Inhalable particles containing isoniazid and rifabutin as adjunct therapy for safe, efficacious and relapse-free cure of experimental animal tuberculosis in one month.

We investigated the preclinical efficacy and safety/tolerability of biodegradable polymeric particles containing isoniazid (INH) and rifabutin (RFB) dry powder for inhalation (DPI) as an adjunct to oral first-line therapy. Mice and guinea pigs infected with Mycobacterium tuberculosis H37Rv (Mtb) were treated with ∼80 and ∼300 µg of the DPI, respectively, for 3-4 weeks starting 3, 10, and 30 days post-infection. Adjunct combination therapy eliminated culturable Mtb from the lungs and spleens of all but one of 52 animals that received the DPI. Relapse-free cure was not achieved in one mouse that received DPI + oral, human-equivalent doses (HED) of four drugs used in the Directly Observed Treatment, Short Course (DOTS), starting 30 days post-infection. Oral doses (20 mg/Kg/day, each) of INH + RFB reduced Mtb burden from ∼106 to ∼103 colony-forming units. Combining half the oral dose with DPI prevented relapse of infection four weeks after stopping the treatment. The DPI was safe in rodents, guinea pigs, and monkeys at 1, 10, and 100 µg/day doses over 90 days. In conclusion, we show the efficacy and safety/tolerability of the DPI as an adjunct to oral chemotherapy in three different animal models of TB.

DNA damage induced by industrial solid waste leachates in Drosophila melanogaster: a mechanistic approach.

Genomic stability requires that error-free genetic information be transmitted from generation to generation, a process that is dependent upon efficient DNA repair. Industrial leachates which contain mixtures of diverse chemicals are a major environmental concern. The interaction between these chemicals may have synergistic, antagonistic, or simply additive effects on biological systems. In the present study, the Comet assay was used to measure the DNA damage produced by leachates of solid wastes from flashlight battery, pigment, and tanning factories in the midgut cells and brain ganglia of Drosophila melanogaster mutants deficient in DNA repair proteins. Larvae were allowed to feed for 48 or 72 hr on diets containing 0.1, 0.5, and 2.0% (v/v) of the leachates. Physicochemical analysis run on the solid wastes, leachates, and treated larvae detected elevated levels of heavy metals. Leachates produced significantly greater levels of DNA damage in mutant strains mei41 (deficient in cell cycle check point protein), mus201 (deficient in excision repair protein), mus308 (deficient in postreplication repair protein), and rad54 (deficient in double strand break repair protein) than in the OregonR(+) wild-type strain. Larvae of the ligaseIV mutant (deficient in double strand break repair protein) were hypersensitive only to the pigment plant waste leachate. Conversely, the dnase2 mutant (deficient in protein responsible for degrading fragmented DNA) was more sensitive to DNA damage induction from the flashlight battery and tannery waste leachates. Our data demonstrate that repair of DNA damage in organisms exposed to leachates is dependent upon several DNA repair proteins, indicative of the involvement of multiple overlapping repair pathways. The study further suggests the usefulness of the Comet assay for studying the mechanisms of DNA repair in Drosophila.

DNA damage induced in human peripheral blood lymphocytes by industrial solid waste and municipal sludge leachates.

Exposure of humans to toxic compounds occurs mostly in the form of complex mixtures. Leachates, consisting of mixtures of many chemicals, are a potential risk to human health. In the present study, leachates of solid wastes from a polyfiber factory (PFL), an aeronautical plant (AEL), and a municipal sludge leachate (MSL) were assessed for their ability to induce DNA damage in human peripheral blood lymphocytes using the alkaline Comet assay. The leachates also were examined for their physical and chemical properties. Lymphocytes were incubated with 0.5-15.0% concentrations (pH range 7.1-7.4) of the test leachates for 3 hr at 37 degrees C, and treatment with 1 mM ethyl methanesulfonate served as a positive control. All three leachates induced significant (P < 0.05), concentration-dependent increases in DNA damage compared with the negative control, as measured by increases in Olive tail moment (arbitrary units), tail DNA (%), and tail length (mum). A comparison of these variables among the treatment groups indicated that the MSL induced the most DNA damage. Inductively coupled plasma emission spectrometry analysis of the leachates indicated that they contained high concentrations of heavy metals, viz. iron, manganese, nickel, zinc, cadmium, chromium, and lead. The individual, synergistic, or antagonistic effects of these chemicals in the leachates may be responsible for the DNA damage. Our data indicate that the ever-increasing amounts of leachates from waste landfill sites have the potential to induce DNA damage and suggest that the exposure of human populations to these leachates may lead to adverse health effects.

Induction of biochemical stress markers and apoptosis in transgenic Drosophila melanogaster against complex chemical mixtures: role of reactive oxygen species.

The study was aimed to investigate the effect of leachates of solid waste from a flashlight battery factory and a pigment plant on 70 kDa heat shock protein (Hsp70) expression, generation of reactive oxygen species (ROS), antioxidant enzymes activities and apoptosis in Drosophila. Third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) were fed on diet mixed with leachates of solid wastes (0.05-2.0%, v/v) released from two industrial plants at three different pHs (7.00, 4.93 and 2.88) for 2-48 h. A concentration- and time-dependent significant change in Hsp70 expression, ROS generation, antioxidant enzymes activities and MDA content was observed in the exposed larvae preceding the antioxidant enzymes activities. Mitochondria-mediated, caspase-dependent apoptotic cell death in the larvae exposed to 1.0 and 2.0% leachates of flashlight battery factory was concurrent with a significant regression in Hsp70 expression and a higher ROS generation. A positive correlation drawn between ROS generation and apoptotic markers and a negative correlation between apoptotic markers and Hsp70 expression in these groups indicated the important role of ROS in the leachate-induced cellular damage. Hsp70 along with antioxidant enzymes offered protection to the organisms exposed to all the tested concentrations of the leachates of pigment plant waste and 0.5% leachate of flashlight battery factory in a cooperative manner when ROS generation was less induced. Conversely, higher levels of ROS generation in the organisms treated with 1.0 and 2.0% leachate of flashlight battery factory after 24 and 48 h resulted in regression of Hsp70 expression in them leading to cell death. The study suggests that (1) leachates of flashlight battery factory waste more adversely affected the organisms in comparison to the leachates of pigment plant waste. (2) Hsp70 may be used as a biomarker of cellular damage in organisms exposed to leachates. (3) Cell based assays using D. melanogaster as an in vivo model may provide important mechanistic information about the adverse effect of xenobiotics.

In vivo genotoxic effects of industrial waste leachates in mice following oral exposure.

Contamination of ground water by industrial waste poses potential health hazards for man and his environment. The improper disposal of toxic wastes could allow genotoxic chemicals to percolate into ground waters, and these contaminated ground waters may produce toxicity, including mutation and eventually cancer, in exposed individuals. In the present study, we evaluated the in vivo genotoxic potential of leachates made from three different kinds of industrial waste (tannery waste, metal-based waste, and waste containing dyes and pigments) that are disposed of in areas adjoining human habitation. Three different doses of test leachates were administered by oral gavage for 15 consecutive days to Swiss albino mice; their bone marrow cells were examined for chromosome aberrations (CAs), micronucleated polychromatic erythrocytes (MNPCEs), and DNA damage using the alkaline Comet assay. Exposure to the leachates resulted in significant (P < 0.05 or P < 0.001) dose-dependent increases in chromosome and DNA damage. Fragmented chromosomes and chromatid breaks were the major CAs observed. Chemical analysis of the leachates indicated that chromium and nickel were elevated above the limits established by health organizations. The highest levels of genotoxicity were produced by the metal-based leachate and the tannery-waste leachate, while the dye-waste leachate produced weaker genotoxic responses. The cytogenetic abnormalities and DNA damage produced by the leachates indicate that humans consuming water contaminated with these materials are at increased risk of developing adverse health consequences.

Nitrous oxide related behavioral and histopathological changes may be related to oxidative stress.

Nitrous oxide (N2O) toxicity can result in myelin loss and hyperhomocysteinemia similar to cobalamin (Cbl) deficiency. Studies on N2O exposure can help in understanding the mechanism of demyelination. In view of paucity of studies on N2O toxicity in rats this study was undertaken. Six male wistar rats were exposed to 1.5L/min N2O with 1:1 O2 for 90 min daily for 1 month. After 1-month exposure blood homocysteine (HCY) and oxidative stress parameters glutathione (GSH) and total antioxidant capacity (TAC) were measured. Brain and spinal cord was subjected to histopathological examination. The neurobehavioral changes, oxidative stress parameters and histopathological changes were correlated with serum B12 and HCY level. After 1-month exposure, the rats appeared sluggish, lethargic and developed predominantly hind limb weakness for 1-1.5h. In the exposed group, the total distance traveled (2001.66 ± 118.27 cm; p=0.037), time moving (80.16 ± 5.7s; p=0.028), number of rearing (10.33 ± 1.45; p=0.014) and grip strength (1042.40 ± 51.3N; p=0.041) were significantly decreased whereas, resting time significantly increased (219.83 ± 5.7s; p=0.030) compared to controls. Serum HCY level was significantly increased (20.56 ± 1.296 µm/ml; p=0.0007) in the exposed group. However, serum B12 and folic acid levels were not significantly different. GSH significantly decreased (2.21 ± 0.60 mg/dl; p=0.018) along with TAC (0.76 ± 0.16 Trolox_Eq_mmol/l; p=0.036). The histopathological studies revealed shrinkage and vacuolation of neurons in cerebral cortex, focal myelin loss, vacuolation in subcortical white matter and spinal cord. N2O exposure results in behavioral alterations, hyperhomocysteinemia, cortical and spinal cord demyelination which were associated with decrease GSH and TAC highlighting pathophysiological role of oxidative stress.

Fast agitated directly suspended droplet microextraction technique for the rapid analysis of eighteen organophosphorus pesticides in human blood.

A new sample preparation technique named as fast agitated directly suspended droplet microextraction (FA-DSDME) was proposed as an improved version of directly suspended droplet microextraction (DSDME) for the extraction and pre-concentration of wide-range organophosphorus pesticides (OPPs) from human blood prior to liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis. In this method, instead of protecting the unwanted rupturing of extraction droplet (organic solvent), it was deliberately splintered into fine droplets by providing automated high-speed agitation to the biphasic extraction system (extraction solvent and sample solution). Fine organic droplets were then recollected into one, not by using a centrifuge machine but just by giving a very slow stirring to the bottom of the extraction system. The present method has surmounted the problem of prolonged extraction time associated with old DSDME. Under optimum extraction conditions, the method showed good sensitivity with low detection limits ranging from 0.0009 to 0.122µgL(-1). Mean recoveries were achieved in the range of 86-109% at three levels of spiking concentration (low, middle and high) from linearity range of individual analyte. Intra-day and inter-day precisions were ≤4.68 and ≤9.57 (%RSD) respectively. Enrichment factor (EF) for each analyte varied from 30 to 132 which prove the ability of this technique to pre-concentrate the extracted analytes up to a good extent. The sample matrices have shown an insignificant influence on method's sensitivity. The proposed method may find immense use in epidemiological, toxicological, regulatory and forensic laboratories.

Optimization and validation of an extraction method for the analysis of polycyclic aromatic hydrocarbons in chocolate candies.

UNLABELLED: Chocolate is a key ingredient in many foods such as milk shakes, candies, bars, cookies, and cereals. Chocolate candies are often consumed by mankind of all age groups. The presence of polycyclic aromatic hydrocarbons (PAHs) in chocolate candies may result in health risk to people. A rapid, precise, and economic extraction method was optimized and validated for the simultaneous determination of polycyclic aromatic hydrocarbons in chocolate candy by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GS-MS) as a confirmatory technique. The method was optimized by using different solvents for liquid-liquid extraction, varying volume of de-emulsifying agent, and quantity of silica gel used for purification. The HPLC separation of 16 PAHs was carried out by C-18 column with mobile phase composed of acetonitrile : water (70 : 30) in isocratic mode with runtime of 20 min. Limit of detection, limit of quantification (LOQ), and correlation coefficients were found in the range of 0.3 to 4 ng g⁻¹, 0.9 to 12 ng g⁻¹, and 0.9109 to 0.9952, respectively. The exploration of 25 local chocolate candy samples for the presence of PAHs showed the mean content of benzo[a]pyrene as 1.62 ng g⁻¹, which representing the need to evaluate effective measures to prevent more severe PAHs contamination in chocolate candies in future. PRACTICAL APPLICATION: Chocolate is one of the most favorite food items among people, especially children. Chocolate candies are often consumed by mankind of all age groups. Chocolate candies are often consumed by children in large quantities. The presence PAHs in chocolate candies may result in health risk to people. In the present study, a precise and cost effective rapid method was employed for the determination of PAHs, which can be employed for daily routine analysis of PAHs in chocolate products.

Adverse effect of tannery waste leachates in transgenic Drosophila melanogaster: role of ROS in modulation of Hsp70, oxidative stress and apoptosis.

Leachate is a complex chemical mixture of chemicals produced as a result of leaching of solid wastes. The potential toxicity of leachates is a major environmental health concern. The present study evaluated the role of ROS in tannery leachates induced Hsp70 expression, antioxidant enzymes and apoptosis in Drosophila. Different concentrations (0.05-2.0%) of leachates prepared from tannery waste at different pH (7.00, 4.93 and 2.88) were mixed with Drosophila food and fed to the larvae for 2-48 h to examine the different stress and apoptotic markers. A concentration- and time-dependent significant increase in Hsp70 expression, ROS generation, antioxidant enzymes activities and MDA content were observed in the exposed larvae. Activities of antioxidant enzymes were delayed compared with Hsp70 expression and MDA level in the exposed organisms. Apoptotic cell death was observed in the exposed larvae at higher concentrations concurrent with a significant regression in Hsp70 along with a higher level of ROS generation. A positive correlation drawn between ROS generation and apoptotic markers and a negative correlation between apoptotic markers and Hsp70 expression at these concentrations indicated the important role of ROS in the induction of cellular damage in the exposed organisms. There was a significant generation of ROS in the larvae exposed to 0.5% of leachates which did not interfere with the protection of their cells by Hsp70 and antioxidant enzymes. However, generation of significantly higher levels of ROS in the larvae exposed to 1.0% and 2.0% leachates may decrease Hsp70 expression thus leading to mitochondria-mediated caspase-dependent apoptotic cell death.

Hydrochemistry of wet atmospheric precipitation over an urban area in northern indo-gangetic plains.

Rain water samples were collected to study the chemical composition of wet atmospheric precipitation (first event) over the Lucknow city in the northern Indo-gangetic alluvial plains. The samples were collected in the month of July, 2005 from different sites. The wet precipitation samples were analyzed for pH, EC, major ions (HCO(-3), Cl(-), SO4(2-), NO(-3), PO4(-3), F(-), Na(+), K(+), Ca(2+), Mg(2+), NH4+) and heavy metals (As, Cd, Al, Co, Cr, Cu, Fe, Mn, Mo, Ni, Hg, Pb, Se, Sn, Ti, V, Zn). The pH values of wet precipitation samples ranged between 6.5 and 8.7. The analysis of linear regression applied to the set of studied variables and computation of neutralization factors showed that neutralization occurred in precipitation samples and Ca(2+) had the maximum neutralization capacity. It was found that Cl(-), [Formula: see text], Ca(2+), Mg(2+), Na(+) and K(+) in the precipitation samples originated mainly from crustal/anthropogenic sources in the region. On an average Fe, and Al accounted for >72% of the total concentration of trace metals in the wet precipitation samples followed by Zn (>10%). Enrichment factors calculated for heavy metals over reference background level in seawater and Earth's crust showed relatively higher enrichment of Zn. The principal component analysis (PCA) identified the possible sources of ionic species and heavy metals in the wet precipitation samples.