Arginine decarboxylase: A novel biological target of mercury compounds identified in PC12 cells.

Mercury compounds are well-known toxic environmental pollutants and potently induce severe neurotoxicological effects in human and experimental animals. Previous studies showed that one of the mechanisms of mercury compounds neurotoxicity arose from the over-activation of the N-methyl d-aspartate (NMDA)-type glutamate receptor induced by increased glutamate release. In this work, we aimed to investigate the molecular mechanisms of Hg compounds neurotoxicities by identifying their biological targets in cells. Firstly, the inhibitory effects of four Hg compounds, including three organic (methyl-, ethyl- and phenyl-mercury) and one inorganic (Hg2+) Hg compounds, on the activity of arginine decarboxylase (ADC), a key enzyme in the central agmatinergic system, were evaluated. They were found to inhibit the ADC activity significantly with methylmercury (MeHg) being the strongest (IC50=7.96nM). Furthermore, they showed remarkable inhibitory effects on ADC activity in PC12 cells (MeHg>EtHg>PhHg>HgCl2), and led to a marked loss in the level of agmatine, an endogenous neuromodulatory and neuroprotective agent that selectively blocks the activation of NMDA receptors. MeHg was detected in the immunoprecipitated ADC from the cells, providing unequivocal evidence for the direct binding of MeHg with ADC in the cell. Molecular dynamics simulation revealed that Hg compounds could form the coordination bond not only with cofactor PLP of ADC, but also with substrate arginine. Our finding indicated that MeHg could attenuate the neuroprotective effects of agmatine by the inhibition of ADC, a new cellular target of MeHg, which might be implicated in molecular mechanism of MeHg neurotoxicity.

Effect of mercury on the seminiferous epithelium of the fowl testis.

Phenylmercuric chloride was applied in three doses (5 ppm, 30 ppm Hg, and 30 ppm Hg + 4 ppm Se) via the food for 60 days. The effect of Hg with an without Se was studied histologically and the data of a shortened spermatogram were evaluated. Treatment with 30 ppm Hg resulted in hypospermia, occurrence of abnormally maturing spermatozoa, reduction of the volume of semen, and decrease in the number of spermatozoa. The dose of 5 ppm Hg only resulted in the appearance of abnormally developing cells and decreased sperm motility. The addition of Se maintained spermatogenesis and the values of semen on the control level.

Potentiating effects of organomercuries on clastogen-induced chromosome aberrations in cultured Chinese hamster cells.

Mercury compounds are among the most serious environmental pollutants. In this communication, the potentiating effects of organic and inorganic mercuries on clastogen-induced chromosome aberrations were studied in Chinese hamster CHO K1 cells. Post-treatment with monoalkylated mercuries--methyl mercuric chloride (MeHgCl) and ethyl mercuric chloride (EtHgCl)--increased the number of breakage- and exchange-type aberrations induced by 4-nitroquinoline 1-oxide (4NQO) and methyl methanesulfonate. With the DNA crosslinking agents mitomycin C (MMC) and cisplatin, MeHgCl enhanced both types of aberrations while EtHgCl enhanced breakage-type aberrations only. Since these monoalkylated mercuries did not show clastogenic effects by themselves under the present experimental conditions, the increases in chromosome aberrations were not additive. Dialkylated mercuries (dimethyl mercury and diethyl mercury) and inorganic mercuries (HgCl and HgCl2) did not show any potentiating effects. When MMC- or 4NQO-treated cells were post-treated with MeHgCl during the G1 phase, both breakage- and exchange-type aberrations were enhanced. Treatment with EtHgCl during the G1 phase also enhanced both types of aberrations induced by 4NQO. With MMC, however, G1 treatment with EtHgCl did not show any potentiating effect. MeHgCl and EtHgCl treatments during the G2 phase enhanced breakage-type aberrations only. Based on these results, the following possible mechanisms for potentiation of clastogenicity by monoalkylated mercuries were suggested; (1) they interfere with repair of base lesions induced by 4NQO and MMS during the pre-replicational stage, thereby increasing unrepaired DNA lesions which convert into DNA double-strand breaks in S phase, (2) MeHgCl (but not EtHgCl) also inhibits repair of crosslinking lesions during the pre-replicational stage, and (3) their G2 effects enhance breakage-type aberrations only.

Mechanisms of mercurial and arsenical inhibition of tyrosine absorption in intestine of the winter flounder Pseudopleuronectus americanus.

Effects of HgCl2 (100 microM) para-chloromercuribenzene sulfonate (PCMBS) (1 mM), and oxophenylarsine (OPA) (250 microM) were determined on (a) the rate of Na pump activity in intact winter flounder intestine; (b) activity of Na-K-ATPase in tissue homogenates; and (c) Na-dependent and Na-independent uptake of tyrosine in brush border membrane vesicles. Initial rate of uptake (influx) of 86Rb from the serosal solution of tissues mounted in Ussing chambers, a measure of Na-K-ATPase activity in the intact cell, was inhibited by all three agents with differing time courses. Rapidly permeating HgCl2 inhibited influx to the same degree as ouabain at 30 min, whereas the effects of PCMBS and OPA required 90 min. Cell potassium was also measured as an indirect indicator of ATPase activity and cell membrane permeability. All three agents decreased cell K, although effects on cell K lagged behind those for inhibition of the ATPase. At the concentrations used in the Ussing chamber (or at one-tenth concentration), all agents completely inhibited Na-K-ATPase activity in enzyme assays performed with tissue homogenates. In contrast, only HgCl2 decreased Na-dependent uptake of tyrosine by brush border membrane vesicles. These results suggest that mercurial and arsenical effects on tyrosine absorption are due to inhibition of the Na-K-ATPase thus decreasing the driving force for the cellular uptake by the Na-tyrosine cotransport system. Direct effects on Na-tyrosine cotransport may play a role in the inhibition observed with HgCl2, but not for PCMBS or OPA.

Quantitative analysis of rat activity in the home cage by infrared monitoring. Application to the acute toxicity testing of acetanilide and phenylmercuric acetate.

A new method for continuous in-cage monitoring of the activity of rats using passive sensors to detect the animals' infrared emissions, and its usefulness for toxicological screening tests is described. In order to obtain baseline values for toxicological tests, the differences between day and night activity, adaptation to changing day/night rhythm, the effects of handling the animals (measurement of body weight, food and water consumption) and the effect on activity patterns of the female estrus cycle were investigated. In two separate experiments, single doses of acetanilide and phenylmercuric acetate were administered. Passive infrared monitoring of motor activity proved to be a sensitive and discriminating method suitable for quantitative and qualitative assessment of acute toxic effects.

[The modification of the toxic effects of phenyl mercury by the adaptive reactions of the rat kidney. 1. Timely development of adaptation]. / Beeinflussung toxischer Wirkungen von Phenylquecksilber durch adaptive Reaktionen der Rattenniere. 1. Mitteilung: Zeitliche Entwicklung der Anpassung.

Doses of 0.5 mg/kg body weight, 1.5 mg/kg, 3.0 mg/kg, 7.5 mg/kg, and 15.0 mg/kg of phenylmercury acetate (PMA), as a model substance, were administered to Ico:WIST rats, over periods of 1, 5, 20, and 40 days, for the purpose of studying time-dependent and dose-related buildup of adaptive alterations in kidney. Substance-related damage to kidneys was assessed with reference to functional and morphological parameters. Application of organic PMA resulted exclusively in accumulation in kidneys of inorganic Hg, as had been shown by determination of residues. One single application of a toxic dose of phenylmercury acetate caused severe structural and functional necrobiotic damage. Repetitive applications caused renal damage in response to lower daily doses but caused also adaptation of kidneys to effect of higher doses. Adaptation was characterised by unambiguous reduction in necrosis, increase in regenerative cells in tubular epithelium, limited enzyme excretion in urine as well as restitution of impaired functional parameters and proteinuria. These findings are discussed in some detail, with reference being made to effects of other Hg compounds and possible mechanisms of adaptation.

[Modification of the toxic effects of phenylmercury by adaptive reactions of the rat kidney. 2. Adaptation to phenylmercury acetate, cadmium chloride and cupric chloride]. / Beeinflussung toxischer Wirkungen von Phenylquecksilber durch adaptive Reaktionen der Rattenniere. 2. Mitteilung: Anpassung an Phenylquecksilberacetat, Cadmium- und Kupferchlorid.

ICO:WIST rats were pretreated over 20 days, using oral administration of subtoxic doses of 1.5 mg/kg body weight of phenylmercury acetate (PMA) 15 mg/kg of CdCl2 and 15 mg/kg of CuCl2, to investigate correlations between nephrotoxic effects of PMA, on the one hand, and buildup through various extraneous substances of adaptive reactivity, on the other. This was followed by application of an acutely toxic dose of 15 mg/kg of PMA. Its effect was compared by morphological and functional investigations with conditions in unchallenged animals. The method, according to all findings, did not cause major renal damage, on completion of PMA, CdCl2, and CuCl2 pretreatment. Nephrotoxic effects induced to pretreated animals (1.5 mg/kg of PMA) by 15 mg/kg of PMA were clearly below those induced to untreated animals. However, PMA effectiveness on kidneys was much higher in animals which had received CdCl2 or CuCl2 pretreatment. Such possible controllability of toxic PMA efficacy through adaptation to various extraneous substances is likely to underline the need for differentiated assessment of progressive and regressive elements related to adaptive reaction.

[Embryotoxic and teratogenic action of phenylmercuric acetate (Falisan-Nassbeize)]. / Embriotoksichno i teratogenno deistvie na fenilmerkuri atsetat (Falisan-Nassbeize).

Tested was the embryotoxic and teratogenic action of the organic mercury compounds (phenyl mercuric acetate), containing 2 per cent mercury, on albino rats. The preparation was introduced orally during pregnancy in the form of a 2 per cent water solution at the following rates: I group--1/8 LD50 (= 4 mg Hg/kg body mass) on the 4th and 5th day of pregnancy; II group--1/3 LD50 (= 10 mg Hg/kg) on the 4th and 5th day too; III group--1/8 LD50 (= 4 mg Hg/kg) from the 3rd to the 19th day; IV group--control animals. The preparation proved to be highly toxic with the animals of the II group, with high mortality rate (42.85 per cent). No teratogenic effect with malformations was produced with the three test groups during embryogenesis.

[The action of phenylmercuriacetate and nitrate in combined application in rats: the thyroid gland, liver enzymes and morphologic findings in the brain and kidney]. / Zur Wirkung von Phenylquecksilberacetat und Nitrat bei kombinierter Verabreichung an Ratten: Schilddrüse, Enzyme der Leber, morphologische Befunde an Gehirn und Nieren.

Rats were exposed to nitrate (NO3-) in drinking water, to phenylmercuryacetate (PMA) by gavage and to NO3- and PMA together during 4 different experiments. PMA impaired kidneys, NO3- thyroid gland, and NO3- and PMA together both organs. In the last case a synergistic effect on the thyroid gland was shown. The lowest effective concentration of NO3- was 40 mg/l. It resulted in histomorphological changes of the thyroid epithelial cells. That low effective dose of NO3- and possible synergistic effects should give a further impulse to take into consideration not only a low iodide intake but also goitrogenic environmental chemicals when evaluating the endemic goitre prevalence.

[Comparative studies of 2 organic mercury compounds alone or in combination with ethanol following pre- or postnatal application in mice]. / Vergleichende Untersuchungen mit zwei organischen Quecksilberverbindungen allein oder in Kombination mit Ethanol nach prä- oder postnataler Applikation an Müsen.

Studies on pre- and/or postnatal effects on mice were carried out with their identical exposure (2 mg Hg/kg body mass) to phenyl mercuric acetate or N,N'-bis-(dimethylmercury)-p-toluol-sulphonamide. Parameters of morphology, of residues, and behaviour teratology were taken into consideration. Potentiating combination effects were only observed with behaviour toxicological investigations after prenatal exposure to methylmercury. That concerned the breeding results of the P-generation and the capacity of the F1-generation to learn on the 30th and 31st day p.n. during a swimming test in a labyrinth. Synergistic effects were observed with both the mercury compounds in the swimming test on the 12th day p.n. after pre- as well as postnatal exposure.

[Embryotoxic and teratogenic action of the organomercury fungicide falizan on chick embryos]. / Embriotoksichno i teratogenno deistvie na organozhivachniia fungitsid falizan na kokoshi embrioni.

Tested was the embryotoxic and teratogenic action of the organomercurial preparation falizan (a phenyl-mercury acetate), containing 2 per cent mercury, in chick embryos. It was introduced into the viteline sac of 3-day-old embryos in the form of a water solution as follows: Group I--0.1 cm3 per egg of a 13 per cent solution of falizan (= 260 micrograms Hg); group II--0.1 cm3 per egg of a 6.5 per cent solution (= 130 micrograms Hg); group III--0.1 cm3 per egg of redistilled water (biologic control); and group IV--the eggs were left untreated (negative control). The amount of the preparation was adjusted to eggs of 65 + 1 g weight. The optimal growth and development of the embryos was guaranteed with the use of an Optima hatching unit, providing humidity of up to 65 per cent = 89 F. The embryotoxic and teratogenic effect of the preparation was recorded on the 14th and the 20th day of incubation. A dose of 260 micrograms Hg was found to be strongly toxic, mortality rate reaching up to 48 per cent from the 7th to the 14th day. Teratogenically, there were no malformations in the test groups both on the 14th and the 20th day of embryogenesis.

Plasma and platelet fibrin-stabilizing factor activity in rats intoxicated with phenylmercuric acetate.

Effect of phenylmercuric acetate on the activity of rat plasma and platelet fibrin-stabilizing factor (FSF) was studied. The mercurial was administered to the animals intragastrically in a single dose of 17.9 mg Hg/kg or in repeated doses of 1.79 mg Hg/kg, twice a week for 152 days. The activities of the investigated fibrin-stabilizing factors were significantly increased in the case of intoxicated rats. Such an effect was observed in both types of the exposure.

Effects of mercury on acetylcholinesterase (E.C. 3.1.1.7.) activity of erythrocytes and bone marrow in rats.

The studies were carried out on male Wistar rats, where the activity of acetylcholinesterase (AchE-E.C. 3.1.1.7) was determined in red cells and bone marrow under the influence of organic and anorganic mercury compounds. A marked decline in the activity of the enzyme could be noted, along with a more pronounced effect of the organic mercury compound on AchE.

Mercury and the activity of erythrocyte and bone marrow glutathione reductase (E.C. 1.6.4.2.) and glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) in rats.

These studies were carried out on male Wistar rats. Erythrocyte and bone marrow activity of glutathione reductase (GR - E.C. 1.6.4.2) and glucose-6-phosphate dehydrogenase (G6PD - E.C. 1.1.1.49) were determined as affected by mercury. Under physiological conditions the above enzymes were found to be markedly more active in the bone marrow than in erythrocytes. The present experiments have revealed that within the first days there will be an increase in enzyme activity in the bone marrow as well as in glutathione reductase in erythrocytes. Simultaneously, a decline in the activity of erythrocytes could be observed which began within the very first days of the experiments as well as a decrease in the bone marrow, which occurred later in the course of the experiment.

Activity of chosen indicator enzymes in blood serum of guinea pigs exposed to ethyl- and phenylmercuric chloride alone or jointly with sodium selenite.

The effect of sodium selenite on the activity of the selected enzymes in blood serum and on mercury concentration in some tissues of guinea pigs exposed to ethyl- (EtHg) or phenylmercuric chloride (PhHg) was investigated. Every second day for a 3-month period animals were given intragastrically a solution of mercuric compounds (2.5 mg Hg/kg) with or without sodium selenite (1 mg Se/kg). The activity of malate dehydrogenase (MDH, EC 1.1.1.37), phosphohexoizomerase (PHI, EC 5.3.1.9), and gamma-glutamyltranspeptidase (GGTP, EC 2.3.2.2) in blood serum of control animals was ca. 3.8, 325, and 48 IU. After 10 weeks of exposure to EtHg and PhHg, the activities (IU) of the above enzymes were, respectively, 5.9 and 6.5 (MDH), 585 and 600 (PHI) and 211 and 86.5 (GGTP). Sodium selenite administered with mercuric compounds did not prevent in increases in enzyme activity. During the experiment the level of inorganic as well as organic mercury accumulated in kidneys and liver was estimated. After a 12-week exposure, sodium selenite decreased the level of total mercury in the liver (in the case of both EtHg and PhHg: from 47.0 to 31.8 and from 41.3 to 25.4 micrograms Hg/g tissue, respectively). It also slightly decreased the mercury level in the kidneys of animals exposed to PhHg (from 889 to 73.3 micrograms Hg/g tissue) but did not change the mercury concentration in the kidneys of guinea pigs exposed to ethylmercuric chloride.