Assessing and managing risks arising from exposure to endocrine-active chemicals

Managing risks to human health and the environment produced by endocrine-active chemicals (EAC) is dependent on sound principles of risk assessment and risk management, which need to be adapted to address the uncertainties in the state of the science of EAC. Quantifying EAC hazard identification, mechanisms of action, and dose-response curves is complicated by a range of chemical structure/toxicology classes, receptors and receptor subtypes, and nonlinear dose-response curves with low-dose effects. Advances in risk science including toxicogenomics and quantitative structure-activity relationships (QSAR) along with a return to the biological process of hormesis are proposed to complement existing risk assessment strategies, including that of the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC 1998). EAC represents a policy issue that has captured the public's fears and concerns about environmental health. This overview describes the process of EAC risk assessment and risk management in the context of traditional risk management frameworks, with emphasis on the National Research Council Framework (1983), taking into consideration the strategies for EAC management in Canada, the United States, and the European Union.     

Development of a Theory-based,Sociocultural Instrument to Assess Black Maternal Intentions to Vaccinate Their Daughters Aged 9 to 12 Against HPV

The human papillomavirus (HPV) vaccine could assist in reducing the cervical cancer disparity existing between Black and White women. Understanding factors influencing Black maternal intentions to vaccinate their daughter is essential in improving vaccination uptake. However, existing instruments do not comprehensively assess factors (e.g., culture) influencing maternal intentions. This paper describes the development of the Human Papillomavirus Vaccination Survey for Black Mothers with Girls Aged 9 to 12 (HPVS-BM), the first instrument to measure knowledge, attitudes, subjective norms, and cultural beliefs relating to Black maternal intentions to vaccinate their daughters aged 9 to 12 years against HPV. The items and scales were refined using content review by experts, as well as cognitive interviews and pilot testing with target audience participants. The final version of the HPVS-BM was administered to 242 Black mothers with adolescent daughters. Internal reliability was determined using Cronbach’s alpha. An a priori hypothetical model was developed to determine convergent and discriminant validity. All scales of the HPVS-BM had an acceptable internal reliability of 0.70 or higher. The intention scale of HPVS-BM was significantly correlated (p?<?.05) with perceived benefits, perceived barriers, and subjective norms, supporting strong convergent validity. Moderate discriminant construct validity was also demonstrated. Exhibiting good psychometrics, this instrument could be used by healthcare researchers and professionals to develop programs to increase HPV vaccination among Black adolescent females aimed at reducing the racial disparities in cervical cancer. Further psychometric testing of this survey tool for understanding factors influencing maternal intentions is warranted.     

Carbonic anhydrase IV and XIV knockout mice: roles of the respective carbonic anhydrases in buffering the extracellular space in brain

Previous studies have implicated extracellular carbonic anhydrases (CAs) in buffering the alkaline pH shifts that accompany neuronal activity in the rat and mouse hippocampus. CAs IV and XIV both have been proposed to mediate this extracellular buffering. To examine the relative importance of these two isozymes in this and other physiological functions attributed to extracellular CAs, we produced CA IV and CA XIV knockout (KO) mice by targeted mutagenesis and the doubly deficient CA IV/XIV KO mice by intercrossing the individual null mice. Although CA IV and CA XIV null mice both are viable, the CA IV nulls are produced in smaller numbers than predicted, indicating either fetal or postnatal losses, which preferentially affect females. CA IV/XIV double KO mice are also produced in fewer numbers than predicted and are smaller than WT mice, and many females die prematurely before and after weaning. Electrophysiological studies on hippocampal slices on these KO mice showed that either CA can mediate buffering after synaptic transmission in hippocampal slices in the absence of the other, but that eliminating both is nearly as effective as the CA inhibitor, benzolamide, in blocking the buffering seen in the WT mice. Thus, both CA IV and CA XIV contribute to extracellular buffering in the central nervous system, although CA IV appears to be more important in the hippocampus. These individual and double KO mice should be valuable tools in clarifying the relative contributions of each CA to other physiological functions where extracellular CAs have been implicated.     

Strand invasion promoted by recombination protein beta of coliphage lambda

Studies of phage lambda in vivo have indicated that its own recombination enzymes, beta protein and lambda exonuclease, are capable of catalyzing two dissimilar pathways of homologous recombination that are widely distributed in nature: single-strand annealing and strand invasion. The former is an enzymatic splicing of overlapping ends of broken homologous DNA molecules, whereas the latter is characterized by the formation of a three-stranded synaptic intermediate and subsequent strand exchange. Previous studies in vitro have shown that beta protein has annealing activity, and that lambda exonuclease, acting on branched substrates, can produce a perfect splice that requires only ligation for completion. The present study shows that beta protein can initiate strand invasion in vitro, as evidenced both by the formation of displacement loops (D-loops) in superhelical DNA and by strand exchange between colinear single-stranded and double-stranded molecules. Thus, beta protein can catalyze steps that are central to both strand annealing and strand invasion pathways of recombination. These observations add beta protein to a set of diverse proteins that appear to promote recognition of homology by a unitary mechanism governed by the intrinsic dynamic properties of base pairs in DNA.     

In vivo imaging of immuno-spin trapped radicals with molecular magnetic resonance imaging in a diabetic mouse model

Oxidative stress plays a major role in diabetes. In vivo levels of membrane-bound radicals (MBRs) in a streptozotocin-induced diabetic mouse model were uniquely detected by combining molecular magnetic resonance imaging (mMRI) and immunotrapping techniques. An anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibody (Ab) covalently bound to an albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent (anti-DMPO probe), and mMRI, were used to detect in vivo levels of DMPO-MBR adducts in kidneys, livers, and lungs of diabetic mice, after DMPO administration. Magnetic resonance signal intensities, which increase in the presence of a Gd-based molecular probe, were significantly higher within the livers, kidneys, and lungs of diabetic animals administered the anti-DMPO probe compared with controls. Fluorescence images validated the location of the anti-DMPO probe in excised tissues via conjugation of streptavidin-Cy3, which targeted the probe biotin moiety, and immunohistochemistry was used to validate the presence of DMPO adducts in diabetic mouse livers. This is the first report of noninvasively imaging in vivo levels of MBRs within any disease model. This method can be specifically applied toward diabetes models for in vivo assessment of free radical levels, providing an avenue to more fully understand the role of free radicals in diabetes.     

Novel Ser79Leu and Ser81Ile substitutions in the quinolone resistance-determining regions of ParC topoisomerase IV and GyrA DNA gyrase subunits from recent fluoroquinolone-resistant Streptococcus pneumoniae clinical isolates

Resistance of Streptococcus pneumoniae to fluoroquinolones is caused predominantly by amino acid substitutions at positions Ser79 of ParC and Ser81 of GyrA to either Phe or Tyr encoded in the quinolone resistance-determining regions of the parC topoisomerase IV and gyrA DNA gyrase genes. Analysis of highly resistant clinical isolates identified novel second-step substitutions, Ser79Leu (ParC) and Ser81Ile (GyrA). To determine contributions of these new mutations to fluoroquinolone resistance either alone or in combination with other Ser79/81 alleles, the substitutions Ser79Leu/Phe/Tyr in ParC and Ser81Ile/Phe/Tyr in GyrA were introduced into the R6 background, resulting in 15 isogenic strains. Their level of fluoroquinolone resistance was determined by susceptibility testing for ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, gemifloxacin, garenoxacin, and norfloxacin. Leu79 and Ile81 alone as well as 79/81Phe/Tyr substitutions did not contribute significantly to resistance, with fluoroquinolone MICs increasing two- to fourfold compared to wild type for all agents tested. Fluoroquinolone MICs for double transformants ParC Ser79Phe/Tyr/Leu-GyrA Ser81Phe/Tyr were uniformly increased by 8- to 64-fold regardless of pairs of amino acid substitutions. However, combinations including Ile81 conferred two- to fourfold-higher levels of resistance than did combinations including any other Ser81 GyrA substitution, thus demonstrating the differential effects of diverse amino acid substitutions at particular hotspots on fluoroquinolone MICs.     

A New Water-Soluble Thermosensitive Star-Like Copolymer as a Promising Carrier of the Chemotherapeutic Drug Doxorubicin

A new water-soluble thermosensitive star-like copolymer, dextran-graft-poly-N-iso-propilacrylamide (D-g-PNIPAM), was created and characterized by various techniques (size-exclusion chromatography, differential scanning calorimetry, Fourier-transform infrared (FTIR) spectroscopy, and dynamic light scattering (DLS) spectroscopy). The viability of cancer cell lines (human transformed cervix epithelial cells, HeLa) as a model for cancer cells was studied using MTT and Live/Dead assays after incubation with a D-g-PNIPAM copolymer as a carrier for the drug doxorubicin (Dox) as well as a D-g-PNIPAM + Dox mixture as a function of the concentration. FTIR spectroscopy clearly indicated the complex formation of Dox with the D-g-PNIPAM copolymer. The size distribution of particles in Hank’s solution was determined by the DLS technique at different temperatures. The in vitro uptake of the studied D-g-PNIPAM + Dox nanoparticles into cancer cells was demonstrated by confocal laser scanning microscopy. It was found that D-g-PNIPAM + Dox nanoparticles in contrast to Dox alone showed higher toxicity toward cancer cells. All of the aforementioned facts indicate a possibility of further preclinical studies of the water-soluble D-g-PNIPAM particles’ behavior in animal tumor models in vivo as promising carriers of anticancer agents.