Comparative proteomics analysis of sarcosine insoluble outer membrane proteins from clarithromycin resistant and sensitive strains of Helicobacter pylori.

Helicobacter pylori causes disease manifestations in humans including chronic gastric and peptic ulcers, gastric cancer, and lymphoid tissue lymphoma. Increasing rates of H. pylori clarithromycin resistance has led to higher rates of disease development. Because antibiotic resistance involves modifications of outer membrane proteins (OMP) in other Gram-negative bacteria, this study focuses on identification of H. pylori OMP's using comparative proteomic analyses of clarithromycin-susceptible and -resistant H. pylori strains. Comparative proteomics analyses of isolated sarcosine-insoluble OMP fractions from clarithromycin-susceptible and -resistant H. pylori strains were performed by 1) one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis protein separation and 2) in-gel digestion of the isolated proteins and mass spectrometry analysis by Matrix Assisted Laser Desorption Ionization-tandem mass spectrometry. Iron-regulated membrane protein, UreaseB, EF-Tu, and putative OMP were down-regulated; HopT (BabB) transmembrane protein, HofC, and OMP31 were up-regulated in clarithromycin-resistant H. pylori. Western blotting and real time PCR, respectively, validated UreaseB subunit and EF-Tu changes at the protein level, and mRNA expression of HofC and HopT. This limited proteomic study provides evidence that alteration of the outer membrane proteins' profile may be a novel mechanism involved in clarithromycin resistance in H. pylori.

Combined analysis of prenatal (maternal hair and blood) and neonatal (infant hair, cord blood and meconium) matrices to detect fetal exposure to environmental pesticides.

OBJECTIVE: The aim of this study was to determine optimum biomarkers to detect fetal exposure to environmental pesticides by the simultaneous analysis of maternal (hair and blood) and infant (cord blood, infant hair or meconium) matrices and to determine if a combination of these biomarkers will further increase the detection rate. PATIENTS AND METHODS: Pregnant women were prospectively recruited from an agricultural site in the Philippines with substantial use at home and in the farm of the following pesticides: propoxur, cyfluthrin, chlorpyrifos, cypermethrin, pretilachlor, bioallethrin, malathion, diazinon and transfluthrin. Maternal hair and blood were obtained at midgestation and at delivery and infant hair, cord blood and meconium were obtained after birth. All samples were analyzed by gas chromatography/mass spectrometry (GC/MS) for the above pesticides and some of their metabolites. RESULTS: A total of 598 mother/infant dyads were included in this report. The highest rates of pesticide exposure were detected in meconium (23.2% to propoxur, 2.0% to pretilachlor, 1.7% to cypermethrin, 0.8% to cyfluthrin, 0.7% to 1,1,1-trichloro-2,2-bis, p-chlorophenylethane (DDT) and 0.3% to malathion and bioallethrin) and in maternal hair (21.6% to propoxur, 14.5% to bioallethrin, 1.3% to malathion, 0.8% to DDT, 0.3% to chlorpyrifos and 0.2% to pretilachlor). Combined analysis of maternal hair and meconium increased detection rate further to 38.5% for propoxur and to 16.7% for pyrethroids. Pesticide metabolites were rarely found in any of the analyzed matrices. CONCLUSIONS: There is significant exposure of the pregnant woman and her fetus to pesticides, particularly to the home pesticides, propoxur and pyrethroids. Analysis of meconium for pesticides was the single most sensitive measure of exposure. However, combined analysis of maternal hair and meconium significantly increased the detection rate. A major advantage of analyzing maternal hair is that prenatal pesticide exposure in the mother can be detected and intervention measures can be initiated to minimize further exposure of the fetus to pesticides.

A comparison of infant hair, cord blood and meconium analysis to detect fetal exposure to environmental pesticides.

OBJECTIVE: The detection of fetal exposure to environmental pesticides is important because many of the pesticides are neurotoxicants and fetal exposure to these compounds can adversely affect prenatal and subsequent neurodevelopment. The aim of this study was to determine, by the comparative analysis of infant hair, cord blood and meconium, the most sensitive matrix to detect fetal exposure to pesticides. PATIENTS AND METHODS: Pregnant women were prospectively recruited from an agricultural site in the Philippines where a preliminary survey indicated a substantial use at home and in the farm of the following pesticides: propoxur, cyfluthrin, chlorpyrifos, cypermethrin, pretilachlor, bioallethrin, malathion, diazinon and transfluthrin. Infant hair, cord blood and meconium were obtained after birth and were analyzed by gas chromatography/mass spectrometry for the above compounds, including lindane and 1,1,1-trichloro-2,2-bis, p-chlorophenylethane (DDT) and some of their known metabolites. RESULTS: A total of 638 infants were included in the study. The highest exposure rate to pesticides was detected in meconium (23.8% to propoxur, 1.9% to pretilachlor, 1.9% to cypermethrin, 0.8% to cyfluthrin, 0.6% to DDT and 0.3% to malathion and bioallethrin). Cord blood was only positive for propoxur (1.9%) whereas infant hair was only positive for chlorpyrifos (0.2%). The highest exposure was to household pesticide (propoxur). The frequency and concentration of pesticides were compared in the three matrices and there was a significantly higher frequency and concentration of propoxur, pretilachlor, DDT, cyfluthrin and cypermethrin in meconium compared to cord blood and infant hair. Pesticide metabolites were not found in any of the matrices analyzed, except in one meconium sample which was positive for 4,4' dichlorodiphenyldichloro ethylene (DDE), a DDT metabolite. CONCLUSIONS: There is significant exposure of the pregnant woman and her fetus to pesticides, particularly to the home pesticide, propoxur. Our study has demonstrated that among cord blood, meconium or infant hair, meconium is the most sensitive matrix to analyze for fetal exposure to pesticides. The accumulation of pesticides in meconium, the ease of meconium collection and the large amount of meconium that could be collected are factors that contribute to the increased sensitivity of this matrix.

Quantitative determination of paraquat in meconium by sodium borohydride-nickel chloride chemical reduction and gas chromatography/mass spectrometry (GC/MS).

The objective of this study was to develop a procedure for the GC/MS assay of paraquat in meconium as a biomarker of fetal exposure to paraquat. The method involved a sodium borohydride-nickel chloride reduction procedure, liquid-liquid extraction of the perhydrogenated product, concentration, and GC/MS assay. The method demonstrated good overall recovery (102.56%) with %CV (inter-assay) of less than 13%, and a limit of detection of 0.0156microg/g. Analysis of meconium samples from a study population in the Philippines (n=70) showed a 2.8% prevalence of fetal exposure to paraquat.

Association between prenatal pesticide exposures and the generation of leukemia-associated T(8;21).

BACKGROUND: This study was designed to investigate the relationship between prenatal pesticide exposures and the generation of leukemia-associated t(8;21)(q22;q22), one of the most common cytogenetic abnormalities in childhood acute myeloid leukemia (AML). PROCEDURE: Gas chromatography/mass spectrometry (GC/MS) was used to quantitatively detect different pesticides (propoxur and cypermethrin) in meconium from 49 newborn babies from the Philippines. The generation of t(8;21) was determined in the corresponding umbilical cord blood samples by detection of the AML1-ETO fusion transcripts derived from t(8;21) using nested RT-PCR. Levels for the AML1-ETO fusion transcripts were quantitated by real-time RT-PCR in the t(8;21) positive cord blood samples. AML1-ETO fusion transcript forms were characterized by RT-PCR amplification and DNA sequencing. RESULTS: In the present study using umbilical cord blood samples obtained from infants whose prenatal exposure to the pesticide, propoxur, was determined by meconium analysis, we showed that (i) incidence of t(8;21) in the exposed group was two-fold higher than that in the unexposed group; and (ii) the levels for AML1-ETO fusion transcripts resulting from t(8;21) positively correlated with propoxur concentrations in meconium. Similar heterogeneity in the fusion transcripts was detected in the t(8;21) positive cord blood samples as in our previous study with t(8;21) AML patients. CONCLUSION: These results further confirm the prenatal origin of t(8;21) and establish a significant correlation between prenatal pesticide exposures and the generation of t(8;21). They suggest that prenatal pesticide exposures may be causal factors for the generation of leukemia-associated chromosomal translocations.

Maternal hair--an appropriate matrix for detecting maternal exposure to pesticides during pregnancy.

The detection of exposure of pregnant women to toxicants in the environment is important because these compounds can be harmful to the health of the woman and her fetus. The aim of this study was to analyze for pesticides/herbicides in paired maternal hair and blood samples to determine the most appropriate matrix for detecting maternal exposure to these compounds. A total of 449 pregnant women were prospectively recruited at midgestation from an agricultural site in the Philippines where a preliminary survey indicated significant use at home and on the farm of the following compounds: propoxur, cyfluthrin, chlorpyrifos, cypermethrin, pretilachlor, bioallethrin, malathion, diazinon, and transfluthrin. Paired maternal hair and blood samples were obtained from each subject upon recruitment into the study (midgestation) and at birth and were analyzed for the above compounds, as well as lindane and DDT [1,1,1-trichloro-2-2-bis(p-chlorophenyl) ethane], and some of their known metabolites by gas chromatography/mass spectrometry. The highest exposure rate was seen for propoxur and bioallethrin and maternal hair analysis provided the highest detection rate for these two compounds, compared to blood, at both time periods: (1) At midgestation, 10.5% positive for propoxur in hair compared to 0.7% in blood (P<0.001) and for bioallethrin, 11.9% positive in hair compared to 0% in blood (P < or = 0.001), and (2) at birth, 11.8% positive for propoxur in hair compared to 4% in blood (P < or = 0.001) and for bioallethrin, 7.8% in hair compared to 0% in blood (P < or = 0.001). A small number of maternal hair samples were also positive for malathion, chlorpyrifos, pretilachlor, and DDT. Only a few of the pesticide metabolites were detected, principally 3-phenoxybenzoic acid, malathion monocarboxylic acid, and DDE [1,1,dichloro-2-2-bis(p-chlorophenyl)ethylene], and they were mostly found in maternal blood. There was a significant association between the use of the home spray pesticide, Baygon, and propoxur in maternal hair at birth (P=0.001) and between the use of a slow-burning mosquito coil and the presence of bioallethrin in maternal hair at midgestation and at birth (P=0.001, P < or = 0.041, respectively). There is significant exposure of the pregnant woman to pesticides, particularly to pesticides that are used at home. Our study demonstrates the advantages of analyzing maternal hair as a readily available biologic matrix for studying maternal exposure to toxicants in the environment, compared to blood. For propoxur, there was a 3- to 15-fold higher detection rate of the pesticide in maternal hair as compared to blood. As for the other pesticides, bioallethrin, malathion, chlorpyrifos, and DDT were exclusively found in maternal hair compared to blood. On the other hand, pesticide metabolites were infrequently found in maternal hair or maternal blood. Pesticides in blood most likely represent acute exposure, whereas pesticides in hair represent past and/or concurrent exposure. The high sensitivity, wide window of exposure, availability, and ease of hair collection are distinct advantages in using hair to detect exposure to pesticides among pregnant women. However, pesticides in maternal hair may also be secondary to passive exposure and therefore not truly representative of the internal pesticide dose. Finally, the analysis of maternal hair for pesticides as an index of maternal exposure to pesticides in the environment allows the institution of measures to prevent further exposure during pregnancy.