Property of arylsulfatase and ß-glucuronidase extracted from digestive tracts of Cipangopaludina chinensis and their cleavage performance on conjugated natural estrogens.

Arylsulfatase and ß-glucuronidase are the two substantial enzymes having a significant role in the cleavage of conjugated natural estrogens (C-NEs). The present study reports that arylsulfatase and ß-glucuronidase have been abundantly found in the digestive tracts of Cipangopaludina chinensis; in which, their corresponding activities were 60 and 5 U/g wet waste, respectively. The arylsulfatase from Cipangopaludina chinensis could show high activity at low temperatures. Hence, its activity still remained at 53.2% of maximal activity even at an extremely low temperature of 4 ℃; while the corresponding activities of arylsulfatase from Helix pomatia or activated sludge were less than 20% and 10%, respectively. The arylsulfatase and ß-glucuronidase from Cipangopaludina chinensis could efficiently cleave C-NEs suggesting that they could be alternative enzymes derived from Helix pomatia that are used for cleavage of conjugated compounds in environmental or biological sample analysis. Meanwhile, they might also be used to enhance the cleavage of C-NEs in municipal wastewater.

Activity measurement of arylsulfatase and ß-glucuronidase in activated sludge: HPLC-based versus classical spectrophotometric method.

Arylsulfatase and ß-glucuronidase are two important enzymes in wastewater and surface water, which play important roles on cleavage of sulfate/glucuronide estrogens. In this work, a high-performance liquid chromatography (HPLC)-based new method was firstly established for arylsulfatase/ß-glucuronidase with determination of p-nitrophenyl sulfate (pNPS)/p-nitrophenyl-ß-D-glucuronide (pNPG). The limits of detections (LODs) of the developed method for pNPS and pNPG were 0.164 and 0.098 µM, respectively. Intraday and interday reproducibility expressed as relative standard deviation (RSD) values of retention times and peak areas was 0.39%-3.68% and 0.23%-4.74%, respectively. The respective recovery efficiencies of this HPLC-based method spiking at three different concentrations for p-nitrophenol (pNP), pNPS, and pNPG in activated sludge were 76.5%-88.1%, 79.2%-93.1%, and 84.2%-96.1%, with RSD below 3.9%. The HPLC-based method was finally applied to estimate the enzyme activity of arylsulfatase/ß-glucuronidase in one activated sludge system and along which the classical spectrophotometric method was also evaluated. Compared with the classic spectrophotometric analytical method, the HPLC-based new method could simultaneously measure arylsulfatase/ß-glucuronidase one time, which was convenient and time-saving. Moreover, the developed method could effectively avoid possible underestimation that the spectrophotometric method might encounter. PRACTITIONER POINTS: A new HPLC-based method for activity estimation of arylsulfatase and ß-glucuronidase was developed. The HPLC-based method can simultaneously estimate enzyme activity of both arylsulfatase and ß-glucuronidase. The HPLC-based method can avoid possible underestimation that spectrophotometric method may encounter.

Sulfite may disrupt estrogen homeostasis in human via inhibition of steroid arylsulfatase.

Steroid arylsulfatase is an important enzyme in human, which plays an important role in dynamic equilibrium of natural estrogens. On the other hand, sulfite can be endogenously produced as a consequence of human body's metabolism of sulfur-containing amino acids, while its main sources to human are mainly derived from food as it is a widely used additive. Sulfite-sensitivity is a well-known phenomenon to a small proportion of populations. However, its potential adverse effects on healthy individuals have been hardly reported. It was for the first time reported in this study that sulfite could effectively inhibit arylsulfatase, and its IC50 values for the snail- and human urine-derived arylsulfatase were determined to be 71.9 and 142.8 µM, which were lower than the concentration of sulfite in some healthy population. Consequently, it appears that sulfite might disrupt estrogen homeostasis in human, and this deserves further investigation.

Inhibition Properties of Arylsulfatase and ß-Glucuronidase by Hydrogen Peroxide, Hypochlorite, and Peracetic Acid.

Arylsulfatase and ß-glucuronidase are two important enzymes in humans, which play an important role in the dynamic equilibrium of steroidal estrogens. This work probably for the first time reported that hydrogen peroxide (H2O2), hypochlorite, and peracetic acid (PAA) could effectively inhibit the activities of arylsulfatase and/or ß-glucuronidase. The 50% of inhibitions (IC50) of H2O2, hypochlorite, and PAA on arylsulfatase were found to be 142.90 ± 9.00, 91.83 ± 10.01, and 43.46 ± 2.92 µM, respectively. The corresponding IC50 values of hypochlorite and PAA on ß-glucuronidase were 704.90 ± 41.40 and 23.26 ± 0.82 µM, whereas H2O2 showed no inhibition on ß-glucuronidase. The inhibitions of arylsulfatase and/or ß-glucuronidase by these three chemicals were pH-dependent. It was further revealed that the inhibitions of hypochlorite on both arylsulfatase and ß-glucuronidase were irreversible. On the contrary, the inhibitions by H2O2 and PAA were reversible. In addition, the inhibition by H2O2 was competitive and that by PAA was noncompetitive. In general, H2O2 and hypochlorite can be endogenously produced in humans, which suggested that the two compounds are potential endocrine disruption compounds (EDCs) as they can cause endocrine disruption via the inhibition of arylsulfatase and ß-glucuronidase. This work further indicated that any agent that can induce the production of H2O2 or hypochlorite in humans is a potential EDC, which explains why some EDCs with very weak or no estrogenic potency can cause endocrine disruption, which is confirmed in epidemiological studies.

Strategy for effective inhibition of arylsulfatase/ß-glucuronidase to prevent deconjugation of sulfate and glucuronide conjugates in wastewater during sample collection and storage.

Arylsulfatase and ß-glucuronidase are two important enzymes that are responsible for deconjugation of estrogen conjugates. It is important to keep estrogen conjugates intact during sample collection and storage, while the effective inhibition conditions for arylsulfatase and ß-glucuronidase remain unknown. To elucidate these conditions, inhibition experiments were performed by adding several inhibitors or by introducing extreme pH conditions. This work confirms that arylsulfatase and ß-glucuronidase can tolerate some extremes, including high concentrations of mercury dichloride, ethanol, and EDTA, while low pH (<3) or high pH (>11) can effectively inhibit their activities. The high tolerance of arylsulfatase and ß-glucuronidase for mercury dichloride explains why estrogen conjugates in wastewater samples were deconjugated, even in the extremely unfavorable condition with a high concentration of mercury dichloride. Although low pH (<3) can effectively inhibit arylsulfatase/ß-glucuronidase, deconjugation of sulfate conjugates by acid hydrolysis readily occurs; thus, a high pH of 11 is an appropriate storage condition for the effective inhibition of arylsulfatase/ß-glucuronidase. This appropriate storage condition was confirmed and validated with diluted and sterilized activated sludge samples in which arylsulfatase/ß-glucuronidase inhibition was effective for 48 h at room temperature and with a high pH of 11. The developed appropriate storage condition for effective inhibition of arylsulfatase/ß-glucuronidase has wide application potential not only for estrogen conjugates but also for all conjugates of other organic micropollutants.