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Bridges, Zalups
Molecular mimicry as a mechanism for the uptake of cysteine S-conjugates of methylmercury and inorganic mercury.Bridges CC, Zalups RK. Chem Res Toxicol. 2006 Sep;19(9):1117-8; author reply 1118-20. [citation only]
System b0,+ and the transport of thiol-s-conjugates of methylmercury.Bridges CC, Zalups RK. J Pharmacol Exp Ther. 2006 Nov;319(2):948-56. Epub 2006 Aug 22. [abstract only]
"Methylmercury (CH(3)Hg(+)) is a clinically relevant toxicant that is the most abundant form of mercury found in the environment. After exposure, it accumulates in the kidneys, liver, and central nervous system. The mechanisms by which this toxicant is taken up by target cells are only now beginning to be understood. Some experimental data support a hypothesis involving molecular mimicry, whereby thiol conjugates of methylmercury (especially a cysteine S-conjugate) mimic one or more amino acids and are transported into target cells by amino acid transporters. In the present study, we tested the hypothesis that Cys and homocysteine (Hcy) S-conjugates of methylmercury (CH(3)Hg-S-Cys and CH(3)Hg-S-Hcy, respectively) mimic one or more amino acids at the site of the Na(+)-dependent amino acid transporter, system B(0,+). In the kidneys, system B(0,+) is situated on the luminal plasma membrane of proximal tubular epithelial cells. To test our hypothesis, we measured uptake of CH(3)Hg-S-Cys and CH(3)Hg-S-Hcy in Xenopus laevis oocytes injected with water or capped RNA encoding mouse ATB(0,+). Analyses of time course, substrate specificity, and saturation kinetics showed that the uptake of CH(3)Hg-S-Cys and CH(3)Hg-S-Hcy was 5- to 10-fold greater in oocytes expressing ATB(0,+) than in corresponding water-injected controls. Moreover, the transport of CH(3)Hg-S-Cys and CH(3)Hg-S-Hcy was inhibited by substrates transported by system B(0,+). Finally, our data indicate that CH(3)Hg-S-Cys and CH(3)Hg-S-Hcy may mimic of one or more amino acids (e.g., methionine) that are normally transported by system B(0,+). To our knowledge, this is the first report implicating system B(0,+) in the transport of any mercuric species."
Cystine alters the renal and hepatic disposition of inorganic mercury and plasma thiol status.Zalups RK, Lash LH. Toxicol Appl Pharmacol. 2006 Jul 1;214(1):88-97. Epub 2006 Feb 8. [abstract only]
"In the present study, we determined whether cystine can inhibit, under certain conditions, the renal tubular uptake of inorganic mercury in vivo. We co-injected (i.v.) cystine with a non-toxic dose of mercuric chloride to rats and then studied the disposition of inorganic mercury during the next 24 h. We also determined if pretreatment with cystine influences the disposition of administered inorganic mercury. Moreover, plasma thiol status was examined after the intravenous administration of cystine with or without mercuric chloride. During the initial hour after co-injection, the renal tubular uptake of mercuric ions was diminished significantly relative to that in control rats. The inhibitory effects of cystine were evident in both the renal cortex and outer stripe of the outer medulla. In contrast, the renal accumulation of mercury increased significantly between the 1st and 12th hour after co-treatment. Urinary excretion and fecal excretion of mercury were greatly elevated in the rats co-treated with cystine and mercuric chloride. Thus, when cystine and mercury are administered simultaneously, cystine can serve as an inhibitor of the renal tubular uptake of mercury during the initial hour after co-treatment. In rats pretreated with cystine, the renal uptake of inorganic mercury was enhanced significantly relative to that in rats not pretreated with cystine. This enhanced accumulation of inorganic mercury correlated with the increased circulating concentrations of the reduced cysteine and glutathione. Additionally, the present findings indicate that thiol status is an important determinant of renal and hepatic disposition, and urinary and fecal excretion, of inorganic mercury."
Role of organic anion and amino acid carriers in transport of inorganic mercury in rat renal basolateral membrane vesicles: influence of compensatory renal growth.Lash LH, Hueni SE, Putt DA, Zalups RK. Toxicol Sci. 2005 Dec;88(2):630-44. Epub 2005 Sep 14.
"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."
Handling of the homocysteine S-conjugate of methylmercury by renal epithelial cells: role of organic anion transporter 1 and amino acid transporters.Zalups RK, Ahmad S. J Pharmacol Exp Ther. 2005 Nov;315(2):896-904. Epub 2005 Aug 4.
"Recently, the activity of the organic anion transporter 1 (OAT1) protein has been implicated in the basolateral uptake of inorganic mercuric species in renal proximal tubular cells. Unfortunately, very little is known about the role of OAT1 in the renal epithelial transport of organic forms of mercury, such as methylmercury (CH(3)Hg(+)). Homocysteine (Hcy) S-conjugates of methylmercury [(S)-(3-amino-3-carboxypropylthio)(methyl)mercury (CH(3)Hg-Hcy)] have been identified recently as being potentially important biologically relevant forms of mercury. Thus, the present study was designed to characterize the transport of CH(3)Hg-Hcy in Madin-Darby canine kidney (MDCK) cells (which are derived from the distal nephron) that were transfected stably with the human isoform of OAT1 (hOAT1). Data on saturation kinetics, time dependence, substrate specificity, and temperature dependence demonstrated that CH(3)Hg-Hcy is a transportable substrate of hOAT1. However, substrate-specificity data from the control MDCK cells also showed that CH(3)Hg-Hcy is a substrate of one or more transporter(s) that is/are not hOAT1. Additional findings indicated that at least one amino acid transport system was probably responsible for this transport. It is noteworthy that the activity of amino acid transporters accounted for the greatest level of uptake of CH(3)Hg-Hcy in the hOAT1-expressing cells. Furthermore, rates of survival of the hOAT1-transfected MDCK cells were significantly lower than those of corresponding control MDCK cells when they were exposed to cytotoxic concentrations of CH(3)Hg-Hcy. Collectively, the present data indicate that CH(3)Hg-Hcy is a transportable substrate of OAT1 and amino acid transporters and, thus, is probably a transportable mercuric species taken up in vivo by proximal tubular epithelial cells."
Handling of cysteine S-conjugates of methylmercury in MDCK cells expressing human OAT1.Zalups RK, Ahmad S. Kidney Int. 2005 Oct;68(4):1684-99. [abstract only]
"BACKGROUND: The activity of the organic anion transporter 1 (OAT1) has been implicated recently in the basolateral uptake of thiol conjugates of inorganic mercury in renal proximal tubular cells. However, very little is known about the role of OAT1 in the renal epithelial transport of organic forms of mercury, such as methylmercury (CH(3)Hg(+)), especially when it is in the form of the cysteine (Cys) S-conjugate of methylmercury (CH(3)Hg-Cys), which is believed to be a biologically relevant form of mercury.
METHODS: Accordingly, the present study, was designed to characterize the transport of CH(3)Hg-Cys in Madin-Darby canine kidney (MDCK) cells transfected stably with the human isoform of OAT1 (hOAT1) and in proximal tubular-derived NRK-52E cells.
RESULTS: Data on saturation kinetics, time dependency, substrate specificity, and temperature dependency demonstrate that CH(3)Hg-Cys is transported by hOAT1. Substrate-specificity data from the control cells also show that CH(3)Hg-Cys is a substrate of one or more transporter(s) that is/are not hOAT1. Additional findings indicate that at least one amino acid transport system is involved in the uptake of CH(3)Hg-Cys in MDCK cells. Furthermore, in the presence of cytotoxic concentrations of CH(3)Hg-Cys, rates of survival were lower in hOAT1-transfected cells than in wild-type control cells.
CONCLUSION: The present data demonstrate clearly that CH(3)Hg-Cys is indeed a transportable substrate of OAT1. Moreover, the collective findings from the MDCK cells and NRK-52E cells infer that CH(3)Hg-Cys is a likely transportable mercuric species in proximal tubular epithelial cells that is taken up in vivo by both OAT1 and amino acid transporters."
Transport of N-acetylcysteine s-conjugates of methylmercury in Madin-Darby canine kidney cells stably transfected with human isoform of organic anion transporter 1.Zalups RK, Ahmad S. J Pharmacol Exp Ther. 2005 Sep;314(3):1158-68. Epub 2005 May 20.
"Recent studies have implicated the activity of the organic anion transporter 1 (OAT1) protein in the basolateral uptake of inorganic mercuric species in renal proximal tubular epithelial cells. However, very little is known about the potential role of OAT1 (and other OATs) in the renal epithelial transport of organic forms of mercury such as methylmercury (CH(3)Hg(+)). The present investigation was designed to study the transport of N-acetyl cysteine (NAC) S-conjugates of both methylmercury (CH(3)Hg-NAC) and inorganic mercury (NAC-Hg-NAC) in renal epithelial cells [Madin-Darby canine kidney (MDCK) cells] stably transfected with the human isoform of OAT1 (hOAT1). These mercuric species were studied because numerous mercapturates have been shown to be substrates of OATs. Data on saturation kinetics, time dependence, substrate specificity, and temperature dependence for the transport of CH(3)Hg-NAC and NAC-Hg-NAC indicate that both of these two mercuric species are indeed transportable substrates of hOAT1. Substrate specificity data also show that CH(3)Hg-NAC is a substrate of a transporter in MDCK cells that is not hOAT1. These data indicate that an amino acid carrier system is a likely candidate responsible for this transport. Furthermore, the rates of survival of the hOAT1-transfected MDCK cells were significantly lower than those of corresponding control MDCK cells when they were exposed to cytotoxic concentrations of CH(3)Hg-NAC or NAC-Hg-NAC. Collectively, the present data support the hypothesis that CH(3)Hg-NAC and NAC-Hg-NAC are transportable substrates of OAT1 and thus potentially transportable mercuric species taken up in vivo at the basolateral membrane of proximal tubular epithelial cells."
Molecular and ionic mimicry and the transport of toxic metals.Bridges CC, Zalups RK. Toxicol Appl Pharmacol. 2005 May 1;204(3):274-308. Review.
"Despite many scientific advances, human exposure to, and intoxication by, toxic metal species continues to occur. Surprisingly, little is understood about the mechanisms by which certain metals and metal-containing species gain entry into target cells. Since there do not appear to be transporters designed specifically for the entry of most toxic metal species into mammalian cells, it has been postulated that some of these metals gain entry into target cells, through the mechanisms of ionic and/or molecular mimicry, at the site of transporters of essential elements and/or molecules. The primary purpose of this review is to discuss the transport of selective toxic metals in target organs and provide evidence supporting a role of ionic and/or molecular mimicry. In the context of this review, molecular mimicry refers to the ability of a metal ion to bond to an endogenous organic molecule to form an organic metal species that acts as a functional or structural mimic of essential molecules at the sites of transporters of those molecules. Ionic mimicry refers to the ability of a cationic form of a toxic metal to mimic an essential element or cationic species of an element at the site of a transporter of that element. Molecular and ionic mimics can also be sub-classified as structural or functional mimics. This review will present the established and putative roles of molecular and ionic mimicry in the transport of mercury, cadmium, lead, arsenic, selenium, and selected oxyanions in target organs and tissues."
Homocysteine, system b0,+ and the renal epithelial transport and toxicity of inorganic mercury.Bridges CC, Zalups RK. Am J Pathol. 2004 Oct;165(4):1385-94.
"Proximal tubular epithelial cells are major sites of homocysteine (Hcy) metabolism and are the primary sites for the accumulation and intoxication of inorganic mercury (Hg(2+)). Previous in vivo data from our laboratory have demonstrated that mercuric conjugates of Hcy are transported into these cells by unknown mechanisms. Recently, we established that the mercuric conjugate of cysteine [2-amino-3-(2-amino-2-carboxy-ethylsulfanylmercuricsulfanyl)propionic acid; Cys-S-Hg-S-Cys], is transported by the luminal, amino acid transporter, system b(0,+). As Cys-S-Hg-S-Cys and the mercuric conjugate of Hcy (2-amino-4-(3-amino-3-carboxy-propylsulfanylmercuricsulfanyl)butyric acid; Hcy-S-Hg-S-Hcy) are similar structurally, we hypothesized that Hcy-S-Hg-S-Hcy is a substrate for system b(0,+). To test this hypothesis, we analyzed the saturation kinetics, time dependence, temperature dependence, and substrate specificity of Hcy-S-Hg-S-Hcy transport in Madin-Darby canine kidney (MDCK) cells stably transfected with system b(0,+). MDCK cells are good models in which to study this transport because they do not express system b(0,+). Uptake of Hg(2+) was twofold greater in the transfectants than in wild-type cells. Moreover, the transfectants were more susceptible to the toxic effects of Hcy-S-Hg-S-Hcy than wild-type cells. Accordingly, our data indicate that Hcy-S-Hg-S-Hcy is transported by system b(0,+) and that this transporter likely plays a role in the nephropathy induced after exposure to Hg(2+). These data are the first to implicate a specific, luminal membrane transporter in the uptake and toxicity of mercuric conjugates of Hcy in any epithelial cell."
Homocysteine and the renal epithelial transport and toxicity of inorganic mercury: role of basolateral transporter organic anion transporter 1.Zalups RK, Ahmad S. J Am Soc Nephrol. 2004 Aug;15(8):2023-31.
"The epithelial cells that line the renal proximal tubule have been shown to be the primary cellular targets where mercuric ions gain entry, accumulate, and induce pathologic effects in vivo. Recent data have implicated at least one of the organic anion transport systems in the basolateral uptake of inorganic mercury (Hg). With the use of a line of type II MDCK cells transfected stably with the human organic anion transporter 1 (hOAT1), the hypothesis that hOAT1 can transport mercuric conjugates of homocysteine (Hcy) was tested. Indeed, MDCK II cells expressing a functional form of hOAT1 gained the ability to transport the mercuric conjugate 2-amino-4-(3-amino-3-carboxy-propylsulfanylmercuricsulfanyl) butyric acid (Hcy-S-Hg-S-Hcy). In addition, p-aminohippurate and the dicarboxylates adipate and glutarate (but not succinate or malonate) inhibited individually the uptake of Hcy-S-Hg-S-Hcy in a concentration-dependent manner. Furthermore, a direct relationship between the uptake of Hcy-S-Hg-S-Hcy and the induction of cellular injury and death was demonstrated in the hOAT1-expressing MDCK II cells only. These data represent the first line of direct evidence implicating one of the organic anion transporters in the uptake of a mercuric conjugate of Hcy in a mammalian cell. Thus, mercuric conjugates of Hcy are potential transportable substrates of OAT1. More important, the findings from the present study implicate the activity of OAT1 in the uptake and toxicity of Hg (when in the form of Hcy-S-Hg-S-Hcy in the extracellular compartment) in proximal tubular epithelial cells in vivo."
Human organic anion transporter 1 mediates cellular uptake of cysteine-S conjugates of inorganic mercury.Zalups RK, Aslamkhan AG, Ahmad S. Kidney Int. 2004 Jul;66(1):251-61. [abstract only]
"BACKGROUND: The epithelial cells lining the renal proximal tubule have been shown to be the primary cellular targets where mercuric ions gain entry, accumulate, and induce pathologic effects in vivo. Recent data have implicated at least one of the organic anion transport systems in the basolateral uptake of inorganic mercury (Hg(2+)).
METHODS: Using a line of Madin-Darby canine kidney (MDCK) II cells transfected stably with the human organic anion transporter 1 (hOAT1), and oocytes from Xenopus laevis microinjected with cRNA for hOAT1, we tested the hypothesis that hOAT1 can transport biologically relevant mercuric conjugates of cysteine (Cys).
RESULTS: Indeed, MDCK II cells expressing a functional form of hOAT1 gained the ability to transport the mercuric conjugate 2-Amino-3-(2-amino-2-carboxy-ethylsulfanyl-mercuricsulfanyl)-propionic acid (Cys-S-Hg-S-Cys), but not the corresponding di-glutathione S-conjugate of Hg(2+) (G-S-Hg-S-G). Moreover, p-aminohippurate (PAH), adipate, and glutarate (but not succinate or malonate) inhibited individually the uptake of Cys-S-Hg-S-Cys in a dose-dependent manner. Uptake of Cys-S-Hg-S-Cys, but not G-S-Hg-S-G, was also documented in Xenopus oocytes expressing hOAT1.
CONCLUSION: These data represent ostensibly the most direct line of evidence implicating a specific membrane protein (i.e., hOAT1) in the transport of a biologically relevant molecular species of Hg(2+) in a mammalian cell. Moreover, these data indicate that the organic anion transporter(s) likely play a prominent role in the basolateral transport of mercuric ions by proximal tubular cells and in the nephropathy induced by Hg(2+)."
Mercuric conjugates of cysteine are transported by the amino acid transporter system b(0,+): implications of molecular mimicry.Bridges CC, Bauch C, Verrey F, Zalups RK. J Am Soc Nephrol. 2004 Mar;15(3):663-73.
"Humans and other mammals continue to be exposed to various forms of mercury in the environment. The kidneys, specifically the epithelial cells lining the proximal tubules, are the primary targets where mercuric ions accumulate and exert their toxic effects. Although the actual mechanisms involved in the transport of mercuric ions along the proximal tubule have not been defined, current evidence implicates mercuric conjugates of cysteine, primarily 2-amino-3-(2-amino-2-carboxyethylsulfanylmercuricsulfanyl)propionic acid (Cys-S-Hg-S-Cys), as the most likely transportable species of inorganic mercury (Hg(2+)). Because Cys-S-Hg-S-Cys and the amino acid cystine (Cys-S-S-Cys) are structurally similar, it was hypothesized that Cys-S-Hg-S-Cys might act as a molecular mimic of cystine at one or more of the amino acid transporters involved in the luminal absorption of this amino acid. One such candidate is the Na(+)-independent heterodimeric transporter system b(0,+). Therefore, the transport of Cys-S-Hg-S-Cys and cystine was studied in MDCK II cells that were or were not stably transfected with b(0,+)AT-rBAT. Transport of Cys-S-Hg-S-Cys and cystine across the luminal plasma membrane was similar in the transfected cells, indicating that Cys-S-Hg-S-Cys can behave as a molecular mimic of cystine at the site of system b(0,+). Moreover, only the b(0,+)AT-rBAT transfectants became selectively intoxicated during exposure to Cys-S-Hg-S-Cys. These findings indicate that system b(0,+) likely contributes to the nephropathy induced by Hg(2+) in vivo. These data represent the first direct molecular evidence for the participation of a specific transporter in the luminal uptake of a large divalent metal cation in proximal tubular cells."
Human renal organic anion transporter 1-dependent uptake and toxicity of mercuric-thiol conjugates in Madin-Darby canine kidney cells.Aslamkhan AG, Han YH, Yang XP, Zalups RK, Pritchard JB. Mol Pharmacol. 2003 Mar;63(3):590-6.
"Mercuric ions are highly reactive and form a variety of organic complexes or conjugates in vivo. The renal proximal tubule is a primary target for mercury uptake and toxicity, and circumstantial evidence implicates organic anion transporters in these processes. To test this hypothesis directly, the transport and toxicity of mercuric-thiol conjugates were characterized in a Madin-Darby canine kidney cell line stably transfected with the human organic anion transporter 1 (hOAT1). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bromide assays (for mitochondrial dehydrogenase) confirmed that mercuric conjugates of the thiols N-acetylcysteine (NAC), cysteine, or glutathione were more toxic in hOAT1-transfected cells than in the nontransfected cells. The NAC-Hg(2+) conjugate was most cytotoxic, inducing greater than 50% cellular death over 18 h at a concentration of 100 microM. The cytotoxic effects were fully reversed by probenecid (an OAT1 inhibitor) and partially reversed by p-aminohippurate (an OAT1 substrate). Toxicity of this conjugate was reduced by the OAT1-exchangeable dicarboxylates alpha-ketoglutarate, glutarate, and adipate, but not by succinate, a nonexchangeable dicarboxylate. (203)Hg-uptake studies showed probenecid-sensitive uptake of mercury-thiol conjugates in the hOAT1-transfected cells. The apparent K(m) for the NAC-Hg(2+) conjugate was 44 +/- 9 microM. Uptake of the NAC-Hg(2+) conjugate was cis-inhibited by glutarate, but not by methylsuccinate, paralleling their effects on toxicity. Probenecid-sensitive transport of the NAC-Hg(2+) conjugate was also shown to occur in Xenopus laevis oocytes expressing the hOAT1 or the rOAT3 transporters, suggesting that OAT3 may also transport thiol-Hg(2+) conjugates. Thus, renal accumulation and toxicity of thiol-Hg(2+) conjugates may depend in part on the activity of the organic transport system."
Simultaneous coexposure to inorganic mercury and cadmium: a study of the renal and hepatic disposition of mercury and cadmium.Zalups RK, Barfuss DW. J Toxicol Environ Health A. 2002 Oct 11;65(19):1471-90. [abstract only]
"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."
Renal organic anion transport system: a mechanism for the basolateral uptake of mercury-thiol conjugates along the pars recta of the proximal tubule.Zalups RK, Barfuss DW. Toxicol Appl Pharmacol. 2002 Aug 1;182(3):234-43. [abstract only]
"The basolateral handling of 20 microM inorganic mercury (Hg(2+)), in the form of mercuric conjugates of cysteine (Cys), N-acetylcysteine (NAC), or glutathione (GSH), was studied in isolated perfused S2 segments of the rabbit proximal tubule. One of the primary aims of the present study was to determine in a direct manner whether basolateral uptake of Hg(++) occurs in the pars recta of the proximal tubule and, more importantly, whether the p-aminohippurate-sensitive (PAH) organic anion transport system is involved in this process. Basolateral uptake and accumulation of Hg(++) occurred when the basolateral membrane of the tubular segments was exposed to mercuric conjugates of Cys, NAC, or GSH. Net basolateral uptake of Hg(++) was more than twice as great in the tubules exposed to mercuric conjugates of Cys or NAC than in the tubules exposed to mercuric conjugates of GSH, indicating that mercuric conjugates of Cys or NAC are transported more efficiently than mercuric conjugates of GSH. When PAH (1 mM) was added to the basolateral compartment (bath) surrounding a perfused S2 segment, the net uptake of Hg(++) (in the form of the mercuric conjugates) was reduced by 60-70%. In addition, when glutarate (4 mM), a transportable substrate for both the sodium-dependent dicarboxylate transporter and the dicarboxylate/organic anion exchanger (OAT1), was added to the basolateral compartment, there was a significant reduction in the uptake and accumulation of Hg(++) in the form of mercuric conjugates of Cys. Overall, these data indicate that Hg(++), in the form of biologically relevant mercuric conjugates of Cys, NAC, or GSH, is taken up significantly at the basolateral membrane of pars recta segments of the proximal tubule, and this uptake is mediated mainly by the actions of the PAH-sensitive organic anion transport system."
Amino acid transporters involved in luminal transport of mercuric conjugates of cysteine in rabbit proximal tubule.Cannon VT, Zalups RK, Barfuss DW. J Pharmacol Exp Ther. 2001 Aug;298(2):780-9.
"The primary aim of the present study was to test the hypothesis that amino acid transport systems are involved in absorptive transport of dicysteinylmercury (cysteine-Hg-cysteine). Luminal disappearance flux [JD, fmol x min(-1) (mm tubular length)(-1)] of inorganic mercury (Hg2+), in the form of dicysteinylmercury, was measured in isolated perfused S2 segments with various amino acids or amino acid analogs in the luminal compartment under one of two conditions, in the presence or absence of Na+. The control perfusion fluid contained 20 microM dicysteinylmercury. Replacing Na+ in both the bathing and perfusing solutions with N-methyl-D-glucamine reduced the JD of Hg2+ by about 40%. Nine amino acids and two amino acid analogs were coperfused individually (at millimolar concentrations) with dicysteinylmercury. The amino acids and amino acid analogs that had the greatest effect on the JD of Hg2+ were L-cystine, L-serine, L-histidine, L-tryptophan, and 2-(-)-endoamino-bicycloheptane-2-carboxylic acid. The greatest reduction (76%) in the total JD of Hg2+ occurred when L-cystine was coperfused with dicysteinylmercury in the presence of Na+. Overall, the current findings indicate that Hg2+ is transported from the lumen into proximal tubular epithelial cells via amino acid transporters that recognize dicysteinylmercury. In addition, the data indicate that multiple amino acid transporters are involved in the luminal uptake of dicysteinylmercury, including the Na+-dependent low-affinity L-cystine, B(0), and ASC systems and the Na+-independent L-system. Furthermore, the transport data obtained when L-cystine was added to the luminal fluid indicate strongly that dicysteinylmercury is likely transported as a molecular homolog of L-cystine."
Temporal changes in metallothionein gene transcription in rat kidney and liver: relationship to content of mercury and metallothionein protein.Zalups RK, Koropatnick J. J Pharmacol Exp Ther. 2000 Oct;295(1):74-82.
"Metallothioneins are encoded by a family of genes that are induced by inorganic mercury. Despite the well-characterized acute response of metallothionein (MT) genes in the kidneys and liver after a single exposure to inorganic mercury, relatively little is known about the activity of these genes and the content of MT protein during prolonged periods after exposure. Rats treated with inorganic mercury accumulate mercury rapidly in kidneys and liver during the first 24 h after exposure, but only in the kidneys does the content of mercury remain elevated throughout the initial 2 weeks. We report herein that transcription of MT genes in response to treatment with inorganic mercury differs dramatically between the kidneys and liver. MT gene transcription and levels of MT protein remained elevated in the kidneys throughout 14 days after treatment. In contrast, the initially high rates of MT gene transcription and enhanced content of MT protein in the liver fell to control levels by 14 days. In the liver, the rates of MT gene transcription and levels of MT protein were strongly correlated with each other and with the content of mercury. In the kidneys, however, these correlations were very weak or absent. Our data indicate that hepatic levels of MT protein are determined primarily by MT gene transcription, but that post-transcriptional events are important in determining the renal content of MT protein during the initial weeks after exposure. This has important implications in understanding differences in mechanisms controlling MT expression in the kidneys and liver."
Molecular homology and the luminal transport of Hg2+ in the renal proximal tubule.Cannon VT, Barfuss DW, Zalups RK. J Am Soc Nephrol. 2000 Mar;11(3):394-402.
"The aim of this study was to define mechanisms involved in the luminal uptake of inorganic mercury in the kidney using isolated perfused straight (S2) segments of the proximal tubule. When mercuric conjugates of glutathione (GSH), cysteinylglycine. or cysteine (containing 203Hg2+) were perfused through the lumen, the rates of luminal disappearance flux (JD) of inorganic mercury were approximately 39, 53, and 102 fmol/min per' min, respectively. Thus, the rates of luminal uptake of mercury are greater when the mercury is in the form of a mercuric conjugate of cysteine than in the form of a mercuric conjugate of cysteinylglycine or GSH. Addition of acivicin to the perfusate, to inhibit activity of the y-glutamyltransferase, caused significant reductions in the J,, for mercury in tubules perfused with mercuric conjugates of GSH. Addition of cilastatin, an inhibitor of dehydropeptidase- l (cysteinylglycinase) activity, caused significant reductions in the uptake of mercury in tubules perfused with mercuric conjugates of cysteinylglycine. These findings indicate that a significant amount of the luminal uptake of mercury, when mercuric conjugates of GSH are present in the lumen, is dependent on the activity of both y-glutamyltransferase and cysteinylglycinase. Finally, the JD for mercury in tubules perfused with mercuric conjugates of cysteine was reduced by approximately 50% when 3.0 mM L-lysine or 5.0 mM cycloleucine was added to the perfusate. It is concluded that these findings indicate that at least some of the luminal uptake of mercuric conjugates of cysteine occurs at the site of one or more amino acid transporters via a mechanism involving molecular homology."
Molecular interactions with mercury in the kidney.Zalups RK. Pharmacol Rev. 2000 Mar;52(1):113-43. Review.
"Mercury is unique among the heavy metals in that it can exist in several physical and chemical forms, including elemental mercury, which is a liquid at room temperature. All forms of mercury have toxic effects in a number of organs, especially in the kidneys. Within the kidney, the pars recta of the proximal tubule is the most vulnerable segment of the nephron to the toxic effects of mercury. The biological and toxicological activity of mercurous and mercuric ions in the kidney can be defined largely by the molecular interactions that occur at critical nucleophilic sites in and around target cells. Because of the high bonding affinity between mercury and sulfur, there is particular interest in the interactions that occur between mercuric ions and the thiol group(s) of proteins, peptides and amino acids. Molecular interactions with sulfhydryl groups in molecules of albumin, metallothionein, glutathione, and cysteine have been implicated in mechanisms involved in the proximal tubular uptake, accumulation, transport, and toxicity of mercuric ions. In addition, the susceptibility of target cells in the kidneys to the injurious effects of mercury is modified by a number of intracellular and extracellular factors relating to several thiol-containing molecules. These very factors are the theoretical basis for most of the currently employed therapeutic strategies. This review provides an update on the current body of knowledge regarding the molecular interactions that occur between mercury and various thiol-containing molecules with respect to the mechanisms involved in the renal cellular uptake, accumulation, elimination, and toxicity of mercury."
Relationships between alterations in glutathione metabolism and the disposition of inorganic mercury in rats: effects of biliary ligation and chemically induced modulation of glutathione status.Zalups RK, Barfuss DW, Lash LH. Chem Biol Interact. 1999 Dec 15;123(3):171-95. [abstract only]
"Influences of biliary ligation and systemic depletion of glutathione (GSH) or modulation of GSH status on the disposition of a low, non-nephrotoxic i.v. dose of inorganic mercury were evaluated in rats in the present study. Renal and hepatic disposition, and the urinary and fecal excretion, of inorganic mercury were assessed 24 h after the injection of a 0.5-micromol/kg dose of mercuric chloride in control rats and rats pretreated with acivicin (two 10-mg/kg i.p. doses in 2 ml/kg normal saline, 90 min apart, 60 min before mercuric chloride), buthionine sulfoximine (BSO; 2 mmol/kg i.v. in 4 ml/kg normal saline, 2 h before mercuric chloride) or diethylmaleate (DEM; 3.37 mmol/kg i.p. in 2 ml/kg corn oil, 2 h before mercuric chloride) that either underwent or did not undergo acute biliary ligation prior to the injection of mercury. Among the groups that did not undergo biliary ligation, the pretreatments used to alter GSH status systemically had varying effects on the disposition of inorganic mercury in the kidneys, liver, and blood. Biliary ligation caused the net renal accumulation of mercury to decrease under all pretreatment conditions. By contrast, biliary ligation caused significant increases in the hepatic burden of mercury in all pretreatment groups except in theacivicin-pretreated group. Blood levels of mercury also increased as a result of biliary ligation, regardless of the type of pretreatment used. The present findings indicate that biliary ligation combined with methods used to modulate GSH status systemically have additive effects with respect to causing reductions in the net renal accumulation of mercury. Additionally, the findings indicate that at least some fraction of the renal accumulation of inorganic mercury is linked mechanistically to the hepato-biliary system."
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