The metabolism and molecular toxicology of chloroprene

Chloroprene (2-chloro-1,3-butadiene, 1) is oxidised by cytochrome P450 enzymes in mammalian liver microsomes to several metabolites, some of which are reactive towards DNA and are mutagenic. Much less of the metabolite (1-chloroethenyl)oxirane (2a/2b) was formed by human liver microsomes compared with microsomes from Sprague-Dawley rats and B6C3F1 mice. Epoxide (2a/2b) was a substrate for mammalian microsomal epoxide hydrolases, which showed preferential hydrolysis of the (S)-enantiomer (2b). The metabolite 2-chloro-2-ethenyloxirane (3a/3b) was rapidly hydrolysed to 1-hydroxybut-3-en-2-one (4) and in competing processes rearranged to 1-chlorobut-3-en-2-one (5) and 2-chlorobut-3-en-1-al (6). The latter compound isomerised to (Z)-2-chlorobut-2-en-1-al (7). In microsomal preparations from human, rat and mouse liver, compounds 4, 5 and 7 were conjugated by glutathione both in the absence and presence of glutathione transferases. There was no evidence for the formation of a chloroprene diepoxide metabolite in any of the microsomal systems. The major adducts from the reaction of (1-chloroethenyl)oxirane (2a/2b) with calf thymus DNA were identified as N7-(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (20) and N3-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyuridine (23), with the latter being derived by alkylation at N-3 of 2'-deoxycytidine, followed by deamination. Adducts in DNA were identified by comparison with those derived from individual deoxyribonucleosides. The metabolite (Z)-2-chlorobut-2-en-1-al (7) formed principally two adducts with 2'-deoxyadenosine which were identified as a pair of diastereoisomers of 3-(2'-deoxy-beta-d-ribofuranosyl)-7-(1-hydroxyethyl)-3H-imidazo[2,1-i]purine (25). The chlorine atom of chloroprene thus leads to different intoxication and detoxication profiles compared with those for butadiene and isoprene. The results infer that in vivo oxidations of chloroprene catalysed by cytochrome P450 are more important in rodents, whereas hydrolytic processes catalysed by epoxide hydrolases are more pronounced in humans. The reactivity of chloroprene metabolites towards DNA is important for the toxicology of chloroprene, especially when detoxication is incomplete.     

Two functionally distinct Axin-like proteins regulate canonical Wnt signaling in C. elegans

Axin is a central component of the canonical Wnt signaling pathway that interacts with the adenomatous polyposis coli protein APC and the kinase GSK3beta to downregulate the effector beta-catenin. In the nematode Caenorhabditis elegans, canonical Wnt signaling is negatively regulated by the highly divergent Axin ortholog PRY-1. Mutation of pry-1 leads to constitutive activation of BAR-1/beta-catenin-dependent Wnt signaling and results in a range of developmental defects. The pry-1 null phenotype is however not fully penetrant, indicating that additional factors may partially compensate for PRY-1 function. Here, we report the cloning and functional analysis of a second Axin-like protein, which we named AXL-1. We show that despite considerable sequence divergence with PRY-1 and other Axin family members, AXL-1 is a functional Axin ortholog. AXL-1 functions redundantly with PRY-1 in negatively regulating BAR-1/beta-catenin signaling in the developing vulva and the Q neuroblast lineage. In addition, AXL-1 functions independently of PRY-1 in negatively regulating canonical Wnt signaling during excretory cell development. In contrast to vertebrate Axin and the related protein Conductin, AXL-1 and PRY-1 are not functionally equivalent. We conclude that Axin function in C. elegans is divided over two different Axin orthologs that have specific functions in negatively regulating canonical Wnt signaling.     

Genome-wide studies of histone demethylation catalysed by the fission yeast homologues of mammalian LSD1

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1(+) gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1Delta strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression.     

Development and validation of an HPLC method for the determination of dibenzoylmethane in rat plasma and its application to the pharmacokinetic study

A highly sensitive and simple high-performance liquid chromatographic (HPLC) assay has been developed and validated for the quantification of dibenzoylmethane (DBM) in rat plasma. DBM and internal standard (I.S.) 1-(5-chloro-2-hydroxy-4-methylphenyl)-3-phenyl-1,3-propanedione (CHMPP) were extracted from rat plasma by ethyl acetate/methanol (95:5, v/v) and analyzed using reverse-phase gradient elution with a Phenomenex Gemini C18 5-mum column. A gradient of mobile phase (mobile phase A: water/methanol (80:20, v/v) with 0.1% TFA and mobile phase B: acetonitrile with 0.1% TFA) at a flow rate of 0.2 mL/min, and ultraviolet (UV) detection at 335 nm were utilized. The lower limit of quantification (LLOQ) using 50 microL rat plasma was 0.05 microg/mL. The calibration curve was linear over a concentration range of 0.05-20 microg/mL. The mean recoveries were 80.6+/-5.7, 83.4+/-1.6 and 77.1+/-3.4% with quality control (QC) level of 0.05, 1 and 20 microg/mL, respectively. Intra- and inter-day assay accuracy and precision fulfilled US FDA guidance for industry bioanalytical method validation. Stability studies showed that DBM was stable in rat plasma after 4h incubation at room temperature, one month storage at -80 degrees C and three freeze/thaw cycles, as well as in reconstitute buffer for 48 h at 4 degrees C. The utility of the assay was confirmed by the successful analysis of plasma samples from DBM pharmacokinetics studies in the rats after oral and intravenous administrations.     

Comparison of Emulsifying Properties of Plant and Animal Proteins in Oil-in-Water Emulsions: Whey,Soy, and RuBisCo Proteins

There is increasing interest within the food industry in replacing animal-derived ingredients with plant-derived alternatives. In this study, we compared the emulsifying properties of an emerging plant protein (RuBisCo protein) with those of a well-established plant (soy protein) and animal (whey protein) protein. The RuBisCo protein (ribulose 1,5-bisphosphate carboxylase) was isolated from duckweed (lemna minor), which is an abundant plant material with a higher protein yield and biomass per unit area than most other plant protein sources. The ability of the three proteins to form and stabilize 10 wt% soybean oil-in-water emulsions was examined. The minimum amount of protein required to produce small droplets (d <?350 nm) decreased in the following order: RuBisCo > soy > whey protein. This effect was mainly attributed to the fact that the molar mass of the proteins decreased in the same order. Even so, the RuBisCo proteins were able to form stable emulsions when used at sufficiently high concentrations (≥ 1%). All three types of protein-coated oil droplets aggregated at pH values near their isoelectric points and at high ionic strengths but there were differences between them. In the absence of added salt, extensive droplet aggregation occurred from pH 4 to 5 for whey protein, from pH 2 to 5 for soy protein, and from pH 2 to 6 for RuBisCo protein. The isoelectric points of all three protein-coated droplets were around pH 5, but the magnitude of the surface potential at low and high pH values was higher for whey protein than for the two plant proteins. At pH 7, extensive droplet aggregation occurred at ≥100 mM NaCl for RuBisCo- and soy protein-coated droplets, but only at ≥400 mM NaCl for whey protein-coated ones. The RuBisCo-coated oil droplets were more prone to flocculation when heated, especially in the presence of salt (100 mM NaCl). Overall, these results show that RuBisCo protein can be used to form and stabilize oil-in-water emulsions, but the pH, salt, and temperature conditions must be carefully controlled to avoid droplet aggregation. We should note that droplet aggregation is advantageous in some applications because it leads to an increase in emulsion viscosity or gelation.

     

A Protocol for the Fluorometric Quantification of mGFP5-ER and sGFP(S65T) in Transgenic Plants

The Green Fluorescent Protein (GFP) from Aequorea victoria has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluor^TM Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 ± 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 g/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 g and 2.11 g sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfp5-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 g mGFP5-ER per mg extractable protein.     

Spirilloxanthin is released by detergent from Rubrivivax gelatinosus reaction center as an aggregate with unusual spectral properties

A preparation containing spirilloxanthin has been isolated from Rubrivivax gelatinosus SC2, a mutant devoid of the reaction center-associated tetraheme cytochrome c, after solubilisation of membranes with lauryl-di-methyl-amine oxide. It was purified by ammonium sulfate precipitation and gel filtration, and analyzed by SDS-gel electrophoresis. Spirilloxanthin was shown to be aggregated in large particles (apparent M_W > 600 kDa) and was not associated with a specific protein. This aggregate was characterized by absorption, circular dichroism and resonance Raman spectroscopies. The absorption spectrum contained two UV bands at 370 and 300 nm, and did not present the visible bands of spirilloxanthin, which however reappeared when spirilloxanthin was extracted from the aggregate with organic solvents. Resonance Raman spectra indicated that at least four different populations of spirilloxanthin were present in the preparation as a mixture of different trans and cis configurations. These properties are similar to those described for a so-called carotenoprotein solubilized with sodium dodecyl sulfate from Rhodospirillum rubrum membranes [Schwenker et al. (1974) Biochim Biophys Acta 351: 246-260; Kito et al. (1983) Photochem Photobiophys 5: 209-217]. We further observed absorption spectra of pure spirilloxanthin dissolved in mixtures of water, polar solvents and detergent, in the absence of protein, resembling those of the.aggregate. We conclude that the aggregate is not a carotenoprotein, but rather an artefact due to the release of spirilloxanthin from the reaction center, leading to the isomerization and association of spirilloxanthin molecules in a detergent particle. We propose the same interpretation for the complex isolated from Rhodospirillum rubrum.