Vol.
185,
No.
June
30.
1992
3, 1992
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
NONOXIDATIVE FATN
AND
ACID ETHYL
ETHANOL
METABOLISM:
ESTER SYNTHASE-III
Keith E. Isenberg*$&
Puran S. Bora*‘r,
EXPRESSION
IN CULTURED
938-943
OF
NEURAL
CELLS
Xia Zhou*, Xiaolin Wut,
Blake W. Moore* and Louis G. Lange*? * Department
of Psychiatry, Washington University School of Medicine, 4940 Children’s Place, St. Louis, MO 63110
i- Department
of Cardiology Research and *Department
Jewish Hospital of St. Louis, 216 S. Kingshighway, Received
May 1,
of Psychiatry,
St. Louis, MO 63110
1992
SUMMARY: Alcohol metabolism in the human brain has been characterized as essentially nonoxidative in nature, with the esterification of ethanol with fatty acids via fatty acid ethyl ester synthase. This pathway of ethanol metabolism is related to end organ damage in the brain but the neural cell type expressing FAEES has not been identified. In this study human and rodent neuroblastoma and glioma cell lines are assayed for fatty acid ethyl ester synthase activity. Cells with neuronal properties demonstrated higher activity than glioma cell lines. We confirmed the presence of the mRNA for one type of synthase, fatty acid ethyl ester synthase-III in three neuronal cell lines - NlEllS cells, PC12 cells, and SK-NMC cells. These results support the hypothesis that FAEES activity is expressed chiefly in cells with neuronal properties and suggest that non-oxidative ethanol metabolism is Press,II potentially related to the toxic effect of ethanol on the human brain. 0 1992Academic
The mechanisms by which ethanol and its metabolites induce end-organ toxicity are frequently unclear.
Oxidative metabolism of ethanol to its toxic metabolite
acetaldehyde
via the enzyme alcohol dehydrogenase (ADH) is a well described pathway in organs such as the liver. Alcohol dehydrogenases can be divided into at least three classes (1). Human class I isoenzymes metabolize ethanol at pharmacological concentrations but have not been identified in the brain. Only one class of ADH isoenzyme has been demonstrated in human brain, the class III ADH (2) but this isoenzyme oxidizes ethanol very poorly and is detected $To whom reprint requests should be addressed. Abbreviations: FAEE, Fatty Acid Ethyl Ester; FAEES, Fatty Acid Ethyl Ester Synthase. 0006-291X/92 Copyright Ail rights
$4.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
938
Vol.
BIOCHEMICAL
185, No. 3, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
only when pentanol or aliphatic alcohols of longer chain length are used to measure activity. Thus the minimal ability of the brain to oxidatively metabolize ethanol (2) implies that other metabolic pathways may underlie the toxic effects of ethanol on the brain. An important
nonoxidative pathway involves the synthesis of fatty acid ethyl esters
(FAEE) from fatty acids and ethanol. The reaction, catalyzed by the enzyme fatty acid ethyl ester synthase (FAEES),
is detectable in many tissues susceptible to ethanol toxicity
including human brain (3-13). The activity is concentrated in gray matter (5,14) suggesting the localization of activity in neurons.
Elevated levels of activity are seen in the brains of
alcoholics compared with nonalcoholic controls (5) correlating FAEES with ethanol toxicity. The present study investigates the role of FAEE in ethanol-induced demonstrates a neural cell model that appears appropriate
neurotoxicity
and
for such studies by reporting
FAEES activity for a variety of neural cells. Selected cell lines were used for the expression of FAEES-III
mRNA.
MATERIAL
AND METHODS
Cell culture: C, rat glioma cells (clone C,,, originally from S.E. Pfeiffer, University of Connecticut), PC12 rat pheochromocytoma cells (provided by J.H. Steinbach, Department of Anesthesiology, Washington University School of Medicine), and NlE115 mouse neuroblastoma cells (provided by Dr. Stan Misler, Jewish Hospital, St. Louis, MO) are seeded onto 100 mM tissue culture plates (Falcon) at a density of lo6 cells/plate and grown in Dulbecco’s Modified Eagles Medium (Gibco) supplemented with L-glutamine (2mM), penicillin (100 units/ml), streptomycin (lOOug/ml) and fetal calf serum (10%) until the cells are confluent (7 to 10 days). SK-N-MC, a human neuroblastoma cell line (ATCC), SK-NSH, a human neuroblastoma cell line (ATCC), U-87MG, a human glioma cell line (ATCC) and U-373 MG, a human glioma cell line (ATCC) are plated in the same manner, but grown in Minimal Essential Medium (Gibco) supplemented with L-glutamine (2mM), penicillin (100 units/ml), streptomycin (100 &ml) and fetal calf serum (10%). Media is changed every two to three days. Cell viability is determined by trypan blue exclusion, cell counts determined by a hemocytometer based method, and protein concentrations determined spectrophotometrically (15). Cells are grown to confluence for assay. Enzvme Assavs: cell and tissue homogenates are mixed with [14C] oleate in a 65 mM phosphate buffer, pH 7.2 for 45 min. at 37°C. The reactions are terminated by the addition of 2 ml cold acetone containing a known amount of ethyl rH] oleate as a yield marker and ethyl oleate as a carrier. Reaction products are separated by chromatography on silica plates in a petroleum ether/diethyl ether/acetic acid (75/5/l) solvent and visualized by iodine vapor and scraped off for liquid scintillation counting. Counts for [‘“Cl are adjusted according to yield as determined by recovery of rH] counts and, after subtracting the buffer blank, results are expressed as nmoles of FAEE formed/ml/hr (8). Quantification of protein concentration permitted conversion to nmoles FAEE formed/mg protein/ hr. Statistical analysis was performed with GB Stat (Version 2.0, Dynamic Microsystems). FAEES-III Northern Blot Analysis (16): cellular RNA is prepared from cells by the acid-phenol method; poly A+ RNA is separated from total cellular RNA by affinity chromatography on an oligo dT column. RNA is electrophoretically fractionated on a 1% 939
Vol.
185,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
agarose 2.2M formaldehyde gel and transferred to nitrocellulose by the capillary method. FAEES-III cDNA (14) is labelled with t3*P] dCTP by “random priming” (Boerhinger Mannheim) and hybridization performed at 60°C. Post hybridization washes in 1 x SSC, 0.1% SDS at 60°C removed nonspecific reaction products. FAEES-III mRNA is visualized by exposure of the filter to x-ray film (Kodak XAR) and enhancing screen for up to 3 days.
RESULTS AND DISCUSSION We sought to clarify the role of ethanol metabolites in neural toxicity by studying FAEES in cultured neural cells. The cultured human neuroblastoma and SK-N-SH
cells, a mouse
pheochromocytoma
neuroblastoma
cell lines, SK-N-MC
cell line NlE115
cells and a rat
cell line PC 12 cells have more FAEES activity (Table 1) than rat C,
glioma cells, human US7 MG and U373 MG glioma cells. These results support the conclusion that neuronal cells express significantly more FAEES activity than other neural cell types.
The relatively greater abundance of FAEES activity in gray matter (which
TABLE FAEES
ACTIVITY
1
IN NEURAL
Cell Lines
CELL
LINES
FAEES Activity (nmole/mg proteirdhr) -c S.D
Glial cell lines C, cells
2.5 f 0.7 3.8 2 0.2 0.8 k 0.1
U87 MG U373 MG
Neuroblastoma cell lines SK-N-MC SK-N-SH NlE115
8.8 rt 5.0 9.4 5 7.9 131 + 21.9
Pheochromocvtoma cell line PC 12 FAEES
105 -c 57.7 activity
is concentrated
in cells
with
neuronal
properties. Clonal cell lines with glial properties (rat C6 cells, human U87 MG cells, and human U373 MG cells) and neuronal properties (human SK-N-MC cells, human SK-N-SH cells, mouse NlE115 cells, and rat PC 12 cells) were grown to confluence and assayed for FAEES activity. Enzyme activity is expressed in nanomoles of product formed per milligram of protein per hour; FAEES activity is the mean k the standard deviation. PC 12 cells express substantial amounts of FAEES activity, and C6 cells express little FAEES activity. 940
Vol.
BIOCHEMICAL
185, No. 3, 1992
28s 18s
AND BIOPHYSICAL
-
+
28s
-
-
18s
A
RESEARCH COMMUNICATIONS
B
Fig.. Cells with neuronal properties express FAEES-III mRNA and a cell line with glial properties does not. Polyadenylated RNA (panel A) or total RNA (panel B) was prepared from PC 12 cells, NlE115 cells, C6 cells, SK-N-SH cells and SK-N-MC cells, electrophoretically separated under denaturing conditions and transferred to nitrocellulose. The Northern blots were probed with a radioactive probe for FAEES-III; the blot was exposed to film after washes removed nonspecific probe binding. The migration of markers is indicated on the side of the panels. The FAEES-III probe detected the same 0.7 kb RNA species in both experiments, the expected size. FAEES-III mRNA appears particularly abundant in PC 12 cells.
contains an abundance of neuronal cell bodies) compared with white matter (composed
chiefly of myelinated processes) previously reported (5) agrees with this conclusion. Northern blots of poly A+ RNA and total RNA reveal abundant expression of the 0.7 kb FAEES-III FAEES-III
mRNA in PC12 cells, NlE115 cells and SK-N-MC
cells (Fig. 1). No
mRNA is detectable in C, cells, supporting the activity data. The PC12 cells
have significant amounts of FAEES activity and also express the 0.7 kb mRNA.
Three
isoforms of FAEES (designated I, II, and III) have been reported (9). The absence of FAEES-III
mRNA in SK-N-SH cells suggests that either FAEES-II, FAEES-I, or both may
be responsible for the observed FAEES activity.
FAEES-I and FAEES-II
are currently
being cloned in our laboratory and will be used as probes to pinpoint the presence of these isoforms. Ethanol is a common cause of dementia (17) and is felt to have a toxic effect independent of alcoholism associated malnutrition
(such as Wernicke-Korsakoff
syndrome).
Imaging studies suggest acute and chronic effects of ethanol; abstinent chronic alcoholics 941
Vol.
185,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
may have diminished cerebral glucose metabolism, reflecting diminished neuronal activity and neuronal loss (18). Q uantitative neuropathological
studies of alcoholics (19) suggest
selective loss of neurons as well as shrinkage. Studying the mechanism of ethanol toxicity in the human brain is complicated by the relative inaccessibility of the brain. The cortex and cerebellar vermis are most susceptible to the effects of ethanol on neurons (18, 19) suggesting regional specificity of ethanol toxicity.
Understanding
the toxic effects of ethanol on the brain is difficult in the intact
animal due to the toxic effects of ethanol on other organs as well as the systemic distribution
of toxic metabolites from organs like the liver. The cell lines employed in this
study are well characterized
and considered useful for neurotoxicology
studies (20).
Cultured neural cell lines are an excellent model system for studying the effects of ethanol and its metabolites. Confirmation
that neurons are able to nonoxidatively metabolize ethanol supports
the hypothesis that FAEES contributes to the central nervous system toxicity of ethanol observed in humans. mitochondrial
FAEE,
products
of the nonoxidative
pathway,
function and induce damage to the brain (21). Moreover,
can impair FAEE inhibit
cholesterol esterification (22) potentially disrupting myelin metabolism and hence impairing nerve conduction.
Studies assessing the genetic linkage of the FAEES pathway to end
organ susceptibility to ethanol toxicity seem warranted.
ACKNOWLEDGMENTS This research was supported by grants from NIH/NIAAA
(AA07569 to Blake W.
Moore; AA06989 to Louis G. Lange; AA08247 to Louis G. Lange); an American Heart Grant In Aid # 890891 (Louis G. Lange); and a grant from the Alcoholic Beverage Medical Research Foundation
(Louis G. Lange).
REFERENCES 1. 2.
Boleda, M.D., Julia, P., Moreno, A., and Pares, X. (1989) Arch. Biochem. Biophys. 274, 74-81. Beisswenger, T.B., Holmquist, B., and Vallee, B.L. (1985) Proc. Natl. Acud. Sci. USA 82, 8369-8373.
3. 4. 5.
Hamamoto, T., Yamada, S., and Hirayama, C. (1990) B&hem. Pharmacol. 39,241245. Laposata, E.A., and Lange, L.G. (1986) Science 231, 497-499. Laposata, E.A., Scherrer, D.E., Mazow, C., and Lange, L.G. (1987) J. Biol. Chem. 262, 4653-4657. 942
Vol.
6.
185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
Laposata, E.A., Scherrer, D.E., and Lange, L.G.
RESEARCH COMMUNICATIONS
(1989) Arch. Pathol. Lab. Med.
113, 762-766. 7. 8.
Bora, P.S., Spilburg, C.A., and Lange, L.G. (1989) FEBS Lett. 258, 236-239. Bora, P.S., Spilburg, C.A., and Lange, L.G. (1989) Proc. Natl. Acd. Sci., U.S.A. 86,
9. 10. 11.
Bora, P.S., Spilburg, C.A., and Lange, L. G. (1989) J. Clin. Invest. 84, 1942-1946. Bora, P.S., and Lange, L.G. (1991) Ann. NYAcad. Sci. 625, 827-829. Bora, P.S., and Lange, L.G. (1991) In Alcohol and Drug Abuse Reviews: Liver Pathology and Drugs of Abuse. (R. R. Watson, Ed.), ~01.11, pp. 241-257. Humana Press, Totowa, NJ. Bora, P.S., and Lange, L.G. (1991) Alcohol Health Research World 14, 27-35. Bora, P.S., Bora, N.S. Wu, X., and Lange, L.G. (1991) .Z.Biol. Chem. 266, 1677416777. Bora, P.S., and Lange, L.G. (1992) Alcohol: Clin. Exp. Rex 16, 220-225. Freshney, R.I. (1987) Culture of Animal Cells: A Manual of Basic Technique (Ed. 2). p. 397. Wiley-Liss, New York. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Ed. 2) 3 vol. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Van Horn, G. (1987) Am. J. Med. 83, 101-110. Wik, G., Borg, S., Sjogren, I., Wiesel, F.-A., Blomquist, G., Borg, J., Greitz, T., Nyback, H., Sedvall, G., Stone-Elander, S., and Widen, L. (1988) Acta. Psych. Stand. 78, 234-241. Harper, C., Kril, J., and Daly, J. (1987) Brit. Med. J. 194, 534-536. Veronesi, B. (1992) In Neurotoxicology. (H. A. Tilson and C.L. Mitchell, Eds.), pp. 21-49. Raven Press, New York. Lange, L.G., and Sobel, B.E. (1983) .Z. Clin. Invest. 72, 724-731. Lange, L.G. (1982) Proc. Nat. Acad. Sci. USA. 79, 3954-3957.
70-4473.
12. 13. 14. 15. 16. 17. 18. 19. 20.
21. 22.
943
185,
No.
June
30.
1992
3, 1992
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
NONOXIDATIVE FATN
AND
ACID ETHYL
ETHANOL
METABOLISM:
ESTER SYNTHASE-III
Keith E. Isenberg*$&
Puran S. Bora*‘r,
EXPRESSION
IN CULTURED
938-943
OF
NEURAL
CELLS
Xia Zhou*, Xiaolin Wut,
Blake W. Moore* and Louis G. Lange*? * Department
of Psychiatry, Washington University School of Medicine, 4940 Children’s Place, St. Louis, MO 63110
i- Department
of Cardiology Research and *Department
Jewish Hospital of St. Louis, 216 S. Kingshighway, Received
May 1,
of Psychiatry,
St. Louis, MO 63110
1992
SUMMARY: Alcohol metabolism in the human brain has been characterized as essentially nonoxidative in nature, with the esterification of ethanol with fatty acids via fatty acid ethyl ester synthase. This pathway of ethanol metabolism is related to end organ damage in the brain but the neural cell type expressing FAEES has not been identified. In this study human and rodent neuroblastoma and glioma cell lines are assayed for fatty acid ethyl ester synthase activity. Cells with neuronal properties demonstrated higher activity than glioma cell lines. We confirmed the presence of the mRNA for one type of synthase, fatty acid ethyl ester synthase-III in three neuronal cell lines - NlEllS cells, PC12 cells, and SK-NMC cells. These results support the hypothesis that FAEES activity is expressed chiefly in cells with neuronal properties and suggest that non-oxidative ethanol metabolism is Press,II potentially related to the toxic effect of ethanol on the human brain. 0 1992Academic
The mechanisms by which ethanol and its metabolites induce end-organ toxicity are frequently unclear.
Oxidative metabolism of ethanol to its toxic metabolite
acetaldehyde
via the enzyme alcohol dehydrogenase (ADH) is a well described pathway in organs such as the liver. Alcohol dehydrogenases can be divided into at least three classes (1). Human class I isoenzymes metabolize ethanol at pharmacological concentrations but have not been identified in the brain. Only one class of ADH isoenzyme has been demonstrated in human brain, the class III ADH (2) but this isoenzyme oxidizes ethanol very poorly and is detected $To whom reprint requests should be addressed. Abbreviations: FAEE, Fatty Acid Ethyl Ester; FAEES, Fatty Acid Ethyl Ester Synthase. 0006-291X/92 Copyright Ail rights
$4.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
938
Vol.
BIOCHEMICAL
185, No. 3, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
only when pentanol or aliphatic alcohols of longer chain length are used to measure activity. Thus the minimal ability of the brain to oxidatively metabolize ethanol (2) implies that other metabolic pathways may underlie the toxic effects of ethanol on the brain. An important
nonoxidative pathway involves the synthesis of fatty acid ethyl esters
(FAEE) from fatty acids and ethanol. The reaction, catalyzed by the enzyme fatty acid ethyl ester synthase (FAEES),
is detectable in many tissues susceptible to ethanol toxicity
including human brain (3-13). The activity is concentrated in gray matter (5,14) suggesting the localization of activity in neurons.
Elevated levels of activity are seen in the brains of
alcoholics compared with nonalcoholic controls (5) correlating FAEES with ethanol toxicity. The present study investigates the role of FAEE in ethanol-induced demonstrates a neural cell model that appears appropriate
neurotoxicity
and
for such studies by reporting
FAEES activity for a variety of neural cells. Selected cell lines were used for the expression of FAEES-III
mRNA.
MATERIAL
AND METHODS
Cell culture: C, rat glioma cells (clone C,,, originally from S.E. Pfeiffer, University of Connecticut), PC12 rat pheochromocytoma cells (provided by J.H. Steinbach, Department of Anesthesiology, Washington University School of Medicine), and NlE115 mouse neuroblastoma cells (provided by Dr. Stan Misler, Jewish Hospital, St. Louis, MO) are seeded onto 100 mM tissue culture plates (Falcon) at a density of lo6 cells/plate and grown in Dulbecco’s Modified Eagles Medium (Gibco) supplemented with L-glutamine (2mM), penicillin (100 units/ml), streptomycin (lOOug/ml) and fetal calf serum (10%) until the cells are confluent (7 to 10 days). SK-N-MC, a human neuroblastoma cell line (ATCC), SK-NSH, a human neuroblastoma cell line (ATCC), U-87MG, a human glioma cell line (ATCC) and U-373 MG, a human glioma cell line (ATCC) are plated in the same manner, but grown in Minimal Essential Medium (Gibco) supplemented with L-glutamine (2mM), penicillin (100 units/ml), streptomycin (100 &ml) and fetal calf serum (10%). Media is changed every two to three days. Cell viability is determined by trypan blue exclusion, cell counts determined by a hemocytometer based method, and protein concentrations determined spectrophotometrically (15). Cells are grown to confluence for assay. Enzvme Assavs: cell and tissue homogenates are mixed with [14C] oleate in a 65 mM phosphate buffer, pH 7.2 for 45 min. at 37°C. The reactions are terminated by the addition of 2 ml cold acetone containing a known amount of ethyl rH] oleate as a yield marker and ethyl oleate as a carrier. Reaction products are separated by chromatography on silica plates in a petroleum ether/diethyl ether/acetic acid (75/5/l) solvent and visualized by iodine vapor and scraped off for liquid scintillation counting. Counts for [‘“Cl are adjusted according to yield as determined by recovery of rH] counts and, after subtracting the buffer blank, results are expressed as nmoles of FAEE formed/ml/hr (8). Quantification of protein concentration permitted conversion to nmoles FAEE formed/mg protein/ hr. Statistical analysis was performed with GB Stat (Version 2.0, Dynamic Microsystems). FAEES-III Northern Blot Analysis (16): cellular RNA is prepared from cells by the acid-phenol method; poly A+ RNA is separated from total cellular RNA by affinity chromatography on an oligo dT column. RNA is electrophoretically fractionated on a 1% 939
Vol.
185,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
agarose 2.2M formaldehyde gel and transferred to nitrocellulose by the capillary method. FAEES-III cDNA (14) is labelled with t3*P] dCTP by “random priming” (Boerhinger Mannheim) and hybridization performed at 60°C. Post hybridization washes in 1 x SSC, 0.1% SDS at 60°C removed nonspecific reaction products. FAEES-III mRNA is visualized by exposure of the filter to x-ray film (Kodak XAR) and enhancing screen for up to 3 days.
RESULTS AND DISCUSSION We sought to clarify the role of ethanol metabolites in neural toxicity by studying FAEES in cultured neural cells. The cultured human neuroblastoma and SK-N-SH
cells, a mouse
pheochromocytoma
neuroblastoma
cell lines, SK-N-MC
cell line NlE115
cells and a rat
cell line PC 12 cells have more FAEES activity (Table 1) than rat C,
glioma cells, human US7 MG and U373 MG glioma cells. These results support the conclusion that neuronal cells express significantly more FAEES activity than other neural cell types.
The relatively greater abundance of FAEES activity in gray matter (which
TABLE FAEES
ACTIVITY
1
IN NEURAL
Cell Lines
CELL
LINES
FAEES Activity (nmole/mg proteirdhr) -c S.D
Glial cell lines C, cells
2.5 f 0.7 3.8 2 0.2 0.8 k 0.1
U87 MG U373 MG
Neuroblastoma cell lines SK-N-MC SK-N-SH NlE115
8.8 rt 5.0 9.4 5 7.9 131 + 21.9
Pheochromocvtoma cell line PC 12 FAEES
105 -c 57.7 activity
is concentrated
in cells
with
neuronal
properties. Clonal cell lines with glial properties (rat C6 cells, human U87 MG cells, and human U373 MG cells) and neuronal properties (human SK-N-MC cells, human SK-N-SH cells, mouse NlE115 cells, and rat PC 12 cells) were grown to confluence and assayed for FAEES activity. Enzyme activity is expressed in nanomoles of product formed per milligram of protein per hour; FAEES activity is the mean k the standard deviation. PC 12 cells express substantial amounts of FAEES activity, and C6 cells express little FAEES activity. 940
Vol.
BIOCHEMICAL
185, No. 3, 1992
28s 18s
AND BIOPHYSICAL
-
+
28s
-
-
18s
A
RESEARCH COMMUNICATIONS
B
Fig.. Cells with neuronal properties express FAEES-III mRNA and a cell line with glial properties does not. Polyadenylated RNA (panel A) or total RNA (panel B) was prepared from PC 12 cells, NlE115 cells, C6 cells, SK-N-SH cells and SK-N-MC cells, electrophoretically separated under denaturing conditions and transferred to nitrocellulose. The Northern blots were probed with a radioactive probe for FAEES-III; the blot was exposed to film after washes removed nonspecific probe binding. The migration of markers is indicated on the side of the panels. The FAEES-III probe detected the same 0.7 kb RNA species in both experiments, the expected size. FAEES-III mRNA appears particularly abundant in PC 12 cells.
contains an abundance of neuronal cell bodies) compared with white matter (composed
chiefly of myelinated processes) previously reported (5) agrees with this conclusion. Northern blots of poly A+ RNA and total RNA reveal abundant expression of the 0.7 kb FAEES-III FAEES-III
mRNA in PC12 cells, NlE115 cells and SK-N-MC
cells (Fig. 1). No
mRNA is detectable in C, cells, supporting the activity data. The PC12 cells
have significant amounts of FAEES activity and also express the 0.7 kb mRNA.
Three
isoforms of FAEES (designated I, II, and III) have been reported (9). The absence of FAEES-III
mRNA in SK-N-SH cells suggests that either FAEES-II, FAEES-I, or both may
be responsible for the observed FAEES activity.
FAEES-I and FAEES-II
are currently
being cloned in our laboratory and will be used as probes to pinpoint the presence of these isoforms. Ethanol is a common cause of dementia (17) and is felt to have a toxic effect independent of alcoholism associated malnutrition
(such as Wernicke-Korsakoff
syndrome).
Imaging studies suggest acute and chronic effects of ethanol; abstinent chronic alcoholics 941
Vol.
185,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
may have diminished cerebral glucose metabolism, reflecting diminished neuronal activity and neuronal loss (18). Q uantitative neuropathological
studies of alcoholics (19) suggest
selective loss of neurons as well as shrinkage. Studying the mechanism of ethanol toxicity in the human brain is complicated by the relative inaccessibility of the brain. The cortex and cerebellar vermis are most susceptible to the effects of ethanol on neurons (18, 19) suggesting regional specificity of ethanol toxicity.
Understanding
the toxic effects of ethanol on the brain is difficult in the intact
animal due to the toxic effects of ethanol on other organs as well as the systemic distribution
of toxic metabolites from organs like the liver. The cell lines employed in this
study are well characterized
and considered useful for neurotoxicology
studies (20).
Cultured neural cell lines are an excellent model system for studying the effects of ethanol and its metabolites. Confirmation
that neurons are able to nonoxidatively metabolize ethanol supports
the hypothesis that FAEES contributes to the central nervous system toxicity of ethanol observed in humans. mitochondrial
FAEE,
products
of the nonoxidative
pathway,
function and induce damage to the brain (21). Moreover,
can impair FAEE inhibit
cholesterol esterification (22) potentially disrupting myelin metabolism and hence impairing nerve conduction.
Studies assessing the genetic linkage of the FAEES pathway to end
organ susceptibility to ethanol toxicity seem warranted.
ACKNOWLEDGMENTS This research was supported by grants from NIH/NIAAA
(AA07569 to Blake W.
Moore; AA06989 to Louis G. Lange; AA08247 to Louis G. Lange); an American Heart Grant In Aid # 890891 (Louis G. Lange); and a grant from the Alcoholic Beverage Medical Research Foundation
(Louis G. Lange).
REFERENCES 1. 2.
Boleda, M.D., Julia, P., Moreno, A., and Pares, X. (1989) Arch. Biochem. Biophys. 274, 74-81. Beisswenger, T.B., Holmquist, B., and Vallee, B.L. (1985) Proc. Natl. Acud. Sci. USA 82, 8369-8373.
3. 4. 5.
Hamamoto, T., Yamada, S., and Hirayama, C. (1990) B&hem. Pharmacol. 39,241245. Laposata, E.A., and Lange, L.G. (1986) Science 231, 497-499. Laposata, E.A., Scherrer, D.E., Mazow, C., and Lange, L.G. (1987) J. Biol. Chem. 262, 4653-4657. 942
Vol.
6.
185, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
Laposata, E.A., Scherrer, D.E., and Lange, L.G.
RESEARCH COMMUNICATIONS
(1989) Arch. Pathol. Lab. Med.
113, 762-766. 7. 8.
Bora, P.S., Spilburg, C.A., and Lange, L.G. (1989) FEBS Lett. 258, 236-239. Bora, P.S., Spilburg, C.A., and Lange, L.G. (1989) Proc. Natl. Acd. Sci., U.S.A. 86,
9. 10. 11.
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