Journal of Alcoholism Drug Abuse & Substance Dependence Category: Medical Type: Research Article
Does Vitamin E Protect Against Hepatic Oxidative Stress During Alcohol Metabolism in Rodent Liver Cell Lines? - An EPR Study
- Obih P1, Soblosky J2, Barry J Potter3*
- 1 College Of Pharmacy, Xavier University Of Louisiana, New Orleans, United States
- 2 Department Of Physiology, School Of Medicine, Louisiana Health, New Orleans, LA, United States
- 3 Department Of Physiology, School Of Medicine, Louisiana Health, 1901 Perdido Street, New Orleans, LA 70112-1393, United States
*Corresponding Author:Barry J Potter
Department Of Physiology, School Of Medicine, Louisiana Health, 1901 Perdido Street, New Orleans, LA 70112-1393, United States
Received Date: Jul 23, 2015 Accepted Date: Sep 07, 2015 Published Date: Sep 30, 2015
While the liver cells contain anti-oxidant molecules such as glutathione, vitamins C and E, and antioxidant enzymes, these become depleted with continuous alcohol intake [5,6]. The consequential change in redox imbalance caused by ROS generation has been observed in patients with alcoholic liver disease  but attempts to ameliorate these disturbances through the administration of antioxidants has met with mixed results [1,8,9]. In contrast to this, many animal model studies (principally with rodents) have shown that these antioxidants and vitamin E in particular do have mitigating effects on free radical damage of the liver related to alcohol abuse [10-14]. Much of the damage appears to be associated with peroxidation of lipid membranes as use of N,N-Diphenyl-P-Phenlylenediamine (DPPD) a lipid radical scavenger has been shown to prevent oxidation and liver cell death in α-tocopherol (a form of vitamin E) deficient rats and cultured hepatocytes [3,15].
Antioxidant supplementation for a wide variety of diseases states has been widely practiced. However, this continues to this day to be controversial. Vitamin E has been extensively studied as a cardio-protectant for many years, but there is little agreement as to its effectiveness [9,16-19]. In general, plasma antioxidants are low in patients with alcoholic liver disease  and with non-alcoholic fatty liver . While clearly an antioxidant, vitamin E has shown mixed results in clinical trials on a variety of liver disease patients [21-25]. Experimental models tend to show a clearer picture, with vitamin E reducing lipid peroxidation [26,27] but have usually either studied acute or overnight effects and have not dissected the liver cell populations to determine whether there is a differential effect. It was the aim of this study, therefore to examine the effects of a stressor (tBH) on cells representing hepatocytes and non-parenchymal cells (Kupffer cells) incubated in alcohol containing medium and to assess both the short-term (4 hours) to long-term (7 days) protective effect of a range of concentrations of vitamin E.
MATERIALS AND METHODS
Free radical generation
Experiments to determine the efficacy of vitamin E (α-tocopherol) as an antioxidant were then performed on these cell cultures at time points ranging from acute (2 hours ) incubations up to 7 days in cell culture. The optimal range of concentrations of vitamin E was determined from an initial acute study using FtO2B cells and a 2 hour incubation (data not shown). 12-well plates were then incubated for either 4 hours, 16 hours (overnight) or 7 days in the presence or absence of ethanol and/or a range of vitamin E concentrations (0 - 100 µM). 10 wells of each plate were used for free radical determinations and 2 for viability assays. Experiments were also carried out using the same protocol but adding a range of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid - Hoffman-LaRoche) concentrations (0 -250 µM) , a more hydrophilic analog of vitamin E, instead of vitamin E. Medium in all culture wells was changed daily.
Free radical determinations
The standard procedure for ROS determination was to incubate the wells containing FtO2B or RAW 264.7 cells with tBH and POBN for 30 minutes at 37°C and then remove the entire contents of the wells and snap-freeze them in Liquid Nitrogen (LN2). Samples were then stored in LN2 until EPR spectroscopy was performed. For EPR measurements, snap-frozen samples were rapidly thawed at 37°C and aspirated into glass capillaries (ID 1mm) and spectral intensity read at room temperature using a Bruker EMX spectrometer & quantitation of this by double integration of the spectra. The EPR spectrum settings were as follows: modulation amplitude 1.0 gauss, scan time 83 seconds, time constant 163 msec and microwave power 40 mW, field sweep 60 gauss, microwave frequency 9.78 GHz (X-band), receiver gain 5 x 103, center field 3485 gauss, as described previously . ROS quantification from the EPR spectra was determined by double integration of the peaks, Day-to-day reproducibility was maintained through use of a 100 µM solution of TEMPOL (4-Hydroxy-2,2,6,6-tetramethylpiperidinyloxy - Enzo Life Sciences), a natural spin adduct. All results were normalized to the sample protein concentration. Values are expressed as arbitrary values or % control values.
Figure 1: Ethanol metabolism by FtO2B cells in culture.
B. 12 well plates containing FtO2B cells grown to 60% confluence were incubated at 37°C in a 5% CO2atmosphere in medium containing 100mM ethanol for 1-7 days. The alcohol containing medium was changed daily. Aliquots were aspirated and ethanol concentrations assayed using a commercial kit (Sigma-Aldrich). Loss through ethanol evaporation was determined using plates containing ethanol and medium alone. Values represent the mean + SEM of 10 determinations. The other 2 wells were chosen randomly and viability of the cells assessed by trypan blue exclusion. Cells were then scraped, washed twice with phosphate-buffered saline and total protein per well quantitated.
Antioxidant effects of vitamin E
FtO2B cells; RAW 264.7 cells. *p <0.05 vs no antioxidant; # p < 0.05 vs no antioxidant and RAW 26.4.7 cell data.
Antioxidant effects of a vitamin E mimetic
Because of these inconsistent results, we have studied the effect of vitamin E supplementation in tissue culture using two cell lines, to investigate possible differential cell responses. This study is part of a larger investigation into the effects of free radical generation following alcohol intoxication and the transition from cell signaling at low concentrations to cellular damage at higher levels using Electron Paramagnetic Resonance (EPR) techniques. The 2 cell lines were chosen to reflex the two major cell populations in the liver; parenchymal cells (FtO2B) and non-parenchymal cells (RAW 264.7). While these are cell lines & not primary cultures of liver cell populations, both cell lines clearly demonstrate most normal liver cell functions and have been used by many investigators in hepatology research. Since the liver hepatocytes are the primary site of ethanol metabolism, we examined the rate of disappearance from the media (adjusted for normal evaporation). The FtO2B cells in this study clearly metabolized ethanol at a constant rate throughout the 7 day study. The rate of ethanol metabolism is similar to that seen in rat hepatocytes . Furthermore, this rate of catabolism appeared constant over 7 days of cell culture with minimal cell death (< 10%). However, a dose of 400 µM tBH was chosen from a dose response curve for the oxidative stress because it had been previously shown to result in 40-50% loss of viability following 120 minutes incubation (Obih et al.,). tBH itself has been used in lipid per oxidation studies and is often used in antioxidant experiments because of its relatively slow generation of free radicals [42,43]. The relationship between free radical generation and alcohol metabolism becomes apparent when ethanol metabolic inhibitors are added: the addition of 4-methylpyrazole to cells cultured in ethanol significantly lowers the free radical generation but not cyanamide. As this is inhibiting alcohol dehydrogenase, but not acetaldehyde dehydrogenase, this implies that free radical production does not occur during the first step of alcohol catabolism, or oxidative stress in these cells is unaffected by ethanol breakdown.
The addition of vitamin E to FtO2B cells in culture clearly has a significant direct protective effect at all concentrations used. Surprisingly it was most effective after an overnight culture and slightly less effective at 7 days. This probably reflects a slower distribution within the cells to where it can exert its beneficial action. For RAW 264.7 cells, however, 5 µM had no protective effect when incubated for 4 hours. After that period, overnight incubation resulted in a dose-dependent tendency for protection against free radicals. After 7 days incubation, protection against free radicals was effectively the same as for the FtO2B cells. These findings correlate well with those observed by Jordao and co-workers in rats , but not in patients with alcoholic cirrhosis .
Because of the time lag for maximum protection in the FtO2B cells, trolox was substituted for α-tocopherol in one set of experiments with FtO2B and RAW 264.7 cells. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) is a water-soluble vitamin E mimetic that will nevertheless partition into liposomes  and has been used as a commercial standard for antioxidant activity. Unlike α-tocopherol, it had no protective effect at concentrations from 10-250 µM when incubated with FtO2B and RAW 264.7 cells for 4 hours in the presence of ethanol. Overnight culture resulted in some protection for FtO2B cells at concentrations of 10 and 50 µM, but pro-oxidant activity for the RAW 264.7 cells at the same concentrations. Why this occurred is not clear, but it is known that trolox can exhibit pro-oxidant activity in the presence of transition metals  and free iron may be released from the cells to generate a Fenton-type reaction. Cell cultures of tumorigenic cells have also shown minimal protection against ROS and impaired cell survival .
From these data it is clear that protection from free radicals in alcoholic liver disease is dependent not only on the antioxidant, but also the cell type. Naturally occurring vitamin E is a composite of 4 tocopherols and 4 tocotrienols, of which a-tocopherol is the most abundant. Based on our data, it is possible that one of the other 7 compounds may prove to be equally effective, if not more so. Furthermore, cell differences in handling of vitamin E may make true protection problematic unless it can be targeted. Further research is clearly needed into these findings from the above research.
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Citation:Obih PO, Soblosky J, Potter BJ (2015) Does Vitamin E Protect Against Hepatic Oxidative Stress During Alcohol Metabolism in Rodent Liver Cell Lines? - An EPR Study. J Alcohol Drug Depend Subst Abus 1: 002.
Copyright: © 2015 Obih P, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.