Renal expression of organic anion transporters is modified after mercuric chloride exposure: Gender-related differences.

Mercuric ions (Hg+2) gain access to proximal tubule cells primarily by the Organic Anion Transporter 1 (Oat1) and 3 (Oat3) in the basolateral plasma membrane. The removal process of Hg+2 ions from cells into the lumen involves an efflux process mainly mediated by the Multidrug Resistance-Associated Protein 2 (Mrp2). The aim of this study was to compare the sex-related differences in the renal expression of Oat1, Oat3, and Mrp2 after mercuric chloride (HgCl2) treatment and analyze their relevance in the mercury-induced nephrotoxicity. Control and Hg-treated male and female Wistar rats were used. Animals received a dose of HgCl2 (4 mg/kg bw, ip) 18 h before the experiments. Tubular injury was assessed by histopathological studies. The renal expression of Oat1, Oat3, and Mrp2 was analyzed by Western Blotting. Mercury levels were determined in urine by cold vapour atomic absorption spectroscopy. HgCl2 treatment increased the expression of renal Oat1 and Mrp2 in both sexes, being more evident in females than in males. The Oat3 renal expression only increased in female rats. The higher expressions of Oat1, Oat3, and Mrp2 could explain the higher renal excretion of mercury and consequently, the lesser renal tubular damage in female rats than in male rats.

Dietary luteolin protects against HgCl2-induced renal injury via activation of Nrf2-mediated signaling in rat.

Luteolin (Lut) belongs to the flavonoid family with various beneficial bioactivities. Here, we investigated whether Lut attenuate mercuric chloride (HgCl2)-induced renal injury in rat. We found that oral gavage administration of Lut (80mg/kg) alleviated anemia and renal histology upon HgCl2 treatment (80mg/L). Lut also significantly reduced HgCl2-induced oxidative stress and inflammatory, presenting as the reduced malondialdehyde (MDA) formation, increased glutathione (GSH) level, and inhibited activation of nuclear factor kappa B (NF-κB). Moreover, Lut protected renal cells from HgCl2-induced apoptosis, as assessed by Terminal deoxynucleotidyl transferase dUNT nick end labeling (TUNEL) assay and the protein levels of B-cell lymphoma gene 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), Bcl-2-associated X protein (Bax), and p53. Interestingly, Lut reduced renal mercuric accumulation in rat. Furthermore, Lut increased nuclear translocation of the nuclear factor-erythroid-2-related factor 2 (Nrf2), and subsequent protein expression of the antioxidant enzymes, heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphatase: quinone-acceptor 1 (NQO1). Our results suggest that Lut suppress HgCl2-induced renal injury via activation of Nrf2 signaling pathway.

Automatic Spectroscopic Data Categorization by Clustering Analysis (ASCLAN): A Data-Driven Approach for Distinguishing Discriminatory Metabolites for Phenotypic Subclasses.

We propose a novel data-driven approach aiming to reliably distinguish discriminatory metabolites from nondiscriminatory metabolites for a given spectroscopic data set containing two biological phenotypic subclasses. The automatic spectroscopic data categorization by clustering analysis (ASCLAN) algorithm aims to categorize spectral variables within a data set into three clusters corresponding to noise, nondiscriminatory and discriminatory metabolites regions. This is achieved by clustering each spectral variable based on the r(2) value representing the loading weight of each spectral variable as extracted from a orthogonal partial least-squares discriminant (OPLS-DA) model of the data set. The variables are ranked according to r(2) values and a series of principal component analysis (PCA) models are then built for subsets of these spectral data corresponding to ranges of r(2) values. The Q(2)X value for each PCA model is extracted. K-means clustering is then applied to the Q(2)X values to generate two clusters based on minimum Euclidean distance criterion. The cluster consisting of lower Q(2)X values is deemed devoid of metabolic information (noise), while the cluster consists of higher Q(2)X values is then further subclustered into two groups based on the r(2) values. We considered the cluster with high Q(2)X but low r(2) values as nondiscriminatory, while the cluster with high Q(2)X and r(2) values as discriminatory variables. The boundaries between these three clusters of spectral variables, on the basis of the r(2) values were considered as the cut off values for defining the noise, nondiscriminatory and discriminatory variables. We evaluated the ASCLAN algorithm using six simulated (1)H NMR spectroscopic data sets representing small, medium and large data sets (N = 50, 500, and 1000 samples per group, respectively), each with a reduced and full resolution set of variables (0.005 and 0.0005 ppm, respectively). ASCLAN correctly identified all discriminatory metabolites and showed zero false positive (100% specificity and positive predictive value) irrespective of the spectral resolution or the sample size in all six simulated data sets. This error rate was found to be superior to existing methods for ascertaining feature significance: univariate t test by Bonferroni correction (up to 10% false positive rate), Benjamini-Hochberg correction (up to 35% false positive rate) and metabolome wide significance level (MWSL, up to 0.4% false positive rate), as well as by various OPLS-DA parameters: variable importance to projection, (up to 15% false positive rate), loading coefficients (up to 35% false positive rate), and regression coefficients (up to 39% false positive rate). The application of ASCLAN was further exemplified using a widely investigated renal toxin, mercury II chloride (HgCl2) in rat model. ASCLAN successfully identified many of the known metabolites related to renal toxicity such as increased excretion of urinary creatinine, and different amino acids. The ASCLAN algorithm provides a framework for reliably differentiating discriminatory metabolites from nondiscriminatory metabolites in a biological data set without the need to set an arbitrary cut off value as applied to some of the conventional methods. This offers significant advantages over existing methods and the possibility for automation of high-throughput screening in "omics" data.

Bioavailability study of arsenic and mercury in traditional Chinese medicines (TCM) using an animal model after a single dose exposure.

Traditional Chinese medicines (TCM) are increasingly being used as alternative medicines in many countries, and this has caused concern because of adverse health effects from toxic metal bioavailability such as mercury (Hg) and arsenic (As). The aim of this study was to investigate the bioavailability of As and Hg from TCM after a single exposure dose using an animal model of female Sprague-Dawley rats. The rats were divided into 6 groups which included four groups treated with sodium arsenite (NaAsO2), arsenic sulfide (As2S3), mercuric chloride (HgCl2), mercuric sulfide (HgS), and two groups treated with TCM containing high Hg or As (Liu Shen Wan: As 7.7-9.1% and Hg 1.4-5.0%; Niuhang Jie du Pian: As 6.2-7.9% and Hg <0.001%). The samples of urine, faeces, kidney and liver were collected for analysis and histological assay. The results indicated that relatively low levels of As and Hg from these TCM were retained in liver and kidney tissues. The levels of As in these tissues after TCM treatment were consistent with the levels from the As sulphide treated group. With the exception of the mercuric chloride treated group, the levels of Hg in urine from other groups were very low, and high levels of As and Hg from TCM were excreted in faeces. The study showed poor bioavailability of As and Hg from TCM as indicated by low relative bioavailability of As (0.60-1.10%) and Hg (<0.001%). Histopathological examination of rat kidney and liver tissues did not show toxic effects from TCM.

The protective role of procyanidins and lycopene against mercuric chloride renal damage in rats.

OBJECTIVE: This study aims to investigate the protection of procyanidins and lycopene from the renal damage induced by mercuric chloride. METHODS: Rats were treated with either procyanidins or lycopene 2h before HgCl(2) subcutaneously injection, once daily treatment for 2 successive days. RESULTS: In comparison with HgCl(2) group, markers of renal function such as blood urea nitrogen in serum and urinary protein were decreased to (18.45±11.63) mmol/L and (15.93±9.36) mmol/L, (4.54±0.78) g/(g·Cr) and (4.40±1.12) g/(g·Cr). N-acetyl-beta-D-glucosaminidase, lactate dehydrogenase, alkaline phosphatase in urine were depressed to (125.49±11.68) U/(g·Cr), (103.73±21.79) U/(g·Cr), (101.99±12.28) U/(g·Cr), and (113.19±23.74) U/(g·Cr), (71.14±21.80) U/(g·Cr), (73.64±21.51) U/(g·Cr) in procyanidins and lycopene groups. Indicators of oxidative stress, for example, Glutathion was reduced to (45.58±9.89) µmol/(g·pro) and (45.33±5.90) µmol/(g·pro), and antioxidant enzymes such as superoxide dismutase, glutathione-peroxidase were enhanced to (43.07±10.97) U/(mg·pro) and (39.94±6.04) U/(mg·pro), (83.85±18.48) U/(mg·pro), and (85.62±12.68) U/(mg·pro). Malondialdehyde was lowered to (0.95±0.12) (µmol/g·pro) and (1.03±0.12) µmol/(g·pro) in procyanidins and lycopene groups. ROS generation was decreased by 27.63% and 16.40% and apoptosis was also decreased in procyanidins and lycopene groups respectively. Pathological changes were much better as well. CONCLUSION: Procyanidins and Lycopene play some protective role against mercury kidney damage.

MRP2 and the handling of mercuric ions in rats exposed acutely to inorganic and organic species of mercury.

Mercuric ions accumulate preferentially in renal tubular epithelial cells and bond with intracellular thiols. Certain metal-complexing agents have been shown to promote extraction of mercuric ions via the multidrug resistance-associated protein 2 (MRP2). Following exposure to a non-toxic dose of inorganic mercury (Hg²+), in the absence of complexing agents, tubular cells are capable of exporting a small fraction of intracellular Hg²+ through one or more undetermined mechanisms. We hypothesize that MRP2 plays a role in this export. To test this hypothesis, Wistar (control) and TR(-) rats were injected intravenously with a non-nephrotoxic dose of HgCl2 (0.5 µmol/kg) or CH3HgCl (5 mg/kg), containing [²°³Hg], in the presence or absence of cysteine (Cys; 1.25 µmol/kg or 12.5mg/kg, respectively). Animals were sacrificed 24 h after exposure to mercury and the content of [²°³Hg] in blood, kidneys, liver, urine and feces was determined. In addition, uptake of Cys-S-conjugates of Hg²+ and methylmercury (CH3Hg+) was measured in inside-out membrane vesicles prepared from either control Sf9 cells or Sf9 cells transfected with human MRP2. The amount of mercury in the total renal mass and liver was significantly greater in TR⁻ rats than in controls. In contrast, the amount of mercury in urine and feces was significantly lower in TR⁻ rats than in controls. Data from membrane vesicles indicate that Cys-S-conjugates of Hg²+ and CH3Hg+ are transportable substrates of MRP2. Collectively, these data indicate that MRP2 plays a role in the physiological handling and elimination of mercuric ions from the kidney.

Toxicometabolomics approach to urinary biomarkers for mercuric chloride (HgCl2)-induced nephrotoxicity using proton nuclear magnetic resonance (¹H NMR) in rats.

The primary objective of this study was to determine and characterize surrogate biomarkers that can predict nephrotoxicity induced by mercuric chloride (HgCl2) using urinary proton nuclear magnetic resonance (¹H NMR) spectral data. A procedure for (1)H NMR urinalysis using pattern recognition was proposed to evaluate nephrotoxicity induced by HgCl2 in Sprague-Dawley rats. HgCl2 at 0.1 or 0.75 mg/kg was administered intraperitoneally (i.p.), and urine was collected every 24 h for 6 days. Animals (n=6 per group) were sacrificed 3 or 6 days post-dosing in order to perform clinical blood chemistry tests and histopathologic examinations. Urinary ¹H NMR spectroscopy revealed apparent differential clustering between the control and HgCl2 treatment groups as evidenced by principal component analysis (PCA) and partial least square (PLS)-discriminant analysis (DA). Time- and dose-dependent separation of HgCl2-treated animals from controls was observed by PCA of ¹H NMR spectral data. In HgCl2-treated rats, the concentrations of endogenous urinary metabolites of glucose, acetate, alanine, lactate, succinate, and ethanol were significantly increased, whereas the concentrations of 2-oxoglutarate, allantoin, citrate, formate, taurine, and hippurate were significantly decreased. These endogenous metabolites were selected as putative biomarkers for HgCl2-induced nephrotoxicity. A dose response was observed in concentrations of lactate, acetate, succinate, and ethanol, where severe disruption of the concentrations of 2-oxoglutarate, citrate, formate, glucose, and taurine was observed at the higher dose (0.75 mg/kg) of HgCl2. Correlation of urinary (1)H NMR PLS-DA data with renal histopathologic changes suggests that ¹H NMR urinalysis can be used to predict or screen for HgCl2-induced nephrotoxicity.

Seventy-five percent nephrectomy and the disposition of inorganic mercury in 2,3-dimercaptopropanesulfonic acid-treated rats lacking functional multidrug-resistance protein 2.

In the present study, we evaluated the disposition of inorganic mercury (Hg(2+)) in sham-operated and 75% nephrectomized (NPX) Wistar and transport-deficient (TR(-)) rats treated with saline or the chelating agent meso-2,3-dimercaptosuccinic acid (DMSA). Based on previous studies, DMSA and TR(-) rats were used as tools to examine the potential role of multidrug-resistance protein 2 (MRP2) in the disposition of Hg(2+) during renal insufficiency. All animals were treated with a low dose (0.5 mumol/kg i.v.) of mercuric chloride (HgCl(2)). At 24 and 28 h after exposure to HgCl(2), matched groups of Wistar and TR(-) rats received normal saline or DMSA (intraperitoneally). Forty-eight hours after exposure to HgCl(2), the disposition of Hg(2+) was examined. A particularly notable effect of 75% nephrectomy in both strains of rats was enhanced renal accumulation of Hg(2+), specifically in the outer stripe of the outer medulla. In addition, hepatic accumulation, fecal excretion, and blood levels of Hg(2+) were enhanced in rats after 75% nephrectomy, especially in the TR(-) rats. Treatment with DMSA increased both the renal tubular elimination and urinary excretion of Hg(2+) in all rats. DMSA did not, however, affect hepatic content of Hg(2+), even in the 75% NPX TR(-) rats. We also show with real-time polymerase chain reaction that after 75% nephrectomy and compensatory renal growth, expression of MRP2 (only in Wistar rats) and organic anion transporter 1 is enhanced in the remaining functional proximal tubules. We conclude that MRP2 plays a significant role in the renal and corporal disposition of Hg(2+) after a 75% reduction of renal mass.

Chemometric models for toxicity classification based on NMR spectra of biofluids.

1H NMR spectroscopic and pattern recognition (PR)-based methods were used to investigate the biochemical variability in urine obtained from control rats and from rats treated with a hydrazine (a model hepatotoxin) or HgCl(2) (a model renal cortical toxin). The 600 MHz (1)H NMR spectra of urine samples obtained from vehicle- or toxin-treated Han-Wistar (HW) and Sprague-Dawley (SD) rats were acquired, and principal components analysis (PCA) and soft independent modeling of class analogy (SIMCA) analysis were used to investigate the (1)H NMR spectral data. Variation and strain differences in the biochemical composition of control urine samples were assessed. Control urine (1)H NMR spectra obtained from the two rat strains appeared visually similar. However, chemometric analysis of the control urine spectra indicated that HW rat urine contained relatively higher concentrations of lactate, acetate, and taurine and lower concentrations of hippurate than SD rat urine. Having established the extent of biochemical variation in the two populations of control rats, PCA was used to evaluate the metabolic effects of hydrazine and HgCl(2) toxicity. Urinary biomarkers of each class of toxicity were elucidated from the PC loadings and included organic acids, amino acids, and sugars in the case of mercury, while levels of taurine, beta-alanine, creatine, and 2-aminoadipate were elevated after hydrazine treatment. SIMCA analysis of the data was used to build predictive models (from a training set of 416 samples) for the classification of toxicity type and strain of rat, and the models were tested using an independent set of urine samples (n = 124). Using models constructed from the first three PCs, 98% of the test samples were correctly classified as originating from control, hydrazine-treated, or HgCl(2)-treated rats. Furthermore, this method was sensitive enough to predict the correct strain of the control samples for 79% of the data, based upon the class of best fit. Incorporation of these chemometric methods into automated NMR-based metabonomics analysis will enable on-line toxicological assessment of biofluids and will provide a tool for probing the mechanistic basis of organ toxicity.

Influence of chronic mercury poisoning upon the connective tissue in rats. I. Effect of mercuric chloride on glycosaminoglycan levels in tissues, serum and urine.

Rats were intoxicated with mercuric chloride (1mg/kg b.w.) daily, for 12 weeks. A decrease in total glycosaminoglycan content was shown in the skin, the lungs, the liver and the heart muscle. These changes were accompanied by a slight alteration of the glycosaminoglycan pattern, a decrease in hyaluronic acid in the skin, the lungs and the heart muscle and an enhancement of heparan sulphate level in the kidneys. In serum of mercury-intoxicated rats, an increase in total glycosaminoglycan levels was shown. This enhancement was caused by elevation of almost all fractions. Urine output of glycosaminoglycans was higher in mercury-treated animals as compared to the controls.

Effect of different renal glutathione levels on renal mercury disposition and excretion in the rat.

Mercury renal disposition has been studied following HgCl2 injection (5.0 mg/kg body wt., s.c.) in controls, diethylmaleate and N-acetylcysteine-treated rats. The different treatments were used to generate statistically different degrees of non-protein sulfhydryls concentration in kidneys. Diethylmaleate (4 mmol/kg body wt., i.p.) diminished kidney glutathione levels to 25% and N-acetylcysteine (2 mmol/kg body wt., i.p.) increased kidney non-protein sulfhydryls levels up to 75% compared with new controls. The amount of mercury in the kidneys, the mercury excretion rate in urine and the mercury plasma disappearance curves were calculated during 3 h post HgCl2 injection. BUN was measured in plasma at the same time period to determine the onset of kidney damage. The results indicate a higher HgCl2 renal clearance in N-acetylcysteine-treated rats compared to controls and less renal mercury accumulation. The data agree with diminished renal toxicity. On the other hand, renal mercury accumulation was higher and mercury renal clearance lower in diethylmaleate-treated animals, associated with higher renal toxicity. The results suggest that non-protein sulfhydryl levels (principally glutathione) might determine renal accumulation of mercury as well as its elimination rate and hence might enhance or mitigate the nephrotoxicity induced by the metal.

Physiological model for the pharmacokinetics of methyl mercury in the growing rat.

We describe a physiological pharmacokinetic model for methyl mercury and its metabolite mercuric mercury in the growing rat. Demethylation appears to occur in both host tissues and gastrointestinal flora with elimination dominated by biliary secretion of inorganic mercury and by transport of methyl mercury into the gut lumen followed by substantial bacterial metabolism. Biliary transport of both organic and inorganic mercury is modeled in terms of the known secretion of glutathione from the hepatic pool. At 98 days following an oral tracer dose of 203Hg-labeled methyl mercury chloride, 65% of the administered dose had been recovered in the feces as inorganic mercury and 15% as organic mercury. Urinary excretion is a minor elimination route, accounting for less than 4% of the dose as methyl mercury and 1% of the dose as inorganic mercury. Irreversible incorporation of the mercurials into hair is a significant route of elimination. Ten percent of the administered dose was contained in the hair shed during the 98 days and over 12% of the dose (almost 90% of the body burden) remained in the hair at the end of that time period. Apparent ingestion of hair by the rats during grooming represents a novel form of toxin recirculation. Transport of both chemical species between blood and tissues is bidirectional and symmetric with relatively slow movement into and out of the brain. Transport mechanisms for both mercurial species are discussed in the context of capillary transport physiology and the blood-brain barrier to small molecules and proteins.

Disposition and retention of mercuric chloride in mice after oral and parenteral administration.

The present study compares effects of dose size on whole-body retention and relative organ distribution of 203HgCl2, after oral and intraperitoneal administration to female mice of two strains (inbred CBA/Bom and outbred Bom:NMRI). Using whole-body retention data of oral and intraperitoneal administration, an estimated "true absorption" of a single oral dose of inorganic mercury was calculated to be about 20% at two different dose levels. At the highest oral dose, a delay in fecal elimination of nonabsorbed mercury was observed, indicating a decreased peristaltic rate. The relative hepatic deposition was larger after oral than after intraperitoneal administration, presumably due to a first-pass effect, and a correspondingly lower relative renal deposition was seen. Increasing doses at both exposure routes resulted in increasing relative deposition in liver, stomach, intestines, and spleen but decreasing relative deposition in lungs and kidneys. Bom:NMRI mice deposited a larger fraction of the whole-body burden in the kidneys and a smaller fraction in the livers than did CBA/Bom mice. Comparison to a previous study with male mice (Nielsen and Andersen, 1989) demonstrates that male and female mice deposit similar fractions of their body burden in the liver, while male mice deposit significantly larger amounts of mercury in the kidneys and smaller amounts in the carcass than do female mice. Thus, the toxicokinetics of inorganic mercury in mice depend on dose size, administration route, and sex; the mouse strain is of less importance than the other factors investigated. The absorption of inorganic mercury was estimated to be about 20%, that is, twice as high as earlier estimates.

Renal handling of inorganic mercury in mice. The early excretion phase following a single intravenous injection of mercuric chloride studied by the Silver Amplification method.

The early renal excretion of mercuric mercury was studied in male BALB/c mice between 15 seconds and 30 min following a single intravenous injection of 3 mg HgCl2/kg body weight. The cytochemical Silver Amplification method applied at the light and electron microscopical levels showed mercury to be excreted by glomerular filtration and reabsorbed by proximal tubular epithelial cells by means of adsorptive endocytosis. Mercury was rapidly demonstrated in the lysosomal vacuome of proximal tubular epithelial cells. No uptake was observed from the peritubular side, and there was no evidence of tubular secretion of mercury. It is proposed that mercury is excreted in the form of mercury-protein complexes, assisted by the physiological proteinuria in mice, which is enhanced by mercury-induced damage to the glomerular structures.