Ethanol and acetaldehyde disturb TNF-alpha and IL-6 production in cultured astrocytes

Abstract

Ethanol disturbs astroglial growth and differentiation and causes functional alterations. Furthermore, many signalling molecules produced by astrocytes contribute to these processes. The aim of the present study was to investigate the influence of ethanol and its primary metabolite, acetaldehyde, on TNF-alpha and IL-6 production in a rat cortical astrocyte primary culture. We are the first to report that both ethanol and acetaldehyde can modulate TNF-alpha and IL-6 secretion from cultured astrocytes. Long-term exposure (7 days) to ethanol and acetaldehyde was more toxic than an acute (24 hours) exposure. However, both compounds showed a biphasic, hormestic effect on the IL-6 secretion after the acute as well as the long-term exposure, and the maximum stimulation was reached for 50-mM ethanol and 1-mM acetaldehyde after 7-day exposure. In contrast, both compounds reduced the TNF-alpha secretion, where the effect was concentration-dependent. The catalase inhibitor 2-amino-1,2,4 triazole significantly reduced the ethanol toxicity in the cultured astrocytes after the acute as well as the long-term exposure. In conclusion, both ethanol and acetaldehyde affect the production of IL-6 and TNF-alpha in cultured astrocytes. The effect depends on the concentration of the compounds and the duration of the exposure. Acetaldehyde is a more potent toxin than ethanol, and ethanol’s toxicity in the brain is at least partially due to its primary metabolite, acetaldehyde.

Keywords

ethanol acetaldehyde astrocytes TNF-alpha IL-6

Introduction

Ethanol consumption has for a long time been associated with brain damage. Experimental studies and necropsy examinations of chronic alcoholics have shown a variety of structural and functional alterations in the neurons as well as in the glial cells.^{1

–12} Astroglial cells represent the majority of the brain-cell population; they play an important role in the developmental guidance of migrating neurons, in the regulation of neurotransmitter and ion levels, in the nutrition of neurons, and in the production of neurotrophic factors. Astrocytes also represent the major site for the detoxification or bioactivation of neurotoxins and contribute importantly to the creation of the immune response in the brain.^{13

–17} Recent studies have shown that some of the effects of ethanol, including psychopharmacological and neurotoxic effects, are mediated through its primary metabolite, acetaldehyde.^{18

–21} Acetaldehyde, derived from a peripheral metabolism of ethanol, penetrates from the blood stream into the brain with difficulty due to the metabolic barriers presented by alcohol- and aldehyde-dehydrogenase.²² However, the localization and relevance of ethanol metabolism in the brain remains controversial. The adult mammalian brain contains three enzyme systems for oxidizing ethanol to acetaldehyde: cytochrome P450 (CYP) 2E1, catalase and alcohol-dehydrogenase.²³ In a naive rat brain, the crucial role of catalase in the oxidative degradation of ethanol to acetaldehyde was established. The presence of catalase was confirmed in glial cells, in oligodendrocytes and in astroglial cells using confocal laser-scanning microscopy.²⁴ The catalase activity was found in aminergic neurone perikaryons and in glial cells.²⁵ Metyrapone (a CYP inhibitor) or pyrazole (an alcohol-dehydrogenase inhibitor) did not influence the formation of acetaldehyde from ethanol in rat-brain homogenates; at the same time, the presence of the catalase inhibitors 2-amino-1,2,4 triazole (AMT), cyanamide or sodium azide, significantly reduced the acetaldehyde formation.^{26
–28} Several disorders in the central nervous system (CNS) related to excessive ethanol consumption seem to be associated with altered cytokine production.^{29

–33} Among them, the pro-inflammatory cytokines TNF-alpha and IL-6 play important roles.^{34

–37} In the brain, TNF-alpha is synthesized in the astrocytes, microglia and some neurones.³⁸ Astrocytes are the major source of IL-6.³⁹ TNF-alpha has an important function in neurotoxicity, synaptic transmission and synaptic plasticity. In addition, TNF-alpha may have a pivotal role in augmenting intracerebral immune response and inflammatory demyelination due to its diverse functional effects on glial cells, such as oligodendrocytes and astrocytes themselves.⁴⁰ Unlike TNF-alpha, which is a prototypical pro-inflammatory cytokine, IL-6 affects inflammation and neuronal regeneration via a number of mechanisms.⁴¹ In this sense, besides its immunoregulatory role, IL-6 can also promote neuronal survival and neurite growth.^42,43

Ethanol can modulate cytokine production in several experimental systems. It has been reported, for example, that ethanol enhances both IL-6 production and hepatic TNF-alpha level in a female rat liver, which promote an inflammatory response. On the other hand, ethanol significantly suppresses TNF-alpha synthesis in a model of chronic alcoholism on alveolar macrophages, murine splenocytes and thymocytes.^{44
–46} In hepatic stellate cells and HepG2, ethanol and acetaldehyde significantly increases the level of IL-6 secretion.^47,48 An elevated level of IL-6 was also confirmed in the peripheral blood of chronic alcoholics, with or without significant liver disease.⁴⁹

It has already been established that ethanol disturbs the astroglial function. Although astrocytes are an important source of several cytokines and contribute significantly to the immune response in the brain, there are no data about ethanol’s influence on TNF-alpha and IL-6 secretion from astrocytes. Additionally, the role of ethanol’s primary metabolite, acetaldehyde, in this process is not elucidated. Thus, in the present work, we have attempted to determine and to compare the influence of different concentrations of ethanol and its primary metabolite, acetaldehyde, on the TNF-alpha and IL-6 secretion in cultured astrocytes after acute and long-term exposure.

Materials and methods

Materials

L-15 Leibowitz medium, foetal bovine serum (FBS), Dulbecco's modified Eagle medium and Ham's nutrient mixture F-12 (DMEM/F12), Penicillin-Streptomycin (10,000 IU/mL−10,000 UG/mL; P/S), Dulbecco's phosphate buffered saline (PBS) were purchased from Gibco BRL, Life Technologies, Paisley, Scotland. Ethanol and acetaldehyde were from Merck, Darmstadt, Germany. 2-amino-1,2,4 triazole (AMT), staurosporine, polymyxin B sulphate, trypan blue and bovine serum albumin were obtained from Sigma Chemical Co., St Louis, Missouri, USA. Bio-Rad protein assay was from Bio-Rad Laboratories, Munich, Germany. Endogen Rat IL-6 ELISA Kit and Endogen Rat TNF-alpha ELISA Kit were obtained from Pierce Biotechnology, Rockford, Illinois, USA.

Animals

Newborn Wistar rats (postnatal day 2) were obtained from our own breeding colony. The animals were maintained under constant environmental conditions, with an ambient temperature of 22 ± 1 C, relative humidity 55% ± 10% and a natural light-dark cycle. The breeding colony was kept in Ehret type-4 cages (Germany). The bedding material was Lignocel 3/4. The colony received a standard rodent diet (Altormin, Germany), and had free access to food and water. We used four newborn animals for each experiment.

All the animal procedures were approved by the National Animal Ethical Committee of the Republic of Slovenia (licence number 323-02-232/2005/2) and were conducted in accordance with the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (ETS 123).

Astrocyte culture preparation

Primary cultures of rat cortical astrocytes were prepared from the brain of new-born Wistar rats. The new-born rats (postnatal day 2) were decapitated and the brains removed aseptically. After removal of the meninges, the cortices were transferred to a Petri dish containing the L-15 (Leibowitz) medium. The cortices were then mechanically dissociated into 10 mL of culture medium, consisting of DMEM/F12 (1:1), 10% (vol/vol) FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The cell suspension was triturated and plated into tissue-culture flasks. The cells were grown at 37°C in a water-saturated air environment, containing 10% CO_2. In order to purify the cultures, when they became confluent they were shaken at 150 rpm for 18 hours, to remove microglial cells. After the shaking, the medium was changed and the cells were trypsinized and cultured for 24 hours in the presence of 10-μM cytosine arabinoside that only allowed the growth of astrocytes. After reaching confluence again, the cells were subcultured onto 35-mm Petri dishes for the treatment with either ethanol or acetaldehyde.

The purity of the culture was checked using immunocytochemical staining for the glial fibrillary acidic protein, which is the major component of the astrocyte cytoskeleton.

Treatment of the cells

Acute (24 hours) treatment: After the cultures became confluent, the culture medium was replaced with 1 mL of fresh medium and the cells were treated with different concentrations of either ethanol (in the presence or absence of 10-mM AMT) or acetaldehyde for 24 hours. AMT was added to the culture medium 6 hours before the treatment with ethanol.

Long-term (7 days) treatment: After the plating onto the Petri dishes, the cells were grown in media, containing different concentrations of either ethanol (in the presence or absence of 10 mM AMT) or acetaldehyde for the next 7 days. In order to minimize the decline of the ethanol and acetaldehyde concentrations in the culture medium due to evaporation, the media were changed every 48 hours and tightly closed in the Petri dishes, which allowed a reduction of the ethanol and acetaldehyde concentration in the culture medium of not more than 10%.

The concentrations of ethanol and acetaldehyde used in the present study were based on our previous studies where a dose-response relationship for the ethanol and acetaldehyde on cell viability and cell proliferation was studied.¹² Only concentrations below the threshold of significant viability decrease were used. Similar ethanol concentrations were also noticed during clinical toxicology practice; recovery was reported in patients with blood-ethanol levels even greater than 327.8 mM.⁵⁰

The experiments were performed under lipopolysaccharide-free conditions. In addition, polymyxin B (10 μg/mL) was added to the culture medium before the treatment, to avoid lipopolysaccharide contamination. The control cells 1 were grown under the same conditions, in the absence of ethanol or acetaldehyde. The control cells 2 were grown under the same conditions and treated with 10-mM AMT only. The concentration of the AMT used in the present study was obtained from our dose-response studies (data not shown) and is similar to previously published data.⁵¹ After the treatment, the cells from the individual dishes were used either for viability testing or protein determination. The protein determination was performed according to the Bradford method, using bovine serum albumin as a standard.⁵² The culture medium was collected, frozen and used for the TNF-alpha and IL-6 determinations.

Cell-viability determination

The viability of the cells was determined using the trypan blue exclusion test. The viable cells were counted after they were treated with different concentrations of either ethanol or acetaldehyde for 24 hours. The positive control cells were treated with 1 µM staurosporine.

TNF-alpha and IL-6 secretion determination

The TNF-alpha and IL-6 levels in the culture media were determined by enzyme immunoassay using the Pierce Biotechnology ELISA protocol.

Statistical analysis

The results are shown as means ± the standard error of the mean (SEM) of three independent assays. The one-way ANOVA with the Tukey post test were used to calculate the significance of the differences between the means. A p value of <0.05 was considered significant.

Results

The cell viability evaluated by the trypan blue test was not affected by ethanol concentrations of below 300 mM. The presence of AMT did not have any statistically significant influence on the cell viability, whereas acetaldehyde did not have any statistically significant influence on the cell viability below 5 mM (Figure 1A and B).

Figure 1.

Ethanol (EtOH) in concentrations of below 300 mM does not influence the viability of astrocytes in primary rat cultures. The presence of AMT does not modulate this effect (A). Acetaldehyde (ACH) in concentrations of below 5 mM does not influence the viability of astrocytes in primary rat cultures (B). Each point is the mean ± SEM of three independent determinations, p < 0.001 versus control c1. c1, control cells − growth medium; c2, control cells, treated with 10 mM AMT; c3, control cells, treated with 1 μM staurosporine.

The estimated EC₅₀ for the influence on the cell viability evaluated by trypan blue test is for ethanol 847.9 mM and 47.2 mM for acetaldehyde.¹²

Ethanol’s influence on the TNF-alpha and IL-6 secretion from cultured astrocytes after acute and chronic exposure

The results showed that ethanol can reduce the secretion of TNF-alpha from cultured astrocytes. The inhibitory effect of the ethanol was both dose- and time-dependent. During acute exposure, there was no significant effect on the TNF-alpha secretion between 50 and 200 mM of ethanol (Figure 2A), whereas the presence of ethanol in the growing media over 7 days strongly inhibited the secretion of the TNF-alpha at much lower concentrations, starting at 25-mM ethanol (Figure 2B). The presence of a catalase inhibitor AMT did not influence the inhibitory effect of ethanol after 24 hours of exposure (Figure 2A). On the contrary, after 7 days of exposure, the AMT significantly reduced the inhibitory effect of ethanol on the TNF-alpha secretion (Figure 2B).

Figure 2.

Ethanol (EtOH) reduces the secretion of TNF-alpha from astrocytes in primary rat cultures in dose-dependent manner after 24 hours (A) and 7 days (B). The presence of 2-amino-1,2,4 triazole (AMT) modulates this effect in 7-day exposure. Each point is the mean ± SEM of three independent determinations. *p < 0.05 versus control c1; °p < 0.05 versus values with the same ethanol concentration in the absence of AMT. c1, control cells − growth medium; c2, control cells, treated with 10 mM AMT.

In the next set of experiments, we determined the influence of different concentrations of ethanol on the IL-6 secretion from cultured astrocytes. We found that low concentrations of ethanol strongly stimulated the IL-6 secretion. After 24 hours of exposure, the maximum increase in the IL-6 secretion was observed between 50 mM and 100 mM. At concentrations higher than 200-mM ethanol, the secretion of IL-6 started to decrease. The maximum secretion of IL-6 represented an approximately 5-fold enhancement over the IL-6 secretion in comparison to the secretion from the control cells (Figure 3A). We also noted such a biphasic, hormestic effect after 7 days of exposure, where the stimulatory effect of the ethanol was shifted to lower concentrations and the maximum stimulation of the IL-6 release was reached at 50-mM ethanol (Figure 3B). The catalase inhibitor AMT significantly reduced the stimulatory effect of the ethanol on the IL-6 secretion from cultured astrocytes after 24 hours as well as after 7 days of exposure (Figure 3A and B).

Figure 3.

Low concentrations of ethanol (EtOH) stimulate the IL-6 secretion from astrocytes in primary rat cultures; at higher concentrations the secretion of IL-6 started to decrease after 24 hours (A) and 7 days of exposure (B). The presence of AMT modulates this effect. Each point is the mean ± SEM of three independent determinations. *p < 0.05 versus control c1; °p < 0.05 versus values with the same ethanol concentration in the absence of AMT. c1, control cells − growth medium; c2, control cells, treated with 10 mM AMT.

Acetaldehyde's influence on the TNF-alpha and IL-6 secretion from cultured astrocytes after acute and chronic exposure

We found that acetaldehyde influenced the TNF-alpha and IL-6 secretion from cultured astrocytes in a pattern similar to that of ethanol. Moreover, acetaldehyde appeared to be a much more potent compound compared with ethanol. The results showed that acetaldehyde strongly inhibited the secretion of the TNF-alpha from the cultured astrocytes after 24 hours of exposure as well as after 7 days of exposure. The strongest inhibitory effect was found between 0.25-mM and 1-mM acetaldehyde concentrations. At a 1-mM concentration of acetaldehyde, approximately 50% inhibition was observed after 24 hours and 70% after 7 days of exposure in comparison with the control (Figure 4A).

Figure 4.

Acetaldehyde (ACH) reduces the TNF-alpha secretion from astrocytes in primary rat cultures after 24 hours and 7 days of exposure (A). Low concentrations of acetaldehyde (ACH) stimulate IL-6 secretion from astrocytes in primary rat cultures, at higher concentrations the secretion of IL-6 started to decrease after 24 hours as well as after 7 days of exposure (B). Each point is the mean ± SEM of three independent determinations. *p < 0.05 versus control c1; °p < 0.05 versus values treated for 24 hours. c1, control cells − growth medium.

The influence of acetaldehyde on IL-6 production showed a quite similar pattern. Acute exposure of the cultured astrocytes to acetaldehyde resulted in a strong stimulation of the IL-6 secretion. The maximum stimulation of IL-6 after 24 hours of exposure was reached at 1-mM acetaldehyde and represents a 7-fold enhancement in comparison to the control. A long-term exposure to acetaldehyde shifted the dose-response curve to the left. Thus, a consecutively stimulatory effect on the IL-6 secretion was observed at 0.25-mM and 0.50-mM acetaldehyde only (Figure 4B).

Discussion

Ethanol causes structural and functional alterations to the CNS, and thus predisposes to autoimmunity, neoplasm and infection. It was already established that ethanol, as well as its primary metabolite, acetaldehyde, disturbs astroglial growth and differentiation and influence the physiological processes that lead to an altered immune response in a rat.^{4
–6,19
–21,29
–31} Although inflammatory processes have been implicated in the pathogenesis of certain neurodegenerative disorders, the mechanisms involved in the neuropathogenesis remain elusive. TNF-alpha and IL-6 are involved in the brain's response to different neurodegenerative and/or neuroregenerative processes, like inflammatory demyelination, neuronal regeneration, neuronal survival etc.^{41
–43} Thus, we studied the influence of ethanol and its primary metabolite, acetaldehyde, on the production of those two cytokines in an astrocyte cell culture.

We found in our study that ethanol in concentrations up to 100 mM strongly inhibits the TNF-alpha secretion from cultured astrocytes after 7 days of exposure, whereas 24 hours of exposure did not significantly influence the TNF-alpha secretion.

In comparison with ethanol, acetaldehyde was a much more potent suppressor of the TNF-alpha secretion from astrocytes. These results are in accordance with acute oral-toxicity data from rats, where acetaldehyde showed a higher level of toxicity than ethanol.⁵⁰ Our study clearly showed that ethanol is an important suppressor of TNF-alpha secretion from astroglial cells and that the suppression of the TNF-alpha secretion from cultured astrocytes is at least partially due to acetaldehyde. However, the involved cellular mechanisms remain unknown. In the literature there are several assumptions about the ethanol impairment of inflammatory and immune responses due to the interference with calcium mobilization in phagocytes, the suppression of lymphocyte activity, the up-regulation of the secretion of immunosuppressant corticosteroids, signal transduction mechanisms, the down-regulation of cytokine and nitric oxide release and the cytokine binding to cell membranes.^{53

–62}

Furthermore, we established that both ethanol and acetaldehyde are potent stimulators of the IL-6 secretion from cultured astrocytes. Similarly, an increased level of IL-6 secretion was observed in hepatic stellate cells and HepG2 cells after exposure to ethanol and acetaldehyde.^47,48 The stimulatory effect of ethanol was effectively reduced by the catalase inhibitor AMT. After 24 hours of exposure to different ethanol concentrations, we obtained a bell-shaped curve (Figure 3); the smallest concentration (50 mM) did not show any influence on the IL-6 secretion, whereas 100-mM and 200-mM ethanol induced abundant progressive secretion of the IL-6, which started to regress at 200-mM ethanol. After 7 days of exposure, we observed the maximum stimulation of the IL-6 secretion from the cells at a much lower concentration (50 mM) of ethanol (Figure 3). The dose-response curve of cultured rat astrocytes exposed to ethanol and acetaldehyde is hormestic. The hormesis is characterised as a continuum, stimulatory at low doses and inhibitory at high doses, resulting in biphasic dose-response curve.^{63

–66} Such a dose-response curve was also noticed in our previous study, where the influence of ethanol and acetaldehyde on astrocytes proliferation was examined. We observed an increase of the protein content after exposure to low concentrations of ethanol and acetaldehyde (up to 50 mM and 0.5 mM, respectively) whereas higher concentrations of both compounds had an inhibitory effect on the protein content.¹²

Acetaldehyde showed a similar response pattern at much lower concentrations, which is in line with Pentreath and Slamon’s conclusion that most gliotoxic agents have a biphasic dose-response curve: the response increases at low, subtoxic doses, which is followed by decreases at higher, cytotoxic doses.⁶⁷ The concentration range over which the biphasic response occurred could vary by several orders of magnitude for different markers of the same toxicant.

In our study, acetaldehyde was a more potent substance than ethanol. The stimulatory effect of acetaldehyde on the IL-6 secretion started at much lower concentration in comparison with the ethanol. After long-term exposure, acetaldehyde was even more effective in comparison to acute exposure, shifting the dose-response curve to the left (Figure 4B).

Inflammation is primarily a protective response of the target organism to a noxis. On the other hand, excessive or long-lasting inflammation is often followed by degenerative processes. The stimulatory effect of ethanol and acetaldehyde on IL-6 secretion seems to be involved in both neuroregenerative and survival processes as well as in neurodegeneration. The obtained hormestic dose-response relationship indicates that higher concentrations and long-term exposure could lead to a neurodegenerative direction, whereas low concentrations may act as neuroprotective.

Unlike TNF-alpha, which is responsible for the induction of multiple pro-inflammatory genes, IL-6 often fails to induce these genes. Moreover, IL-6 can down-regulate the expression of TNF-alpha.⁴² This is in line with our results, where we found the first significant decrease in the TNF-alpha level at the highest level of IL-6 after a long-term exposure to ethanol.

We found that the pre-treatment of the cells with the catalase inhibitor AMT (10 mM) significantly reduced the effect of the ethanol on the TNF-alpha and IL-6 secretion (Figure 2B and 3), which confirmed the role of acetaldehyde in the ethanol toxicity. Our results are in accordance with one of the rare reports found in the literature concerning acetaldehyde production in cultured astrocytes, where 10-mM AMT decreased ethanol degradation to acetaldehyde by 52%.⁵¹ Catalase is involved in several metabolic processes. In addition to ethanol metabolism in rat astrocytes, catalase is involved in the neuroprotective action of reactive oxygen substances, i.e. H₂O₂, in conditions of low oxygen supply in the rat brain. In the presence of higher concentrations of AMT, the neuroprotective effect of catalase is blocked.⁶⁸ Among other mechanisms, the intracellular concentration of H₂O₂, as a ubiquitous second messenger in subtoxic condition, is maintained at a constant level by catalase also.^{69

–74} We were able to conclude that catalase acts at the same time on acetaldehyde production and hydrogen peroxide removal and it plays a key role in the modulation of the effects due to long-term ethanol exposure.

In summary, we are the first to report that both ethanol and its primary metabolite, acetaldehyde, are potent modulators of TNF-alpha and IL-6 secretion from rat astroglial cells in primary cultures. Long-term exposure to ethanol and acetaldehyde results in more pronounced effects. Since the obtained dose-response relationship is non-linear and non-threshold but hormestic, it seems that the concentration of ethanol or acetaldehyde to which astrocytes have been exposed plays a crucial role in the extent of the inflammatory response. The duration of the exposure and the extent of the inflammatory response determine the outcome of the exposure, being either neuroprotective or neurodegenerative. Our findings will contribute to a better understanding of the effects of ethanol exposure on neurodegenerative and immunosuppressive processes and the regulatory role of TNF-alpha and IL-6 in the CNS.

Footnotes

The work was supported by the research grant P3-0067 from the Slovenian Research Agency, Republic of Slovenia.

References

Thomson

Pratt

Jeyasingham

Shaw

. Alcohol and brain damage. Hum Toxicol 1988; 7: 455–463.

Singh

Lai

Khan

. Ethanol-induced single-strand DNA breaks in rat brain cells. Mutat Res 1995; 345: 191–196.

Barret

Soubeyran

Usson

Eysseric

Saxod

. Characterization of the morphological variations of astrocytes in culture following ethanol exposure. Neurotoxicology 1996; 17: 497–507.

Holownia

Ledig

Copin

Tholey

. The effect of ethanol on HSP70 in cultured rat glial cells and in brain areas of rat pups exposed to ethanol in utero. Neurochem Res 1995; 20: 875–878.

Holownia

Ledig

Mapoles

Ménez

. Acetaldehyde-induced growth inhibition in cultured rat astroglial cells. Alcohol 1996; 13: 93–97.

Holownia

Ledig

Ménez

. Ethanol-induced cell death in cultured rat astroglia. Neuro Toxicol Teratol 1997; 19: 141–146.

Holownia

Ledig

Braszko

Ménez

. Acetaldehyde cytotoxicity in cultured rat astrocytes. Brain Res 1999; 833: 202–208.

Lamarche

Gonthier

Signorini

Eysseric

Barret

. Acute exposure of cultured neurones to ethanol results in reversible DNA single-strand breaks; whereas chronic exposure causes loss of cell viability. Alcohol Alcohol 2003; 38: 550–558.

Soltaninejad

Kebriaeezadeh

Minaiee

Ostad

Hosseini

Azizi

Biochemical and ultrastructural evidences for toxicity of lead through free radicals in rat brain. Hum Exp Toxicol 2003; 22: 417–423.

10.

Guizzetti

Costa

. Cholesterol homeostasis in the developing brain: a possible new target for ethanol. Hum Exp Toxicol 2007; 26: 355–360.

11.

Sarc

Lipnik-Stangelj

. Influence of ethanol and its first methabolite acetaldehyde on the central nervous system. Zdrav Vestn 2009; 78: 91–96.

12.

Sarc

Lipnik-Stangelj

. Comparison of ethanol and acetaldehyde toxicity in rat astrocytes in primary culture. Arh Hig rada Toksikol 2009; 60: 297–304.

13.

Kimelberg

Norenberg

. Astocytes. Sci Am 1989; 260: 66–76.

14.

Di Monte

Royland

Irwin

Langston

. Astrocytes as the site for bioactivation of neurotoxins. Neurotoxicology 1996; 17: 697–703.

15.

Monnet-Tschudi

Zurich

Honegger

. Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures. Hum Exp Toxicol 2007; 26: 339–346.

16.

Calabrese

. Astrocytes: adaptive responses to low doses of neurotoxins. Crit Rev Toxicol 2008; 38: 463–471.

17.

Calabrese

. Pharmacological enhancement of neuronal survival. Crit Rev Toxicol 2008; 38: 349–389.

18.

Quertemont

Eriksson

Zimatkin

Pronko

Diana

Pisano

Is ethanol a pro-drug? Acetaldehyde contribution to brain ethanol effects. Alcohol Clin Exp Res 2005; 29: 1514–1521.

19.

Quertemont

Tambour

Bernaerts

Zimatkin

Tirelli

. Behavioral characterization of acetaldehyde in C57BL/6J mice: locomotor, hypnotic, anxiolytic and amnesic effects. Psychopharmacology 2004; 177: 84–92.

20.

Deitrich

. Acetaldehyde: déjà vu du jour. J Stud Alcohol 2004; 65: 557–572.

21.

Luke

. Ethanol. In: Goldfrank

Flomenbaum

Lewin

Howland

Hoffman

Nelson

(eds) Goldfrank’s Toxicologic Emergencies. 7th ed. New York: McGraw-Hill, 2005, 1147–1161.

22.

Zimatkin

Dietrich

. Ethanol metabolism in the brain. Addiction Biol 2007; 2: 387–413.

23.

Hunt

. Role of acetaldehyde in the actions of ethanol on the brain. Alcohol 1996; 13: 147–151.

24.

Schad

Fahimi

Völkl

Baumgart

. Expression of catalase mRNA and protein in adult rat brain: detection by nonradioactive in situ hybridization with signal amplification by catalyzed reporter deposition (ISH-CARD) and immunohistochemistry (IHC)/immunofluorescence (IF). J Histochem Cytochem 2003; 51: 751–760.

25.

Zimatkin

Buben

. Ethanol oxidation in the living brain. Alcohol Alcohol 2007; 42: 529–532.

26.

Aragon

Rogan

Amit

. Studies on ethanol-brain catalase interaction: evidence for central ethanol oxidation. Alcoh Clin Exp Res 1991; 15: 165–169.

27.

Gill

Menez

Lucas

Deitrich

. Enzymatic production of acetaldehyde from ethanol in rat brain tissue. Alcohol Clin Exp Res 1992; 16: 910–915.

28.

Aragon

Amit

. Differences in ethanol-induced behaviors in normal and acatalasemic mice: systematic examination using a biobehavioral approach. Pharmacol Biochem Behav 1993; 44: 547–554.

29.

Vallés

Blanco

Pascual

Guerri

. Chronic ethanol treatment enhances inflammatory mediators and cell death in the brain and in astrocytes. Brain Pathol 2004; 14: 365–371.

30.

Davis

Syapin

. Acute ethanol exposure modulates expression of inducible nitric-oxide synthase in human astroglia: evidence for a transcriptional mechanism. Alcohol 2004; 32: 195–202.

31.

Blanco

Guerri

. Ethanol intake enhances inflammatory mediators in brain: role of glial cells and TLR4/IL-1RI receptors. Front Biosci 2007; 12: 2616–2630.

32.

Szabo

Mandrekar

Oak

Mayerle

Effect of ethanol on inflammatory responses. Implications for pancreatitis. Pancreatology 2007; 7: 115–123.

33.

Goral

Karavitis

Kovacs

. Exposure-dependent effects of ethanol on the innate immune system. Alcohol 2008; 42: 237–247.

34.

Szabo

Mandrekar

Girouard

Catalano

. Regulation of human monocyte functions by acute ethanol treatment: decreased tumor necrosis factor-alpha, interleukin-1 beta and elevated interleukin-10, and transforming growth factor-beta production. Alcohol Clin Exp Res 1996; 20: 900–907.

35.

Zisman

Strieter

Kunkel

Tsai

Wilkowski

Bucknell

Ethanol feeding impairs innate immunity and alters the expression of Th1- and Th2-phenotype cytokines in murine Klebsiella pneumonia. Alcohol Clin Exp Res 1998; 22: 621–627.

36.

Spies

Tønnesen

Andreasson

Helander

Conigrave

. Perioperative morbidity and mortality in chronic alcoholic patients. Alcohol Clin Exp Res 2001; 25: 164–170.

37.

Jerrells

. Association of alcohol consumption and exaggerated immunopathologic effects in the liver induced by infectious organism. Front Biosci 2002; 7: 487–493.

38.

Morganti-Kossman

Lenzlinger

Hans

Stahel

Csuka

Ammann

Production of cytokines following brain injury: beneficial and deleterious for the damaged tissue. Mol Psychiatry 1997; 2: 133–136.

39.

Gruol

Nelson

. Physiological and pathological roles of interleukin-6 in the central nervous system. Mol Neurobiol 1997; 15: 307–339.

40.

Chung

Benveniste

EN.

TNF-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1 beta. J Immunol 1990; 144: 2999–3007.

41.

John

Lee

Brosnan

. Cytokines: powerful regulators of glial cell activation. Neuroscientist 2003; 9: 10–22.

42.

Shrikant

Benveniste

. The central nervous system as an immunocompetent organ: role of glial cells in antigen presentation. J Immunol 1996; 157: 1819–1822.

43.

Bonni

Sun

Nadal-Vicens

Bhatt

Frank

Rozovsky

Regulation of gliogenesis in the central nervous system by the JAK-STAT signaling pathway. Science 1997; 278: 477–483.

44.

D’Souza

Nelson

Summer

Deaciuc

. Expression of tumor necrosis factor-alpha and interleukin-6 cell-surface receptors of the alveolar macrophage in alcohol-treated rats. Alcohol Clin Ex Res 1994; 18: 1430–1435.

45.

Wang

Huang

Giger

Watson

. Influence of chronic dietary ethanol on cytokine production by murine splenocytes and thymocytes. Alcohol Clin Exp Res 1994; 18: 64–70.

46.

Xie

Kolls

Bagby

Greenberg

. Independent suppression of nitric oxide and TNF alpha in the lung of conscious rats by ethanol. FASEB J 1995; 9: 253–261.

47.

Quiroz

Bucio

Souza

Hernandez

Gonzalez

Gomez-Quiroz

. Effect of endotoxin pretreatment on hepatic stellate cell response to ethanol and acetaldehyde. J Gastroenterol Hepatol 2001; 16: 1267–1273.

48.

Gutierrez-Ruiz

Gomez Quiroz

Hernandez

Bucio

Souza

Llorente

. Cytokine response and oxidative stress produced by ethanol, acetaldehyde and endotoxin treatment in HepG2 cells. Isr Med Assoc J 2001; 3: 131–136.

49.

Latvala

Hietala

Koivisto

Jarvi

Anttila

Niemela

. Immune responses to ethanol metabolites and cytokine profiles differentiate alcoholics with or without liver disease. Am J Gastroenterol 2005; 100: 1303–1310.

50.

Poisindex (2008) (CD ROM). Ethanol. Greenwood Village, USA: Thompson Micromedex; 2009.

51.

Eysseric

Gonthier

Soubeyran

Richard

Daveloose

Barret

. Effects of chronic ethanol exposure on acetaldehyde and free radical production by astrocytes in culture. Alcohol 2000; 21: 117–125.

52.

Bradford

. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248–254.

53.

Sei

McIntyre

Skolnick

Arora

. Ethanol inhibits mitogen-induced calcium mobilization in mouse splenocytes. Life Sci 1992; 50: 419–426.

54.

Roselle

Mendenhall

. Ethanol-induced alterations in lymphocyte function in the guinea pig. Alcohol Clin Exp Res 1984; 8: 62–67.

55.

McIntyre

Oxenkrug

. Chronic alcohol administration increases corticosterone resistance to suppression by dexamethasone. Biol Psychiatry 19: 1725–9.

56.

Deaciuc

D'Souza

Bagby

Lang

Spitzer

. Effect of acute alcohol administration on TNF-alpha binding to neutrophils and isolated liver plasma membranes. Alcohol Clin Exp Res 1992; 16: 533–538.

57.

Surrenti

Galli

. Molecular mechanisms of alcohol-induced liver injury. Gastroenterol Dietol 2003; 49: 95–105.

58.

Happel

Nelson

. Alcohol, immunosuppression, and the lung. Proc Am Thorac Soc 2005; 2: 428–432.

59.

Nath

Szabo

. Alcohol-induced modulation of signaling pathways in liver parenchymal and nonparenchymal cells: implications for immunity. Semin Liver Dis 2009; 29: 166–177.

60.

Goral

Karavitis

Kovacs

. Exposure-dependent effects of ethanol on the innate immune system. Alcohol 2008; 42: 237–247.

61.

Seth

D'Souza El-Guindy

Apte

Mari

Dooley

Neuman

. Alcohol, signaling, and ECM turnover. Alcohol Clin Exp Res 2010; 34: 4–18.

62.

Fernandez-Lizarbe

Pascual

Guerri

. Critical role of TLR4 response in the activation of microglia induced by ethanol. J Immunol 2009; 183: 4733–4744.

63.

Calabrese

Baldwin

. Hormesis: a generalizable and unifying hypothesis. Crit Rev Toxicol 2001; 31: 353–424.

64.

Calabrese

Baldwin

. Ethanol and hormesis. Crit Rev Toxicol 2003; 33: 407–424.

65.

Calabrese

. Neuroscience and hormesis: overview and general findings. Crit Rev Toxicol 2008; 38: 249–252.

66.

Calabrese

. Dose-response features of neuroprotective agents: an integrative summary. Crit Rev Toxicol 2008; 38: 253–348.

67.

Pentreath

Slamon

. Astrocyte phenotype and prevention against oxidative damage in neurotoxicity. Hum Exp Toxicol 2000; 19: 641–649.

68.

Nisticò

Piccirilli

Cucchiaroni

Armogida

Guatteo

Giampà

. Neuroprotective effect of hydrogen peroxide on an in vitro model of brain ischaemia. Br J Pharmacol 2008; 153: 1022–1029.

69.

Moreno

Mugnaini

Cerù

. Immunocytochemical localization of catalase in the central nervous system of the rat. J Histochem Cytochem 1995; 43: 1253–1267.

70.

Halliwell

. Antioxidant defence mechanisms: from the beginning to the end (of the beginning). Free Radic Res 1999; 31: 261–272.

71.

Finkel

. Oxygen radicals and signaling. Curr Opin Cell Biol 1998; 10: 248–253.

72.

Sharpe

Ollosson

Stewart

Clark

. Oxidation of nitric oxide by oxomanganese-salen complexes: a new mechanism for cellular protection by superoxide dismutase/catalase mimetics. Biochem J 2002; 366: 97–107.

73.

Safiulina

Afzalov

Khiroug

Cherubini

Giniatullin

. Reactive oxygen species mediate the potentiating effects of ATP on GABAergic synaptic transmission in the immature hippocampus. J Biol Chem 2006; 281: 23464–23470.

74.

Mander

Jekabsone

Brown

. Microglia proliferation is regulated by hydrogen peroxide from NADPH oxidase. J Immunol 2006; 176: 1046–1052.