Cytotoxicity assessment of gliotoxin and penicillic acid in Tetrahymena pyriformis.

Various studies have documented the associations between mold exposure and effects on health. Mycotoxins, which occur in spores and mold fragments, can be involved in processes that have pathological effects, such as adynamia of the immune system, recurrent infections of the respiratory tract, or asthma. Using Tetrahymena pyriformis, a single-cell organism well established as a suitable model for human respiratory epithelium-cell functionalities, we investigated dose-response relationships of the mycotoxins gliotoxin and penicillic acid. Our study focused on the viability (cell count, MTT assay), energy levels (adenosine-5'-triphosphate content), energy-providing processes (MTT reduction per cell), and cell respiration (oxygen consumption). Both mycotoxins acted as cytotoxins in a dose-dependent manner. Gliotoxin had a stronger inhibitory effect (EC50 0.38 microM) than did penicillic acid (EC50 343.19 microM). The energy-providing processes were not inhibited or were only weakly inhibited under the influence of gliotoxin, whereas penicillic acid caused stimulation of the physiological parameters. Summarizing the results, it is clear that the two investigated mycotoxins must have different modes of action. They are not only different in the strength of their toxic effects but also in a variety of physiological aspects. In addition, T. pyriformis showed differences in its ability to overcome the negative effects of particular mycotoxin exposures.

Energization of Comamonas testosteroni ATCC 17454 for indicating toxic effects of chlorophenoxy herbicides.

The toxicity of chlorophenoxy herbicides to a bacterium, strongly related to the well-known species Delftia (formerly Comamonas) acidovorans that are able to detoxify these xenobiotics, was investigated. The oxidation of n-hexanol via alcohol dehydrogenases, coupled with the generation of ATP by electron transport phosphorylation (ETP), was used as an indicator for energy-toxic effects on the growth of Comamonas testosteroni ATCC 17454. Uncoupling--reductions in ATP synthesis accompanied by increased respiration--was found to be induced by 1 mM of the classic uncoupler 2,4-dinitrophenol (2,4-DNP) at pH 7.0 and 8.0. At pH 5.4 and 6.0, the ATP synthesis and respiration were strongly inhibited by both 2,4-DNP and the chlorophenoxy herbicides tested. In contrast, 5 mM of 2,4-dichlorophenoxyacetic acid (2,4-D) and of 2-(2,4-dichlorophenoxy)-propanoic acid (2,4-DCPP) were required for detectable uncoupling effects--reduction of the P/O ratios by about 30%--at pH 7.0. These chemicals may have less uncoupling power because the concentration of their protonated (undissociated) forms (pKa values 2.7 and 3.0) is an order of magnitude lower than that of 2,4-DNP (pKa = 4.0) at this pH value. Strong uncoupling accompanied by increased respiration, like that induced by 1 mM 2,4-DNP, was also caused by 5 mM 4-(2,4-dichlorophenoxy)-butyric acid (2,4-DCPB), which correlates with its high pKa value of 4.6. The order of toxicity of the chlorophenoxy herbicides (2,4-D < 2,4-DCPP < 2,4-DCPB) to the ETP, which correlates well with the lipophilicity of their undissociated forms (log P 2.7 < 3.4 < 3.5, respectively), was confirmed by measuring their capacity to inhibit the growth of Comamonas testosteroni ATCC 17454. The results show that energization via alcohol dehydrogenases can be used as an indicator for investigating energy-toxic effects of organics on the ETP and growth of chlorophenoxy herbicide-detoxifying bacteria.

Suitability of the trans/cis ratio of unsaturated fatty acids in Pseudomonas putida NCTC 10936 as an indicator of the acute toxicity of chemicals.

This study explored the suitability of using the trans/cis ratio of unsaturated fatty acids as an indicator of the acute toxicity of membrane active hazardous chemicals. The conversion of cis into trans fatty acids in Pseudomonas putida NCTC 10936 in response to 4-chlorophenol and temperature changes was compared with the results from another kind of toxicity test using the same organism, based on the sensitivity of its xylose oxidation-driven ATP synthesis to uncoupling. The response of both indicators is believed to be largely due to changes in the fluidity of the cytoplasmic membrane. However, the electron transport phosphorylation reacted faster and more sensitively to the fluidizing effect of 4-chlorophenol than the isomerization of unsaturated fatty acids. Therefore, measuring the trans/cis ratio does not provide as good early warning signals of acute toxicity as monitoring the response of the electron transport phosphorylation. If used as an indicator of chemostress, with Pseudomonas species as test organisms, the ratio should only be used in conjunction with other parameters reflecting the energetic state of the cells.

Protein synthesis patterns in Acinetobacter calcoaceticus induced by phenol and catechol show specificities of responses to chemostress.

The proteins induced in Acinetobacter calcoaceticus by the potentially toxic growth substrates phenol and catechol were analyzed by 2D-electrophoresis of cell extracts and compared with those induced by heat shock and oxidative stress. Although both aromatic compounds are quite similar, the only difference being that catechol has an additional hydroxyl group, the responses obtained differed considerably. Phenol has greater lipophilicity and mainly induced heat shock proteins, whereas catechol, which causes the production of reactive oxygen species, predominantly induced oxidative stress proteins. Furthermore, some special proteins were induced by phenol or catechol, which might be useful as biomarkers for chemostress, and could be involved in the catalytic degradation of potentially toxic compounds.

Flow cytometric techniques to characterise physiological states of Acinetobacter calcoaceticus.

Monitoring biotechnological processes involves acquiring information about key metabolic events and, ideally, single cell states should be determined to obtain comprehensive data on the physiological status of the surveyed population. In this paper, growth stages of the strain Acinetobacter calcoaceticus 69-V were characterised at the single cell level using flow cytometry. Four methods for analysing bacterial cellular characteristics by fluorescence were compared with respect to their sensitivity to changes in the physiological states induced by changing micro-environmental conditions. DNA analysis was confirmed to be highly informative with regard to the multiplication activity of the population. Measuring the membrane potential related fluorescence intensity (MPRFI) and the rRNA content were found to be useful for describing high-active cell states. A method for the measurement of the fluidity related fluorescence intensity (FRFI) was developed, since it allowed changes in the fluidity of the bacterial membrane to be detected, and thereby provided a valuable means of tracking adaptation of the population to micro-environmental deviations from optimal growth conditions.

Induction of heat shock proteins in response to primary alcohols in Acinetobacter calcoaceticus.

Cells of Acinetobacter calcoaceticus 69-V, a species able to metabolize a range of aliphatic hydrocarbons and alcohols, were confronted with ethanol, butanol, hexanol or heat shock during growth on acetate as sole source of carbon and energy. The primary alcohols and the heat shock led to the synthesis of new proteins or amplified expression of specific, common and general proteins, which were detected by silver staining after two-dimensional gel electrophoresis. Some of the alcohol-inducible proteins were identified as heat shock proteins by comparing protein patterns of alcohol-shocked cells with those of heat-shocked cells, and by N-terminal amino acid sequencing. DnaK was found to be amplified after all treatments, but GroEI only after heat shock and ethanol treatment. The N-terminal amino acid sequence of the protein, which was considerably amplified after alcohol treatment and heat shock, shows homology to HtpG (high temperature protein G). Some of the heat shock proteins induced by ethanol differ from those induced by butanol and hexanol, suggesting there are at least two different signals for the induction of some heat shock proteins by primary alcohols. This could be due to the different localization of ethanol, butanol and hexanol in the membrane, or because higher cytoplasmic concentrations of ethanol than of butanol or hexanol were applied in these tests in order to keep concentrations of the alcohols in the membrane roughly similar. Besides heat shock proteins, a group of proteins were observed which were only induced by butanol and hexanol, possibly indicating the existence of a further defense mechanism against high concentrations of hydrophobic substrates preventing protein denaturation and membrane damage.

The toxicity of substituted phenolic compounds to a detoxifying and an acetic acid bacterium.

In the detoxifying bacterium Acinetobacter calcoaceticus 69-V and in the acetic acid bacterium Acetobacter methanolicus MB 58, glucose and xylose are oxidized, respectively, via PQQ-dependent membrane-bound dehydrogenases, which are linked to the respiratory chain in a manner enabling energy conservation via electron transport phosphorylation (ETP) in the cytoplasmic membrane. Neither the glucose and gluconic acid nor the xylose and xylonic acid are metabolized. Therefore, measurements of sugar oxidation-driven ATP syntheses ought not to be disturbed by ATP drainage caused by anabolic processes. Studying the effect of substituted phenolic compounds on these energization processes reveals that their toxicity increases with an increasing degree of chlorination and that A. calcoaceticus 69-V is more stable than A. methanolicus MB 58 against chlorinated phenols. On the other hand, A. methanolicus MB 58 is more stable against 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D), especially in the acidic pH range, in which the sensitivity of ATP synthesis to the uncouplers is higher than that of respiration. The toxicity caused by protonophoric activities ought to be barely detectable by respiratory and dehydrogenase tests. The luminescence system of Photobacterium phosphoreum tested in the luminescent bacteria test was much more sensitive. This test system should be used as a screening tool and the effects measured must be confirmed by toxicity tests evaluating the stability of bacteria themselves involved in processes of detoxification as well as the production of toxic metabolites, monitored with respect to their velocity and efficiency.

Chemicals and heat generate different protein patterns in Acinetobacter calcoaceticus.

The effect of exposing Acinetobacter calcoaceticus 69-V to DNP-stress and heat shock was examined by two-dimensional gel electrophoresis of proteins, which were detected either by autoradiography or by silver staining. Both DNP stress and heat shock led to altered patterns of protein synthesis or concentration. About 10% of the proteins which were synthesized newly or at an increased rate and about 25% of those which were found newly or with an increased concentration after DNP treatment were identified after heat shock, too.

The glucose dehydrogenase-mediated energization of Acinetobacter calcoaceticus as a tool for evaluating its susceptibility to, and defence against, hazardous chemicals.

Cells of Acinetobacter calcoaceticus 69-V could be energized by glucose oxidation after the growth on acetate, ethanol, hexanol and benzoate. The velocities of glucose oxidation-driven ATP syntheses were relatively constant in the range from pH 5.4 to 7.5. With decreasing pH values (7.0, 6.0, 5.4) ATP synthesis was inhibited more strongly by the action of 2,4-dinitrophenol and at the same pH value glucose oxidation was nearly unimpaired or inhibited more weakly. This finding is expressed by a decrease of the P/O ratios, indicating the uncoupling of the electron-transport phosphorylation by 2,4-dinitrophenol. The sensitivity towards this uncoupling effect was higher in ethanol-grown cells of Acinetobacter calcoaceticus 69-V than in hexanol- or acetate-grown cells. This increase in sensitivity was accompanied by a decrease of the ratio of saturated (mainly C16:0) to unsaturated (C16:1, C18:1) fatty acids in ethanol-grown cells compared with hexanol-grown ones. The knowledge of such differences in the susceptibility and its molecular background, e.g. possible substrate-induced changes of the fatty acid composition of the cytoplasmic membranes, should help elucidate mechanisms of poisoning by membrane-active hazardous chemicals and develop defence strategies.

[Regulation of PEP-carboxylase of the facultative methylotrop Acetobacter sp. MB 58]. / Regulation der PEP-Carboxylase des fakultativ methylotrophen Acetobacter sp. MB 58.

Acetobacter sp. MB 58 assimilates methanol via the fructose-1,6-bisphosphate variant of the hexulose phosphate pathway. Glyceraldehyde-3-phosphate originates as net product of an assimilation loop involving the regeneration of the C1-acceptor ribulose-5-phosphate and must be available for the de novo synthesis of the C1-acceptor as well as for the oxidative glycolysis. It is made probable in a regulatory model that this is accomplished via alternating anabolic and catabolic phases which are controlled by concerted action of PEP-carboxylase and pyruvate kinase. Whereas Ac-CoA is a crucial effector and alpha-ketoglutarate and aspartate are inhibitors for the PEP-carboxylase, the pyruvate kinase is assumed to be regulated by energy charge.