Methylmercury Hydroxide Research Articles

Liver protein synthesis during the acute response to methylmercury administration

1. 1. After administration of methylmercury hydroxide to rats for 3 days, liver homogenate fractions were prepared for studying protein synthesis in vivo and in vitro. 2. 2. The treatment caused a significant increase in the incorporation of [ 14C]leucine into the protein of the microsomal fraction 12 and 30 min following [ 14C]leucine injection. 3. 3. The specific activity of the serum proteins was also greatly increased by methylmercury, especially proteins of mobilities in sodium dodecylsulphate electrophoresis of mol. wt 42,000–45,000 and 56,000–66,000. 4. 4. Postmicrosomal supernatant fractions and ribosomal fractions were used to assay protein synthesis in vitro and both of these fractions showed increased activity when they were prepared from the liver homogenates of rats that had received methylmercury. 5. 5. Evidence was obtained that methylmercury administration to normal rats or to hypophysectomized rats increased the capacity for peptide elongation in the cytosolic fractions.

Catfish vitellogenin and its messenger RNA are smaller than their chicken and Xenopus counterparts

Injection of male bullheads ( Ameiurus nebulosus) with estradiol induced the production of a major serum phosphoprotein of molecular weight 145 000. This protein was immunoprecipitable by antisera raised against lipovitellin from bullhead eggs and was absent from the serum of control males. Production of this serum protein coincided with changes in the liver mRNA population, which suggested that estradiol had induced the synthesis of additional mRNA sequences in the high-frequency class. Agarose gel electrophoresis in the presence of methyl mercury hydroxide showed that this mRNA population contained at least one species which was not present in the liver of uninjected males. This new RNA was the major polyadenylated species present in the total cellular RNA and its size relative to ribosomal RNAs and locust vitellogenin mRNA was estimated as 5800 nucleotides. When the liver total RNAs were translated in the mRNA-dependent rabbit reticulocyte lysate system the major translation product from the induced fish had the same molecular weight (145 000) as the serum phosphoprotein and was immunoprecipitable by antilipovitellin antisera. This translation product was not coded for by RNA from control fish. These observations are consistent with the induction of vitellogenesis by estradiol as reported in other egg-laying vertebrates and they show that bullhead vitellogenin and its mRNA are significantly smaller than their avian and amphibian counterparts.

Arrangement of messenger RNAs and protein coding sequences in the major late transcription unit of adenovirus 2

The messenger RNAs present in HeLa cells late in adenovirus 2 infection have been characterized utilizing three complementary methods of analysis. In each case the mRNAs were defined functionally, by identification of the polypeptides they encode when translated in a reticulocyte cell-free system. The RNAs were examined by (1) electrophoretic fractionation in agarose gels containing methyl-mercury hydroxide to estimate the sizes of the functional RNAs; (2) selection by annealing to viral DNA fragments immobilized on nitrocellulose to define the genomic origin of their constituent sequences; (3) hybrid-arrested translation using viral DNA fragments to locate their protein coding sequence. The results allow the positioning of both the coding and non-coding regions of all the late mRNAs with respect to the DNA. They demonstrate that the mature mRNAs originating in the major late transcription unit fall into five families. Within each family the mRNAs overlap in a staggered fashion, sharing the same 3′ terminus but differing at their 5′ termini. The data also reveal the existence of two new late proteins of 52,000 and 55,000 M r encoded between map co-ordinates 30 to 34 and permit the positioning of the late RNAs, according to the polypeptides they encode, as follows: 52, 55K · IIIa: III · pVII · V: pVI · II: 100 K/33 K · pVIII: IV. (The colons signify the ends of families; centre dots the junctions within families; the slash an overlap of coding sequences; and the comma, coding sequences which have not been separated in this analysis. A 55 K protein has M r = 55,000.) Those mRNAs which are either the only transcripts from a specific region (such as those for polypeptides IX, IVa2 and IV) or are the 3′-terminal transcripts within a family (such as those coding for polypeptides IIIa, V, II and pVIII) migrate as discrete species upon electrophoresis in agarose gels containing methyl-mercury hydroxide. However, the 5′-proximal RNAs within a family (such as those encoding polypeptides 52, 55 K, III, pVII, pVI, 100 K and 33 K) exhibit a broad distribution of molecular weights.

Comparison of potential nuclear precursors for prolactin and growth hormone messenger RNA.

Recombinant DNA plasmids containing the coding sequence for rat prolactin or rat growth hormone have been used to investigate the presence of possible precursors for prolactin and growth hormone mRNA. Cytoplasmic and nuclear RNA was prepared from either rat pituitaries or fromthe GC pituitary cell line. RNA was electrophoresed on agarose gels containing methylmercury hydroxide and then transferred to diazobenzyloxymethyl paper. The paper was then hybridized to prolactin or growth hormone recombinant DNA probes labeled in vitro with 32P. The prolactin probe hybridized to RNA species of 7.0, 6.4, 3.8, 1.7, and 1.0 kilobases in nuclear RNA and only to a 1.0-kilobase species in cytoplasmic RNA. Hybridization with a growth hormone probe demonstrated nuclear RNA species of 6.7, 5.6, 2.3, and 1.0 kilobases. These findings demonstrate the presence of multiple species of prolactin and growth hormone RNA which are larger larger than the mature cytoplasmic mRNAs. The large nuclear RNAs are likely precursors for prolactin and growth hormone mRNA.

Acute methyl mercury intoxication in mice--effect on the immune system.

Effect of organic mercury on the immune system was studied in experimental acute methyl mercury intoxication in mice. After 7 daily injections of methyl mercury hydroxide, marked atrophy of the thymic cortex and splenic lymphoid follicles were observed, associated with decrease of PHA- and LPS-responsiveness of splenic lymphocytes as well as conversely increased both PHA- and LPS-responsiveness of thymocytes. In addition, apparent decrease of antibody formation against sheep red blood cells was observed at 7 day after the last injection. However, all these changes restored to normal level within 4 weeks after the last injection. Histochemically, mercury deposits were massively found in the renal tubules and slightly in macrophages throughout the body at 7 day after the last injection. The mercury deposits in macrophages of the thymus, lymph nodes and liver increased in concentration thereafter, with concomitant decrease of those in renal tubules. Presence of mercury element in these deposits was confirmed by electron probe analysis. It was suggested that the organic mercury gave direct cytotoxic effect on lymphocytes, but the effect appeared to be soon detoxicated by macrophages.

Methylmercury hydroxide enhancement of translation and transcription of ovalbumin and conalbumin mRNA's.

Translation of total mRNA in heterologous protein-synthesizing systems is often employed as an indirect means of assessing relative mRNA concentrations. However, it is well known that the efficiency of translation of specific mRNAs differs. One such example is the poor translational efficiency of conalbumin mRNA relative to ovalbumin mRNA. In this report we have studied the translation of conalbumin and ovalbumin mRNAs in crude mRNA preparations and with highly purified mRNA preparations. We find that treatment of RNA with methylmercury hydroxide prior to translation improves the translational efficiency of both mRNAs and preferentially improves translational efficiency of conalbumin mRNA to the point where it more correctly reflects the relative concentration of these two mRNAs in crude mRNA preparations. Conalbumin mRNA is also a poor template for the synthesis of full length cDNA synthesis by avian myeloblastosis virus reverse transcriptase, and treatment of this mRNA with methylmercury hydroxide increases the size of DNA sequences synthesized. We conclude that treatment with methylmercury hydroxide produces a partial denaturation of mRNA complexed with either itself or with other RNA molecules and results in more efficient utilization in both translational assays and DNA polymerization reactions.

Changes in expression of albumin and alpha-fetoprotein genes during rat liver development and neoplasia.

Albumin mRNA was isolated and purified from rat liver polysomes by a combination of immunoprecipitation of specific polysomes, poly(U)-Sepharose 4B chromatography, and fractionation of the resulting poly(A)-containing RNA on a sucrose gradient. alpha-Fetoprotein (AFP) mRNA was isolated from Morris hepatoma 7777 by a similar procedure. The purity of the mRNA preparations was determined by analytical gel electrophoresis under denaturing conditions, analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides synthesized in a wheat germ cell-free system, and the kinetics of hybridization to cDNA transcribed from albumin mRNA and AFP mRNA. The albumin mRNA possessed a chain length of approximately 2265 nucleotides and the AFP mRNA possesed a length of approximately 2235 nucleotides when examined under stringent denaturing conditions on agarose gels containing 10 mM methylmercury hydroxide. Analysis of poly(A) content by a hybridization assay with [3H]poly(U) revealed the presence in albumin mRNA of a poly(A) region containing approximately 100 adenosine residues. The AFP mRNA preparation was found to contain an average poly(A) tract of approximately 190 bases. Thus, albumin mRNA appears to contain approximately 330 untranslated nucleotides, and AFP mRNA appears to contain a similar number (approximately 285) of noncoding, nonpoly(A) bases. The purified albumin and AFP mRNA's were used as templates for synthesis of full-length cDNA hybridization probes. Both of the probes selectively hybridized to their templates with kinetics expected for single RNA species the sizes of albumin and AFP mRNA. ROt analysis was used to quantitate albumin and AFP mRNA sequences during normal liver postnatal development and liver oncogenesis. The number of polysomal AFP mRNA molecules per liver was found to drastically decrease during the first weeks of postnatal life, concomitant with a decline in the AFP synthetic capacity of the livers and in the serum concentrations of AFP. During this period, the concentration of albumin mRNA molecules per cell in the liver remained at high, approximately constant levels. In Morris hepatoma 7777, the concentration of AFP-specifying sequences was at least 10(3)-fold higher than that found in normal adult liver, whereas the content of albumin nRNA was four- to five-fold lower. These changes in concentration of albumin and AFP mRNA sequences closely correlated with a parallel variation in the specific protein synthetic capacity of the tissues.

Mercury Circulation in Aquatic Environment. Part 2: Metabolism of Methyl and Phenyl Mercury in Phytoplankton

AbstractBiotransformation of phenyl and methyl mercury chloride was studied in experiments with planktonic algae Chlorella kessleri, Scenedesmus obliquus and blue‐green algae Microcystis incerta. To algal cultures cultivated in an inorganic medium the labelled phenyl or methyl mercury hydroxide was added separately in the phase of the logarithmic growth, the resultant concentration being 3.2 × 10−7 M. In intervals of 0, 1, 3, 7, 10 and 14 days samples of the algae and medium were analysed for the total amount of mercury and for the presence of phenyl or methyl mercury. It was found that both methyl and phenyl mercury had accumulated very rapidly in the algae and that up to 70% of 203Hg added was present in the algae during the first 72 hours of the experiment. From the 2nd day onwards, however, methyl and phenyl mercury changed rapidly into inorganic mercury so that by the end of the experiment almost all of the mercury present in algae and the medium was in the form of inorganic mercury.

A nuclear precursor to human γ-globin messenger RNA

RNA isolated from human foetal liver at an erythroid stage of development was transferred from methyl mercury hydroxide agarose gels to diazobenzyloxymethyl paper. A 16S RNA species containing gamma-globin mRNA sequences was identified by hybridisation to 32P-labelled human gamma-globin cDNA recombinant plasmid pHgamma GI. This RNA is found only in the cell nucleus and is polyadenylated. Human globin gene transcription is thus analagous to that in mouse: interventing sequences are transcribed and processed from the pre-mRNA in the nucleus after polyadenylation.

Transcription of structural and intervening sequences in the ovalbumin gene and identification of potential ovalbumin mRNA precursors

Structural sequences that are extensively separated by nonstructural intervening sequences in the natural ovalbumin gene are coordinately expressed in target and nontarget tissue. The intervening sequences, which consist of unique sequences in the chick genome, are transcribed in their entirety. The amount of nuclear RNA corresponding to these sequences, however, is approximately 10 times less than that observed for structural sequences. The accumulation of RNA corresponding to structural and intervening sequences during acute estrogen stimulation suggests either that there are different rates of transcription for these regions of the ovalbumin gene or that RNA sequences corresponding to the intervening sequences are preferentially processed and degraded. Comparison of the in vitro expression of portions of the ovalbumin gene in nuclei isolated from chronically stimulated oviducts indicates that both structural and intervening sequences are preferentially transcribed in vitro at rates approximately 500 times greater than expected for random transcription of the haploid chick genome. In addition, electrophoresis of oviduct nuclear RNA on agarose gels containing methylmercury hydroxide reveals multiple species of RNA that are from 1.3 to over 4 times larger than ovalbumin mRNA and hybridize to both structural and intervening sequences of the ovalbumin gene. These results are consistent with transcription of the entire ovalbumin gene into a large precursor molecule followed by excision of the intervening sequences and appropriate ligation of the structural sequences to form the mature mRNA.

The efficacy of 2,3-dimercaptopropanol and D-penicillamine on methyl mercury induced neurological signs and weight loss

In order to test the effectiveness of complexing therapy on methyl mercury induced neurotoxicity, female rats were treated for five days with either 2,3-dimercaptopropanol (BAL) or D-penicillamine (DPA) beginning 1 or 12 days after the final dose of methyl mercury hydroxide (MMOH). MMOH was administered orally at a dose of 13.3 mg/kg once each day for 3 successive days. BAL, dissolved in peanut oil, was administered sc in a dose of 30 mg/kg twice each treatment day. DPA was dissolved in water and administered sc in a dose of 1200 mg/kg once each treatment day. Therapy begun 1 day after the last MMOH administration was prior to the appearance of toxic signs but therapy begun 12 days later was after signs had developed. The ability of BAL or DPA therapy to alter the distribution of radio-labelled ( 203Hg) MMOH was determined. Both DPA and BAL significantly reduced tissue concentrations of mercury when administered beginning 1 day after MMOH, but only DPA was effective in removing mercury from tissues when treatment was begun 12 days after the last dose of MMOH. In a separate experiment when either BAL or DPA therapy was initiated as above 1 day after the last dose of MMOH, the appearance of neurological signs of toxicity was prevented and weight loss was reversed. When therapy was initiated the twelfh day after the last MMOH dose, neither BAL nor DPA was effective in reversing either neurotoxic signs or weight loss. Therefore, therapy is ineffective in reversing neurological signs if it is delayed too long after MMOH administration, even though it is effective in reducing tissue mercury levels.

Synthesis of full length cDNAs from four partially purified oviduct mRNAs.

Total poly(A)-containing RNA prepared from hen oviduct and centrifuged on an isokinetic sucrose gradient displays four peaks of optical absorbance. These have been identified by translation in vitro as lysozyme, ovomucoid, ovalbumin, and conalbumin mRNAs. Isolation and recentrifugation of the peaks results in partial purification of each mRNA. Molecular weights have been determined for the mRNAs on agarose gels containing 20 mM methylmercury hydroxide. Each mRNA possesses a number of apparently untranslated nucleotides ranging from approximately 900 bases for ovalbumin and conalbumin mRNAs to 200 bases for ovomucoid and lysozyme mRNAs. The mRNAs have been copied with avian myeloblastosis virus reverse transcriptase. Each mRNA with the exception of conalbumin gives rise to a high proportion of full length cDNA. Several parameters previously reported to influence the size distribution of cDNA had no effect on the length of cDNA made from any mRNA fraction. The proportion of full length copy does depend on the reverse transcriptase lot.

Chronic Methylmercury Exposure in the Monkey (Macaca mulatta)

Small daily doses of methylmercury hydroxide were administered to rhesus monkeys for periods of up to 17 months. Behavioral tests of peripheral vision and of the accuracy and rapidity of hand movements did not disclose any early subtle deficits preceding the onset of obvious signs of neurotoxicity. These signs appeared suddenly and involved reduced food intake (anorexia), clumsiness of jumping, loss of fine control of the digits, and uncoordinated mastication. With a constant daily dose of 0.1 mg/kg or less, blood concentration of mercury reached a peak after about 2 months, and then decreased to about half the peak value. Subsequently, increasing the daily dose level above 0.1 mg/kg (range of 0.12-0.21 mg/kg) produced an increase of blood concentration which tended to stabilize in the range of 2.0-2.5 ppm. After several months at these elevated concentrations all animals exhibited signs of neurotoxicity.

Kinetik sehr schneller Methylquecksilber‐Übertragungsreaktionen zwischen pyridinhaltigen Chelatliganden und dem Hydroxid‐Ion

Kinetics of very fast Methylmercury‐Transfer Reactions between Pyridine‐containing Chelating Ligands and the Hydroxide IonThe kinetics of the reactions between methylmercury hydroxide and bipyridine, terpyridine, phenanthroline and pyridine‐2‐azo‐4‐dimethylaniline have been studied by means of the temperature jump relaxation method. These bi‐ and tridentate ligands form methylmercury chelates. Their formation is faster than the formation of the pyridine complex. The chelate complexes show an unusually high reactivity. Their reaction rates with hydroxide ions reach almost the diffusion controlled limit.

Crystal structure of (DL-methioninato)methylmercury(II)

The synthesis of (DL-methioninato)methylmercury(II) from methylmercury hydroxide and DL-methionine is described. Crystals of the title compound are monoclinic, space group P21/c with a= 7.048(9), b= 5.826(4), c= 25.323(53)A, β= 93.2(2)°, Z= 4. The structure was solved by the heavy-atom method by use of 1 027 observed diffractometer-measured reflections and refined to R 0.099. The amino-acid is bound to the methylmercury moiety via the amino-group. Important metal–ligand distances are: Hg–N 2.06(4) and Hg–C(6) 2.11(5)A. Two additional weak interactions to carboxylate oxygen atoms are present. The vibrational spectra and n.m.r. spectra are discussed in the light of the solid-state structure and previous deductions concerning the Interaction of methionine with inorganic and organo-mercury compounds.

Embryocidal and teratogenic effects of methylmercury in mice

Embryocidal and teratogenic effects of methylmercury hydroxide (MeHgOH) were determined in three strains of mice, C57BL 6J , CD, and 129 SvSl . In single dose experiments, a dose of 0, 2, 4, 8, or 12 mg/kg of MeHgOH was administered by various routes to all three strains of pregnant mice on Day 10 of gestation. For multiple dose experiments, a daily dose of 0, 2, 4, 6, 8, or 10 mg/kg was injected sc to 129 SvSl mice from Days 7 to 12 of gestation. The fetotoxic effects were examined on Day 18 of gestation. Embryocidal and teratogenic effects were noted in all three strains of mice. Cleft palate was the most frequently observed teratogenic malformation. Other malformations, such as anophthalmia, micrognathia, exencephaly, missing limbs, and facial deformity, were seen infrequently. Multiple dose administration of MeHg to pregnant dams caused a higher incidence of MeHg teratogenesis with a lower incidence of maternal toxicity in comparison to single dose administration. Present findings indicate that all three species of mice investigated are susceptible to the teratogenic effects of MeHg.

Behavioral effects on the progeny of mice treated with methylmercury

The effects of prenatal methylmercury (MeHg) exposure on the behavior of postnatal offspring were determined in 129 SvSl mice. A single dose of 0, 6, 8, or 12 mg/kg of methylmercury hydroxide (MeHgOH) was injected sc to pregnant mice on Day 10 of gestation. The following three types of behavioral tests were conducted on offspring from these mothers at various ages: (1) exploratory behavior by open-field test at age 23 days old, (2) spontaneous locomotor activity at ages 24, 44, and 64 days old, and (3) convulsive behavior to a convulsant, flurothyl at age 70 days old. Another group of 33-day-old offspring whose mothers had received three multiple doses of MeHgOH or daily doses of 0 or 4 mg/kg from Day 10 to Day 12 of gestation were also used for the open-field behavioral study. Results from the openfield test indicated that prenatal MeHg exposure decreases exploratory behavior as indicated by longer center square latency, lower number of peripheral squares traversed, lower frequency of rearing and grooming, lower frequency of urination, and higher frequency of backing, in treated groups in comparison to control groups. Depression in spontaneous locomotor activity was also observed in offspring prenatally exposed to MeHg; however, the effect diminished gradually as the animals grew older. In the flurothyl convulsion behavior test, it was found that animals prenatally treated with MeHg demonstrated significantly lower threshold to preliminary clonic convulsion as well as maximal clonic-tonic convulsion; thus, indicating an increase in susceptibility to flurothyl-induced convulsions. The observed decrease in exploratory behavior and spontaneous locomotor activity with an increase in convulsive susceptibility are not necessarily inconsistent findings and may be due to MeHg affecting two independent mechanisms. Therefore, it has been demonstrated that behavioral effects can be induced in offspring prenatally exposed to MeHg. Furthermore, behavioral effects of MeHg can be detected in offspring having no detectable gross congenital malformations.

Postnatal behavioral effects in mice after prenatal exposure to methylmercury

CFW mice were injected with methylmercury hydroxide (1, 2, 3, 5 or 10 mg/kg as mercury) on Day 8 of gestation. Mice treated with 3, 5 or 10 mg/kg averaged 1 3 fewer pups than controls. Pups from these treated animals weighed less than controls and the weight differences persisted through weaning but were no longer significant at 56 days of age. Mice exposed to methylmercury in utero showed significant differences from controls in their behavior in a 2-way active avoidance shuttle box and in a punishment situation but not when tested in an open field, a water escape runway or a conditioned suppression paradigm. Neither the mothers nor progency of the mice exposed prenatally to methylmercury showed behavioral deficits.

Methylmercury-cholinesterase interactions in rats.

The interaction of methylmercury hydroxide (MMH) and cholinesterases was studied in male and female rats. MMH administered subcutaneously in doses of 10 mg/kg for 2 days reduced the level of plasma cholinesterase (ButChE) by 68% in females and 47% in males while brain acetylcholinesterase (AChE) was unaffected. Normal females had higher but more variable ButChE levels than normal males. In a time-course experiment, a single dose of MMH (10 mg/kg) reduced ButChE levels when mercury levels reached 22 mug/ml in the blood. A 10% reduction in brain AChE was observed at 72 hours; however, mercury reached a concentration of only 2.0 mug/g in brain tissue. The determination of the Michaelis constant Km and maximum velocity value Vmax for butyrylcholine and ButChE in control and MMH-treated (1 mg/kg) animals indicated that MMH reduced Vmax only. Since no loss in ButChE activity occurred when MMH and control plasma were incubated in vitro, MMH is not a direct inhibitor of ButChE. Because only the inactive monomeric form of ButChE contains free sulfhydryl groups, it is postulated that MMH combines covalently with the sulfur, preventing formation of active enzyme. By analogy, it is believed this is also the case with AChE.

Neurotoxicity of methylmercury in squirrel monkeys. Cerebral cortical pathology, interference with scotopic vision, and changes in operant behavior.

Blood mercury was raised to levels exceeding 1,000 ng/gm by weekly oral doses of methylmercury hydroxide to squirrel monkeys. Operant behavior tests were employed and neurological signs were recorded on film. Sudden visual disturbance occured with subacute exposure. Prolonged exposure resulted in impaired coordination with impairment of scotopic vision and possibly also sensory disturbances. Scotopic vision was tested by determining the critical fusion intensity (cfi) at 10 cps. An increase in cfi was the earliest neurological sign appearing in some monkeys months before other signs could be detected. Poisoned monkeys exhibited typical cerebral cortical lesions. The visual cortex was invariably involved. Extension to adjacent cortical areas increased with increasing duration of exposure and increasing brain mercury. The microgram per gram brain tissue was the lowest concentration of methylmercury seen wiht morphological lesions.