Phenylmercuric Chloride Research Articles

Interfacing nanoliter liquid chromatography and inductively coupled plasma mass spectrometry with an in-column high-pressure nebulizer for mercury speciation

Metal speciation analysis of microsamples in the clinical, biological and forensic fields is important for elucidating the metals’ toxicity, mobility and metabolic behaviors of metals. Such analysis may be achieved by nanoliter high-performance liquid chromatography (nanoHPLC) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). In this work, an in-column high-pressure nebulizer (ICHPN) was developed to enhance the sensitivity and the separation efficiency of the sheathless nanoHPLC-ICP-MS coupling. The ICHPN consists of two concentric fused-silica capillaries with tapered tips connected via a PEEK tee, where C18 silica particles with a size of 5 μm were packed in the tapered inner capillary based on the keystone effect. Combining a heated single pass spray chamber with a makeup gas, the ICHPN was capable of independently optimizing the nebulization efficiency and the transport efficiency. The ICHPN offered high sensitivities, low detection limits and good precision at nanoflow rates. Compared with commercial nebulizers, the ICHPN fabrication was simple, rapid, reproducible and inexpensive. By implementing the ICHPN, we achieved rapid separation of four mercury species, i.e., Hg2+ and methyl-, ethyl- and phenylmercuric chloride, within 8.0 min with good resolution (2.0–13.9). Detection limits of 0.044-0.13 μg L−1 were obtained with precisions of peak heights and areas ranging from 1.5 to 3.5% for a 50 μg L−1 standard solution. Good agreement between the determined and certified values of mercury species in a certified reference material of caprine blood (SRM 955c) together with good recoveries (93–101%) validated the accuracy of the method.

Synthesis and characterization of a mercury-containing trimetalloboride.

The reaction of phenylmercuric chloride with an anionic dimanganese borylene (Li(+)[Cp2(CO)4Mn2B](-)) led to the formation of a trimetalloboride featuring the first reported bond between mercury and a non-cluster boron atom. Examination by (199)Hg NMR indicated (11)B-(199)Hg scalar coupling. Theoretical calculations indicated the nature of bonding to be σ-donation from a B-Mn π-orbital to Hg, in conjunction with weak Hgd→π* back-donation.

Abstract B42: Structure-activity study of tubulin precipitation by thiol-reactive compounds

Abstract Tubulin, a major constituent of microtubules, has been established as an important chemotherapeutic target. Its expression is tightly regulated at the transcriptional level and not affected by tubulin targeting agents including taxanes and vinca alkaloids. Recently, we reported that benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), two isothiocyanates (ITCs) derived from cruciferous vegetables, induce rapid and significant precipitation and degradation of both α- and β-, but not γ-, tubulin at 10 μM through covalent binding to cysteine residues. In contrast, sulforaphane and erucin, two hydrophilic ITCs, and N-methylphenethylamine, an analog of PEITC without electrophilic functional group, show no activity as well as little downstream effects such as cell cycle arrest at mitosis and apoptosis induction at the same concentration. Because ITC are electrophiles, these findings suggest that thiol-reactivity may be the triggering event, the structure of compounds is associated with its activity, and tubulin precipitation and degradation are biologically important. In this study, we report that other thiol-reactive compounds, such as N-benzylmaleimide, phenylarsine oxide, and phenylmercuric chloride, also induce significant tubulin precipitation. They were much more potent than their hydrophilic counterparts, such as N-maleoyl-beta-alanine, arsenic oxide, and mercury chloride. These results shed lights onto structure-activity relationships. More importantly, their potencies of precipitating tubulin correlate well with not only their affinity to tubulin cysteines but also induction of G2/M phase arrest and apoptosis in human lung cancer cells A549. The results support the notion that cysteine modification is an upstream event for tubulin instability and cytotoxicity. The study presents critical knowledge on the rational design and screening of thiol-reactive compounds targeting the stability of tubulin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B42.

Effects of ortho‐substituents in SE1 protonolysis of phenylmercuric chloride

AbstractThe kinetics of the reactions of o‐substituted phenylmercuric chlorides, o‐RC6H4HgCl (R = CH3, H, C2H5O, CH3O, C6H5, F, COOC2H5, Cl, Br, CF3, NO2), with hydrochloric acid in 80% aqueous dioxane in the presence of NaI were studied. The reactions are of the first order. The rate constant at 40°C decreases in the order of R: CH3 > H > C2H5O > CH3O > C6H5 > F > COOC2H5 > Cl > Br > CF3 > NO2. The analysis of effects of those o‐substitutes is carried out through multiple regression of log k/kH with the corresponding inductive substituent constants σI and the various resonance substituent constants σ, σR(BA), σ, σ and σx, and the corresponding Swain–Lupton field effect constant $\Im $ and resonance effect constant $\Re $. The results showed that o‐substituent intramolecular coordination with the neighbor mercury (field effect) is the main effect in effects of o‐substituents on rate of the SE1 protonolysis. Copyright © 2007 John Wiley & Sons, Ltd.

Contact Allergy in Agricultural Workers

Agricultural workers (n = 121) referred to the Nofer Institute of Occupational Medicine for suspected occupational disease were subjected to dermatological and allergological examinations. All were patch and prick tested with standard occupational and environmental allergen sets. Contact dermatitis was diagnosed in 60 (49.6%) patients. In the women, the dominant allergy was that to nickel, benzalkonium, palladium, cobalt, thimerosal, fragrances and balsam of Peru, while the men were most frequently sensitive to chromates, cobalt, 4-phenylenediamine, fragrances, captan, formaldehyde, 4-aminoazobenzene, wool alcohols and cinnamic alcohol. Phenylmercuric chloride caused allergic reactions in 6 women and 2 men, while 6 workers were sensitive to neomycin. Allergy to glutaraldehyde was diagnosed in 3 workers, to lysol in 4, to chlorhexidine in 1, to chloramine in 1. Three patients reacted to thiurams, 3 to diphenylguanidine, 3 to mercaptobenzothiazole. Of the 9 workers sensitive to 4-phenylenediamine, only 2 men reacted additionally to N-isopropyl-N-4-phenylenediamine. Ziram (zinc dimethyldithiocarbamate) and copper sulfate caused allergy in 1 agricultural worker each. Immediate allergy was diagnosed in 43 (35.5%) patients. The dominant allergens in that category included straw dust, hay dust, wheat threshing, rye pollen and cow epithelium. The final diagnosis was based on the clinical picture and the results of the patch and prick tests. Allergic contact dermatitis was diagnosed in 27 (22.3%), atopic dermatitis in 27, irritant contact dermatitis in 15 (12.4%) and urticaria in 9 (7.5%) agricultural workers. Other dermal diseases were diagnosed in single patients. The clinical examinations showed no dermal lesions in 31 (25.6%) patients.

The effect of selenium on phenyl mercury toxicity and mercury retention in chicken

Phenylmercuric chloride in a dose 30 ppm of mercury alone and the same dose supplemented with 4 ppm of selenium were fed to chickens of both sexes for 8 weeks. Body weight, mortality rate, pathological changes and the level of mercury in the muscle, liver, and kidneys of the laying hens and cocks were analysed. After the dosage period, the mean body weight of the hens was unaffected, while in the group of cocks fed 30 ppm Hg without selenium addition, a decrease occured. In the group with Se addition no significant change in body weight of either sex was observed. Differences in the affinity of some organs to bind Hg were observed. Moreover, supplementation with 4 ppm of selenium resulted in significantly increased mercury levels in the organs analysed. The highest increase was found in the liver, but there was the same increasing tendency in the muscle and kidneys.

The effect of selenium on mercury retention in the offspring of treted hens

The levels of mercury in the chicken organs were determined after long term oral administration of mercury to laying hens. Phenylmercuric chloride was fed to laying hens and cocks for 28 and 56 days in a dose 30 ppm of mercury alone and the same dose supplemented with 4 ppm of selenium. The mortality rate of their offspring and the level of mercury in the chick muscle, liver and kidneys after seven days of rearing were analyzed. The mercury accumulated in the fertilized eggs was transferred from the hens to their offspring. The mercury level in the offspring of both experimental groups was highest in the kidneys, followed by liver and muscle. The addition of selenium to the diet for laying hens resulted in a different rate of a decrease of mercury level in their offspring in all organs analyzed.

Isokinetic correlation analysis of the series of electrophilic substitution reactions ofo-substituted phenylmercurials

Isokinetic relationships of the series of electrophilic substitution reactions of o-substi- tuted phenylmercurials are studied, and the results show, for the first time, that isokinetic correlation analysis can reveal the dominant factor in the system of the reactions. The analysis of S E 2 of o -substituted phenylmercuric chlorides indicates that the steric effect of o -substituents is the predominant factor when I 2 is used as electrophilic agent, however, the electronic effect of o-substituents would become the predominant factor when HCl is used as the electrophilic agent. In S E 1 of o-substituted phenylmercuric chlorides, the results of the analysis display that the predominant factor is the field effects of o-substituents. The determination of predominant factors in those reactions would provide further scientific basis for those reaction mechanisms.

Effects of ortho-Substituents in Protonolysis of Arylmercuric Chlorides with Hydrochloric Acid

The kinetics of reaction of ortho-substituted phenylmercuric chlorides, 2-R-C6H4HgCl (R = H, CH3, CH3O, C2H5O, CF3, COOC2H5, Cl, Br, F, NO2), with hydrochloric acid in absolute ethanol in the presence of sodium iodide has been studied. The reaction is of the overall second order, first order with respect to each reactant. The rate constant determined at a fixed temperature decreases in the order of R: C2H5O > CH3O > CH3 > H > COOC2H5 > F > Cl > Br > CF3 > NO2. The role of inductive, resonance and field effects of the ortho-substituents, and the relation between steric demands of the ortho-substituents and volume of attacking species are discussed. The underlying mechanism is proposed on the basis of experimental results.

Synthesis and characterization of the linear MHgM trimetallic complexes [ η5-Bz 5C 5(CO 3M] 2Hg and [ η5-HO 2CC 5H 4(CO 3M] 2Hg(M = Cr, Mo, W). The X-ray crystal structure of η5-Bz 5C 5(CO) 3W] 2Hg

The η 5-Bz 5C 5 ligand-containing trimetallic complexes [ η 5-Bz 5C 5(CO) 3M] 2Hg(M = Cr, Mo, W) were synthesized from the in situ reaction of phenylmercuric chloride with corresponding transition metal lithium salts [ η 5-Bz 5C 5(CO) 3M] Li in THF, whereas the η 5-HO 2CC 5H 4 ligand-containing trimetallic complexes [ η 5-HO 2CC 5H 4(CO) 3M] 2Hg were synthesized through hydrolysis of their corresponding esters in the presence of KOH/EtOH/H 2O, followed by acidification. These compounds were characterized by elemental analysis, IR, 1H NMR and MS spectroscopy, while the structure of [ η 5-Bz 5C 5(CO) 3W] 2Hg was determined by an X-ray diffraction analysis.

Characterization of interstitial collagenases in jaw cyst wall.

Neutral salt extracts of 14 specimens of jaw cysts were prepared. Histopathological analysis showed that the specimens consisted of 6 radicular cysts, 6 dentigerous cysts, 1 residual cyst, and 1 odontogenic keratocyst. One periapical granuloma, 1 dental follicle and a sample of clinically healthy oral mucosa were similarly processed and used as controls. Measurement of collagenase activity by monitoring the formation of specific degradation products of type I and II collagen in solution by SDS-PAGE demonstrated that all the cyst extracts contained collagenase, some of which was endogenously activated. Cyst wall collagenase preferably degraded type I over type II collagen, which suggests that the degradation was due to MMP-1 (matrix metalloproteinase-1) rather than the MMP-8 type. This was further supported by the doxycycline-inhibition profile of cyst collagenase, which was similar to that of MMP-1. Part of the cyst wall collagenase was in latent proenzyme form and probably derived, at least in part, from the newly synthesized intracellular collagenase pool. Latent cyst collagenase was efficiently activated with phenylmercuric chloride and to a lesser extent by gold (I) thioglucose and NaOCl. Western-blotting, using specific antibodies against collagenase from human polymorphonuclear neutrophilic leukocytes (MMP-8) and from fibroblasts (MMP-1), revealed a typical 55/45 kDa doublet; also MMP-8 in the latent 80 kDa form and fragmented to 65 kDa active species were found. These results suggest the presence of MMP-1 and, to a lesser extent, MMP-8 type collagenase in the cyst wall.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and characterization of trimetallic complexes [η 5-RC 5H 4(CO) 3E] 2Hg (E = Cr, Mo, W ; R = Me, Et, n-Bu). Crystal structure of [η 5-EtC 5H 4(CO) 3Cr] 2Hg

The synthesis of six linear trimetallic compounds of the type [η 5-RC 5H 4 (CO) 3E] 2Hg (E = Cr, Mo, W ; R = Me, Et, n-Bu), from phenylmercuric chloride and corresponding transition metal lithium salts [η 5-RC 5H 4(CO) 3E]Li is described. The new compounds were characterized by elemental analysis, MS, IR and 1H NMR spectroscopy and the structure of [η 5-EtC 5H 4(CO) 3Cr] 2Hg as determined by X-ray diffraction. The CrHgCr skeleton of the molecule is diagonal with some departure from linearity and the coordination geometry around each chromium atom approximates to a square-based pyramid with one substituted cyclopentadienyl ring occupying the apical position.

Role of Mesenchymal Collagenase in the Loosening of Total Hip Prosthesis

Fibroblast-type interstitial collagenase (E.C. 3.4.24.7) was associated with loosening of total hip prostheses in eight patients: there were four cemented stems and one cementless stem with the common type of loosening and two cemented stems and one cementless acetabular component with aggressive granulomatous lesions. The authors used a specific, well-characterized, heterologous, affinity-purified, polyclonal rabbit anti-human fibroblast collagenase antiserum applied in avidin-biotin-peroxidase-complex (ABC) staining. In the aggressive granulomatous type of loosening, collagenase was found in most of the fibroblast- and macrophagelike cells, including multinuclear giant cells and epithelioid cells in periprosthetic tissue. Collagenase-positive cells also were found in the periprosthetic tissue associated with common loosening. Collagenase was also found in capillary and postcapillary venule endothelial cells in the richly vascularized aggressive granulomatous tissue. Collagenase was extracted directly from the tissue samples and incubated with soluble Type I collagen. Collagen degradation products then were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the three-fourths length degradation product quantitated by gel scanning densitometry. In both aggressive granulomatosis and the common type of loosening, extractable collagenase was found in tissue. No significant differences between the sample groups were detected in respect to total measurable collagenase, however. The extractable collagenase was present in a latent form that could be activated by the organomercurial procollagenase activator, phenylmercuric chloride (PMC). It is likely that interstitial collagenase contributes to rapid growth of reactive infiltrative tissue, loosening of the prosthesis associated with aggressive granulomatosis, and the periprosthetic lytic process associated with the common type of hip prosthesis loosening.

Heat sensitivity of mercuric ion and organomercurial degrading enzymes of aquatic, mercury-resistant bacteria

Activities of both Hg(2+)-reductase and organomercurial lyase of Hg-resistant aquatic bacteria were stable at 20°C for several days within whole cells. Organomercurial lyase activity degrading specifically thimersol, an organomercurial, was more stable than the corresponding enzyme activity degrading other organomercurials like phenylmercuric acetate (PMA) and methoxyethyl mercuric chloride (MEMC). Hg(2+)-reductases of Gram-negative bacteria in cell-free extract were more heat resistant than those of Gram-positive bacteria, except for those of two species of Bacillus, which were as heat resistant as those of the Gram-negative bacteria. Differences in the heat resistances of organomercurial lyases of two different broad-spectrum Hg-resistant bacteria indicate the presence of two enzyme systems for degrading thimersol, PMA and MEMC.

Organomercury Compounds. XXXI. Preparations and 199Hg N.M.R. Spectra of Organomercury Derivatives of 2-Phenylpyridine, Benzo[h]quinoline, 1-Phenylpyrazole and 3,4,5-Trimethyl-1-phenylpyrazole, and the X-Ray Crystal Structure of Bis[2-(pyridin-2'-yl)phenyl]mercury

2-(Pyridin-2'-yl) phenylmercuric acetate has been prepared by mercuration of 2-phenylpyridine. Symmetrization of the corresponding chloride by alkaline sodium stannite gave bis [2-(pyridin-2'-yl)phenyl]mercury, which was also prepared from 2-(2'-aminophenyl)pyridine by the diazo method and treatment of the initial product with copper powder and aqueous ammonia. Mercuration of benzo [h] quinoline and 3,4,5-trimethyl-1-phenylpyrazole with mercuric acetate followed by treatment with lithium chloride yielded benzo [h]quinolin-10-ylmercuric chloride and 2-(3',4',5'-trimethylpyrazol-1'-yl) phenylmercuric chloride respectively. Treatment of the former product with tribromide ions gave 10-bromobenzo[h] quinoline. The exchange Grignard reaction between 1-phenylpyrazole and ethylmagnesium bromide to give 2-(pyrazol-1'-yl) phenylmagnesium bromide has been monitored by reactions with benzonitrile and D2O to establish optimum conditions for reaction with mercuric bromide giving bis [2-(pyrazol-1'-yl)phenyl]mercury. The 199Hg n.m.r. chemical shifts of the majority of mercurials are shifted substantially downfield relative to the corresponding simple phenylmercurials consistent with weak intramolecular coordination by the heterocyclic nitrogen donor atoms, but a small upfield shift is observed for bis [2-(pyrazol-1'-yl)phenyl]mercury. The X-ray crystal structure of bis [2-(pyridin-2′-yl)phenyl]mercury [monoclinic, space group P21/n, a 12.746(2), b 11.660(2), c 5.698(1) Ǻ, β 92.81(1)′, V 845.8 Ǻ3] shows a centrosymmetric molecule with strong linear two coordination [Hg-C 2.098(8) Ǻ; C-Hg-C 180.0°] and significant but much weaker Hg-N interactions [Hg-N 2.798(7) Ǻ; N-Hg-N 180.0°] giving overall distorted square planar stereochemistry. The phenyl rings are mutually coplanar, whilst the two pyridin-2'-yl rings are parallel and inclined at 10.8° to the phenyl groups.

Gas chromatography of chloride and bromide as phenylboronic acid/mercuric nitrate derivatives with microwave induced plasma atomic emission detection

Capillary column gas chromatography (GC) with flame ionization detection (FID) or microwave induced plasma atomic emission detection (MIP-AED) were examined for the determination of chloride, using phenylboronic acid as derivatizing reagent. Phenylboronic acid and mercuric nitrate were used in a 1:1 molar ratio to produce phenylmercuric chloride (PMC). The limit of detection for chloride as PMC using FID was 3.3·10 −10 g and using AED, measuring the mercury response at 185 nm, it was 6.25·10 −11 g. Under the conditions used, chloride and bromide (as PMC and PMB) failed to separate adequately, but they could be determined by GC-AED using the wavelenghts 478 nm and 479 nm for bromide and chloride, respectively. The specific detection limits for bromide and chloride obtained from bromine and chlorine responses were 2.9·10 −9 g and 1.8·10 −9 g per injection.

Substrate Specificity and Activation Mechanisms of Collagenase from Human Rheumatoid Synovium

Substrate specificity studies of collagenase extracted from human rheumatoid synovium suggest that synovial pannus tissue overlying articular cartilage may not be particularly active in degradation of cartilage type II collagen, which, considering the poor inherent healing capacity of the articular hyaline cartilage, may exert a protective function against inadvertant tissue damage. Rheumatoid synovial tissue was also used to establish synovial fibroblast cell lines. Treatment of these cells in monolayer cultures with IL-1 leads to collagenase gene activation, increased collagenase production and an almost complete autoactivation of secreted collagenase. Interleukin-1 also activated stromelysin gene suggesting this as a possible mechanism effecting autoactivation. Latent human fibroblast and macrophage collagenase purified from culture medium were efficiently activated by phenylmercuric chloride but also by gold thioglucose, gold sodium thiomalate and HCIO. These new observations support the Cys73 switch activation mechanism. In contrast to neutrophil collagenase, the activation by gold(I) compounds and HCIO was associated with a change in the apparent molecular weight of the fibroblast procollagenase. In addition, gold(I) compounds rendered collagenase more susceptible to thermal denaturation. Thus the fibroblast-type interstitial collagenase, probably derived from fibroblast- and macrophage-like synoviocytes, seems to provide the predominant collagenolytic potential in human rheumatoid synovial tissue. Furthermore, the conditions in synovitis tissue may be such as to favor at least initial activation of collagenase synthesized and secreted in situ.

Hypochlorous acid induced activation of human neutrophil and gingival crevicular fluid collagenase can be inhibited by ascorbate.

Interstitial collagenase either obtained from human neutrophils by phorbol myristate acetate (PMA) induced degranulation or isolated from human gingival crevicular fluid was found to be activated by addition of an oxidative agent, hypochlorous acid (HOCl). Collagenase released by PMA stimulated neutrophils was completely in latent form but underwent partial autoactivation during 16 h incubation at 22 degrees C in the presence of soy bean trypsin inhibitor. The partial autoactivation was potentiated to complete activation of released collagenase after addition of exogenous HOCl. Ascorbate prevented this activation of neutrophil collagenase. Isolated human gingival crevicular fluid collagenase represented an apparent Mr of 70 kD in completely latent form, whereas 70/54 kD enzyme species were detected for partially autoactive form of the enzyme. Western blot analysis of gingival crevicular fluid using a polyclonal antibody raised against purified human neutrophil collagenase revealed the same 70/54 kD molecular forms of the enzyme. The latent gingival crevicular fluid collagenase was also activated by HOCl and this activation could be prevented by ascorbate. Activation of the 70 kD latent collagenase by HOCl as well as by other non-proteolytic activators such as an organomercurial compound (phenylmercuric chloride) and a gold(I) compound (gold thioglucose) was not associated with detectable changes in apparent Mr, whereas trypsin activation resulted in fragmentation of 70 kD enzyme to 54 kD species. Our results provide further evidence for the neutrophil origin of gingival crevicular fluid collagenase and suggest that, in addition to proteolytic activation, oxidative and antioxidative agents seem to be able to regulate neutrophil collagenase activity.

Collagenase Reserves in Polymorphonuclear Neutrophil Leukocytes from Synovial Fluid and Peripheral Blood of Patients with Rheumatoid Arthritis

Degradation of cartilage in rheumatoid arthritis (RA) may be in part due to release of collagenase from specific granules of polymorphonuclear neutrophil leukocytes (PMNs) during degranulation. We decided to study, not synovial fluid (SF) collagenase, but PMN collagenase reserves. PMN were isolated from parallel SF and peripheral blood (PB) samples obtained from 7-arthritis patients. PMNs were stimulated in vitro by tetradecanoyl-phorbol-13-acetate (TPA). Collagenase activity in the supernatant without and with phenylmercuric chloride activation was studied. Compared to PB PMNs, there was no consistent decrease in the total collagenase reserves in the inflammatory SF PMNs. This suggests that the release of collagenase in the inflammatory synovial fluid does not deplete SF PMNs of the collagenase synthesized at the myelocyte stage. The role of PMN collagenase in pathogenesis of cartilage destruction would then seem to be more dependent on local release and autoactivation at cartilage surface by adherent PMNs and not excessive collagenase release from free floating SF PMNs at single cell level. Furthermore, under the experimental conditions used the proportion of collagenase released in active form was higher in SF PMN specimens than in PB PMN specimens (p less than 0.01). The predominant collagenous component of adult cartilage, native type II collagen, was degraded by PMN collagenase as fast as native type I collagen. These findings suggest an important role for this enzyme in destruction of the free cartilage surface in RA.

ChemInform Abstract: Synthesis of Chiral Adenosine Receptor Recognition Units via a Sharpless Asymmetric Epoxidation Procedure.

AbstractButadiene monoxide (II) reacts with phenylmercuric chloride (I) in the presence of a Pd catalyst to form trans‐4‐phenyl‐2‐buten‐1‐ol (III) which is oxidized with peracids.