Epidermal growth factor attenuates tubular necrosis following mercuric chloride damage by regeneration of indigenous,not bone marrow‐derived cells

To assess effects of epidermal growth factor (EGF) and pegylated granulocyte colony‐stimulating factor (P‐GCSF; pegfilgrastim) administration on the cellular origin of renal tubular epithelium regenerating after acute kidney injury initiated by mercuric chloride (HgCl₂). Female mice were irradiated and male whole bone marrow (BM) was transplanted into them. Six weeks later recipient mice were assigned to one of eight groups: control, P‐GCSF+, EGF+, P‐GCSF+EGF+, HgCl₂, HgCl₂+P‐GCSF+, HgCl₂+EGF+ and HgCl₂+P‐GCSF+EGF+. Following HgCl₂, injection tubular injury scores increased and serum urea nitrogen levels reached uraemia after 3 days, but EGF‐treated groups were resistant to this acute kidney injury. A four‐in‐one analytical technique for identification of cellular origin, tubular phenotype, basement membrane and S‐phase status revealed that BM contributed 1% of proximal tubular epithelium in undamaged kidneys and 3% after HgCl₂ damage, with no effects of exogenous EGF or P‐GCSF. Only 0.5% proximal tubular cells were seen in S‐phase in the undamaged group kidneys; this increased to 7–8% after HgCl₂ damage and to 15% after addition of EGF. Most of the regenerating tubular epithelium originated from the indigenous pool. BM contributed up to 6.6% of the proximal tubular cells in S‐phase after HgCl₂ damage, but only to 3.3% after additional EGF. EGF administration attenuated tubular necrosis following HgCl₂ damage, and the major cause of this protective effect was division of indigenous cells, whereas BM‐derived cells were less responsive. P‐GCSF did not influence damage or regeneration.     

An in vitro test for measuring cytotoxicity of mercuric chloride to human kidney epithelial cells by specific enzyme release

Mercuric compound toxicity is well documented in animals and man for practically all organs. The recent development of cell culture techniques appeared as a novel fruitful tool in toxicology, especially in renal toxicology. Heavy metal induced renal cell alterations can be evaluated by membrane permeability damages.The present study evaluates mercuric chloride nephrotoxic effect in human kidney epithelial cells by measuring the release of two specific nephrotoxicity marker enzymes, Gamma Glutamyl Transferase (GGT) and Alkaline Phosphatase (ALP) in the culture medium. Cultured kidney epithelial cells were exposed to different HgCl₂ concentrations (5, 10, 20, and 50 g). Cultures were examined after 6 and 24 hours exposure. A good correlation between mercury dose and toxic effect, and exposure time and toxic effect was found. Enzymes were significantly released into the culture medium for 5 g and 10 g HgCl₂/ml after 6 hours exposure; and after 24 hours exposure, enzymes were released for 5 g/ml only.It appears that the specific tubular enzyme release in the culture medium is a good in vitro test for quantification of specific tubular damage.     

Mercury leads to abnormal red blood cell adhesion to laminin mediated by membrane sulfatides

Exposure to mercury is associated with numerous health problems, affecting different parts of the human body, including the nervous and cardiovascular systems in adults and children; however, the underlying mechanisms are yet to be fully elucidated. We investigated the role of membrane sulfatide on mercuric ion (Hg²⁺) mediated red blood cell (RBC) adhesion to a sub-endothelial matrix protein, laminin, using a microfluidic system that mimics microphysiological flow conditions. We exposed whole blood to mercury (HgCl₂), at a range of concentrations to mimic acute (high dose) and chronic (low dose) exposure, and examined RBC adhesion to immobilized laminin in microchannels at physiological flow conditions. Exposure of RBCs to both acute and chronic levels of Hg²⁺ resulted in elevated adhesive interactions between RBCs and laminin depending on the concentration of HgCl₂ and exposure duration. BCAM-Lu chimer significantly inhibited the adhesion of RBCs that had been treated with 50 μM of HgCl₂ solution for 1 h at 37 °C, while it did not prevent the adhesion of 3 h and 24 h Hg²⁺-treated RBCs. Sulfatide significantly inhibited the adhesion of RBC that had been treated with 50 μM of HgCl₂ solution for 1 h at 37 °C and 0.5 μM of HgCl₂ solution for 24 h at room temperature (RT). We demonstrated that RBC BCAM-Lu and RBC sulfatides bind to immobilized laminin, following exposure of RBCs to mercuric ions. The results of this study are significant considering the potential associations between sulfatides, red blood cells, mercury exposure, and cardiovascular diseases.     

Role of Oxidative Stress,Apoptosis, and Intracellular Homeostasis in Primary Cultures of Rat Proximal Tubular Cells Exposed to Cadmium

Cadmium (Cd) is a known nephrotoxic element. In this study, the primary cultures of rat proximal tubular (rPT) cells were treated with low doses of cadmium acetate (2.5 and 5 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability, together with a significant increase in the number of apoptotic and necrotic cells, were seen in the experiment. Simultaneously, elevation of intracellular [Ca²⁺]i and reactive oxygen species (ROS) levels, significant depletion of mitochondrial membrane potential(Δ Ψ) and cellular glutathione (GSH), intracellular acidification, and inhibition of Na⁺, K⁺-ATPase and Ca²⁺-ATPase activities were revealed in a dose-dependent manner during the exposure, while the cellular death and the apoptosis could be markedly reversed by N-acetyl-l-cysteine (NAC). Also, the calcium overload and GSH depletion were significantly affected by NAC. In conclusion, exposure of rPT cells to low-dose cadmium led to cellular death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death might be the chief mechanism, which may be mediated by oxidative stress. Also, a disorder of intracellular homeostasis induced by oxidative stress and mitochondrial dysfunction is a trigger of apoptosis in rPT cells.     

The Role of Intracellular Glutathione in Inorganic Mercury-Induced Toxicity in Neuroblastoma Cells

It is well known that antioxidants containing sulfhydryl (−SH) groups are protective against the toxic effects of mercury. The current study was designed to elucidate the mechanism(s) of the cytoprotective effects of glutathione (GSH) and N-acetylcysteine (NAC) against the toxicity of inorganic mercury (HgCl₂) in neuroblastoma cells (N-2A). The obtained results demonstrated the protective effects of these compounds in a dose dependant manner up to 95 and 74% cell viability, respectively as compared to the control of HgCl₂ of 10%. The administration of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, increased the toxicity of HgCl₂ in a dose dependent manner. Moreover, BSO treatment attenuated the levels of the cellular free −SH concentrations at low concentrations (1–100 μM) of HgCl₂. The data also show that cellular thiol concentrations were augmented in the presence of GSH and NAC and these compounds were cytoprotective against HgCl₂ and this is due to up regulating of GSH synthesis. A reduction in intracellular levels of GSH was observed with treatment of HgCl₂. In addition, the ratio of GSH/GSSG increased from 16:1 to 50:1 from 1 to 10 μM concentration of HgCl_2. The ratio of GSH/GSSG then decreased from 4:1 to 0.5:1 with the increase of concentration of HgCl₂ between 100 μM and 1 mM due to the collapse of the N-2A cells. It was of interest to note that the synthesis of GSH was stimulated in cells exposed to low concentration of HgCl₂ when extra GSH is available. These data support the idea that the loss of GSH plays a contributing role to the toxic effects of HgCl₂ and that inorganic mercury adversely affects viability, through altering intracellular −SH concentrations. The data further indicate that the availability of GSH to the cells may not be sufficient to provide protection against mercury toxicity and the de novo synthesis of intracellular GSH is required to prevent the damaging effects of mercury.     

Role of Reactive Oxygen Species and Glutathione in Inorganic Mercury-Induced Injury in Human Glioma Cells

The present study was undertaken to examine the role of reactive oxygen species (ROS) and glutathione (GSH) in glia cells using human glioma cell line A172 cells. HgCl₂ caused the loss of cell viability in a dose-dependent manner. HgCl₂-induced loss of cell viability was not affected by H₂O₂ scavengers catalase and pyruvate, a superoxide scavenger superoxide dismutase, a peroxynitrite scavenger uric acid, and an inhibitor of nitric oxide N^G-nitro-arginine Methyl ester. HgCl₂ did not cause changes in DCF fluorescence, an H₂O₂-sensitive fluorescent dye. The loss of cell viability was significantly prevented by the hydroxyl radical scavengers dimethylthiourea and thiourea, but it was not affected by antioxidants DPPD and Trlox. HgCl₂-induced loss of cell viability was accompanied by a significant reduction in GSH content. The GSH depletion was almost completely prevented by thiols dithiothreitol and GSH, whereas the loss of viability was partially prevented by these agents. Incubation of cells with 0.2 mM buthionine sulfoximine for 24 hr, a selective inhibitor of -glutamylcysteine synthetase, resulted in 56% reduction in GSH content without any change in cell viability. HgCl² resulted in 34% reduction in GSH content, which was accompanied by 59% loss of cell viability. These results suggest that HgCl₂-induced cell death is not associated with generation of H₂O₂ and ROS-induced lipid peroxidation. In addition, these data suggest that the depletion of endogenous GSH itself may not play a critical role in the HgCl₂-induced cytotoxicity in human glioma cells.     

Expression of mercuric reductase from Bacillus megaterium MB1 in eukaryotic microalga Chlorella sp. DT: an approach for mercury phytoremediation

A eukaryotic microalga, Chlorella sp. DT, was transformed with the Bacillus megaterium strain MB1 merA gene, encoding mercuric reductase (MerA), which mediates the reduction of Hg²⁺ to volatile elemental Hg⁰. The transformed Chlorella cells were selected first by hygromycin B and then by HgCl₂. The existence of merA gene in the genomic DNA of transgenic strains was shown by polymerase chain reaction amplification, while the stable integration of merA into genomic DNA of transgenic strains was confirmed by Southern blot analysis. The ability to remove Hg²⁺ in merA transgenic strains was higher than that in the wild type. The merA transgenic strains showed higher growth rate and photosynthetic activity than the wild type did in the presence of a toxic concentration of Hg²⁺. Cultured with Hg²⁺, the expression level of superoxide dismutase in transgenic strains was lower than that in the wild type, suggesting that the transgenic strains faced a lower level of oxidative stress. All the results indicated that merA gene was successfully integrated into the genome of transgenic strains and functionally expressed to promote the removal of Hg²⁺.     

Mercuric chloride induces increases in both cytoplasmic and nuclear free calcium ions through a protein phosphorylation-linked mechanism

The mechanism of the lymphocyte stimulatory action of sulfhydryl group-reactive mercuric ions was studied with respect to its potential ability to induce a protein tyrosine phosphorylation-linked signal for mobilization of free Ca²⁺ into cytoplasm and nucleus of the cell. Exposure of human leukamic T cell line (Jurkat) cells to high (1 mM) and low (0.01 mM) concentrations of HgCl₂ induced tyrosine phosphorylation of multiple proteins in a concentration-dependent manner. Confocal microscopy directly visualized the time course localization of Ca²⁺ inside the cells after exposure to HgCl₂. The onset and level of Ca²⁺ mobilization following HgCl₂ exposure were in parallel to those of protein tyrosine phosphorylation. Interestingly, by either concentration of HgCl₂, Ca²⁺ was mobilized in both cytoplasm and nucleus almost simultaneously, and the level of Ca²⁺ mobilization in the nucleus was more than that in the cytoplasm. All the HgCl₂-mediated Ca²⁺ mobilization was prevented by addition of protein kinase inhibitor staurosporin prior to HgCl₂. These results suggest that heavy metal stress triggers a protein tyrosine phosphorylation-linked signal that leads to a nuclear event-dominant Ca²⁺ mobilization.     

HgCl2-induced alteration of actin filaments in cultured primary rat proximal tubule epithelial cells labelled with fluorescein phalloidin

When proximal tubule epithelial cells are exposed to HgCl₂, cytoplasmic blebs are formed. These represent on early, potentially reversible response to injury. These blebs are accompanied by reorganization of cytoskeletal proteins, and pre-sumably by alternations in cytoskeletal-plasma membrane interactions. Ca²⁺-activated proteinases, such as calpain, are known to affect cytoskeletal proteins and to be involved in diverse cellular processes. However, the role of calpains in cytotoxicity d due to HgCl₂ is unknown. To determine the relationship between Factin, calpain, and HgCl₂ toxicity, cells were stained with fluorescein phalloidin before and after treatment with HgCl₂. Cells were grown on coverslips and exposed to HgCl₂ (10 or 25 M) in the presence or absence of the calpain inhibitor, leupeptin. Untreated cells were flat, polygonal, and contained many fluorescent-stained cables of actin filaments. Generally, cells exposed to HgCl₂ became pleomorphic and contracted as the blebs formed. These cells showed fewer actin cables and fluorescence was seen mostly as either compact areas of dense stain or as peripheral rings. In many cells, actin cables and filaments were completely absent. Disappearance of F-actin was initially seen by 2 min after exposure to HgCl₂. Thus, disruption of the actin cytoskeleton and blebbing were found to be early events in HgCl₂ toxicity. When leupeptin was used with HgCl₂ treatment, the actin staining appeared similar to that of untreated cells. These findings clearly illustrate that HgCl₂ injury to proximal tubule epithelial cells causes rearrangement and alteration of F-actin which may involve the activation of calpain.Abbreviations HgCl₂ mercuric chloride - PTE proximal tubule epithelium - [Ca²⁺]_i cytosolic ionized calcium - [Ca²⁺]_e extracellular calcium - PBS phosphate buffered saline Supported by Navy N00014-88-K-0427 & NIH DK15440. This is contribution No. 2905 from the Cellular Pathobiology Laboratory.     

Staphylococcal enterotoxin-B (SEB) alters [14C]-choline transport and phosphatidylcholine metabolism in cultured human kidney proximal tubular cells

We studied the effects of SEB on [¹⁴C]-choline transport and metabolism of choline containing phospholipids in cultured human kidney proximal tubular (PT) cells. SEB increased the uptake of [¹⁴C]-choline in PT cells as a function of toxin concentration, incubation time, and pH. The maximum increase in uptake (3.5–5-fold compared to control) was observed at a toxin concentration of 10 ug/10⁴ cells, at 4 h and at pH 7.4. Two toxins structurally related to SEB, Staphylococcal enterotoxin-A and toxic shock toxin (TST-1) failed to alter [¹⁴C]-choline uptake in PT cells, a finding which indicates that SEB-mediated alteration in choline uptake in PT cells has high specificity.We found that SEB markedly and significantly increased the incorporation of [¹⁴C]-choline into phosphatidylcholine, Iysophosphatidylcholine and sphingomyelin, but not into phosphatidylethanolamine. Maximum increase in the incorporation of [¹⁴C]-choline into phosphatidlycholine (3-fold compared to control) was observed at 4 h after incubation with toxin. In contrast, SEB did not alter the incorporation of [¹⁴C]-choline in phosphatidylethanolamine. The cellular level of phosphatidylcholine was also increased (2-fold compared to control) in PT cells incubated with SEB. This was accompanied by a 3-to-4-fold increase in CTP: phosphocholine, cytidyltransferase activity.In sum, SEB specifically stimulates phosphatidylcholine synthesis in PT cells by increasing choline uptake or by activating CTP: phosphocholine, cytidyltransferase, or both. We believe this is the first-ever report indicating that a toxin can increase phosphatidylcholine synthesis. This high order of specificity may be in part due to the presence of a glycosphingolipid receptor in PT cells that specifically binds SEB but not SEA or TST-1. Accordingly, it is tempting to speculate that the receptor may somehow be involved in the SEB-mediated regulation of phosphatidylcholine synthesis.Abbreviations SEB Staphylococcal entertoxin-B - SEA Staphylococcal enterotoxin-A - TST-1 Toxic shock syndrome toxin-1 - PT Proximal tubular - PC Phosphatidylcholine - SM Sphingomyelin - LPC Lysophosphatidyl-choline - CT Cytidyltransferase     

Effect of Celecoxib on Ca2+ Fluxes and Proliferation in MDCK Renal Tubular Cells

The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular Ca^{2 +} concentration ([Ca^{2 +}]_i) and proliferation was examined by using the Ca^{2 +}-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (≥1 μ M) caused an increase of [Ca^{2 +}]_i in a concentration-dependent manner. Celecoxib-induced [Ca^{2 +}]_i increase was partly reduced by removal of extracellular Ca^{2 +}. Celecoxib-induced Ca^{2 +} influx was independently suggested by Mn^{2 +} influx-induced fura-2 fluorescence quench. In Ca^{2 +}-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca^{2 +}-ATPase, caused a monophasic [Ca^{2 +}]_i increase, after which celecoxib only induced a tiny [Ca^{2 +}]_iincrease; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [Ca^{2 +}]_i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [Ca^{2 +}]_i increases. Overnight incubation with 1 or 10 μ M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [Ca^{2 +}]_i increase in renal tubular cells by stimulating both extracellular Ca^{2 +} influx and intracellular Ca^{2 +} release and is highly toxic to renal tubular cells in vitro.     

Comparative studies of zinc metabolism in cultured chinese hamster cells with differing metallothionein-induction capacities

Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cd^r2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd²⁺ compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd²⁺-resistant Cd^r2C10 subline to induce synthesis of the Cd²⁺- and Zn²⁺-binding protein(s), metallothionein(s) (MT). Evidence that Cd²⁺ behaves as an analog of the essential trace metal, Zn²⁺, especially as an inducer of MT synthesis, suggested that the Cd^r and CHO cell types could be employed to investigate cellular Zn²⁺ metabolism. In the present study, measurements were made to compare CHO and Cd^r cell types for (a) growth as a function of the level of ZnCl₂ added to the culture medium, (b) uptake and subcellular distribution of Zn²⁺, and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cd^r cell types grew normally (T _d≊16–18 h) during exposures to Zn²⁺ at levels up to 100 μM added to the growth medium, but displayed abrupt growth inhibition at higher Zn²⁺ levels, (b) Cd^r cells incorporate fourfold more Zn²⁺ during a 24-h exposure to the maximal subtoxic level of Zn²⁺ and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cd^r cell is proficient in this response during exposure to the maximal subtoxic Zn²⁺ level. These findings suggest that (a) the CHO and Cd^r cell systems will be useful in further studies of cellular Zn²⁺ metabolism, especially in comparisons of Zn²⁺ metabolism in the presence and absence of induction of the Zn²⁺-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular Zn²⁺ uptake.     

Detoxification of mercury and organomercurials by nitrogen-fixing soil bacteria

Minimal inhibitory concentration values of HgCl₂ and 5 organomercurials were determined against 24 mercury-resistant N₂-fixing soil bacteria previously isolated from soil and identified in our laboratory. These bacterial strains also displayed multiple antibiotic resistant properties. Typical growth pattern of a highly mercury-resistantBeijerinckia sp (KDr₂) was studied in liquid broth supplemented with toxic levels of mercury compounds. Four bacterial strains were selected for determining their ability to volatilize mercury and their Hg-volatilizing capacity was different. Cell-free extracts prepared from overnight mercury-induced cells catalyzed Hg²⁺-induced NADPH oxidation. Specific activities of Hg²⁺-reductase which is capable of catalyzing conversion of Hg²⁺ →Hg(o) of 10 Hg-resistant bacterial strains are also reported.     

DMPS and N-acetylcysteine induced renal toxicity in mice exposed to mercury

Acute effects of mercuric chloride (HgCl₂) were evaluated on mice. Mice received a single dose of HgCl₂ (4.6 mg/kg, subcutaneously) for three consecutive days. Thirty minutes after the last injection with HgCl₂, mice received one single injection of 2,3-dimercapto-1-propanesulfonic acid (DMPS) or N-acetylcysteine (NAC) or diphenyl diselenide (PhSe)₂. DMPS, NAC and (PhSe)₂ were utilized as therapy against mercury exposure. At 24 h after the last HgCl₂ injection, blood, liver and kidney samples were collected. δ-Aminolevulinate dehydratase (δ-ALA-D) and Na⁺, K_-⁺ ATPase activities, thiobarbituric acid-reactive substances (TBARS), non-protein thiols (NPSH) and ascorbic acid concentrations were evaluated. Plasma aspartate (AST) and alanine (ALT) aminotransferase activities, as well as urea and creatinine levels were determined. The group of mice exposed to Hg + (PhSe)₂ presented 100% of lethality. Exposure with HgCl₂ caused a decrease on the body weight gain and treatments did not modify this parameter. δ-ALA-D, AST and ALT activities, TBARS, ascorbic acid levels and NPSH (hepatic and erythrocytic) levels were not changed after HgCl₂ exposure. HgCl₂ caused an increase in renal NPSH content and therapies did not modify these levels. Mice treated with (PhSe)₂, Hg + NAC and Hg + DMPS presented a reduction in plasma NPSH levels. Creatinine and urea levels were increased in mice exposed to Hg + NAC, while Hg + DMPS group presented an increase only in urea level. Na⁺, K_-⁺ ATPase activity was inhibited in mice exposed to Hg + DMPS and Hg + NAC. In conclusion, therapies with (PhSe)₂, DMPS and NAC following mercury exposure must be better studied because the formation of more toxic complexes with mercury, which can mainly damage renal tissue.     

Gentamicin-induced alterations in phospholipid metabolism in cultured human proximal tubular cells

The effects of gentamicin on phospholipid levels and metabolism and the uptake of phosphatidylcholine (PC) adsorbed to low-density lipoprotein (LDL) were investigated in cultured human proximal tubular (PT) cells. Cells incubated with gentamicin (0.3 mM) for one to 21 days had a similar increase in the cell number and protein as compared to control cells. However, the cellular levels of phosphatidylcholine (PC) and sphingomyelin (SM), but not other phospholipids, increased in a time-dependent manner. Incubation of gentamicin (0.3 to 3.0 mM) resulted in a concentration-dependent increase in the cellular levels of PC (50% to 320%) and SM (20% to 40%). Gentamicin stimulated the incorporation of [¹⁴C]-acetate into diacylglycerol, PC, and SM in the order of 300%, 66%, and 20%, respectively, but not into lysophosphatidylcholine (LPC). Similarly, gentamicin stimulated the incorporation of [¹⁴C]-choline into PC and SM in the order of 300% and 172%, respectively, but not into LPC as compared to control cells. In addition, gentamicin also stimulated the incorporation of [¹⁴C]-choline into cytidine diphosphocholine (CDP-choline). However, the endocytosis of [¹⁴C]-PC-LDL was lower in cells incubated with gentamicin than in control cells. Thus, exogenously derived PC on LDL does not contribute to the increased cellular levels of PC in PT cells incubated with gentamicin. The activity of cytidine triphosphate (CTP):phosphocholine cytidyltransferase was moderately lower in cells incubated with gentamicin as compared to control. By contrast, the activity of phospholipase A₁ and phospholipase C was twofold lower in cells incubated with gentamicin for 21 days as compared to control. Thus, increased incorporation of [¹⁴C]-acetate and [¹⁴C]-choline into PC in cells incubated with gentamicin may not only be due to increased endogenous synthesis but to decreased catabolism of newly synthesized PC. We conclude that gentamicin impairs the lysosomal catabolism of PC, leading to its accumulation in PT cells. This phenomenon may be an indication of gentamicin-induced nephrotoxicity in man.     

Redox-active complexes formed during the interaction between glutathione and mercury and/or copper ions

Prompted by the recently reported capacity of the physiologically occurring Cu(I)-[glutathione]₂ complex (Cu(I)-[GSH)]₂) to reduce oxygen, the effect of various GSH-binding metals (Co²⁺, Cd²⁺, Zn²⁺, Pb²⁺, Al³⁺, Hg²⁺ and Ni²⁺) on the superoxide-generating capacity of such complex was investigated. Amongst all tested metals, only Hg²⁺ was able to substantially affect the capacity of Cu(I)-[GSH]₂ to generate superoxide. When Hg²⁺ and Cu(I)-[GSH]₂ were mixed equimolarly, the superoxide formation, assessed through the cytochrome c reduction and dihydroethidium oxidation, was increased by over 50%. Such effect was totally inhibitable by SOD. Based on the reportedly higher affinity of Hg²⁺ for GSH and the observed ability of Hg²⁺ to lower the concentration of Cu(I)-[GSH]₂ (spectroscopically assessed), we suggest that Hg²⁺ displaces Cu(I) from Cu(I)-[GSH]₂, to release Cu(I) ions and form a Hg(II)-[GSH]₂ complex. The latter species would account for the Hg²⁺-induced exacerbation of the superoxide production. In fact, the present study provides first time evidence that a preformed Hg(II)-[GSH]₂ complex is able to concentration-dependently reduce oxygen. Such redox-activity was evidenced using cytochrome c and confirmed by EPR studies using DMPO (5,5-dimethyl-1-pyrroline N-oxide, a spin-trapping agent). Considering this novel ability of Hg(II)-[GSH]₂ to generate superoxide, a further characterization of its redox-activity and its potential to affect superoxide-susceptible biological targets appears warranted.     

Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis

Macroautophagy/autophagy protects against cellular stress. Renal sublethal injury-triggered tubular epithelial cell cycle arrest at G₂/M is associated with interstitial fibrosis. However, the role of autophagy in renal fibrosis is elusive. Here, we hypothesized that autophagy activity in tubular epithelial cells is pivotal for inhibition of cell cycle G₂/M arrest and subsequent fibrogenic response. In both renal epithelial cells stimulated by angiotensin II (AGT II) and the murine kidney after unilateral ureteral obstruction (UUO), we observed that occurrence of autophagy preceded increased production of COL1 (collagen, type I). Pharmacological enhancement of autophagy by rapamycin suppressed COL1 accumulation and renal fibrosis. In contrast, genetic ablation of autophagy by proximal tubular epithelial cell-specific deletion of Atg5, with reduction of the LC3-II protein level and degradation of SQSTM1/p62, showed marked cell cycle arrest at the G₂/M phase, robust COL1 deposition, and severe interstitial fibrosis in a UUO model, as compared with wild-type mice. In vitro, AGT II exposure triggered autophagy preferentially in the G₁/S phase, and increased COL1 expression in the G₂/M phase in renal epithelial cells. Stimulation of Atg5-deficient primary proximal tubular cells with AGT II also resulted in elevated G₂/M arrest and COL1 production. Pharmacological or genetic inhibition of autophagy increased AGT II-mediated G₂/M arrest. Enhanced expression of ATG5, but not the autophagy-deficient ATG5 mutant K130R, rescued the G₂/M arrest, suggesting the regulation of cell cycle progression by ATG5 is autophagy dependent. In conclusion, Atg5-mediated autophagy in proximal epithelial cells is a critical host-defense mechanism that prevents renal fibrosis by blocking G₂/M arrest.     

Biotransformation of mercury in pH-stat cultures of eukaryotic freshwater algae

Eukaryotic algae were studied to determine their ability to biotransform Hg^II under aerated and pH controlled conditions. All algae converted Hg^II into β-HgS and Hg⁰ to varying degrees. When Hg^II was administered as HgCl₂ to the algae, biotransformation by species of Chlorophyceae (Selenastrum minutum and Chlorella fusca var. fusca) was initiated with β-HgS synthesis (K _1/2 of hours) and concomitant Hg° evolution occurred in the first hour. Hg° synthesis was impeded by the formation of β-HgS and this inhibition was released in C. fusca var. fusca when cellular thiols were oxidized by the addition of dimethylfumarate (DMF). The diatom, Navicula pelliculosa (Bacillariophyceae), converted a substantially greater proportion of the applied Hg^II into Hg⁰, whereas the thermophilic alga, Galdieria sulphuraria (Cyanidiophyceae), rapidly biotransformed as much as 90% of applied Hg^II into β-HgS (K _1/2 ≈ 20 min). This thermophile was also able to generate Hg⁰ even after all exogenously applied HgCl₂ had been biotransformed. The results suggest that β-HgS may be the major dietary mercurial for grazers of contaminated eukaryotic algae.