Toxicological safety evaluation of 3,3'-diselenodipropionic acid (DSePA), a pharmacologically important derivative of selenocystine.

Diselenodipropionic acid (DSePA), a pharmacologically important derivative of selenocystine was evaluated for acute toxicity, mutagenic safety and metabolic stability. The estimated median oral lethal dose (LD50) cut-off of DSePA in mice and rat models was ∼200 mg/kg and ∼25 mg/kg respectively, which is considerably higher than the reported oral LD50 dose of its parent compound. Subsequently DSePA treatment in absence and presence of rat liver S9 fraction was found to be non-mutagenic at the tested doses up to 1 mM in rifampicin resistance assay and up to 6 mM in Ames test. In vitro degradation studies indicated that DSePA was more stable in S9 fraction of human compared to rat. The kinetic parameters Km and Vmax of DSePA degradation estimated using rat S9 fraction was 9.81 µM and 1.06 nmol/ml/min respectively. Further, DSePA treatment (1-50 µM) with or without rat S9 fraction did not induce any toxicity in human intestinal epithelial cells (Int 407) while showing comparable bioactivity of glutathione peroxidase (GPx) level. In conclusion, superior metabolic stability of DSePA in human S9 fraction with a concomitant lack of mutagenic effects suggests that it may be a suitable derivative of selenocytine for future biological studies.

Diketo modification of curcumin affects its interaction with human serum albumin.

Curcumin isoxazole (CI) and Curcumin pyrazole (CP), the diketo modified derivatives of Curcumin (CU) are metabolically more stable and are being explored for pharmacological properties. One of the requirements in such activities is their interaction with circulatory proteins like human serum albumin (HSA). To understand this, the interactions of CI and CP with HSA have been investigated employing absorption and fluorescence spectroscopy and the results are compared with that of CU. The respective binding constants of CP, CI and CU with HSA were estimated to be 9.3×105, 8.4×105 and 2.5×105M-1, which decreased with increasing salt concentration in the medium. The extent of decrease in the binding constant was the highest in CP followed by CI and CU. This revealed that along with hydrophobic interaction other binding modes like electrostatic interactions operate between CP/CI/CU with HSA. Fluorescence quenching studies of HSA with these compounds suggested that both static and dynamic quenching mechanisms operate, where the contribution of static quenching is higher for CP and CI than that for CU. From fluorescence resonance energy transfer studies, the binding site of CU, CI and CP was found to be in domain IIA of HSA. CU was found to bind in closer proximity with Trp214 as compared to CI and CP and the same was responsible for efficient energy transfer and the same was also established by fluorescence anisotropy measurements. Furthermore docking simulation complemented the experimental observation, where both electrostatic as well as hydrophobic interactions were indicated between HSA and CP, CI and CU. This study is useful in designing more stable CU derivatives having suitable binding properties with proteins like HSA.

Effect of Low-Dose Selenium Supplementation on the Genotoxicity, Tissue Injury and Survival of Mice Exposed to Acute Whole-Body Irradiation.

The aim of the present study is to evaluate the radioprotective effect of low-dose selenium supplementation (multiple administrations) on radiation toxicities and mortality induced by lethal dose of whole-body irradiation (WBI). For this, BALB/c mice received sodium selenite (4 µg/kg body wt) intraperitoneally for five consecutive days and subjected to WBI at an absorbed dose of 8 Gy (60Co, 1 Gy/min). Administration of sodium selenite was continued even during the post irradiation days three times a week till the end of the experiment. The radioprotective effect was evaluated in terms of the improvement in 30 days post irradiation survival, protection from DNA damage, and biochemical and histological changes in radiosensitive organs. The results indicated that low-dose sodium selenite administration did not protect the mice from radiation-induced hematopoietic and gastrointestinal injuries and subsequent mortality. However, it significantly prevented the radiation-induced genotoxicity or DNA damage in peripheral leukocytes. Further sodium selenite administration modulated the messenger RNA (mRNA) expression of GPx1, GPx2, and GPx4 in the spleen and intestine differentially and led to a significant increase in GPx activity (∼1.5 to 2-folds) in these organs. In line with this observation, sodium selenite administration reduced the level of lipid peroxidation in the intestine. In conclusion, our study shows that low-dose sodium selenite supplementation can be an effective strategy to prevent WBI-induced genotoxicity but may not have an advantage against mortality sustained during nuclear emergencies.

Alkyl chain modulated cytotoxicity and antioxidant activity of bioinspired amphiphilic selenolanes.

A series of amphiphilic conjugates of dihydroxy selenolane (DHS) and monoamine selenolane (MAS), which we had previously reported to inhibit lipid peroxidation and assist the oxidative protein folding reaction respectively in cell free systems, were evaluated for cytotoxicity, associated mechanisms and antioxidant effects in cells. Our results indicated that a fatty acid/alkyl group of variable chain lengths (C6-14) as a lipophilic moiety of the DHS/MAS conjugates not only improved their ability to incorporate within the plasma membrane of cells but also modulated their cytotoxicity. In the concentration range of 1-50 µM, C6 conjugates were non-toxic whereas the long chain (≥C8) conjugates showed significant cytotoxicity. The induction of toxicity investigated by the changes in membrane leakage, fluidity, mitochondrial membrane potential and annexin-V-propidium iodide (PI) staining by using flow cytometry revealed plasma membrane disintegration and subsequent induction of necrosis as the major mechanism. Further, the conjugates of DHS and MAS also showed differential as well as nonlinear tendency in cytotoxicity with respect to chain lengths and this effect was attributed to their self-aggregation properties. Compared with the parent compounds, C6 conjugates not only exhibited better antioxidant activity in terms of the induction of selenoproteins such as glutathione peroxidase 1 (GPx1), GPx4 and thioredoxin reductase 1 (TrxR1) but also protected cells from the AAPH induced oxidative stress. In conclusion, the present study suggests the importance of hydrophilic-lipophilic balance (HLB) in fine tuning the toxicity and activity of bioinspired amphiphilic antioxidants.

DNA binding, photoactivated DNA cleavage and cytotoxic activity of Cu(II) and Co(II) based Schiff-base azo photosensitizers.

A new class of Cu(II) and Co(II) complexes of azo-containing Schiff base of the type [Cu(L1)2] and [Co(L1)2], where L1=4-[(E)-{2-hydroxy-3-[(E)-(4-bromophenyl)diazenyl]benzylidene}amino]benzoic acid have been synthesized and characterized. Extension of conjugation and the presence of free carboxylic acid group of the ligand L1 increased the wavelength of the complexes from visible region to the near IR region (620-850 nm). The Cu(II) and Co(II) complexes interacted with CT-DNA via intercalative mode with the respective Kb value of 3.2×10(4) M(-1) and 2.9×10(4) M(-1) and acted as proficient photocleavers of SC pUC19 DNA in UV-A light, forming (1)O2 as the reactive oxygen species with the quantum yield of 0.38 and 0.36, respectively. Furthermore, the Cu(II) and Co(II) complexes showed photocytotoxicity toward two selected tumor cell lines MCF-7 and A549 by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method, and the Cu(II) complex exhibits higher photocytotoxicity than Co(II) complex against each of the selected cell lines, this result is identical with their DNA binding ability order.

Metal based photosensitizers of tetradentate Schiff base: promising role in anti-tumor activity through singlet oxygen generation mechanism.

In the present investigation, a Schiff base N'(1),N'(3)-bis[(Z)-(2-hydroxynapthyl)methylidene]benzene-1,3-dicarbodihydrazide (L1) and its Co(II), Ni(II) and Cu(II) complexes have been synthesized and characterized as novel photosensitizing agents for photodynamic therapy (PDT). The interaction of these complexes with calf thymus DNA (CT DNA) has been explored using absorption, thermal denaturation and viscometric studies. The experimental results revealed that Co(II) and Ni(II) complexes on binding to CT DNA imply a covalent mode, most possibly involving guanine N7 nitrogen of DNA, with an intrinsic binding constant Kb of 4.5×10(4)M(-1) and 4.2×10(4)M(-1), respectively. However, interestingly, the Cu(II) complex is involved in the surface binding to minor groove via phosphate backbone of DNA double helix with an intrinsic binding constant Kb of 5.7×10(4)M(-1). The Co(II), Ni(II) and Cu(II) complexes are active in cleaving supercoiled (SC) pUC19 DNA on photoexposure to UV-visible light of 365nm, through (1)O2 generation with quantum yields of 0.28, 0.25 and 0.30, respectively. Further, these complexes are cytotoxic in A549 lung cancer cells, showing an enhancement of cytotoxicity upon light irradiation.

Solvent dependent photophysical properties of dimethoxy curcumin.

Dimethoxy curcumin (DMC) is a methylated derivative of curcumin. In order to know the effect of ring substitution on photophysical properties of curcumin, steady state absorption and fluorescence spectra of DMC were recorded in organic solvents with different polarity and compared with those of curcumin. The absorption and fluorescence spectra of DMC, like curcumin, are strongly dependent on solvent polarity and the maxima of DMC showed red shift with increase in solvent polarity function (Δf), but the above effect is prominently observed in case of fluorescence maxima. From the dependence of Stokes' shift on solvent polarity function the difference between the excited state and ground state dipole moment was estimated as 4.9 D. Fluorescence quantum yield (φ(f)) and fluorescence lifetime (τ(f)) of DMC were also measured in different solvents at room temperature. The results indicated that with increasing solvent polarity, φ(f) increased linearly, which has been accounted for the decrease in non-radiative rate by intersystem crossing (ISC) processes.

Cobalt(II), Nickel(II) and Copper(II) complexes of a tetradentate Schiff base as photosensitizers: Quantum yield of 1O2 generation and its promising role in anti-tumor activity.

In the present investigation, a Schiff base N'1,N'3-bis[(E)-(5-bromo-2-hydroxyphenyl)methylidene]benzene-1,3-dicarbohydrazide and its metal complexes have been synthesized and characterized. The DNA-binding studies were performed using absorption spectroscopy, emission spectra, viscosity measurements and thermal denatuaration studies. The experimental evidence indicated that, the Co(II), Ni(II) and Cu(II) complexes interact with calf thymus DNA through intercalation with an intrinsic binding constant Kb of 2.6×10(4) M(-1), 5.7×10(4) M(-1) and 4.5×10(4) M(-1), respectively and they exhibited potent photodamage abilities on pUC19 DNA, through singlet oxygen generation with quantum yields of 0.32, 0.27 and 0.30 respectively. The cytotoxic activity of the complexes resulted that they act as a potent photosensitizers for photochemical reactions.

Differential antioxidant/pro-oxidant activity of dimethoxycurcumin, a synthetic analogue of curcumin.

Dimethoxycurcumin (Dimc), a metabolically stable analogue of curcumin, is under investigation as an anti-tumour agent. Recently a number of studies have been performed on Dimc in this laboratory and also by others. In the present article, all these results have been summarized and wherever possible compared with those of curcumin. Rate constant for reactions of Dimc with superoxide radicals was comparable with that of curcumin, while its reaction with peroxyl radicals was much slower. These results were further supported by the observations on the scavenging of basal ROS levels in lymphocytes and evaluation of antioxidant activities. In line with the earlier reports on curcumin, Dimc was a pro-oxidant and generated ROS in tumour cells. Both curcumin and Dimc were non-toxic to lymphocytes, while exhibiting comparable cytotoxicity to tumour cells. Additionally, these compounds showed higher uptake in tumour cells than in normal lymphocytes. Fluorescence studies on both the compounds revealed their binding to genomic DNA, similar sub-cellular distribution and nuclear localization. All these studies suggested that methylation of the phenolic-OH group in curcumin, although decreasing the antioxidant activity marginally, showed comparable pro-oxidant activity, making it a promising anti-tumour agent.

Comparative study of copper(II)-curcumin complexes as superoxide dismutase mimics and free radical scavengers.

Two stoichiometrically different copper(II) complexes of curcumin (stoichiometry, 1:1 and 1:2 for copper:curcumin), were examined for their superoxide dismutase (SOD) activity, free radical-scavenging ability and antioxidant potential. Both the complexes are soluble in lipids and DMSO. The formation constants of the complexes were determined by voltammetry. EPR spectra of the complexes in DMSO at 77K showed that the 1:2 Cu(II)-curcumin complex is square planar and the 1:1 Cu(II)-curcumin complex is distorted orthorhombic. Cu(II)-curcumin complex (1:1) with larger distortion from square planar structure shows higher SOD activity. These complexes inhibit gamma-radiation induced lipid peroxidation in liposomes and react with DPPH acting as free radical scavengers. One-electron oxidation of the two complexes by radiolytically generated azide radicals in Tx-100 micellar solutions produced phenoxyl radicals, indicating that the phenolic moiety of curcumin in the complexes participates in free radical reactions. Depending on the structure, these two complexes possess different SOD activities, free radical neutralizing abilities and antioxidant potentials. In addition, quantum chemical calculations with density functional theory have been performed to support the experimental observations.

Pyrroloquinoline-quinone: a reactive oxygen species scavenger in bacteria.

Transgenic Escherichia coli expressing pyrroloquinoline-quinone (PQQ) synthase gene from Deinococcus radiodurans showed superior survival during Rose Bengal induced oxidative stress. Such cells showed significantly low levels of protein carbonylation as compared to non-transgenic control. In vitro, PQQ reacted with reactive oxygen species with rate constants comparable to other well known antioxidants, producing non-reactive molecular products. PQQ also protected plasmid DNA and proteins from the oxidative damage caused by gamma-irradiation in solution. The data suggest that radioprotective/oxidative stress protective ability of PQQ in bacteria may be consequent to scavenging of reactive oxygen species per se and induction of other free radical scavenging mechanism.