Release Of Hydrogen Cyanide Research Articles

Trade‐offs between direct and indirect defences of lima bean (Phaseolus lunatus)

Summary Plant defence theory predicts trade‐offs among defence traits as a result of resource limitation or pleiotropic effects. Although theoretically widely accepted, empirical demonstrations of such trade‐offs are surprisingly scarce and mechanistic explanations are usually lacking. We quantified cyanogenesis (the release of hydrogen cyanide (HCN)) as a direct defence and the emission of volatile organic compounds (VOCs) as an indirect defence against herbivores. To elucidate whether the trade‐offs occur at the genetic or phenotypic level we investigated cultivated and wild‐type accessions of lima bean (Fabaceae: Phaseolus lunatus L.) and compared different leaf developmental stages. Genetic relationships among the accessions were studied using amplified fragment length polymorphism (AFLP) analysis. Cyanogenesis and the release of VOCs differed significantly among the accessions and were negatively correlated: high cyanogenic accessions released low amounts of VOCs and vice versa. The same remained true for the ontogenetic stages, since primary leaves of all accessions hardly ever produced HCN at all, yet regularly showed high release rates of VOCs. Low and high cyanogenic accessions of lima bean formed distinct clades in an AFLP‐based dendrogram, while wild‐types and cultivars did not separate. The first pattern indicates that the underlying defensive syndromes are genetically conserved, while the latter is likely to be caused by a multiple origin of cultivated lima beans or an extensive gene flow among cultivated and wild plants. Synthesis. Trade‐offs between cynogenesis and VOC release were obvious both between accessions and at the ontogenetic level, and thus cannot be explained by pleiotropy. We contend that allocation restrictions and/or adaptations to different enemy pressures are most likely to explain why lima bean can invest into cyanogenesis or VOCs, but not both.

Hydroxynitrile glucosides

β- and γ-Hydroxynitrile glucosides are structurally related to cyanogenic glucosides (α-hydroxynitrile glucosides) but do not give rise to hydrogen cyanide release upon hydrolysis. Structural similarities and frequent co-occurrence suggest that the biosynthetic pathways for these compounds share common features. Based on available literature data we propose that oximes produced by CYP79 orthologs are common intermediates and that their conversion into β- and γ-hydroxynitrile glucosides is mediated by evolutionary diversified multifunctional orthologs to CYP71E1. We designate these as CYP71 βγ and CYP71 αβγ; in combination with the classical CYP71 α (CYP71E1 and orthologs) these are able to hydroxylate any of the carbon atoms present in the amino acid and oxime derived nitriles. Subsequent dehydration reactions and hydroxylations and a final glycosylation step afford the unsaturated β- and γ-hydroxynitrile glucosides. This scheme would explain the distribution patterns of α-, β- and γ-hydroxynitrile glucosides found in plants. The possible biological functions of these hydroxynitriles are discussed.

RESOURCE AVAILABILITY AND THE ABUNDANCE OF AN N-BASED DEFENSE IN AUSTRALIAN TROPICAL RAIN FORESTS

Plant defense theories predict that relatively resource-rich environments (those with more fertile soil) will support a greater abundance of plants with nitrogen-based chemical defense, but this has yet to be adequately tested. We tested this prediction by measuring the diversity and contribution to total biomass of cyanogenic plants (those that release hydrogen cyanide from endogenous cyanide-containing compounds) in the Australian tropical rain forest. We examined 401 species in thirty 200-m2 plots, six at each of five sites, for cyanogenesis. In upland/highland rain forest, two pairs of sites similar in rainfall and altitude, but differing in soil nutrients, were selected, as well as one site in lowland rain forest. Sites differed markedly in species composition and foliar N was positively related to soil fertility. Holding altitude constant, we did not detect significant differences in the proportion of cyanogenic species with soil fertility, nor did we consistently detect significant increases in the contribution of cyanogenic species to total biomass on higher nutrient sites. Thus we found no clear evidence that soil fertility affects the distribution and prevalence of species investing in a constitutive N-based defense at the community level.

Cyanogenesis in plants and arthropods

Cyanogenic glucosides are phytoanticipins known to be present in more than 2500 plant species. They are regarded as having an important role in plant defense against herbivores due to bitter taste and release of toxic hydrogen cyanide upon tissue disruption, but recent investigations demonstrate additional roles as storage compounds of reduced nitrogen and sugar that may be mobilized when demanded for use in primary metabolism. Some specialized herbivores, especially insects, preferentially feed on cyanogenic plants. Such herbivores have acquired the ability to metabolize cyanogenic glucosides or to sequester them for use in their own defense against predators. A few species of arthropods (within diplopods, chilopods and insects) are able to de novo biosynthesize cyanogenic glucosides and some are able to sequester cyanogenic glucosides from their food plant as well. This applies to larvae of Zygaena (Zygaenidae). The ratio and content of cyanogenic glucosides is tightly regulated in Zygaena filipendulae, and these compounds play several important roles in addition to defense in the life cycle of Zygaena. The transfer of a nuptial gift of cyanogenic glucosides during mating of Zygaena has been demonstrated as well as the involvement of hydrogen cyanide in male attraction and nitrogen metabolism. As more plant and arthropod species are examined, it is likely that cyanogenic glucosides are found to be more widespread than formerly thought and that cyanogenic glucosides are intricately involved in many key processes in the life cycle of plants and arthropods.

Evolution of heteromeric nitrilase complexes in Poaceae with new functions in nitrile metabolism

Members of the nitrilase 4 (NIT4) family of higher plants catalyze the conversion of beta-cyanoalanine to aspartic acid and asparagine, a key step in cyanide detoxification. Grasses (Poaceae) possess two different NIT4 homologs (NIT4A and NIT4B), but none of the recombinant Poaceae enzymes analyzed showed activity with beta-cyanoalanine, whereas protein extracts of the same plants clearly posses this activity. Sorghum bicolor contains three NIT4 isoforms SbNIT4A, SbNIT4B1, and SbNIT4B2. Individually, each isoform does not possess enzymatic activity whereas the heteromeric complexes SbNIT4A/B1 and SbNIT4A/B2 hydrolyze beta-cyanoalanine with high activity. In addition, the SbNIT4A/B2 complex accepts additional substrates, the best being 4-hydroxyphenylacetonitrile. Corresponding NIT4A and NIT4B isoforms from other Poaceae species can functionally complement the sorghum isoforms in these complexes. Site-specific mutagenesis of the active site cysteine residue demonstrates that hydrolysis of beta-cyanoalanine is catalyzed by the NIT4A isoform in both complexes whereas hydrolysis of 4-hydroxyphenylacetonitrile occurs at the NIT4B2 isoform. 4-Hydroxyphenylacetonitrile was shown to be an in vitro breakdown product of the cyanogenic glycoside dhurrin, a main constituent in S. bicolor. The results indicate that the SbNIT4A/B2 heterocomplex plays a key role in an endogenous turnover of dhurrin proceeding via 4-hydroxyphenylacetonitrile and thereby avoiding release of toxic hydrogen cyanide. The operation of this pathway would enable plants to use cyanogenic glycosides as transportable and remobilizable nitrogenous storage compounds. Through combinatorial biochemistry and neofunctionalizations, the small family of nitrilases has gained diverse biological functions in nitrile metabolism.

Quantitative Effects of Cyanogenesis on an Adapted Herbivore

Plant cyanogenesis means the release of gaseous hydrogen cyanide (HCN) in response to cell damage and is considered as an effective defense against generalist herbivores. In contrast, specialists are generally believed not to be affected negatively by this trait. However, quantitative data on long-term effects of cyanogenesis on specialists are rare. In this study, we used lima bean accessions (Fabaceae: Phaseolus lunatus L.) with high quantitative variability of cyanogenic features comprising cyanogenic potential (HCNp; concentration of cyanogenic precursors) and cyanogenic capacities (HCNc; release of gaseous HCN per unit time). In feeding trials, we analyzed performance of herbivorous Mexican bean beetle (Coleoptera: Coccinellidae: Epilachna varivestis Mulsant) on selected lines characterized by high (HC-plants) and low HCNp (LC-plants). Larval and adult stages of this herbivore feed on a narrow range of legumes and prefer cyanogenic lima bean as host plant. Nevertheless, we found that performance of beetles (larval weight gain per time and body mass of adult beetles) was significantly affected by lima bean HCNp: Body weight decreased and developmental period of larvae and pupae increased on HC-plants during the first generation of beetles and then remained constant for four consecutive generations. In addition, we found continuously decreasing numbers of eggs and larval hatching as inter-generational effects on HC-plants. In contrast to HC-plants, constantly high performance was observed among four generations on LC-plants. Our results demonstrate that Mexican bean beetle, although preferentially feeding on lima bean, is quantitatively affected by the HCNp of its host plant. Effects can only be detected when considering more than one generation. Thus, cyanide-containing precursors can have negative effects even on herbivores adapted to feed on cyanogenic plants.

Molecular evolution of the Li/li chemical defence polymorphism in white clover (Trifolium repens L.)

White clover (Trifolium repens) is naturally polymorphic for cyanogenesis (hydrogen cyanide release following tissue damage). The ecological factors favouring cyanogenic and acyanogenic plants have been examined in numerous studies over the last half century, making this one of the best-documented examples of an adaptive polymorphism in plants. White clover cyanogenesis is controlled by two, independently segregating Mendelian genes: Ac/ac controls the presence/absence of cyanogenic glucosides; and Li/li controls the presence/absence of their hydrolysing enzyme, linamarase. In this study, we examine the molecular evolution and population genetics of Li as it relates to the cyanogenesis polymorphism. We report here that Li exists as a single-copy gene in plants possessing linamarase activity, and that the absence of enzyme activity in li/li plants is correlated with the absence of much or all of the gene from the white clover genome. Consistent with this finding, we confirm by reverse transcription-polymerase chain reaction that Li gene expression is absent in plants lacking enzyme activity. In a molecular population genetic analysis of Li and three unlinked genes using a worldwide sample of clover plants, we find an absence of nucleotide variation and statistically significant deviations from neutrality at Li; these findings are consistent with recent positive directional selection at this cyanogenesis locus.

Ligand displacement, headspace single-drop microextraction, and capillary electrophoresis for the determination of weak acid dissociable cyanide

A new method involving ligand displacement, headspace single-drop microextraction (SDME) with in-drop derivatization and capillary electrophoresis (CE) was developed for the determination of weak acid dissociable (WAD) cyanide. WAD metal–cyanide complexes (Ag(CN) 2 −, Cd(CN) 4 2−, Cu(CN) 3 2−, Hg(CN) 2, Hg(CN) 4 2−, Ni(CN) 4 2− and Zn(CN) 4 2−) are decomposed with ligand-displacing reagent and the released hydrogen cyanide is extracted from neutral solution (pH 6.5) with an aqueous microdrop (5 μl) containing Ni(II)–NH 3 as derivatization agent. The hydrogen cyanide extracted reacts with Ni 2+ to form a stable and highly UV absorbing tetracyanonickelate anion which is then determined by CE. Among the three different ligand-displacing reagents (i.e., ethylenediamine, dithizone and polyethileneimine) studied none of the reagents used alone releases cyanide completely from all WAD cyanide complexes. Complete recoveries were obtained by the extraction of WAD cyanide from 0.4 mol l −1 ethylenediamine chloride buffer (pH 6.5) containing 0.001% (wt) dithizone. Proposed system was applied to determine WAD cyanide in industrial wastewater and river waters samples with spiked recoveries in the range of 95.8–104.7%.

Study of an Enzyme Activity in Extracts of Ginkgo biloba Leaves

Biocatalyst is a strong tool for preparation of enantiopure compounds through kinetic resolution or direct synthesis. However, enzymes do not always satisfy the needs of researchers for this purpose because they often do not have enough activity or selectivity toward unnatural substrates. To solve the problems, it is important to find new enzymes from nature, although researchers have tried to change enzyme activity or selectivity by mutagenesis such as random mutagenesis and rational design. Researchers have isolated a variety of enzymes from various sources such as bacteria, fungi, mammals, and plants. Diabetic enzymes such as lipases and proteases from bacteria and mammals are well characterized and utilized successfully in academic and industrial areas. The enzymes involved in mechanisms for protecting plants from insect or fungal attack are also useful in organic synthesis. Such enzymes generally catalyze reactions to produce toxic chemicals in the last step of secondary metabolism to kill predators. As an example, hydroxynitrilases from cyanogenic plants catalyze the biodegradation of cyanogenic glycosides to release hydrogen cyanide. The reverse reaction is interesting in organic synthesis because hydroxynitrilases catalyze formation of enantiopure cyanohydrins from aldehydes or ketones and hydrogen cyanide. Searching enzymes involved in the secondary metabolism of plants may provide useful biocatalysts in organic synthesis because the enzymes have a key role for synthesis of many biologically active compounds. Ginkgo biloba leaves have high anti-insect activity and biological activity. Thus, they might contain an interesting enzyme involved in the secondary metabolism and the enzyme can be useful for organic synthesis. We have chosen Ginkgo biloba leaves for finding a new enzyme because Ginkgo biloba leaves potentially contain enzymes involved in the secondary metabolism and also the study of the enzymatic activity of extracts from Ginkgo leaves has not been focused while the chemicals in them have been well characterized. Herein, we represent an enzyme activity in Ginkgo biloba leaves. Most enzymes are classified into six groups, such as oxidoreductase, transferases, hydrolases, lyases, isomerases, and ligases. Activities for oxidoreductase, hydrolase, and lyase were chosen for screening enzyme activities in Ginkgo biloba leaves because these enzymes are important in organic synthesis. We assayed the activities of peroxidase for oxidoreductase, β-amylase, nitrilase, and esterase for hydrolase, and hydroxynitrilase for lyase. Ginkgo biloba leaves were ground by a homogenizer and washed with ethyl acetate to remove organic components. Ginkgo powders were extracted with the reaction buffers for activity screening. Under the assay condition tested, Ginkgo extracts showed only the peroxidase activity (Table 1). In the assay condition, peroxidases oxidize pyrogallol to purpurogallin using hydrogenperoxide (eq. 1). The absorbance change can be monitored at 420 nm. The reaction coordinate showed clear difference between the reaction by Ginkgo extracts and the blank reaction (Figure 1).

Hydrogen cyanide release during feeding of generalist and specialist lepidopteran larvae on a cyanogenic plant, Passiflora capsularis

Abstract The hydrogen cyanide‐based interaction of a strongly cyanogenic plant, Passiflora capsularis, and larvae of two insect herbivores, a generalist (Spodoptera frugiperda) and a specialist (Heliconius erato), is examined in terms of the combined kinetics of the feeding process and the simultaneous hydrogen cyanide (HCN) liberation, as compared with the natural kinetics of hydrogen cyanide evolution by plant‐leaf tissue. There are marked differences in acceptance of P. capsularis by third‐instar larvae of specialist and generalist species. The former, H. erato, display a parsimonious ingestion rate of 0.74 ± 0.15 mg (fresh weight) min−1 comprising 18% active feeding time, whereas S. frugiperda larvae show a more erratic and restrained feeding involving 4% of the time at 0.45 ± 0.14 mg min−1. These S. frugiperda larvae ingest 124.4 ± 8.3 mg (fw) of the non‐cyanogeneic Spinacia oleracea leaves in 24 h compared with only 74.7 ± 20.1 mg of P. capsularis in the same period. The total hydrogen cyanide released naturally from wild specimens of P. capsularis plants is in the range 326–3901 μg g−1. Hydrogen cyanide evolution from macerated P. capsularis leaves takes place along a hyperbolic function with time and initial velocities of cyanide evolution are a linear function of total hydrogen cyanide. When feeding on P. capsularis leaves, H. erato releases only a minor fraction relative to total hydrogen cyanide (0.09%) and to the anticipated cyanide from the initial velocity (7%). By contrast, S. frugiperda evokes 5.8‐fold more than the anticipated hydrogen cyanide release from the plant. The findings are interpreted as diverging strategies by generalist and specialist insects in the utilization of hydrogen cyanide in cyanogenic plants.

Oviposition Choice of Mexican Bean Beetle (Epilachna varivestis) Depends on Host Plants Cyanogenic Capacity

The choice of insect females as to where to lay their eggs strongly affects progeny survival and, thus, fitness. We conducted choice experiments with female Mexican bean beetles, which were offered lima bean plants differing in their cyanogenic capacity (HCNc), i.e., in the intensity of hydrogen cyanide release per time unit from damaged plant tissue. Females preferred to lay their eggs on plants with low HCNc. In contrast, the mere concentration of cyanide-containing precursors (the cyanogenic potential, HCNp) did not affect oviposition choices. Plant cyanogenesis occurs after tissue damage, which brings specific beta-glucosidases in contact with their substrates, cyanogenic glycosides, from which they are separated by compartmentation in the intact plant tissue. Plants commonly store toxic compounds in an inactive form. Our results demonstrate that for cyanogenesis, which is widespread in plants, it is the toxic product itself rather than the precursor that affects oviposition choices of a specialized herbivore.

Reconstitution of cyanogenesis in barley (Hordeum vulgare L.) and its implications for resistance against the barley powdery mildew fungus

Barley (Hordeum vulgare L.) produces a leucine-derived cyanogenic beta-D-glucoside, epiheterodendrin that accumulates specifically in leaf epidermis. Barley leaves are not cyanogenic, i.e. they do not possess the ability to release hydrogen cyanide, because they lack a cyanide releasing beta-D-glucosidase. Cyanogenesis was reconstituted in barley leaf epidermal cells through single cell expression of a cDNA encoding dhurrinase-2, a cyanogenic beta-D-glucosidase from sorghum. This resulted in a 35-60% reduction in colonization rate by an obligate parasite Blumeria graminis f. sp. hordei, the causal agent of barley powdery mildew. A database search for barley homologues of dhurrinase-2 identified a (1,4)-beta-D-glucan exohydrolase isozyme betaII that is located in the starchy endosperm of barley grain. The purified barley (1,4)-beta-D-glucan exohydrolase isozyme betaII was found to hydrolyze the cyanogenic beta-D-glucosides, epiheterodendrin and dhurrin. Molecular modelling of its active site based on the crystal structure of linamarase from white clover, demonstrated that the disposition of the catalytic active amino acid residues was structurally conserved. Epiheterodendrin stimulated appressoria and appressorial hook formation of B. graminis in vitro, suggesting that loss of cyanogenesis in barley leaves has enabled the fungus to utilize the presence of epiheterodendrin to facilitate host recognition and to establish infection.

Kinetics of the Natural Evolution of Hydrogen Cyanide in Plants in Neotropical Pteridium arachnoideum and its Ecological Significance

The time-dependent natural release of hydrogen cyanide (HCN) was studied quantitatively using young croziers of the neotropical bracken fern Pteridium arachnoideum. HCN production was quantified in crushed tissue using a flow reactor at 30.0 +/- 0.1 degrees C. Released HCN was carried into appropriate traps with a moist air flow. Aliquots were drawn from the traps at fixed time intervals, and the HCN concentration was evaluated spectroscopically. All available prunasin (Pru), the only cyanogenic glycoside present, underwent decomposition into HCN in less than 1200 min. Fiddleheads (N = 76) contained 1.84-107.70 mg Pru g(1) dw in a continuous fashion suggesting genetic polymorphism. Acyanogenic morphs were rare (1/77). From the kinetics of the samples with Pru content near the median histographic distribution (N = 46), accumulated HCN formation as a function of time, initial velocities, average HCN production rate, and corresponding rate equations were obtained. Initial and average velocities correlated well with total Pru content. The yield of cyanide liberation varied widely between 0.51 and 47.86 microg HCN min(-1) g(-1) dw and was a linear function of [Pru]t. However, the beta-glucosidase enzyme involved in this reaction was not rate limiting and occurs in excess in the natural system. Enzyme activity was found to be independent of [Pru]t. The contribution of HCN as an allomone-upon-request against herbivores was assessed quantitatively. Bracken fiddleheads produced a pulse of HCN soon after tissue injury that waned rapidly, leaving a large portion of intact prunasin to decompose more slowly in the herbivore's lumen. The balance between the external and internal courses was found to depend on the concentration of prunasin in the plant, the amount of crozier eaten, and the time used to consume it.

Influence of two selective factors on cyanogenesis polymorphism ofTrifolium repens L. in Darjeeling Himalaya

Cyanogenesis-the production of toxic hydrogen cyanide (HCN) by damaged tissue-inTrifolium repens L. (white clover), a type of most important pasture legume, has been studied at different elevations of Darjeeling Himalaya (latitude-27° 2′ 57″ N, longitude-88° 15′ 45″ E). Release of HCN takes place due to reaction between cyanogenic glucosides stored in vacuoles of the leaf cell and the corresponding enzyme β-glucosidase present in another compartment, often cell wall. Cyanogenesis, a defense system in plant, protects the clover from herbivore and inhibits grazing. Biochemical analysis showed the presence and absence of the cyanogenesis trait within the population in different proportions at different elevations. Acyanogenic individuals also showed variations with respect to presence or absence of either cyanogenic glucosides or β-glucosidase enzyme or both. The distribution of cyanogenic and acyanogenic plants was found in all places, but at lower altitudes (2084–2094 m) the dominating plants were cyanogenic whereas in higher altitude (2560 m) the dominating plants were acyanogenic. It was observed that blister beetle (Mylabris pustalata Thunb.) and the mollusc (Macrochlamys tusgurium Benson.) were the most common consumer of leaflets ofT. repens. Six categories of damage on white clover leaf by these animals were recorded. Our results suggest that the two selective factors or forces i.e. very cold temperature (harmful to cyanogenic plants) at higher altitude as well as indiscriminate but preferential predation (harmful to acyanogenic plants) interact to affect the system of cyanogenesis and also to cause the stable and protective polymorphism inT. repens rather than genotypic differences present among the plants.

Characterization of a New Pseudomonas aeruginosa Strain NJ-15 as a Potential Biocontrol Agent

Phylogenetic characterization of soil isolate NJ-15, based on sequence homology of a partial 746-bp fragment of 16SrDNA amplicon, with the ribosomal database sequences (http://www.msu.edu/RDP/cgis/phylip.cgi), validated the strain as Pseudomonas aeruginosa. The strain NJ-15 produced a substantial amount of indole acetic acid (IAA) in tryptophan-supplemented medium. Besides, the strain also exhibited significant production of both the siderophore and hydrogen cyanide (HCN) on chrome azurol S and King's B media, respectively. The data revealed lower HCN production under iron-limiting conditions vis-à-vis higher HCN release with iron stimulation. Significant growth inhibition of phytopathogenic fungi occurred in the order as Fusarium oxysporum > Trichoderma herizum > Alternaria alternata > Macrophomina phasiolina upon incubation with strain NJ-15 cells. Thus, the secondary metabolites producing new Pseudomonas aeruginosa strain NJ-15 exhibited innate potential of plant growth promotion and biocontrol activities in vitro.

Rapid quantitation of cyanide in whole blood by automated headspace gas chromatography

Cyanide (CN), a chemical asphyxiant, is a rapidly acting and powerful poison. We have developed a sensitive, rapid, simple, and fully automated method for measuring CN in whole blood. The assay is based on the use of gas chromatography (GC) with nitrogen–phosphorus detection and acetonitrile as an internal reference. Following the automated addition of phosphoric acid to the blood sample, the released hydrogen cyanide is analyzed using a fully automated headspace GC system. The assay, validated on human blood samples spiked with potassium cyanide and on clinical samples from fire victims who had smoke inhalation injury, can detect CN at a wide range of concentrations (30–6000 μg/l) in about 17 min (including incubation and GC run time, and <2 min for manual sample preparation). This automated, high-throughput, simple, and sensitive method is suitable for the rapid diagnosis of CN in clinical and forensic specimens.

Determination of cyanide by a pervaporation–UV photodissociation–potentiometric detection approach

An automated system for the determination of total cyanide in water samples was developed. Stable metal–cyanide complexes such as Fe(CN)63− are photodissociated in an acidic medium with a UV lamp. The released hydrogen cyanide (HCN) is pervaporated and collected in dilute NaOH solution acting as an acceptor. The cyanide ion is isolated from interferents such as sulfide and determined potentiometrically using an ion-selective electrode. The content of cyanide in three wastewater samples obtained with the new method was compared with the results provided by the EPA method OIA-1677 for the determination of total cyanide in water samples. Excellent agreement was obtained in all instances. The method can be applied over a wide range of concentrations (0.4–200 μg l−1 CN− and 0.05–50 mg l−1 CN− using injection volumes of 200 and 50 μl, respectively). The sample throughput of the system is 40 h−1 and the detection limit for total cyanide is 0.01 μg l−1.

Toxicity of Fire Smoke

This review is an attempt to present and describe the major immediate toxic threats in fire situations. These are carbon monoxide, a multitude of irritating organic chemicals in the smoke, oxygen depletion, and heat. During the past 50 years, synthetic polymers have been introduced in buildings in very large quantities. Many contain nitrogen or halogens, resulting in the release of hydrogen cyanide and inorganic acids in fire smoke as additional toxic threats. An analysis of toxicological findings in fire and nonfire deaths and the results of animal exposures to smoke from a variety of burning materials indicate that carbon monoxide is still likely to be the major toxicant in modern fires. However, the additional toxic threats mentioned above can sometimes be the principal cause of death or their addition can result in much lower than expected carboxyhemoglobin levels in fire victims. This analysis also revealed that hydrogen cyanide is likely to be present in appreciable amounts in the blood of fire victims in modern fires. The mechanisms of action of acute carbon monoxide and hydrogen cyanide poisonings are reviewed, with cases presented to illustrate how each chemical can be a major contributor or how they may interact. Also, lethal levels of carboxyhemoglobin and cyanide in blood are suggested from an analysis of the results of a large number of fire victims from different fire scenarios. The contribution of oxygen depletion and heat stress are more difficult to establish. From the analysis of several fire scenarios, they may play a major role in the room of origin at the beginning of a fire. The results in animal studies indicate that when major oxygen depletion (<10%) is added to lethal or sublethal levels of carbon monoxide or hydrogen cyanide its major role is to substantially reduce the time to death. In these experiments the carboxyhemoglobin level at death was slightly reduced from the expected level with exposure to carbon monoxide alone. However, blood cyanide was reduced by a factor of ten from the expected level with exposure to hydrogen cyanide alone. This is another factor (among many other presented) complicating the task of establishing the contribution of cyanide in the death of fire victims, from its analysis in their blood. Finally the role of ethanol intoxication, as it may influence carboxyhemoglobin levels at death, is reviewed. Its role is minor, if any, but the data available on ethanol in brain tissue and blood of fire victims confirmed that brain ethanol level is an excellent predictor of blood ethanol.

Temporal and spatial variation in cyanogenic glycosides in Eucalyptus cladocalyx

The release of hydrogen cyanide from endogenous cyanide-containing compounds in plants is an effective herbivore deterrent. We investigated temporal and spatial variations in cyanogenic glycoside concentration in greenhouse-grown seedlings and 6-year-old plantation trees of Eucalyptus cladocalyx F. Muell., which allocates up to 20% of leaf nitrogen to the cyanogenic glycoside, prunasin. The highest cyanogenic glycoside concentrations were in the young, developing vegetative and reproductive tissues. Both the overall cyanogenic glycoside concentration and the proportion of nitrogen allocated to cyanogenic glycoside decreased as tissues matured. Cyanogenic glycoside and nitrogen concentrations were similar at all positions on the leaf blade. There was no change in concentration of cyanogenic glycosides either diurnally or following wounding of the tissue, suggesting that these compounds are constitutive. Cyanogenic glycoside concentration varied seasonally in young leaf tips of field-grown E. cladocalyx, but not in mature, fully expanded leaves. Although some of the changes in cyanogenic glycoside concentration in young leaf tips may have been driven by changes in leaf nitrogen, there was a significant decrease in the proportion of nitrogen allocated to cyanogenic glycosides in young leaves during the summer, coinciding with the peak flowering period. Mobilization of cyanogenic glycosides may have occurred to provide nitrogen for reproduction. Most of the observed temporal and spatial variations in cyanogenic glycosides are consistent with the optimal use of resources, particularly nitrogen.

Spectrophotometer-aided evaluation of cyanogenic potential in white clover (Trifolium repens L.)

In comparison with ordinary methods of colorimetric evaluation of cyanogenic potential based on visual evaluation of the alkaline picrate reaction, a spectrophotometer-aided method could be more accurate (since it determines the exact amount of hydrogen cyanide released by the plant material), and less time-consuming as it can be performed on bulk material rather than on a number of individual plants. Ten white clover populations were evaluated by a spectrophotometer-aided method and by two visual evaluation criteria. All methods indicated the presence of large variation between populations. Visual methods gave almost identical results and allowed only for the distinction between cyanogenic and substantially acyanogenic populations. The results were only moderately consistent with those obtained by the spectrophotometer-aided method, which could detect the presence of variation also between cyanogenic populations. The effects of various incubation times (from 4 to 48 h) and of the addition of β-glucosidase on hydrogen cyanide release were also investigated. Comparable results for ranking of populations could be obtained over a range of incubation times, but at least 24 h were needed for a reliable estimation of the hydrogen cyanide produced by plants. The addition of enzyme did not increase the released cyanide. The effect of season and/or conditions of evaluation was marked on mean cyanogenic potential but limited on ranking of populations. Copyright © 2000 John Wiley & Sons, Ltd.