Mannose-specific plant and microbial lectins as antiviral agents: A review.

Lectins are non-immunological carbohydrate-binding proteins classified on the basis of their structure, origin, and sugar specificity. The binding specificity of such proteins with the surface glycan moiety determines their activity and clinical applications. Thus, lectins hold great potential as diagnostic and drug discovery agents and as novel biopharmaceutical products. In recent years, significant advancements have been made in understanding plant and microbial lectins as therapeutic agents against various viral diseases. Among them, mannose-specific lectins have being proven as promising antiviral agents against a variety of viruses, such as HIV, Influenza, Herpes, Ebola, Hepatitis, Severe Acute Respiratory Syndrome Coronavirus-1 (SARS-CoV-1), Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) and most recent Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The binding of mannose-binding lectins (MBLs) from plants and microbes to high-mannose containing N-glycans (which may be simple or complex) of glycoproteins found on the surface of viruses has been found to be highly specific and mainly responsible for their antiviral activity. MBLs target various steps in the viral life cycle, including viral attachment, entry and replication. The present review discusses the brief classification and structure of lectins along with antiviral activity of various mannose-specific lectins from plants and microbial sources and their diagnostic and therapeutic applications against viral diseases.

Structure-based pharmacophore modeling, virtual screening and simulation studies for the identification of potent anticancerous phytochemical lead targeting cyclin-dependent kinase 2.

Cyclin-dependent kinases are of critical importance in directing various cell cycle phases making them as potential tumor targets. Cyclin-dependent kinase 2 (CDK2) in particular plays a significant part during cell cycle events and its imbalance roots out tumorogenic environment. Herein, we built a structure-based pharmacophore model complementing the ATP pocket site of CDK2 with four pharmacophoric features, using a series of structures obtained from cluster analysis during MD simulation assessment. This was followed by its validation and further database screening against Taiwan indigenous plants database (5284 compounds). The screened compounds were subjected toward Lipinski's rule (RO5) and ADMET filter followed by docking analysis and simulation study. In filtering hits (10 compounds) via molecular docking against CDK2, Schinilenol with -8.1 kcal/mol fetched out as a best lead phytoinhibitor in the presence of standard drug (Dinaciclib). Additionally, pharmacophore mapping analysis also indicated relative fit values of dinaciclib and schinilenol as 2.37 and 2.31, respectively. Optimization, flexibility prediction and the stability of CDK2 in complex with the ligands were also ascertained by means of molecular dynamics for 50 ns, which further proposed schinilenol having better binding stability than dinaciclib with RMSD values ranging from 0.31 to 0.34 nm. Reactivity site, biological activity detection and cardiotoxicity assessment also proposed schinilenol as a better phytolead inhibitor than the existing dinaciclib. Abbreviations: CDK2: Cyclin dependent kinase2; ATP: Adenosine triphosphate; MD: Molecular dynamics, RO5: Rule of five; ADMET: Absorption, distribution, metabolism, and excretion; RMSD: Root mean square deviation; DS: Discovery Studio; SOM: Site of metabolism; RBPM: receptor based pharmacophore model; TIP: Schinilenol; hERG: human Ether-à-go-go - Related GeneCommunicated by Ramaswamy H. Sarma.

Natural Products as Anti-Cancerous Therapeutic Molecules Targeted towards Topoisomerases.

Topoisomerases are reported to resolve the topological problems of DNA during several cellular processes, such as DNA replication, transcription, recombination, and chromatin remodeling. Two types of topoisomerases (Topo I and II) accomplish their designated tasks by introducing single- or double-strand breaks within the duplex DNA molecules, and thus maintain the proper structural conditions of DNA to release the topological torsions, which is generated by unwinding of DNA to access coded information, in the course of replication, transcription, and other processes. Both the topoisomerases have been looked at as crucial targets against various types of cancers such as lung, melanoma, breast, and prostate cancers. Conceptually, targeting topoisomerases will disrupt both DNA replication and transcription, thereby leading to inhibition of cell division and consequently stopping the growth of actively dividing cancerous cells. Since the discovery of camptothecin (an alkaloid) as an inhibitor of Topo I in 1958, a number of derivatives of camptothecin were developed as potent inhibitors of Topo I. Two such derivatives of camptothecin, namely, topotecan and irinotecan, have been commonly used as US Food and Drug Administration (FDA) approved drugs against Topo I. Similarly, the first Topo II inhibitor, namely, etoposide, an analogue of podophyllotoxin, was developed in 1966 and got FDA approval as an anti-cancer drug in 1983. Subsequently, several other inhibitors of Topo II, such as doxorubicin, mitoxantrone, and teniposide, were developed. These drugs have been reported to cause accumulation of cytotoxic non-reversible DNA double-strand breaks (cleavable complex). Thus, the present review describes the anticancer potential of plant-derived secondary metabolites belonging to alkaloids, flavonoids and terpenoids directed against topoisomerases. Furthermore, in view of the recent advances made in the field of computer-aided drug design, the present review also discusses the use of computational approaches such as ADMET, molecular docking, molecular dynamics simulation and QSAR to assess and predict the safety, efficacy, potency and identification of these potent anti-cancerous therapeutic molecules.

Genotoxicity of dichlorvos in strains of Drosophila melanogaster defective in DNA repair.

Dichlorvos (DDVP), an organophosphate pesticide, is reported to be genotoxic at high concentrations. However, the roles of DNA damage repair pathways in DDVP genotoxicity are not well characterized. To test whether pre- and post-replication pathways are involved, we measured changes in DNA migration (Comet assay) in the midgut cells of Drosophila melanogaster Oregon R+ larvae and in some mutants of pre- (mei-9, mus201, and mus207) and post- (mei-41 and mus209)replication DNA repair pathways. Insects were exposed to environmentally relevant concentrations of DDVP (up to 15ng/ml) for 48h. After insect exposure to 0.15ng/ml DDVP, we observed greater DNA damage in pre-replication repair mutants; effects on Oregon R+ and post-replication repair mutants were insignificant. In contrast, significant DNA damage was observed in the post-replication repair mutants after their exposure to 1.5 and 15ng/ml DDVP. The pre-replication repair mutant mus207 showed maximum sensitivity to DDVP, suggestive of alkylation damage to DNA. We also examined mutants (SOD- and urate-null) that are sensitive to oxidative stress and the results indicate that significant oxidative DNA damage occurs in DDVP-exposed mutants. This study suggests involvement of both pre- and post-replication repair pathways against DDVP-induced DNA damage in Drosophila, with oxidative DNA damage leading to genotoxicity.

Tinospora cordifolia extract modulates COX-2, iNOS, ICAM-1, pro-inflammatory cytokines and redox status in murine model of asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora cordifolia (Willd.) Miers is an important constituent of several ayurvedic medicinal preparations. In Ayurveda it is mentioned as "rasayan" and traditionally used for the treatment of asthma, chronic cough besides other ailments. This study was carried out to study the mechanisms involved in protection accorded by extract of Tinospora cordifolia (Tc) stem to asthmatic mice by regulation of oxidative stress, pro-inflammatory mediator release and redox signaling involving NFκB. MATERIALS AND METHODS: BALB/c mice were sensitized with intraperitoneal (i.p.) Ovalbumin (Ova) on days 0 and 14, followed by intranasal Ovalbumin (Ova) challenge on days 24 and 27 to generate an in vivo asthma model. Tc extract (hydroalcoholic, 100 mg/kg) and dexamethasone (1 mg/kg) were given orally from day 15 to 23 to the Tc+Ova treated group and Dex+Ova treated group respectively. Oxidative stress parameters e.g. activity of superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx) and catalase, lipid peroxidation, GSH/GSSG ratio, protein carbonyl content, eosinophil peroxidase, myeloperoxidase activity, and NO release were measured in tissue, blood and bronchoalveolar lavage fluid (BALF). Estimation of cytokines was done in BALF. Western blot analysis was done for IκB α, iNOS, COX-2, iCAM-1 and pJNK MAPKs along with histopathology. RESULTS: Tc extract treated mice showed decreased airway hyper-responsiveness, eosinophil count and IgE content in blood as compared to Ova treated asthmatic mice. Increase in activities of SOD, catalase, glutathione reductase, glutathione peroxidase as well as GSH/GSSG ratio was observed while a decrease in MDA formation, protein carbonyl content, eosinophil peroxidase, myeloperoxidase activity and NO release in BALF was seen in Tc treated mice. In BALF, levels of cytokines IL-4 and TNF-α were reduced and IFN-γ levels increased in extract treated mice. At the same time Tc treatment of Ova-challenged mice significantly increased the level of IκB α, cytosolic inhibitor of redox sensitive transcription factor NFκB. Immunoblot analysis revealed considerable decrease in the levels of COX-2, ICAM-1, iNOS, and pJNK. Histopathology and PAS staining also indicate a protective effect of Tc extract in inflammation and mucus hyper-secretion due to goblet cell hyperplasia. CONCLUSION: The results suggest a protective effect of Tc extract against oxidative stress, pro-inflammatory mediator release and redox signaling in the murine model of asthma. The Tc extract shows therapeutic potential for management of asthmatic inflammation and other lung inflammatory conditions.

Purification and characterization of a zinc-dependent cinnamyl alcohol dehydrogenase from Leucaena leucocephala, a tree legume.

A cinnamyl alcohol dehydrogenase (CAD) from the secondary xylem of Leucaena leucocephala has been purified to homogeneity through successive steps of ammonium sulfate fractionation, DEAE cellulose, Sephadex G-75, and Blue Sepharose CL-6B affinity column chromatographies. CAD was purified to 514.2 folds with overall recovery of 13 % and specific activity of 812. 5 nkat/mg. Native and subunit molecular masses of the purified enzyme were found to be ∼76 and ∼38 kDa, respectively, suggesting it to be a homodimer. The enzyme exhibited highest catalytic efficiency (Kcat/Km 3.75 µM(-1) s(-1)) with cinnamyl aldehyde among all the substrates investigated. The pH and temperature optima of the purified CAD were pH 8.8 and 40 °C, respectively. The enzyme activity was enhanced in the presence of 2.0 mM Mg(2+), while Zn(2+) at the same concentration exerted an inhibitory effect. The inclusion of 2.0 mM EDTA in the assay system activated the enzyme. The enzyme was inhibited with caffeic acid and ferulic acid in a concentration-dependent manner, while no inhibition was observed with salicylic acid. Peptide mass analysis of the purified CAD by MALDI-TOF showed a significant homology to alcohol dehydrogenases of MDR superfamily.

Transcriptomic analysis provides insights on hexavalent chromium induced DNA double strand breaks and their possible repair in midgut cells of Drosophila melanogaster larvae.

Hexavalent chromium [Cr(VI)] is a well known mutagen and carcinogen. Since genomic instability due to generation of double strand breaks (DSBs) is causally linked to carcinogenesis, we tested a hypothesis that Cr(VI) causes in vivo generation of DSBs and elicits DNA damage response. We fed repair proficient Drosophila melanogaster (Oregon R(+)) larvae Cr(VI) (20.0µg/ml) mixed food for 24 and 48h and observed a significant (p<0.05) induction of DSBs in their midgut cells after 48h using neutral Comet assay. Global gene expression profiling in Cr(VI)-exposed Oregon R(+) larvae unveiled mis-regulation of DSBs responsive repair genes both after 24 and 48h. In vivo generation of DSBs in exposed Drosophila was confirmed by an increased pH2Av immunostaining along with the activation of cell cycle regulation genes. Analysis of mis-regulated genes grouped under DSB response by GOEAST indicated the participation of non-homologous end joining (NHEJ) DSB repair pathway. We selected two strains, one mutant (ligIV) and another ku80-RNAi (knockdown of ku80), whose functions are essentially linked to NHEJ-DSB repair pathway. As a proof of principle, we compared the DSBs generation in larvae of these two strains with that of repair proficient Oregon R(+). Along with this, DSBs generation in spn-A and okr [essential genes in homologous recombination repair (HR) pathway] mutants was also tested for the possible involvement of HR-DSB repair. A significantly increased DSBs generation in the exposed ku80-RNAi and ligIV (mutant) larvae because of impaired repair, concomitant with an insignificant DSBs generation in okr and spn-A mutant larvae indicates an active participation of NHEJ repair pathway. The study, first of its kind to our knowledge, while providing evidences for in vivo generation of DSBs in Cr(VI) exposed Drosophila larvae, assumes significance for its relevance to higher organisms due to causal link between DSB generation and Cr(VI)-induced carcinogenesis.

Similarities in diesel exhaust particles induced alterations in expression of cytochrome P-450 and glutathione S-transferases in rat lymphocytes and lungs.

Freshly prepared peripheral blood lymphocytes (PBL) are known to express cytochrome P450s (CYPs) and glutathione S-transferases (GSTs) involved in the bioactivation and detoxification of organic components of diesel exhaust particles (DEPs). To validate that blood lymphocyte expression profiles could be used as a biomarker to predict exposure to vehicular emissions, similarities in the alterations in the mRNA expression of CYPs and GSTs were studied in PBL and lungs of rats exposed to DEPs. Adult male Wistar rats were treated transtracheally with different doses of DEPs (3.75- or 7.5- or 15- or 30-mg/kg b.wt.). The animals were anaesthetized after 24 h and blood was drawn and lungs were taken out and processed. DEP produced a similar pattern of increase in the mRNA expression of CYPs (CYP1A1, 1A2, 1B1, 2E1), associated arylhydrocarbon receptor (Ahr) and arylhydrocarbon nuclear translocator (Arnt) and GSTs (GSTPi, GSTM1 and GSTM2) at all the doses in lungs and PBL. The protein expression of CYP1A1/1A2 and 2E1 and catalytic activity of CYPs and GSTs also showed a similar pattern of increase in blood lymphocyte and in lungs isolated from DEP treated rats. Our data indicating similarities in the alterations in the expression of carcinogen metabolizing CYPs and GSTs in PBL with the lung enzymes suggests the suitability of using expression profiles of blood lymphocyte CYPs and GSTs as a biomarker to predict exposure to vehicular emissions.

Purification and characterization of Plasmodium yoelii adenosine deaminase.

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K(m) values of 41 µM and 34 µM for adenosine and 2'-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K(i) value of 0.5 µM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional -SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.

Tracing the tracks of genotoxicity by trivalent and hexavalent chromium in Drosophila melanogaster.

Mutagen sensitive strains (mus) in Drosophila are known for their hypersensitivity to mutagens and environmental carcinogens. Accordingly, these mutants were grouped in pre- and post-replication repair pathways. However, studying mutants belonging to one particular repair pathway may not be adequate for examining chemical-induced genotoxicity when other repair pathways may neutralize its effect. To test whether both pre-and post-replication pathways are involved and effect of Cr(III)- and Cr(VI)-induced genotoxicity in absence or presence of others, we used double mutant approach in D. melanogaster. We observed DNA damage as evident by changes in Comet assay DNA migration in cells of larvae of Oregon R(+) and single mutants of pre- (mei-9, mus201 and mus210) and post- (mei-41, mus209 and mus309) replication repair pathways and also in double mutants of different combinations (pre-pre, pre-post and post-post replication repair) exposed to increasing concentrations of Cr(VI) (0.0, 5.0, 10.0 and 20.0 µg/ml) for 48 h. The damage was greater in pre-replication repair mutants after exposure to 5.0 µg/ml Cr(VI), while effects on Oregon R(+) and post replication repair mutants were insignificant. Post-replication repair mutants revealed significant DNA damage after exposure to 20.0 µg/ml Cr(VI). Further, double mutants generated in the above repair categories were examined for DNA damage following Cr(VI) exposure and a comparison of damage was studied between single and double mutants. Combinations of double mutants generated in the pre-pre replication repair pathways showed an indifferent interaction between the two mutants after Cr(VI) exposure while a synergistic interaction was evident in exposed post-post replication repair double mutants. Cr(III) (20.0 µg/ml) exposure to these strains did not induce any significant DNA damage in their cells. The study suggests that both pre- and post-replication pathways are affected in Drosophila by Cr(VI) leading to genotoxicity, which may have consequences for metal-induced carcinogenesis.

Suppression of oxidative stress and pro-inflammatory mediators by Cymbopogon citratus D. Stapf extract in lipopolysaccharide stimulated murine alveolar macrophages.

Exploration of antioxidants of plant origin and their scientific validation for their immense pharmacological potential is emerging as an issue of intense research now-a-days.The effect of Cymbopogon citratus extract was seen on cell viability, oxidative stress markers i.e. ROS production, SOD activity, lipid peroxidation and GSH content of murine alveolar macrophages stressed with lipopolysaccharide. Modulation in release of NO and pro-inflammatory cytokine TNF-α along with alterations in mitochondrial membrane potential under stress were compared with known plant derived antioxidant quercetin. The extract was not found to be cytotoxic at any of the selected doses. At 5 and 10 µg the extract showed significant increase in SOD activity, GSH content (p<0.001), decrease in ROS production as seen by fluorescent dye DCFH-DA and also MDA formation (lipid peroxidation marker) significantly. The extract also showed reduction in the release of pro-inflammatory mediators TNF-α and NO significantly indicating an anti-inflammatory effect. The extract was able to restore mitochondrial membrane potential as estimated by spectrofluorimetry using the fluorescent dye Rhodamine 123. The results suggest potential use of the cytoprotective, antioxidant and anti-inflammatory property of C. citratus in the form of dietary component and also in formulations against lung inflammatory diseases where oxidative stress plays an important role.

Ventilator-associated pneumonia caused by carbapenem-resistant Enterobacteriaceae carrying multiple metallo-beta-lactamase genes.

CONTEXT: Ventilator-associated pneumonia (VAP) is a leading nosocomial infection in the intensive care unit (ICU). Members of Enterobacteriaceae are the most common causative agents and carbapenems are the most commonly used antibiotics. Metallo-beta-lactamase (MBL) production leading to treatment failure may go unnoticed by routine disc diffusion susceptibility testing. Moreover, there is not much information on association of MBL-producing Enterobacteriaceae with ICU-acquired VAP. Therefore, a study was undertaken to find out the association of MBL-producing Enterobacteriaceae with VAP. SETTINGS: This study was conducted in a large tertiary care hospital of North India with an eight-bed critical care unit. MATERIALS AND METHODS: The respiratory samples (bronchoalveolar lavage, protected brush catheter specimens and endotracheal or transtracheal aspirates) obtained from VAP patients (during January 2005-December 2006) were processed, isolated bacteria identified and their antibiotic susceptibilities tested as per standard protocols. The isolates of Enterobacteriaceae resistant to carbapenem were subjected to phenotypic and genotypic tests for the detection of MBLs. RESULTS: Twelve of 64 isolates of Enterobacteriaceae were detected as MBL producers, bla(IMP) being the most prevalent gene. Additionally, in three strains, simultaneous coexistence of multiple MBL genes was detected. CONCLUSION: The coexistence of multiple MBL genes in Enterobacteriaceae is an alarming situation. As MBL genes are associated with integrons that can be embedded in transposons, which in turn can be accommodated on plasmids thereby resulting in a highly mobile genetic apparatus, the further spread of these genes in different pathogens is likely to occur.

Study of genetic polymorphism in solvent exposed population and its correlation to in vitro effect of trichloroethylene on lymphocytes.

Trichloroethylene (TCE) is major industrial pollutant that contaminate environment. Its exposure may lead to hepato-renal toxicity along with the cancer progression. Although extensive research is done on its toxicity still not much is known about its genotoxic potential on humans in relation to genetic polymorphism. Cytochrome P450 (CYP P-450) and glutathione-S-transferases (GSTs) are important in cellular detoxification of TCE. Variations in gene sequences result in population specific regional genetic variations (polymorphism). Genotyping of CYP1A1, GSTM1, GSTT1 and GSTP1 polymorphism was performed in 220 normal and 97 solvent-exposed individuals from northern part of India using real time PCR, PCR and restriction digestion techniques. The parameters examined to study genotoxicity were chromosomal aberration (CA) and cytokinesis block micronucleus assay (CBMN) in lymphocyte culture in vitro. The observed average frequencies for GSTM1 (null) and GSTT1 (null) were 41, 22 and 12.7%, respectively in normal subjects whereas frequencies of CYP1A1/GSTP1 with (ile/ile) or (ile/val) or(val/val) were found to be 76.2/52, 21.4/42.1 and 2.4/5.9% respectively. It was further observed that the frequencies of above genes were found to be similar in solvent exposed groups. The distribution frequencies of GST genes, when compared with other reports from various regions of India show variations. In vitro TCE exposure (2, 4 and or 6 mM) did not show any significant genotoxic effect. TCE maybe toxic due to its metabolite.

Aeromonas caviae septicemia in immunocompetent gastrointestinal carriers

Aeromonas caviae strains have been isolated from blood and stool cultures of three immunocompetent patients, residents of Northern India, who presented with community acquired septicemia without any recent history of diarrhea. Cell culture infectivity test performed on Hep-2 cells have shown substantial degree of invasiveness in the isolated strains. This case unleashes a possibility of asymptomatic gastrointestinal carriage of such strains of A. caviae in a very large population of India, as several areas of India have very high rates of Aeromonas induced acute diarrhea/gastroenteritis (up to 13 percent). It needs to be appraised further in India as well as other countries having high rates of Aeromonas induced acute diarrhea/gastroenteritis.

Aeromonas caviae septicemia in immunocompetent gastrointestinal carriers.

Aeromonas caviae strains have been isolated from blood and stool cultures of three immunocompetent patients, residents of Northern India, who presented with community acquired septicemia without any recent history of diarrhea. Cell culture infectivity test performed on Hep-2 cells have shown substantial degree of invasiveness in the isolated strains. This case unleashes a possibility of asymptomatic gastrointestinal carriage of such strains of A. caviae in a very large population of India, as several areas of India have very high rates of Aeromonas induced acute diarrhea/gastroenteritis (up to 13%). It needs to be appraised further in India as well as other countries having high rates of Aeromonas induced acute diarrhea/gastroenteritis.

Effect of pretreatment of cytochrome P450 (P450) modifiers on neurobehavioral toxicity induced by deltamethrin.

To investigate the involvement of cytochrome P450 (P450) enzyme induction and the effect of different P450 modifiers in the neurobehavioral toxicity of deltamethrin, deltamethrin (10 mg/kg; orally for 1 day) was administered to young male albino Wistar rats, or in rats pretreated with phenobarbital (PB; 80 mg/kg, ip for 5 days), an inducer of P450 2B1/2B2 or 3-methylcholanthrene (MC; 30 mg/kg, ip for 5 days), an inducer of P450 1A1/1A2 or cobalt chloride (CoCl(2); sc for 2 days), a depletor of P450s. The administration of PB or MC or CoCl(2) alone did not produced any symptoms of neurobehavioral toxicity. While a single oral administration of deltamethrin produced tremors in two out of 10 rats and decreased the spontaneous locomotor activity, pretreatment with MC or PB potentiated the deltamethrin induced neurobehavioral toxicity with 50% of the treated rats exhibiting tremors. Half of the animals pretreated with MC prior to exposure to deltamethrin also exhibited choreoathetosis. The decrease in the spontaneous locomotor activity was found to be much more significant in PB- or MC-pretreated animals exposed to deltamethrin. In contrast to the pretreatment with inducers, rats pretreated with CoCl(2) exhibited no symptoms of tremors or choreoathetosis, indicating that a reactive metabolite of deltamethrin is formed by P450 catalysed reactions which is involved in the neurobehavioral toxicity of deltamethrin.

Induction of rat brain cytochrome P450s (P450s) by deltamethrin: regional specificity and correlation with neurobehavioral toxicity.

Oral administration of 5 mg/kg body weight of deltamethrin, an alpha-cyano type II pyrethroid insecticide once a day for 1, 7, 15 and 21 consecutive days to young Druckerey rats (6- 8 weeks old) produced a time dependent increase in the activity of cytochrome P450 (P450) dependent 7-ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) in rat brain microsomes. A significant induction was observed on prolonged exposure of deltamethrin for 15 or 21 days. The induction in the activity of cerebral P450 enzymes was associated with the time dependent increase in the spontaneous locomotor activity indicating accumulation of deltamethrin or its metabolites in brain with the increase in the duration of exposure. Administration of deltamethrin (5 mg/kg) for 21 days produced region specific changes in the dealkylation of ethoxyresorufin and pentoxyresorufin in rat brain with significant induction occurring in the activity of P450 1A1/2 dependent EROD in cerebellum, hippocampus, hypothalamus and medulla-pons and that of P450 2B1/2 mediated PROD in hippocampus, hypothalamus, corpus striatum and mid brain. The data suggests that the differences in the induction of individual P450 isoenzymes in diverse brain regions could play a role in regulating the response of brain to pyrethroid insecticides by modulating their concentration per se or their active metabolites at the target site(s).

Induction of rat brain and liver cytochrome P450 1A1/1A2 and 2B1/2B2 isoenzymes by deltamethrin.

Deltamethrin, an α-cyano type II pyrethroid, administered orally (5, 10 and 15 mg/kg body weight for 7 consecutive days or at 5 mg/kg for further 15 and 21 days) to young albino Wistar rats (approximately 8 weeks old) produced a dose- and time-dependent increase in the activity of cytochrome P450-dependent 7-ethoxyresorufin-O-dealkylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) in rat liver and brain. However, significant induction in the enzyme activities was observed at higher doses or prolonged exposure. The magnitude of induction in rat liver microsomes was less at 15 mg/kg dose as compared to 10 mg/kg dose. Western blot analysis revealed a similar dose-related and time-dependent increase in the expression of P450 2B1/2B2 and 1A1 isoenzymes as indicated by the increased cross-reactivity of liver microsomes isolated from deltamethrin-treated animals with anti-P450 2B1/2B2 and 1A1. Inhibition of EROD and PROD observed after in vitro addition of anti-P450 2B1/2B2 and 1A1/1A2 or organic inhibitors, metyrapone and α-naphthoflavone, to the brain and liver microsomes of deltamethrin-pretreated animals (5 mg/kg×21 days), further provided support that the induction observed in the EROD and PROD activity in brain is due to the increased expression of P450 2B1/2B2 and 1A1/1A2, while, in the liver, isoenzymes other than these are also involved.

Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus.

An aspen lignin-specific O-methyltransferase (bi-OMT; S-adenosyl-L-methionine: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase, EC 2.1.1.68) antisense sequence in the form of a synthetic gene containing the cauliflower mosaic virus 35S gene sequences for enhancer elements, promoter and terminator was stably integrated into the tobacco genome and inherited in transgenic plants with a normal phenotype. Leaves and stems of the transgenes expressed the antisense RNA and the endogenous tobacco bi-OMT mRNA was suppressed in the stems. Bi-OMT activity of stems was decreased by an average of 29% in the four transgenic plants analyzed. Chemical analysis of woody tissue of stems for lignin building units indicated a reduced content of syringyl units in most of the transgenic plants, which corresponds well with the reduced activity of bi-OMT. Transgenic plants with a suppressed level of syringyl units and a level of guaiacyl units similar to control plants were presumed to have lignins of distinctly different structure than control plants. We concluded that regulation of the level of bi-OMT expression by an antisense mechanism could be a useful tool for genetically engineering plants with modified lignin without altering normal growth and development.