The Effect of Dual Bioactive Compounds Artemisinin and Metformin Co-loaded in PLGA-PEG Nano-particles on Breast Cancer Cell lines: Potential Apoptotic and Anti-proliferative Action.

The most prevalent malignancy among women is breast cancer. Phytochemicals and their derivatives are rapidly being recognized as possible cancer complementary therapies because they can modify signaling pathways that lead to cell cycle control or directly alter cell cycle regulatory molecules. The phytochemicals' poor bioavailability and short half-life make them unsuitable as anticancer drugs. Applying PLGA-PEG NPs improves their solubility and tolerance while also reducing drug adverse effects. According to the findings, combining anti-tumor phytochemicals can be more effective in regulating several signaling pathways linked to tumor cell development. The point of the study was to compare the anti-proliferative impacts of combined artemisinin and metformin on cell cycle arrest and expression of cyclin D1 and apoptotic genes (bcl-2, Bax, survivin, caspase-7, and caspase-3), and also hTERT genes in breast cancer cells. T-47D breast cancer cells were treated with different concentrations of metformin (MET) and artemisinin (ART) co-loaded in PLGA-PEG NPs and free form. The MTT test was applied to assess drug cytotoxicity in T47D cells. The cell cycle distribution was investigated using flow cytometry and the expression levels of cyclin D1, hTERT, Bax, bcl-2, caspase-3, and caspase-7, and survivin genes were then determined using real-time PCR. The findings of the MTT test and flow cytometry revealed that each state was cytotoxic to T47D cells in a time and dose-dependent pattern. Compared to various state of drugs (free and nano state, pure and combination state) Met-Art-PLGA/PEG NPs demonstrated the strongest anti-proliferative impact and considerably inhibited the development of T-47D cells; also, treatment with nano-formulated forms of Met-Art combination resulted in substantial downregulation of hTERT, Bcl-2, cyclin D1, survivin, and upregulation of caspase-3, caspase-7, and Bax, in the cells, as compared to the free forms, as indicated by real-time PCR findings. The findings suggested that combining an ART/MET-loaded PLGA-PEG NP-based therapy for breast cancer could significantly improve treatment effectiveness.

Ultra-sensitive speciation analysis of mercury by CE-ICP-MS together with field-amplified sample stacking injection and dispersive solid-phase extraction.

A simple dispersive solid-phase extraction (DSPE) used to extract and preconcentrate ultra-trace MeHg, EtHg and Hg(2+) from water sample, and a sensitive method for the simultaneous analysis of MeHg, EtHg and Hg(2+) by using capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS) with field-amplified sample stacking injection (FASI) were first reported in this study. The DSPE used thiol cotton particles as adsorbent, and is simple and effective. It can be used to extract and preconcentrate ultra-trace mercury compounds in water samples within 30 min with a satisfied recovery and no mercury species alteration during the process. The FASI enhanced the sensitivity of CE-ICP-MS with 25-fold, 29-fold and 27-fold for MeHg, EtHg and Hg(2+) , respectively. Using FASI-CE-ICP-MS together with DSPE, we have successfully determined ultra-trace MeHg, EtHg and Hg(2+) in tap water with a limits of quantification (LOQs) of 0.26-0.45 pg/mL, an RSD (n = 3) < 6% and a recovery of 92-108%. Ultra-high sensitivity, as well as much less sample and reagent consumption and low operating cost, make our method a valuable technique to the speciation analysis of ultra-trace mercury.

Anatomical mercury speciation in bay scallops by thio-bearing chelating resin concentration and GC-electron capture detector determination.

The highly toxic methyl-, ethyl- and phenylmercury species that may exist in the three main anatomical parts - the adductor muscle, the mantle and the visceral mass - of bay scallops (Argopecten irradias) were quantitatively released by cupric chloride, zinc acetate, sodium chloride and hydrochloric acid (HCl) under ultrasonic extraction. After centrifugation, the mercury species in the supernatant were concentrated by thio (SH)-bearing chelating resins, eluted with HClO4 and HCl and extracted with toluene. Separation was achieved by capillary GC equipped with programmed temperatures, a constant nitrogen flow and detected by a micro-electron capture detector (µECD). Under optimised conditions, the LODs for methyl-, ethyl- and phenylmercury in bay scallop samples were 1.1, 0.65 and 0.80 ng g(-1), respectively. The maximum RSD for three replicate determinations of methyl-, ethyl- and phenylmercury in bay scallop samples were 13.7%, 14.0% and 11.2%, respectively. In the concentration range of 4-200 ng g(-1) in bay scallop samples, the calibration graphs were linear with correlation coefficients not less than 0.997. Recoveries for spiked samples were in the range of 92.7-103.5% (methylmercury), 87.5-108.3% (ethylmercury) and 91.6-106.0% (phenylmercury), respectively. The method was verified by the determination of methylmercury in a CRM GBW10029 (Total Mercury and Methyl Mercury in Fish Tissue), with results in good agreement with the certified values. Methylmercury - the only existing species in bay scallops - was successfully determined by the method.

Determination of mercurial species in fish by inductively coupled plasma mass spectrometry with anion exchange chromatographic separation.

This work demonstrated the feasibility of mercury speciation analysis by anion exchange chromatographic separation with inductively coupled plasma mass spectrometry detection. For the first time, by complexing with the mobile phase containing 3-mercapto-1-propanesulfonate into negatively charged complexes, fast separation of inorganic mercury (Hg(2+)), monomethylmercury (MeHg), ethylmercury (EtHg) and phenylmercury (PhHg) was achieved within 5 min on a 12.5-mm strong anion exchange column. The detection limits for Hg(2+), MeHg, EtHg and PhHg were 0.008, 0.024, 0.029 and 0.034 µg L(-1), respectively. The relative standard deviations of peak height and peak area (5.0 µg L(-1) for each Hg species) were all below 3%. The determined contents of Hg(2+), MeHg and total Hg in a certified reference material of fish tissue by the proposed method were in good accordance with the certified values with satisfactory recoveries. The relative errors for determining MeHg and total mercury were -2.4% and -1.2%, respectively, with an acceptable range for spike recoveries of 94-101%. Mercury speciation in 11 fish samples were then analyzed after the pretreated procedure. The mercury contents in all fish samples analyzed were found compliant with the criteria of the National Standards of China.

Toxicology of alkylmercury compounds.

Methylmercury is a global pollutant and potent neurotoxin whose abundance in the food chain mandates additional studies on the consequences and mechanisms of its toxicity to the central nervous system. Formulation of our new hypotheses was predicated on our appreciation for (a) the remarkable affinity of mercurials for the anionic form of sulfhydryl (-SH) groups, and (b) the essential role of thiols in protein biochemistry. The present chapter addresses pathways to human exposure of various mercury compounds, highlighting their neurotoxicity and potential involvement in neurotoxic injury and neurodegenerative changes, both in the developing and senescent brain. Mechanisms that trigger these effects are discussed in detail.

Nowhere to hide: Chemical toxicants and the unborn child.

Contemporary reproductive aged women and their offspring are facing an unprecedented onslaught of toxicant exposures from myriad sources in their day-to-day life. Public health recommendations regarding optimal diet and nutrition in pregnancy must incorporate several considerations including safety of available foodstuffs, cultural practices and lifestyle issues. Gestational consumption of contaminated seafood remains a potential source of toxicant exposure, including mercury, for the developing child. Health care professionals responsible for the care of women and their developing children need to become apprised of: a) risks associated with toxicant bioaccumulation in pregnancy; b) ongoing information emerging in the important field of reproductive toxicology; and c) strategies within the clinical setting to facilitate nutritional sufficiency and precautionary avoidance of adverse exposure among young women.

Synthesis, structure, and reactivity of two-coordinate mercury alkyl compounds with sulfur ligands: relevance to mercury detoxification.

The susceptibility of two-coordinate mercury alkyl compounds of the type X-Hg-R (where X is a monodentate sulfur donor) towards protolytic cleavage has been investigated as part of ongoing efforts to obtain information relevant to understanding the mechanism of action of the organomercurial lyase, MerB. Specifically, the reactivity of the two-coordinate mercury alkyl compounds PhSHgR, [mim(Bu(t))]HgR and {[Hmim(Bu(t))]HgR}(+) (Hmim(Bu(t)) = 2-mercapto-1-t-butylimidazole; R = Me, Et) towards PhSH was investigated, thereby demonstrating that the ability to cleave the Hg-C bond is very dependent on the nature of the system. For example, whereas the reaction of PhSHgMe with PhSH requires heating at 145 degrees C for several weeks to liberate CH(4), the analogous reaction of PhSHgEt with PhSH leads to evolution of C(2)H(6) over the course of 2 days at 100 degrees C. Furthermore, protolytic cleavage of the Hg-C bond by PhSH is promoted by Hmim(Bu(t)). For example, whereas the reaction of {[Hmim(Bu(t))]HgEt}(+) with PhSH eliminates C(2)H(6) at elevated temperatures, the protolytic cleavage occurs over a period of 2 days at room temperature in the presence of Hmim(Bu(t)). The ability of Hmim(Bu(t)) to promote the protolytic cleavage is interpreted in terms of the formation of a higher coordinate species {[Hmim(Bu(t))](n)HgR}(+) that is more susceptible to Hg-C bond cleavage than is two-coordinate {[Hmim(Bu(t))]HgR}(+). These observations support the notion that access to a species with a coordination number greater than two is essential for efficient activity of MerB.

Metal-induced impairment of the cellular immunity of newborn harbor seals (Phoca vitulina).

The cellular immunity of newborn harbor seals and the influence of pollutants are rarely investigated. This study evaluated the lymphocyte proliferation using a lymphocyte proliferation test (LTT) to understand the dynamics of immune response in seal pups of varying ages from the moment they arrived in a seal center after active beaching until their release into wildlife 3 months later after rehabilitation. Moreover, the effect of various metals (Ag, Al, Au, Be, Cd, Co, Cr, Cu, different Hg compounds, Mo, Ni, Pb, Pd, Pt, Sn, Ti) on lymphocyte proliferation in terms of immunosuppression and hypersensitivity was investigated. First, a strong lymphocyte proliferation in newborns as a reflection of relative immunocompetence was found. Second, different metal-induced influences on lymphocyte proliferation such as specific inhibition by Be, Cd, Hg, and Sn as well as stimulation induced by Mo and Ni were determined. For seals tested repeatedly, the suppressive effect was detected in newborns but not found in the same animals when they were older and had become immunologically competent. Summarizing, the lymphocyte proliferation used as a marker in this investigation provided useful immunological information on these developing animals, and its application for toxicological studies on pollutants can be recommended.

Human serum albumin-mercurial species interactions.

Binding of metal ions to the heteroatomic sites of proteins is undoubtedly fundamental to their observed physiological effects. In this paper, the interactions of inorganic mercury (Hg2+), methylmercury (MeHg+), ethylmercury (EtHg+), and phenylmercury (PhHg+) with human serum albumin (HSA) were studied from the electrophoretic behaviors, stoichiometry, thermodynamics, and kinetics by using a new hybrid technique, capillary electrophoresis on-line coupled with electrothermal atomic absorption spectrometry (CE-ETAAS), together with the consequent structural information from circular dichroism and Raman spectroscopy. The stoichiometry (mercurial species to HSA) for the interactions of Hg2+, MeHg+, EtHg+, and PhHg+ with HSA was found to be 6:1, 4:1, 4:1, and 3:1, respectively. Two types of binding sites in HSA were observed for the binding of mercurial species with the orders of magnitude of binding constants of 10(7) and 10(6) L mol-1, respectively, showing strong affinity of mercurial species for HSA. The interactions of mercurial species with both types of binding sites in HSA are exothermic and thermodynamically favorable and are both enthalpically and entropically driven. The binding of mercurial species to HSA follows the first-order kinetics for mercurial species and zero-order kinetics for HSA with the apparent activation energy of 57-59 kJ mol-1. Among the four mercurial species examined, only Hg2+ induces the secondary structure transition of HSA. Mercury-HSA adducts are formed mainly through metal-sulfur binding with participation of C=O and/or C-N groups of amino acid residues in HSA molecules. The present work represents the most comprehensive study on the interactions between various mercurial species with HSA and provides new evidence for and insights into the interactions of mercurial species with HSA for further understanding of the toxicological effects of mercurial species.

Ion exchange preconcentration and separation of mercury species by CE with indirect contactless conductometric detection.

Capillary electrophoretic separation of four mercury species (inorganic Hg(2+), methyl-, ethyl-, and phenylmercury) was achieved in less than 8 min with an electrolyte consisting of 150 mM 2-amino-2-methyl-propanol (AMP) at pH 11.6. The analytes were complexed with 0.1% mercaptopropionic acid (MPA), separated in counter-EOF as anionic complexes, and detected by a contactless conductometric detector. Ion exchange preconcentration with on-column formation of MPA-mercury complexes was developed. Preconcentration factors of 25-150 were achieved and LODs in the range of 2.9-6.9 ng/mL were feasible. This method may prove to be applicable as a rapid screening method for mercury speciation in environmental samples.

1,6-Hexanedithiol monolayer as a receptor for specific recognition of alkylmercury.

1,6-Hexanedithiol monolayer acts as an unusually specific recognition agent for CH3Hg+ when the microcantilever is used as the transducer; the mechanism of the sensor is discussed.

Thimerosal positivities: the rôle of SH groups and divalent ions.

For a better understanding of the mechanistic details of the interactions of organomercury compounds inside the skin, 32 subjects who previously had given positive patch-test reactions to thimerosal (TH) and negative reactions to thiosalicylic acid, were divided into 2 groups. 16 subjects were repatch tested to ethylmercury chloride (EtHgCl) and to solutions containing EtHgCl mixed with L-cysteine and glutathione, respectively. The remaining 16 were repatch tested to EtHgCl and to solutions containing EtHgCl mixed with chlorides of Zn, Mg, and Mn, respectively. The results showed that whilst L-cysteine, glutathione and ZnCl2 were able to abolish or to reduce the positive reactions to EtHgCl, chlorides of Mg and Mn were unable to do so. Patch tests revealed that in causing positive reactions to TH, EtHg probably interacted with thiol groups and with Zn ions, as in biological systems when causing toxic effects. The limited number of TH reactions in the general population, the constant presence of concomitant positive reactions to EtHgCl and MeHgCl, and the lack of cross-reactivity with other organic or inorganic mercury compounds, lead us to speculate that reactions to TH are due to organomercury alkyl compounds, and that positive subjects have a constitutively reduced capability to metabolize organomercury compounds, rather than to reveal previous exposure.

[Determination of alkylmercuric compounds in human body fluids by gas chromatography--atomic absorption spectrophotometry].

In order to detect trace of alkylmercuric compounds in human body fluids, a highly sensitive, selective and rapid method, based on the coupling of gas chromatography with atomic absorption spectrophotometry, has been developed. The transfer-tube was made up of Teflon, with stainless steel tubes in the two sides. The atomization of alkylmercuric compounds was performed in quartz tube. The quantitative determination of alkylmercuric compounds in urine and blood samples collected from the workers exposing to metallic mercury, has been extensively carried out in this work. The optimal Westoo program was used to enrich methylmercuric chloride in samples, Extracts of methylmercuric chloride in benzene were determined by GC-AAS, with a total recovery of more than 90%. Dimethylmercury in samples was extracted once by xylene. Then the extracts were determined, with a total recovery of 65%-70%. The detectable limit for mercury was 0.01ng. The methylation of mercury was confirmed in our experiment. This method can be used for the determination of other metallic compounds.

Drug-protein interactions. Refined structures of three sulfonamide drug complexes of human carbonic anhydrase I enzyme.

N-unsubstituted sulfonamide drugs are widely used for opthalmic disorders. Inhibition of carbonic anhydrase enzyme is believed to be the chief reason for their therapeutic effects. Structures of three such sulfonamide drugs complexed to human carbonic anhydrase I enzyme (HCAI) refined crystallographically at 2 A resolution are reported here. The drug molecules are all bound in the active site of the enzyme, but among themselves show differences in the orientations of the sulfamido groups interacting with the essential zinc ion in the active site. The activity linked solvent molecule coordinated to zinc in the native enzyme is displaced by all the three sulfonamides. The active site loop of Leu198, Thr199 and His200 has been identified to be important for binding of the drug molecules due to their appreciable atomic displacements and intra-molecular hydrogen bonds arising out of their interactions with the sulfonamides. These interactions along with active site charge requirements are proposed to be responsible for the orientational differences of the sulfamido groups and also for differences in the inhibitory powers of the drugs. A hydrogen bond network involving solvent molecules and active site residues His200 and His67 amongst others in the native enzyme, is disrupted upon binding of methazolamide but not in the other two sulfonamides. This is the first crystallographic evidence of the possible involvement of His200 in the inhibition of HCAI. An important role of Thr199 in distinguishing between the substrate and inhibitor binding modes of HCO3- to the enzyme at high pH is also inferred.

[Study of the binding of diuretics by serum proteins according to changes in tryptophan fluorescence]. / Izuchenie sviazyvaniia diuretikov s belkami syvorotki krovi po izmeneniiu triptofanovoi fliuorestsentsii.

The effect of diuretics on tryptophan fluorescence of blood serum proteins divided by electrophoresis was studied. It was shown that ethacrynic acid produces the most significant extinction of tryptophan fluorescence in albumin fraction and novurit in globulin fraction. Furosemide possessing a high affinity for all three obtained fractions of protein does not exhibit a preferential binding to one or another of these fractions. It was also found that furosemide and ethacrynic acid by binding to human serum albumin molecule produce its conformational alterations. Mercury diuretic does not possess such effect.

Crystallographic studies of inhibitor binding sites in human carbonic anhydrase II: a pentacoordinated binding of the SCN- ion to the zinc at high pH.

The binding of four inhibitors--mercuric ion, 3-acetoxymercuri-4-aminobenzenesulfonamide (AMS), acetazolamide (Diamox), and thiocyanate ion--to human carbonic anhydrase II (HCA II) has been studied with X-ray crystallography. The binding of mercury to HCA II at pH 7.0 has been investigated at 3.1 A resolution. Mercuric ions are observed at both nitrogens in the His-64 ring. One of these sites is pointing toward the zinc ion. The only other binding site for mercury is at Cys-206. The binding of the two sulfonamide inhibitors AMS and Diamox, has been reinvestigated at 2.0 and 3.0 A, respectively. Only the nitrogen of the sulfonamide group binds to the zinc ion replacing the hydroxyl ion. The sulfonamide oxygen closest to the zinc ion is 3.1 A away. Thus the tetrahedral geometry of the zinc is retained, refuting earlier models of a pentacoordinated zinc. The structure of the thiocyanate complex has been investigated at pH 8.5 and the structure has been refined at 1.9 A resolution using the least-squares refinement program PROLSQ. The crystallographic R factor is 17.6%. The zinc ion is pentacoordinated with the anion as well as a water molecule bound in addition to the three histidine residues. The nitrogen atom of the SCN- ion is 1.9 A from the zinc ion but shifted 1.3 A with respect to the hydroxyl ion in the native structure and at van der Waals' distance from the O gamma l atom of Thr-199. This is due to the inability of the O gamma l atom of Thr-199 to serve as a hydrogen bond donor, thus repelling the nonprotonated nitrogen. The SCN- molecule reaches into the deep end of the active site cavity where the sulfur atom has displaced the so-called "deep" water molecule of the native enzyme. The zinc-bound water molecule is 2.2 A from the zinc ion and 2.4 A from the SCN- nitrogen. In addition, this water is hydrogen bonded to the O gamma l atom of Thr-199 and to another water molecule. We have observed that solvent and inhibitor molecules have three possible binding sites on the zinc ion and their significance for the catalysis and inhibition of HCA II will be discussed. All available crystallographic data are consistent with a proposed catalytic mechanism in which both the OH moiety and one oxygen of the substrate HCO3- ion are ligated to the zinc ion.

Synthesis of 2,6-anhydro-S-[ethylmercury(II)]-1-thio-D-glycero-L-manno-heptitol+ ++ and bis(2,6-anhydro-1-thio-D-glycero-L-manno-heptitol)mercury(II), and the study of their interaction with beta-D-galactosidase from E. coli.

Two competitive inhibitors of beta-D-galactosidase activity, namely, 2,6-anhydro-S-[ethylmercury(II)]-1-thio-D-glycero-L-manno-heptitol (4) and bis(2,6-anhydro-1-thio-D-glycero-L-manno-heptitol)mercury(II) (6), with inhibition constants of 8.0 X 10(-4) M and 1.9 X 10(-4) M, were synthesized. Compound 6 was incorporated into the crystalline enzyme by cocrystallization. The stoichiometry of the enzyme-inhibitor complex was 1:4, corresponding to one molecule of inhibitor per active site of the enzyme. Compound 4 was found to be unstable against X-ray irradiation, whereas compound 6 was submitted to X-rays for several days without any radiation damage.