements obtained by this method give values similar to those found previously by more cumbersome methods. It can be conveniently used to prepare biological samples for ultratrace analysis. The mean Cr concentration in human lenses varied between 0.345±0.147 μg/g dry wt in the normal population and 0.205±0.160 μg/g dry wt in cataractous lenses.     

Supplementation with chromium picolinate recovers renal Cr concentration and improves carbohydrate metabolism and renal function in type 2 diabetic mice

To study the preventive effect of supplemented chromium picolinate (CrPic) on the development of diabetic nephropathy in mice, we analyzed the effects of CrPic supplementation on renal function and concentrations of serum glucose and tissue chromium (Cr). In experiment 1, male KK-Ay obese diabetic mice were fed either a control diet (control) or a diet supplemented with 2 mg/kg diet (Cr2) or 10 mg/kg diet (Cr10) of Cr for 12 wk. Cr10 significantly ameliorated hyperglycemia after a glucose load, creatinine clearance rates, and urinary microalbumin levels (p<0.05). In experiment 2, the Cr10 diet was fed to male KK-Ay obese diabetic mice and C57BL nondiabetic mice for 4 wk. The CrPic diet reduced urinary albumin excretion in the diabetic mice (p<0.05). Inductively coupled plasma-mass spectrometry analysis revealed that the renal Cr content and the recovery of renal Cr concentration after Cr supplementation were significantly lower in the diabetic mice than in the nondiabetic mice (p<0.01). These observations suggest that Cr supplementation of type 2 diabetic mice reduces the symptoms of hyperglycemia and improves the renal function by recovering renal Cr concentration.     

Determination of 19 elements in human eye lenses

Neutron activation analysis was used to determine the concentrations of 19 elements in normal and senile human cataractous lenses. It was found that the concentrations of Ca, Na, Cl, Eu, Sb, and Fe were significantly higher, and those of K, Rb, Cs, Cr, Mn, Co, Sc, and Ce were significantly lower in senile mature cataractous lenses than those in normal human eye lenses. No changes were found for the concentrations of Se, Zn, Mg, S, and Th in the two groups. Positive correlations between Na, Cl, and Ca and K, Rb, and Cs were found, whereas a significantly negative correlation between na, Ca, Cl and K, Rb, Cs were found. The roles of these elements in the evolution of cataract are discussed.     

The Potential of Cr3 [Triaqua‐μ3‐Oxo‐Hexa‐μ‐Propionatotrichromium(III) Chloride] to Reduce Birth Defects in the Offspring of Diabetic CD‐1 Mice

Diabetes mellitus is a growing concern worldwide and leads to multiple complications during pregnancy. Pharmacologic doses of chromium (Cr) have been linked with improving insulin sensitivity and other positive benefits in the treatment of diabetes in animal models. By using streptozotocin induced hyperglycemia in female CD‐1 mice, reproductive outcomes of diabetic and chromium‐dosed diabetic females were examined. After dosing 10 mg/kg Cr in the form of triaqua‐μ₃‐oxo‐hexa‐μ‐propionatotrichromium(III) chloride or Cr3 during gestation days 8–16 (GD8–GD16), all females were sacrificed on gestation day 17 (GD17) and examined for maternal weight gain. The fetuses were examined for gross malformations and for skeletal malformations. The offspring of Cr3‐dosed females tended to have a reduction in the incidence of supernumerary ribs. While hyperglycemia still had negative impacts on the health of dams and their offspring, administration of Cr led to an apparent trend in the reduction in the number of malformations and incidence of supernumerary ribs compared to those of untreated diabetic mothers     

α-Crystallin chaperone function in diabetic rat and human lenses

This study focussed on the effect of diabetes on the chaperone function of alpha-crystallin. The authors relied on diabetic rats with a wide range of plasma glucose levels and non-diabetic control rats to establish a possible relationship between severity of diabetes and alpha-crystallin chaperone activity. In addition, 52-56 and 63-69 year-old diabetic and non-diabetic human lenses were used to show whether diabetes affects alpha-crystallin chaperone activity in human lenses. Correlation between plasma glucose levels and loss of chaperone activity of the alphaL-crystallin fraction in diabetic rats indicated good correlation. The glycemic threshold, reported before for cataract development in diabetic rats, seems to be valid for the chaperone activity loss as well. Analysis of the human lens alphaL-crystallin showed lower chaperone activity in all the diabetic lenses than in the age-matched control lenses. In the 63-69 age group, the loss in chaperone activity due to diabetes was significantly larger than in the 52-56 age group suggesting a dominant effect of duration of diabetes.     

Determination of calcium, sodium, potassium and magnesium concentrations in human senile cataractous lenses

Cataractous lenses have been found to have a distribution of the intracellular ionic environment, the concentrations of potassium and magnesium decreasing and the concentrations of sodium and calcium increasing relative to the cytosol of most cells. This arises as a result of changes to lens membrane characteristics causing an increase in lens membrane permeability. These changes have been found to be initiated as a result of normal ageing of the human lens. In this study, total Ca2+, K+, Na+ and Mg2+ contents have been determined in human normal and cataractous lenses using atomic absorption and flame emission spectroscopy. The normal human lens Ca2+ is between 0.15 and 0.5 miromol g(-1) fresh lens weight; in senile cataracts the value increased up to 9.31 micromol g(-1) ( p < 0.0001). The normal levels of Na+, Mg2+ and K+ are 20, 5.5 and 60 micromol g(-1) respectively; these changed to 136.10, 3.60 and 9.33 micro mol g(-1), respectively in cataractous senile human lenses ( p < 0.002, p < 0.002 and p < 0.01). The remarkable differences in these elements may play some role in cataractogenesis.     

delta-Aminolevulinic acid dehydratase activity in erythrocytes of diabetic patients

Erythrocytes delta-aminolevulinate dehydratase activity was assayed in 41 diabetic patients and 33 normal controls. It was found that in diabetic patients the erythrocyte ALA-D activity was lower than in controls, and the difference of the mean values was statistically highly significant (P less than 0.001). We found a significant negative correlation (r = - 0.846, P less than 0.001) between ALA-D activity and blood glucose levels. For this reason, using normal adult human whole blood haemolysates, it was investigated the effects in vitro of glucose and insulin on normal erythrocytic ALA-D. No significant difference in ALA-D activity was found in the presence of insulin. On the other hand, there was considerable decrease in the enzyme activity in the blood samples after glucose addition.     

Effect of chromium niacinate and chromium picolinate supplementation on lipid peroxidation, TNF-alpha, IL-6, CRP, glycated hemoglobin, triglycerides, and cholesterol levels in blood of streptozotocin-treated diabetic rats

Chromium (Cr(3+)) supplementation facilitates normal protein, fat, and carbohydrate metabolism, and is widely used by the public in many countries. This study examined the effect of chromium niacinate (Cr-N) or chromium picolinate (Cr-P) supplementation on lipid peroxidation (LP), TNF-alpha, IL-6, C-reactive protein (CRP), glycosylated hemoglobin (HbA(1)), cholesterol, and triglycerides (TG) in diabetic rats. Diabetes (D) was induced in Sprague-Dawley rats by streptozotocin (STZ) (ip, 65 mg/kg BW). Control buffer, Cr-N, or Cr-P (400 microg Cr/kg BW) was administered by gavages daily for 7 weeks. Blood was collected by heart puncture using light anesthesia. Diabetes caused a significant increase in blood levels of TNF-alpha, IL-6, glucose, HbA(1), cholesterol, TG, and LP. Compared with D, Cr-N supplementation lowered the blood levels of TNF-alpha (P=0.04), IL-6 (P=0.02), CRP (P=0.02), LP (P=0.01), HbA(1) (P=0.02), TG (P=0.04), and cholesterol (P=0.04). Compared with D, Cr-P supplementation showed a decrease in TNF-alpha (P=0.02), IL-6 (P=0.02), and LP (P=0.01). Chromium niacinate lowers blood levels of proinflammatory cytokines (TNF-alpha, IL-6, CRP), oxidative stress, and lipids levels in diabetic rats, and appears to be a more effective form of Cr(3+) supplementation. This study suggests that Cr(3+) supplementation can lower the risk of vascular inflammation in diabetes.     

Comparison of the chromium distribution in organs and subcellular fractions of normal and diabetic rats by using enriched stable isotope Cr-50 tracer technique

The enriched stable isotope⁵⁰Cr(III) tracer technique combined with neutron activation analysis was used to examine the intracellular distribution of Cr(III) in the liver, pancreas, testes, and kidney homogenates of both normal and diabetic rats. Our new results showed that the nucleic fraction has the highest Cr concentration in the liver cell of both normal and diabetic rats. The diabetic rats retain more Cr in the mitochondrial and lysosomal fractions of liver homogenate than the normal. This is likely an indication of chromium participating in the glucose or lipid metabolism to compensate the low level of insulin in the body of diabetic rats. The concentrations of Cr in the subcellular fractions of pancreas, testes, and kidney in the normal rats are higher than those in the diabetic rats, which favor the hypothesis that Cr(III) plays its biological function via interaction with the insulin-sensitive tissues or enhancement of the sensitivity of the insulin receptor.     

CRE-decoy oligonucleotide-inhibition of gene expression and tumor growth

The authors prepared water-soluble (WSF), urea-soluble (USF), alkali-soluble (ASF), sonicated (SF), sonicated insoluble (SIF) and membrane (MF) fractions of lens proteins from human senile and diabetic cataractous lenses and age-matched clear lenses. Levels of advanced glycation end products (AGEs) including carboxymethyl lysine (CML), a glycoxidation product, were determined by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA). Distribution of AGEs in the various protein fractions was ascertained by SDS-PAGE and Western blotting. An overall increase in the levels of AGEs in diabetic cataractous lenses as compared to senile cataractous lenses and clear lenses has been observed. ASF and SF , both of which originated from the urea-insoluble fraction, showed the highest levels of AGEs. However, no clear-cut differences in CML levels were seen among clear lenses and senile and diabetic cataractous lenses. AGEs were found to be distributed mostly in the high molecular aggregates in all the fractions. These data suggest that AGEs contribute to protein aggregation and subsequent insolubilization.     

The role of intracellular zinc in chromium(VI)-induced oxidative stress, DNA damage and apoptosis

Several studies have demonstrated that zinc is required for the optimal functioning of the skin. Changes in intracellular zinc concentrations have been associated with both improved protection of skin cells against various noxious factors as well as with increased susceptibility to external stress. Still, little is known about the role of intracellular zinc in hexavalent chromium (Cr(VI))-induced skin injury. To address this question, the effects of zinc deficiency or supplementation on Cr(VI)-induced cytotoxicity, oxidative stress, DNA injury and cell death were investigated in human diploid dermal fibroblasts during 48 h. Zinc levels in fibroblasts were manipulated by pretreatment of cells with 100 microM ZnSO4 and 4 or 25 microM zinc chelator TPEN. Cr(VI) (50, 10 and 1 microM) was found to produce time- and dose-dependent cytotoxicity resulting in oxidative stress, suppression of antioxidant systems and activation of p53-dependent apoptosis which is reported for the first time in this model in relation to environmental Cr(VI). Increased intracellular zinc partially attenuated Cr(VI)-induced cytotoxicity, oxidative stress and apoptosis by enhancing cellular antioxidant systems while inhibiting Cr(VI)-dependent apoptosis by preventing the activation of caspase-3. Decreased intracellular zinc enhanced cytotoxic effects of all the tested Cr(VI) concentrations, leading to rapid loss of cell membrane integrity and nuclear dispersion--hallmarks of necrosis. These new findings suggest that Cr(VI) as a model environmental toxin may damage in deeper regions residing skin fibroblasts whose susceptibility to such toxin depends among others on their intracellular Zn levels. Further investigation of the impact of Zn status on skin cells as well as any other cell populations exposed to Cr(VI) or other heavy metals is warranted.     

Prolonged exposure to particulate Cr(VI) is cytotoxic and genotoxic to fin whale cells

BackgroundHexavalent chromium [Cr(VI)] is a human lung carcinogen and global marine pollutant. High Cr concentrations, resembling the ones observed in occupationally exposed workers, have been observed in fin whales (Balaenoptera physalus) in the Gulf of Maine. This outcome suggests Cr might be disrupting the health of fin whale populations. Indeed, Cr in acute (24 h) exposure does cause toxicity in fin whale cells. However, human cell culture data indicate prolonged exposures (120 h) induce a higher amount of toxicity compared to 24 h exposure due to an inhibition of homologous recombination repair. However, whether prolonged exposure causes similar outcomes in fin whale cells is unknown.ObjectiveDue to the importance of assessing prolonged exposure toxicity, this study focuses on characterizing acute and prolonged exposure of Cr(VI) in male and female fin whale cells.MethodsCytotoxicity was measured by the clonogenic assay, also known as colony forming assay, which measures the ability of cells to proliferate and form colonies after the treatment. DNA double strand breaks were analyzed by neutral comet assay. Clastogenicity was measured using the chromosome aberration assay. Intracellular Cr levels were measured with Graphite Furnace Atomic Absorption Spectrometry (GFAAS) with Syngistix Software.ResultsIn this study, we demonstrate that particulate Cr(VI) induces cytotoxicity and genotoxicity in a treatment-dependent manner after 24 h and 120 h exposures. Cytotoxicity levels were generally low with relative survival above 64 %. DNA double strand break data and chromosome aberration data were elevated after a 24 h exposure, but decreased after a 120 h exposure. While cytotoxicity was similar after 24 h and 120 h exposures, less DNA double strand breaks and chromosomal instability occurred with prolonged exposure.ConclusionParticulate Cr(VI) is cytotoxic and genotoxic to fin whale cells after acute and prolonged exposures. The reduction of genotoxicity we have observed after 120 h exposure may be partly explained by lower intracellular Cr levels after 120 h. However, the decrease in intracellular levels is not reflected by a similar decrease in chromosome aberrations suggesting other mechanisms may be at play. Male fin whale cells appear to be more susceptible to the genotoxic effects of particulate Cr(VI) while female cells are less susceptible possibly due to increased cell death of damaged cells, but more work is needed to clarify if this outcome reflects a sex difference or interindividual variability. Overall, the study shows particulate Cr(VI) does induce toxicity at both acute and prolonged exposures in fin whales cells indicating Cr(VI) exposure is a health risk for this species.     

A new method for the purification of human eosinophils and neutrophils, and a comparison of the ability of these cells to damage schistosomula of Schistosoma mansoni.

Centrifugation of human white blood cells over either Ficoll-Hypaque or slightly hypertonic Metrizamide discontinuous gradients reliably produces separate fractions that are enriched for either neutrophilic or eosinophilic granulocytes. This single step purification routinely yields 90 to 100% pure neutrophils and 85 to 100% pure eosinophils. Metrizamide gradients, in particular, reproducibly provide high yields of 90 to 100% pure eosinophils from normal subjects with 2 to 3% eosinophils in their peripheral blood. The method does not damage cells as judged by morphologic or functional criteria. The purified cell populations were tested for their ability to damage antibody-coated schistosomula either by the measurement of 51Cr release from labeled organisms, or by direct morphologic assessment. Neutrophils were superior in their ability to release 51Cr from labeled organisms, but eosinophils adhered to the organisms to a greater extent and induced microscopically detectable damage.     

Comparative cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human and sperm whale (Physeter macrocephalus) skin cells

Chromium (Cr) is a global marine pollutant, present in marine mammal tissues. Hexavalent chromium [Cr(VI)] is a known human carcinogen. In this study, we compare the cytotoxic and clastogenic effects of Cr(VI) in human (Homo sapiens) and sperm whale (Physeter macrocephalus) skin fibroblasts. Our data show that increasing concentrations of both particulate and soluble Cr(VI) induce increasing amounts of cytotoxicity and clastogenicity in human and sperm whale skin cells. Furthermore, the data show that sperm whale cells are resistant to these effects exhibiting less cytotoxicity and genotoxicity than the human cells. Differences in Cr uptake accounted for some but not all of the differences in particulate and soluble Cr(VI) genotoxicity, although it did explain the differences in particulate Cr(VI) cytotoxicity. Altogether, the data indicate that Cr(VI) is a genotoxic threat to whales, but also suggest that whales have evolved cellular mechanisms to protect them against the genotoxicity of environmental agents such as Cr(VI).     

Cadmium- and chromium-induced oxidative stress, DNA damage, and apoptotic cell death in cultured human chronic myelogenous leukemic K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells

Sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] are both cytotoxic and mutagenic. This study examined the toxic and apoptotic potentials of these two cations on three cell types in vitro, namely, human chronic myelogenous leukemic (CML) K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells. The cells were incubated with 0-100 microM concentrations of the two cations for 0, 24, or 48 hours at 37 degrees C. Both Cr(VI) and Cd(II) induced changes in intracellular oxidized states of cells, which were detected using laser scanning confocal microscopy. Cell cycle modulation and apoptosis of the K562 cells by Cr(VI) and Cd(II) were determined by flow cytometry. Significant decreases in the G2/M phase were observed in the Cr(VI) and Cd(II) treated CML cells compared with untreated cells. At 12.5 microM, Cr(VI) induced greater apoptosis in K562 cells as compared with Cd(II). In the K562 cells, 2.2- and 3.0-fold increases in DNA fragmentation were observed following incubation with 12.5 and 25 microM Cr(VI), respectively, and 1.2- and 1.7-fold increases in DNA fragmentation were observed with Cd(II). Furthermore, approximately 2.7- and 4.9-fold increases in cytochrome c reduction were observed following incubation with 12.5 and 25 microM Cr(VI), respectively, and 1.6- and 3.3-fold increases in cytochrome c reduction were observed with Cd(II), demonstrating enhanced production of superoxide anion. Approximately 3.1 to 6-fold increases in hydroxyl radical production were observed following incubation of the K562 cells with these cations at 12.5 and 25 microM concentrations. These results in K562 cells were compared with promyelocytic leukemic HL-60 cells and normal human peripheral blood mononuclear cells. More pronounced effects were observed on K562 and HL-60 cells, and much lesser effects were observed on normal human peripheral blood mononuclear cells. The results demonstrate that both cations are toxic, producing oxidative tissue damage and apoptosis. Furthermore, more drastic effects were observed on K562 and HL-60 cells as compared with normal human peripheral blood mononuclear cells.     

Effects of dietary chromium chloride supplementation on performance,some serum parameters,and immune response in broilers

This study was conducted to investigate the effect of increasing dietary levels of inorganic chromium (CrCl₃·6H₂O) on the performance, blood chemistry, and immune response of broilers. Eighty newly hatched Ross PM3 broiler chicks were evenly distributed to five groups of 16 chicks each. Two groups (control and only sheep red blood cell inoculated) were fed the basal diet containing 2.2 and 4.5 mg Cr/kg and the remaining groups were fed 20, 40, or 80 mg/kg Cr-supplemented diets for 44 d. Chicks in all groups, except in the control, at 3 and 5 wk of age, were injected intraperitonally with sheep red blood cell for determining the primary and secondary antibody responses, respectively. When the chicks were 4 wk of age, a delayed-type hypersensitivity test was performed. White blood cells were differentiated. Blood samples were collected for the determination of serum proteins, glucose, cholesterol, cortisol, minerals, and alkaline phosphatase activity and for antibody response. Chromium had no effect on weight gain, but 20 mg/kg supplemental Cr resulted in 18.57% reduction in feed consumption and improved feed efficiency by 16.77%. Chromium did not affect serum cholesterol and P levels but reduced serum glucose and increased serum protein, Cr, Ca, and Mg levels, and ALP activity. A slight reduction was observed with Cr supplementation in cortisol levels. Slight but not significant increases were observed with Cr in serum Zn and Cu. Chromium increased the ratio of bursa of Fabricius and liver to body weight. Heterophil and monocyte counts and heterophil/lymphocyte ratio were reduced and lymphocyte counts, total antibody, IgG, and IgM titers were increased by supplemental Cr. All levels of Cr increased the cell-mediated response to phytohemagglutinin. No alterations in tissues were observed by histopathological examinations.     

Modeling simultaneous hexavalent chromium reduction and phenol degradation by a defined coculture of bacteria

Based on the kinetics of Cr(VI) reduction by Escherichia coli ATCC 33456 and phenol degradation by Pseudomonas putida DMP-1, a mathematical model is developed to describe simultaneous Cr(VI) reduction and phenol degradation in the coculture of the two species. The developed model incorporates the toxicity effects of Cr(VI) and phenol on phenol degradation and Cr(VI) reduction in the coculture. The model illustrates the inhibitory effects of phenol on Cr(VI) reduction and Cr(VI) toxicity toward phenol degradation. The model also reveals the recoveries of the activities of the repressed bacterial cells with continuous Cr(VI) reduction and phenol degradation in the coculture. The model is capable of predicting simultaneous Cr(VI) reduction and phenol degradation within a broad range of Cr(VI) and phenol concentrations and under an appropriate composition of populations. However, the model simulates lower concentrations of phenol than experimental observations once Cr(VI) is reduced to a low level (<7 mg/L). The model simulation for Cr(VI) also deviates from experimental data when P. putida is outnumbered by E. coli by a ratio of 1:5. (c) 1995 John Wiley & Sons, Inc.     

Hair,serum, and urine chromium concentrations in former employees of the leather tanning industry

In this study, we measured concentrations of Cr in hair, serum, and urine of men who were previously employed in the leather tanning industry. Concentrations of Cr in hair and serum were significantly lower than corresponding values obtained during their employment and were comparable to levels obtained for controls in a previous study. These results suggest that Cr III accumulated from employment in the leather tanning industry does not result in longterm elevation of Cr concentrations in the body     

Glucosylation of human lens protein and cataractogenesis.

Examination of glucosylation of lens protein was conducted utilizing tritiated BH₄^?. The overall results indicate that approximately 0.20 moles of tritium were incorporated per mole of protein. Similar results were obtained with normal and senile cataractous lenses with varying degrees of opacity. Furthermore no difference in the ³H incorporation was observed between soluble and insoluble protein fractions derived from these lenses. Investigation of selected polypeptides isolated from the senile cataracts gave comparable results. Protein isolated from diabetic lenses had only slightly higher levels of tritium incorporation, giving an average value of 0.27 moles per mole of protein. Analyses of the tritiated products indicate that approximately 50% of the incorporation is probably due to reduction of other types of compounds. These results suggest that glucosylation does not appear to be a primary factor in cataract formation.