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Toxic but sublethal oral doses of 125 mg/kg (1.1 mmol/kg) of the cruciferous nitrile, 1-cyano-3,4-epithiobutane (CEB), or 175 mg/kg (2.1 mmol/kg) of its synthetic saturated analogue, n-valeronitrile (VN), were given by gavage to male CDF (F-344/CrlBr) rats once daily for 1, 2 or 3 days, in order to compare target tissues and to observe structure-activity relationships between the nitriles. CEB-induced changes included degeneration and necrosis of the pars recta of the renal proximal tubules, ulceration and necrosis in the forestomach, a mild increase (4.5-fold) in daily urinary thiocyanate (SCN-) excretion (only in rats treated for 3 days) and 1.5- to 2.4-fold increases in hepatic and pancreatic non-protein thiol (RSH) concentrations (in all CEB-treated groups). In VN-treated rats, there were no consistent histological changes but 95- to 170-fold increases in daily urinary SCN- excretion, delayed clinical signs of cyanide toxicity and minimal effects on tissue RSH concentrations. These results indicate different toxic mechanisms for VN and CEB. The nephrotoxic effects of CEB were very similar to those of 1-cyano-2-hydroxy-3,4-epithiobutane, suggesting a role for the epithio group in the nephrotoxicity of these nitriles. The relatively low SCN- excretion in CEB-treated rats also suggested that cyanide played only a minimal role in CEB toxicity, while the high SCN- excretion, clinical signs of cyanide poisoning and lack of histological changes imply a greater role for metabolically-derived cyanide in VN toxicity. The enhancement of tissue RSH by CEB treatment with indications of enhanced tissue glutathione concentrations suggested the involvement of glutathione in the detoxication of CEB and/or its reactive metabolites.
A mechanism-based model for prediction of acute nitrile toxicity was developed using octanol-water partition coefficients (log P) and estimated rates of alpha-hydrogen atom abstraction as variables. Relative rates of hydrogen atom abstraction were derived from differences in heats of formation for ground-state and radical geometries and radical ionization potentials. Calculated energies of activation for all potential sites of oxidation for a given nitrile were used to estimate partitioning of metabolites among multiple oxidative pathways. logP and the resulting corrected rate constants for alpha-carbon oxidation were effective variables in an acute toxicity model of structurally diverse nitriles. The pharmacokinetics of substrate disposition is discussed in the context of multiple metabolic pathways. Structure-toxicity relationships are also discussed.
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The dissociation constants for reversible covalent binding of twelve peptide nitrile inhibitors to the active site of papain have been measured by means of fluorescence titration. The binding constants generally parallel the kinetic specificity constants (kcat/Km) for related papain substrates, supporting earlier suggestions that peptide nitriles behave as transition state analog inhibitors of papain. In ten cases the temperature dependence of binding was analyzed to determine the enthalpic and entropic contributions to the binding energy. A compensation plot of delta H vs. T delta S resulted in two parallel lines, one for 'specific' nitriles (i.e., N-Ac-L-aa-NHCH2CN; aa = Phe, Leu, Met) and the other for 'non-specific' nitriles (e.g., N-Ac-D-Phe-NHCH2CN, PhCH2CH2CONHCH2CN hippurylnitrile, etc.). For both specific and nonspecific nitriles representing an 1800-fold range of Kd values (0.27 microM-490 microM), the solvent deuterium isotope effect on binding (Kd(H2O)/Kd(D2O) = DKd) was very close to 2.0. This isotope effect could be accounted for entirely by the simple protonic change which occurs upon the reversible addition of the active site sulfhydryl of papain to the nitrile group of the peptide derivative to form a covalent thioimidate linkage. In contrast, six closely related non-nitrile ligands containing identical peptide side chains but having C-terminal groups incapable of binding covalently to papain had unmeasureably high dissociation constants. Collectively, these results indicate that strong binding of peptide nitrile substrate analogs to papain requires a combination of (1) hydrophobic interaction (especially at the P2 position), (2) specific intermolecular hydrogen bonding and (3) covalent interaction of the nitrile with the active site sulfhydryl group.
Reports from several laboratories agree that many, but not all, aliphatic nitriles undergo hepatic biotransformation in mice and rats to release free cyanide, but the mechanisms at work in these reactions remain in doubt. We have used primarily n-butyronitrile, propionitrile, and their respective alpha-carbon-hydroxylated homologs, propionaldehyde cyanohydrin and lactonitrile, to examine this question in mice. Pretreatment of mice with the hepatic microsomal enzyme inducers, pregnenolone-16 alpha-carbonitrile, troleandomycin, and isosafrole, or with the cytochrome P-450-depleting agent, cobaltous chloride, did not influence the mortality of mice given single doses of nitriles. Repeated injections of aspirin or sodium salicylate in water failed to protect mice against death by the nitriles. Dimethyl sulfoxide, however, was effective in reducing mortality after nitrile administration. Repeated injections of 4-methylpyrazole or disulfiram protected mice against death after nitriles. Most of the treatment regimens successful against the nitriles also protected against death due to the cyanohydrins. The cyanohydrins were more acutely toxic than their parent nitriles, produced death much more rapidly, and resulted in the same toxic signs, suggesting that they are intermediates in the bioactivation pathway leading to free cyanide. The cyanohydrins appeared to serve as weak substrates for yeast alcohol dehydrogenase, however, incubation of them with either yeast or horse liver alcohol dehydrogenase did not increase the rate of cyanide release over that in incubations where the enzymes were absent. The slow rate of cyanide release due to spontaneous hydrolysis interfered with the determinations of alcohol dehydrogenase activity, but it cannot account for the rapid action and high toxicity of the cyanohydrins in vivo, or for the efficacy of the treatment regimens which protected against death. It appears unlikely that prostaglandin synthetase or alcohol dehydrogenase are importantly involved in nitrile bioactivation. The same active process, however, appears to be responsible both for alpha-carbon hydroxylation and for the subsequent degradation of the resulting cyanohydrins to release free cyanide. It is far more efficient in mediating the latter reaction than the former.
The effect of ethanol on metabolism of 20 nitriles was studied in vivo and in vitro in mice. The hepatic microsomal metabolizing activity for nitriles was at a maximum 13 h after ethanol dosing (4.0 g/kg). Using microsomes from mice pretreated with ethanol under the above conditions, enhancement of the in vitro metabolism of nitriles was 1.00-1.83 compared with the glucose-treated control. When mice were orally given nitriles 13 h after dosing with either ethanol (4.0 g/kg) or glucose (7.0 g/kg), the hepatic metabolizing activity of nitriles for the ethanol-treated group was always higher than that for the glucose group, although no change in the content of hepatic microsomal P-450 was observed between the two groups. However, ethanol added to the incubation mixture inhibited the in vitro metabolism of most nitriles. The results in the present study suggest that ethanol can enhance the acute toxicity of nitriles.
Bacteria were enriched from soil samples with succinate as a carbon source and racemic naproxen nitrile [2-(6-methoxy-2-naphthyl)propionitrile] as sole source of nitrogen. Since naproxen nitrile was only poorly soluble in water media amended with different water-immiscible organic phases were used for the enrichments. With pristane (2,6,10,14-tetramethylpentadecane) as the organic phase two bacterial strains were isolated (strain C3II and strain MP50) which were identified as rhodococci. Cells of both strains converted naproxen nitrile via naproxen amide to naproxen. From racemic naproxen nitrile Rhodococcus sp. C3II formed S-naproxen amide and subsequently S-naproxen. Racemic naproxen amide was hydrolysed to S-naproxen. Rhodococcus sp. MP50 converted racemic naproxen nitrile predominantly to R-naproxen amide and racemic naproxen amide to S-naproxen. With both strains racemic naproxen amide was converted to S-naproxen with an enantiomeric excess > 99% at a conversion rate up to 80% of the theoretical value. In strain C3II the enzymes which hydrolysed naproxen nitrile and naproxen amide were present only at a low constitutive level. In contrast, in Rhodococcus sp. MP50 these activities were induced when grown in the presence of various nitriles.
In order to investigate structure-activity relationships that influence metabolism of nitriles to CN-, thiocyanate was measured, as an index of CN- release, in urine of rats given equimolar doses of nitriles. Significantly more SCN- was excreted after po than after ip administration of saturated (C2-C5) nitriles, but SCN- excretion was the same after both routes for n-hexanenitrile. Among saturated nitriles, SCN- excretion was maximal for the C3 and C4 compounds, propionitrile, n-butyronitrile, and isobutyronitrile, after both po and ip administration. SCN- excretion was not elevated after administration of the tertiary nitrile trimethylacetonitrile. Administration (po) of the unsaturated nitriles acrylonitrile, crotonitrile, and 3-butenenitrile yielded 37%, 5.6%, and 29% of the dose as SCN-, whereas after ip injection 4.5%, 4.6%, and 18% of the doses were excreted as SCN-, respectively. After iv injection of acrylonitrile, urinary SCN- content was not elevated, whereas 45% of an iv dose of the saturated analog propionitrile was excreted as SCN-. These results suggest that length of the carbon chain, presence of substituents at the alpha-carbon, position of double bonds, and, for some compounds, route of administration, are important factors influencing the release of CN- from nitriles.
A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (alpha subunit 26 kDa, beta subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the beta subunit. However, the amino acid sequence of the alpha subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.
Nitrile-hydrating enzymes produced by bacteria and fungi catalyse the conversion of a large number of chemically diverse nitriles, including many economically important compounds used industrially for chemical synthesis of amides and acids. This paper presents data on two new, highly different nitrile-hydrolysing enzymes which were isolated in connection with our studies on enzymic nitrile transformations. Particular attention was paid to the enzymes' substrate specificities and sensitivity to substrate/product inhibition. One of our microbial isolates was a Rhodococcus sp. (strain CH5). This strain produces a constitutive hydratase that has a broad substrate spectrum, including aliphatic and aromatic nitriles, mononitriles and dinitriles, hydroxynitriles and amino-nitriles. It also produces a constitutive amidase of equally low substrate specificity. The hydratase/amidase system catalysed the hydrolysis of D,L-aminonitriles into racemic mixtures of amino acids. Strain CH5 is able to produce high concentrations of malonic acid monoamide from malononitrile and malonamide. The other isolate, Alcaligenes sp. (strain I4), can convert high concentrations of cyanoacetate into malonic acid, presumably by means of an aliphatic nitrilase that is specific for cyanoacetate. Enzyme kinetic experiments have shown that this enzyme is very resistant to both substrate and product inhibition.
Several pseudokinases, previously regarded as dead enzymes due to the lack of catalytic residues, catalyze nucleotidylation. While they often utilize macromolecular substrates such as proteins and RNAs in primary metabolism, those acting on non-macromolecules in specialized metabolisms are limited. Calyculin A, a cytotoxic natural product produced by an uncultured sponge symbiont, possesses a unique nitrile group at the end of its tetraene tail. Even though its biosynthetic gene cluster (BGC) has been identified, the enzyme responsible for nitrile formation remains unknown. Herein, through a comparative analysis of the BGCs for calyculin derivatives in symbiotic bacteria from distinct sources, we identified a novel nitrile-forming enzyme, CalN. While CalN lacks sequence homology with other known nitrile-forming enzymes, it is structurally similar to pseudokinases. In vitro enzymatic reactions demonstrated that CalN specifically catalyzes nitrile formation through the adenylation of an amide substrate, calyculinamide A. In silico analyses and mutational experiments showed that CalN's structure features a unique insertion that plays critical roles in ATP recognition and the spatial coordination of catalytic residues. This study not only identifies a new family of nitrile-forming enzymes but also expands the variety of chemical reactions mediated by pseudokinases in nature.
Studies on the metabolic fate of aromatic nitriles, in contrast to their aliphatic counterparts, have been minimal and the subject of controversy. The in vitro metabolic fate of several aromatic nitriles with varying substituents was investigated by using rat liver subcellular fractions, with a particular emphasis on the nitrile moiety. Benzonitriles and 4-cyanophenols underwent oxidative metabolism to produce ring-hydroxylated metabolites. On the other hand, 2-cyanophenol was resistant to metabolism. o-Tolunitrile was metabolized and produced o-cyanobenzyl alcohol and phthalide. Phthalide, however, was chemically derived from o-cyanobenzyl alcohol, the initial metabolite. 4-Nitrobenzonitrile was resistant to oxidation on the ring, but was readily reduced to the corresponding amine metabolite under both aerobic and anaerobic conditions. Nitroxynil (3-iodo-4-hydroxy-5-nitrobenzonitrile) was metabolized to produce 3-iodo-4-hydroxy-5-nitrobenzamide and 3-iodo-4-hydroxy-5-nitrobenzoic acid. The enzyme(s) responsible for this hydrolytic metabolism was primarily localized in the cytosol. Among the nitriles tested, o-tolunitrile and nitroxynil produced metabolites in which the nitrile moiety was modified. Nitroxynil, however, was the only compound that was directly metabolized on the nitrile moiety by the rat liver enzyme(s).
N-(N-acetyl-1-phenylalanyl)aminoacetronitrile is an inhibitor of papain. With 13C NMR spectroscopy we have shown that a reversible covalent adduct is formed with papain. The reversible nature of the covalent-adduct formation was demonstrated with NMR saturation-transfer technique using a DANTE pulse for selective excitation. In addition the covalent adduct was displaced with an aldehyde inhibitor to regenerate the nitrile compound. No hydrolysis of the nitrile was observed. The covalent adduct is most likely a thioimidate formed between the essential thiol and the nitrile. Several p-nitroanilide substrates and their corresponding nitrile inhibitors were examined. A correlation between Ki and kcat/Km was observed. This finding together with the fact that the pH dependence of Ki parallels that of kcat/Km suggests that the interaction of nitriles and papain has considerable transition-state character. In contrast, a nitrile was shown to be an ineffective inhibitor of alpha-chymotrypsin.