Novel risk assessment of reactive metabolites from discovery to clinical stage.

This study was aimed to predict drug-induced liver injury caused by reactive metabolites. Reactive metabolites covalently bind to proteins and could result in severe outcomes in patients. However, the relation between the extent of covalent binding and clinical hepatotoxicity is still unclear. From a perspective of body burden (human in vivo exposure to reactive metabolites), we developed a risk assessment method in which reactive metabolite burden (RM burden), an index that could reflect the body burden associated with reactive metabolite exposure, is calculated using the extent of covalent binding, clinical dose, and human in vivo clearance. The relationship between RM burden and hepatotoxicity in humans was then investigated. The results indicated that this RM burden assessment exhibited good predictability for sensitivity and specificity, and drugs with over 10 mg/day RM burden have high-risk for hepatotoxicity. Furthermore, a quantitative trapping assay using radiolabeled trapping agents ([35S]cysteine and [14C]KCN) was also developed, to detect reactive metabolite formation in the early drug discovery stage. RM burden calculated using this assay showed as good predictability as RM burden calculated using conventional time- and cost-consuming covalent binding assays. These results indicated that the combination of RM burden and our trapping assay would be a good risk assessment method for reactive metabolites from the drug discovery stage.

Evaluation of the Removal of Potassium Cyanide and its Toxicity in Green Algae (Chlorella vulgaris).

To evaluate the removal of potassium cyanide (KCN) and its toxicity in algae, an initial comprehensive analysis was performed with Chlorella vulgaris. The algae showed potential removal capability for KCN, with the maximal removal rate of 61%. Moreover, effects of KCN on growth, cellular morphology and antioxidant defense system of C. vulgaris were evaluated. Cell number and chlorophyll a content decreased in most cases, with the maximal inhibition rates of 48% and 99%, respectively. The 100 mg L- 1 KCN seriously damaged the algal cell membrane. Additionally, activity of superoxide dismutase (SOD) was promoted by KCN exposure among 0.1-50 mg L- 1 and inhibited by 100 mg L- 1 KCN, while the malondialdehyde (MDA) content gradually decreased in C. vulgaris with increasing exposure concentration compared to the control. The present study reveals that C. vulgaris is useful in bio-treatment of cyanide-contaminated aquatic ecosystem, except in high concentrations which would cause overwhelming effects.

Nitrile-synthesizing enzyme: Gene cloning, overexpression and application for the production of useful compounds.

One of the nitrile-synthesizing enzymes, ß-cyano-L-alanine synthase, catalyzes ß-cyano-L-alanine (ß-CNAla) from potassium cyanide and O-acetyl-L-serine or L-cysteine. We have identified this enzyme from Pseudomonas ovalis No. 111. In this study, we cloned the ß-CNAla synthase gene and expressed it in Escherichia coli and Rhodococcus rhodochrous. Furthermore, we carried out co-expression of ß-CNAla synthase with nitrilase or nitrile hydratases in order to synthesize aspartic acid and asparagine from KCN and O-acetyl-L-serine. This strategy can be used for the synthesis of labeled amino acids by using a carbon-labeled KCN as a substrate, resulting in an application for positron emission tomography.

Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

Characterization of rock bream (Oplegnathus fasciatus) cytosolic Cu/Zn superoxide dismutase in terms of molecular structure, genomic arrangement, stress-induced mRNA expression and antioxidant function.

Superoxide dismutases (SODs) are dedicated to scavenge and dismutate the superoxide anions in order to protect the cells from oxidative stress by establishing the redox homeostasis. In this study, we describe a cytosolic Cu/ZnSOD, the second SOD member from rock bream Oplegnathus fasciatus (Of-cCu/ZnSOD) at molecular, genomic structural-, transcriptional- and functional-levels. The determination of genomic arrangement of Of-cCu/ZnSOD by means of a BAC library revealed that its primary transcript is represented by five exons and encoded a peptide of 154 amino acids. In silico investigation of Of-cCu/ZnSOD indicated the presence of several family characteristics including two Cu/ZnSOD signatures, seven metal liganding residues and eight ß-sheets forming a ß-barrel topology. Alignment and modeling studies confirmed the conservation of Cu/ZnSOD at primary and tertiary levels. While invertebrate Cu/ZnSOD members mainly demonstrate a tetraexonic structure, the vertebrate members have acquired an additional intron in the third exon resulting in a quinquepartite arrangement with class-specific exon lengths. Although, teleost Cu/ZnSOD members resembled the mammalian orthologs in their genomic organization, they shared a proximal position with molluscan members in the phylogeny. The antioxidant (AO) activity of Of-cCu/ZnSOD was affirmed by a recombinant protein which was also used to examine the biophysical and biochemical properties. The pronounced activity was detected when the rOf-cCu/ZnSOD was expressed with the Cu(2+) and Zn(2+) supplementation. The optimum activities were observed at pH10 and 25°C, and KCN strongly inhibited the activity of the rOf-cCu/ZnSOD. Furthermore, a constitutive mRNA expression of Of-cCu/ZnSOD with higher levels in blood>liver>heart and brain was observed, which was consistent with the transcriptional profile of Of-mMnSOD, suggesting important physiological role(s). This idea was further strengthened by the temporal assessment of Of-cCu/ZnSOD transcripts in animals under pathological (bacteria- or viral-induced) and physiological (H2O2-induced oxidative) stress conditions using qPCR, in which it exhibited significantly up-regulated levels. Screening of Of-cCu/ZnSOD 5'-flanking region revealed the presence of several important transcription factor binding sites that potentially govern the Cu/ZnSOD expression. These findings conjointly contribute to expand our understanding regarding the piscine Cu/ZnSODs and; in particular, the AO enzyme network of rock bream.

Inhibition of photosynthetic CO2 fixation in the coral Pocillopora damicornis and its relationship to thermal bleaching.

Two inhibitors of the Calvin-Benson cycle [glycolaldehyde (GA) and potassium cyanide (KCN)] were used in cultured Symbiodinium cells and in nubbins of the coral Pocillopora damicornis to test the hypothesis that inhibition of the Calvin-Benson cycle triggers coral bleaching. Inhibitor concentration range-finding trials aimed to determine the appropriate concentration to generate inhibition of the Calvin-Benson cycle, but avoid other metabolic impacts to the symbiont and the animal host. Both 3 mmol l(-1) GA and 20 µmol l(-1) KCN caused minimal inhibition of host respiration, but did induce photosynthetic impairment, measured by a loss of photosystem II function and oxygen production. GA did not affect the severity of bleaching, nor induce bleaching in the absence of thermal stress, suggesting inhibition of the Calvin-Benson cycle by GA does not initiate bleaching in P. damicornis. In contrast, KCN did activate a bleaching response through symbiont expulsion, which occurred in the presence and absence of thermal stress. While KCN is an inhibitor of the Calvin-Benson cycle, it also promotes reactive oxygen species formation, and it is likely that this was the principal agent in the coral bleaching process. These findings do not support the hypothesis that temperature-induced inhibition of the Calvin-Benson cycle alone induces coral bleaching.

Toxicokinetic aspects of thiocyanate after oral exposure to cyanide in female Wistar rats in different physiological states.

Cyanide (CN) is an ion that has been well studied in toxicology and has been associated with several intoxication episodes: the ingestion of contaminated foods and water, chemical war, suicides, homicides, occupational exposures and the use of certain medicines. The aim of the present study was to determine the toxicokinetic parameters of thiocyante (SCN), the main metabolite of CN, after oral administration of potassium cyanide (KCN) to female rats at diestrus, gestational and lactational periods. Female Wistar rats were divided into three equal groups: virgins in the diestrus phase of the estrus cycle, females at the 14th day of gestation and females at the 14th day of lactation. Each group of rats received 3.0 mg of potassium cyanide per kilogram (KCN/kg body weight) by gavage, and blood was collected at several time points. We also collected amniotic fluid from pregnant rats and milk from the nursing rats to analyze thiocyanate concentration. The results showed that SCN levels were significantly increased in serum, milk and amniotic fluid after administration of KCN. In conclusion, the results of the present study evidence that the metabolism of CN varies greatly considering the physiologic state of the female rat, being females at estrus probably more exposed by these substances than at gestation and lactation because in these states there are other compartments, fetus and milk, which may capture these substances, as demonstrated by the V(d) values.

The putative thiosulfate sulfurtransferases PspE and GlpE contribute to virulence of Salmonella Typhimurium in the mouse model of systemic disease.

The phage-shock protein PspE and GlpE of the glycerol 3-phosphate regulon of Salmonella enterica serovar Typhimurium are predicted to belong to the class of thiosulfate sulfurtransferases, enzymes that traffic sulfur between molecules. In the present study we demonstrated that the two genes contribute to S. Typhimurium virulence, as a glpE and pspE double deletion strain showed significantly decreased virulence in a mouse model of systemic infection. However, challenge of cultured epithelial cells and macrophages did not reveal any virulence-associated phenotypes. We hypothesized that their contribution to virulence could be in sulfur metabolism or by contributing to resistance to nitric oxide, oxidative stress, or cyanide detoxification. In vitro studies demonstrated that glpE but not pspE was important for resistance to H2O2. Since the double mutant, which was the one affected in virulence, was not affected in this assay, we concluded that resistance to oxidative stress and the virulence phenotype was most likely not linked. The two genes did not contribute to nitric oxid stress, to synthesis of essential sulfur containing amino acids, nor to detoxification of cyanide. Currently, the precise mechanism by which they contribute to virulence remains elusive.

Aerobic degradation of phenolics and aromatic hydrocarbons in presence of cyanide.

Present study focused on the degradation of a mixture of phenol, cresol, xylenol, quinoline, and indole along with cyanide, commonly found in coke oven wastewater, using aerobic mixed culture. It was found that xylenol and indole were difficult to degrade, when the concentrations were above 250 mg/L. It was observed that free cyanide (2.5mg/L and above) has the potency to holdup the oxidation of organics (250 mg/L) until the cyanide concentration drops to a minimum level. Final TOC in the mixed pollutant system was less than 4 mg/L, indicating the absence of other organic byproducts. Experimental results highlight effect of free cyanide on removal of organics and the combined toxic influence of cyanide and organics on the microbes treating coking wastewater. The proposed mathematical model was able to predict the biodegradation of mixed pollutant system satisfactorily.

Rhodanese activity in different tissues of the ostrich.

1. The purpose of this investigation was to determine the activity, and compare the pattern of distribution, of rhodanese (thiosulphate: cyanide sulphurtransferase, EC. 2.8.1.1) in different tissues of male and female ostriches. 2. Tissue samples from male and female Blue Neck ostriches were assayed for rhodanese activity by the determination of thiocyanate formed by the action of the enzyme on thiosulphate and KCN. 3. Rhodanese was present in all tissues, and the highest activity was observed in the kidney and liver. Other tissues which contained significant activities of rhodanese were the duodenum, pancreas, heart, caecum and rectum. 4. Unlike other birds, the proventiculus does not appear to have an important role in cyanide detoxification in ostrich and, like mammals, the kidney and liver perform this function. 5. The results suggest that the main organs harbouring high rhodanese activity in the ostrich are associated with sites likely to be required in rhodanese mediated cyanide detoxification.

On the role of ß-cyanoalanine synthase (CAS) in metabolism of free cyanide and ferri-cyanide by rice seedlings.

A study was conducted to investigate the contribution of ß-cyanoalanine synthase (CAS) to the botanical metabolism of free cyanide and iron cyanides. Seedlings of rice (Oryza sativa L. cv. XZX 45) were grown hydroponically and then amended with free cyanide (KCN) or ferri-cyanide [K(3)Fe(CN)(6)] into the growth media. Total cyanide, free cyanide, and Fe(3+)/Fe(2+) in aqueous solution were analyzed to identify the speciation of K(3)Fe(CN)(6). Activity of CAS in different parts of the rice seedlings was also assayed in vivo and results indicated that dissociation of K(3)Fe(CN)(6) to free cyanide in solution was negligible. Almost all of the applied KCN was removed by rice seedlings and the metabolic rates were concentration dependent. Phyto-transport of K(3)Fe(CN)(6) was apparent, but appreciable amounts of cyanide were recovered in plant tissues. The metabolic rates of K(3)Fe(CN)(6) were also positively correlated to the concentrations supplied. Rice seedlings exposed to KCN showed a considerable increase in the CAS activity and roots had higher CAS activity than shoots, indicating that CAS plays an important role in the botanical assimilation of KCN. However, no measurable change of CAS activity in different parts of rice seedlings exposed to K(3)Fe(CN)(6) was detected, suggesting that K(3)Fe(CN)(6) is likely metabolized by rice directly through an unknown pathway rather than the ß-cyanoalanine pathway.

A mitochondrial membrane exopolyphosphatase is modulated by, and plays a role in, the energy metabolism of hard tick Rhipicephalus (Boophilus) microplus embryos.

The physiological roles of polyphosphates (polyP) recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX) and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (P(i)) and this activation was much greater using polyP(3) than polyP(15). After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP(3) was 10 times stronger than that for polyP(15). Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 µg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5'-triphosphate synthesis in hard tick embryos.

Characterization of cyanide-trapped methylated metabonates formed during reactive drug metabolite screening in vitro.

Reactive metabolites are estimated to be one of the main reasons behind unexpected drug-induced toxicity, by binding covalently to cell proteins or DNA. Due to their high reactivity and short lifespan, reactive metabolites are analyzed after chemical trapping with nucleophilic agents such as glutathione or cyanide. Recently, unexplained and uncharacterized methylated reaction products were reported in a human liver microsome based reactive metabolite trapping assay utilizing potassium cyanide as a trapping agent. Here, a similar assay was utilized to produce mono- or dimethylated and further cyanide-trapped reaction products from propranolol, amlodipine and ciprofloxacin, followed by ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS) and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) experiments for their more detailed structural elucidation. Formation of all observed cyanide-trapped products was clearly NADPH-dependent and thus metabolism-mediated. The suggested reaction pathways included N-methylation leading to iminium formation in primary and/or secondary amines preceded by cytochrome P450 (CYP)-mediated reactions. As the methylation reaction was suggested to be involved in formation of the actual reactive iminium ion, the observed cyanide-trapped products were experimental artifacts rather than trapped reactive metabolites. The results stress that to avoid overestimating the formation of reactive metabolites in vitro, this methylation phenomenon should be taken into account when interpreting the results of cyanide-utilizing reactive metabolite trapping assays. This in turn emphasizes the importance of identification of the observed cyano conjugates during such studies. Yet, metabolite identification has a high importance to avoid overestimation of in vitro metabolic clearance in the cases where this kind of metabonate formation has a high impact in the disappearance rate of the compound.

The cost and risk of using sodium nitroprusside as a NO donor in chlorophyll fluorescence experiments.

Sodium nitroprusside (SNP) is a widely used nitric oxide (NO) donor chemical, although it has been reported to release cyanide as well as NO during its photolysis. The aim of this work was to examine this potential side effect of SNP. Chlorophyll fluorescence experiments with pea leaves showed that SNP modifies photosynthetic electron transport. The SNP-induced changes were only partially restored in the presence of a NO-specific scavenger. Moreover, a stoichiometric KCN treatment mimicked the outcome caused by the joint application of the NO donor and NO scavenger. These results confirm the cyanide release of SNP and show that both of its photolytic products reduce the photochemical activity of photosystem II in vivo.

Palmitate-induced NO production has a dual action to reduce cell death through NO and accentuate cell death through peroxynitrite formation.

The objective of this study was to determine the role of palmitate-induced stimulation of nitric oxide synthase (NOS) on palmitate-induced cell death, specifically distinguishing the effects of the subtype NOS2 from NOS3, defining the effect of NO on mitochondria death pathways, and determining whether palmitate induces peroxynitrite formation which may impact cardiomyocyte cell survival. Cardiomyocytes from embryonic chick hearts were treated with palmitate 300-500 microM. Cell death was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The ability of palmitate to induce NO production and its consequences were tested by using the NOS inhibitor 7-nitroindazole (7-N) and the peroxynitrite scavenger (5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) chloride) (FeTPPS). The effect of palmitate on the mitochondria was assessed by Western blotting for cytochrome c release into the cytosol, and assessment of mitochondrial transmembrane potential (DeltaPsi(m)) by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide staining and immunocytochemistry. The NOS inhibitor 7-N, which is selective for NOS2 and not for NOS3, significantly (p<0.05) increased palmitate-induced cell death. In contrast, 7-N did not alter cell death produced by the combination of potassium cyanide and deoxyglucose, which, respectively, inhibit glycolysis and oxidative phosphorylation. The mitochondrial actions of palmitate, specifically palmitate-induced translocation of mitochondrial cytochrome c to cytosol and loss of mitochondrial transmembrane potential, were not altered by pretreatment with 7-N. FeTPPS, which isomerizes peroxynitrite to nitrate and thereby reduces the toxic effects of peroxynitrite, produced a significant reduction in palmitate-induced cell death. In summary, these data suggest that palmitate stimulates NO production, which has a dual action to protect against cell death or to induce cell death. Palmitate-induced cell death is mediated, in part, through NO generation, which leads to peroxynitrite formation. The protective effect of NO is operative through stimulation of NOS2 but not NOS3. The actions of NO on palmitate-induced cell death are independent of mitochondrial cell death pathways.

Application of immobilized cells to the treatment of cyanide wastewater.

Cyanide is highly toxic to living organisms, particularly in inactivating the respiration system by tightly binding to terminal oxidase. To protect the environment and water bodies, wastewater containing cyanide must be treated before discharging into the environment. Biological treatment is a cost-effective and environmentally acceptable method for cyanide removal compared with the other techniques currently in use. Klebsiella oxytoca (K. oxytoca), isolated from cyanide-containing industrial wastewater, has been shown to be able to biodegrade cyanide to non-toxic end products. The technology of immobilized cells can be applied in biological treatment to enhance the efficiency and effectiveness of biodegradation. In this study, potassium cyanide (KCN) was used as the target compound and both alginate (AL) and cellulose triacetate (CTA) techniques were applied for the preparation of immobilized cells. Results from this study show that KCN can be utilized as the sole nitrogen source by K. oxytoca. The free suspension systems reveal that the cell viability was highly affected by initial KCN concentration, pH, and temperature. Results show that immobilized cell systems could tolerate a higher level of KCN concentration and wider ranges of pH and temperature, especially in the system with CTA gel beads. Results show that a longer incubation period was required for KCN degradation using immobilized cells compared to the free suspended systems. This might be due to internal mass transfer limitations. Results also indicate that immobilized systems can support a higher biomass concentration. Complete KCN degradation was observed after the operation of four consecutive degradation experiments with the same batch of immobilized cells. This suggests that the activity of the immobilized cells can be maintained and KCN can be used as the nitrogen source throughout KCN degradation experiments. Results reveal that the application of immobilized cells of K. oxytoca is advantageous to the maintenance of KCN degradation efficiency. Thus, it is conceivable that the immobilized cells of K. oxytoca would be applicable to the treatment of cyanide-containing wastewaters.

Regulated ATP release from astrocytes through lysosome exocytosis.

Release of ATP from astrocytes is required for Ca2+ wave propagation among astrocytes and for feedback modulation of synaptic functions. However, the mechanism of ATP release and the source of ATP in astrocytes are still not known. Here we show that incubation of astrocytes with FM dyes leads to selective labelling of lysosomes. Time-lapse confocal imaging of FM dye-labelled fluorescent puncta, together with extracellular quenching and total-internal-reflection fluorescence microscopy (TIRFM), demonstrated directly that extracellular ATP or glutamate induced partial exocytosis of lysosomes, whereas an ischaemic insult with potassium cyanide induced both partial and full exocytosis of these organelles. We found that lysosomes contain abundant ATP, which could be released in a stimulus-dependent manner. Selective lysis of lysosomes abolished both ATP release and Ca2+ wave propagation among astrocytes, implicating physiological and pathological functions of regulated lysosome exocytosis in these cells.

[Adaptation of aerobic methylobacteria to dichloromethane degradation].

A shortening of the lag phase in dichloromethane (DCM) consumption was observed in the methylobacteria Methylopila helvetica DM6 and Albibacter methylovorans DM10 after prior growth on methanol with the presence of 1.5% NaCI. Neither heat nor acid stress accelerated methylobacterium adaptation to DCM consumption. Sodium azide (1 mM) and potassium cyanide (1 mM) inhibited consumption of DCM by these degraders but not by transconjugants Methylobacterium extorquens AM1, expressing DCM dehalogenase but unable to grow on DCM. This indicates that the degrader strains possess energy-dependent systems of transport of DCM or chloride anions produced during DCM dehalogenation. Inducible proteins were found in the membrane fraction of A. methylovorans DM10 cells adapted to DCM and elevated NaCl concentration.

The impact of bioaugmentation on metal cyanide degradation and soil bacteria community structure.

Metal cyanides are significant contaminants of many soils found at the site of former industrial activity. In this study we isolated bacteria capable of degrading ferric ferrocyanide and K2Ni(CN)4. One of these bacteria a Rhodococcus spp. was subsequently used to bioaugment a minimal medium broth, spiked with K2Ni(CN)4, containing 1 g of either an uncontaminated topsoil or a former coke works site soil. Degradation of the K2Ni(CN)4 was observed in both soils, however, bioaugmentation did not significantly impact the rate or degree of K2Ni(CN)4 removal. Statistical analysis of denaturing gradient gel electrophoresis profiles showed that the topsoil bacterial community had a higher biodiversity, and its structure was not significantly affected by either K2Ni(CN)4 or bioaugmentation. In contrast, profiles from the coke works site indicated significant changes in the bacterial community in response to these additions. Moreover, in both soils although bioaugmentation did not affect rates of biodegradation the Rhodococcus spp. did become established in the communities in broths containing both top and coke works soil. We conclude that bacterial communities from contaminated soils with low biodiversity are much more readily perturbed through interventions such as contamination events or bioaugmentation treatments and discuss the implications of these findings for bioremediation studies.

Nitric oxide reduces seed dormancy in Arabidopsis.

Dormancy is a property of many mature seeds, and experimentation over the past century has identified numerous chemical treatments that will reduce seed dormancy. Nitrogen-containing compounds including nitrate, nitrite, and cyanide break seed dormancy in a range of species. Experiments are described here that were carried out to further our understanding of the mechanism whereby these and other compounds, such as the nitric oxide (NO) donor sodium nitroprusside (SNP), bring about a reduction in seed dormancy of Arabidopsis thaliana. A simple method was devised for applying the products of SNP photolysis through the gas phase. Using this approach it was shown that SNP, as well as potassium ferricyanide (Fe(III)CN) and potassium ferrocyanide (Fe(II)CN), reduced dormancy of Arabidopsis seeds by generating cyanide (CN). The effects of potassium cyanide (KCN) on dormant seeds were tested and it was confirmed that cyanide vapours were sufficient to break Arabidopsis seed dormancy. Nitrate and nitrite also reduced Arabidopsis seed dormancy and resulted in substantial rates of germination. The effects of CN, nitrite, and nitrate on dormancy were prevented by the NO scavenger c-PTIO. It was confirmed that NO plays a role in reducing seed dormancy by using purified NO gas, and a model to explain how nitrogen-containing compounds may break dormancy in Arabidopsis is presented.