Novel palladium(II) complexes containing a sulfur ligand: structure and biological activity on HeLa cells

Two novel palladium(II) complexes with a thiosalicylic acid (HSC₆H₄CO₂H) ligand, with the formulas [Pd(TSA)(L)]·mH₂O (TSA is thiosalicylic acid; in complex 1, L is 1,10-phenanthroline and m = 1; in complex 2, L is 2,2′-bipyridine and m = 2), have been synthesized and characterized. The coordination geometry of both palladium atoms is square planar; they are four-coordinated and each is coordinated in an N,N,O⁻,S⁻ mode. There is a sigmoid oxygen chain in complex 1, but an oxygen ring in complex 2. The competitive binding of the complexes to HeLa cell DNA (HL-DNA) has been investigated by fluorescence spectroscopy. The results show that the two complexes have the ability to bind with HL-DNA. Viscosity studies suggest that the complexes bind to DNA by intercalation. Gel electrophoresis assay demonstrated the ability of the complexes to cleave the HL-DNA. The two complexes exhibit cytotoxic specificity and a significant cancer cell inhibitory rate. The apoptosis tests indicated that the complexes have an apoptotic effect. Furthermore, complex 1 exhibits more biological activity than complex 2, which is mainly because the area of the aromatic ring of 1,10-phenanthroline is larger than that of 2,2′-bipyridine.     

DNA molecular recognition and cellular selectivity of anticancer metal(II) complexes of ethylenediaminediacetate and phenanthroline: multiple targets

Abstract  

By inhibiting only two or three of 12 restriction enzymes, the series of [M(phen)(edda)] complexes [M(II) is Cu, Co, Zn; phen is 1,10-phenanthroline; edda is N,N′-ethylenediaminediacetate] exhibit DNA binding specificity. The Cu(II) and Zn(II) complexes could differentiate the palindromic sequences 5′-CATATG-3′ and 5′-GTATAC-3′, whereas the Co(II) analogue could not. This and other differences in their biological properties may arise from distinct differences in their octahedral structures. The complexes could inhibit topoisomerase I, stabilize or destabilize G-quadruplex, and lower the mitochondrial membrane potential of MCF7 breast cells. The pronounced stabilization of G-quadruplex by the Zn(II) complex may account for the additional ability of only the Zn(II) complex to induce cell cycle arrest in S phase. On the basis of the known action of anticancer compounds against the above-mentioned individual targets, we suggest the mode of action of the present complexes could involve multiple targets. Cytotoxicity studies with MCF10A and cisplatin-resistant MCF7 suggest that these complexes exhibit selectivity towards breast cancer cells over normal ones.     

Combined arene ruthenium porphyrins as chemotherapeutics and photosensitizers for cancer therapy

Mononuclear 5-(4-pyridyl)-10,15,20-triphenylporphyrin and 5-(3-pyridyl)-10,15,20-triphenylporphyrin as well as tetranuclear 5,10,15,20-tetra(4-pyridyl)porphyrin (tetra-4-pp) and 5,10,15,20-tetra(3-pyridyl)porphyrin) (tetra-3-pp) arene ruthenium(II) derivatives (arene is C₆H₅Me or p-Pr ⁱ C₆H₄Me) were prepared and evaluated as potential dual photosensitizers and chemotherapeutics in human Me300 melanoma cells. In the absence of light, all tetranuclear complexes were cytotoxic (IC₅₀ ≤ 20 μM), while the mononuclear derivatives were not (IC₅₀ ≥ 100 μM). Kinetic studies of tritiated thymidine and tritiated leucine incorporations in cells exposed to a low concentration (5 μM) of tetranuclear p-cymene derivatives demonstrated a rapid inhibition of DNA synthesis, while protein synthesis was inhibited only later, suggesting arene ruthenium–DNA interactions as the initial cytotoxic process. All complexes exhibited phototoxicities toward melanoma cells when exposed to laser light of 652 nm. At low concentration (5 μM), LD₅₀ of the mononuclear derivatives was between 5 and 10 J/cm², while for the tetranuclear derivatives LD₅₀ was approximately 2.5 J/cm² for the [Ru₄(η⁶-arene)₄(tetra-4-pp)Cl₈] complexes and less than 0.5 J/cm² for the [Ru₄(η⁶-arene)₄(tetra-3-pp)Cl₈] complexes. Examination of cells under a fluorescence microscope revealed the [Ru₄(η⁶-arene)₄(tetra-4-pp)Cl₈] complexes as cytoplasmic aggregates, whereas the [Ru₄(η⁶-arene)₄(tetra-3-pp)Cl₈] complexes were homogenously dispersed in the cytoplasm. Thus, these complexes present a dual synergistic effect with good properties of both the arene ruthenium chemotherapeutics and the porphyrin photosensitizer.     

Biological activity of enantiomeric complexes [PtCl2L2] (L2 is aromatic bisphosphanes and aromatic diamines)

Enantiomeric complexes of formula [PtCl₂L₂] [L₂ is (R)-(+)-BINAP and (S)-(−)-BINAP, where BINAP is 2,2′-bis(diphenylphosphane)-1,1′-binaphthyl, and (R)-(+)-DABN and (S)-(−)-DABN, where DABN is 1,1′-binaphthyl-2,2′-diamine], were tested for their cytotoxic activity against three cancer cell lines and for their ability to bind to the human telomeric sequence folded in the G-quadruplex structure. Similar experiments were carried out on prototypal complexes cisplatin and cis-[PtCl₂(PPh₃)₂] for comparison. Platinum complexes containing phosphanes proved less cytotoxic to cancer cell lines and less likely to interact with the nucleobases of the G-quadruplex than those containing amines; in both cases the S-(−) isomer was more active than the R-(+) counterpart. More specifically, whereas all the platinum complexes were able to platinate the G-quadruplex structure from the human telomeric repeat, the extent and sites of platination depended on the nature of the ligands. Complexes containing (bulky) phosphanes interacted only with the adenines of the loops, whereas those containing the less sterically demanding amines interacted with adenines and some guanines of the G-quartet.     

Type 1 copper site synthetic model complexes with increased redox potentials

Reactions of NaSCPh₃ with (R₃tacn)Cu(OTf)₂ (R is Me, iPr; tacn is 1,4,7-triazacyclononane; OTf is CF₃SO₃ ⁻) yield blue complexes identified as ((R₃tacn)CuSCPh₃)(OTf) on the basis of UV–vis, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy and electrospray ionization mass spectrometry. These complexes exhibit spectroscopic properties typical of type 1 copper sites in proteins, including diagnostic Sπ → Cu(d_{x² - y²} ) (d_{{x^{2} - y^{2} }} ) ligand-to-metal charge transfer transitions at approximately 610–630 nm and small A _|| values in EPR spectra of less than 100 × 10⁻⁴ cm⁻¹. Cyclic voltammetry experiments revealed redox potentials for the complexes similar to those of several low-potential type 1 copper proteins (e.g., azurin, stellacyanin) and approximately 0.5 V higher than those of previously reported model compounds. Thus, the new complexes mimic key aspects of both the structure and the function of type 1 copper sites.     

Evaluation of novel <Superscript>99m</Superscript>Tc(I)-labeled homobivalent α-melanocyte-stimulating hormone analogs for melanocortin-1 receptor targeting

Abstract  

Aiming to apply the multivalency concept to melanoma imaging, we have assessed the in vivo melanocortin type 1 receptor (MC1R)-targeting properties of ^99mTc(I)-labeled homobivalent peptide conjugates which contain copies of the α-melanocyte-stimulating hormone (α-MSH) analog [Ac-Nle⁴, Asp⁵, d-Phe⁷, Lys¹¹]α-MSH_4–11 separated by linkers of different length (L ² nine atoms and L ³ 14 atoms). The MC1R-binding affinity of L ² and L ³ is significantly higher than that of the monovalent conjugate L ¹ . Metallation of these conjugates yielded the complexes fac-[M(CO)₃(k³-L)]⁺ (M is ^99mTc/Re; 1/1a, L is L ¹ ; 2/2a, L is L ² ; 3/3a, L is L ³ ), with IC₅₀ values in the subnanomolar and nanomolar range. The MC1R-mediated internalization of 2 and 3 is higher than that of 1 in B16F1 melanoma cells. Biodistribution studies in melanoma-bearing mice have shown low nonspecific accumulation with a tumor uptake that correlates with IC₅₀ values. However, no correlation between tumor uptake and valency was found. Nevertheless, 2 displayed the highest tumor retention, and the best tumor to nontarget organ ratios.     

Assessing the in vitro toxicity of the lunar dust environment using respiratory cells exposed to Al<Subscript>2</Subscript>O<Subscript>3</Subscript> or SiO<Subscript>2</Subscript> fine dust particles

Prior chemical and physical analysis of lunar soil suggests a composition of dust particles that may contribute to the development of acute and chronic respiratory disorders. In this study, fine Al₂O₃ (0.7 μm) and fine SiO₂ (mean 1.6 μm) were used to assess the cellular uptake and cellular toxicity of lunar dust particle analogs. Respiratory cells, murine alveolar macrophages (RAW 264.7) and human type II epithelial (A549), were cultured as the in vitro model system. The phagocytic activity of both cell types using ultrafine (0.1 μm) and fine (0.5 μm) fluorescent polystyrene beads was determined. Following a 6-h exposure, RAW 264.7 cells had extended pseudopods with beads localized in the cytoplasmic region of cells. After 24 h, the macrophage cells were rounded and clumped and lacked pseudopods, which suggest impairment of phagocytosis. A549 cells did not contain beads, and after 24 h, the majority of the beads appeared to primarily coat the surface of the cells. Next, we investigated the cellular response to fine SiO₂ and Al₂O₃ (up to 5 mg/ml). RAW 264.7 cells exposed to 1.0 mg/ml of fine SiO₂ for 6 h demonstrated pseudopods, cellular damage, apoptosis, and necrosis. A549 cells showed slight toxicity when exposed to fine SiO₂ for the same time and dose. A549 cells had particles clustered on the surface of the cells. Only a higher dose (5.0 mg/ml) of fine SiO₂ resulted in a significant cytotoxicity to A549 cells. Most importantly, both cell types showed minimal cytotoxicity following exposure to fine Al₂O₃. Overall, this study suggests differential cellular toxicity associated with exposure to fine mineral dust particles.     

Chiral Ruthenium(II) Polypyridyl Complexes: Stabilization of G-Quadruplex DNA,Inhibition of Telomerase Activity and Cellular Uptake

Two ruthenium(II) complexes, Λ-[Ru(phen)₂(p-HPIP)]²⁺ and Δ-[Ru(phen)₂(p-HPIP)]²⁺, were synthesized and characterized via proton nuclear magnetic resonance spectroscopy, electrospray ionization-mass spectrometry, and circular dichroism spectroscopy. This study aims to clarify the anticancer effect of metal complexes as novel and potent telomerase inhibitors and cellular nucleus target drug. First, the chiral selectivity of the compounds and their ability to stabilize quadruplex DNA were studied via absorption and emission analyses, circular dichroism spectroscopy, fluorescence-resonance energy transfer melting assay, electrophoretic mobility shift assay, and polymerase chain reaction stop assay. The two chiral compounds selectively induced and stabilized the G-quadruplex of telomeric DNA with or without metal cations. These results provide new insights into the development of chiral anticancer agents for G-quadruplex DNA targeting. Telomerase repeat amplification protocol reveals the higher inhibitory activity of Λ-[Ru(phen)₂(p-HPIP)]²⁺ against telomerase, suggesting that Λ-[Ru(phen)₂(p-HPIP)]²⁺ may be a potential telomerase inhibitor for cancer chemotherapy. MTT assay results show that these chiral complexes have significant antitumor activities in HepG2 cells. More interestingly, cellular uptake and laser-scanning confocal microscopic studies reveal the efficient uptake of Λ-[Ru(phen)₂(p-HPIP)]²⁺ by HepG2 cells. This complex then enters the cytoplasm and tends to accumulate in the nucleus. This nuclear penetration of the ruthenium complexes and their subsequent accumulation are associated with the chirality of the isomers as well as with the subtle environment of the ruthenium complexes. Therefore, the nucleus can be the cellular target of chiral ruthenium complexes for anticancer therapy.     

Electrophysiological and Structural Aspects in the Frontal Cortex After the Bee (<Emphasis Type="Italic">Apis mellifera</Emphasis>) Venom Experimental Treatment

The aim of this study is to evaluate the bioelectrical and structural–functional changes in frontal cortex after the bee venom (BV) experimental treatments simulating both an acute envenomation and a subchronic BV therapy. Wistar rats were subcutaneously injected once with three different BV doses: 700 μg/kg (T₁ group), 2100 μg/kg (T₃ group), and 62 mg/kg (sublethal dose—in T_SL group), and repeated for 30 days with the lowest dose (700 μg/kg—in T_S group). BV effects were assessed by electrophysiological, histological, histochemical, and ultrastructural methods. Single BV doses produced discharges of negative and biphasic sharp waves, and epileptiform spike-wave complexes. The increasing frequency of these elements suggested a dose-dependent neuronal hyperexcitation or irritation. As compared to the lower doses, the sublethal dose was responsible for a pronounced toxic effect, confirmed by ultrastructural data in both neurons and glial cells that underwent extensive, irreversible changes, triggering the cellular death. Subchronic BV treatment in T_S group resulted in a slower frequency and increased amplitude of cortical activity suggesting neuronal loss. However, neurons were still stimulated by the last BV dose. Structural–functional data showed a reduced cellular density in frontal cortex of animals in this group, while the remaining neurons displayed both specific (stimulation of neuronal activity) and unspecific modifications (moderate alterations to necrotic phenomena). Molecular mechanisms involved in BV interactions with the nervous tissue are also discussed. We consider all these data very important for clinicians who manage patients with multiple bee stings, or who intend to set an appropriate BV therapy.     

Spectroscopic investigations on the interactions of potent platinum(II) anticancer agents with bovine serum albumin

The interactions of three platinum(II)-based anticancer complexes [(5,6-dimethyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)]²⁺, [(5,6-dimethyl-1,10-phenanthroline)(1R,2R-diaminocyclohexane)platinum(II)]²⁺, and [(5,6-dimethyl-1,10-phenanthroline)(1,2-diaminoethane)platinum(II)]²⁺ (56MEEN) with BSA have been examined by circular dichroism (CD), fluorescence and ¹H pulsed gradient spin–echo (PGSE) diffusion NMR spectroscopy. The number of association constants and sites differed depending upon the spectroscopic method. This may be because each technique monitors different types of interaction/s and/or as a consequence of the different concentration ranges required for each technique. The titration of BSA with the achiral 56MEEN as monitored by CD indicates a reduction in the α-helical nature of the albumin, with the association constant calculated to be ~5 × 10⁶ M⁻¹ for one site. Due to the chiral nature of the other two complexes, their association with albumin was not monitored using CD but was examined using fluorescence and PGSE diffusion NMR. Titration of BSA with any of the three metal complexes resulted in quenching of fluorescence, with the number of association sites calculated to be ~1.1, with an association constant of ~2 × 10⁵ M⁻¹. PGSE diffusion NMR provided insights into interactions occurring with the BSA in its entirety, rather than with individual regions. Metal complex binding sites were estimated (~10 equivalent) from the diffusion data, with the average association constant for all sites ~10²–10³M⁻¹. These experiments highlight the information that can be elucidated from complementary spectroscopic techniques and demonstrate the usefulness of PGSE diffusion NMR in monitoring multiple weak binding sites, which is of great importance in studying drug-biomolecule interactions.     

The influence of bisnaphthalimidopropyl polyamines on DNA instability and repair in Caco-2 colon epithelial cells

Bisnaphthalimido compounds bis-intercalate to DNA via the major groove and are potentially potent cancer therapeutics. Previously, we incorporated natural polyamines as linkers connecting the two naphthalimido ring moieties to create a series of soluble bisnaphthalimidopropyl polyamines (BNIPPs). Here, extending earlier work on bisnaphthalimidopropylspermidine (BNIPSpd)-induced apoptosis in colon adenocarcinoma Caco-2 cells, we compare the cytotoxicity and genotoxicity of BNIPSpd relative to the spermine and oxaspermine derivatives, bisnaphthalimidopropylspermine (BNIPSpm) and bisnaphthalimidopropyloxaspermine (BNIPOSpm). The order of cytotoxicity after 24 h was BNIPSpd (IC₅₀ = 0.47 μM) > BNIPSpm (IC₅₀ = 10.04 μM) > BNIPOSpm (IC₅₀ >50 μM). After a 72-h BNIPOSpm exposure, an IC₅₀ = 10.25 μM was achieved. With 4-h exposure to BNIPSpd or BNIPSpm or 12-h exposure to BNIPOSpm, concentrations ≥1 μM induced a significant dose-dependent increase in DNA damage as measured by alkaline single-cell gel electrophoresis. The longer incubation times required for BNIPOSpm to induce DNA strand breaks reflect a slower rate of BNIPOSpm cellular distribution as monitored via BNIPP fluorescence within the cells. Moreover, exposure to a non-genotoxic concentration of BNIPSpd, BNIPSpm (0.1 μM for 4 h) or BNIPOSpm (0.1 μM for 12 h) induced a significant decrease in repair of oxidative DNA damage induced by hydrogen peroxide. In conclusion, BNIPP exposure in Caco-2 cells is associated with significant induction of DNA damage and inhibition of DNA repair at non-genotoxic concentrations. The latter is a novel consequence of BNIPP–cell interactions which adds to the spectrum of therapeutically relevant activities that may be exploited for the design and development of naphthalimide-based therapeutics.     

Phosphate ester cleavage promoted by a tetrameric iron(III) complex

Abstract  

The purple acid phosphatases (PAPs) are the only binuclear metallohydrolases where the necessity for a heterovalent active site [Fe(III)–M(II) (M is Fe, Zn or Mn)] for catalysis has been established. The paradigm for the construction of PAP biomimetics, both structural and functional, is that the ligands possess characteristics which mimic those of the donor sites of the metalloenzyme and permit discrimination between trivalent and divalent metal ions. The donor atom set of the ligand 2-((2-hydroxy-5-methyl-3-((pyridin-2-ylmethylamino)methyl)benzyl)(2-hydroxybenzyl)amino)acetic acid (H₃HPBA) mimics that of the active site of PAP although the iron(III) complex of this ligand has been characterized as the tetramer [Fe₄(HPBA)₂(μ-CH₃COO)₂(μ-O)(μ-OH)(OH₂)₂]ClO₄·5H₂O. The phosphoesterase-like activity of the complex in 1:1 acetonitrile/water has now been investigated using the substrate 2,4-bis(dinitrophenyl)phosphate. The pH dependence of the catalytic rate revealed a non-symmetric bell-shaped profile, with a finite but non-zero rate at high pH. Unlike the traditional approach usually employed to analyse these bell-shaped profiles, the approach used here involved incorporating additional species which contribute to the overall activity. Employing this approach, we show that the complex has a k _cat of 1.6 (±0.2) × 10⁻³ s⁻¹, three kinetically relevant pK _a values of 5.3, 6.2 and 8.4, with K _M of 7.4 ± 0.6 mM. The kinetic parameters are similar to those reported for heterovalent PAP biomimetics. Additionally, it is observed that, unlike the enzyme, the oxidation state is not the determining factor for catalytic activity.     

Several Conserved Positively Charged Amino Acids in OATP1B1 are Involved in Binding or Translocation of Different Substrates

OATP1B1 and 1B3 are related transporters mediating uptake of numerous compounds into hepatocytes. A putative model of OATP1B3 with a “positive binding pocket” containing conserved positively charged amino acids was predicted (Meier-Abt et al. J Membr Biol 208:213–227, 2005). Based on this model, we tested the hypothesis that these positive amino acids are important for OATP1B1 function. We made mutants and measured surface expression and uptake of estradiol-17β-glucuronide, estrone-3-sulfate and bromosulfophthalein in HEK293 cells. Two of the mutants had low surface expression levels: R181K at 10% and R580A at 30% of wild-type OATP1B1. A lysine at position 580 (R580K) rescued the expression of R580A. Mutations of several amino acids resulted in substrate-dependent effects. The largest changes were seen for estradiol-17β-glucuronide, while estrone-3-sulfate and bromosulfophthalein transport were less affected. The wild-type OATP1B1 K _m value for estradiol-17β-glucuronide of 5.35 ± 0.54 μM was increased by R57A to 30.5 ± 3.64 μM and decreased by R580K to 0.52 ± 0.18 μM. For estrone-3-sulfate the wild-type high-affinity K _m value of 0.55 ± 0.12 μM was increased by K361R to 1.8 ± 0.47 μM and decreased by R580K to 0.1 ± 0.04 μM. In addition, R580K reduced the V _max values for all three substrates to <25% of wild-type OATP1B1. Mutations at intracellular K90, H92 and R93 mainly affected V _max values for estradiol-17β-glucuronide uptake. In conclusion, the conserved amino acids R57, K361 and R580 seem to be part of the substrate binding sites and/or translocation pathways in OATP1B1.     

Microperoxidase 11: a model system for porphyrin networks and heme–protein interactions

We measured the circular dichroism (CD) and absorption spectra of the B-band region of microperoxidase 11 (MP11) as a function of temperature and peptide concentration. At micromolar concentrations, small MP11 dimers or trimers lead to excitonic coupling between low-spin and high-spin heme groups, to which the NH₂ group of the MP11 N-terminal and H₂O are bound as a sixth ligand, respectively. These aggregates convert into monomers with hexacoordinated high-spin heme groups with increasing temperature. This transition can be described by a two-state model. Aggregation becomes more extended at 50 μM concentration and causes some B-band hyperchromism, which reflects a J-type arrangement of heme groups linked together in the aggregates formed. At near-millimolar concentration, the CD and absorption spectra of the B-band region suggest the existence of even more extended and thermally stable aggregates, which might involve μ-oxo dimers of the heme groups. The degree of aggregation at 50 and 500 μM concentration increases substantially if the sample is freed from most of its oxygen in a N₂ atmosphere. The CD spectrum of the monomeric high-spin species is reminiscent of that observed for the unfolded alkaline conformation of the intact protein. Finally, we investigated the binding of acetylmethionine (AcM) ligands to the heme at aggregation-supporting conditions (500 μM concentration). The data suggest that the ligand prevents any substantial aggregation. As a surprising result, our data reveal that AcM–MP11 complexes exhibit a high-spin/low-spin mixture, with the high-spin configuration being stabilized at high temperatures.     

Investigation of the Cytotoxicity of Antimicrobial Peptide P40 on Eukaryotic Cells

The in vitro cytotoxicity of the antimicrobial peptide P40 was investigated. The food grade bacteriocin nisin was also analyzed for comparison. VERO cells were treated with different concentrations (0.02–2.5 μg ml⁻¹) of nisin and P40, and cell viability and plasma membrane integrity were checked by MTT, neutral red uptake (NRU), and lactate dehydrogenase (LDH) assays. In MTT and NRU assays the EC₅₀ to the purified peptide P40 were 0.30 and 0.51 μg ml⁻¹, while values found to nisin were 0.35 and 0.79 μg ml⁻¹, respectively. In the LDH assay, the EC₅₀ was 0.57 and 0.62 μg ml⁻¹ for P40 and nisin, respectively. The peptide P40 revealed higher hemolytical activity (19%) when compared to nisin (4.9%) at the highest concentration tested (2.5 μg ml⁻¹). Relatively few studies about the cytotoxicity of antimicrobial peptides are available. The determination of the cytotoxicity of antimicrobial peptides is an essential step to warrant their safe use.     

Enantiodifferent Proton Exchange in Alanine and Asparagine in the Presence of H 2 17 O

Using Time Domain ¹H Nuclear Magnetic Resonance with H₂¹⁷O (H₂¹⁷O-TD-¹HNMR), we found [H₂¹⁷O]- and pH-controlled chiral differences in proton exchange properties in alanine (Ala) and asparagine (Asn). To minimize and equalize chemical impurities, Asn enantiomers were purified by crystallization from racemic solution. At <0.1 M H₂¹⁷O, a shift in isoelectric pH (pI) occurred, ~1.14 kJ mol⁻¹ l-d-Asn ΔΔG ^o′ in the 5.91–6.42 pH range. One potential source for this asymmetry is the enantio-different magnetic moments (lμ↑ ≠ dμ↓) produced by neutral ring currents in the chiral center, leading to enantio-different nuclear spin organization and charge distribution in the amino group. At ≥pI, dissimilar interactions may occur in the hydration of the amino group with H₂¹⁷O (NH₂/H₂¹⁷O ≠ NH₂/H₂¹⁶O; NH₃ ⁺/H₂¹⁷O ≠ NH₂/H₂¹⁷O; l-*C-NH₂/H₂¹⁷O ≠ d-*C-NH₂/H₂¹⁷O). As lμ↑ ≠ dμ↓, the l-*C-amino and the d-*C-amino groups are diastereo spin-isomers. The nuclear spin of ¹⁷O may be parallel or antiparallel with the ortho-¹H¹H pair; hence two ortho-H₂¹⁷O molecules exist, also diastereo spin-isomers. As the pK of H₂¹⁷O is different from H₂¹⁶O, dissimilarities between l-*C- and d-*C-amino groups are converted into proton exchange differences. During H₂¹⁷O-TD-¹HNMR, the H₂¹⁷O molecule is a “probe” of the state of the amino group. Regarding prebiotic evolution: prebiotic chirality may not require stochastic symmetry breaking or preexisting chiral conditions; chemical chiral effects due to lμ↑ ≠ dμ↓ are small and need chiral amplification to generate an enantiomeric excess significant for prebiotic evolution; and prebiotic symmetry breaking was homochiral because the effect of lμ↑ and dμ↓ on the amino group should be similar in all alpha amino acids.     

In vitro effect of malathion and diazinon on oocytes fertilization and embryo development in porcine

Diazinon and malathion are commonly used organophosphate insecticides in agriculture, industry, and in veterinary medicine as an ectoparasiticide. The importance to carry out in vitro reproductive toxicology assays lies on the need of knowing the alterations these insecticides may cause at cellular level, since they are endocrine disruptors that interfere with reproductive functions. The aim of this study was to evaluate in vitro oocyte viability, fertilization, and embryo development with different concentrations of diazinon and malathion. For in vitro fertilization (IVF), porcine oocytes and sperm were co-incubated for 7 h with increasing concentrations (50, 100, and 500 μM) of diazinon and malathion. For embryo development, fertilized oocytes were cultured in medium containing the same insecticide concentrations during 96 h for embryo development and 144 h for morulae formation. Diazinon did not affect oocyte viability and embryo divisions but decreased IVF (fertilization inhibition₅₀ = 502 μM) and morulae formation (morulae inhibition₅₀ = 344 μM). Malathion affected all the studied parameters: lethal concentration₅₀ = 1 mM, fertilization inhibition₅₀ = 443 μM, development inhibition₅₀ = 375 μM, and morulae inhibition₅₀ = 216 μM. The results of this study indicate that diazinon and malathion used in commercial formulation could be toxic, producing impairment in in vitro fertilization and embryo development. This is an approach for further investigations to find out cell damage mechanisms produced by these widely used insecticides.     

Pyrophosphate-bridged complexes with picomolar toxicity

Recently, we have observed the emergence of a new series of pyrophosphate-bridged coordination complexes. Such complexes have been prepared by overcoming the ready hydrolysis of the pyrophosphate moiety. To date, no exploration has been conducted on the cytotoxicity of such complexes. Three pyrophosphate-bridged complexes, namely {[Ni(phen)₂]₂(μ-P₂O₇)}·27H₂O, {[Cu(phen)(H₂O)]₂(μ-P₂O₇)}·8H₂O and {[Co(phen)₂]₂(μ-P₂O₇)}·6MeOH, (where phen is 1,10′-phenanthroline) were chosen for their comparative structural similarities and suitable aqueous solubility. Cytotoxicity studies in the adriamycin-resistant ovarian cancer cell line A2780/AD demonstrated highly significant efficacy, with values as low as 160 pM for the cobalt complex at 72 h. The underlying mechanism for such exceptional toxicity is investigated focusing on DNA interactions, topoisomerase I enzyme inhibition and oxidative stress (followed by intracellular glutathione levels). The role of hydrolysis in uptake and toxicity is also explored (followed by electronic absorption spectroscopy, ³¹P NMR, and confocal microscopy) and the complexes are compared to cisplatin controls. Overall a clear picture of the extraordinary toxicity emerged. The results demonstrate a new class of prodrugs with significant potential for future development for the treatment of drug-resistant cancer cell lines.     

Interaction of polyethyleneimine‐anchored copper(II) complexes with tRNA studied by spectroscopy methods and biological activities

Ultraviolet–visible, emission and circular dichroism spectroscopic methods were used in transfer RNA (tRNA) interaction studies performed for polyethyleneimine–copper(II) complexes [Cu(phen)(l ‐Tyr)BPEI]ClO₄ (where phen =1,10‐phenanthroline, l ‐Tyr = l ‐tyrosine and BPEI = branched polyethyleneimine) with various degrees of coordination (x = 0.059, 0.149, 0.182) in the polymer chain. The results indicated that polyethyleneimine–copper(II) complexes bind with tRNA mostly through surface binding, although other binding modes, such as hydrogen bonding and van der Waals interactions, might also be present. Dye‐exclusion, sulforhodamine B and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assays of a polyethyleneimine–copper(II) complex with a higher degree of coordination against different cancer cell lines proved that the complex exhibited cytotoxic specificity and a significant cancer cell inhibition rate. Antimicrobial screening showed activity against some human pathogens.     

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X<Subscript>7</Subscript>-KO mice

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X₇-invalidated (KO) mice was tested. Low concentrations (1–100 μM) of ATP transiently increased the intracellular concentration of calcium ([Ca²⁺]_i) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X₇ receptors. One millimolar ATP provoked a sustained increase in the [Ca²⁺]_i only in WT mice. The response to 10 μM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K⁺]_i) and stimulated the secretion of interleukin-1β (IL-1β) only in cells from WT mice. Ten micromolar ATP in combination with 3 μM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1β was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 μM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 μM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 μM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X₄ receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K⁺]_i triggered the secretion of IL-1β. Pore formation was also triggered by activation of P2X₄ receptors. Higher concentrations of ATP elicited similar responses after binding to P2X₇ receptors. The expression of the P2X₇ receptors was also coupled to a better response to P2Y receptors.