Contamination of Phthalate Esters (PAEs) in Typical Wastewater-Irrigated Agricultural Soils in Hebei,North China

The Wangyang River (WYR) basin is a typical wastewater irrigation area in Hebei Province, North China. This study investigated the concentration and distribution of six priority phthalate esters (PAEs) in the agricultural soils in this area. Thirty-nine soil samples (0–20 cm) were collected along the WYR to assess the PAE residues in soils. Results showed that PAEs are ubiquitous environmental contaminants in the topsoil obtained from the irrigation area. The concentrations of Σ₆PAEs range from 0.191 μg g⁻¹ dw to 0.457 μg g⁻¹ dw with an average value of 0.294 μg g⁻¹ dw. Di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DnBP) are the dominant PAE species in the agricultural soils. Among the DEHP concentrations, the highest DEHP concentration was found at the sites close to the villages; this result suggested that dense anthropogenic activities and random garbage disposal in the rural area are possible sources of PAEs. The PAE concentrations were weakly and positively correlated with soil organic carbon and soil enzyme activities; thus, these factors can affect the distribution of PAEs. This study further showed that only dimethyl phthalate (DMP) concentrations exceeded the recommended allowable concentrations; no remediation measures are necessary to control the PAEs in the WYR area. However, the PAEs in the topsoil may pose a potential risk to the ecosystem and human health in this area. Therefore, the exacerbating PAE pollution should be addressed.     

Phthalic Acid Esters in Soils from Vegetable Greenhouses in Shandong Peninsula,East China

Soils at depths of 0 cm to 10 cm, 10 cm to 20 cm, and 20 cm to 40 cm from 37 vegetable greenhouses in Shandong Peninsula, East China, were collected, and 16 phthalic acid esters (PAEs) were detected using gas chromatography-mass spectrometry (GC-MS). All 16 PAEs could be detected in soils from vegetable greenhouses. The total of 16 PAEs (Σ₁₆PAEs) ranged from 1.939 mg/kg to 35.442 mg/kg, with an average of 6.748 mg/kg. Among four areas, including Qingdao, Weihai, Weifang, and Yantai, the average and maximum concentrations of Σ₁₆PAEs in soils at depths of 0 cm to 10 cm appeared in Weifang, which has a long history of vegetable production and is famous for extensive greenhouse cultivation. Despite the different concentrations of Σ₁₆PAEs, the PAE compositions were comparable. Among the 16 PAEs, di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DnOP), di-n-butyl phthalate (DnBP), and diisobutyl phthalate (DiBP) were the most abundant. Compared with the results on agricultural soils in China, soils that are being used or were used for vegetable greenhouses had higher PAE concentrations. Among PAEs, dimethyl phthalate (DMP), diethyl phthalate (DEP) and DnBP exceeded soil allowable concentrations (in US) in more than 90% of the samples, and DnOP in more than 20%. Shandong Peninsula has the highest PAE contents, which suggests that this area is severely contaminated by PAEs.     

Newly Identified Thermostable Esterase from Sulfobacillus acidophilus: Properties and Performance in Phthalate Ester Degradation

EstS1, a newly identified thermostable esterase from Sulfobacillus acidophilus DSM10332, was heterologously expressed in Escherichia coli and shown to enzymatically degrade phthalate esters (PAEs) to their corresponding monoalkyl PAEs. The optimal pH and temperature of the esterase were found to be 8.0 and 70°C, respectively. The half-life of EstS1 at 60°C was 15 h, indicating that the enzyme had good thermostability. The specificity constant (k_cat/K_m) of the enzyme for p-nitrophenyl butyrate was as high as 6,770 mM⁻¹ s⁻¹. The potential value of EstS1 was demonstrated by its ability to effectively hydrolyze 35 to 82% of PAEs (10 mM) within 2 min at 37°C, with all substrates being completely degraded within 24 h. At 60°C, the time required for complete hydrolysis of most PAEs was reduced by half. To our knowledge, this enzyme is a new esterase identified from thermophiles that is able to degrade various PAEs at high temperatures.     

Purification and characterization of an intracellular esterase from a Fusarium species capable of degrading dimethyl terephthalate

Esterase is the key enzyme involved in microbial degradation of phthalate esters (PAEs). In this study, an intracellular esterase was purified from a coastal sediment fungus Fusarium sp. DMT-5-3 capable of utilizing dimethyl terephthalate (DMT) as a substrate. The purified enzyme is a polymeric protein consisting of two identical subunits with a molecular mass of about 84 kDa. The enzyme showed a maximum esterase activity at 50 °C and was stable below 30 °C. The optimal pH was 8.0 and the enzyme was stable between pH 6.0 and 10.0. The esterase activity was inhibited by Cr³⁺, Hg²⁺, Cu²⁺, Zn²⁺, Ni²⁺, and Cd²⁺. Substrate specificity analysis showed that the enzyme was specific to DMT hydrolysis, but had no effect on other isomers of dimethyl phthalate esters (DMPEs) or monomethyl phthalate esters (MMPEs). These findings suggest that the phthalate esterase produced by Fusarium sp. DMT-5-3 is inducible and distinctive esterases involved in hydrolysis of the two carboxylic ester linkages of DMPEs.     

A Mono-2-Ethylhexyl Phthalate Hydrolase from a Gordonia sp. That Is Able To Dissimilate Di-2-Ethylhexyl Phthalate

Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The K_m and V_max values for mono-2-ethylhexyl phthalate were 26.9 ± 4.3 μM and 18.1 ± 0.9 μmol/min · mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.     

Potential for Biodegradation of Phthalic Acid Esters in Marine Regions

Di-(2-ethylhexyl) phthalate was the major phthalic acid ester in the Mississippi River estuary, with mean levels of 0.1 μg/g (dry weight) in surface sediments, 1.0 μg/liter in river water, and 0.7 μg/liter in delta water. Bacteria that grew aerobically on dibutyl phthalate and o-phthalic acid were readily detected in the sediments and water. Pure cultures of bacteria were isolated on seven different phthalic acid esters from freshwater and marine sources. The marine isolates were taxonomically diverse and grew on a variety of phthalic acid esters. Dibutyl phthalate and o-phthalic acid supported growth in full-strength synthetic sea-water medium, but Na⁺ -dependent catabolism was demonstrable only for o-phthalic acid.     

Persistent Associations between Maternal Prenatal Exposure to Phthalates on Child IQ at Age 7 Years

Background

Prior research reports inverse associations between maternal prenatal urinary phthalate metabolite concentrations and mental and motor development in preschoolers. No study evaluated whether these associations persist into school age.

Methods

In a follow up of 328 inner-city mothers and their children, we measured prenatal urinary metabolites of di-n-butyl phthalate (DnBP), butylbenzyl phthalate (BBzP), di-isobutyl phthalate (DiBP), di-2-ethylhexyl phthalate and diethyl phthalate in late pregnancy. The Wechsler Intelligence Scale for Children, 4th edition was administered at child age 7 years and evaluates four areas of cognitive function associated with overall intelligence quotient (IQ).

Results

Child full-scale IQ was inversely associated with prenatal urinary metabolite concentrations of DnBP and DiBP: b = −2.69 (95% confidence interval [CI] = −4.33, −1.05) and b = −2.69 (95% CI = −4.22, −1.16) per log unit increase. Among children of mothers with the highest versus lowest quartile DnBP and DiBP metabolite concentrations, IQ was 6.7 (95% CI = 1.9, 11.4) and 7.6 (95% CI = 3.2, 12.1) points lower, respectively. Associations were unchanged after control for cognition at age 3 years. Significant inverse associations were also seen between maternal prenatal metabolite concentrations of DnBP and DiBP and child processing speed, perceptual reasoning and working memory; DiBP and child verbal comprehension; and BBzP and child perceptual reasoning.

Conclusion

Maternal prenatal urinary metabolite concentrations measured in late pregnancy of DnBP and DiBP are associated with deficits in children’s intellectual development at age 7 years. Because phthalate exposures are ubiquitous and concentrations seen here within the range previously observed among general populations, results are of public health significance.     

Comparison of β-Glucosidase Activities in Different Streptomyces Strains

Cellobiase (β-glucosidase) production was compared for two streptomycetes: Streptomyces flavogriseus, a known producer of cellulase complex, and Streptomyces sp. strain CB-12, a strain isolated for its rapid growth on cellobiose. The optimal conditions for enzyme activity were established in relation to pH, temperature, enzyme stability, and substrate affinity. The production of β-glucosidase by the two strains depended on the carbon substrate in the medium. Cellobiose was found to repress the biosynthesis of the enzyme in S. flavogriseus and to stimulate its production in strain CB-12. The biosynthesis of the enzyme correlated well with the accumulation of glucose in the culture filtrates. The combined action of the β-glucosidases produced by the two Streptomyces strains might allow a better utilization of the reaction products which arise during the biodegradation of cellulose.     

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDO_KF1 from Comamonas testosteroni KF1 and found that it had an apparent k_cat/K_m for phthalate of 0.58 ± 0.09 μM⁻¹s⁻¹, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α₃ trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDO_KF1 larger than that of other characterized ROs. Complexes of PDO_KF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.     

Thermophilic, reversible gamma-resorcylate decarboxylase from Rhizobium sp. strain MTP-10005: purification, molecular characterization, and expression

We found the occurrence of thermophilic reversible γ-resorcylate decarboxylase (γ-RDC) in the cell extract of a bacterium isolated from natural water, Rhizobium sp. strain MTP-10005, and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be about 151 kDa by gel filtration, and that of the subunit was 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; in other words, the enzyme was a homotetramer. The enzyme was induced specifically by the addition of γ-resorcylate to the medium. The enzyme required no coenzyme and did not act on 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3,5-dihydroxybenzoate, 2-hydroxybenzoate, or 3-hydroxybenzoate. It was relatively thermostable to heat treatment, and its half-life at 50°C was estimated to be 122 min; furthermore, it catalyzed the reverse carboxylation of resorcinol. The values of k_cat/K_m (mΜ⁻¹·s⁻¹) for γ-resorcylate and resorcinol at 30°C and pH 7 were 13.4 and 0.098, respectively. The enzyme contains 327 amino acid residues, and sequence identities were found with those of hypothetical protein AGR C 4595p from Agrobacterium tumefaciens strain C58 (96% identity), 5-carboxyvanillate decarboxylase from Sphingomonas paucimobilis (32%), and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylases from Bacillus cereus ATCC 10987 (26%), Rattus norvegicus (26%), and Homo sapiens (25%). The genes (graA [1,230 bp], graB [888 bp], and graC [1,056 bp]) that are homologous to those in the resorcinol pathway also exist upstream and downstream of the γ-RDC gene. Judging from these results, the resorcinol pathway also exists in Rhizobium sp. strain MTP-10005, and γ-RDC probably catalyzes a reaction just before the hydroxylase in it does.     

A Novel 2-Aminomuconate Deaminase in the Nitrobenzene Degradation Pathway of Pseudomonas pseudoalcaligenes JS45

2-Aminomuconate, an intermediate in the metabolism of tryptophan in mammals, is also an intermediate in the biodegradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45. Strain JS45 hydrolyzes 2-aminomuconate to 4-oxalocrotonic acid, with the release of ammonia, which serves as the nitrogen source for growth of the microorganism. As an initial step in studying the novel deamination mechanism, we report here the purification and some properties of 2-aminomuconate deaminase. The purified enzyme migrates as a single band with a molecular mass of 16.6 kDa in 15% polyacrylamide gel electrophoresis under denaturing conditions. The estimated molecular mass of the native enzyme was 100 kDa by gel filtration and 4 to 20% gradient nondenaturing polyacrylamide gel electrophoresis, suggesting that the enzyme consists of six identical subunits. The enzyme was stable at room temperature and exhibited optimal activity at pH 6.6. The K_m for 2-aminomuconate was approximately 67 μM, and the V_max was 125 μmol · min⁻¹ · mg⁻¹. The N-terminal amino acid sequence of the enzyme did not show any significant similarity to any sequence in the databases. The purified enzyme converted 2-aminomuconate directly to 4-oxalocrotonate, rather than 2-hydroxymuconate, which suggests that the deamination was carried out via an imine intermediate.     

Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. ¹³C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.     

Biodegradation of diethyl phthalate by an organic-solvent-tolerant Bacillus subtilis strain 3C3 and effect of phthalate ester coexistence

The contamination and distribution of phthalate esters - synthetic compounds widely used in plastic product production, including food and medical packaging - has raised safety concerns due to their endocrine-disrupting activity and mandated to be treated. Bacillus subtilis strain 3C3, isolated as an organic-solvent-tolerant bacterium, was capable of utilizing diethyl phthalate as a sole carbon source. Biodegradation of diethyl phthalate occurred constitutively without lag period, and its kinetics followed a first-order model. The biodegradability was significantly enhanced with the supplementation of yeast extract as a co-metabolic substrate. In the presence of Tween-80 as a solubilizing agent, cells rapidly degrade a range of short-chain phthalate esters at high concentrations (up to 1000 mg l⁻¹ for diethyl phthalate). The biodegradation of short-chain phthalates in the binary, ternary and quaternary substrate system revealed that the coexistence of other short-chain phthalates had no significant influence on the biodegradation of diethyl phthalate, and vice versa. These results substantiated that B. subtilis strain 3C3 has potential application as a bioaugmented bacterial culture for bioremediation of phthalates.     

Purification and some properties of a phthalate ester hydrolyzing enzyme from Nocardia erythropolis

Summary A phthalate ester hydrolyzing enzyme has been purified from the culture broth of Nocardia erythropolis, a Gram-positive bacterium capable of degrading phthalate esters rapidly. The purified enzyme appeared homogeneous on polyacrylamide gel disc-electrophoresis, and its molecular weight was estimated to be about 15,000. The optimal pH and temperature were pH 8.6 and 42°C, respectively. The enzyme was stable in a pH range from 7.0 to 8.0 and below 30°C. The enzyme activity was stimulated by Ca²⁺ and taurocholate, but inhibited by several metals such as Hg²⁺. Most of the phthalate esters tested were hydrolyzed to phthalate and alcohols regardless of the type of side-chain. In addition, the enzyme rapidly hydrolyzed olive oil and tributyrin. This enzyme from N. erythropolis may be a novel type of lipase with broad substrate specificity.Microbial degradation of phthalate esters. Part X     

Children’s Phthalate Intakes and Resultant Cumulative Exposures Estimated from Urine Compared with Estimates from Dust Ingestion,Inhalation and Dermal Absorption in Their Homes and Daycare Centers

Total daily intakes of diethyl phthalate (DEP), di(n-butyl) phthalate (DnBP), di(isobutyl) phthalate (DiBP), butyl benzyl phthalate (BBzP) and di(2-ethylhexyl) phthalate (DEHP) were calculated from phthalate metabolite levels measured in the urine of 431 Danish children between 3 and 6 years of age. For each child the intake attributable to exposures in the indoor environment via dust ingestion, inhalation and dermal absorption were estimated from the phthalate levels in the dust collected from the child’s home and daycare center. Based on the urine samples, DEHP had the highest total daily intake (median: 4.42 µg/d/kg-bw) and BBzP the lowest (median: 0.49 µg/d/kg-bw). For DEP, DnBP and DiBP, exposures to air and dust in the indoor environment accounted for approximately 100%, 15% and 50% of the total intake, respectively, with dermal absorption from the gas-phase being the major exposure pathway. More than 90% of the total intake of BBzP and DEHP came from sources other than indoor air and dust. Daily intake of DnBP and DiBP from all exposure pathways, based on levels of metabolites in urine samples, exceeded the Tolerable Daily Intake (TDI) for 22 and 23 children, respectively. Indoor exposures resulted in an average daily DiBP intake that exceeded the TDI for 14 children. Using the concept of relative cumulative Tolerable Daily Intake (TDI_cum), which is applicable for phthalates that have established TDIs based on the same health endpoint, we examined the cumulative total exposure to DnBP, DiBP and DEHP from all pathways; it exceeded the tolerable levels for 30% of the children. From the three indoor pathways alone, several children had a cumulative intake that exceeded TDI_cum. Exposures to phthalates present in the air and dust indoors meaningfully contribute to a child’s total intake of certain phthalates. Such exposures, by themselves, may lead to intakes exceeding current limit values.     

Diethylhexyl Phthalates Is Associated with Insulin Resistance via Oxidative Stress in the Elderly: A Panel Study

Background

Insulin resistance (IR) is believed to be the underlying mechanism of metabolic syndrome and type 2 diabetes mellitus (DM). Recently, a few studies have demonstrated that phthalates could cause oxidative stress which would contribute to the development of IR. Therefore, we evaluated whether exposure to phthalates affects IR, and oxidative stress is involved in the phthalates-IR pathway.

Methods

We recruited 560 elderly participants, and obtained blood and urine samples during repeated medical examinations. For the determination of phthalate exposure, we measured urinary levels of mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) as metabolites of diethylhexyl phthalates (DEHP), and mono-n-butyl phthalate (MnBP) as a metabolite of di-butyl phthalate (DBP). Malondialdehyde (MDA), an oxidative stress biomarker, was also measured in urine samples. We measured serum levels of fasting glucose and insulin, and derived the homeostatic model assessment (HOMA) index to assess IR. A mixed-effect model and penalized regression spline were used to estimate the associations among phthalate metabolites, MDA, and IR.

Results

The molar sum of MEHHP and MEOHP (∑DEHP) were significantly associated with HOMA (β = 0.26, P = 0.040), and the association was apparent among participants with a history of DM (β = 0.88, P = 0.037) and among females (β = 0.30, P = 0.022). However, the relation between MnBP and HOMA was not found. When we evaluated whether oxidative stress is involved in increases of HOMA by ∑DEHP, MDA levels were significantly associated with increases of ∑DEHP (β = 0.11, P<0.001) and HOMA (β = 0.49, P = 0.049).

Conclusions

Our study results suggest that exposure to DEHP in the elderly population increases IR, which is related with oxidative stress, and that participants with a history of DM and females are more susceptible to DEHP exposure.     

Role of ςB in Heat, Ethanol, Acid, and Oxidative Stress Resistance and during Carbon Starvation in Listeria monocytogenes

To determine the contribution of sigma B (ς^B) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50°C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45°C, 5% ethanol, or pH 4.5). The ΔsigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the ΔsigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the ΔsigB strain. These results suggest the existence in L. monocytogenes of both a ς^B-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. ς^B contributed to resistance to both oxidative stress and carbon starvation in L. monocytogenes. The ΔsigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the ΔsigB strain lost viability more rapidly than the parent strain. ς^B contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that ς^B plays a role in protecting L. monocytogenes against environmental adversities.     

An Integrated Approach Combining Chemical Analysis and an In Vivo Bioassay to Assess the Estrogenic Potency of a Municipal Solid Waste Landfill Leachate in Qingdao

Various adverse effects related to landfill leachate have made leachates an important issue in past decades, and it has been demonstrated that landfill leachate is an important source of environmental estrogens. In this study, we employed chemical analysis of some already evaluated estrogenic substances, in combination with a bioassay using several specific biomarkers (e.g., plasma vitellogenin and sex steroids, enzyme activity of gonad gamma-glutamyl transpeptidase, and gonadosomatic index) to evaluate the estrogenic activities in outlets from different stages of the leachate treatment process. The results indicated that 5 environmental estrogens (4-t-octylphenol, bisphenol A, di-ethyl phthalate, di-n-butyl phthalate, and diethylhexyl phthalate) were detected by a gas chromatography-mass spectrometry, and the concentrations in leachate samples were 6153 ng/L, 3642 ng/L, 2139 ng/L, 5900 ng/L, and 9422 ng/L, respectively. Leachate (1∶200 diluted) induced the synthesis of plasma vitellogenin and led to decreased enzyme activity of gonad gamma-glutamyl transpeptidase and gonadosomatic index in male goldfish (Carassius auratus) after a 28-day exposure, while increased circulating 17β-estradiol level was also observed in males exposed to treated effluent. Although the target EEs were partially removed with removal rates varying from 87.2% to 99.77% by the “membrane bioreactor+reverse osmosis+aeration zeolite biofilter” treatment process, the treated effluent is still estrogenic to fish. The method combined chemical techniques with the responses of test organisms allowing us to identify the group of estrogen-like chemicals so that we were able to evaluate the overall estrogenic effects of a complex mixture, avoiding false negative assessments.     

Molecular Ageing of Alpha- and Beta-Synucleins: Protein Damage and Repair Mechanisms

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[³H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated ³H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of ³H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.