Genetic polymorphisms of GSTM1, GSTT1 and GSTP1 and susceptibility to DNA damage in workers occupationally exposed to organophosphate pesticides

GSTM1, T1 and P1 are important enzymes of glutathione S-transferases (GSTs), involved in the metabolism of many endogenous and exogenous compounds. Individual genetic variation in these metabolizing enzymes may influence the metabolism of their substrates. The present study was designed to determine the genotoxic effects using DNA damage and its association with GSTM1, GSTT1, and GSTP1 (Ile105Val) genetic polymorphisms in workers occupationally exposed to organophosphate pesticides (OPs). We examined 230 subjects including 115 workers occupationally exposed to OPs and an equal number of normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using individual PCR or PCR-RFLP. Significantly higher DNA tail moment (TM) was observed in workers as compared to control subjects (14.41 ± 2.25 vs. 6.36 ± 1.41 tail % DNA, p<0.001). The results revealed significantly higher DNA TM in workers with GSTM1 null genotype than those with GSTM1 positive (15.18 vs. 14.15 tail % DNA, p=0.03). A significantly higher DNA TM was also observed in workers with homozygous Ile-Ile GSTP1 genotype than heterozygous (Ile-Val) and mutant (Val-Val) GSTP1 genotype (p=0.02). In conclusion, the results show that null deletion of GSTM1 and homozygote wild GSTP1 genotype could be related to inter-individual differences in DNA damage arises from the gene-environment interactions in workers occupationally exposed to OPs.     

Characterization and Distribution of the Selected Metals in the Scalp Hair of Cancer Patients in Comparison with Normal Donors

Eighteen metals were estimated in the scalp hair samples from cancer patients (n = 111) and normal donors (n = 113). Nitric acid–perchloric acid wet digestion procedure was used for the quantification of the selected metals by flame atomic absorption spectrophotometry. In the scalp hair of cancer patients, highest average levels were found for Ca (861 μg/g), followed by Na (672 μg/g), Zn (411 μg/g), Mg (348 μg/g), Fe (154 μg/g), Sr (129 μg/g), and K (116 μg/g), whereas in comparison, the dominant metals in the scalp hair of normal donors were Ca (568 μg/g), Zn (177 μg/g), Mg (154 μg/g), Fe (110 μg/g), and Na (103 μg/g). The concentrations of Ca, Cd, Co, Cr, Fe, K, Mg, Mn, Na, Ni, Pb, Sb, Sr, and Zn were notably higher in the hair of cancer patients as compared with normal donors, which may lead to a number of physiological disorders. Strong positive correlations were found in Mn–Pb (0.83), Cd–Cr (0.82), Cd–Li (0.57), Fe–Pb (0.56), and Fe–Mn (0.55) in the hair of cancer patients whereas Na–Cd, Li–Cr, Li–Co, Co–Cd, Li–Cd, Na–Co, Na–Li, Ca–Mg and Na–Cr exhibited strong relationships (r > 0.50) in the hair of normal donors. Principal Component Analysis (PCA) of the data revealed seven PCs, both for cancer patients and normal donors, but with significantly different loadings. Cluster Analysis (CA) was also used to support the PCA results. The study evidenced significantly different pattern of metal distribution in the hair of cancer patients in comparison with normal donors. The role of trace metals in carcinogenesis was also discussed.     

Targeted elimination of peroxisome proliferator-activated receptor gamma in beta cells leads to abnormalities in islet mass without compromising glucose homeostasis

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is an important regulator of lipid and glucose homeostasis and cellular differentiation. Studies of many cell types in vitro and in vivo have demonstrated that activation of PPAR gamma can reduce cellular proliferation. We show here that activation of PPAR gamma is sufficient to reduce the proliferation of cultured insulinoma cell lines. We created a model with mice in which the expression of the PPARG gene in beta cells was eliminated (beta gamma KO mice), and these mice were found to have significant islet hyperplasia on a chow diet. Interestingly, the normal expansion of beta-cell mass that occurs in control mice in response to high-fat feeding is markedly blunted in these animals. Despite this alteration in beta-cell mass, no effect on glucose homeostasis in beta gamma KO mice was noted. Additionally, while thiazolidinediones enhanced insulin secretion from cultured wild-type islets, administration of rosiglitazone to insulin-resistant control and beta gamma KO mice revealed that PPAR gamma in beta cells is not required for the antidiabetic actions of these compounds. These data demonstrate a critical physiological role for PPAR gamma function in beta-cell proliferation and also indicate that the mechanisms controlling beta-cell hyperplasia in obesity are different from those that regulate baseline cell mass in the islet.     

Multicellular Tumour Spheroid as a model for evaluation of [18F]FDG as biomarker for breast cancer treatment monitoring

Background  

In order to explore a pre-clinical method to evaluate if [¹⁸F]FDG is valid for monitoring early response, we investigated the uptake of FDG in Multicellular tumour spheroids (MTS) without and with treatment with five routinely used chemotherapy agents in breast cancer.     

Tuftsin-mediated immunoprophylaxis against an isolate of Aspergillus fumigatus shows less in vivo susceptibility to amphotericin B

In the present study, we evaluated the immunopotentiating efficacy of tuftsin against experimental murine aspergillosis in both normal and immunodebilitant BALB/c mice. The animals were challenged with an isolate of Aspergillus fumigatus (1x10(8) cfu/mouse) that was showing less susceptibility to lower doses of amphotericin B in murine animal model. Co-administration of the immunomodulator tuftsin and liposomised-amphotericin B was found to be highly effective in the treatment of systemic infection of A. fumigatus in both immunocompetent and leukopenic mice. Moreover, pre-treatment of mice with liposomised-tuftsin prior to challenging them with A. fumigatus infection and subsequent treatment with tuftsin-bearing liposomised-amphotericin B was found to be extremely efficient in successful elimination of fungal pathogen. In another set of experiments, tuftsin-mediated antigen-specific memory antibody response was also assessed by immunizing the animals with A. fumigatus cytosolic antigen. The animals that received a booster 150 days after the first immunization with tuftsin-liposomes-antigen showed more resistance to A. fumigatus infection in comparison with the na?ve animals.     

Cytotoxic mannich bases of 1-arylidene-2-tetralones

Various 1-arylidene-2-tetralones 1 had been shown previously to possess moderate cytotoxic properties unaccompanied by murine toxicity. The objective of the present investigation was to undertake different molecular modifications of representative members of series 1 with a view to discerning those structural features leading to increased potencies. All compounds were evaluated using human Molt 4/C8 and CEM T-lymphocytes as well as murine P388 and L1210 leukemic cells. The Mannich bases 2, 4, 5 and 7 possessed increased potencies compared to the corresponding unsaturated ketones 1 and in general were potent cytotoxics having IC50 values in the 0.2-10 microM range. QSAR using the cytotoxicity data for 2a-e suggested that potency was positively correlated with the size of the substituents in the arylidene aryl ring. Compounds 2a-f were evaluated using a panel of approximately 53 human tumour cell lines and, when all cell lines were considered, were more potent than the reference drug melphalan. In particular, marked antileukemic activity was displayed. Molecular modeling was utilized in order to evaluate whether the shapes of the different compounds contributed to the varying potencies observed. Representative compounds demonstrated minimal or no inhibiting properties towards human N-myristoyltransferase (NMT) and did not bind to calf thymus DNA. This study has revealed a number of unique lead molecules as candidate anti-neoplastic agents serving as prototypes for future development.     

Sperm status and DNA dose play key roles in sperm/ICSI‐mediated gene transfer in caprine

In relation to the growing recent interest in the establishment of sperm‐mediated gene transfer (SMGT) technology as a convenient and effective method for the simple production of transgenic animals, in this study the possibility of using SMGT to produce transgenic caprine embryos was investigated for the first time. Buck sperm were directly incubated with different concentrations (0–500 ng) of pcDNA/his/Lac‐Z plasmid and used for IVF or ICSI. Sperm used for ICSI were categorized into motile or live‐immotile group before being injected into oocytes. In a separate experiment, dead sperm prepared by repeated freezing/thawing were used for DNA‐incubation before ICSI. Sham injection was carried out by intracytoplasmic injection of approximately the same volume of media containing different doses of DNA using an ICSI needle. Transgene expression and transmission were detected by X‐Gal staining and PCR analysis of developed embryos, respectively. A reasonable blastocyst rate was observed in all the groups. Only embryos in the sham group were negative for transgene transmission. Transgene expression was completely dependent on the delivery technique and status of sperm, and was only observed in the live‐immotile and dead ICSI groups. The results of this study showed that the technique (IVF vs. ICSI vs. sham injection), sperm status (motile vs. live‐immotile vs. dead) and to some extent DNA concentration affect embryo development, transgene transmission and expression. Mol. Reprod. Dev. 77:868–875, 2010. © 2010 Wiley‐Liss, Inc.     

Comparison of Trace Elements in the Scalp Hair of Malignant and Benign Breast Lesions Versus Healthy Women

Trace elements including Al, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Sb, Sr, and Zn were analyzed in the scalp hair samples of women with malignant breast lesions, women with benign breast lesions, and healthy donors using atomic absorption spectrophotometric method. In the scalp hair of malignant-tumor patients, the highest average concentration was shown by Ca (1,187 μg/g), followed by Na (655 μg/g), Mg (478 μg/g), Zn (391 μg/g), Sr (152 μg/g), Fe (114 μg/g), and K (89.8), while in the case of benign-tumor patients, the average estimated element levels were 1,522, 1,093, 572, 457, 217, 80.4, and 74.7 μg/g, respectively. Most of the elements exhibited non-normal distribution evidenced by large spread, standard error, and skewness values. Mean concentrations of Ca (634 μg/g), Zn (206 μg/g), Mg (162 μg/g), Fe (129 μg/g), and Na (82.1 μg/g) were noteworthy in the scalp hair of healthy women. Average levels of Na, Sr, K, Cd, Co, Pb, Mg, Ca, Zn, Ni, Sb, and Mn were revealed to be significantly higher in the hair of malignant and benign patients compared to the healthy women; however, Fe, Cu, Al, and Cr were not significantly different in the scalp hair of the three groups. The quartile distributions of Ca, Cd, Co, Cr, K, Mg, Mn, Na, Ni, Pb, Sb, and Sr revealed maximum spread in the scalp hair of malignant and benign groups; nevertheless, Al, Cu, Fe, and Zn exhibited almost comparable quartile levels in the three groups. Strong correlation coefficients were found between Fe and Cd, Al and Na, Mn and Sr, Co and Cr, Cd and Cr, Pb and K, Pb and Mn, Cu and Na, and Al and Fe in the scalp hair of malignant-tumor patients, while Fe and K, Cd and Co, Na and Co, and Cr and Pb showed strong correlations in the scalp hair of benign-tumor patients, both of which were significantly different compared with the healthy subjects. Multivariate cluster analysis also revealed divergent clustering of the elements in the scalp hair of malignant and benign patients in comparison with the healthy women.     

Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 μg/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.