Potent mutagenicity of an azide, 3-azido-1,2-propanediol, in human TK6 cells.

Sodium azide is a strong mutagen that has been successfully employed in mutation breeding of crop plants. In biological systems, it is metabolically converted to the proximate mutagen azidoalanine, which requires further bioactivation to a putative ultimate mutagen that remains elusive. The nature of the DNA modifications induced by azides leading to mutations is also unknown. Other mutagenic organic azido compounds seem to share the same bioactivation pathway to the ultimate mutagenic species as they induce point mutations dependent on the same DNA repair pathways. We investigated mutations induced by the representative mutagen 3-azido-1,2-propanediol (azidoglycerol, AZG) in the human TK6 cell line. Until now, azides have been considered to be non-mutagens and non-carcinogens in mammals, including humans, as judged only by the conventional clastogenicity chromosomal aberration types of bioassays. Here, we show the potent mutagenicity of AZG in cultured human cells, comparable to alkylating agents such as methyl methanesulfonate at concentrations with similar lethality. The potent ability of an organic azide to induce base substitutions in a mammalian system raises an alert with respect to human exposure to organic and inorganic azido compounds.

The Influence of Metabolic Inhibitors, Antibiotics, and Microgravity on Intact Cell MALDI-TOF Mass Spectra of the Cyanobacterium Synechococcus Sp. UPOC S4.

The aim and novelty of this paper are found in assessing the influence of inhibitors and antibiotics on intact cell MALDI-TOF mass spectra of the cyanobacterium Synechococcus sp. UPOC S4 and to check the impact on reliability of identification. Defining the limits of this method is important for its use in biology and applied science. The compounds included inhibitors of respiration, glycolysis, citrate cycle, and proteosynthesis. They were used at 1-10 µM concentrations and different periods of up to 3 weeks. Cells were also grown without inhibitors in a microgravity because of expected strong effects. Mass spectra were evaluated using controls and interpreted in terms of differential peaks and their assignment to protein sequences by mass. Antibiotics, azide, and bromopyruvate had the greatest impact. The spectral patterns were markedly altered after a prolonged incubation at higher concentrations, which precluded identification in the database of reference spectra. The incubation in microgravity showed a similar effect. These differences were evident in dendrograms constructed from the spectral data. Enzyme inhibitors affected the spectra to a smaller extent. This study shows that only a long-term presence of antibiotics and strong metabolic inhibitors in the medium at 10-5 M concentrations hinders the correct identification of cyanobacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).

Toxicological analysis of azide and cyanide for azide intoxications using gas chromatography.

Azide is a highly toxic chemical agent to human being. Accidental, but also intentional exposure to azide occurs. To be able to confirm azide ingestion, we developed a method to identify and quantify azide in biological matrices. Cyanide was included in the method to evaluate suggested in vivo production of cyanide after azide ingestion. Azide in biological matrices was first derivatized by propionic anhydride to form propionyl azide. Simultaneously, cyanide was converted into hydrogen cyanide. After thermal rearrangement of propionyl azide, ethyl isocyanate was formed, separated together with hydrogen cyanide by gas chromatography (GC) and detected using a nitrogen phosphorous detector (NPD). The method was linear from 1.0-100 µg/mL for both analytes, and azide was stable in human plasma at -20°C for at least 49 days. Azide was measured in the gastric content of two cases of suspected azide ingestion (case 1:1.2 mg/mL, case 2:1.5 mg/mL). Cyanide was only identified in the gastric content of case 1 (approximately 1.4 µg/mL). Furthermore, azide was quantified in plasma (19 µg/mL), serum (24 µg/mL), cell pellet (21 µg/mL) and urine (3.0 µg/mL) of case 2. This method can be used to confirm azide and cyanide exposure, and azide concentrations can be quantified in several biological matrices.

Assessing modulators of cytochrome c oxidase activity in Galleria mellonella larvae.

Caterpillars of the greater wax moth, Galleria mellonella, are shown to be a useful invertebrate organism for examining mitochondrial toxicants (inhibitors of electron transport) and testing putative antidotes. Administration of sodium azide, sodium cyanide, or sodium (hydro)sulfide by intra-haemocoel injection (through a proleg) results in a dose-dependent paralysed state in the larvae lasting from <1 to ~40 min. The duration of paralysis is easily monitored, because if turned onto their backs, the larvae right themselves onto their prolegs once they are able to move again. The efficacy of putative antidotes to the three toxicants can routinely be assessed by observing shortened periods of paralysis with larvae given toxicant and antidote compared to larvae administered only the same dose of toxicant. The validity of the approach is demonstrated with agents previously shown to be antidotal towards cyanide intoxication in mice; namely, sodium nitrite and CoN4[11.3.1] (cobalt(II/III) 2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]-heptadeca-1(7)2,11,13,15-pentaenyl cation). These same compounds are shown to be antidotal towards all three toxicants in the G. mellonella caterpillars; findings that may prove important in relation to azide and sulfide poisonings, for which there are currently no effective antidotes available. The observation that sodium nitrite ameliorates cyanide toxicity in the larvae is additionally interesting because it unambiguously demonstrates that the antidotal action of nitrites does not require the involvement of methemoglobin, contributing to the resolution of an ongoing controversy.

A ratiometric Raman probe for live-cell imaging of hydrogen sulfide in mitochondria by stimulated Raman scattering.

Stimulated Raman Scattering (SRS) coupled with alkyne tags has been an emerging imaging technique to visualize small-molecule species with high sensitivity and specificity. Here we describe the development of a ratiometric Raman probe for visualizing hydrogen sulfide (H2S) species in living cells as the first alkyne-based sensor for SRS microscopy. This probe uses an azide unit as a selective reactive site, and it targets mitochondria with high specificity. The SRS ratiometric images show a strong response to H2S level changes in living cells.

Knock-out of a mitochondrial sirtuin protects neurons from degeneration in Caenorhabditis elegans.

Sirtuins are NAD⁺-dependent deacetylases, lipoamidases, and ADP-ribosyltransferases that link cellular metabolism to multiple intracellular pathways that influence processes as diverse as cell survival, longevity, and cancer growth. Sirtuins influence the extent of neuronal death in stroke. However, different sirtuins appear to have opposite roles in neuronal protection. In Caenorhabditis elegans, we found that knock-out of mitochondrial sirtuin sir-2.3, homologous to mammalian SIRT4, is protective in both chemical ischemia and hyperactive channel induced necrosis. Furthermore, the protective effect of sir-2.3 knock-out is enhanced by block of glycolysis and eliminated by a null mutation in daf-16/FOXO transcription factor, supporting the involvement of the insulin/IGF pathway. However, data in Caenorhabditis elegans cell culture suggest that the effects of sir-2.3 knock-out act downstream of the DAF-2/IGF-1 receptor. Analysis of ROS in sir-2.3 knock-out reveals that ROS become elevated in this mutant under ischemic conditions in dietary deprivation (DD), but to a lesser extent than in wild type, suggesting more robust activation of a ROS scavenging system in this mutant in the absence of food. This work suggests a deleterious role of SIRT4 during ischemic processes in mammals that must be further investigated and reveals a novel pathway that can be targeted for the design of therapies aimed at protecting neurons from death in ischemic conditions.

Best practices for application of attachment cells to in vitro micronucleus assessment by flow cytometry.

This work seeks to provide users with guidance on cell culture, treatment, processing and analytical conditions for achieving optimal performance of the in vitro micronucleus assay using the In Vitro MicroFlow(®) method. Experimental data are provided to support the advice described. The information provided covers specific topics or issues that are identified as critical to the methodology and thus is meant to work with instruction manuals, published papers and other references, and not as a replacement for these documents. The content is divided into several sections. Cell culture and treatment describes conditions for routine maintenance of cells as well as treatment with test articles. Preparation and processing of samples details steps found to be critical in execution of the procedure. Instrument parameters and analysis covers set-up of the flow cytometer and evaluation of the samples. General assay considerations and interpretation of results describes examination of data in terms of assay validity, viability and genotoxicity assessment. The goal is to educate users and enable them to design, conduct and interpret flow cytometric in vitro micronucleus (MN) studies. Readers should obtain an understanding of specific cell culture practices, options for assay formatting and execution and the information required to successfully integrate and validate the in vitro MN assay into their existing safety program.

Root aquaporins contribute to whole plant water fluxes under drought stress in rice (Oryza sativa L.).

Aquaporin activity and root anatomy may affect root hydraulic properties under drought stress. To better understand the function of aquaporins in rice root water fluxes under drought, we studied the root hydraulic conductivity (Lpr) and root sap exudation rate (Sr) in the presence or absence of an aquaporin inhibitor (azide) under well-watered conditions and following drought stress in six diverse rice varieties. Varieties varied in Lpr and Sr under both conditions. The contribution of aquaporins to Lpr was generally high (up to 79% under well-watered conditions and 85% under drought stress) and differentially regulated under drought. Aquaporin contribution to Sr increased in most varieties after drought, suggesting a crucial role for aquaporins in osmotic water fluxes during drought and recovery. Furthermore, root plasma membrane aquaporin (PIP) expression and root anatomical properties were correlated with hydraulic traits. Three chromosome regions highly correlated with hydraulic traits of the OryzaSNP panel were identified, but did not co-locate with known aquaporins. These results therefore highlight the importance of aquaporins in the rice root radial water pathway, but emphasize the complex range of additional mechanisms related to root water fluxes and drought response.

Effects of azide on electron transport of exoelectrogens in air-cathode microbial fuel cells.

The effects of azide on electron transport of exoelectrogens were investigated using air-cathode MFCs. These MFCs enriched with azide at the concentration higher than 0.5mM generated lower current and coulomb efficiency (CE) than the control reactors, but at the concentration lower than 0.2mM MFCs generated higher current and CE. Power density curves showed overshoot at higher azide concentrations, with power and current density decreasing simultaneously. Electrochemical impedance spectroscopy (EIS) showed that azide at high concentration increased the charge transfer resistance. These analyses might reflect that a part of electrons were consumed by the anode microbial population rather than transferred to the anode. Bacterial population analyses showed azide-enriched anodes were dominated by Deltaproteobacteria compared with the controls. Based on these results it is hypothesized that azide can eliminate the growth of aerobic respiratory bacteria, and at the same time is used as an electron acceptor/sink.

Differential resistance of drinking water bacterial populations to monochloramine disinfection.

The impact of monochloramine disinfection on the complex bacterial community structure in drinking water systems was investigated using culture-dependent and culture-independent methods. Changes in viable bacterial diversity were monitored using culture-independent methods that distinguish between live and dead cells based on membrane integrity, providing a highly conservative measure of viability. Samples were collected from lab-scale and full-scale drinking water filters exposed to monochloramine for a range of contact times. Culture-independent detection of live cells was based on propidium monoazide (PMA) treatment to selectively remove DNA from membrane-compromised cells. Quantitative PCR (qPCR) and pyrosequencing of 16S rRNA genes was used to quantify the DNA of live bacteria and characterize the bacterial communities, respectively. The inactivation rate determined by the culture-independent PMA-qPCR method (1.5-log removal at 664 mg·min/L) was lower than the inactivation rate measured by the culture-based methods (4-log removal at 66 mg·min/L). Moreover, drastic changes in the live bacterial community structure were detected during monochloramine disinfection using PMA-pyrosequencing, while the community structure appeared to remain stable when pyrosequencing was performed on samples that were not subject to PMA treatment. Genera that increased in relative abundance during monochloramine treatment include Legionella, Escherichia, and Geobacter in the lab-scale system and Mycobacterium, Sphingomonas, and Coxiella in the full-scale system. These results demonstrate that bacterial populations in drinking water exhibit differential resistance to monochloramine, and that the disinfection process selects for resistant bacterial populations.

Guanosine protects C6 astroglial cells against azide-induced oxidative damage: a putative role of heme oxygenase 1.

Guanosine, a guanine-based purine, is an extracellular signaling molecule that is released from astrocytes and shows neuroprotective effects in several in vivo and in vitro studies. Our group recently showed that guanosine presents antioxidant properties in C6 astroglial cells. The heme oxygenase 1 signaling pathway is associated with protection against oxidative stress. Azide, an inhibitor of the respiratory chain, is frequently used in experimental models to induce oxidative and nitrosative stress. Thus, the goal of this study was to investigate the effect of guanosine on azide-induced oxidative damage in C6 astroglial cells. Azide treatment of these cells resulted in several detrimental effects, including induction of cytotoxicity and mitochondrial dysfunction, increased levels of reactive oxygen/nitrogen species, inducible nitric oxide synthase expression and NADPH oxidase, decreased glutamate uptake and EAAC1 glutamate transporter expression, decreased glutathione (GSH) levels, and decreased activities of glutamine synthetase (GS), superoxide dismutase and catalase (CAT). The treatment also increased nuclear factor-κB activation and the release of proinflammatory cytokines tumor necrosis factor α and IL-1ß. Guanosine strongly prevented these effects, protecting glial cells against azide-induced cytotoxicity and modulating glial, oxidative and inflammatory responses through the activation of the heme oxygenase 1 pathway. These observations reinforce and support the role of guanosine as an antioxidant molecule against oxidative damage. Guanosine protects against azide-induced oxidative damage in C6 astroglial cells. Azide-induced mitochondrial dysfunction (1); increased reactive oxygen species/reactive nitrogen species levels (2); decreased glutamate uptake (3), GS activity (4), GSH levels (5), and SOD (6) and CAT (7) activities; increased glutathione peroxidase (GPx) (8) and NADPH oxidase (9) activities and cellular superoxide levels (10); increased NF-κB activation (11), TNF-α and IL-1ß levels (12); and induced iNOS expression (13). Guanosine prevented these effects through the HO1 signaling pathway, thus our findings support the antioxidant effects of guanosine.

Novel nucleoside analogue FNC is effective against both wild-type and lamivudine-resistant HBV clinical isolates.

BACKGROUND: HBV infection causes major public health problems worldwide. The clinical limitation of current antiviral drugs for HBV, such as lamivudine, is causing rapid emergence of drug-resistant viral strains during prolonged antiviral therapy. Therefore, new antiviral drugs are urgently needed to prevent or delay the selection of drug-resistant HBV mutants. A novel cytidine analogue, FNC (2'-deoxy-2'-ß-fluoro-4'-azidocytidine), was recently shown to strongly inhibit human HBV and duck HBV (DHBV) replication in vitro and in vivo, respectively. The present study was designed to evaluate the in vitro antiviral activity of FNC against clinical wild-type and lamivudine-resistant HBV isolates in transiently transfected cells. METHODS: HBV DNA was extracted from serum samples collected both before lamivudine therapy and at the time of viral breakthrough and was amplified by PCR. The amplicon was cloned into a novel expression vector, pHY106, which can initiate the intracellular HBV replication cycle after cell transfection. Following transfection of the cloned amplicon into HepG2 cells, a drug susceptibility assay was performed. Quantitative real-time PCR was used for determining the amount of intracellular HBV DNA, and the effective concentration required to reduce HBV replication by 50% (EC(50)) was calculated. RESULTS: FNC inhibited the replication of both wild-type and lamivudine-resistant HBV clinical isolates in a dose-dependent manner, with mean ±SD EC(50) values of 0.12 ±0.01 µM and 0.27 ±0.01 µM, respectively. CONCLUSIONS: FNC is a potential antiviral agent against both wild-type and lamivudine-resistant HBV clinical isolates, and therefore deserves further evaluation for the treatment of HBV infection.

Evaluation of a ricin vaccine candidate (RVEc) for human toxicity using an in vitro vascular leak assay.

To protect against ricin intoxication, a genetically derived ricin A chain vaccine candidate (RVEc) was developed lacking the toxic N-glycosidase activity (Olson et al., 2004). The vaccine protects animals against an aerosolized ricin holotoxin (RT) challenge (Carra et al., 2007). In the current study, the RVEc vaccine was evaluated for its interaction and effect on human endothelial cells. RVEc was tested in an in vitro cellular-based bioassay, consisting of primary human endothelial cells cultured on collagen-coated inserts, to which concentrations of the vaccine candidate (0.6, 2, 2.5 or 9 µM) were added. RVEc showed no signs of adverse activity on the cells (e.g., cytotoxicty activity) as measured by changes in trans-endothelial electrical resistance (TEER). In contrast, ricin toxin (RT) cytotoxicity was observed at all concentrations tested. Under light microscopy, no cytotoxicity was visible at 24h with 0.6 or 9 µM of RVEc. However, cytotoxicity was observed for RT and to a lesser degree for RTA. Flow cytometric analysis showed binding of RT, slight binding of RTA, and no binding of the RVEc vaccine to endothelial cells. The presence of RTB as a contaminant contributing to the cytotoxicity in the RTA preparation was ruled out by a RTB-specific ELISA. In addition, RTA at 9 µM produced a cytotoxic activity that could not be explained exclusively by the presence of azide in the RTA buffer. In the current study, the model demonstrated no discernable adverse events of the RVEc vaccine on human endothelial cells, when compared to the toxicity caused by holotoxin or native RTA preparations.

Genotoxicity assessment of two hypergolic energetic propellant compounds.

Recognition of the occupational hazards from exposure to the propellants hydrazine and monomethylhydrazine (MMH) has led to research into less toxic alternatives. Two hypergolic compounds, dimethylamino-2-ethylazide (DMAZ) and N,N,N',N'-tetramethylethanediamine (TMEDA), have been identified as possible replacements for MMH. We have obtained genotoxicity data for these compounds from in vitro and in vivo studies. DMAZ did not produce any mutagenic effects at concentrations up to 5mg/plate in the TA98 and TA1537 strains of Salmonella typhimurium and in an Escherichia coli (WP2 uvrA) strain, with or without metabolic activation, but did produce a positive response in the TA100 and TA1535 strains, both with and without metabolic activation. TMEDA was found not to be mutagenic in any of the bacterial strains tested (Salmonella TA98, TA100, TA1535, TA1537 and E. coli, WP2 uvrA), with or without metabolic activation. DMAZ did not induce structural chromosomal aberrations at levels up to 5mg/mL in Chinese hamster ovary (CHO) cells, with or without metabolic activation. TMEDA produced a positive response in this system, with or without metabolic activation, but only at the highest concentration, 5mg/mL. However, according to the OECD guideline TG 473, the compound is considered to be negative in the CHO chromosomal aberration assay, since the compound was not clastogenic at 0.01M (1.140mg/mL). DMAZ and TMEDA, when tested in vivo in the CD-1 mouse at doses up to 500 and 250mg/kg, respectively, did not induce micronuclei in bone marrow erythrocytes. These studies demonstrate that DMAZ is mutagenic in specific strains of Salmonella. However, both compounds were negative for induction of chromosomal aberrations in CHO cells in vitro and in the in vivo mouse micronucleus assay.

Drp1 dephosphorylation in ATP depletion-induced mitochondrial injury and tubular cell apoptosis.

Recent studies revealed a striking morphological change of mitochondria during apoptosis. Mitochondria become fragmented and notably, the fragmentation contributes to mitochondrial outer membrane permeabilization and consequent release of apoptotic factors. In renal tubular cells, mitochondrial fragmentation involves the activation of Drp1, a key mitochondrial fission protein. However, it is unclear how Drp1 is regulated during tubular cell apoptosis. In this study, we examined Drp1 regulation during tubular cell apoptosis following ATP depletion. Rat kidney proximal tubular cells (RPTC) were subjected to azide treatment or severe hypoxia in glucose-free medium to induce ATP depletion. During ATP depletion, Drp1 was shown to be dephosphorylated at serine-637. Drp1 dephosphorylation could be suppressed by cyclosporine A and FK506, two calcineurin inhibitors. Importantly, cyclosporine A and FK506 could also prevent mitochondrial fragmentation, Bax accumulation, cytochrome c release, and apoptosis following ATP depletion in RPTC. The results suggest that calcineurin-mediated serine-637 dephosphorylation is involved in Drp1 activation during ATP depletion in renal tubular cells. Upon activation, Drp1 contributes to mitochondrial fragmentation and outer membrane permeabilization, resulting in the release of apoptogenic factors and apoptosis.

Mitochondrial uncouplers act synergistically with the fumigant phosphine to disrupt mitochondrial membrane potential and cause cell death.

Phosphine is the most widely used fumigant for the protection of stored commodities against insect pests, especially food products such as grain. However, pest insects are developing resistance to phosphine and thereby threatening its future use. As phosphine inhibits cytochrome c oxidase (complex IV) of the mitochondrial respiratory chain and reduces the strength of the mitochondrial membrane potential (DeltaPsi(m)), we reasoned that mitochondrial uncouplers should act synergistically with phosphine. The mitochondrial uncouplers FCCP and PCP caused complete mortality in populations of both wild-type and phosphine-resistant lines of Caenorhabditis elegans simultaneously exposed to uncoupler and phosphine at concentrations that were individually nonlethal. Strong synergism was also observed with a third uncoupler DNP. We have also tested an alternative complex IV inhibitor, azide, with FCCP and found that this also caused a synergistic enhancement of toxicity in C. elegans. To investigate potential causes of the synergism, we measured DeltaPsi(m), ATP content, and oxidative damage (lipid hydroperoxides) in nematodes subjected to phosphine-FCCP treatment and found that neither an observed 50% depletion in ATP nor oxidative stress accounted for the synergistic effect. Instead, a synergistic reduction in DeltaPsi(m) was observed upon phosphine-FCCP co-treatment suggesting that this is directly responsible for the subsequent mortality. These results support the hypothesis that phosphine-induced mortality results from the in vivo disruption of normal mitochondrial activity. Furthermore, we have identified a novel pathway that can be targeted to overcome genetic resistance to phosphine.

Light-activated destruction of cancer cell nuclei by platinum diazide complexes.

A possible way to avoid dose-limiting side effects of platinum anticancer drugs is to employ light to cause photochemical changes in nontoxic platinum prodrugs that release active antitumor agents. This strategy could be used in the treatment of localized cancers accessible to irradiation (e.g., bladder, lung, esophagus, and skin). We report here that nontoxic photolabile diam(m)ino platinum(IV) diazido complexes inhibit the growth of human bladder cancer cells upon irradiation with light, and are non-crossresistant to cisplatin. Their rate of photolysis closely parallels that of DNA platination, indicating that the photolysis products interact directly, and rapidly, with DNA. Photoactivation results in a dramatic shrinking of the cancer cells, loss of adhesion, packing of nuclear material, and eventual disintegration of their nuclei, indicating a different mechanism of action from cisplatin.

Severe cytochrome c oxidase inhibition in vivo does not induce a pyrimidine deficiency; neuroprotective action of oral uridine prodrug PN401 requires supraphysiological levels of uridine.

It has been hypothesized that mitochondrial respiratory chain dysfunction leads to a pyrimidine deficiency since the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase is coupled to the electron transport chain. The uridine prodrug triacetyluridine (PN401) is neuroprotective in several models of neurodegenerative disease involving respiratory chain toxins. Therefore, the therapeutic effects of PN401 might involve the correction of a pyrimidine deficiency secondary to respiratory chain impairment. We infused mice with the cytochrome c oxidase inhibitor azide, which inhibited brain complex IV activity. Chronic infusion of azide for 2 or 14 days induced significant toxicity and mortality but did not cause a pyrimidine deficit in the brain. In contrast, the pyrimidine synthesis inhibitor N-phosphonoacetyl-l-aspartate (PALA) produced a pyrimidine deficit with minimal mortality. Treatment with 6% PN401 decreased mortality and cerebrocortical apoptosis caused by azide. Previously, we found that optimal neuroprotection against mitochondrial complex II inhibition required 4-6% PN401. PN401 at 1, 3, 6 and 10% in chow induced nonlinear increases in plasma uridine with 6% PN401 elevating plasma uridine up to 80 muM, and these higher micromolar uridine levels were also required for neuroprotection in chemical hypoxia models in vitro. Our results indicate that severe complex IV inhibition in vivo does not lead to a pyrimidine deficiency, and therefore the protective effect of PN401 in the azide toxin model is not mediated through the correction of a pyrimidine deficiency. Furthermore, supraphysiological levels of uridine are required to produce optimal protective effects in disorders involving impairment of mitochondrial respiratory complex II or IV.