Aryl Hydrocarbon Receptor Facilitates DNA Strand Breaks and 8-Oxo-2��-deoxyguanosine Formation by the Aldo-Keto Reductase Product Benzo[a]pyrene-7,8-dione

Polycyclic aromatic hydrocarbon (PAH) o-quinones produced by aldo-keto reductases are ligands for the aryl hydrocarbon receptor (AhR) (Burczynski, M. E., and Penning, T. M. (2000) Cancer Res. 60, 908–915). They induce oxidative DNA lesions (reactive oxygen species-mediated DNA strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) formation) in human lung cells. We tested whether the AhR enhances PAH o-quinone-mediated oxidative DNA damage by translocating these ligands to the nucleus. Using the single cell gel electrophoresis (comet) assay to detect DNA strand breaks in murine hepatoma Hepa1c1c7 cells and its AhR- and aryl hydrocarbon receptor nuclear translocator-deficient variants, benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione) produced fewer DNA strand breaks in AhR-deficient cells compared with aryl hydrocarbon receptor nuclear translocator-deficient and wild type Hepa1c1c7 cells. Decreased DNA strand breaks were also observed in human bronchoalveolar H358 cells in which the AhR was silenced by siRNA. The antioxidant α-tocopherol and the iron chelator/antioxidant desferal decreased the formation of B[a]P-7,8-dione-mediated DNA strand breaks indicating that they were reactive oxygen species-dependent. By coupling the comet assay to 8-oxoguanine glycosylase (hOGG1), which excises 8-oxo-Gua, strand breaks dependent upon this lesion were measured. hOGG1 treatment produced more DNA single strand breaks in B[a]P-7,8-dione-treated Hepa cells and H358 cells than in its absence. The levels of hOGG1-dependent DNA strand breaks mediated by B[a]P-7,8-dione were lower in AhR-deficient Hepa and AhR knockdown H358 cells. The AhR antagonist α-naphthoflavone also attenuated B[a]P-7,8-dione-mediated DNA strand breaks. The decrease in 8-oxo-dGuo levels in AhR-deficient Hepa cells and AhR knockdown H358 cells was validated by immunoaffinity capture stable isotope dilution ([¹⁵N₅]8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry. We conclude that the AhR shuttles PAH o-quinone genotoxins to the nucleus and enhances oxidative DNA damage.Polycyclic aromatic hydrocarbons (PAHs)² are ubiquitous environmental pollutants that include over 200 compounds with two or more fused benzene rings. PAHs are formed as a result of incomplete combustion of fossil fuels (e.g. coal and oil) and are present in car and diesel exhaust and smoked or charbroiled food (1–3). They are also found in cigarette smoke condensate and tobacco products and are suspect agents in the causation of human lung cancer (4, 5). PAHs must be metabolically activated to reactive genotoxins to cause their mutagenic and carcinogenic effects.Two major metabolic activation pathways are possible starting from the proximate PAH carcinogen (−)B[a]P-7,8-trans-dihydrodiol (Fig. 1). The P4501A1/1B1 pathway converts (−)B[a]P-7,8-trans-dihydrodiol to yield (+)-anti-7,8-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydroB[a]P (6–8). This diol epoxide forms stable N²-2′-deoxyguanosine (dGuo) adducts in vitro and in vivo (9, 10) and leads to mutation in H-ras (11) and may account for mutations in “hot spots” in p53 observed in lung cancer (12). The G to T transversions most often observed in these genes might arise because of the action of one or more trans-lesional by-pass DNA polymerases that read through stable diol-epoxide DNA adducts with low processivity and fidelity (13, 14).Open in a separate windowFIGURE 1.PAH activation by AKRs to cause oxidative DNA damage.As an alternative, human aldo-keto reductases (AKR1A1 and AKR1C1-AKR1C4) catalyze the NADP⁺-dependent oxidation of (±)B[a]P-7,8-trans-dihydrodiol to produce the electrophilic and redox-active B[a]P-7,8-dione (15, 16). In this pathway, AKRs convert B[a]P-7,8-trans-dihydrodiol to form a ketol that rearranges to a catechol. The catechol then undergoes two subsequent one-electron oxidations to yield the fully oxidized o-quinone. Once formed, B[a]P-7,8-dione amplifies reactive oxygen species (ROS) by entering futile redox cycles that deplete cellular reducing equivalents (e.g. NADPH) (17). PAH o-quinones can undergo 1,4- or 1,6-Michael addition with guanine and adenine bases to form stable N²-dGuo and N⁶-dAdo adducts in vitro (18–20). They can also react with the N7 position of guanine to yield depurinating adducts (21). It is possible that these covalent PAH o-quinone adducts could give to G to T transversion mutations.PAH o-quinones also cause oxidative DNA damage in vitro and in vivo (22–25). Nanomolar concentrations of PAH o-quinones under redox cycling conditions (NADPH and Cu(II)) lead to significant 8-oxo-dGuo formation in bulk DNA, and the responsible oxidant was found to be singlet oxygen (¹O₂) (24, 26). Under these conditions, PAH o-quinones produced 8-oxo-dGuo as the most dominant lesion among the three types of oxidative lesions measured (abasic sites, 8-oxo-dGuo, and oxidized pyrimidines) (26). In a yeast reporter gene assay, which scored loss-of-function mutations in p53, PAH o-quinones were found to be highly mutagenic but only under redox cycling conditions. The dominant mutation observed was a G to T transversion that was suppressed by ROS scavengers (27). Subsequent HPLC analysis coupled with electrochemical detection showed that there was a linear correlation between 8-oxo-dGuo formation in p53 and mutation frequency, indicating that 8-oxo-dGuo was the likely adduct responsible for the G to T transversions observed (28). These data suggest that oxidative DNA lesions caused by PAH o-quinones are more relevant in causing mutation than covalent PAH o-quinone-DNA adducts. In the latter case even if these adducts form, they do not appear to be mutagenic on p53.Recently, using either a hOGG1-coupled comet assay or an immunoaffinity capture-stable isotope dilution liquid chromatography/electrospray ionization/multiple reaction monitoring/mass spectrometry (LC/ESI/MRM/MS) assay, it was shown that both the AKR substrate (B[a]P-7,8-trans-dihydrodiol) and the AKR product (B[a]P-7,8-dione) caused significant DNA strand breaks and 8-oxo-dGuo formation in human lung adenocarcinoma A549 cells (25). Similar results were not observed with (+)-anti-7,8-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydroB[a]P or the regioisomer B[a]P-4,5-trans- dihydrodiol in these AKR-expressing cells. Subsequent use of the fluorescent dye dichlorofluorescein diacetate revealed that B[a]P-7,8-dione generated ROS in the nuclear compartment of the cells, suggesting that the PAH o-quinone was transported into the nucleus to increase the ROS-mediated DNA strand breaks and 8-oxo-dGuo (25). In addition, earlier disposition studies detected significant amounts of [³H]B[a]P-7,8-dione in the cell pellets of primary rat hepatocytes within 0.5 h, which caused extensive strand scission of the genomic DNA (29), suggesting that B[a]P-7,8-dione reached the nucleus. However, how PAH o-quinones gain entry into the nucleus and induce oxidative DNA damage is currently unknown.PAH o-quinones are ligands for the aryl hydrocarbon receptor (AhR) (30). These quinones can promote translocation of AhR to nucleus to induce P4501A1 expression. Upon binding with PAH o-quinones, the AhR dissociates from heat shock protein 90 and is rapidly translocated into nucleus where it dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) (31, 32). The quinone-bound AhR·ARNT complex then binds to the xenobiotic response element (XRE) and robustly activates the expression of AhR-regulated genes (30). These data raise the possibility that oxidative DNA damage caused by PAH o-quinones occurs because of their transportation and concentration in the nucleus mediated by the AhR. However, this hypothesis has not been formally tested.We now show that B[a]P-7,8-dione produces AhR-dependent DNA strand breaks and 8-oxo-dGuo formation using murine Hepa1c1c7 cells but not in its AhR-deficient variant. Similar results were obtained in human bronchoalveolar carcinoma H358 cells, but these effects were attenuated when the AhR was knocked down with siRNA. DNA lesions were measured by using the comet assay, which was coupled with hOGG1. These results were also confirmed by LC-ESI/MRM/MS assay for 8-oxo-dGuo. Our finding shows that PAH o-quinones produced by AKRs can cause ROS-mediated genotoxicity via an AhR-dependent mechanism, and this might contribute to PAH-mediated carcinogenesis.     

Characterisation of the tryptophan synthase alpha subunit in maize

Background  

In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP) by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA) and β (TSB) homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS) complex in Arabidopsis. On the other hand maize (Zea mays) expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB.     

Soil respiration does not acclimatize to warmer temperatures when modeled over seasonal timescales

Soil warming studies have generally demonstrated an ephemeral response of soil respiration to warming suggesting acclimatization to increased temperatures. Many of these studies depict acclimatization as an empirical temperature-respiration model with data collected from late spring through early autumn. We examined the apparent temperature sensitivity of soil respiration in chronically warmed soils over three different timescales: annually, during the growing season, and seasonally during winter, spring, summer, and fall. Temperature sensitivity was evaluated by fitting exponential and flexible temperature functions as mixed effects models. From model coefficients, we estimated annual, growing season, and season-specific Q ₁₀ values, and assessed the ability of model coefficients to predict daily soil respiration rates over a two-year period. We found that respiration in warmed soils can exhibit characteristics of acclimatized temperature sensitivity depending on the timeframe and the function (exponential or flexible) used. Models using growing season data suggested acclimatization while models using data collected in winter or spring indicated enhanced temperature sensitivity with 5 °C of warming. Differences in temperature sensitivity affected predicted daily soil respiration rates, particularly in winter and spring. Models constructed over longer timescales overestimated daily respiration rates by as much 10–40 % whereas season-specific predictions were generally within 2 % of actual values. Failure to use season-specific models to depict changes in temperature dependence may over- or under-estimate carbon losses due to climate warming, especially during the colder months of the year.     

Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non‐electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science‐based process validation strategies to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009; 104: 371–380 © 2009 Wiley Periodicals, Inc.     

Mobile larynx in Mongolian gazelle: Retraction of the larynx during rutting barks in male Mongolian gazelle (Procapra gutturosa Pallas, 1777)

This study provides the first evidence of pronounced temporary laryngeal descent in a bovid species. An elaborate acoustic display is prominent in male courtship behavior of polygynous Mongolian gazelle. During rut, rounding up of females is accompanied by continuous head‐up barking by dominant males. Throughout the rut their evolutionarily enlarged larynx descends to a low mid‐neck resting position. In the course of each bark the larynx is additionally retracted toward the sternum by 30% of the resting vocal tract length. A geometric model of active larynx movements was constructed by combining results of video documentation, dissection, skeletonization, and behavioral observation. The considerable distance between resting position and maximal laryngeal descent suggests a backward tilting of the hyoid apparatus and an extension of the thyrohyoid connection during the retraction phase. Return to the resting position is effected by strap muscles and by the elastic recoil of the pharynx and the thyrohyoid connection. An intrapharyngeal inflation of the peculiar palatinal pharyngeal pouch of adult males is inferred from a short‐time expansion of the ventral neck region rostral to the laryngeal prominence. The neck of adult dominant males is accentuated by long gray guard hairs during the rut. The passive swinging of the heavy larynx of adult males during locomotion gives the impression of a handicap imposed on rutting males. Apparently, this disadvantage becomes outweighed by the profits for reproductive success. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Non-Selective Evolution of Growing Populations

Non-selective effects, like genetic drift, are an important factor in modern conceptions of evolution, and have been extensively studied for constant population sizes (Kimura, 1955; Otto and Whitlock, 1997). Here, we consider non-selective evolution in the case of growing populations that are of small size and have varying trait compositions (e.g. after a population bottleneck). We find that, in these conditions, populations never fixate to a trait, but tend to a random limit composition, and that the distribution of compositions “freezes” to a steady state. This final state is crucially influenced by the initial conditions. We obtain these findings from a combined theoretical and experimental approach, using multiple mixed subpopulations of two Pseudomonas putida strains in non-selective growth conditions (Matthijs et al, 2009) as model system. The experimental results for the population dynamics match the theoretical predictions based on the Pólya urn model (Eggenberger and Pólya, 1923) for all analyzed parameter regimes. In summary, we show that exponential growth stops genetic drift. This result contrasts with previous theoretical analyses of non-selective evolution (e.g. genetic drift), which investigated how traits spread and eventually take over populations (fixate) (Kimura, 1955; Otto and Whitlock, 1997). Moreover, our work highlights how deeply growth influences non-selective evolution, and how it plays a key role in maintaining genetic variability. Consequently, it is of particular importance in life-cycles models (Melbinger et al, 2010; Cremer et al, 2011; Cremer et al, 2012) of periodically shrinking and expanding populations.     

Flow and Diffusion in Channel-Guided Cell Migration

Collective migration of mechanically coupled cell layers is a notable feature of wound healing, embryonic development, and cancer progression. In confluent epithelial sheets, the dynamics have been found to be highly heterogeneous, exhibiting spontaneous formation of swirls, long-range correlations, and glass-like dynamic arrest as a function of cell density. In contrast, the flow-like properties of one-sided cell-sheet expansion in confining geometries are not well understood. Here, we studied the short- and long-term flow of Madin-Darby canine kidney (MDCK) cells as they moved through microchannels. Using single-cell tracking and particle image velocimetry (PIV), we found that a defined averaged stationary cell current emerged that exhibited a velocity gradient in the direction of migration and a plug-flow-like profile across the advancing sheet. The observed flow velocity can be decomposed into a constant term of directed cell migration and a diffusion-like contribution that increases with density gradient. The diffusive component is consistent with the cell-density profile and front propagation speed predicted by the Fisher-Kolmogorov equation. To connect diffusion-mediated transport to underlying cellular motility, we studied single-cell trajectories and occurrence of vorticity. We discovered that the directed large-scale cell flow altered fluctuations in cellular motion at short length scales: vorticity maps showed a reduced frequency of swirl formation in channel flow compared with resting sheets of equal cell density. Furthermore, under flow, single-cell trajectories showed persistent long-range, random-walk behavior superimposed on drift, whereas cells in resting tissue did not show significant displacements with respect to neighboring cells. Our work thus suggests that active cell migration manifests itself in an underlying, spatially uniform drift as well as in randomized bursts of short-range correlated motion that lead to a diffusion-mediated transport.     

Weathering-Associated Bacteria from the Damma Glacier Forefield: Physiological Capabilities and Impact on Granite Dissolution

Several bacterial strains isolated from granitic rock material in front of the Damma glacier (Central Swiss Alps) were shown (i) to grow in the presence of granite powder and a glucose-NH₄Cl minimal medium without additional macro- or micronutrients and (ii) to produce weathering-associated agents. In particular, four bacterial isolates (one isolate each of Arthrobacter sp., Janthinobacterium sp., Leifsonia sp., and Polaromonas sp.) were weathering associated. In comparison to what was observed in abiotic experiments, the presence of these strains caused a significant increase of granite dissolution (as measured by the release of Fe, Ca, K, Mg, and Mn). These most promising weathering-associated bacterial species exhibited four main features rendering them more efficient in mineral dissolution than the other investigated isolates: (i) a major part of their bacterial cells was attached to the granite surfaces and not suspended in solution, (ii) they secreted the largest amounts of oxalic acid, (iii) they lowered the pH of the solution, and (iv) they formed significant amounts of HCN. As far as we know, this is the first report showing that the combined action of oxalic acid and HCN appears to be associated with enhanced elemental release from granite, in particular of Fe. This suggests that extensive microbial colonization of the granite surfaces could play a crucial role in the initial soil formation in previously glaciated mountain areas.Glaciers in alpine regions are highly sensitive to changes in climatic conditions (29). Increasing global atmospheric temperatures over the last decades have resulted in the recession of alpine glaciers (18). Forefields of temperate alpine glaciers provide unique opportunities to study initial soil formation as well as microbial and plant succession along the chronosequences (12, 26, 34, 36). The forefields close to the glacier terminus are initially vegetation free and consist mainly of rock material with high fractions of silt-sized grains with low C and N content and small amounts of available nutrients (14). Mineral weathering is a key process in the formation of soils (1, 26), and the crucial importance of microbially promoted mineral weathering for nutrient acquisition is increasingly recognized (2, 4, 39, 46). Recently exposed rock surfaces can be considered primary ecosystems where only a few microbes that are adapted due to their mineral-weathering abilities can grow (17). Some cations of rock-forming minerals are essential for proper cell functions. However, our understanding of geochemically significant microbes in forefields of temperate alpine glacier is still very limited but is crucial for increasing our knowledge of nutrient mobilization and the buildup of organic matter that is essential for the development of macroorganisms.The area of the Damma glacier in Central Switzerland is characterized by a relatively homogenous granitic rock basement and is used as field site of the interdisciplinary research project “Biosphere-Geosphere interactions: Linking climate change, weathering, soil formation and ecosystem evolution (BigLink)” (5). In the frame of this research project, we studied the functional roles of granite-colonizing microbes as biotic weathering agents in previously glaciated areas. So far, relatively little is known about microbe-granite interactions, especially regarding the release of trace elements. Several studies have examined the dissolution of specific granite-forming minerals in the presence of actively metabolizing bacteria or compounds that simulate metabolic activity (24, 30, 31, 37, 38, 44). There is a general agreement that microbially produced organic acids, siderophores, and extracellular polysaccharides can all promote dissolution of minerals. Previous dissolution experiments have mainly been performed with (i) commercially obtained minerals (23, 45), (ii) model microorganisms that were commercially obtained from culture collections (3, 35, 45), or (iii) laboratory strains, such as those of Bacillus subtilis (23) and Burkholderia fungorum (47). Most studies have focused on individual mineral specimens rather than on the mixture of minerals that are present in granite rock (47). Few studies observed mineral weathering of collected rock and bacteria isolated from volcanic areas covered with vegetation (30, 31). Moreover, there are no studies on microbial weathering for such immediately deglaciated environments combining functional and taxonomic investigations, probably due to the difficulties in obtaining heterotrophic bacterial isolates from granitic glacier forefields. In spite of this, a comprehensive culture collection containing approximately 500 bacterial strains, which were isolated from the glacier tongue of the Damma glacier, was established. Full-length 16S rRNA gene sequences of 120 isolates revealed that many isolates obtained from oligotrophic media were closely related to readily cultivable heterotrophic bacteria (e.g., Arthrobacter sp., Collimonas sp., Paenibacillus sp., and Pseudomonas sp.). These bacteria have been found to enhance mineral dissolution (39).Our aim was to characterize the impact of microorganisms on granite weathering. We performed laboratory dissolution experiments with sterile crushed granite rock material, 12 bacterial strains, and 1 algal strain. To investigate the potential weathering abilities of these isolates, granite dissolution experiments were performed abiotically with model agents, such as HCl for proton-promoted weathering or oxalate and citrate and KCN for ligand-promoted weathering.