The aging kidney and the nephrotoxic effects of mercury.

Owing to advances in modern medicine, life expectancies are lengthening and leading to an increase in the population of older individuals. The aging process leads to significant alterations in many organ systems, with the kidney being particularly susceptible to age-related changes. Within the kidney, aging leads to ultrastructural changes such as glomerular and tubular hypertrophy, glomerulosclerosis, and tubulointerstitial fibrosis, which may compromise renal plasma flow (RPF) and glomerular filtration rate (GFR). These alterations may reduce the functional reserve of the kidneys, making them more susceptible to pathological events when challenged or stressed, such as following exposure to nephrotoxicants. An important and prevalent environmental toxicant that induces nephrotoxic effects is mercury (Hg). Since exposure of normal kidneys to mercuric ions might induce glomerular and tubular injury, aged kidneys, which may not be functioning at full capacity, may be more sensitive to the effects of Hg than normal kidneys. Age-related renal changes and the effects of Hg in the kidney have been characterized separately. However, little is known regarding the influence of nephrotoxicants, such as Hg, on aged kidneys. The purpose of this review was to summarize known findings related to exposure of aged and diseased kidneys to the environmentally relevant nephrotoxicant Hg.

Mechanisms involved in the transport of mercuric ions in target tissues.

Mercury exists in the environment in various forms, all of which pose a risk to human health. Despite guidelines regulating the industrial release of mercury into the environment, humans continue to be exposed regularly to various forms of this metal via inhalation or ingestion. Following exposure, mercuric ions are taken up by and accumulate in numerous organs, including brain, intestine, kidney, liver, and placenta. In order to understand the toxicological effects of exposure to mercury, a thorough understanding of the mechanisms that facilitate entry of mercuric ions into target cells must first be obtained. A number of mechanisms for the transport of mercuric ions into target cells and organs have been proposed in recent years. However, the ability of these mechanisms to transport mercuric ions and the regulatory features of these carriers have not been characterized completely. The purpose of this review is to summarize the current findings related to the mechanisms that may be involved in the transport of inorganic and organic forms of mercury in target tissues and organs. This review will describe mechanisms known to be involved in the transport of mercury and will also propose additional mechanisms that may potentially be involved in the transport of mercuric ions into target cells.

Toxicological significance of renal Bcrp: Another potential transporter in the elimination of mercuric ions from proximal tubular cells.

Secretion of inorganic mercury (Hg(2+)) from proximal tubular cells into the tubular lumen has been shown to involve the multidrug resistance-associated protein 2 (Mrp2). Considering similarities in localization and substrate specificity between Mrp2 and the breast cancer resistance protein (Bcrp), we hypothesize that Bcrp may also play a role in the proximal tubular secretion of mercuric species. In order to test this hypothesis, the uptake of Hg(2+) was examined initially using inside-out membrane vesicles containing Bcrp. The results of these studies suggest that Bcrp may be capable of transporting certain conjugates of Hg(2+). To further characterize the role of Bcrp in the handling of mercuric ions and in the induction of Hg(2+)-induced nephropathy, Sprague-Dawley and Bcrp knockout (bcrp(-/-)) rats were exposed intravenously to a non-nephrotoxic (0.5 µmol · kg(-1)), a moderately nephrotoxic (1.5 µmol · kg(-1)) or a significantly nephrotoxic (2.0 µmol · kg(-1)) dose of HgCl2. In general, the accumulation of Hg(2+) was greater in organs of bcrp(-/-) rats than in Sprague-Dawley rats, suggesting that Bcrp may play a role in the export of Hg(2+) from target cells. Within the kidney, cellular injury and necrosis was more severe in bcrp(-/-) rats than in controls. The pattern of necrosis, which was localized in the inner cortex and the outer stripe of the outer medulla, was significantly different from that observed in Mrp2-deficient animals. These findings suggest that Bcrp may be involved in the cellular export of select mercuric species and that its role in this export may differ from that of Mrp2.

Structural characterization of 1,3-propanedithiols that feature carboxylic acids: Homologues of mercury chelating agents.

The molecular structures of a series of 1,3-propanedithiols that contain carboxylic acid groups, namely rac- and meso-2,4-dimercaptoglutaric acid (H4DMGA) and 2-carboxy-1,3-propanedithiol (H3DMCP), have been determined by X-ray diffraction. Each compound exhibits two centrosymmetric intermolecular hydrogen bonding interactions between pairs of carboxylic acid groups, which result in a dimeric structure for H3DMCP and a polymeric tape-like structure for rac- and meso-H4DMGA. Significantly, the hydrogen bonding motifs observed for rac- and meso-H4DMGA are very different to those observed for the 1,2-dithiol, rac-2,3-dimercaptosuccinic acid (rac-H4DMSA), in which the two oxygen atoms of each carboxylic acid group hydrogen bond to two different carboxylic acid groups, thereby resulting in a hydrogen bonded sheet-like structure rather than a tape. Density functional theory calculations indicate that 1,3-dithiolate coordination to mercury results in larger S-Hg-S bond angles than does 1,2-dithiolate coordination, but these angles are far from linear. As such, κ2-S2 coordination of these dithiolate ligands is expected to be associated with mercury coordination numbers of greater than two. In vivo studies demonstrate that both rac-H 4 DMGA and H3DMCP reduce the renal burden of mercury in rats, although the compounds are not as effective as either 2,3-dimercaptopropane-1-sulfonic acid (H3DMPS) or meso-H4DMSA.

New insights into the metabolism of organomercury compounds: mercury-containing cysteine S-conjugates are substrates of human glutamine transaminase K and potent inactivators of cystathionine γ-lyase.

Anthropogenic practices and recycling in the environment through natural processes result in release of potentially harmful levels of mercury into the biosphere. Mercury, especially organic forms, accumulates in the food chain. Mercury reacts readily with sulfur-containing compounds and often exists as a thiol S-conjugate, such as the l-cysteine (Cys)-S-conjugate of methylmercury (CH(3)Hg-S-Cys) or inorganic mercury (Cys-S-Hg-S-Cys). These S-conjugates are structurally similar to l-methionine and l-cystine/l-cystathionine, respectively. Bovine and rat glutamine transaminase K (GTK) catalyze transamination of sulfur-containing amino acids. Recombinant human GTK (rhGTK) has a relatively open catalytic active site, and we report here that this enzyme, like the rat and bovine enzymes, can also utilize sulfur-containing l-amino acids, including l-methionine, l-cystine, and l-cystathionine as substrates. The current study extends this list to include mercuric S-conjugates, and shows that CH(3)Hg-S-Cys and Cys-S-Hg-S-Cys are substrates and reversible inhibitors of rhGTK. The homocysteine S-conjugates, Hcy-S-Hg-S-Hcy and CH(3)Hg-S-Hcy, are also inhibitors. Finally, we show that HgCl(2), CH(3)Hg-S-Cys and Cys-S-Hg-S-Cys are potent irreversible inhibitors of rat cystathionine γ-lyase. The present study broadens our knowledge of the biochemistry of mercury compounds by showing that Cys S-conjugates of mercury interact with enzymes that catalyze transformations of biologically important sulfur-containing amino acids.

Relationships between the renal handling of DMPS and DMSA and the renal handling of mercury.

Within the body of this review, we provide updates on the mechanisms involved in the renal handling mercury (Hg) and the vicinal dithiol complexing/chelating agents, 2,3-bis(sulfanyl)propane-1-sulfonate (known formerly as 2,3-dimercaptopropane-1-sulfonate, DMPS) and meso-2,3-bis(sulfanyl)succinate (known formerly as meso-2,3-dimercaptosuccinate, DMSA), with a focus on the therapeutic effects of these dithiols following exposure to different chemical forms of Hg. We begin by reviewing briefly some of the chemical properties of Hg, with an emphasis on the high bonding affinity between mercuric ions and reduced sulfur atoms, principally those contained in protein and nonprotein thiols. A discussion is provided on the current body of knowledge pertaining to the handling of various mercuric species within the kidneys, focusing on the primary cellular targets that take up and are affected adversely by these species of Hg, namely, proximal tubular epithelial cells. Subsequently, we provide a brief update on the current knowledge on the handling of DMPS and DMSA in the kidneys. In particular, parallels are drawn between the mechanisms participating in the uptake of various thiol S-conjugates of Hg in proximal tubular cells and mechanisms by which DMPS and DMSA gain entry into these target epithelial cells. Finally, we discuss factors that permit DMPS and DMSA to bind intracellular mercuric ions and mechanisms transporting DMPS and DMSA S-conjugates of Hg out of proximal tubular epithelial cells into the luminal compartment of the nephron, and promoting urinary excretion.

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.

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.

Mercury toxicokinetics--dependency on strain and gender.

Mercury (Hg) exposure from dental amalgam fillings and thimerosal in vaccines is not a major health hazard, but adverse health effects cannot be ruled out in a small and more susceptible part of the exposed population. Individual differences in toxicokinetics may explain susceptibility to mercury. Inbred, H-2-congenic A.SW and B10.S mice and their F1- and F2-hybrids were given HgCl2 with 2.0 mg Hg/L drinking water and traces of (203)Hg. Whole-body retention (WBR) was monitored until steady state after 5 weeks, when the organ Hg content was assessed. Despite similar Hg intake, A.SW males attained a 20-30% significantly higher WBR and 2- to 5-fold higher total renal Hg retention/concentration than A.SW females and B10.S mice. A selective renal Hg accumulation but of lower magnitude was seen also in B10.S males compared with females. Differences in WBR and organ Hg accumulation are therefore regulated by non-H-2 genes and gender. Lymph nodes lacked the strain- and gender-dependent Hg accumulation profile of kidney, liver and spleen. After 15 days without Hg A.SW mice showed a 4-fold higher WBR and liver Hg concentration, but 11-fold higher renal Hg concentration, showing the key role for the kidneys in explaining the slower Hg elimination in A.SW mice. The trait causing higher mercury accumulation was not dominantly inherited in the F1 hybrids. F2 mice showed a large inter-individual variation in Hg accumulation, showing that multiple genetic factors influence the Hg toxicokinetics in the mouse. The genetically heterogeneous human population may therefore show a large variation in mercury toxicokinetics.

Transport of inorganic mercury and methylmercury in target tissues and organs.

Owing to the prevalence of mercury in the environment, the risk of human exposure to this toxic metal continues to increase. Following exposure to mercury, this metal accumulates in numerous organs, including brain, intestine, kidneys, liver, and placenta. Although a number of mechanisms for the transport of mercuric ions into target organs were proposed in recent years, these mechanisms have not been characterized completely. This review summarizes the current literature related to the transport of inorganic and organic forms of mercury in various tissues and organs. This review identifies known mechanisms of mercury transport and provides information on additional mechanisms that may potentially play a role in the transport of mercuric ions into target cells.

MRP2 involvement in renal proximal tubular elimination of methylmercury mediated by DMPS or DMSA.

2, 3-Dimercaptopropane-1-sulfonic acid (DMPS) and meso-2, 3-Dimercaptosuccinic acid (DMSA) are dithiols used to treat humans exposed to methylmercury (CH(3)Hg(+)). After treatment, significant amounts of mercury are eliminated rapidly from the kidneys and are excreted in urine. In the present study, we extended our previous studies by testing the hypothesis that MRP2 mediates the secretion of DMPS or DMSA S-conjugates of CH(3)Hg(+). To test this hypothesis, the disposition of mercury was assessed in control and Mrp2-deficient (TR(-)) rats exposed intravenously to a 5.0-mg/kg dose of CH(3)HgCl. Twenty-four and 28 h after exposure, groups of four control and four TR(-) rats were injected with saline, DMPS, or DMSA. Tissues were harvested 48 h later. Renal and hepatic contents of mercury were greater in saline-injected TR(-) rats than in controls. In contrast, the amounts of mercury excreted in urine and feces by TR(-) rats were less than those by controls. DMPS and DMSA significantly reduced the renal and hepatic content of mercury in both groups of rats, with the greatest reduction in controls. A significant increase in urinary and fecal excretion of mercury (which was greater in the controls) was also observed. Our findings in inside-out membrane vesicles prepared from hMRP2-transfected Sf9 cells show that uptake of DMPS and DMSA S-conjugates of CH(3)Hg(+) was greater in the vesicles containing hMRP2 than in control vesicles. Overall, these dispositional findings indicate that MRP2 does play a role in DMPS- and DMSA-mediated elimination of mercury from the kidney.

Multidrug resistance proteins and the renal elimination of inorganic mercury mediated by 2,3-dimercaptopropane-1-sulfonic acid and meso-2,3-dimercaptosuccinic acid.

Current therapies for inorganic mercury (Hg(2+)) intoxication include administration of a metal chelator, either 2,3-dimercaptopropane-1-sulfonic acid (DMPS) or meso-2,3-dimercaptosuccinic acid (DMSA). After exposure to either chelator, Hg(2+) is rapidly eliminated from the kidneys and excreted in the urine, presumably as an S-conjugate of DMPS or DMSA. The multidrug resistance protein 2 (Mrp2) has been implicated in this process. We hypothesize that Mrp2 mediates the secretion of DMPS- or DMSA-S-conjugates of Hg(2+) from proximal tubular cells. To test this hypothesis, the disposition of Hg(2+) was examined in control and Mrp2-deficient TR(-) rats. Rats were injected i.v. with 0.5 mumol/kg HgCl(2) containing (203)Hg(2+). Twenty-four and 28 h later, rats were injected with saline, DMPS, or DMSA. Tissues were harvested 48 h after HgCl(2) exposure. The renal and hepatic burden of Hg(2+) in the saline-injected TR(-) rats was greater than that of controls. In contrast, the amount of Hg(2+) excreted in urine and feces of TR(-) rats was less than that of controls. DMPS, but not DMSA, significantly reduced the renal and hepatic content of Hg(2+) in both groups of rats, with the greatest reduction in controls. A significant increase in urinary and fecal excretion of Hg(2+), which was greater in the controls, was also observed following DMPS treatment. Experiments utilizing inside-out membrane vesicles expressing MRP2 support these observations by demonstrating that DMPS- and DMSA-S-conjugates of Hg(2+) are transportable substrates of MRP2. Collectively, these data support a role for Mrp2 in the DMPS- and DMSA-mediated elimination of Hg(2+) from the kidney.

Mouse monocytes (RAW CELLS) and the handling of cysteine and homocysteine S-conjugates of inorganic mercury and methylmercury.

Although there is evidence indicating that mononuclear phagocytes can take up mercury by some forms of endocytosis, very little is known about the potential for the uptake of mercuric species by carrier-mediated processes. Thus, we hypothesized that monocytes also possess mechanisms allowing these cells to take up inorganic mercury (Hg2+) and/or methylmercury (CH3Hg+) as cysteine (Cys) and/or homocysteine (Hcy) S-conjugates by certain membrane transport proteins. The specific thiol S-conjugates were chosen for study because our laboratory and those of some other investigators have demonstrated that these species of mercury are indeed transportable substrates for several membrane transport proteins in certain types of epithelial cells. We chose to use RAW 264.7 cells for our experiments. These cells represent an adherent line of mouse monocytes. Kinetic analyses for the uptake of Cys-Hg-Cys, CH3Hg-Cys, Hcy-Hg-Hcy, and CH3Hg-Hcy revealed that uptake occurred by a saturable, concentration-dependent mechanism, displaying Michaelis-Menten properties. Interestingly, in the cells exposed to the Cys or Hcy S-conjugate of Hg2+, significantly more Hg2+ was taken up in the presence of 140 mM sodium chloride (NaCl) than in the presence of 140 mM N-methyl-D-glucamine (NMDG), indicating that Na-dependent processes play more of a role in the uptake of these species of Hg2+ than sodium-independent ones. With respect to the uptake of CH3Hg+, rates of uptake of the Cys and Hcy S-conjugates of CH3Hg+ were similar under both Na-dependent and Na-independent conditions, although the levels of uptake of these mercuric species far exceeded the levels of uptake of the corresponding S-conjugate of Hg2+. Uptake of Hg2+ and CH3Hg+, as the Cys or Hcy S-conjugates, was also time-dependent. We also showed that when the temperature in the bathing medium was reduced to 4 degrees C, uptake of the Cys S-conjugates Hg2+ or CH3Hg+ was for the most part reduced to negligible levels in the RAW cells; indicating that the preponderance of uptake at 37 degrees C was not due primarily to simple diffusion and/or non-specific binding. Overall, the present findings strongly suggest that the uptake of the Cys and Hcy S-conjugates of Hg2+ and/or CH3Hg+ occurs in monocytes by one or more mechanisms involving carrier proteins.

Influence of compensatory renal growth on susceptibility of primary cultures of renal cells to chemically induced injury.

Primary cultures of rat renal proximal tubular (PT) and distal tubular (DT) cells from control and uninephrectomized (NPX) Sprague-Dawley rats were established to study whether the altered toxicological responses identified in freshly isolated cells are maintained in culture. Previous work showed that primary cultures of PT cells from hypertrophied rat kidneys maintained their differentiated properties, as evidenced by their high respiratory rate, active transport function, transport and metabolism of glutathione, and their hypertrophic phenotype. In the present study, primary cultures of PT cells from NPX rat kidneys, but to a much lesser extent DT cells, were more susceptible to cellular injury induced by either mercuric chloride, KCN, or tert-butyl hydroperoxide (tBH), than corresponding cells from normal rat kidneys. Direct comparisons of cytotoxicity and lipid peroxidation induced by tBH in freshly isolated renal cells showed that the primary cultures of cells from NPX rat kidneys retained their altered susceptibility relative to cells from control rats. These results show that primary cultures of PT cells from NPX rats are more sensitive to cellular injury induced by three mechanistically distinct toxicants, demonstrating their usefulness in the study of the molecular and biochemical basis for the altered phenotype of compensatory renal growth. This is the first report validating the use of a mammalian renal cell culture model to study the toxicological effects of compensatory renal cellular hypertrophy.

Compensatory Renal Hypertrophy and the Uptake of Cysteine S-Conjugates of Hg2+ in Isolated S2 Proximal Tubular Segments.

Chronic kidney disease is characterized by a progressive and permanent loss of functioning nephrons. In order to compensate for this loss, the remaining functional nephrons undergo significant structural and functional changes. We hypothesize that luminal uptake of inorganic mercury (Hg2+), as a conjugate of cysteine (Cys; Cys-S-Hg-S-Cys), is enhanced in S2 segments of proximal tubules from the remnant kidney of uninephrectomized (NPX) rabbits. To test this hypothesis, we measured uptake and accumulation of Cys-S-Hg-S-Cys in isolated perfused S2 segments of proximal tubules from normal (control) and NPX rabbits. The remnant kidney in NPX rabbits undergoes significant hypertrophy during the initial 3 weeks following surgery. Tubules isolated from NPX rabbits were significantly larger in diameter and volume than those from control rabbits. Moreover, real-time PCR analyses of proximal tubules indicated that the expression of selected membrane transporters was greater in kidneys of NPX animals than in kidneys of control animals. When S2 segments from control and NPX rabbits were perfused with cystine or Cys-S-Hg-S-Cys, we found that the rates of luminal disappearance and tubular accumulation of Hg2+ were greater in tubules from NPX animals. These increases were inhibited by the addition of various amino acids to the perfusate. Taken together, our data suggest that hypertrophic changes in proximal tubules lead to an enhanced ability of these tubules to take up and accumulate Hg2.

Compensatory renal hypertrophy and the handling of an acute nephrotoxicant in a model of aging.

Aging often results in progressive losses of functioning nephrons, which can lead to a significant reduction in overall renal function. Because of age-related pathological changes, the remaining functional nephrons within aged kidneys may be unable to fully counteract physiological and/or toxicological challenges. We hypothesized that when the total functional renal mass of aged rats is reduced by 50%, the nephrons within the remnant kidney do not fully undergo the functional and physiological changes that are necessary to maintain normal fluid and solute homeostasis. We also tested the hypothesis that the disposition and handling of a nephrotoxicant are altered significantly in aged kidneys following an acute, 50% reduction in functional renal mass. To test these hypotheses, we examined molecular indices of renal cellular hypertrophy and the disposition of inorganic mercury (Hg(2+)), a model nephrotoxicant, in young control, young uninephrectomized (NPX), aged control and aged NPX Wistar rats. We found that the process of aging reduces the ability of the remnant kidney to undergo compensatory renal growth. In addition, we found that an additional reduction in renal mass in aged animals alters the disposition of Hg(2+) and potentially alters the risk of renal intoxication by this nephrotoxicant. To our knowledge, this study represents the first report of the handling of a nephrotoxicant in an aged animal following a 50% reduction in functional renal mass.

Basal and zinc-induced metallothionein in resistance to cadmium, cisplatin, zinc, and tertbutyl hydroperoxide: studies using MT knockout and antisense-downregulated MT in mammalian cells.

Metallothioneins (MTs) mediate resistance to metal and non-metal toxicants. To differentiate the role of MTs from other protective factors, resistance to zinc (Zn), cadmium (Cd), tertbutyl hydroperoxide (tBH), and cisplatin (CDDP) was compared in renal cell lines from wild type (MT-WT) and MT-1/MT-2 knockout (MT-KO) mice. MT-WT cells were more resistant to tBH than MT-KO cells but, unexpectedly, were more sensitive to Zn, Cd, and CDDP. Thus, basal expression of MT conferred resistance to tBH, but not to Cd or CDDP. Pretreatment with Zn increased MT expression and enhanced resistance to Cd and CDDP only in MT-WT cells, indicating a critical role for MT in this form of resistance. By contrast, Zn-pretreatment increased resistance to subsequent Zn exposure, but did not alter resistance to tBH, regardless of MT-status. Therefore, Zn-induced resistance to subsequent exposure to Zn (but not to Cd or CDDP) was mediated by non-MT factors, and neither Zn-induced MT nor other factors affected tBH sensitivity. Furthermore, antisense down-regulation of MT in human HeLa cells reduced basal MT levels and resistance to TBH, but not to Cd or CDDP. Therefore, basal MT alone can mediate resistance to TBH (but not to Cd or CDDP) in mouse and human cells. These data suggest that MT can mediate resistance to toxicants by different mechanisms, some of which correlate with the cellular content of MT protein. Moreover, resistance to some agents (Cd and CDDP) can be enhanced by inducing MT. Resistance to other agents (tBH) requires only basal (non-induced) MT levels.

Role of organic anion and amino acid carriers in transport of inorganic mercury in rat renal basolateral membrane vesicles: influence of compensatory renal growth.

Susceptibility to renal injury induced by inorganic mercury (Hg(2+)) increases significantly as a result of compensatory renal growth (following reductions of renal mass). We hypothesize that this phenomenon is related in part to increased basolateral uptake of Hg(2+) by proximal tubular cells. To determine the mechanistic roles of various transporters, we studied uptake of Hg(2+), in the form of biologically relevant Hg(2+)-thiol conjugates, using basolateral membrane (BLM) vesicles isolated from the kidney(s) of control and uninephrectomized (NPX) rats. Binding of Hg(2+) to membranes, accounted for 52-86% of total Hg(2+) associated with membrane vesicles exposed to HgCl(2), decreased with increasing concentrations of HgCl(2), and decreased slightly in the presence of sodium ions. Conjugation of Hg(2+) with thiols (glutathione, L-cysteine (Cys), N-acetyl-L-cysteine) reduced binding by more than 50%. Under all conditions, BLM vesicles from NPX rats exhibited a markedly lower proportion of binding. Of the Hg(2+)-thiol conjugates studied, transport of Hg-(Cys)(2) was fastest. Selective inhibition of BLM carriers implicated the involvement of organic anion transporter(s) (Oat1 and/or Oat3; Slc22a6 and Slc22a8), amino acid transporter system ASC (Slc7a10), the dibasic amino acid transporter (Slc3a1), and the sodium-dicarboxylate carrier (SDCT2 or NADC3; Slc13a3). Uptake of each mercuric conjugate, when factored by membrane protein content, was higher in BLM vesicles from uninephrectomized (NPX) rats, with specific increases in transport by the carriers noted above. These results support the hypothesis that compensatory renal growth is associated with increased uptake of Hg(2+) in proximal tubular cells and we have identified specific transporters involved in the process.

Disposition of inorganic mercury in pregnant rats and their offspring.

Environmental toxicants such as methylmercury have been shown to negatively impact fetal health. Despite the prevalence of inorganic mercury (Hg(2+)) in the environment and the ability of methylmercury to biotransform into Hg(2+), little is known about the ability of Hg(2+) to cross the placenta into fetal tissues. Therefore, it is important to understand the handing and disposition of Hg(2+) in the reproductive system. The purpose of the current study was to assess the disposition and transport of Hg(2+) in placental and fetal tissues, and to test the hypothesis that acute renal injury in dams can alter the accumulation of Hg(2+) in fetal tissues. Pregnant Wistar rats were injected intravenously with 0.5 or 2.5 µmol kg(-1) HgCl2 for 6 or 48 h and the disposition of Hg(2+) was measured. Accumulation of Hg(2+) in the placenta was rapid and dose-dependent. Very little Hg(2+) was eliminated during the initial 48 h after exposure. When dams were exposed to the low dose of HgCl2, fetal accumulation of Hg(2+) increased between 6h and 48 h, while at the higher dose, accumulation was similar at each time point. Within fetal organs, the greatest concentration of Hg(2+) (nmol/g) was localized in the kidneys, followed by the liver and brain. A dose-dependent increase in the accumulation of Hg(2+) in fetal organs was observed, suggesting that continued maternal exposure may lead to increased fetal exposure. Taken together, these data indicate that Hg(2+) is capable of crossing the placenta and gaining access to fetal organs in a dose-dependent manner.

Simultaneous coexposure to inorganic mercury and cadmium: a study of the renal and hepatic disposition of mercury and cadmium.

This study was designed to evaluate the effects of simultaneous coexposure to inorganic mercury and cadmium on the renal and hepatic disposition of each metal. Dispositional changes were assessed in rats 1 h and 24 h after the coexposure to relatively low doses of the metals (which individually are nonnephrotoxic in rats). The rational for studying mercury and cadmium is that both of these metals are encountered frequently in the same contaminated areas. Coadministration of a 0.5- micromol/kg dose of mercuric chloride with a 10- micromol/kg dose of cadmium chloride resulted in a decrease in the net renal accumulation of inorganic mercury at 1 and 24 h after exposure. Assessment of the disposition of both metals in renal zones indicates that the decreased renal accumulation of inorganic mercury was due specifically to changes in the accumulation of mercury in the renal cortex. Coexposure to inorganic mercury and cadmium also caused both the hepatic accumulation of mercury and the urinary excretion of mercury to increase during the initial 24 h after coexposure. During the initial 1 h after coexposure, the content of mercury in the blood was enhanced significantly. However, by the end of the first 24 h after exposure, the content of mercury in the blood was lower than that in animals treated with only inorganic mercury, likely due to the increased urinary excretion of mercury. Interestingly, with the exception of decreased fecal excretion of cadmium, no other changes in the disposition of cadmium were detected in the animals treated with both mercury and cadmium. These novel findings indicate that at the doses of inorganic mercury and cadmium used in the present study, cadmium has profound effects on the renal and hepatic handling of mercury. Based on the present findings, it appears that cadmium [by some currently unknown mechanism(s)] interferes with the luminal and/or basolateral uptake and/or net accumulation of mercury along S1 and S2 segments of the proximal tubules, which results in an overall decrease in the renal burden of mercury and an increased rate in the urinary excretion of mercury.