Nephrotoxicity of simultaneous exposure to mercury and uranium in comparison to individual effects of these metals in rats

Both inorganic mercury and uranium are known nephrotoxicants in mammals. In this study, the renal toxicity of a concurrent exposure to inorganic mercury and uranium was compared with the nephrotoxic effects of the individual metals in a rat model. Eight groups of rats, 10 animals per group, were subcutaneously given a single administration of mercuric chloride (HgCl₂, 0.34 mg/kg and 0.68 mg/kg), uranyl acetate dihydrate (UAD, 2.5 mg/kg and 5 mg/kg), or combinations of both compounds at the same doses. A ninth group of rats received sc injections of 0.9% saline and was designated as the control group. Necrosis of proximal tubules, which was observed in all experimental groups, was the most relevant morphologic abnormality. Marked changes, which were remarkably greater than those induced by the individual elements, were noted in some urinary parameters in the groups concurrently exposed to HgCl₂ and UAD. It could be an indicator of a synergistic interaction between mercury and uranium. In contrast, compared with the urinary levels found after individual administration of the highest doses of mercury and uranium, significant reductions in the urinary concentrations of these elements were noted following simultaneous exposure to both metals. At these doses, the reduction in the urinary metal excretion was also accompanied by significant decreases in the renal content of mercury and uranium. Whereas the results of some parameters pointed out a possible synergistic interaction between mercury and uranium, other measures hinted that an antagonistic interaction between these elements is also present.     

An evaluation ofmer-specified reduction of ionic mercury as a remedial tool of a mercury-contaminated freshwater pond

Summary The potential former-mediated reduction/volatilization of ionic mercury as a tool in the decontamination of a freshwater pond was evaluated using laboratory incubations and a microcosm simulation. In flask assays inoculations with ionic mercury-resistant bacteria (10⁵–10⁷ cells ml^–1) isolated from the pond, significantly increased the rate of mercury loss (MANOVA,P0.05) relative to uninoculated controls. The effects of cell density, mercuric mercury concentration, addition of nutrients and supplementation with the sulfhydryl reagent -mercaptoethanol on the rate of mercury loss, were investigated. Inoculation (by 10⁵ cells ml^–1) of a flow-through microcosm that simulated the cycling of mercury in the contaminated pond, stimulated by more than 4-fold the formation of volatile elemental mercury. Thus, biological formation of volatile mercury may hold a promise as a remedial tool of contaminated natural waters.Publication no. 906 of the US Environmental Research Laboratory, Gulf Breeze, FL 32561, USA.Mention of trade names or commercial products does not constitute endorsement on recommendation for use.     

Mercuric reductase in environmental Gram-positive bacteria sensitive to mercury

According to existing data, mercury resistance operons (mer operons) are in general thought to be rare in bacteria, other than those from mercury-contaminated sites. We have found that a high proportion of strains in environmental isolates of Gram-positive bacteria express mercuric reductase (MerA protein): the majority of these strains are apparently sensitive to mercury. The expression of MerA was also inducible in all cases. These results imply the presence of phenotypically cryptic mer resistance operons, with both the merA (mercuric reductase) and merR (regulatory) genes still present, but the possible absence of the transport function required to complete the resistance mechanism. This indicates that mer operons or parts thereof are more widely spread in nature than is suggested by the frequency of mercury-resistant bacteria.     

Mercury resistance in aromatic compound degrading Pseudomonas strains

Abstract: Six of ten Pseudomonas strains selected from environmental samples for their ability to degrade aromatic compounds were found to be mercury resistant. Mercury detoxification proceeded through Hg²⁺ volatilization and the genes involved were chromosomally located. All the mercury resistant strains proved able to degrade aromatic compounds in the presence of Hg²⁺.     

Inhibitory effects of HgCl2 on excitation-secretion coupling at the motor nerve terminal and excitation-contraction coupling in the muscle cell

Summary 1. Indirect and direct twitch (0.1-Hz) stimulation of the rat phrenic nerve-diaphragm disclosed that the inhibitory effect of HgCl₂, 3.7 × 10^–5M, on the neuromuscular transmission and in the muscle cell, was accelerated by 10-sec periods of 50-Hz tetanic stimulation every 10 min. This activity-dependent enhancement suggested an inhibitory mechanism of HgCl₂ related to the development of fatigue, like membrane depolarization or decreased excitability, decreased availability of transmitter, or interference with the factors controlling excitation-secretion coupling of the nerve terminal, i.e. (Ca²⁺)₀ or (Ca²⁺)_i, and excitation-contraction coupling in the muscle cell, i.e., (Ca²⁺)_i.2. During both indirect and direct stimulation, HgCl₂-induced inhibition was enhanced markedly by pretreatment with caffeine, which releases Ca²⁺ from endoplasmic and sarcoplasmic reticulum in the nerve terminal and muscle cell, respectively. This caffeine-induced enhancement was completely antagonized by dantrolene, which inhibits the caffeine-induced release. However, dantrolene alone did not antagonize the HgCl₂-induced inhibition.3. Since caffeine depletes the intracellular Ca²⁺ stores of the smooth endoplasmic reticulum, HgCl₂ probably inhibits by binding to SH groups of transport proteins conveying the messenger function of (Ca²⁺)_i. In the muscle cell this leads to inhibition of contraction. In the nerve terminal, an additional enhancement of the HgCl₂-induced inhibition, by inhibiting reuptake of choline by TEA and tetanic stimulation, suggested that HgCl₂ inhibited a (Ca²⁺)_i signal necessary for this limiting factor in resynthesis of acetylcholine.4. The (Ca²⁺)₀ signal necessary for stimulus-induced release of acetylcholine was not affected by HgCl₂. Hyperpolarization in K⁺-free solution antagonized the inhibitory effect of HgCl₂ at indirect stimulation, and Ca²⁺-free solution enhanced the inhibitory effect at direct stimulation. K⁺ depolarization, membrane electric field increase with high Ca²⁺, membrane stabilization with lidocaine, and half-threshold stimulation, did not change the inhibitory effect of HgCl CH₃HgCl, 1.85 × 10^–5M, disclosed a synergistic interaction with caffeine during direct, but not during indirect, stimulation.     

Comparison of the effectiveness of 2,3-dimercaptopropanol (BAL) andmeso-2,3-dimercaptosuccinic acid (DMSA) as protective agents against mercuric chloride-induced nephrotoxicity in rats

The effectiveness of 2,3-dimercaptopropanol (BAL) andmeso-2,3-dimercaptosuccinic acid (DMSA) on HgCl₂-induced nephrotoxicity was studied in the rat. Seven groups of adult male rats were given a single sc toxic dose of HgCl₂ (0.68 mg/kg) followed by 0.9% saline (positive control group), BAL (15, 30, and 60 mg/kg) or DMSA (50, 100, and 200 mg/kg) administered ip at 0, 24, 48, and 72 h thereafter. Although the renal function of HgCl₂-exposed rats was slightly improved after BAL administration, Hg concentrations in the kidney were only reduced at 60 mg/kg. In addition, the protective effect of BAL was not dose-related. In contrast to BAL, DMSA was effective in increasing the urinary excretion of Hg and in reducing the renal Hg content. These results show that DMSA would be more effective than BAL in preventing or in protecting against inorganic Hg-induced nephrotoxicity.     

Organomercurial resistance determinants in Pseudomonas K-62 are present on two plasmids

Pseudomonas strain K-62 was found to contain six plasmids. A mutant derivative cured of the 26-kb plasmid showed a higher sensitivity to mercurials; however, the strain was still able to volatilize them. Loss of the 68-kb plasmid.in addition to the 26-kb plasmid abolished the ability of mercury volatilization in this strain and led to a further decrease in the level of mercurial resistance. These results are the first to demonstrate that the organomercurial resistance of Pseudomonas strain K-62 is plasmid-based, and that both the 26- and 68-kb plasmids are required for full expression of the mercurial resistance. Probes specific for the mer genes merA, merB, and merR strongly hybridized with the 26-kb plasmid, but not with the 68-kb plasmid. Two fragments of the 26-kb plasmid that hybridized with the mer genes were cloned and expressed in Escherichia coli. One recombinant plasmid (pMRA17) inducibly encoded a typical broad-spectrum mercurial resistance, whereas the other recombinant plasmid (pMRB01) constitutively conferred hypersensitivity to phenylmercury in the absence of mercuric reductase activity. The results suggest that the two organomercurial lyases in the cells are transcribed from different operator-promoters.     

Functional studies of rat hydroxymethylbilane synthase

The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Hydroxymethylbilane synthase catalyzes a rate-limiting step for the biosyntheses of tetrapyrrolic natural products. We carried out extensive studies of rat hydroxymethylbilane synthase in the present investigation. The enzymatic reaction rate of the holoenzyme was found to be lower than those of the enzyme-intermediate complexes, which corrected the previous theoretical analysis result. Several mutants were constructed, purified and characterized. D44 was found to play an important role in the disassembly of the enzyme-intermediate complexes. E63 and H78 were important for maintaining the activity of the enzyme at high temperature. Four substrate analogs with variation of porphobilinogen side-chain were synthesized and incubated with the enzyme. Three analogs were found to be weak substrates of the enzyme. All four analogs can be used for the preparation of uroporphyrin I analogs.     

Lipid peroxidation in rats administrated with mercuric chloride

Parenteral administration of mercuric chloride (HgCl₂) to rats enhanced lipid peroxidation in liver, kidney, lung, testis, and serum (but not in heart, spleen, or muscle), as measured by the thiobarbituric acid reaction for malondialdehyde (MDA) in fresh tissue homogenates and body fluids. After sc injection of HgCl₂ (5 mg/kg body wt), MDA concentrations in liver and kidney became significantly increased by 9 h and reached peak values at 24 h. Dose-response studies were carried out with male albino rats of the Fisher-344 strain (body wt 170–280 g) injected with 1, 3, 5 mg Hg/kg as HgCl₂ and sacrificed after 24 h. In time-response studies, animals were administered 5 mg Hg/kg as HgCl₂ and sacrificed after 3, 9, 18, 24, and 48 h. Studies in the authors' laboratory have shown that (1) concentrations of MDA are increased in targets (liver, kidney, lung, and testis) of HgCl₂-treated rats; (2) severity of hepatotoxicity and nephrotoxicity is generally consistent with the elevation of Hg and MDA concentrations, based upon the time-course and dose-effect relationships observed after administration of HgCl₂ to rats; and (3) concentrations of MDA are reduced in target tissues after pretreatment with antioxidants and chelators to HgCl₂-treated rats. The results of this study implicate that the lipid peroxidation is one of the molecular mechanisms for cell injury in acute HgCl₂ poisoning.     

Lactating and non-lactating rats differ in sensitivity to HgCl2: Protective effect of ZnCl2

This work investigated zinc (Zn) and mercury (Hg) effects on oxidative parameters, markers of toxicity and metal levels in different tissues from non-lactating rats (NLR) and lactating rats (LR). Adult NLR and LR received ZnCl₂ (27 mg/kg) or saline (0.9%) subcutaneously and after 24 h they received HgCl₂ (5 mg/kg) or saline (0.9%). Twenty four hours later, they were sacrificed and the preparation of biological material and biochemical analyses were performed. With respect to oxidative parameters, Hg exposure decreased kidney total SH levels from NLR and LR and hepatic catalase activity (not statistically significant) in NLR. Zinc pre-treatment partly prevented the decrease of kidney total SH levels in LR. Zinc per se increased hepatic non-protein SH levels of NLR and LR. Regarding toxicity markers, Hg exposure inhibited the δ-aminolevulinic acid dehydratase (δ-ALA-D) activity from kidney and liver of NLR, inhibited serum alanine aminotransferase (ALT) activity of LR and increased serum creatinine and urea levels of NLR and LR. Zinc pre-exposure prevented the enzymatic alterations caused by Hg. NLR and LR Hg exposed presented accumulation of mercury in the kidney, liver, blood and urine. Zinc pre-treatment prevented this accumulation partly in NLR liver and blood and completely in LR kidney and liver. These results show that NLR and LR are differently sensitive to HgCl₂ and that ZnCl₂ showed a promising effect against Hg toxicity.     

Probing the active site of rat porphobilinogen synthase using newly developed inhibitors

The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Porphobilinogen synthase catalyzes a rate-limiting step for the biosyntheses of tetrapyrrolic natural products. In the present study, a variety of new substrate analogs and reaction intermediate analogs were synthesized, which were used as probes for studying the active site of rat porphobilinogen synthase. The compounds 1, 3, 6, 9, 14, 16, and 28 were found to be competitive inhibitors of rat porphobilinogen synthase with inhibition constants ranging from 0.96 to 73.04 mM. Compounds 7, 10, 12, 13, 15, 17, 18, and 26 were found to be irreversible enzyme inhibitors. For irreversible inhibitors, loose-binding inhibitors were found to give stronger inactivation. The amino group and carboxyl group of the analogs were found to be important for their binding to the enzyme. This study increased our understanding of the active site of porphobilinogen synthase.     

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans

Summary A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cm^s), arginine auxotrophic (Arg^–) mutant of Streptomyces lividan 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cm^s Arg^– mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.     

Endotoxin-challenges precocial neonates and iron changes

In adult mammals fever is associated with the reduction of blood plasma iron level. Immature mammals, however, show either a decrease (precocial animals such as guinea pig neonates) or a lack of reduction (altricial animals such as human neonates) of plasma iron in response to endotoxin. In order to determine whether this difference is connected with maturity just after delivery, plasma iron concentration, hematocrit, body temperature and body mass were measured in rat pups injected with E. coli endotoxin in doses of 50 or 200 μg kg⁻¹. Rat pups, like human neonates, are altricial animals. In 7-day-old rats injection of LPS led to a dose-dependent decrease in plasma iron level. The fall in plasma iron was accompanied by changes in body temperature and body mass. The results showed that plasma iron response to endotoxin in altricial rat neonates is similar to that observed in precocial guinea pig pups. Accepted: 4 October 1996