A Yeast Toxic Mutant of HET-s(218-289) Prion Displays Alternative Intermediates of Amyloidogenesis

Amyloids are thought to be involved in various types of neurodegenerative disorders. Several kinds of intermediates, differing in morphology, size, and toxicity, have been identified in the multistep amyloidogenesis process. However, the mechanisms explaining amyloid toxicity remain unclear. We previously generated a toxic mutant of the nontoxic HET-s_(218-289) amyloid in yeast. Here we report that toxic and nontoxic amyloids differ not only in their structures but also in their assembling process. We used multiple and complementary methods to investigate the intermediates formed by these two amyloids. With the methods used, no intermediates were observed for the nontoxic amyloid; however, under the same experimental conditions, the toxic mutant displayed visible oligomeric and fibrillar intermediates.     

From PCBs to highly toxic metabolites by the biphenyl pathway

The degradation of polychlorobiphenyls (PCBs) by diverse bacteria, including Burkholderia sp. LB400, is incomplete with a concomitant accumulation of metabolic intermediates. In this study, the toxicity of diverse (chloro)biphenyls and of their biotransformation into the first two metabolic intermediates of the biphenyl pathway, were determined for the model bacterium Escherichia coli. Recombinant E. coli strains expressing different subsets of bph genes of strain LB400 accumulated metabolic intermediates from (chloro)biphenyls. During biotransformation of these compounds into metabolic intermediates, the viability and metabolic kinetics were determined. The toxicity of biotransformation of (chloro)biphenyls into different metabolic intermediates of (chloro)biphenyls varied. Dihydrodiols and dihydroxybiphenyls are very toxic metabolites for bacteria even after short incubation times, affecting the cell viability much more than (chloro)biphenyls. When bacteria transformed 2-CB into dihydrodiol or dihydroxybiphenyl, a great decrease of intact cells and abundant cell lysis was observed by transmission electronic microscopy. Cell viability of Burkholderia sp. LB400 and of E. coli exposed directly to 2,3-dihydroxybiphenyl decreased also drastically. The toxicity of metabolites generated during oxidation of PCBs may partly explain the recalcitrance to biodegradation of these pollutants. Conversion of less toxic compounds into products with increased toxicity resembles the bioactivation of xenobiotics in higher organisms.     

Free radical metabolism of antiparasitic agents

In recent years it has been apparent that many of the known antiparasitic drugs produce free radicals. Intracellular reduction followed by autooxidation yielding O.-2 and H2O2 has been suggested as the mode of action of nifurtimox on Trypanosoma cruzi and as the basis of its toxicity in mammals. On the other hand, free radical intermediates that do not generate oxygen-reduction products under physiological conditions have been found in the metabolic pathways of other antiparasitic nitro compounds (benznidazole, metronidazole, and other 5-nitroimidazoles) used in the treatment of diseases such as Chagas' disease, trichomoniasis, giardiasis, balantidiasis, amebiasis, and schistosomiasis. In these cases, as well as in the case of niridazole (used in the treatment of schistosomiasis), covalent binding or other interactions of the intermediates of nitroreduction with parasite macromolecules are possibly involved in their toxicity. Redox cycling of these compounds under aerobic conditions appears to be a detoxification reaction by inhibiting net reduction of the drugs.     

Huntingtin spheroids and protofibrils as precursors in polyglutamine fibrilization

The pathology of Huntington's disease is characterized by neuronal degeneration and inclusions containing N-terminal fragments of mutant huntingtin (htt). To study htt aggregation, we examined purified htt fragments in vitro, finding globular and protofibrillar intermediates participating in the genesis of mature fibrils. These intermediates were high in beta-structure. Furthermore, Congo Red, a dye that stains amyloid fibrils, prevented the assembly of mutant htt into mature fibrils, but not the formation of protofibrils. Other proteins capable of forming ordered aggregates, such as amyloid beta and alpha-synuclein, form similar intermediates, suggesting that the mechanisms of mutant htt aggregation and possibly htt toxicity may overlap with other neurodegenerative disorders.     

Effects of nimesulide and its metabolites or manufacturing intermediates on the viability and growth of the human hepatoma HepG2 cell line.

Hepatitis and fulminant hepatic failure have, infrequently, been associated with nimesulide. To establish if nimesulide or its analogues have direct cytotoxic activity on liver cells, experiments were undertaken to investigate the effects of nimesulide and its principal metabolites and production intermediates on the viability and growth of the human hepatoma cell line, HepG2, in vitro. The parent drug, metabolites or production intermediates as well as formulations of nimesulide were incubated for 6-48 hr with HepG2 cells and the extent of toxicity determined using the mitochondrial selective redox dye 3-4,5-dimethylthazol-2-yl)-2,4-diphenyl tetrazolium bromide (MTT). The results showed that there was no appreciable cytotoxic activity exhibited by nimesulide and its principle metabolites or production intermediates on HepG2 cells.     

FURTHER STUDIES ON THE BREAKDOWN OF 2:4-DICHLOROPHENOXYACETIC ACID BY A SOIL BACTERIUM

Bacterial growth rates and the kinetics of detoxication have been followed during the course of breakdown of 2:4-dichlorophenoxyacetic acid in a liquid mineral salt medium by cultures of Bacterium globiforme isolated from garden soil. It was found that toxicity persisted apparently unchanged long after bacterial growth had ceased, and finally disappeared suddenly after a lag period comparable in duration with those obtained in soil perfusion experiments. These results can best be explained by the production of highly phytotoxic intermediates in the first stages of 2:4-D breakdown. Preliminary experiments involving paper partition chromatography indicate that there may be at least two such intermediates.     

Bioactivation of xenobiotics by formation of toxic glutathione conjugates

Evidence has been accumulating that several classes of compounds are converted by glutathione conjugate formation to toxic metabolites. The aim of this review is to summarize the current knowledge on the biosynthesis and toxicity of glutathione S-conjugates derived from halogenated alkanes, halogenated alkenes, and hydroquinones and quinones. Different types of toxic glutathione conjugates have been identified and will be discussed in detail: (i) conjugates which are transformed to electrophilic sulfur mustards, (ii) conjugates which are converted to toxic metabolites in an enzyme-catalyzed multistep mechanism, (iii) conjugates which serve as a transport form for toxic quinones and (iv) reversible glutathione conjugate formation and release of the toxic agent in cell types with lower glutathione concentrations. The kidney is the main, with some compounds the exclusive, target organ for compounds metabolized by pathways (i) to (iii). Selective toxicity to the kidney is easily explained due to the capability of the kidney to accumulate intermediates formed by processing of S-conjugates and to bioactivate these intermediates to toxic metabolites. The influences of other factors participating in the renal susceptibility are discussed.     

Chromium (V) and hydroxyl radical formation during the glutathione reductase-catalyzed reduction of chromium (VI)

Electron spin resonance measurements provide evidence for the formation of long-lived Cr(V) intermediates in the reduction of Cr(VI) by glutathione reductase in the presence of NADPH and for the hydroxyl radical formation during the glutathione reductase catalyzed reduction of Cr(VI). Hydrogen peroxide suppresses Cr(V) and enhances the formation of hydroxyl radicals. Thus Cr(V) intermediates catalyze generation of hydroxyl radicals from hydrogen peroxide through a Fenton-like reaction. Thus the mechanism of Cr(VI) toxicity might involve the interaction between macromolecules and the hydroxyl radicals.     

TRPM7 regulates polarized cell movements

TFM (L-trifluoromethionine), a potential prodrug, was reported to be toxic towards human pathogens that express MGL (L-methionine γ-lyase; EC 4.4.1.11), a pyridoxal phosphate-containing enzyme that converts L-methionine into α-oxobutyrate, ammonia and methyl mercaptan. It has been hypothesized that the extremely reactive thiocarbonyl difluoride is produced when the enzyme acts upon TFM, resulting in cellular toxicity. The potential application of the fluorinated thiomethyl group in other areas of biochemistry and medicinal chemistry requires additional studies. Therefore a detailed investigation of the theoretical and experimental chemistry and biochemistry of these fluorinated groups (CF?S? and CF?HS?) has been undertaken to trap and identify chemical intermediates produced by enzyme processing of molecules containing these fluorinated moieties. TvMGL (MGL from Trichomonas vaginalis) and a chemical model system of the reaction were utilized in order to investigate the cofactor-dependent activation of TFM and previously uninvestigated DFM (L-difluoromethionine). The differences in toxicity between TFM and DFM were evaluated against Escherichia coli expressing TvMGL1, as well as the intact human pathogen T. vaginalis. The relationship between the chemical structure of the reactive intermediates produced from the enzymatic processing of these analogues and their cellular toxicity are discussed.     

Nitric oxide-induced homologous recombination in Escherichia coli is promoted by DNA glycosylases

Nitric oxide (NO*) is involved in neurotransmission, inflammation, and many other biological processes. Exposure of cells to NO* leads to DNA damage, including formation of deaminated and oxidized bases. Apurinic/apyrimidinic (AP) endonuclease-deficient cells are sensitive to NO* toxicity, which indicates that base excision repair (BER) intermediates are being generated. Here, we show that AP endonuclease-deficient cells can be protected from NO* toxicity by inactivation of the uracil (Ung) or formamidopyrimidine (Fpg) DNA glycosylases but not by inactivation of a 3-methyladenine (AlkA) DNA glycosylase. These results suggest that Ung and Fpg remove nontoxic NO*-induced base damage to create BER intermediates that are toxic if they are not processed by AP endonucleases. Our next goal was to learn how Ung and Fpg affect susceptibility to homologous recombination. The RecBCD complex is critical for repair of double-strand breaks via homologous recombination. When both Ung and Fpg were inactivated in recBCD cells, survival was significantly enhanced. We infer that both Ung and Fpg create substrates for recombinational repair, which is consistent with the observation that disrupting ung and fpg suppressed NO*-induced recombination. Taken together, a picture emerges in which the action of DNA glycosylases on NO*-induced base damage results in the accumulation of BER intermediates, which in turn can induce homologous recombination. These studies shed light on the underlying mechanism of NO*-induced homologous recombination.     

Effect of the insect phenoloxidase on the metabolism of l‐DOPA

Insect prophenoloxidase (PPO) induces melanization around pathogens. Before melanization, PPO is cleaved into phenoloxidase (PO) by serine proteases. Insect PPO can also be activated by exogenous proteases secreted by pathogens as well as by other compounds, such as ethanol and cetylpyridinium chloride (CPC). However, the effect of these activators on the activity of PO is unclear. In this study, the insect endogenous serine protease AMM1, α‐chymotrypsin, and ethanol were used to activate recombinant Drosophila PPO1 (rPPO1), and the PO activity differed depending on the activator applied. The PO‐induced intermediates during melanization also varied markedly in their numbers and abundances. Therefore, this study indicates that the mechanism of PPO activation influences PO activity. It also suggests that PO‐induced different intermediates may affect the antibacterial activity during melanization due to their toxicity.     

Toxic oxygen species and protective systems of the chloroplast

Salin, M. L. 1988. Toxic oxygen species and protective systems of the chloroplast. -Physiol. Plant. 72: 681–689.
As a consequence of living in an environment enriched in oxygen, which they themselves at least partially generate, photosynthetic organisms are exposed to large fluxes of oxyradicals and reactive oxygen species. Among these are superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen. These highly reactive intermediates pose the threat of toxicity unless neutralized by scavenger substrates or enzymes. The production of oxyradicals and intermediates by chloroplasts as well as the means of protection are discussed in this review.     

Formation of chlorocatechol meta cleavage products by a pseudomonad during metabolism of monochlorobiphenyls.

Pseudomonas cepacia P166 was able to metabolize all monochlorobiphenyls to the respective chlorobenzoates. Although they transiently accumulated, the chlorobenzoate degradation intermediates were further metabolized to chlorocatechols, which in turn were meta cleaved. 2- and 3-Chlorobiphenyl both produced 3-chlorocatechol, which was transformed to an acyl halide upon meta cleavage. 3-Chlorocatechol metabolism was toxic to the cells and impeded monochlorobiphenyl metabolism. In the case of 2-chlorobiphenyl, toxicity was manifested as a diminished growth rate, which nevertheless effected rapid substrate utilization. In the case of 3-chlorobiphenyl, which generates 3-chlorocatechol more rapidly than does 2-chlorobiphenyl, toxicity was manifested as a decrease in viable cells during substrate utilization. 4-Chlorobenzoate was transformed to 4-chlorocatechol, which was metabolized by a meta cleavage pathway leading to dehalogenation. Chloride release from 4-chlorocatechol metabolism, however, was slow and did not coincide with rapid 4-chlorocatechol turnover. Growth experiments with strain P166 on monochlorobiphenyls illustrated the difficulties of working with hydrophobic substrates that generate toxic intermediates. Turbidity could not be used to measure the growth of bacteria utilizing monochlorobiphenyls because high turbidities were routinely measured from cultures with very low viable-cell counts.     

Comparative toxicity and mutagenicity of N-hydroxy-2-acetylaminofluorene and 7-acetyl-N-hydroxy-2-acetylaminofluorene in human lymphoblasts.

Exponentially growing TK6 human lymphoblasts were exposed to either 0-50 microM N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or 0-10 microM 7-acetyl-N-hydroxy-2-acetylaminofluorene (7-acetyl-N-OH-AAF) in both the absence and presence of a partially purified preparation of hamster-liver N-arylhydroxamic acid N,O-acyltransferase (AHAT). Neither N-arylhydroxamic acid was toxic to the lymphoblasts, nor mutagenic at the thymidine kinase (tk) locus, in the absence of AHAT over the concentration range examined. In the presence of AHAT, an enzyme that activates N-arylhydroxamic acids to electrophilic N-acetoxyarylamine intermediates, both compounds caused toxicity and mutagenicity in TK6 cells. The 7-acetyl-N-OH-AAF was approximately 10-fold more toxic and mutagenic than the unsubstituted N-OH-AAF. These data demonstrate that metabolism of these N-arylhydroxamic acids, presumably to N-acetoxyarylamine intermediates by AHAT, is a key event in the biological activity of these agents. In addition, the presence of electron-withdrawing 7-acetyl substituent that is thought to stabilize N-acetoxy intermediates, appears to enhance the biological activity of the unsubstituted N-OH-AAF.     

Target cells for methylsulphonyl-2,6-dichlorobenzene in the olfactory mucosa in mice

Previously we reported that methylsulphonyl-2,6-dichlorobenzene, 2, 6-(diCl-MeSO(2)-B), was irreversibly bound to the olfactory mucosa of mice and induced necrosis of the Bowman's glands with subsequent neuroepithelial degeneration and detachment. In this study, autoradiography and histopathology were used to determine tissue-localization and toxicity of 2,6-(diCl-MeSO(2)-B) in the olfactory mucosa of control mice and animals pretreated with cytochrome P450 (CYP) and glutathione (GSH) modulators. The Bowman's glands of the olfactory mucosa were the major target sites of non-extractable binding of 2,6-(diCl-(14)C-MeSO(2)-B), whereas the olfactory neuroepithelium and nerve bundles showed only background levels of silver grains. Metyrapone pretreatment slightly decreased binding in the Bowman's glands and markedly decreased toxicity in the olfactory mucosa after 2,6-(diCl-MeSO(2)-B) administration. These results support that a CYP-mediated activation of 2, 6-(diCl-MeSO(2)-B) takes place in the Bowman's glands giving rise to toxic reactive intermediates. In mice pretreated with the GSH-depleting agent phorone, a marked increase of irreversible binding of 2,6-(diCl-(14)C-MeSO(2)-B) in the Bowman's glands was observed. Tape-section autoradiograms also revealed a significant increase of uptake of radioactivity in the olfactory bulb. As determined by histopathology, GSH-depletion increased both the extent and severity of the lesion in the mucosa. These results imply that 2,6-(diCl-MeSO(2)-B)-reactive intermediates are conjugated with GSH. The amount of irreversible binding and toxicity in the olfactory mucosa seems to be associated with the level of 2, 6-(diCl-MeSO(2)-B)-reactive intermediates.     

Soluble oligomers are sufficient for transmission of a yeast prion but do not confer phenotype

Amyloidogenic proteins aggregate through a self-templating mechanism that likely involves oligomeric or prefibrillar intermediates. For disease-associated amyloidogenic proteins, such intermediates have been suggested to be the primary cause of cellular toxicity. However, isolation and characterization of these oligomeric intermediates has proven difficult, sparking controversy over their biological relevance in disease pathology. Here, we describe an oligomeric species of a yeast prion protein in cells that is sufficient for prion transmission and infectivity. These oligomers differ from the classic prion aggregates in that they are soluble and less resistant to SDS. We found that large, SDS-resistant aggregates were required for the prion phenotype but that soluble, more SDS-sensitive oligomers contained all the information necessary to transmit the prion conformation. Thus, we identified distinct functional requirements of two types of prion species for this endogenous epigenetic element. Furthermore, the nontoxic, self-replicating amyloid conformers of yeast prion proteins have again provided valuable insight into the mechanisms of amyloid formation and propagation in cells.     

Roles of apoplastic peroxidases, laccases, and lignification in the manganese tolerance of hyperaccumulator Phytolacca americana

We investigated the response of Mn-hyperaccumulator Phytolacca americana L. to manganese excess as well as the relationships between lignin deposition in the plant’s leaves, peroxidase and laccase activities in the leaf apoplast, and Mn toxicity. The exceptionally high tolerance of P. americana to Mn, both in solution and in tissue, was confirmed. No visible brown spot was observed in the leaves of plants treated with ≤10,000 μM Mn for 10 days. Mn treatment significantly increased lignin content and laccase activity in the apoplastic washing fluid (AWF) of P. americana leaves. In contrast, an increase in the Mn supply was paralleled by a significant decrease in the concentration of total phenolic compounds (TPCs) and in water-soluble guaiacol peroxidase (SPOD) activity in leaf AWF. This result suggested that an increase in lignin deposition decreased the concentration of apoplastic TPCs that are available to generate potentially toxic intermediates by acting as peroxidase substrates. Thus, data of the present study indicate that lignin formation by laccase activities reduces Mn toxicity and increases Mn tolerance of P. americana by depressing SPOD-mediated formation of toxic intermediates from TPCs.     

Toxicity of terpenes to spores and mycelium of Penicillium digitatum

Spores, although often considered metabolically inert, catalyze a variety of reactions. The use of spores instead of mycelium for bioconversions has several advantages. In this paper, we describe the difference in susceptibility of mycelium and spores against toxic substrates and products. A higher resistance of spores toward the toxic effects of bioconversion substrates and products is an advantage that has not been studied in detail until now. This paper shows that spores of Penicillium digitatum ATCC 201167 are on average over 2.5 times more resistant than mycelium toward the toxicity of substrates, intermediates, and products of the geraniol bioconversion pathway. Furthermore, the higher resistance of spores to citral was shown as an advantage in its biotransformation by P. digitatum. Using three different approaches the toxicity of the compounds were tested. The order of toxicity toward P. digitatum was, starting with the most toxic, citral > nerol/geraniol > geranic acid > methylheptenone > acetaldehyde.     

Assessment of toxicity of volatile fatty acids to Photobacterium phosphoreum

The toxicity of four volatile fatty acids (VFAs) as anaerobic digestion (AD) intermediates was investigated at pH 7. Photobacterium phosphoreum T3 was used as an indicator organism. Binary, ternary and mixtures of AD intermediates were designated by letters A (acetic acid + propionic acid), B (acetic acid + butyric acid), C (acetic acid + ethanol), D (propionic acid + butyric acid), E (propionic acid + ethanol), F (butyric acid + ethanol), G (acetic acid + propionic acid + butyric acid), H (acetic acid + propionic acid + ethanol), I (acetic acid + butyric acid+ ethanol), J (propionic acid + butyric acid + ethanol) and K (acetic acid + propionic acid + butyric acid + ethanol) to assess the toxicity through equitoxic mixing ratio method. The IC₅₀ values of acetic acid, propionic acid, butyric acid and ethanol were 9.812, 7.76, 6.717 and 17.33 g/L respectively, displaying toxicity order of: butyric acid > propionic acid > acetic acid > ethanol being additive in nature. The toxic effects of four VFAs could be designated as synergistic and one additive in nature.     

Synthesis and SAR of vinca alkaloid analogues

Versatile intermediates 12′-iodovinblastine, 12′-iodovincristine and 11′-iodovinorelbine were utilized as substrates for transition metal based chemistry which led to the preparation of novel analogues of the vinca alkaloids. The synthesis of key iodo intermediates, their transformation into final products, and the SAR based upon HeLa and MCF-7 cell toxicity assays is presented. Selected analogues 27 and 36 show promising anticancer activity in the P388 murine leukemia model.