Abjal Pasha Shaik

Effect of Organophosphorus and Organochlorine Pesticides (Monochrotophos, Chlorpyriphos, Dimethoate, and Endosulfan) on Human Lymphocytes In‐Vitro

The toxicological profile of the four pesticides described herein characterizes its effects on lymphocytes from peripheral blood from healthy donors. The exposure to all pesticides was by direct interaction/incubation with varying concentrations of the pesticide with blood sample in‐vitro. The dose response relationship in each case was calculated by applying log tables as LC50 values. Cytotoxicity of these pesticides on lymphocytes was measured using the trypan blue dye exclusion technique. Based on LC50 value, all the four pesticides were found to be highly toxic to lymphocyte culture, among them, monocrotophos and endosulfan were the most toxic and dimethoate was the least toxic. The genotoxicity of the pesticides was also determined by comet assay. The results revealed that the pesticides caused increase in the tail length indicating DNA damage. This study suggests that these pesticides have the capacity to alter the genetic material particularly chromosomes in mammalian cultures. The comet assay used in this study was found to be a sensitive and rapid method to detect genotoxicity of pesticide compounds.

Cytotoxicity and Genotoxicity Induced by the Pesticide Profenofos on Cultured Human Peripheral Blood Lymphocytes

Profenofos is a broad-spectrum organophosphate insecticide and acaricide used widely for agricultural and household purposes. The aim of this investigation was to determine its toxicity profile in vitro, using lymphocytes from peripheral blood samples of healthy human donors. We found the IC50 of profenofos to be 3.5 μM as measured by Trypan blue dye exclusion method. Chromosomal analyses of the metaphase plates of the samples treated with sublethal concentrations of profenofos revealed satellite associations and chromatid breaks and gaps, indicating its effect on chromosomes. The results were further supported by comet assay, where single-strand breaks in DNA were observed as comet tail lengths. The results were statistically significant (p < 0.01, ANOVA). Hence, it may be proposed that in vitro assays like the comet assay and chromosomal aberrations test, which indicate genetic damage, could be used to study the effect of organophosphorus pesticide poisoning in humans.

Magnetic single-walled carbon nanotubes as efficient drug delivery nanocarriers in breast cancer murine model: noninvasive monitoring using diffusion-weighted magnetic resonance imaging as sensitive imaging biomarker.

Purpose Targeting doxorubicin (DOX) by means of single-walled carbon nanotube (SWCNT) nanocarriers may help improve the clinical utility of this highly active therapeutic agent. Active targeting of SWCNTs using tumor-specific antibody and magnetic attraction by tagging the nanotubes with iron oxide nanoparticles can potentially reduce the unnecessary side effects and provide enhanced theranostics. In the current study, the in vitro and in vivo efficacy of DOX-loaded SWCNTs as theranostic nanoprobes was evaluated in a murine breast cancer model. Methods Iron-tagged SWCNTs conjugated with Endoglin/CD105 antibody with or without DOX were synthetized and extensively characterized. Their biocompatibility was assessed in vitro in luciferase (Luc2)-expressing 4T1 (4T1-Luc2) murine breast cancer cells using TiterTACS™ Colorimetric Apoptosis Detection Kit (apoptosis induction), poly (ADP-ribose) polymerase (marker for DNA damage), and thiobarbituric acid-reactive substances (oxidative stress generation) assays, and the efficacy of DOX-loaded SWCNTs was evaluated by measuring the radiance efficiency using bioluminescence imaging (BLI). Tumor progression and growth were monitored after 4T1-Luc2 cells inoculation using noninvasive BLI and magnetic resonance imaging (MRI) before and after subsequent injection of SWCNT complexes actively and magnetically targeted to tumor sites. Results Significant increases in apoptosis, DNA damage, and oxidative stress were induced by DOX-loaded SWCNTs. In addition, a tremendous decrease in bioluminescence was observed in a dose- and time-dependent manner. Noninvasive BLI and MRI revealed successful tumor growth and subsequent attenuation along with metastasis inhibition following DOX-loaded SWCNTs injection. Magnetic tagging of SWCNTs was found to produce significant discrepancies in apparent diffusion coefficient values providing a higher contrast to detect treatment-induced variations as noninvasive imaging biomarker. In addition, it allowed their sensitive noninvasive diagnosis using susceptibility-weighted MRI and their magnetic targeting using an externally applied magnet. Conclusion Enhanced therapeutic efficacy of DOX delivered through antibody-conjugated magnetic SWCNTs was achieved. Further, the superiority of apparent diffusion coefficient measurements using diffusion-weighted MRI was found to be a sensitive imaging biomarker for assessment of treatment-induced changes.

Estimation of Apoptosis and Necrosis Caused by Pesticides In Vitro on Human Lymphocytes Using DNA Diffusion Assay

Organophosphorus pesticides like monocrotophos, profenofos, chlorpyrifos, and acephate are most commonly used in India for agriculture and public health programs. Previous studies have revealed that at low doses, organophosphorus pesticides not only act as genotoxic agents but also affect several other biochemical pathways. The aim of the current investigation was to assess apoptosis and necrosis caused by these pesticides on human peripheral blood lymphocytes under in vitro conditions using the DNA diffusion assay. Our studies have revealed that all the above pesticides induced apoptosis and necrosis in cultured human peripheral blood lymphocytes in in vitro conditions. The results are statistically significant (p < 0.001). Data on these alterations of immune cells are required for understanding the subchronic effects mediated by pesticides on nontarget organisms.

Lead-Induced Genotoxicity in Lymphocytes from Peripheral Blood Samples of Humans: In Vitro Studies

Lead is a known toxicant that has been implicated in encephalopathy in children and may affect the gastrointestinal and hematopoietic and other systems in adults. In fact, lead has been shown to compete with calcium for entry into the synaptosome and induce toxic effects. The aim of the current study was to evaluate the cytotoxic and genotoxic effects of lead by using lymphocytes from human peripheral blood in vitro. The LC50 for lead nitrate as determined by Trypan blue dye exclusion technique was found to be 3.14 mM. Chromosomal aberration frequency at sublethal doses (1/10 of LC50) as determined by examining the metaphase chromosomes (karyotyping) did not show significant aberrations except for some aneuploidy and about 2–4% gaps, breaks (3–4%), and about 5% satellite associations. However, significant DNA damage was determined by SCGE (Comet assay). The comet tail length proportionately increased with increasing lead nitrate concentration. Thus, Pb can induce single-strand DNA breaks, possibly by competing with metal binding sites.

Meta-analysis study of glutathione-S-transferases (GSTM1, GSTP1, and GSTT1) gene polymorphisms and risk of acute myeloid leukemia.

To investigate the association of glutathione-S-transferase (GST) polymorphisms with the risk of acute myeloid leukemia (AML), a meta-analysis of case–control studies published between 1998 and 2009 was performed. Pooled odds ratios (ORs) were assessed using both fixed- and random-effects models. Heterogeneity across studies was calculated, and funnel plots were constructed to test for publication bias. Overall, the random-effects OR with GSTM1 null genotype, GSTP1 Val105 allele and GSTT1 null genotype were 1.30 (95% confidence intervals (CI) 1.04–1.62, p = 0.018), 1.03 (95% CI 0.80–1.33, p = 0.80) and 1.24 (95% CI 0.98–1.58, p = 0.06), respectively. Statistically, significant increased risk of AML was observed with GSTM1 while borderline significance was seen with GSTT1 null genotypes. However, fixed-effects model showed significant risk of AML in the presence of null genotypes of GSTM1 and GSTT1(p < 0.05). Significant heterogeneity was found between studies relating to GSTP1 (p = 0.162), however, no heterogeneity was seen in studies that evaluated GSTM1 (Q-value = 44; I2 = 70.9; p-value < 0.01]; and GSTT1 (Q-value = 26.03; I2 = 57.74; p-value < 0.01] polymorphisms. From the limited studies on the association of GSTP1 with risk of AML, the role of this gene cannot be ascertained fully. Significant association of these three genes with risk of AML must be evaluated further with respect to population, smoking, eating habits, ethnicity, and race.

A meta-analysis of eNOS and ACE gene polymorphisms and risk of pre-eclampsia in women

A meta-analyses of endothelial nitric oxide synthase (eNOS) and angiotensin-converting enzyme (ACE) gene polymorphisms in pre-eclampsia was performed. We shortlisted 33 studies (17 for ACE; 16 for eNOS gene polymorphisms), of which 29 articles (16 for ACE and 15 for eNOS) were analysed. Overall, 1,620 cases with pre-eclampsia and 2,158 controls were analysed for intron 16 insertion–deletion polymorphism in ACE gene. A total of 1,610 subjects with pre-eclampsia and 2,875 controls were analysed for the Glu298Asp in eNOS gene. Overall, the random-effects odds ratio (OR) with Glu298Asp in eNOS gene was 0.958 (95% confidence intervals, CI 0.747–1.228, p > 0.05), and for the insertion–deletion/ACE polymorphism was 0.987 (95% CI 0.698–1.395, p > 0.05). Significant heterogeneity was observed in the studies that evaluated polymorphisms in ACE (Q value = 55.6; I2 = 73; p value = 0.000); and eNOS (Q value = 37.2; I2 = 62.4; p value = 0.001) polymorphisms. No significant risk of pre-eclampsia was observed in both eNOS and ACE genes with these polymorphisms.

Individual susceptibility and genotoxicity in workers exposed to hazardous materials like lead

The present study was undertaken to investigate lead-induced toxicity in occupationally exposed humans and to evaluate whether genetic damage can be correlated with the known clinical indicators of lead poisoning. For this purpose, genotoxicity biomarkers along with some clinical indices of lead poisoning were determined in blood samples of battery plant workers and compared with healthy control subjects. Workers had significantly increased chromosomal aberrations, micronuclei and DNA damage compared to the controls. Increased blood lead levels (BLLs), decreased hemoglobin, PCV and symptoms of lead poisoning were used as clinical indices of lead toxicity. In addition gene polymorphisms in ALAD and MGP gene were investigated and correlated with BLL and hemoglobin content. Our results showed no significant effects of the ALAD G177C polymorphism on BLL concentrations and BLL concentrations varied to levels much above the normal reference ranges independent of the genotype. Although, significance could not be achieved, ALAD 1-2/2-2 type subjects had numerically higher BLLs (76.2-89.1 microg/dl), compared to ALAD 1-1 volunteers (21.8-79.1 microg/dl). Similarly, this study also aimed to identify the relation of some SNPs with emphasis on lead toxicity and since MGP gene is an important biomarker associated with calcium metabolism; it was hypothesized that it may be associated with lead toxicity. However, we did not find any significant association of MGP T-138C and lead poisoning. Further studies on the role of gene polymorphisms over a larger population along with genotoxicity parameters and biochemical analyses may serve to understand lead toxicity.

Polymorphisms in MGP gene and their association with lead toxicity

Matrix γ-carboxy glutamic acid protein (MGP) is a 10-kDa secreted protein containing five residues of the vitamin K-dependent calcium binding amino acid γ-carboxyglutamic acid (Gla). This study was carried out to examine the effects of MGP gene promoter polymorphism (T-138C) on blood lead levels (BLL) and hematological parameters in 113 battery manufacturing unit workers occupationally exposed to lead and 102 controls. Genotypes for the MGP T-138C polymorphism were determined by PCR and restriction fragment length digestion. BLL were determined by Anode Stripping Voltammetry using ESA Model 3010B Lead analyzer. Complete blood picture (CBP) was analyzed using ADVIA Cell counter for each sample. The frequencies of MGP–TT, CT and CC genotypes in our population were 38.6%, 44.3%, and 17.2%, respectively. The frequencies for T and C alleles were 0.612 and 0.386, respectively. Although BLL did not differ significantly among genotypes; they were higher in workers with TT/CT genotype compared to CC genotype subjects (76–88 μg/dL vs 22–45 μg/dL, p > 0.05). About 29.2% of volunteers (n = 33) from the occupationally exposed group had hemoglobin levels below 10.0 gms/dl. There was no significant difference in total white cell count and platelet count between occupational and non-exposed groups. The possible role of SNPs in the promoter region of MGP gene with relation to lead toxicity was investigated for the first time in the Indian population; although significance could not be achieved in this study, further assessments over a larger population size may help in better understanding of the consequences of lead exposure.

Evaluation of cytotoxicity and genotoxicity of pesticide mixtures on lymphocytes

Abstract The cytotoxicity and genotoxicity of pesticide mixtures viz. endosulfan + chlorpyrifos, chlorpyrifos + profenofos, and endosulfan + profenofos were evaluated on cultured human peripheral blood lymphocytes using assays for cell viability, and genotoxicity using chromosomal aberrations test and comet assay. The LC50 values for cytotoxicity were 3.50 μM, 4.18 μM, and 10.5 μM for profenofos, endosulfan, and chlorpyrifos respectively. When combined in equimolar concentrations, the LC50 values for cytotoxicity were 1.4 μM, 1.8 μM, and 2.0 μM for endosulfan + chlorpyrifos, chlorpyrifos + profenofos, and endosulfan + profenofos, respectively. Higher concentrations of individual pesticides (0.5–4.0 μM) but very low concentrations of pesticide mixtures caused significant DNA damage. Additive index values indicated a synergistic effect of toxicity for endosulfan + chlorpyrifos combination (1.12 TTU). The binary mixture of chlorpyrifos + profenofos showed an additive toxicity (0.46 TTU) while an antagonistic effect was observed for endosulfan + profenofos combination. Synergism could be due to these complementary pesticides simultaneously acting in different ways, magnifying their efficacy, whereas an additive interaction would imply that the chemicals are acting by the same mechanism and at the same target. Analysis of toxicity of pesticide mixtures may serve as important biomarker for occupational and household exposure to pesticides, with different modes of action.