Luminescent bacteria toxicity assay in the study of mercury speciation

The toxicities of solutions of 10 mercury compounds to luminescent bacteria were measured using the Microtox Toxicity Bioassay. The aim of this study was to assess the influence that the counter-ions have on the aquatic toxicity of mercury salts. The toxicities of these mercury compounds were very similar, except for mercurous tannate and mercuric salicylate. This can be attributed to differences in the ionization and speciation patterns of these compounds relative to the other compounds tested. In general, the toxicity of the solutions at pH 5 was not significantly different from the toxicity of these solutions at pH 6, but a clear reduction in toxicity was observed when the pH of the solution was adjusted to pH 9. Significant differences were found between the toxicity of Hg(I) and Hg(II) salts of the same anion at pH 9. When cysteine was added to a mercuric nitrate solution (at pH 6), a reduction in the toxicity was observed. This can be explained in terms of the strong binding of mercury to cysteine, thus reducing the concentration of mercury species available to cause an observable toxic effect to the bioluminescent bacteria.     

Development of a toxicity test system using primary rat liver cells

Summary A model in vitro rat liver parenchymal cellular toxicity system employing cells obtained by the in situ collagenase perfusion technique has been developed to detect potential liver toxicants. The initial evaluation of this test system was accomplished using cadmium chloride, chromium chloride, cobalt chloride, mercuric chloride, nickelous chloride, sodium arsenite, sodium selenite, and ammonium vanadate. Linear regression analysis of the dose response curves was used to determine the effective concentration at which the viability was reduced to 50% (EC₅₀). The relative toxicity of the compounds was as follows: Cd>V=As>Se>Hg>Cr=Co>Ni. Since several of the compounds with very similar EC_50S had significantly different dose response slopes, an additional parameter, lowest effective concentration tested (LECT) was employed to assess the relative toxicity. The LECT was determined using the Williams test and the relative toxicity of the compounds was found to be Cd=Se>V>As=Hg>Co>Cr=Ni. The primary objective in developing this rat liver cellular toxicity test system was to employ an in vitro test system utilizing metabolically active primary cells from a potential target organ. This study demonstrates the utility of this test system in determining the relative liver cell toxicity of a series of inorganic agents of differing toxicity.     

Affinity-based inhibition of beta-amyloid toxicity

Strategies for interfering with protein aggregation are important for elucidating and controlling the pathologies of amyloid diseases. We have previously identified compounds that block the cellular toxicity of the beta-amyloid peptide, but the relationship between their ability to inhibit toxicity and their affinity for A beta is unknown. To elucidate this relationship, we have developed an assay capable of measuring the affinities of small molecules for beta-amyloid peptide. Our approach employs immobilized beta-amyloid peptide at low density to minimize the problems that arise from variability in the beta-amyloid aggregation state. We found that low-molecular weight (MW of 700-1700) ligands for beta-amyloid can be identified readily by using surface plasmon resonance. The best of these bound effectively (K(d) approximately 40 microM) to beta-amyloid. The affinities measured for peptides in the SPR assay correspond to results from A beta cell toxicity assays. The most potent ligands for immobilized beta-amyloid are the most potent inhibitors of the neuronal cell toxicity of beta-amyloid. Compounds with dissociation constants above approximately 100 microM did not show significant activity in the cell toxicity assays. Our data support the hypothesis that ligands exhibiting greater affinity for the beta-amyloid peptide are effective at altering its aggregation and inhibiting cell toxicity.     

Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms

Cryoprotectant toxicity is a fundamental obstacle to the full potential of artificial cryoprotection, yet it remains in general a poorly understood phenomenon. Unfortunately, most relevant biochemical studies to date have not met the basic criteria required for demonstrating mechanisms of toxicity. A model biochemical study of cryoprotectant toxicity was that of Baxter and Lathe, which demonstrated that alteration of a specific enzyme (fructose diphosphatase, or FDPase) was the cause of impaired glycolysis after treatment with and removal of dimethyl sulfoxide (D). FDPase alteration by D was reported to be preventable by the simultaneous presence of amides. This protection could be due to a "counteracting solute" effect similar to that employed by nature, but we find no meaningful correlation between the general protein stabilizing or destabilizing tendency of the cryoprotectant medium and its toxicity. Baxter and Lathe postulated that the effect of D arises from hydrogen bonding between D and the epsilon amino groups of surface lysine residues on FDPase, and it was found that molecules which resembled this group could block the alteration induced by D, presumably by competing with lysine residues for association with D. However, we find that the interaction between D and lysine in the presence of water is actually thermochemically repulsive, and that the presence of formamide does not affect the interaction between D and lysine, implying no useful complex formation between formamide and D. We were also unable to demonstrate that the blocking compounds consistently reduce toxicity when added to D rather than substituting for D, contrary to predictions based on complex formation between blocking compounds and D. In summary, it seems that present concepts of cryoprotectant toxicity are in need of serious revision.     

PCNA indexing as a preclinical immunohistochemical biomarker for testicular toxicity

It is well known that toxicants such as cyclophosphamide and ethanol can have deleterious effects on normal spermatogenesis. End points such as testis weight and sperm counts have been used widely to assess gross structural and functional changes in testes resulting from toxicant exposure. Histopathological assessments are more sensitive measures of testicular health, but generally they are neither quantitative nor sensitive enough to detect early toxicity. Recently, immunolabeling cells with proliferating cell nuclear antigen (PCNA) has been used to identify proliferating spermatogonia; however, there have been no systematic attempts to quantify these changes. We have developed a sensitive, reliable and quantitative assay using immunohistochemistry on formalin fixed, paraffin embedded rat testes to assess the degree of proliferation-related toxicity. An indexing scheme was derived based on the determination of radially positioned PCNA-positive cells within similarly staged seminiferous tubules presenting a single layer of PCNA-positive cells along the basement membrane of the basal tubular compartment. An average of 60 tubules in the testes were counted per animal. Our results show significant decreases in the PCNA index in rats treated with an experimental compound that has been shown to produce testicular histopathology. The analysis provides a quick, reliable, sensitive, and quantitative means for assessing early testicular toxicity. The assay has potential utility as an in vivo biomarker for detecting early testicular toxicity of experimental compounds in preclinical development as well as for refining follow-up compounds with reduced testicular toxicity.     

Separation and aquatic toxicity of enantiomers of 1-(substituted phenoxyacetoxy)alkylphosphonate herbicides

A series of organophosphorous compounds (OPs), 1-(substituted phenoxyacetoxy)alkylphosphonates containing a chiral carbon atom, show notable herbicidal activities. In this study, the enantioselective separation and biological toxicity of all these compounds were investigated. The enantioselective separation on the columns of Chiralpak AD, Chiralpak AS, Chiralcel OD, and Chiralcel OJ were compared under various chromatographic conditions. All the analytes investigated obtained baseline resolution (R(s) > 1.5) on Chiralpak AD column, which showed best chiral separation capacity. Further investigation was carried out on Chiralpak AD to evaluate the influence of the mobile phase composition and column temperature. The effect of the structural features on discrimination was also examined. The resolved enantiomers were distinguished by their signs of circular dichroism. The acute aquatic toxicity of enantiomers and racemate to Daphnia magna (D. magna) were assessed. The in vivo assays showed that compound 3 was about 2-148.5 times more toxic than the other four analogues to D. magna. The racemates of compounds 3 and 5 showed intermediate toxicity compare to their enantiomers, while those of compounds 1, 2, and 4 showed synergistic or antagonistic effect. These results suggest that the biological toxicity of chiral OPs to nontarget organisms is enantioselective and therefore should be evaluated with their pure enantiomers.     

Examination of a rodent limb bud micromass assay as a prescreen for developmental toxicity

The mouse limb bud micromass assay is one of many short-term tests proposed as preliminary screens for potential developmental toxicity. Previous efforts to validate this assay have used too few "nonteratogens." The purpose of this study was to examine additional compounds, most of which, based on the literature, were perceived to have low potential for developmental toxicity in vivo. In addition, a method of data analysis was sought that would identify selective developmental toxins in the micromass assay, i.e., those that are effective at dosages not maternally toxic. The concentration of each of 23 compounds that produced a 50% inhibition (IC50) of radiolabeled thymidine (T) and sulfate (S) incorporation was determined and used to calculate a T/S ratio. The T/S ratio may be a useful measure of developmental hazard, since T incorporation measures toxicity toward a general cell function (DNA synthesis) and S incorporation measures mainly toxicity toward a developmentally specific cell activity (chondroitin sulfate synthesis). All compounds tested produced T/S ratios of less than 2.0. Since 22 of these 23 compounds are classified as "nonteratogens" or nonselective developmental toxins in vivo, a low T/S ratio in this in vitro assay system may be capable of discriminating potential for developmental hazard in vivo.     

Implications of a statistical occurrence model for mixture toxicity estimation

This study provides a method for characterizing the effects of concentration variability and correlation among co-acting compounds on mixture toxicity, considering the implications of missing chemical data. The method is explored by developing a set of multiple occurrence scenarios for mixtures of related chemicals. The calculations are performed for hypothetical mixtures of a group of ten synthetic antibiotics that have been tested on marine bacterium to fit dose-response relationships for long-term bioluminescence inhibition of Vibrio fischeri. Mixture toxicities are computed and compared for the assumptions of independent joint action theory and concentration/dose addition theory. The study results show that higher variability in concentrations is associated with higher effective (average) mixture toxicity, in this application by as much as a factor of ten for mixtures with highly variable component concentrations. Moreover, omitting the most toxic compounds caused mixture toxicities to be underestimated by a factor of approximately two. We recommend a pre-assessment of the effect of different chemical occurrence patterns and variability on mixture toxicity to help prioritize needs for further co-occurrence data and toxicity studies.     

Radioprotective effectiveness and toxicity of 4,5-disubstituted tryptamines

In experiments on mice a study was made of different substituents in the 4th position of the indole ring of 5-methoxytryptamines (5-MOT) on toxicity and radioprotective efficiency of the compounds of this class. It was shown that the administration of the amino-group to a mexamine molecule increased the preparation toxicity; the nitro-group somewhat diminished the toxic properties, and the acetylamino group did not change 5-MOT toxicity. A 5-MOT derivative with a nitro group possessed the strongest radioprotective action. The radioprotective efficiency of these compounds persisted for 1-2 h.     

CP5484, a novel quaternary carbapenem with potent anti-MRSA activity and reduced toxicity

A new series of 1beta-methyl carbapenems possessing a 6,7-disubstituted imidazo[5,1-b]thiazol-2-yl group directly attached to the C-2 position of the carbapenem nucleus was prepared, and the activities of these compounds against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. To study the effect of basic moieties on anti-MRSA activity, we introduced an amino, or imino, or amidino group at the 6-position of imidazo[5,1-b]thiazole in place of the carbamoylmethyl moiety of CP5068. Anti-MRSA activities of almost all basic group-substituted carbapenems were improved, though some of the compounds showed stronger acute toxicity in mice than IPM. In order to decrease the toxicity without decreasing the activity, we introduced various additional functionalities around the basic moiety. Finally, we obtained CP5484, which has excellent anti-MRSA activity and low acute toxicity.     

Structure-function relationships for inhibitors of beta-amyloid toxicity containing the recognition sequence KLVFF.

Beta-amyloid (Abeta), the primary protein component of Alzheimer's plaques, is neurotoxic when aggregated into fibrils. We have devised a modular strategy for generating compounds that inhibit Abeta toxicity. These compounds contain a recognition element, designed to bind to Abeta, linked to a disrupting element, designed to interfere with Abeta aggregation. On the basis of this strategy, a hybrid peptide was synthesized with the sequence KLVFF (residues 16-20 of Abeta) as the recognition element and a lysine hexamer as the disrupting element; this compound protects cells in vitro from Abeta toxicity [Pallitto, M. M., et al. (1999) Biochemistry 38, 3570]. To determine if the length of the disrupting element could be reduced, peptides were synthesized that contained the KLVFF recognition element and a sequence of one to six lysines as disrupting elements. All compounds enhanced the rate of aggregation of Abeta, with the magnitude of the effect increasing as the number of lysines in the disrupting element increased. The greatest level of protection against Abeta toxicity was achieved with compounds containing disrupting elements of three or more lysines in sequence. A peptide with an anionic disrupting element, KLVFFEEEE, had activity similar to that of KLVFFKKKK, in both cellular toxicity and biophysical assays, whereas a peptide with a neutral polar disrupting element, KLVFFSSSS, was ineffective. Protective compounds retained activity even at an inhibitor:Abeta molar ratio of 1:100, making these some of the most effective inhibitors of Abeta toxicity reported to date. These results provide critical insight needed to design more potent inhibitors of Abeta toxicity and to elucidate their mechanism of action.     

Compound toxicity screening and structure-activity relationship modeling in Escherichia coli

Synthetic biology and metabolic engineering are used to develop new strategies for producing valuable compounds ranging from therapeutics to biofuels in engineered microorganisms. When developing methods for high-titer production cells, toxicity is an important element to consider. Indeed the production rate can be limited due to toxic intermediates or accumulation of byproducts of the heterologous biosynthetic pathway of interest. Conversely, highly toxic molecules are desired when designing antimicrobials. Compound toxicity in bacteria plays a major role in metabolic engineering as well as in the development of new antibacterial agents. Here, we screened a diversified chemical library of 166 compounds for toxicity in Escherichia coli. The dataset was built using a clustering algorithm maximizing the chemical diversity in the library. The resulting assay data was used to develop a toxicity predictor that we used to assess the toxicity of metabolites throughout the metabolome. This new tool for predicting toxicity can thus be used for fine-tuning heterologous expression and can be integrated in a computational-framework for metabolic pathway design. Many structure-activity relationship tools have been developed for toxicology studies in eukaryotes [Valerio (2009), Toxicol Appl Pharmacol, 241(3): 356-370], however, to the best of our knowledge we present here the first E. coli toxicity prediction web server based on QSAR models (EcoliTox server: http://www.issb.genopole.fr/～faulon/EcoliTox.php).     

Correlation of metal toxicity with in vitro calmodulin inhibition

A fundamental biochemical process that is directly and universally related to the toxicity of metals and metal compounds has yet to be identified. Results of studies reported here indicate that the toxicity of a series of divalent metal cations correlates well with the metals' ability to inhibit the Ca2+ receptor protein calmodulin (r = 0.986). Because calmodulin regulates a variety of cellular enzymes and processes including intracellular Ca2+ concentrations, calmodulin inhibition may have value for predicting metal toxicity and for revealing information about the mechanism by which metals induce toxic effects.     

Use of toxicity assays for enantiomeric discrimination of pharmaceutical substances

Toxicity assays are commonly used as general indicators of environmental water pollution. In the study described here, selected toxicity tests have been used to evaluate the different toxicity levels of enantiomers of different pharmaceutical drugs that can be found as potential contaminants in water environments. Isomers of dopa, fluoxetine, and atenolol were tested with three aquatic organisms corresponding to different trophic levels: Daphnia magna (a crustacean), Pseudokirchneriella subcapitata (a microalga), and Tetrahymena thermophila (a protozoan). Different levels of toxicity were observed for each enantiomer, suggesting that significant enantioselectivity occurs in aquatic toxicity and that such enantiomeric differences must be considered when evaluating the ecological effects of these compounds.     

Background and overview of current sediment toxicity identification evaluation procedures

Laboratory bioassays can provide an integrated assessment of the potential toxicity of contaminated sediments to aquatic organisms; however, toxicity as a sole endpoint is not particularly useful in terms of identifying remedial options. To focus possible remediation (e.g., source control), it is essential to know which contaminants are responsible for toxicity. Unfortunately, contaminated sediments can contain literally thousands of potentially toxic compounds. Methods which rely solely on correlation to identify contaminants responsible for toxicity are limited in several aspects: (a) actual compounds causing toxicity might not be measured, (b) concentrations of potentially toxic compounds may covary, (c) it may be difficult to assess the bioavailability of contaminants measured in a sediment, and (d) interactions may not be accounted for among potential toxicants (e.g., additivity). Toxicity identification evaluation (TIE) procedures attempt to circumvent these problems by using toxicity-based fractionation procedures to implicate specific contaminants as causative toxicants. Phase I of TIE characterizes the general physio-chemical nature of sample toxicants. Phase II employs methods to measure toxicants via different analytical methods, and Phase III consists of techniques to confirm that the suspect toxicants identified in Phases I and II of the TIE actually are responsible for toxicity. These TIE procedures have been used to investigate the toxicity of a variety of samples, including sediments. Herein we present a brief conceptual overview of the TIE process, and discuss specific considerations associated with sediment TIE research. Points addressed include: (a) selection and preparation of appropriate test fractions, (b) use of benthic organisms for sediment TIE work, and (c) methods for the identification of common sediment contaminants.     

The toxicity of plant alkaloids: an Ecogeographic perspective

The toxicity of alkaloids was sought in relation to the geography of alkaloid-bearing plants. Relative toxicity was inferred from the structure of the compounds, which exhibit regular structure-activity relationships. The toxicity of alkaloids from tropical plants is much greater than those from temperate plants. No significant differences were found between herbaceous and woody plants. The alkaloid content of leaves tends to be a positive function of alkaloid toxicity. The ecogeographical distribution of toxicity is interpreted in terms of plant defense.     

The direct toxicity of alpha-naphthylisothiocyanate in cell culture

α-naphthylisothiocyanate (ANIT) kills rat liver cells in culture. Testing of a variety of related compounds revealed that toxicity depended upon the isothiocyanate group. The toxicity of ANIT was similar on several lines of cultured cells and in the presence of inhibitors and inducers of microsomal enzyme systems. The addition of a microsomal metabolizing system from rat liver to the cell cultures greatly enhanced the toxicity of 2 hepatotoxins requiring metabolism, but did not affect the toxicity of ANIT. It is concluded therefore that ANIT is toxic without biotransformation.