In vitro assessment of the toxicity of metal compounds

A review of in vitro mutagenesis assessment of metal compounds in mammalian and nonmammalian test systems has been compiled. Prokaryotic assays are ineffective or inconsistent in their detection of most metals as mutagens, with the notable exception of hexavalent chromium. Mammalian assay systems appear to be similarly inappropriate for the screening of metal compounds based upon the limited number of studies that have employed those compounds having known carcinogenic activity. Although of limited value as screening tests for the detection of potentially carcinogenic metal compounds, the well-characterized in vitro mutagenesis systems may prove to be of significant value as a means to elucidate mechanisms of metal genotoxicity.     

The oxo/peroxo debate: a nonheme iron perspective

The oxygen activation mechanisms proposed for nonheme iron systems generally follow the heme paradigm in invoking the involvement of iron-peroxo and iron-oxo species in their catalytic cycles. However, the nonheme ligand environments allow for end-on and side-on dioxygen coordination and impart greater flexibility in the modes of dioxygen activation. The currently available evidence for nonheme iron-peroxo and iron-oxo intermediates is summarized and discussed in light of the ongoing discussion on the nature of the oxidant(s) in heme enzymes.     

Computational Oral Absorption Simulation for Low‐Solubility Compounds

Bile micelles play an important role in oral absorption of low‐solubility compounds. Bile micelles can affect solubility, dissolution rate, and permeability. For the pH–solubility profile in bile micelles, the Henderson–Hasselbalch equation should be modified to take bile‐micelle partition into account. For the dissolution rate, in the Nernst–Brunner equation, the effective diffusion coefficient in bile‐micelle media should be used instead of the monomer diffusion coefficient. The diffusion coefficient of bile micelles is 8‐ to 18‐fold smaller than that of monomer molecules. For permeability, the effective diffusion coefficient in the unstirred water layer adjacent to the epithelial membrane, and the free fraction at the epithelial membrane surface should be taken into account. The importance of these aspects is demonstrated here using several in vivo and clinical oral‐absorption data of low‐solubility model compounds. Using the theoretical equations, the food effect on oral absorption is further discussed.     

Pretubulysin: a new option for the treatment of metastatic cancer

Tubulin-binding agents such as taxol, vincristine or vinblastine are well-established drugs in clinical treatment of metastatic cancer. However, because of their highly complex chemical structures, the synthesis and hence the supply issues are still quite challenging. Here we set on stage pretubulysin, a chemically accessible precursor of tubulysin that was identified as a potent microtubule-binding agent produced by myxobacteria. Although much simpler in chemical structure, pretubulysin abrogates proliferation and long-term survival as well as anchorage-independent growth, and also induces anoikis and apoptosis in invasive tumor cells equally potent to tubulysin. Moreover, pretubulysin posseses in vivo efficacy shown in a chicken chorioallantoic membrane (CAM) model with T24 bladder tumor cells, in a mouse xenograft model using MDA-MB-231 mammary cancer cells and finally in a model of lung metastasis induced by 4T1 mouse breast cancer cells. Pretubulysin induces cell death via the intrinsic apoptosis pathway by abrogating the expression of pivotal antiapoptotic proteins, namely Mcl-1 and Bcl-x_L, and shows distinct chemosensitizing properties in combination with TRAIL in two- and three-dimensional cell culture models. Unraveling the underlying signaling pathways provides novel information: pretubulysin induces proteasomal degradation of Mcl-1 by activation of mitogen-activated protein kinase (especially JNK (c-Jun N-terminal kinase)) and phosphorylation of Mcl-1, which is then targeted by the SCF^Fbw7 E3 ubiquitin ligase complex for ubiquitination and degradation. In sum, we designate the microtubule-destabilizing compound pretubulysin as a highly promising novel agent for mono treatment and combinatory treatment of invasive cancer.     

Plant responses to H2S and SO2 fumigation. I. Effects on growth, transpiration and sulfur content of spinach

Exposure of spinach (Spinacia oleracea L. cv. Monosa) to 0.25 μl l_?1 H₂S reduced the relative growth rate by 26, 47 and 60% at 15, 18 and 25°C, respectively. Shoot to root ratio decreased in plants fumigated at 18 and 25°C. Growth of spinach was not affected by a 2-week exposure to 0.10 or 0.25 μl l_?1 SO₂. Both H₂S and SO₂ fumigation increased the content of sulfhydryl compounds and sulfate. A 2-week exposure to 0.25 μl l_?1 H₂S resulted in an increase in sulfhydryl and sulfate content of 250 to 450% and 63 to 248% in the shoots, respectively, depending on growth temperature. Exposure to 0.15 and 0.30 μl l_?1 H₂S at 20°C for 2 weeks resulted in a 46% increase in sulfate content of the shoots at 0.30 μl l_?1 and no detectable increase at 0.15 μl l_?1 H₂S; the sulfate content of the roots increased by 195 and 145% at 0.15 and 0.30 μl l_?1 H₂S, respectively. Fumigation with 0.25 μl l_?1 SO₂ at 20°C for 2 weeks resulted in an increase in sulfhydryl content and sulfate content in the shoots of 285% and 300 to 1100%. H₂S fumigation during the 12 h light period or only during the dark period resulted in identical growth reduction and accumulation of sulfhydryl compounds; they were about 50 and 67% of those observed in continuously exposed plants. H₂S- and SO₂-exposed plants showed an increased transpiration rate, which was mainly caused by an increased dark-period transpiration. No effect of H₂S and SO₂ on the water uptake of the plants and the osmotic potential of the leaves was detected. Plants fumigated with 0.25 μl l_?1 H₂S for 2 weeks were smaller and differed morphologically from the control plants by slightly more abaxially curved leaf margins. Cross sections of the leaves showed smaller cells at the margins and smaller and fewer air spaces. The increased transpiration in the H₂S-exposed plants is discussed in relation to the observed morphological changes.     

Anaerobic degradation of phenol via carboxylation to 4-hydroxybenzoate: in vitro study of isotope exchange between 14CO2 and 4-hydroxybenzoate

Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U-¹⁴C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn²⁺ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol ¹⁴CO₂ exchange · min^-1 · mg^-1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min^-1 · mg^-1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed.     

Biotransformations of aromatic aldehydes by acetogenic bacteria

Vanillin was subject to O demethylation and supported growth of Clostridium formicoaceticum and Clostridium thermoaceticum. Vanillin was also stimulatory to the CO-dependent growth of Peptostreptococcus productus. The aldehyde substituent of vanillin was metabolized by routes which were dependent upon both the acetogen and a co-metabolizable substrate (e.g. carbon monoxide [CO]). C. formicoaceticum and C. thermoaceticum oxidized the aldehyde group of vanillin to the carboxyl level, while P. productus reduced the aldehyde group of vanillin to the alcohol level. In contrast, during CO-dependent growth, C. thermoaceticum reduced 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol while P. productus both reduced and oxidized 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol and 4-hydroxybenzoate, respectively. These metabolic potentials indicate aromatic aldehydes may affect the flow of reductant during acetogenesis.     

Degradation of diarylethane structures by Pseudomonas fluorescens biovar I

Pseudomonas fluorescens biovar I was isolated from a pulp mill effluent based on its ability to grow on synthetic media containing 1,2-diarylethane structures as the sole carbon and energy source. Analysis of samples taken from cultures of this strain in benzoin or 4,4-dimethoxybenzoin (anisoin), showed that cleavage between the two aliphatic carbons takes place prior to ring fission. Intermonomeric cleavage was also obtained with crude extracts. Substrates of this reaction were only those 1,2-diarylethane compounds that supported growth of the bacterium. The purification and partial characterization of an enzyme that catalyzes the NADH-dependent reduction of the carbonyl group of benzoin and anisoin is also reported.     

Anaerobic degradation of trans-cinnamate and ω-phenylalkane carboxylic acids by the photosynthetic bacterium Rhodopseudomonas palustris: evidence for a β-oxidation mechanism

The mechanism responsible for the initial steps in the anaerobic degradation of trans-cinnamate and -phenylalkane carboxylates by the purple non-sulphur photosynthetic bacterium Rhodopseudomonas palustris was investigated. Phenylacetate did not support growth and there was a marked CO₂ dependence for growth on acids with greater side-chain lengths. Here, CO₂ was presumably acting as a redox sink for the disposal of excess reducing equivalents. Growth on benzoate did not require the addition of exogenous CO₂. Aromatic acids with an odd number of side-chain carbon atoms (3-phenylpropionate, 5-phenylvalerate, 7-phenylheptanoate) gave greater apparent molar growth yields than those with an even number of side-chain carbon atoms (4-phenylbutyrate, 6-phenylhexanoate, 8-phenyloctanoate). HPLC analysis revealed that phenylacetate accumulated and persisted in the culture medium during growth on these latter compounds. Cinnamate and benzoate transiently accumulated in the culture medium during growth on 3-phenylpropionate, and benzoate alone accumulated transiently during the course of trans-cinnamate degradation. The transient accumulation of 4-phenyl-2-butenoic acid occurred during growth on 4-phenylbutyrate, and phenylacetate accumulated to a 1:1 molar stoichiometry with the initial 4-phenylbutyrate concentration. It is proposed that the initial steps in the anaerobic degradation of trans-cinnamate and the group of acids from 3-phenylpropionate to 8-phenyloctanoate involves -oxidation of the side-chain.Abbreviation 3-PP 3-phenylpropionic acid - 4-PB 4-phenylbutyric acid - 5-PV 5-phenylvaleric acid - 6-PH 6-phenylhexanoic acid - 7-PH 7-phenylheptanoic acid - 8-PO 8-phenyloctanoic acid - 4-P2B 4-phenyl-2-butenoic acid - GC/MS Gas chromatography/Mass spectrometry - HPLC High-pressure liquid chromatography     

Anaerobic degradation of hydroaromatic compounds by newly isolated fermenting bacteria

Aerobic organisms degrade hydroaromatic compounds via the hydroaromatic pathway yielding protocatechuic acid which is further metabolized by oxygenase-mediated ring fission in the 3-oxoadipate pathway. No information exists on anaerobic degradation of hydroaromatics so far. We enriched and isolated from various sources of anoxic sediments several strains of rapidly growing gram-negative bacteria fermenting quinic (1,3,4,5-tetrahydroxy-cyclohexane-1-carboxylic acid) and shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) in the absence of external electron acceptors. Quinic and shikimic acid were the only ones utilized of more than 30 substrates tested. The marine isolates formed acetate, butyrate, and H₂, whereas all freshwater strains formed acetate and propionate as typical fermentation products. Aromatic intermediates were not involved in this degradation. Characterization of the isolates, fermentation balances for both hydroaromatic compounds, and enzyme activities involved in one degradation pathway are presented.Abbreviations BV benzyl viologen (1,1-dibenzyl-4,4-bipyridinium dichloride) - CoA coenzyme A - CTAB cetyltrimethylammonium bronide - DCPIP 2,4-dichlorophenolindophenol - DTT 1,4-dithiotheriol - MV methyl viologen (1,1-dimethyl-4,4-bipyridinium dichloride) - Tricine N-[tris-(hydroxymethyl)-methyl]-glycine - Tris tris-(hydroxymethyl)-aminomethane