Gene transfer by polyoma-like particles assembled in a cell-free system.

Empty capsids of polyoma virus interact with DNA in a cell-free system to form polyoma-like particles (PLP). The DNA in these particles is protected from the action of pancreatic deoxyribonuclease. Transfer of genetic information by PLP has been accomplished by using a restriction fragment containing the transforming sequences of polyoma DNA as a model gene. Infection of rat F111 cells by PLP containing these sequences results in DNA-mediated cellular transformation. Gene transfer by PLP is 50 to 150 times more efficient than by the calcium phosphate precipitation method.

Urinary excretion of meso-2,3-dimercaptosuccinic acid in human subjects.

The urinary excretion of meso-2,3-dimercaptosuccinic acid (DMSA), which is an effective chelating agent for lead, was determined after the oral administration of 10 mg DMSA/kg to six normal young men. The DMSA that was absorbed was extensively biotransformed. After 14 hours only 2.53% of the administered DMSA was excreted in the urine as unaltered DMSA and 18.1% as altered forms. The unaltered DMSA was 12% of the total DMSA found in the urine. The altered form(s) of DMSA was 88% of the total urinary DMSA. The altered DMSA can be converted to unaltered DMSA by electrolytic reduction, which indicates that the altered forms of DMSA are disulfides. The excretion of altered DMSA reached a peak between 2 and 4 hours after DMSA administration. There were small but statistically significant increases in the excretion of zinc, copper, and lead after DMSA administration. DMSA did not influence the urinary excretion of 27 other metals and elements.

Determination and metabolism of dithiol chelating agents. IV. Urinary excretion of meso-2,3-dimercaptosuccinic acid and mercaptosuccinic acid in rabbits given meso-2,3-dimercaptosuccinic acid.

The water-soluble dithiol chelating agents meso-2,3-dimercaptosuccinic acid (DMSA) and 2,3-dimercaptopropane-1-sulfonic acid (DMPS) are becoming of increasing importance for the treatment of lead, arsenic and mercury poisoning. There is, however, a paucity of data about their metabolic transformation. Male rabbits were given DMSA (0.20 mmol/kg) i.m., and urine was collected over a 6-hr period. Monobromobimane derivatization, HPLC separation, and fluorescence detection, along with [U-14C]DMSA data, demonstrated that the total 14C found in the urine was distributed as 73% unaltered DMSA, 7% mercaptosuccinic acid and 6 and 14% of two unknowns. Electrolytic reductive treatment of the urine did not increase the urinary content of DMSA, indicating that oxidative biotransformation is not a major pathway for DMSA in the rabbit. This latter result is strikingly different from that for DMPS in rabbit.

Mobilization of mercury and arsenic in humans by sodium 2,3-dimercapto-1-propane sulfonate (DMPS).

Sodium 2,3-dimercapto-1-propane sulfonate (DMPS, Dimaval) is a water-soluble chelating agent that can be given by mouth or systemically and has been used to treat metal intoxication since the 1960s in the former Soviet Union and since 1978 in Germany. To better approximate the body burdens of Hg and As in humans, DMPS-Hg andDMPS-As challenge tests have been developed. The tests involve collecting an overnight urine, administering 300 mg DMPS at zero time, collecting the urine from 0 to 6 hr, and determining the urinary Hg before and after DMPS is given. The challenge test, when applied to normal college student volunteers with and without amalgam restorations in their mouths, indicated that two-thirds of the Hg excreted in the urine after DMPS administration originated in their dental amalgams. In addition, there was a positive linear correlation between the amalgam score (a measure of amalgam surface) and urinary Hg after the challenge test. When the DMPS-Hg challenge test was used to study dental personnel occupationally exposed to Hg, the urinary excretion of Hg was 88, 49, and 35 times greater after DMPS administration than before administration in 10 dental technicians, 5 dentists, and 13 nondental personnel, respectively. DMPS also was used to measure the body burden of humans with a history of drinking water containing 600 microgram As/liter. DMPS administration resulted in a tripling of the monomethylarsonic acid percentage and a halving of the dimethylarsinic acid percentage as related to total urinary As. Because South American animals studied were deficient in arsenite methyltransferase, a hypothesis is presented that arsenite and arsenite methyltransferase may have had a role in the evolution of some South American animals.

Determination of monomethylarsonous acid, a key arsenic methylation intermediate, in human urine.

In this study we report on the finding of monomethylarsonous acid [MMA(III)] in human urine. This newly identified arsenic species is a key intermediate in the metabolic pathway of arsenic biomethylation, which involves stepwise reduction of pentavalent to trivalent arsenic species followed by oxidative addition of a methyl group. Arsenic speciation was carried out using ion-pair chromatographic separation of arsenic compounds with hydride generation atomic fluorescence spectrometry detection. Speciation of the inorganic arsenite [As(III)], inorganic arsenate [As(V)], monomethylarsonic acid [MMA(V)], dimethylarsinic acid [DMA(V)], and MMA(III) in a urine sample was complete in 5 min. Urine samples collected from humans before and after a single oral administration of 300 mg sodium 2,3-dimercapto-1-propane sulfonate (DMPS) were analyzed for arsenic species. MMA(III) was found in 51 out of 123 urine samples collected from 41 people in inner Mongolia 0-6 hr after the administration of DMPS. MMA(III )in urine samples did not arise from the reduction of MMA(V) by DMPS. DMPS probably assisted the release of MMA(III) that was formed in the body. Along with the presence of MMA(III), there was an increase in the relative concentration of MMA(V) and a decrease in DMA(V) in the urine samples collected after the DMPS ingestion.

Arsenic: health effects, mechanisms of actions, and research issues.

A meeting on the health effects of arsenic (As), its modes of action, and areas in need of future research was held in Hunt Valley, Maryland, on 22-24 September 1997. Exposure to As in drinking water has been associated with the development of skin and internal cancers and noncarcinogenic effects such as diabetes, peripheral neuropathy, and cardiovascular diseases. There is little data on specific mechanism(s) of action for As, but a great deal of information on possible modes of action. Although arsenite [As(III)] can inhibit more than 200 enzymes, events underlying the induction of the noncarcinogenic effects of As are not understood. With respect to carcinogenicity, As can affect DNA repair, methylation of DNA, and increase radical formation and activation of the protooncogene c-myc, but none of these potential pathways have widespread acceptance as the principal etiologic event. In addition, there are no accepted models for the study of As-induced carcinogenesis. At the final meeting session we considered research needs. Among the most important areas cited were a) As metabolism and its interaction with cellular constituents; b) possible bioaccumulation of As; c) interactions with other metals; d) effects of As on genetic material; e) development of animal models and cell systems to study effects of As; and f) a better characterization of human exposures as related to health risks. Some of the barriers to the advancement of As research included an apparent lack of interest in the United States on As research; lack of relevant animal models; difficulty with adoption of uniform methodologies; lack of accepted biomarkers; and the need for a central storage repository for stored specimens.

Binding of polonium-210 to liver metallothionein.

The interactions of 210Po at the molecular level in biological systems have received little study even though this alpha-emitting radionuclide occurs widely in nature. Polonium-210 was given subcutaneously to rats and found to be incorporated into liver metallothionein as judged by a number of criteria including heat stability, acetone precipitation, and chromatography. In vitro studies confirmed this binding. The binding of 210Po to metallothionein has implications that may help explain some of the radiation damage 210Po causes intracellularly.

The enigma of arsenic carcinogenesis: role of metabolism.

Inorganic arsenic is considered a high-priority hazard, particularly because of its potential to be a human carcinogen. In exposed human populations, arsenic is associated with tumors of the lung, skin, bladder, and liver. While it is known to be a human carcinogen, carcinogenesis in laboratory animals by this metalloid has never been convincingly demonstrated. Therefore, no animal models exist for studying molecular mechanisms of arsenic carcinogenesis. The apparent human sensitivity, combined with our incomplete understanding about mechanisms of carcinogenic action, create important public health concerns and challenges in risk assessment, which could be met by understanding the role of metabolism in arsenic toxicity and carcinogenesis. This symposium summary covers three critical major areas involving arsenic metabolism: its biodiversity, the role of arsenic metabolism in molecular mechanisms of carcinogenesis, and the impact of arsenic metabolism on human risk assessment. In mammals, arsenic is metabolized to mono- and dimethylated species by methyltransferase enzymes in reactions that require S-adenosyl-methionine (SAM) as the methyl donating cofactor. A remarkable species diversity in arsenic methyltransferase activity may account for the wide variability in sensitivity of humans and animals to arsenic toxicity. Arsenic interferes with DNA methyltransferases, resulting in inactivation of tumor suppressor genes through DNA hypermethylation. Other studies suggest that arsenic-induced malignant transformation is linked to DNA hypomethylation subsequent to depletion of SAM, which results in aberrant gene activation, including oncogenes. Urinary profiles of arsenic metabolites may be a valuable tool for assessing human susceptibility to arsenic carcinogenesis. While controversial, the idea that unique arsenic metabolic properties may explain the apparent non-linear threshold response for arsenic carcinogenesis in humans. In order to address these outstanding issues, further efforts are required to identify an appropriate animal model to elucidate carcinogenic mechanisms of action, and to define dose-response relationships.

Biological chelation: 2,3-dimercapto-propanesulfonic acid and meso-dimercaptosuccinic acid.

Water soluble analogs of British Anti-Lewisite that are active orally and less toxic than BAL are now available. These agents are 2,3-dimercapto-1-propanesulfonic acid and meso-dimercaptosuccinic acid. Evidence for their effectiveness in preventing the lethal effects of sodium arsenite in mice and lewisite in rabbits is presented. These analogs can be expected to replace BAL in the treatment of heavy metal poisoning.

Arsenic binding proteins of mammalian systems: I. Isolation of three arsenite-binding proteins of rabbit liver.

It is well known that arsenite/arsenate (As3+/As5+) administered to rabbits is bound initially to cellular proteins of the liver before methylated arsenic metabolites appear in urine. This protein binding may decrease the in situ toxicity of inorganic arsenic by decreasing its metabolic availability until it is methylated enzymatically. We have investigated the binding of As3+ and As5+ to the cytosolic proteins of rabbit liver. The results indicate that when cytosolic proteins are incubated with inorganic arsenic, the amount of As3+ bound is 13 times greater than that for As5+. Arsenite-specific binding sites on cytosolic proteins were determined to be 67% of the total (specific and non-specific) number of possible binding sites. Ammonium sulfate fractionation, non-denaturing PAGE and gel filtration chromatography indicate that three liver proteins with molecular weights of 100 kDa, 450 kDa and > 2000 kDa strongly bind arsenite. The radioactive profiles after gel filtration chromatography of liver cytosolic proteins are very similar whether As3+ binding occurs in vitro or in vivo. Thus, the in vitro model appears to be valid for further study of these arsenite-binding proteins.

Sodium 2,3-dimercapto-1-propanesulfonate (DMPS) treatment does not redistribute lead or mercury to the brain of rat.

Since there has been concern about whether any of the chelating agents used therapeutically might cause an initial redistribution of heavy metals to the brain and since the sodium salt of 2,3-dimercapto-1-propanesulfonic acid (Dimaval, DMPS) has been used to treat heavy metal intoxication in humans, the hypothesis that DMPS does not redistribute and increase lead or mercuric ions in the brains of rats was tested. Lead acetate at a concentration of 50 mg/l was made available in the drinking water of rats for 86 days. Other rats received intraperitoneal injections of 0.50 mg Hg/kg (as mercuric chloride) each day for 5 days a week for a total of 32 or 41 days. Animals were divided into groups and given, i.p., either 0.27 mmol DMPS/kg body weight or saline, each day for 1, 2, 3 or 4 days. Lead or mercury concentrations of the brain were determined after each group received DMPS for the different number of days. DMPS treatment did not result in any initial increase of lead or mercuric ions in the brain. The mercury content of the kidney decreased. The results of these experiments demonstrated that lead or mercuric ions were not redistributed to or increased in the brains of rats during the initial days of DMPS treatment.

Utilization of renal slices to evaluate the efficacy of chelating agents for removing mercury from the kidney.

Mercury is an environmental contaminant that preferentially accumulates in the kidney. It has been previously shown using proton-induced X-ray emission analysis that mercury (HgCl2) accumulated in precision-cut rabbit renal cortical slices. In this study, the efficacy of seven chelating agents for the removal of Hg from renal slices has been examined. Rabbits were injected with HgCl2 (10 mg/kg) and 3 h later kidneys were sliced, or renal slices were exposed in vitro to a mildly toxic concentration of HgCl2 (5 x 10(-5)M, 4 h). The slices were then treated in vitro with 10 mM concentrations of EDTA, lipoic acid (LA), penicillamine (PA), glutathione (GSH), 1,4-dithiothreitol (DTT), DMSA, or DMPS. DMPS proved to be the most effective in mobilizing Hg from in vivo or in vitro HgCl2-exposed renal tissue ( > 85% of control after 3 h incubation). Relative efficacies for the seven agents were DMPS > DMSA, PA > DTT, GSH > LA, EDTA. The use of renal slices appears to be a useful in vitro tool for assessing the efficacy of chelating agents on mobilizing accumulated Hg from renal tissue.

Mobilization of heavy metals by newer, therapeutically useful chelating agents.

Four chelating agents that have been used most commonly for the treatment of humans intoxicated with lead, mercury, arsenic or other heavy metals and metalloids are reviewed as to their advantages, disadvantages, metabolism and specificity. Of these, CaNa2EDTA and dimercaprol (British anti-lewisite, BAL) are becoming outmoded and can be expected to be replaced by meso-2,3-dimercaptosuccinic acid (DMSA, succimer) for treatment of lead intoxication and by the sodium salt of 2,3-dimercapto-1-propanesulfonic acid (DMPS, Dimaval) for treating lead, mercury or arsenic intoxication. Meso-2,3-DMSA and DMPS are biotransformed differently in humans. More than 90% of the DMSA excreted in the urine is found in the form of a mixed disulfide in which each of the sulfur atoms of DMSA is in disulfide linkage with an L-cysteine molecule. After DMPS administration, however, acyclic and cyclic disulfides of DMPS are found in the urine. The Dimaval-mercury challenge test holds great promise as a diagnostic test for mercury exposure, especially for low level mercurialism. Urinary mercury after Dimaval challenge may be a better biomarker of low level mercurialism than unchallenged urinary mercury excretion.

Central nervous system toxicity of manganese. II: Cocaine or reserpine inhibit manganese concentration in the rat brain.

Manganese concentrates in the ventral mesencephalon of male Sprague-Dawley rats after intrathecal administration of MnCl2. We tested the hypothesis that Mn concentration in the central nervous system (CNS), particularly in the ventral mesencephalon, is decreased by inhibiting dopamine reuptake using cocaine or by decreasing dopamine concentrations using reserpine. The intrathecal administration of Mn (250 micrograms Mn/rat as MnCl2) caused the Mn concentration in the ventral mesencephalon to increase from 0.57 to 31.8 micrograms Mn/g. Cocaine administration (8.6 mg/kg i.p.) thirty minutes prior to MnCl2 decreased ventral mesencephalon Mn to 3.3 micrograms Mn/g. By giving reserpine (5 mg/kg i.p.) 24 hours prior to MnCl2 the ventral mesencephalon Mn concentration was decreased from 29.9 micrograms Mn/g to 3.7 micrograms Mn/g. Intrathecal MnCl2 decreased the dopamine concentration in the caudate putamen by 40% six hours after administration. Cocaine or reserpine decreased the Mn concentration in the ventral mesencephalon, occipital pole, frontal lobe and caudate putamen but did not change the Mn concentration in the cerebellum. The results indicate that the mechanism(s) by which Mn is concentrated in many brain regions can be inhibited by cocaine, a dopamine reuptake inhibitor, or by reserpine, a dopamine depleter, and suggest that the Mn concentration in the CNS is related to dopamine reuptake and/or concentration.

Anti-lewisite activity and stability of meso-dimercaptosuccinic acid and 2,3-dimercapto-1-propanesulfonic acid.

Meso-dimercaptosuccinic acid (DMSA) and the sodium salt of 2,3-dimercapto-1-propanesulfonic acid (DMPS) are analogous in chemical structure to dimercaprol (BAL, British Anti-Lewisite). Dimercaprol was among the first therapeutically useful metal chelating agents and was developed originally as an anti-lewisite agent. Either DMSA or DMPS protects rabbits from the lethal systemic action of dichloro(2-chlorovinyl)arsine (29.7 mumols/kg, also known as lewisite. The analogs are active in this respect when given either sc or po. The stability of each of the three dimercapto compounds in distilled H2O, pH 7.0 at 24 degrees, has been examined for seven days. DMSA retained 82% of its mercapto groups, but no titratable mercapto groups remained in the DMPS or BAL solutions. At pH 5.0, however, there was no striking difference in the stability of the three dimercapto compounds (78-87%) over a seven day period. DMSA and DMPS warrant further investigation as water soluble metal binding agents in both in vivo and in vitro experiments.

Lead chelates of meso- and racemic-dimercaptosuccinic acid.

Lead chelates of racemic- and meso-dimercaptosuccinic acid (DMSA) were synthesized and isolated from aqueous solutions and characterized by potentiometric measurements and infrared spectroscopy. Two types of lead chelates of racemic DMSA were isolated: one in which racemic-DMSA is coordinated to Pb+2 via one oxygen and one sulfur atom and the other in which the Pb2+ is coordinated via two sulfur atoms. The latter form of the chelate is converted into the former upon dissolution in dimethylsulfoxide. Only one type of Pb2+ chelate of the meso form of the ligand was formed. In this case, meso-DMSA is coordinated to Pb+2 via one oxygen and one sulfur atom. Meso- and racemic-DMSA have very different solubilities in aqueous solutions. Meso-DMSA is slightly soluble in water, whereas racemic-DMSA is very soluble in water even in the presence of strong acids. The solubilities of the chelates were found to be pH dependent. When the uncoordinated sulfhydryl and carboxylic acid groups dissociate, the chelates dissolve and remain in aqueous solution. The infrared spectra of meso- and racemic-DMSA show distinct features that can be used to detect the presence of either diastereoisomer.

Enzymatic methylation of arsenic compounds: IV. In vitro and in vivo deficiency of the methylation of arsenite and monomethylarsonic acid in the guinea pig.

Using an in vitro assay which measures the transfer of a radiolabeled methyl moiety of S-[methyl-3H]adenosylmethionine ([3H]SAM) to arsenite or monomethylarsonate (MMA) to yield [methyl-3H]MMA or [methyl-3H]dimethylarsinate (DMA) respectively, guinea pig liver cytosol was found to be deficient in the enzyme activities which methylate these substrates. Moreover, when guinea pigs were given a single intraperitoneal dose of [73As]arsenate (400 micrograms/kg body weight, 25 microCi/kg body weight), very little or no methylated arsenic species were detected in the urine after cation exchange chromatography. The urine collected 0-12 h after arsenate injection contained 98% inorganic arsenic and less than 1% DMA. No MMA was detected in the 0-12 h urine. Urine collected 12-24 h after injection contained approximately 93% inorganic arsenic, 2% MMA and 3% DMA in five of the six animals studied. However, in the 12-24 h urine of one guinea pig, 17% of the radioactivity was DMA, 80% was inorganic arsenic and 3% was MMA. The guinea pig, like the marmoset and tamarin monkeys and unlike most other animals studied thus far, appears to be deficient as far as the enzyme activities that methylate inorganic arsenite. The results of these experiments suggest that there may be a genetic polymorphism associated with the enzymes that methylate inorganic arsenite.

Behavioral effects of low-level exposure to Hg0 among dental professionals: a cross-study evaluation of psychomotor effects.

A Across-study design was used to evaluate the sensitivities of five psychomotor tasks previously used to assess preclinical effects of low-level Hg0 (urinary < or =55 microg/l). Pooling dental professional subject populations from six studies conducted over the last 6 years, a larger study population was obtained with a high degree of uniformity (N = 230). The five psychomotor tests were: Intentional Hand Steadiness Test (IHST); Finger Tapping: The One-Hole Test: NES Simple Reaction Time (SRT); and Hand Tremor. Multivariate analyses were conducted following the hierarchical analysis of multiple responses (HAMR) approach. First, multiple scores of each test were combined into a single-factor (or related summary) variable and its reliability was estimated. Second. multiple regression analyses were conducted including log-transformed [Hg0]U levels, age, gender, and alcohol consumption in each model. Computed were both B and bu, the magnitudes of the log-Hg0 standardized coefficient. respectively uncorrected and corrected for dependent variable attenuation due to unreliability. Results indicated remarkable differences in the effects of relative level of Hg0 on psychomotor performance. Significant associations were found for the IHST factor (B = 0.415, p < 10(-6)), followed by finger tapping, which was relatively meager and insignificant (B 0.141, p = 0.17). The IHST results hold the greatest occupational relevance for dental professionals who rely on manual dexterity in restorative dentistry. Further, this statistical approach is recommended in future studies for condensation of multiple scores into summary scores with enhanced reliabilities useful in correcting for attenuation relationships (B(u)s) with exposure levels.

Diversity of inorganic arsenite biotransformation.

Biotransformation of inorganic arsenic in mammals is catalyzed by three serial enzyme activities: arsenate reductase, arsenite methyltransferase, and monomethylarsonate methyltransferase. Our laboratory has purified and characterized these enzymes in order to understand the mechanisms and elucidate the variations of the responses to arsenate/arsenite challenge. Our results indicate a marked deficiency and diversity of these enzyme activities in various animal species.