Vandana Singh

Pesticide bioaccumulation and plasma sex steroids in fishes during breeding phase from north India

The investigation was done to monitor the total hexachlorocyclohexane (∑HCH) and total dichlorodiphenyltrichloroethane (∑DDT), aldrin, endosulfan and chlorpyrifos in liver, brain and ovary, gonadosomatic index (GSI) and plasma levels of testosterone (T) and estradiol-17β (E2) during breeding season of captured catfishes and carps from the unpolluted ponds of Gujartal, Jaunpur (reference site) and polluted rivers Gomti, Jaunpur and Ganga, Varanasi. Results have indicated that catfishes have higher bioaccumulation of pesticides than the carps, which was beyond the permissible limits for ∑HCH whereas ∑DDT only by catfishes of polluted rivers. The GSI and plasma levels of T and E2 were lowered in the fishes captured from the polluted rivers. In conclusion, the fishes from river Gomti and Ganga showed a high degree of contamination and disrupted reproductive axis when compared to those from the reference site reflecting the degree of pesticide pollution present in those water bodies.

Cypermethrin induced histological changes in gonadotrophic cells, liver, gonads, plasma levels of estradiol-17β and 11-ketotestosterone, and sperm motility in Heteropneustes fossilis (Bloch)

The aim of the present investigation is to assess the impact of cypermethrin on reproductive physiology in catfish, Heteropneustes fossilis during prespawning phase. Results indicate that there is a decrease in the size of gonadotrophic cells with less granulation, pycnosis in the liver, presence of immature oocytes and atretic follicles in the ovaries and gross condensation of spermatogenic cells in testes after cypermethrin exposure at sublethal concentration. The gonado-somatic index (GSI), plasma levels of estradiol-17beta (E2) and 11-ketotestosterone (11-KT) also decreases. The motility of sperm cells is dependent on the dilution (2000 times) and duration of motility is recorded 2min maximally at 90s after post-activation. The dose 0.1 and 0.01ppm is sublethal, while 1ppm is lethal on sperm motility. Results indicate that cypermethrin causes inhibition of reproduction by acting at the hypothalamo-hypophyseal-gonadal axis as is manifest from the histological observations of gonadotrophs along with disruption of follicular wall and spermatogenic cells. Obviously such changes are responsible for decreasing the steroid hormone levels which result in decreasing scale and duration of sperm motility after 45d exposure of cypermethrin in this species.

Pesticide residues and reproductive dysfunction in different vertebrates from north India

Organochlorines (isomers of hexachlorocyclohexane--HCHs and metabolites of dichlorodiphenyltrichloroethane--DDTs, aldrin and endosulfan) and organophosphate (chlorpyrifos) insecticide residues were investigated by gas liquid chromatography in the blood of fish, chick, goat and man. The plasma levels of testosterone (T) and estradiol-17beta (E2) was measured by radioimmunoassay in the catfish Rita rita captured from unpolluted reference site and polluted river Gomti during prespawning phase. Results indicated that in R. rita the SigmaDDT, SigmaHCH, endosulfan, aldrin, chlorpyrifos in blood levels were in preferential order (SigmaDDT>SigmaHCH>endosulfan>aldrin>chlorpyrifos) of their bioaccumulation. The blood levels of SigmaHCH and SigmaDDT also showed high levels in chick, goat and man, and preferential order of bioaccumulation was goat>chick>man>fish. The SigmaDDT also showed preferential order (man>chick>goat>fish) of bioaccumulation. Among the different tissues of fish (blood, liver, brain and ovary) the SigmaDDT was very high as compared to SigmaHCH as well as the rest of tissues which was very selective bioconcentration in different tissues of fish during prespawning phase. The gonado-somatic index, T and E2 declined in the catfish captured from polluted river when compared with the catfish captured from reference site affecting reproductive physiology. Our results indicated that increase of insecticides in blood level in vertebrates causes reproductive dysfunction and suggested that for human beings food like fish, chick and goat containing beyond permissible limit of insecticides must be avoided.

PEG-mediated one-pot multicomponent reactions for the efficient synthesis of functionalized dihydropyridines and their functional group dependent DNA cleavage activity.

Polyethylene glycol (PEG) has been found to be an inexpensive, non-toxic and useful medium for the one pot synthesis of highly functionalized dihydropyridines using multicomponent reactions (MCRs) at room temperature under catalyst free conditions. The notable features of this protocol are: mild reaction condition, applicability to wide range of substrates, reusability of the PEG and good yields. The interaction of the synthesized compounds with pUC19 plasmid DNA was also analyzed. Some of the synthesized compounds showed interesting functional group dependent nuclease activity for plasmid DNA cleavage under physiological conditions.

Sperm motility in the fishes of pesticide exposed and from polluted rivers of Gomti and Ganga of north India.

Investigation of lethal dose of gamma-HCH (gamma isomer of hexachlorocyclohexane), DDT (dichlorodiphenyltrichloroethane) and chlorpyrifos on spermatozoa motility after 40 days exposure in catfish, Heteropneustes fossilis was done under laboratory conditions. The sperm motility was done in the fishes captured from unpolluted ponds of Gujartal considering as reference site and polluted rivers Gomti and Ganga of north India at pre-spermiating stage. Results indicate that 1ppm of gamma-HCH, DDT and chlorpyrifos was lethal dose on sperm motility. The motility of spermatozoa decreased in insecticide exposed fish as well as in the fishes of polluted rivers when compared with their respective controls. The sperm motility was highest at 1:2000 (testicular milt: extender) dilution and duration of sperm motility was 90s after post-activation. The duration of motility also declined in the fishes captured from polluted rivers when compared with the same species captured from the reference site. It is concluded that the insecticides decrease the sperm motility and its duration in exposed fish as well as in the captured fishes from polluted rivers causing the decline in fish population of riverine systems due to influence of xenobiotics on the endocrine system.

Repair efficiency of clustered abasic sites by APE1 in nucleosome core particles is sequence and position dependent

Closely located multiple abasic sites or clustered abasic sites are highly mutagenic and potentially cytotoxic. They have been found to be repair resistant in several in vitro studies. We studied the efficiency of the repair of clustered abasic sites by the APE1 enzyme in nucleosome core particles (NCPs). Sequences having genomic importance as the core sequence of TATA box and CpG islands were used to assemble the NCPs where the abasic clusters are located around the A/T or G/C rich 0.5 positioning site of the NCPs. The thermodynamics of the binding and repair of the A/T or G/C encased clustered abasic sites in the NCPs by APE1 enzyme are reported herein for the first time that was monitored by Isothermal Titration Calorimetry (ITC). The A/T encased clustered abasic sites in the NCP showed greater binding affinity with APE1 than the G/C counterpart. A/T encased abasic sites are also cleaved faster to generate double strand breaks by APE1 enzyme as compared to the CpG island sequence in the NCP, albeit at much slower rate than the linear model. Although, the overall reactivity of the abasic sites is appreciably reduced in the NCPs, distinct differences exist in the processing of the abasic sites that are flanked by A/T or G/C rich sequence. Our study suggests that both sequence effect and nucleosomal positioning are important determinants for the repair efficiency of clustered abasic sites in NCPs.

Condensation of DNA--a putative obstruction for repair process in abasic clustered DNA damage.

Clustered DNA damages are defined as two or more closely located DNA damage lesions that may be present within a few helical turns of the DNA double strand. These damages are potential signatures of ionizing radiation and are often found to be repair resistant. Types of damaged lesions frequently found inside clustered DNA damage sites include oxidized bases, abasic sites, nucleotide dimers, strand breaks or their complex combinations. In this study, we used a bistranded two-lesion abasic cluster DNA damage model to access the repair process of DNA in condensate form. Oligomer DNA duplexes (47 bp) were designed to have two deoxyuridine in the middle of the sequences, three bases apart in opposite strands. The deoxyuridine residues were converted into abasic sites by treatment with UDG enzyme creating an abasic clustered damage site in a precise position in each of the single strand of the DNA duplex. This oligomer duplex having compatible cohesive ends was ligated to pUC19 plasmid, linearized with HindIII restriction endonuclease. The plasmid-oligomer conjugate was transformed into condensates by treating them with spermidine. The efficiency of strand cleavage action of ApeI enzyme on the abasic sites was determined by denaturing PAGE after timed incubation of the oligomer duplex and the oligomer-plasmid conjugate in presence and absence of spermidine. The efficiency of double strand breaks was determined similarly by native PAGE. Quantitative gel analysis revealed that rate of abasic site cleavage is reduced in the DNA condensates as compared to the oligomer DNA duplex or the linear ligated oligomer-plasmid conjugates. Generation of double strand break is significantly reduced also, suggesting that their creation is not proportionate to the number of abasic sites cleaved in the condensate model. All these suggest that the ApeI enzyme have difficulty to access the abasic sites located deep into the condensates leading to repair refractivity of the damages. In addition, we found that presence of a polyamine such as spermidine has no notable effect in the incision activity of ApeI enzyme in linear oligomer DNA duplexes in our experimental concentration.

A quantum dot–MUC1 aptamer conjugate for targeted delivery of protoporphyrin IX and specific photokilling of cancer cells through ROS generation

Non-targeted photosensitizers lack selectivity that undermines the potential use of photodynamic therapy (PDT). Herein, we report the DNA mediated assembly of a ZnSe/ZnS quantum dot (QD)-photosensitizer (PS)-Mucin 1(MUC1) aptamer conjugate for targeting the MUC1 cancer biomarker and simultaneous generation of reactive oxygen species (ROS). A photosensitizer, protoporphyrin IX (PpIX), was conjugated to a single stranded DNA and self-assembled to a complementary strand that was conjugated to a QD and harboring a MUC1 aptamer sequence. A multistep fluorescence resonance energy transfer (FRET) is shown that involves the QD, PpIX and covalently linked CF™ 633 amine dye (CF dye) to the MUC1 peptide that tracks the potency of the aptamer to attach itself with the MUC1 peptide. Since the absorption spectra of the CF dye overlap with the emission spectra of PpIX, the former acts as an acceptor to PpIX forming a second FRET pair when the dye labeled MUC1 binds to the aptamer. The binding of the QD-PpIX nanoassemblies with MUC1 through the aptamer was further confirmed by gel electrophoresis and circular dichroism studies. The selective photodamage of MUC1 expressing HeLa cervical cancer cells through ROS generation in the presence of the QD-PpIX FRET probe upon irradiation is successfully demonstrated.

Direct observation of preferential processing of clustered abasic DNA damages with APE1 in TATA box and CpG island by reaction kinetics and fluorescence dynamics.

Sequences like the core element of TATA box and CpG island are frequently encountered in the genome and related to transcription. The fate of repair of clustered abasic sites in such sequences of genomic importance is largely unknown. This prompted us to investigate the sequence dependence of cleavage efficiency of APE1 enzyme at abasic sites within the core sequences of TATA box and CpG island using fluorescence dynamics and reaction kinetics. Simultaneous molecular dynamics study through steady state and time resolved fluorescence spectroscopy using unique ethidium bromide dye release assay confirmed an elevated amount of abasic site cleavage of the TATA box sequence as compared to the core CpG island. Reaction kinetics showed that catalytic efficiency of APE1 for abasic site cleavage of core CpG island sequence was ∼4 times lower as compared to that of the TATA box. Higher value of Km was obtained from the core CpG island sequence than the TATA box sequence. This suggests a greater binding effect of APE1 enzyme on TATA sequence that signifies a prominent role of the sequence context of the DNA substrate. Evidently, a faster response from APE1 was obtained for clustered abasic damage repair of TATA box core sequences than CpG island consensus sequences. The neighboring bases of the abasic sites in the complementary DNA strand were found to have significant contribution in addition to the flanking bases in modulating APE1 activity. The repair refractivity of the bistranded clustered abasic sites arise from the slow processing of the second abasic site, consequently resulting in decreased overall production of potentially lethal double strand breaks.

Processing of abasic site damaged lesions by APE1 enzyme on DNA adsorbed over normal and organomodified clay.

The efficiency of the apurinic/apyrimidinic endonuclease (APE1) DNA repair enzyme in the processing of abasic site DNA damage lesions at precise location in DNA oligomer duplexes that are adsorbed on clay surfaces was evaluated. Three different forms of clay namely montmorillonite, quaternary ammonium salt modified montmorillonite and its boiled counterpart i.e. partially devoid of organic moiety were used for a comparative study of adsorption, desorption and DNA repair efficiency on their surfaces. The interaction between the DNA and the clay was analysed by X-ray diffraction, Atomic force microscopy, UV-Vis spectroscopy and Infrared spectroscopy. The abasic site cleavage efficiency of APE1 enzyme was quantitatively evaluated by polyacrylamide gel electrophoresis. Apart from the difference in the DNA adsorption or desorption capacity of the various forms of clay, substantial variation in the repair efficiency of abasic sites initiated by the APE1 enzyme on the clay surfaces was observed. The incision efficiency of APE1 enzyme at abasic sites was found to be greatly diminished, when the DNA was adsorbed over organomodified montmorillonite. The reduced repair activity indicates an important role of the pendant surfactant groups on the clay surfaces in directing APE1 mediated cleavage of abasic site DNA damage lesions.