Nitrofen induces apoptosis independently of retinaldehyde dehydrogenase (RALDH) inhibition

BACKGROUND: Nitrofen is a diphenyl ether that induces congenital diaphragmatic hernia (CDH) in rodents. Its mechanism of action has been hypothesized as inhibition of the retinaldehyde dehydrogenase (RALDH) enzymes with consequent reduced retinoic acid signaling. METHODS: To determine if nitrofen inhibits RALDH enzymes, a reporter gene construct containing a retinoic acid response‐element (RARE) was transfected into HEK‐293 cells and treated with varying concentrations of nitrofen in the presence of retinaldehyde (retinal). Cell death was characterized by caspace‐cleavage microplate assays and terminal deoxynucleotidyl transferase dUTP nick end‐labeling (TUNEL) assays. Ex vivo analyses of cell viability were characterized in fetal rat lung explants using Live/Dead staining. Cell proliferation and apoptosis were assessed using fluorescent immunohistochemistry with phosphorylated histone and activated caspase antibodies on explant tissues. Nile red staining was used to identify intracellular lipid droplets. RESULTS: Nitrofen‐induced dose‐dependent declines in RARE‐reporter gene expression. However, similar reductions were observed in control‐reporter constructs suggesting that nitrofen compromised cell viability. These observed declines in cell viability resulted from increased cell death and were confirmed using two independent assays. Ex vivo analyses showed that mesenchymal cells were particularly susceptible to nitrofen‐induced apoptosis while epithelial cell proliferation was dramatically reduced in fetal rat lung explants. Nitrofen treatment of these explants also showed profound lipid redistribution, primarily to phagocytes. CONCLUSIONS: The observed declines in nitrofen‐associated retinoic acid signaling appear to be independent of RALDH inhibition and likely result from nitrofen induced cell death/apoptosis. These results support a cellular apoptotic mechanism of CDH development, independent of RALDH inhibition. Birth Defects Res (Part B) 89:223–232, 2010. © 2010 Wiley‐Liss, Inc.     

Development of sequential multiple comparison procedure for dose response test

We propose a multiple comparison procedure to identify the minimum effective dose level by sequentially comparing each dose level with the zero dose level in the dose finding test. If we can find the minimum effective dose level at an early stage in the sequential test, it is possible to terminate the procedure in the dose finding test after a few group observations up to the dose level. Thus, the procedure is viable from an economical point of view when high costs are involved in obtaining the observations. In the procedure, we present an integral formula to determine the critical values for satisfying a predefined type I familywise error rate. Furthermore, we show how to determine the required sample size in order to guarantee the power of the test in the procedure. In practice, we compare the power of the test and the required sample size for various configurations of the population means in simulation studies and adopt our sequential procedure to the dose response test in a case study.     

Synopsis of the CASIROZ case study: carbon sink strength of Fagus sylvatica L. in a changing environment--experimental risk assessment of mitigation by chronic ozone impact

Databases are needed for the ozone (O(3)) risk assessment on adult forest trees under stand conditions, as mostly juvenile trees have been studied in chamber experiments. A synopsis is presented here from an integrated case study which was conducted on adult FAGUS SYLVATICA trees at a Central-European forest site. Employed was a novel free-air canopy O(3) fumigation methodology which ensured a whole-plant assessment of O(3) sensitivity of the about 30 m tall and 60 years old trees, comparing responses to an experimental 2 x ambient O(3) regime (2 x O(3), max. 150 nl O(3) l (-1)) with those to the unchanged 1 x ambient O(3) regime (1 x O(3)=control) prevailing at the site. Additional experimentation on individual branches and juvenile beech trees exposed within the forest canopy allowed for evaluating the representativeness of young-tree and branch-bag approaches relative to the O(3) sensitivity of the adult trees. The 2 x O(3) regime did not substantially weaken the carbon sink strength of the adult beech trees, given the absence of a statistically significant decline in annual stem growth; a 3 % reduction across five years was demonstrated, however, through modelling upon parameterization with the elaborated database. 2 x O(3) did induce a number of statistically significant tree responses at the cell and leaf level, although the O(3) responsiveness varied between years. Shade leaves displayed an O(3) sensitivity similar to that of sun leaves, while indirect belowground O(3) effects, apparently mediated through hormonal relationships, were reflected by stimulated fine-root and ectomycorrhizal development. Juvenile trees were not reliable surrogates of adult ones in view of O(3) risk assessment. Branch sections enclosed in (climatized) cuvettes, however, turned out to represent the O(3) sensitivity of entire tree crowns. Drought-induced stomatal closure decoupled O(3) intake from O(3) exposure, as in addition, also the "physiologically effective O(3) dose" was subject to change. No evidence emerged for a need to lower the "Critical Level for Ozone" in risk assessment of forest trees, although sensitive tree parameters did not necessarily reflect a linear relationship to O(3) stress. Exposure-based concepts tended to overestimate O(3) risk under drought, which is in support of current efforts to establish flux-related concepts of O(3) intake in risk assessment.     

A genetic map of tetraploid <Emphasis Type="Italic">Paspalum notatum</Emphasis> Flügge (bahiagrass) based on single-dose molecular markers

Paspalum notatum Flügge is a warm-season forage grass with mainly diploid (2n = 20) and autotetraploid (2n = 40) representatives. Diploid races reproduce sexually and require crosspollination due to a self-incompatible mating system, while autotetraploids reproduce by aposporous apomixis. The objectives of this work were to develop a genetic linkage map of Paspalum notatum Flügge at the tetraploid level, identify the linkage/s group/s associated with apomixis and carry out a general characterization of its mode of inheritance. A pseudo test-cross F₁ family of 113 individuals segregating for the mode of reproduction was obtained by crossing a synthetic completely sexual tetraploid plant (Q4188) as female parent with a natural aposporous individual (Q4117) as pollen donor. Map construction was based on single-dose markers (SDAFs) segregating from both parents. Two linkage maps (female and male) were constructed. Within each map, homologous groups were assembled by detecting repulsion-phase linked SDAFs. Putative Q4188 and Q4117 homolog groups were identified by mapping shared single dose markers (BSDF). The Q4188 map consisted of 263 markers distributed on 26 co-segregation groups over a total genetic distance of 1.590.6 cM, while the Q4117 map contained 216 loci dispersed on 39 co-segregation groups along 2.265.7 cM, giving an estimated genome coverage of 88% and 83%, respectively. Seven and 12 putative homologous chromosomes were detected within Q4188 and Q4117 maps, respectively. Afterward, ten female and male homologous chromosomes were identified by mapping BSDFs. In the Q4117 map, a single linkage group was associated with apospory. It was characterized by restriction in recombination and preferential chromosome pairing. A BPSD marker mapping within this group allowed the detection of the female homolog and the putative four male groups of the set carrying apospory.     

Human Risk Assessment for Nonylphenol

This article presents a risk assessment for human exposure to nonylphenol (NP). We critically reviewed and assessed all relevant full-text publications based on a variety of data quality attributes. Two categories of data, environmental monitoring and biomonitoring from exposed individuals, were used to estimate human exposure to NP. Environmental monitoring data included the measurement of NP in food, water, air, and dust. From these data and estimates of human intake rates for the sources, exposures were estimated from each source and source-specific Margins of Exposure (MOEs) calculated. However, the nature of the populations studied prevented the calculation of aggregate exposure calculations from these data. Rather, the most reliable estimates of aggregate exposure to NP were those derived from biomonitoring studies in exposed individuals. Using the daily absorbed dose estimates for NP, MOEs were calculated for these populations. The MOEs were based on the use of a No-Observed-Adverse-Effect-Level (NOAEL) for sensitive toxicological endpoints of interest, that is, systemic and reproductive toxicity from continuous-feeding more than 3.5 generations (13 mg/kg/day). The MOEs were all greater than 1000 (ranging from 2863 to 8.4 × 10⁷), clearly indicating reasonable certainty of no harm for source-specific and aggregate (based on biomonitoring) exposures to NP.     

Monte Carlo simulation of the dose distribution of ICRP adult reference computational phantoms for acquisitions with a 320 detector-row cone-beam CT scanner

PurposeThe purpose of this study was to develop and validate a Monte Carlo (MC) simulation tool for patient dose assessment for a 320 detector-row CT scanner, based on the recommendations of International Commission on Radiological Protection (ICRP). Additionally, the simulation was applied on four clinical acquisition protocols, with and without automatic tube current modulation (TCM).MethodsThe MC simulation was based on EGS4 code and was developed specifically for a 320 detector-row cone-beam CT scanner. The ICRP adult reference phantoms were used as patient models. Dose measurements were performed free-in-air and also in four CTDI phantoms: 150 mm and 350 mm long CT head and CT body phantoms. The MC program was validated by comparing simulations results with these actual measurements acquired under the same conditions. The measurements agreed with the simulations across all conditions within 5%. Patient dose assessment was performed for four clinical axial acquisitions using the ICRP adult reference phantoms, one of them using TCM.ResultsThe results were nearly always lower than those obtained from other dose calculator tools or published in other studies, which were obtained using mathematical phantoms in different CT systems. For the protocol with TCM organ doses were reduced by between 28 and 36%, compared to the results obtained using a fixed mA value.ConclusionsThe developed simulation program provides a useful tool for assessing doses in a 320 detector-row cone-beam CT scanner using ICRP adult reference computational phantoms and is ready to be applied to more complex protocols.     

Cell death via mitochondrial apoptotic pathway due to activation of Bax by lysosomal photodamage

Lysosomal photosensitizers have been used in photodynamic therapy. The combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. Lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, N-aspartyl chlorin e6 (NPe6), an effective photosensitizer that preferentially accumulates in lysosomes, was used to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) after NPe6-photodynamic treatment (NPe6-PDT) was studied using real-time single-cell analysis. Our results demonstrated that NPe6-PDT induced rapid generation of reactive oxygen species (ROS). The photodynamically produced ROS caused a rapid destruction of lysosomes, leading to release of cathepsins, and the ROS scavengers vitamin C and NAC prevent the effects. Then the following spatiotemporal sequence of cellular events was observed during cell apoptosis: Bcl-2-associated X protein (Bax) activation, cytochrome c release, and caspase-9/-3 activation. Importantly, the activation of Bax proved to be a crucial event in this apoptotic machinery, because suppressing the endogenous Bax using siRNA could significantly inhibit cytochrome c release and caspase-9/-3 activation and protect the cell from death. In conclusion, this study demonstrates that PDT with lysosomal photosensitizer induces Bax activation and subsequently initiates the mitochondrial apoptotic pathway.     

A single, low, oral dose of a 5-carbon-linked trioxane dimer orthoester plus mefloquine cures malaria-infected mice

Four 5-carbon-linked trioxane dimer orthoesters (6a-6d) have been prepared in 4 or 5 chemical steps from the natural trioxane artemisinin (1). When administered orally to malaria-infected mice using a single dose of only 6 mg/kg body weight along with 18 mg/kg of mefloquine hydrochloride, trioxane dimer orthoester sulfone 6d completely and safely cured the mice; after 30 days, the cured mice showed no detectable parasitemia, gained at least as much weight as the control mice (no infection), and behaved normally.     

黑麦草根系分泌物剂量对污染土壤芘降解和土壤微生物的影响

模拟根际根系分泌物梯度递减效应,研究了黑麦草根系分泌物剂量对污染土壤中芘降解特征和土壤微生物生态特征的影响.结果表明:污染土壤中芘残留量随根系分泌物添加剂量的增加呈现先下降后上升的非线性变化,达到最低芘残留量的添加剂量是总有机碳(TOC) 32.75 mg·kg-1,说明此浓度下根系分泌物显著促进了芘的降解;土壤微生物生物量碳和微生物熵的变化趋势与污染土壤中芘残留量变化趋势相反,表明土壤微生物与污染土壤 中芘残留量存在密切关系.芘污染土壤中微生物群落以细菌占主导地位,且细菌变化趋势与芘降解变化一致,表明芘以细菌降解为主,根系分泌物主要通过影响细菌数量,进而影响芘的降解.能催化有机物质脱氢反应的土壤微生物胞内酶——脱氢酶活性的变化与土壤微生物变化趋势一致,进一步证明微生物及其生物化学特性变化是污染土壤中芘残留量随根系分泌物添加剂量变化的生态机制.     

On the Definition of Internal Dose Metrics

Internal dose metrics, as computed with pharmacokinetic models, are increasingly used as a means for extrapolating animal toxicological data to humans and to extrapolate across routes of administration. These internal dose metrics are thought to provide a more scientific means of comparing toxicological effects across animal species. The use of internal dose metrics is based on the universal assumption that toxic effects are equal across species if and only if the concentration of the toxic moieties in the target tissue is equal across species. Herein it is shown that this assumption is inconsistent with empirical toxicological data. It is shown that measurement of internal dose metrics in chronological time, as is done for AUC (Area under the concentration curve) and rate of metabolite production per kg of target tissue, does not produce equal toxic effects across species. A consequence of this observation is that the application of pharmacokinetics in risk assessments for such important chemicals as trichloroethylene, vinyl chloride, perchloroethylene, and perchlorate may need reassessment.