Functional proteomics reveals hepatotoxicity and the molecular mechanisms of different forms of chromium delivered by skin administration

Chromium compounds are known to be associated with cytotoxicity and carcinogenicity when applied via the skin. However, there is no perspective research to elucidate the causations between chromium exposure and hepatotoxicity. In the present study, the impact of hexavalent/trivalent chromium on the liver and the underlying pathogenic processes were revealed in the female nude mice model. The liver damage under different treatments was evaluated by histologic examination. Functional proteome tools combined with a network analysis revealed statistically significant candidate protein networks predicted to be changed in the presence of chromium compounds. RNA interference-mediated silencing and immunohistochemistry were used to confirm whether the candidate protein was capable of resulting in hepatotoxicity elicited by chromium. Potassium chromate as the Cr(VI) compound generated greater oxidative stress, apoptosis and hepatotoxicity compared to chromium nitrate [Cr(III)]-treated samples. The most meaningful changes were observed amongst proteins involved in carbohydrate metabolism, endoplasmic reticulum (ER) stress, calcium homeostasis and apoptosis. Furthermore, abrogation of transitional ER ATPase (VCP) led to significant inhibition in hepatic cell viability under Cr(VI) administration, and the expression profiles of cytokeratin were closely correlated with apoptotic degrees of liver tissue. Collectively, our findings suggest that Cr(VI) might induce the accumulation of misfolded proteins and adverse effects leading to cell apoptosis and liver injury. These signature networks represent an approach to discover novel relationships in complex data, and functional proteomics of liver may provide solid evidence of chromium-caused hepatic damage via the skin.     

Stability and absorption of chromium and absorption of chromium histidinate complexes by humans

Increased intake of chromium (Cr) often leads to improvements in glucose, insulin, lipids, and related variables in studies involving humans and experimental and farm animals. However, the results are often variable, depending not only on the selection of subjects but also dietary conditions and the form of supplemental Cr used. Our objective was to find a Cr supplement suitable for humans that was absorbed better than any of those available. Chromium absorption by six adult subjects, three males and three females, was determined based on the amount of Cr excreted in the urine in the initial 2 d following intake of 200 μg of Cr of the various forms of chromium tested. The absorption of the newly synthesized complexes was greatest for those containing histidine. Urinary Cr losses for six control subjects consuming 200 μg of Cr as Cr histidinate increased from basal levels of 256±48 to 3670±338 ng/d compared with 2082±201 ng for Cr picolinate, the currently most popular nutrient supplement, in the 48h following Cr consumption. Chromium histidinate complexes were stable and absorption was similar to the initial values after more than 2 yr. Mixing of some of the complexes with starch, which was postulated to improve Cr absorption, was shown to essentially block Cr absorption within 1 mo. These data demonstrate that urinary Cr losses need to be determined because stability and absorption of the Cr complexes varies widely and could be responsible for the variability in some of the Cr supplementation studies. Chromium ***DIRECT SUPPORT *** A02Q2015 00003 histidinate complexes are absorbed better than any of the Cr complexes currently available and need to be evaluated as Cr nutritional supplements.     

Comparison of acute absorption of commercially available chromium supplements

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

Effects of epidermal turnover on the dynamics of percutaneous drug absorption

A method was proposed to evaluate the effect of desquamation on the time to reach steady-state drug delivery rate and concentration in the skin layers. Evaluation of the effective time constant, in terms of key physicochemical properties of the system, does not require solving the fundamental equations. Laplace transforms and the residue theorem were applied to monitor the evolution of the medicament in the skin layers and subsequent uptake into the systemic circulation. The equilibrium flux and the effective time constant declined with increasing stratum corneum turnover velocity and were not affected by the speed at which cells in the viable epidermis moved toward the surface of the skin. Ninety eight percent of the equilibrium penetration rate was reached after four time constants. This dynamic metric helped measure the extent to which high lipophilicity or molecular weight delayed percutaneous drug absorption.     

An analytical solution for percutaneous drug absorption: application and removal of the vehicle

The methods of Laplace transform were used to solve a mathematical model developed for percutaneous drug absorption. This model includes application and removal of the vehicle from the skin. A system of two linear partial differential equations was solved for the application period. The concentration of the medicinal agent in the skin at the end of the application period was used as the initial condition to determine the distribution of the drug in the skin following instantaneous removal of the vehicle. The influences of the diffusion and partition coefficients, clearance factor and vehicle layer thickness on the amount of drug in the vehicle and the skin were discussed.     

Physiological chromium determination in serum by zeeman graphite furnace atomic absorption spectrometry

Several authors have already underlined chromium implication in glucose and lipids metabolism of humans. In this field, physiological chromium determination in serum could be helpful, but the discrepancies reported in numerous papers are confusing. Here we report some results obtained by Zeeman correction Graphite Furnace Atomic Absorption Spectrometry: This technique includes a dilution of serum with 12.5 mM ultrapure nitric acid and 0.25% Triton X-100 (final concentrations). Some main features can be outlined: (1) the contamination constitutes a serious drawback and (2) the sensitivity of the technique is critical (characteristic mass found: 1.76 pg/0.0044 A.s). Our results obtained from 27 healthy subjects (2.01±0.77 nmol/L) agree with most recent studies and indicate that serum chromium level does not seem to be sex-related.     

A comparison of the bovine uterine and plasma proteome using iTRAQ proteomics

Early embryo loss is a key factor affecting fertility in dairy and beef herds. Prior to implantation, the bovine embryo spends around 16 days free-floating in the uterine environment and is dependent on the composition of uterine fluid for normal growth and development. However, there is a lack of information regarding the protein composition of the bovine uterus and how it relates to plasma. In this study, uterine flushings (UF) (n = 6) and blood plasma (n = 4) were collected from beef heifers on day 7 of the oestrous cycle, albumin depleted and compared using iTRAQ proteomics. A total of 35 proteins were higher and 18 were lower in UF including metabolic enzymes, proteins with anti-oxidant activity and those involved in modulation of the immune response. This study confirms the dynamic nature of the bovine uterine proteome and that it differs from plasma. Factors affecting the uterine proteome and how it impacts on embryo survival warrant further study.     

Proteomic analysis of chromium cytotoxicity in cultured rat lung epithelial cells

Chromium (Cr) has been widely used in industry for more than one century. Exposure to hexavalent Cr compounds is strongly associated with increasing risk of lung cancer. Extensive researches at DNA level indicated that generation of ROS from the reduction of Cr(VI) leading to DNA damage is the major cause of the toxicity and carcinogenicity of Cr(VI). The present study in cellular and protein levels confirmed that Cr(VI) induced apoptosis of lung epithelial cells (LEC) via ROS generation. To view the differentially expressed proteins in the process of Cr(VI) reduction, subcellular proteomics was applied and allowed the identification of more than 30 proteins with expression alteration. Most of those proteins are correlated with ROS-elicited responses, which were further validated by Western blotting analysis, induction of p53 pathway and antioxidative treatment. The current findings provided additional evidence in protein level to support the claim that ROS generated during the process of Cr(VI) reduction are involved in the Cr(VI)-induced toxicity and carcinogenesis.     

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.     

Tissue damage in organic rainbow trout muscle investigated by proteomics and bioinformatics

The response to tissue damage is a complex process, which involves the coordinated regulation of multiple proteins to ensure tissue repair. In order to investigate the effect of tissue damage in a lower vertebrate, samples were taken from rainbow trout (Oncorhynchus mykiss) at day 7 after damage and proteins were separated using 2DE. The experimental design included two groups of rainbow trout, which were fed organic feed either with or without astaxanthin. In total, 96 proteins were found to be affected by tissue damage, clearly demonstrating in this lower vertebrate the complexity and magnitude of the cellular response, in the context of a regenerative process. Using a bioinformatics approach, the main biological function of these proteins were assigned, showing the regulation of proteins involved in processes such as apoptosis, iron homeostasis, and regulation of muscular structure. Interestingly, it was established that exclusively within the astaxanthin feed group, three members of the annexin protein family (annexin IV, V, and VI) were regulated in response to tissue damage.     

Differential expression of specific cellular defense proteins in rat hypothalamus under simulated microgravity induced conditions: Comparative proteomics

Microgravity severely halts the structural and functional cerebral capacity of astronauts especially affecting their brains due to the stress produced by cephalic fluid shift. We employed a rat tail suspension model to substantiate simulated microgravity (SM) in brain. In this study, comparative mass spectrometry was applied in order to demonstrate the differential expression of 17 specific cellular defense proteins. Gamma‐enolase, peptidyl‐prolyl cis‐trans isomerase A, glial fibrillary acidic protein, heat shock protein HSP 90‐alpha, 10 kDa heat shock protein, mitochondrial, heat shock cognate 71 kDa protein, superoxide dismutase 1 and dihydropyrimidinase‐related protein 2 were found to be upregulated by HPLC/ESI‐TOF. Furthermore, five differentially expressed proteins including 60 kDa heat shock protein, mitochondrial, heat shock protein HSP 90‐beta, peroxiredoxin‐2, stress‐induced‐phosphoprotein, and UCHL‐1 were found to be upregulated by HPLC/ESI‐Q‐TOF MS. In addition, downregulated proteins include cytochrome C, superoxide dismutase 2, somatic, and excitatory amino acid transporter 1 and protein DJ‐1. Validity of MS results was successfully performed by Western blot analysis of DJ‐1 protein. This study will not only help to understand the neurochemical responses produced under microgravity but also will give future direction to cure the proteomic losses and their after effects in astronauts.     

Quantitative chemical proteomics in small-scale culture of phorbol ester stimulated basal breast cancer cells

Basal-like breast cancers are commonly negative for expression of estrogen and progesterone receptors and HER-2 (triple-negative breast cancer), which makes this subtype of breast cancers more aggressive and less responsive to standard treatment. We have applied a small-scale chemical proteomics method using bisindolylmaleimide (Bis) class of protein kinase C inhibitors to study the Bis-binding proteome in a cell culture model of basal breast carcinoma (MDA-MB-231). Using MS, we identified 174 proteins captured by the Bis-probe in phorbol ester (PMA) stimulated cells. Gene ontology analysis broadly categorised these proteins as ATP binding (42%), GTP binding (6%) and having nucleoside-triphosphatase activity (21%). Of the 64 enzymes captured by the Bis-probe, the majority had either ATP and/or nucleotide binding functions. Two previously unreported Bis binding protein kinases, serine/arginine-rich protein-specific kinase 1 (SRPK1) and interferon-induced RNA-dependent protein kinase (PKR) were observed. We then incorporated SILAC for quantitation to examine the proteins that were differentially captured by the Bis-probe following 30 and 60 min PMA stimulation. This provided novel evidence for PMA regulation of the enzymes glyceraldehyde-3-phosphate dehydrogenase, nucleolar RNA helicase 2 and Heterogeneous nuclear ribonucleoprotein M.     

Quantitative proteomics of heavy metal stress responses in Sydney rock oysters

Currently, there are few predictive biomarkers in key biomonitoring species, such as oysters, that can detect heavy metal pollution in coastal waterways. Several attributes make oysters superior to other organisms for positive biomonitoring of heavy metal pollution. In particular, they are filter feeders with a high capacity for bioaccumulation. In this study, we used two proteomics approaches, namely label-free shotgun proteomics based on SDS-PAGE gel separation and gas phase fractionation, to investigate the heavy metal stress responses of Sydney rock oysters. Protein samples were prepared from haemolymph of oysters exposed to 100 μg/L of PbCl(2), CuCl(2), or ZnCl(2) for 4 days in closed aquaria. Peptides were identified using a Bivalvia protein sequence database, due to the unavailability of a complete oyster genome sequence. Statistical analysis revealed 56 potential biomarker proteins, as well as several protein biosynthetic pathways to be greatly impacted by metal stress. These have the potential to be incorporated into bioassays for prevention and monitoring of heavy metal pollution in Australian oyster beds. The study confirms that proteomic analysis of biomonitoring species is a promising approach for assessing the effects of environmental pollution, and our experiments have provided insights into the molecular mechanisms underlying oyster stress responses.     

Binding of chromium(VI) to histones: implications for chromium(VI)-induced genotoxicity

The first evidence has been obtained for Cr(VI) (chromate) binding to isolated calf thymus (CT) histones under physiological conditions (pH 7.4, Cl⁻ concentration 152 mM, 310 K). No significant Cr(VI) binding under the same conditions was observed for other extracellular and intracellular proteins, including albumin, apo-transferrin and G-actin, as well as for CT DNA. The mode of Cr(VI) binding to histones was studied by vibrational, electronic and X-ray absorption (X-ray absorption near-edge structure and X-ray absorption fine structure) spectroscopies and molecular mechanics calculations. A proposed binding mechanism includes electrostatic interactions of CrO₄ ²⁻ with protonated Lys and Arg residues of histones, as well as the formation of hydrogen bonds with the protein backbone. Similarly, Cr(VI) can bind to nuclear localization signals (typically, Lys- and Arg-rich fragments) of other nuclear proteins. Selective binding of Cr(VI) to newly synthesized nuclear proteins (including histones) in the cytoplasm is likely to be responsible for the active transport of Cr(VI) into the nuclei of living cells. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma

Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull‐down, and OptiPrep^TM density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep^TM density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.     

On the interaction of compounds of chromium(VI) with hydrogen peroxide. A study of chromium(VI) and (V) peroxides in the acid-basic pH range

A computational study of chromium(VI) and (V) peroxides, which exhibit important genotoxic and mutagenic activity, is reported. Energies and equilibrium geometries for [Cr^VI(O)(O₂)₂(OH)]⁻, [Cr^VI(O)(O₂)₂(OH₂)], [Cr^VI(O)(O₂)₂(py)], [Cr^VI(OH)(O₂)₂(OH₂)]⁺, [Cr^V(O)(O₂)₂(OH₂)]⁻ and species were calculated using molecular mechanics calculations (MMFF94 and MM⁺), quantum calculations with semi-empirical methods (RHF and UHF/PM3) and density functional theory (pBP86/DN* or pBP/DN* and B3LYP/6-31G(d). Equilibrium geometries for the compounds [Cr^V(O₂)₃(OH)]²⁻ and [Cr^V(O₂)₄]³⁻ were determined by molecular mechanics. Vibrational frequencies, standard thermodynamic quantities and electronic spectra were calculated using B3LYP/6-31G(d). The structural relationship between all these species and an explanation of the formation of peroxo species in the acid-basic pH range are given. An experimental study of peroxo species in basic medium was also performed (synthesis, X-ray powder diffraction patterns and infrared spectra of the peroxo complexes isolated) but did not confirm the existence of a tri-peroxo complex in the solid phase.     

Skin aging caused by intrinsic or extrinsic processes characterized with functional proteomics

The skin provides protection against environmental stress. However, intrinsic and extrinsic aging causes significant alteration to skin structure and components, which subsequently impairs molecular characteristics and biochemical processes. Here, we have conducted an immunohistological investigation and established the proteome profiles on nude mice skin to verify the specific responses during aging caused by different factors. Our results showed that UVB‐elicited aging results in upregulation of proliferating cell nuclear antigen and strong oxidative damage in DNA, whereas chronological aging abolished epidermal cell growth and increased the expression of caspase‐14, as well as protein carbonylation. Network analysis indicated that the programmed skin aging activated the ubiquitin system and triggered obvious downregulation of 14‐3‐3 sigma, which might accelerate the loss of cell growth capacity. On the other hand, UVB stimulation enhanced inflammation and the risk of skin carcinogenesis. Collectively, functional proteomics could provide large‐scale investigation of the potent proteins and molecules that play important roles in skin subjected to both intrinsic and extrinsic aging.     

Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry

The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used LC‐MS/MS to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the second day of wandering stage, first day of pupation, ninth day of pupation, and first day as an adult moth. GO annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity‐related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant‐binding, and chemosensory proteins, showed stage‐specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis.     

Functional proteomics reveals the protective effects of saffron ethanolic extract on hepatic ischemia‐reperfusion injury

Hepatic ischemia‐reperfusion (IR) injury is a common clinical problem and ROS may be a contributing factor on IR injury. The current study evaluates the potential protective effect of saffron ethanol extract (SEE) in a rat model upon hepatic IR injury. Caspases 3 and terminal deoxynucleotidyl transferase‐mediated dUTP biotin nick end labeling (TUNEL) results showed increased cell death in the IR samples; reversely, minor apoptosis was detected in the SEE/IR group. Pretreatment with SEE significantly restored the content of antioxidant enzymes (SOD1 and catalase) and remarkably inhibited the intracellular ROS concentration in terms of reducing p47phox translocation. Proteome tools revealed that 20 proteins were significantly modulated in protein intensity between IR and SEE/IR groups. Particularly, SEE administration could attenuate the carbonylation level of several chaperone proteins. Network analysis suggested that saffron extract could alleviate IR‐induced ER stress and protein ubiquitination, which finally lead to cell apoptosis. Taken together, SEE could reduce hepatic IR injury through modulating protein oxidation and our results might help to develop novel therapeutic strategies against ROS‐caused diseases.