Proteomics Identification of Azaspiracid Toxin Biomarkers in Blue Mussels, Mytilus edulis

Azaspiracids are a class of recently discovered algae-derived shellfish toxins. Their distribution globally is on the increase with mussels being most widely implicated in azaspiracid-related food poisoning events. Evidence that these toxins were bound to proteins in contaminated mussels has been shown recently. In the present study characterization of these proteins in blue mussels, Mytilus edulis, was achieved using a range of advanced proteomics tools. Four proteins present only in the hepatopancreas of toxin-contaminated mussels sharing identity or homology with cathepsin D, superoxide dismutase, glutathione S-transferase Pi, and a bacterial flagellar protein have been characterized. Several of the proteins are known to be involved in self-defense mechanisms against xenobiotics or up-regulated in the presence of carcinogenic agents. These findings would suggest that azaspiracids should now be considered and evaluated as potential tumorigenic compounds. The presence of a bacterial protein only in contaminated mussels was an unexpected finding and requires further investigation. The proteins identified in this study should assist with development of urgently required processes for the rapid depuration of azaspiracid-contaminated shellfish. Moreover they may serve as early warning indicators of shellfish exposed to this family of toxins. Molecular & Cellular Proteomics 8: 1811-1822, 2009.

Bioavailability of 2,4-dichlorophenol associated with soil water-soluble humic material

Interaction of organic xenobiotics with soil water-soluble humic material (WSHM) may influence their environmental fate and bioavailability. We utilized bacterial assays (lux-based toxicity and mineralization by Burkholderia sp. RASC) to assess temporal changes in the bioavailability of [¹⁴C]-2,4-dichlorophenol (2,4-DCP) in soil water extracts (29.5 μg mL^-1 2,4-DCP; 840.2 μg mL^-1 organic carbon). HPLC determined and bioavailable concentrations were compared. Gel permeation chromatography (GPC) was used to confirm the association of a fraction (>50%) of [¹⁴C]-2,4-DCP with WSHM. Subtle differences in parameters describing 2,4-DCP mineralization curves were recorded for different soil-2,4-DCP contact times. Problems regarding the interpretation of mineralization data when assessing the bioavailability of toxic compounds are discussed. The lux-bioassay revealed a time-dependent reduction in 2,4-DCP bioavailability: after 7 d, less than 20% was bioavailable. However, GPC showed no quantitative difference in the amount of WSHM-associated 2,4-DCP over this time. These data suggest qualitative changes in the nature of the 2,4-DCP-WSHM association and that associated 2,4-DCP may exert a toxic effect. Although GPC distinguished between free- and WSHM-associated 2,4-DCP, it did not resolve the temporal shift in bioavailability revealed by the lux biosensor. These results stress that assessment of risk posed by chemicals must be considered using appropriate biological assays.

Investigation of organic xenobiotic transfers, partitioning and processing in air-soil-plant systems using a microcosm apparatus. Part I:Microcosm development

A microcosm system was developed to investigate transfers of organic xenobiotics in air-soil-plant systems. This was validated using ¹⁴C labelled 1,2-dichlorobenzene (DCB) as a model compound. Trapping efficiency was 106 ± 3% for volatile compounds and 93.0 ± 2.2% for carbon dioxide in a blank microcosm arrangement. Recovery of 1,2-dichlorobenzene spiked to grassed and unplanted soils was > 90% after 1 week. The predominant DCB loss process was volatilisation with no evidence for mineralisation over 1 week and 20-30% of the added spike remained in soil. Although there was no evidence for root uptake and translocation of added label, foliar uptake of soil volatilised compound was detected. The microcosm showed good potential for study of ¹⁴C labelled and unlabelled organic xenobiotic transfers in air-soil-plant systems with single plants and also intact planted cores.

Re-inoculation of autoclaved soil as a non-sterile treatment for xenobiotic sorption and biodegradation studies

Autoclaved soil is commonly used for the study of xenobiotic sorption and as an abiotic control in biodegradation experiments. Autoclaving has been reported to alter soil physico-chemical and xenobiotic sorption characteristics such that comparison of autoclaved with non-autoclaved treatments in soil aging and bioavailability studies may yield misleading results. Experiments could be improved by using autoclaved soil re-inoculated with indigenous microorganisms as an additional or alternative non-sterile treatment for comparison with the sterile, autoclaved control. We examined the effect of autoclaving (3 x 1 h, 121°C, 103.5 KPa) on the physico-chemical properties of a silt loam soil (pH 7.2, 2.3% organic carbon) and the establishment of indigenous microorganisms reintroduced after autoclaving. Sterilisation by autoclaving significantly (p ≤ 0.05) decreased pH (0.6 of a unit) and increased concentrations of water-soluble organic carbon (WSOC; nontreated = 75 mg kg^-1; autoclaved = 1526 mg kg^-1). The initial first-order rate of ¹⁴C-2,4-dichloro-UL-phenol (2,4-DCP) adsorption to non-treated, autoclaved and re-inoculated soil was rapid (K₁ = 16.8-24.4 h^-1) followed by a slower linear phase (K₂). In comparison with autoclaved soil (0.038% day^-1), K₂ values were higher for re-inoculated (0.095% day^-1) and nontreated (0.181% day^-1) soil. This was attributed to a biological process. The Freundlich adsorption coefficient (K(f)) for autoclaved soil was significantly (p ≤ 0.05) higher than for re-inoculated or non-treated soil. Increased adsorption was attributed to autoclaving-induced changes to soil pH and solution composition. Glucose-induced respiration of autoclaved soil after re-inoculation was initially twice that in the non-treated control, but it decreased to control levels by day 4. This reduction corresponded to a depletion of WSOC. 2,4-DCP mineralisation experiments revealed that the inoculum of nonsterile soil (0.5 g) contained 2,4-DCP-degrading microorganisms capable of survival in autoclaved soil. The lag phase before detection of significant 2,4-DCP mineralisation was reduced (from 7 days to ≤3 days) by pre-incubation of re-inoculated soils for 7 and 14 days before 2,4-DCP addition. This was attributed to the preferential utilisation of WSOC prior to the onset of 2,4-DCP mineralisation. Cumulative ¹⁴CO₂ evolved after 21 days was significantly lower (p ≤ 0.05) from non-treated soil (25.3%) than re-inoculated soils (ca 45%). Experiments investigating sorption-biodegradation interactions of xenobiotics in soil require the physico-chemical properties of sterile and non-sterile treatments to be as comparable as possible. For fundamental studies, we suggest using re-inoculated autoclaved soil as an additional or alternative non-sterile treatment.

Assessment of toxicological interactions of benzene and its primary degradation products (catechol and phenol) using a lux-modified bacterial bioassay

A bacterial bioassay has been developed to assess the relative toxicities of xenobiotics commonly found in contaminated soils, rivers, waters, and ground waters. The assay utilized decline in luminescence of lux- marked Pseudomonas fluorescens on exposure to xenobiotics. Pseudomonas fluorescens is a common bacterium in the terrestrial environment, providing environmental relevance to soil, river, and ground water systems. Three principal environmental contaminants associated with benzene degradation were exposed to the luminescence-marked bacterial biosensor to assess their toxicity individually and in combination. Median effective concentration (EC50) values for decline in luminescence were determined for benzene, catechol, and phenol and were found to be 39.9, 0.77, and 458.6 mg/L, respectively. Catechol, a fungal and bacterial metabolite of benzene, was found to be significantly more toxic to the biosensor than was the parent compound benzene, showing that products of xenobiotic biodegradation may be more toxic than the parent compounds. Combinations of parent compounds and metabolites were found to be significantly more toxic to the bioassay than were the individual compounds themselves. Development of this bioassay has provided a rapid screening system suitable for assessing the toxicity of xenobiotics commonly found in contaminated soil, river, and ground-water environments. The assay can be utilized over a wide pH range and is therefore more applicable to such environmental systems than bioluminescence-based bioassays that utilize marine organisms and can only be applied over a limited pH and salinity range.

Observation of an intact noncovalent homotrimer of detergent-solubilized rat microsomal glutathione transferase-1 by electrospray mass spectrometry

Microsomal glutathione transferase-1 (MGST1) is a membrane-bound enzyme involved in the detoxification of xenobiotics and the protection of cells against oxidative stress. The proposed active form of the enzyme is a noncovalently associated homotrimer that binds one substrate glutathione molecule/trimer. In this study, this complex has been directly observed by electrospray mass spectrometry analysis of active rat liver MGST1 reconstituted in a minimum amount of detergent. The measured mass of the homotrimer is 53 kDa, allowing for the mass of three MGST molecules in complex with one glutathione molecule. Collision-induced dissociation of the trimer complex resulted in the formation of monomer and homodimer ion species. Two distinct species of homodimer were observed, one unliganded and one identified as a homodimer.glutathione complex. Activation of the enzyme by N-ethylmaleimide through modification of Cys(49) (Svensson, R., Rinaldi, R., Swedmark, S., and Morgenstern, R. (2000) Biochemistry 39, 15144-15149) was monitored by the observation of an appropriate increase in mass in both the denatured monomeric and native trimeric forms of MGST1. Together, the data correspond well with the proposed functional organization of MGST1. These results also represent the first example of direct electrospray mass spectrometry analysis of a detergent-solubilized multimeric membrane protein complex in its native state.