Molecular basis of formaldehyde detoxification. Characterization of two S-formylglutathione hydrolases from Escherichia coli, FrmB and YeiG

The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and alpha-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical alpha/beta-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.     

Proteomic and metabolomic analysis reveals rapid and extensive nicotine detoxification ability in honey bee larvae

Despite potential links between pesticides and bee declines, toxicology information on honey bee larvae (Apis mellifera) is scarce and detoxification mechanisms in this development stage are virtually unknown. Larvae are exposed to natural and synthetic toxins present in pollen and nectar through consumption of brood food. Due to the characteristic intensive brood care displayed by honey bees, which includes progressive feeding throughout larval development, it is generally assumed that larvae rely on adults to detoxify for them and exhibit a diminished detoxification ability. We found the opposite. We examined the proteomic and metabolomic responses of in vitro reared larvae fed nicotine (an alkaloid found in nectar and pollen) to understand how larvae cope on a metabolic level with dietary toxins. Larvae were able to effectively detoxify nicotine through an inducible detoxification mechanism. A coordinated stress response complemented the detoxification processes, and we detected significant enrichment of proteins functioning in energy and carbohydrate metabolism, as well as in development pathways, suggesting that nicotine may promote larval growth. Further exploration of the metabolic fate of nicotine using targeted mass spectrometry analysis demonstrated that, as in adult bees, formation of 4-hydroxy-4-(3-pyridyl) butanoic acid, the result of 2′C-oxidation of nicotine, is quantitatively the most significant pathway of nicotine metabolism. We provide conclusive evidence that larvae are capable of effectively catabolising a dietary toxin, suggesting that increased larval sensitivity to specific toxins is not due to diminished detoxification abilities. These findings broaden the current understanding of detoxification biochemistry at different organizational levels in the colony, bringing us closer to understanding the capacity of the colony as a superorganism to tolerate and resist toxic compounds, including pesticides, in the environment.     

Immunotoxins: status and prospects.

Immunotoxins, which are conjugates of cell-binding antibodies and toxins, show considerable promise in the treatment of certain cancers. Genetic engineering is increasingly being used to refine and modify these conjugates, and it is now possible to design, express and purify completely recombinant therapeutic molecules.     

Enhanced formaldehyde detoxification by overexpression of glutathione-dependent formaldehyde dehydrogenase from Arabidopsis

The ADH2 gene codes for the Arabidopsis glutathione-dependent formaldehyde dehydrogenase (FALDH), an enzyme involved in formaldehyde metabolism in eukaryotes. In the present work, we have investigated the potential role of FALDH in detoxification of exogenous formaldehyde. We have generated a yeast (Saccharomyces cerevisiae) mutant strain (sfa1Delta) by in vivo deletion of the SFA1 gene that codes for the endogenous FALDH. Overexpression of Arabidopsis FALDH in this mutant confers high resistance to formaldehyde added exogenously, which demonstrates the functional conservation of the enzyme through evolution and supports its essential role in formaldehyde metabolism. To investigate the role of the enzyme in plants, we have generated Arabidopsis transgenic lines with modified levels of FALDH. Plants overexpressing the enzyme show a 25% increase in their efficiency to take up exogenous formaldehyde, whereas plants with reduced levels of FALDH (due to either a cosuppression phenotype or to the expression of an antisense construct) show a marked slower rate and reduced ability for formaldehyde detoxification as compared with the wild-type Arabidopsis. These results show that the capacity to take up and detoxify high concentrations of formaldehyde is proportionally related to the FALDH activity in the plant, revealing the essential role of this enzyme in formaldehyde detoxification.     

Development of a subunit vaccine for prevention of Clostridium difficile associated diseases: Biophysical characterization of toxoids A and B

Inactivation of bacterial toxins for use in human vaccines traditionally is achieved by treatment with formaldehyde. In contrast, the bivalent experimental vaccine for the prevention of C. difficile infections (CDI) that is currently being evaluated in clinical trials was produced using a different strategy. C. difficile toxins A and B were inactivated using site-directed mutagenesis and treatment with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysulfosuccinimide (EDC/NHS). In the present work we investigate the effect of genetic and chemical modifications on the structure of inactivated toxins (toxoids) A and B. The far-UV circular dichroism (CD) spectra of wild type toxins, mutated toxins, and EDC/NHS-inactivated toxoids reveal that the secondary structure of all proteins is very similar. The near-UV CD spectra show that aromatic residues of all proteins are in a unique asymmetric environment, indicative of well-defined tertiary structure. These results along with the fluorescence emission maxima of 335 nm observed for all proteins suggest that the tertiary structure of toxoids A and B is preserved as well. Analytical ultracentrifugation data demonstrate that all proteins are predominantly monomeric with small fractions of higher molecular weight oligomeric species present in toxoids A and B. Differential scanning calorimetry data reveal that genetic mutations induce thermal destabilization of protein structures. Subsequent treatment with EDC/NHS results either in a minimal (1 °C) increase of apparent thermostability (toxoid B) or no change at all (toxoid A). Therefore, our two-step inactivation strategy is an effective approach for the preparation of non-toxic proteins maintaining native-like structure and conformation.     

Paralytic shellfish poisoning (PSP) toxin profiles and short-term detoxification kinetics in mussels Mytilus galloprovincialis fed with the toxic dinoflagellate Alexandrium tamarense

Mussels (Mytilus galloprovincialis) were experimentally contaminated with paralytic shellfish poisoning (PSP) toxins by being fed with the toxic dinoflagellate Alexandrium tamarense, and changes in toxin content and specific composition during the decontamination period were analyzed by high-performance liquid chromatography (HPLC). Toxins excreted by the mussels into the seawater were also recovered using an activated charcoal column and analyzed by HPLC. The predominant toxins in A. tamarense, mussels, and seawater were the N-sulfocarbamoyl-11-hydrosulfate toxins (C1,2) and carbamate gonyautoxins-1,4 (GTX1,4). There were no remarkable differences in the relative proportions of the predominant toxins within A. tamarense, mussels and seawater. Because the relative proportion of the various toxin analogues excreted by the mussels was similar to that within their tissues during detoxification, it appeared that the selective release of particular toxins by the mussels was unlikely. The total amount of toxin lost from mussels was nearly equal to that which was found dissolved in the seawater, suggesting that, at least the early stages of mussel detoxification, most losses can be accounted for by excretion.     

A comparative study of ricin and diphtheria toxin-antibody-conjugate kinetics on protein synthesis inactivation

Kinetic data on toxin and antibody-toxin-conjugate inactivation of protein synthesis have been used to assess the variables which affect the transport of these toxins into the cytosol compartment. First-order inactivation rate constants of protein synthesis (ki) are compared under conditions of known receptor occupancy. The effect of inclusion of toxin B chains, both homologous and heterologous, in antibody-toxin conjugates is observed, and factors which affect toxin lag periods are studied. The results show that the inclusion of B chains in conjugates increases ki values 3-10-fold, but only if the B chain is homologous with the A chain. In spite of the augmentation of antibody-toxin-conjugate ki values by homologous toxin B chain, these ki values are only 1/20 those observed with unmodified toxins on sensitive cells. A further difference noted between toxins and antibody-toxin conjugates is the presence of a dose-dependent lag when toxins, but not antibody-toxin conjugates, effect sensitive cell types. This lag period for ricin can be shortened by alkalinizing the cell medium. The kinetic data can be fit by assuming a processing step interposed between the binding of ricin to surface receptors and the interaction of the A chain with ribosomes which is first-order in toxin concentration and pH-dependent. The time constant of this event is reflected in the dose-dependent lag period. It is proposed that antibody-toxin conjugates do not participate in this processing event and therefore fail to achieve the high entry levels exhibited by unmodified toxins.     

Inactivation of conserved C. elegans genes engages pathogen- and xenobiotic-associated defenses

The nematode C.?elegans is attracted to nutritious bacteria and is repelled by pathogens and toxins. Here we show that RNAi and toxin-mediated disruption of core cellular activities, including translation, respiration, and protein turnover, stimulate behavioral avoidance of normally attractive bacteria. RNAi of these and other essential processes induces expression of detoxification and innate immune effectors, even in the absence of toxins or pathogens. Disruption of core processes in non-neuronal tissues was sufficient to stimulate aversion behavior, revealing a neuroendocrine axis of control that additionally required serotonergic and Jnk kinase signaling pathways. We propose that surveillance pathways overseeing core cellular activities allow animals to detect invading pathogens that deploy toxins and virulence factors to undermine vital host functions. Variation in cellular surveillance and endocrine pathways controlling behavior, detoxification, and immunity selected by past toxin or microbial interactions could underlie aberrant responses to foods, medicines, and microbes.     

Synthesis of stable hydrazones of a hydrazinonicotinyl-modified peptide for the preparation of 99mTc-labeled radiopharmaceuticals.

Hydrazones of a 6-hydrazinonicotinyl-modified cyclic peptide IIb/IIIa receptor antagonist were prepared in order to protect the hydrazine moiety from reaction with trace aldehyde and ketone impurities encountered during the process of manufacturing and compounding lyophilized kits used in radiolabeling with (99m)Tc. Hydrazones were prepared by either a direct reaction of the 6-hydrazinonicotinyl-modified cyclic peptide with carbonyl compounds or by conjugation of the cyclic peptide with hydrazones of succinimidyl 6-hydrazinonicotinate. Stability of the hydrazones was evaluated by treatment with formaldehyde. Hydrazones derived from simple aliphatic aldehydes underwent an exchange reaction with formaldehyde, while hydrazones of aromatic aldehydes and ketones provided the greatest level of stability when challenged with formaldehyde. We have been successful in protecting 6-hydrazinonicotinyl-modified cyclic peptides from reacting with formaldehyde, while still allowing sufficient reactivity for radiolabeling with (99m)Tc. The hydrazones of succinimidyl 6-hydrazinonicotinate are convenient and general reagents for forming 6-hydrazinonicotinyl conjugates with amino-functionalized bioactive molecules.     

Studies of the mechanism of cell intoxication by diphtheria toxin fragment A-asialoorosomucoid hybrid toxins. Evidence for utilization of an alternative receptor-mediated transport pathway

In order to study the variables important for determining the cytotoxicity of protein-protein hybrid toxins, the disulfide and thioiether conjugates between diphtheria toxin fragment A (DTA) and asialoorosomucoid (ASOR) were synthesized, purified, and tested for cytotoxicity toward isolated rat hepatocytes as monitored by the inhibition of protein synthesis. After 4-h incubation at 37 degrees C, both DTA-ASOR conjugates were nontoxic, but were highly toxic in the presence of an inhibitor of 125I-ASOR degradation. At concentrations inhibiting 125I-ASOR degradation to the same extent, the efficiency of inhibitors to enhance the toxicity of both conjugates varied with the following rank order: colchicine greater than chloroquine greater than NH4Cl greater leupeptin greater than cytochalasin B. In the absence of an inhibitor, both conjugates, after 10-h incubation, also exhibited measurable toxicity at concentrations as low as 10(-9) M, and DTA-S-S-ASOR was 60 times more toxic than DTA-S-ASOR. In a physiological salt solution containing energy source and bovine serum albumin, both conjugates also exhibited partial inhibition of protein synthesis after 4-h incubation and were enhanced by 2 microM colchicine toward complete inhibition with similar C0.5 of 2 X 10(-10) M. The toxicity of the conjugates were prevented in the presence of excess ASOR. These results not only indicate that intracellular degradation is an important factor restricting the toxicity of the conjugates, but also indicate that the toxic entry of DTA activity from these hybrids is independent of lysosomal degradation. Consequently, it appears that conjugate toxicity is dependent on the release of DTA activity from an extralysosomal compartment whose pool size is increased by the inhibitors and the change of medium. The effects of the inhibitors to inhibit 125I-ASOR uptake and degradation were also studied to facilitate interpretation of their enhancement of conjugate toxicity. Specifically, colchicine inhibited both internalization and degradation of the ligand in a saturable fashion with a C0.5 of 0.5 microM and a maximum inhibition of 65%. The data of the present study are interpreted so as to support a hypothesis that two receptor-mediated pathways are involved in the internalization and transport to lysosomes of ASOR and DTA-ASOR conjugates, only one of which is colchicine-sensitive.     

Hormonotoxins. Preparation and characterization of ovine luteinizing hormone-gelonin conjugate

With the aim of targeting toxins to selected cells in the gonad, we have prepared conjugates of ovine luteinizing hormone (oLH) with a single chain ribosome-inactivating protein called gelonin. The two proteins were thiolated by using N-succinimidyl-3-(2-pyridyldithio)propionate and subsequently reacted under appropriate conditions to form oLH-S-S-gelonin complex. A complete biochemical analysis of thiolated oLH and oLH-gelonin conjugates has been performed. The linkage of the hormone to the toxin probably occurred through a single amino group in the alpha-subunit, with the beta-subunit remaining free. Modification of a single amino group on the alpha-subunit reduced receptor binding and immunological reactivity of the thiolated oLH, but subsequent complexing with the toxin-gelonin did not seriously compromise these activities. oLH and gelonin were calculated to be present in a 1:1 ratio in the hormonotoxin preparation. The conjugate retained significant steroidogenic activity in rat granulosa cells. Upon reaction with mouse tumor Leydig cells (MA-10 cells), the toxin component of the complex became internalized to a sufficient degree to effectively inhibit protein synthesis. The studies provide a rational basis for the design and study of large hormonotoxins.     

Functions of ABC transporters in plants

ABC (ATP-binding cassette) proteins are ubiquitously found in prokaryotes and eukaryotes and generally serve as membrane-intrinsic primary active pumps. In higher plants, ABC proteins constitute a large family, grouped phylogenetically into eight clusters, subfamilies ABCA-ABCI (ABCH is not found in plants). ABC transporters shuttle substrates as diverse as lipids, phytohormones, carboxylates, heavy metals, chlorophyll catabolites and xenobiotic conjugates across a variety of biological membranes. To date, the largest proportions of characterized members have been localized to the plasma membrane and the tonoplast, with dominant implications in cellular secretion and vacuolar sequestration, but they are also found in mitochondrial, plastidal and peroxisomal membranes. Originally identified as tonoplast-intrinsic proteins that shuttle xenobiotic conjugates from the cytosol into the vacuole, thus being an integral part of the detoxification machinery, ABC transporters are now recognized to participate in a multitude of physiological processes that allow the plant to adapt to changing environments and cope with biotic and abiotic stresses.     

Physicochemical and immunochemical assays for monitoring consistent production of tetanus toxoid

The detoxification of tetanus toxin by formaldehyde is a crucial step in the production of tetanus toxoid. The inactivation results in chemically modified proteins and it determines largely the ultimate efficacy and safety of the vaccine. Currently, the quality of tetanus toxoid lots is evaluated in potency and safety tests performed in animals. As a possible alternative, this article describes a panel of in vitro methods, which provides detailed information about the quality of tetanus toxoid. Ten experimental lots of tetanus toxoid were prepared using increasing concentrations of formaldehyde and glycine to obtain tetanus toxoids having differences in antigenicity, immunogenicity, residual toxicity and protein structure. The structural properties of each individual toxoid were determined using immunochemical and physicochemical methods, including biosensor analysis, ELISA, circular dichroism, TNBS assay, differential scanning calorimetry, fluorescence and SDS-PAGE. The quality of a tetanus toxoid lot can be assessed by these set of analytical techniques. Based on antigenicity, immunogenicity and residual toxicity data, criteria are formulated that tetanus toxoids lot have to meet in order to have a high quality. The in vitro methods are a valuable selection of techniques for monitoring consistency of production of tetanus toxoid, especially for the detoxification process of tetanus toxin.     

Biochemical aspects of aldehyde fixation and a new formaldehyde fixative.

In this paper, it is assumed that tissue fixation is a process in which the proteins become less soluble and catabolic reactions stop. With this definition in mind, 2.5 and 5% glutaraldehhde and 4% formaldehyde in 0.1 M potassium phosphate buffer, pH 7.4, were compared with a new fixative, bicarbonate-formaldehyde. The following results were obtained. (I) With 2.5 and 5% glutaraldehyde, the solubility of tissue proteins were not decreased unifromly, and tissue glycogen was poorly preserved. (2) 4% formaldehyde in potassium phosphate buffer gave relatively good results. (3) Bicarbonate-formaldehyde decreased the solubility of tissue proteins reliably and preserved tissue glycogen perfectly. Histologically, it yielded excellent results. Since glutaraldehyde alters the properties of proteins substantially (Hopwood, 1972; Habeeb & Hiramoto, 1968), and since the natural appearance of tissues depends on native tissue proteins, formaldehyde-containing fixatives, in particular bicarbonate-formaldehyde, are preferable to glutaraldehyde-containing fixatives for all tissue preparative techniques. However, it is important that the fixation time in formaldehyde is kept short.     

Transport of glutathione-conjugates in human erythrocytes

The last step of detoxification of both endogenous and environmental toxicants is typically a conjugation that produces a bulky hydrophilic molecule. The excretion of such conjugates out of cells is of sufficient biological importance to have led to the evolution of ATP-driven export pumps for this purpose. The substrate specificity of such transporters is broad, and in some cases it has been shown to include not only anionic conjugates but also neutral or weakly cationic drugs. In the present article, we review the molecular identity, functional and structural characteristics of these pumps, mainly on the example of human erythrocytes, and discuss their physiological role in detoxification and in the multidrug resistance phenotype of cancer cells.     

Initial microbial adhesion is a determinant for the strength of biofilm adhesion

Abstract A considerable amount of methylformate accumulated in the culture medium of methanol-grown methylotrophic yeasts. Methylformate is considered as an intermediate in a novel formaldehyde oxidation pathway. Through investigations with Pichia methanolica , methylformate formation was found to be catalysed by a new type of alcohol dehydrogenase, which was named methylformate synthase. When cells were grown on a relatively high concentration of methanol or exposed to a high concentration of formaldehyde, formation of methylformate was enhanced and the level of methylformate synthase in the cells increased. How methylformate synthase is involved in formaldehyde oxidation and formaldehyde detoxification is discussed.     

Inhibition of Cathepsin B1 by E-64, a Thiol Proteinase Inhibitor,and Its Derivatives

Four formaldehyde-resistant yeasts were isolated from soil. Three were tentatively identified as Debaryomyces vanriji and one as Trichosporon penicillatum. These yeasts almost completely consumed formaldehyde at 0.15 to 0.55% in growth medium containing glucose as carbon source, but the carbon of formaldehyde was not incorporated into the cell constituents. In formaldehyde-containing medium, yeast growth occurred after formaldehyde consumption. The yeasts showed relatively high activities of formaldehyde dehydrogenase, S-formylglutathione hydrolase and formate dehydrogenase. The resistance to formaldehyde is attributed to detoxification by oxidation.     

The mechanism of microbial resistance to hexahydro-1,3,5-triethyl-s-triazine

Summary Four strains ofPseudomonas putida and two unidentifiedPseudomonas species that were resistant to hexahydro-1,3,5-triethyl-s-triazine (HHTT) were shown to be resistant to formaldehyde as well. Conjugation experiments revealed that: (a) HHTT and formaldehyde resistance was cotransferred in every case where exconjugants were recovered; (b) in every case HHTT resistance and formaldehyde resistance were expressed to the same level in the exconjugant as in the donor; (c) resistance to either HHTT or formaldehyde alone was never observed; and (d) in instances where HHTT and formaldehyde resistance in the exconjugants was unstable, the exconjugants lost resistance to both agents simultaneously and never to one agent alone. Resistant organisms (e.g.P. putida 3-T-15²) had high levels of formaldehyde dehydrogenase and this enzyme appeared to be constitutively expressed. It was concluded that resistance to HHTT was due to resistance to its degradation product, formaldehyde, via detoxification of formaldehyde by formaldehyde dehydrogenase. HHTT- and formaldehyde-sensitive organisms had barely detectable levels (most likely repressed levels) of formaldehyde dehydrogenase. Although speculative, it is possible that formaldehyde resistance may be due to a mutation resulting in derepression of the gene coding for formaldehyde dehydrogenase. While it could not be discerned whether HHTT resistance and formaldehyde resistance were carried on two separate but closely linked genes or if only one gene was involved, the evidence suggested that only one gene was involved. Similarly, it could not be determined whether HHTT and formaldehyde resistance was encoded by chromosomal or plasmid genes.     

Role of Aldose Reductase in the Metabolism and Detoxification of Carnosine-Acrolein Conjugates

Oxidation of unsaturated lipids generates reactive aldehydes that accumulate in tissues during inflammation, ischemia, or aging. These aldehydes form covalent adducts with histidine-containing dipeptides such as carnosine and anserine, which are present in high concentration in skeletal muscle, heart, and brain. The metabolic pathways involved in the detoxification and elimination of these conjugates are, however, poorly defined, and their significance in regulating oxidative stress is unclear. Here we report that conjugates of carnosine with aldehydes such as acrolein are produced during normal metabolism and excreted in the urine of mice and adult human non-smokers as carnosine-propanols. Our studies show that the reduction of carnosine-propanals is catalyzed by the enzyme aldose reductase (AR). Carnosine-propanals were converted to carnosine-propanols in the lysates of heart, skeletal muscle, and brain tissue from wild-type (WT) but not AR-null mice. In comparison with WT mice, the urinary excretion of carnosine-propanols was decreased in AR-null mice. Carnosine-propanals formed covalent adducts with nucleophilic amino acids leading to the generation of carnosinylated proteins. Deletion of AR increased the abundance of proteins bound to carnosine in skeletal muscle, brain, and heart of aged mice and promoted the accumulation of carnosinylated proteins in hearts subjected to global ischemia ex vivo. Perfusion with carnosine promoted post-ischemic functional recovery in WT but not in AR-null mouse hearts. Collectively, these findings reveal a previously unknown metabolic pathway for the removal of carnosine-propanal conjugates and suggest a new role of AR as a critical regulator of protein carnosinylation and carnosine-mediated tissue protection.