Oxidative Stress and Apoptosis Contributed to Nonylphenol-Induced Cell Damage in Mouse NCTC Clone 1469 Cells

Nonylphenol (NP) is considered an environmental toxicant and endocrine-disrupting compound. The present study aimed to investigate the effects of NP on NCTC Clone 1469, nonparenchymal hepatocytes, and to study the molecular basis of NP-induced liver injury. The results showed that NP decreased cell viability and induced nucleus crenulation and intracellular enzyme leakage in NCTC Clone 1469 cells. Additionally, NP-induced oxidative stress and apoptosis of NCTC Clone 1469 are accompanied by upregulating reactive oxygen species (ROS) production, increase of Bax, decrease of Bcl-2, activation of caspase-3 and caspase-12, and release of cytosolic free Ca2+ in the cells. ROS scavenger, N-acetyl-L-cysteine (NAC), prevented the intracellular enzyme leakage induced by NP. NP induced alteration of estrogen receptor- (ER-) α and ER-β expression, while ER antagonists, ICI 182,780, showed no effect on NP-induced intracellular enzyme leakage. We proposed that NP triggered cell damage via inducing oxidative stress and apoptosis in cells, but not estrogenic effect.

Immobilization of Old Yellow Enzymes via Covalent or Coordination Bonds

Ene-reductases (ERs) belonging to the old yellow enzyme (OYE) family have been thoroughly investigated for the stereospecific reduction of activated prochiral C=C double bonds. In this work, OYE3 was immobilized both by covalent binding on glyoxyl-agarose (OYE3-GA), and by affinity-based adsorption on EziGTM particles (OYE3-EziG). The immobilized OYE3-GA was demonstrated to be active (activity recovery = 52%) and to retain almost 100% of its activity under the enzymatic assay conditions (50 mM phosphate buffer pH 7, 28 °C) for six days, whereas the activity of the non-immobilized enzyme dropped to 50% after two days. In the case of EziGTM, the highest activity recovery (54%) was achieved by using the most hydrophilic carrier (EziGTM Opal) that was selected for the full characterization of this type of enzyme preparation (stability, recycling, re-use, enzyme leakage). OYE3-EziG was slightly less stable than OYE3-GA under the same experimental conditions. OYE3-GA could be recycled and re-used for up to 12 reaction cycles in the bioreduction of α-methyl-trans-cinnamaldehyde; after 12 runs, the highest conversion achieved was 40%. In the case of the co-immobilized OYE3/GDH-EziG, the conversion dropped to 56% after two reaction cycles. No enzyme leakage was detected over 48 h for both OYE3-GA and OYE3/GDH-EziG (50 mM phosphate buffer pH 7, 28 °C). These seed results pave the way for a true optimization of the immobilization of OYE3, as well as for the use of immobilized OYE3 for preparative applications both in batch and continuous flow conditions.

Immobilization of Peroxidase from Cauliflower Stem on Ultrafiltration Membrane for Phenol Removal

The aim of this work is to investigate phenol removal by immobilized peroxidase extracted from cauliflower stem. Peroxidase was partially purified by membrane filtration and diafiltration. Almost four-fold increase in the measured activity of partially purified peroxidase was obtained. The enzyme was then immobilized on to the surface of regenerated cellulose ultrafiltration membrane (molecular weight cut-off 30 kDa) using a dead-end filtration unit. Three different immobilization methods (physical adsorption, cross-linking and covalent-bonding using glutaraldehyde as a membrane activator) were tested. The immobilization and enzymatic reaction efficiency were evaluated in terms of the immobilization yield, the enzyme leakage from the system, the phenol removal and the permeate flux. Results showed that the immobilization methods did not much affect the permeate flux of the membrane. The peroxidase immobilization by covalent-bonding on regenerated cellulose membrane produced the highest immobilization yield and the lowest enzyme leakage. The immobilized enzymatic reaction efficiency on phenol removal was 100% at operational time 60 min and reduced to 96.4% at 600 min.