ABSTRACT. Antioxidant and oxidative stress effects of prooxidants are generally dose-dependent, and these effects depend on the prooxidant species and cell type. However, the cellular response to oxidant challenge is a complicated interplay of events involving cellular expression of phase II detoxification enzymes and cellular metal metabolism. This study demonstrates the effect of tertbutylhydroquinone (t-BHQ)-induced oxidative stress on MDCK cells. Cell toxicity tests were carried out using the crystal violet (CV) assay with the following prooxidants: t-BHQ, diethyl maleate (DEM), hydrogen peroxide (H 2 O 2 ), diquat (DQ) and β-naphthoflavone (β-NF). Except for β-NF, these prooxidants showed dose-dependent cytotoxicity besides the most potent t-BHQ cytotoxicity. Only t-BHQ and DEM caused significant time-dependent expression of ferritin protein as an antioxidant, which segregates Fe 2+ , causing the Fenton reaction. t-BHQ and DEM increased formation of lipid peroxidation, but DQ showed a tendency to decrease lipid peroxidation levels. In XTT assay, even when substantial cell death was observed in the CV assay, t-BHQ appeared to increase cell viability by enhancing XTT reduction, likely through the production of NADPH. Although curcumin, which induces cytoprotective phase II enzymes and chelates metal irons, decreased cell viability, it inhibited t-BHQ cytotoxicity. These results indicate that t-BHQ exhibits strong cytotoxicity against MDCK cells, an effect mitigated by curcumin, and that t-BHQ-induced oxidative stress activates the pentose phosphate pathway.

ABSTRACT. Tetrazolium salts such as XTT and MTT are widely used to produce formazan for cell proliferation and cytotoxicity assays through bioreductase activity. However, the XTT assay showed significant increase in MDBK cell viability when cells were treated with both 50 and 100 M of the pro-oxidant, tert-butylhydroquinone (t-BHQ), although the crystal violet assay showed no cytotoxic effect with these concentrations, and the induction of lipid peroxidation was not observed. We investigated the mechanism of enhancement of XTT substrate reduction after treatment of MDBK cells with t-BHQ, leading to apparent increase in cell viability. t-BHQ caused an increase in absorbance at 340 nm in culture medium, suggesting that t-BHQ increases cellular production and release of NADH and/or NADPH. Although t-BHQ did not change the NADH concentration in cell culture medium, the addition of NADP + -dependent glutathione reductase decreased the XTT reduction to the control level, indicating cellular release of NADPH. t-BHQ also increased intracellular glucose-6-phosphate dehydrogenase activity, producing NADPH. Taken together, our findings indicate that t-BHQ treatment activates NADPH generating enzymes such as glucose-6-phosphate dehydrogenase followed by release of NADPH in the cell culture medium, resulting in direct XTT reduction by NADPH. Oxidative stress is an inevitable problem in almost all living organisms, from microorganisms to mammals, due to the undesirable generation of reactive oxygen species (ROS) during respiratory electron transport energy production [9,22]. Oxidative stress results from an imbalance between oxidant and antioxidant effects, where excess ROS overcome the detoxication capacity of biological antioxidative functions [9,22]. ROS lead to the direct oxidation of cellular components, including lipids, proteins and nucleic acids, which results in the alteration of the structure and function of oxidized molecules [9,14,22].Pro-oxidants such as hydrogen peroxide, tert-butylhydroquinone (t-BHQ), -naphthoflavone induce oxidative stress [7,16,18], whereas these pro-oxidants are characterized as antioxidant to enhance basal transcription of phase II e n z y m e s s u c h a s g l u t a t h i o n e -S -t r a n s f e r a s e , NAD(P)H:(quinone-acceptor) oxidoreductase, -glutamylcysteine synthetase, heme oxygenase, and ferritin [6,13,24,25]. These chemopreventive enzymes which mediate oxidative stress were found to be positively regulated by nuclear factor E2-related factor 2 (Nrf2)-related elcetrophile/antioxidatnt responsive element (EpRE/ARE) activation [6,13,20,24,25]. Therefore, although pro-oxidantinduced cytotoxicity is dose-dependent, lower doses of prooxidants cause cytoprotective responses to a variety of oxidative stressors [11,16]. Various cytotoxicity tests were developed to evaluate cell viability, from cell staining to detection of cellular enzymes and MTT assays, respectively [2,10]. These tetrazolium salts were reduced by endogenous or exogenous reducing enzymes followed by production of f...

Bovine milk α-casein has been identified as an iron- and heme-binding protein. However, the physiological role of its iron-binding remains to be elucidated in more detail. α-Casein was immobilized on CNBr-activated Sepharose 4B beads, and the α-casein agarose beads efficiently bound hemin as well as ferrous ammonium sulfate (Fe(2+)) as compared with control beads. Additionally, α-casein-beads bound bovine holo-lactoferrin (Lf), but not holo-transferrin. Lf caused the release of Fe(2+) which had bound to the α-casein-agarose beads beforehand. These results suggest that bovine α-casein iron-dependently binds holo-bovine Lf more strongly than Fe(2+), and that strong binding between them may play a physiological role in regulating iron homeostasis in the bovine mammary gland.

Key words psychiatric nurses, causal model of aggressive behavior, structural equation model, multiple group analysis, di erences between the sexes AbstractPurpose: We conducted a survey of psychiatric nurses to verify a postulated model for the psychological process leading to anger and aggressive behavior due to poor and insu cient social and related factors.Methods: e analysis was conducting by applying a causal model (postulated in my hypothesis) of con rmed validity explaining the relationship among the process of anger expression and the factors. Using this model, a structural equation (path diagram drawn using the causal model) was generated and path analysis was conducted by applying a structural equation model (SEM) and using the model s goodness-of-t index. We also used nine di erent types of questionnaires, including questionnaires on social support, stress, and sympathy.Results and Conclusion: On analyzing the answers of the 1,001 surveyed nurses ( nal response rate=70.0%), we observed that the results of the analysis indicated that the model best-t occurred at the following parameters: GFI=0.94, AGFI=0.91, CFI=0.92, and RMSEA=0.063. Furthermore, it was revealed that the following model is the best t for the process that leads to the expression of aggressive behavior and the factors that a ect the process: inability to receive social support accumulation of stress concerning the workplace environment, human relations at the workplace, and job contents arousal of anger expression of aggressive behavior. In addition, the results of multiple group analysis indicated clear gender di erences regarding several factors, including a partial correlation between sympathy and job-related stress, etc.