ESPERlhfENTAL
AND
MOLECULAR
Saturated II. Lipid
27, 262-276
PATHOLOGY
Free Fatty
(1977)
Acid
Toxicity
Accumulation, Ultrastructural Alterations, in Mammalian Cells in Culture GERALD
B. GORDON
Department of Pathology, State Unicersity of New Upstate Medical Center, Syracuse, New York Reched
and Toxicity
Februrary
York,
17, 1977
The addition of 3 x lo-” M long-chain saturated free fatty acids (FFA) to suspension tissue cultures of strain L mouse fibroblasts resulted in the inhibition of cell proliferation and eventual cell death. The earliest morphologic alterations associated with this toxicity were characteristic elongated dilatations of the endoplasmic reticulum ( ER). These appeared as angular, slit-like, electron-translucent spaces which displaced the normal granular contents of the ER. Lipid analyses demonstrated the early accumulation of triglyceride and FFA in these cells. Longer incubations produced other morphologic changes associated with cell injury, and elevations of all lipid fractions were found. Unsaturated FFA at the same concentration did not inhibit culture growth. Cells incubated with unsaturated FFA contained numerous cytoplasmic lipid droplets, but no other ultrastructural abnormalities were noted. Intracellular triglyceride was markedly increased, without accumulation of any other major lipid fraction. These findings indicate that saturated FFA toxicity may be due to the deposition of saturated fats within the ER with subsequent interference with cellular metabolism.
INTRODUCTION In previous experiments (Hawley and Gordon, 1976), long-chain saturated free fatty acids (FFA) were shown to cause structural and functional abnormalities in human neutrophils. In c&o incubation of neutrophils with saturated FFA resulted in cleft-like dilatations of the endoplasmic reticulum and perinuclear space. This finding was associated with decreased neutrophil function tests, especially chemotactic ability. Incubation with the same concentration of unsaturated FFA did not produce similar structural or functional alterations. Moskowitz (1967) observed that cells grown in monolayer tissue culture reacted differently to added saturated or unsaturated fatty acid. Unsaturated FFA resulted in the accumulation of numerous lipid-staining cytoplasmic droplets. However, saturated FFA caused the formation of cytoplasmic clefts, which failed to stain with any of the usual histologic lipid stains. Furthermore, it was stated that cells containing these clefts underwent irreversible degenerative changes. In the present experiments, studies on the effect of saturated and unsaturated fatty acids on cell growth, viability, lipid accumulation, and morphology were 262 Copyright All rights
@ 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0014~ -4800
SATURATED
FREE FATTY
ACID
TOXICITY
263
performed in suspension cultured mammalian cells. This report describes the characteristic findings of saturated FFA cellular toxicity including the combination of peculiar ultrastructural alterations of the endoplasmic reticulum and the accumulation of intracellular triglyceride and FFA. MATERIALS
AND METHODS
Preparation of Albumin-Bound FFA Four long-chain fatty acids were chosen for this study: oleic, a monounsaturated fatty acid; linoleic, a polyunsaturate; and two saturated fatty acids, palmitic and stearic. Solutions of albumin-bound FFA were prepared by a modification of the method of Goodman ( 1958). One millimole of FFA (Hormel Institute, Austin, Minnesota) was dissolved in 20 ml of n-heptane and added to 100 ml of Eagle’s minimum essential medium (MEM) containing 3 g of fatty acid-free bovine albumin, fraction V (Miles Laboratories Inc., Elkhart, Indiana). The mixture was shaken gently at room temperature for 48 hr, centrifuged, and the heptane layer was removed by thorough aspiration. Any residual dissolved heptane was removed by evaporation under a stream of nitrogen, The pH was adjusted to 7.0 with 0.1 M NaOH. The solution was then sterilized by filtration through a 0.22-pm membrane filter (Millipore Corp., Bedford, Massachusetts) and stored under a nitrogen atmosphere. Fatty acid-free albumin solutions were prepared in the same manner by incubating with n-heptane without added fatty acid for use in control experiments. No detectable differences on cellular growth, viability, or morphology were observed in cultures receiving either unincubated albumin solutions or n-heptaneincubated albumin solutions. Previously, Goodman (1958) and Spector et al. (1969) reported no apparent physical or chemical alterations in albumin similarly treated with heptane. The prepared solutions were analyzed for FFA concentration by the method of Novak ( 1965). Albumin content was determined by a modified Lowry procedure (Oyama and Eagle, 1956). A molar ratio (Y) of FFA to albumin of approximately 8:l was routinely obtained in the test solutions with an average FFA concentration of 3 X 10e3M. Tissue Culture Studi& Strain L (Earle) mouse fibroblasts were grown in suspension tissue culture in 250-ml Ehrlenmeyer flasks agitated on a rotary shaker at 37°C (Kuchler and Merchant, 1956). Each flask contained 80 ml of culture medium composed of Eagle’s MEM supplemented with 10% horse serum, penicillin (100 units/ml), and streptomycin (100 pg/ml). An appropriate number of cells from stock cultures in logarithmic growth were used to obtain an initial inoculum in the experimental cultures of approximately 250,000 cells/ml. The pH was adjusted to approximately 7.0 by gassingwith filtered 8% CO:! in air. For each experiment, replicate cultures were established and growth curves were determined by daily cell counts and cellular protein determinations. The cell counts were performed using a standard hemocytometer as described previously (King et al., 1959), and cell protein content was determined by the modified Lowry method of Oyama and Eagle (1956). After 24 hr of incuba-
264
GERALD
B. GORDON
tion with the cultures in the logarithmic growth phase, 8 ml of culture medium was removed from each experimental culture and replaced with 8 ml of Eagle’s MEM containing the appropriate albumin-bound long-chain free fatty acid (3 x lo-” hl). Control cultures received 8 ml of medium containing fatty acidfree albumin. The actual media concentration of FFA and protein was estimated by mixing the supernatant removed from each culture with the test FFA solution in the exact proportion used in each experiment. The average media molar ratio (Y) of FFA to albumin in the experimental cultures was determined to be 3.0 -C 0.2 as compared to 0.3 * 0.1 in the controls. In different experiments, cultures were allowed to incubate with the added long-chain free fatty acid for varying intervals: 1, 2, 4, 12, 24, and 48 hr. After incubation, the cells were counted and cell viability was estimated by trypan blue dye exclusion ( McLimans et nl., 1957). From each culture, aliquots of 5 ml for protein determination and 50 ml for lipid studies were taken. The remainder was prepared for electron microscopy. The experiments with each fatty acid were done in triplicate and repeated twice so that the reported data on ultrastructure, cell growth, and lipid analysis each represents the findings of six separate cultures. Statistical analyses for significance were performed by using Student’s t test. I,? .A; ” 4 ‘L, %J Suspension fixation of the L cells was performed as described previously (Gordon et al., 1963). Cold 2% glutaraldehyde in 0.5 M cacodylate buffer, pH 7.2, was added immediately in equal amounts to the aliquots of the cultures taken for electron microscopy. After 10 min at 5”C, the cells were centrifuged at 4OOg for 3 min, and the supernatant was discarded. Additional glutaraldehyde fixative was gently layered over the cell buttons, which were allowed to fix overnight in the cold. The cell buttons were then washed in 0.1 M cacodylate buffer containing 0.2 M sucrose, sliced into l-mm” blocks and postfixed in veronalbuffered 1% osmium tetroxide (Palade, 1952). Dehydration through alcohol and propylene oxide was followed by embedding in Araldite (Luft, 1961). Sections were cut with an LKB ultramicrotome and stained with uranyl acetate and lead citrate prior to examination in a Philips EM 300 electron microscope.
Electron
Microscopy
Lipid Analyses
4 .
AND
MOLECULAR
Saturated II. Lipid
27, 262-276
PATHOLOGY
Free Fatty
(1977)
Acid
Toxicity
Accumulation, Ultrastructural Alterations, in Mammalian Cells in Culture GERALD
B. GORDON
Department of Pathology, State Unicersity of New Upstate Medical Center, Syracuse, New York Reched
and Toxicity
Februrary
York,
17, 1977
The addition of 3 x lo-” M long-chain saturated free fatty acids (FFA) to suspension tissue cultures of strain L mouse fibroblasts resulted in the inhibition of cell proliferation and eventual cell death. The earliest morphologic alterations associated with this toxicity were characteristic elongated dilatations of the endoplasmic reticulum ( ER). These appeared as angular, slit-like, electron-translucent spaces which displaced the normal granular contents of the ER. Lipid analyses demonstrated the early accumulation of triglyceride and FFA in these cells. Longer incubations produced other morphologic changes associated with cell injury, and elevations of all lipid fractions were found. Unsaturated FFA at the same concentration did not inhibit culture growth. Cells incubated with unsaturated FFA contained numerous cytoplasmic lipid droplets, but no other ultrastructural abnormalities were noted. Intracellular triglyceride was markedly increased, without accumulation of any other major lipid fraction. These findings indicate that saturated FFA toxicity may be due to the deposition of saturated fats within the ER with subsequent interference with cellular metabolism.
INTRODUCTION In previous experiments (Hawley and Gordon, 1976), long-chain saturated free fatty acids (FFA) were shown to cause structural and functional abnormalities in human neutrophils. In c&o incubation of neutrophils with saturated FFA resulted in cleft-like dilatations of the endoplasmic reticulum and perinuclear space. This finding was associated with decreased neutrophil function tests, especially chemotactic ability. Incubation with the same concentration of unsaturated FFA did not produce similar structural or functional alterations. Moskowitz (1967) observed that cells grown in monolayer tissue culture reacted differently to added saturated or unsaturated fatty acid. Unsaturated FFA resulted in the accumulation of numerous lipid-staining cytoplasmic droplets. However, saturated FFA caused the formation of cytoplasmic clefts, which failed to stain with any of the usual histologic lipid stains. Furthermore, it was stated that cells containing these clefts underwent irreversible degenerative changes. In the present experiments, studies on the effect of saturated and unsaturated fatty acids on cell growth, viability, lipid accumulation, and morphology were 262 Copyright All rights
@ 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0014~ -4800
SATURATED
FREE FATTY
ACID
TOXICITY
263
performed in suspension cultured mammalian cells. This report describes the characteristic findings of saturated FFA cellular toxicity including the combination of peculiar ultrastructural alterations of the endoplasmic reticulum and the accumulation of intracellular triglyceride and FFA. MATERIALS
AND METHODS
Preparation of Albumin-Bound FFA Four long-chain fatty acids were chosen for this study: oleic, a monounsaturated fatty acid; linoleic, a polyunsaturate; and two saturated fatty acids, palmitic and stearic. Solutions of albumin-bound FFA were prepared by a modification of the method of Goodman ( 1958). One millimole of FFA (Hormel Institute, Austin, Minnesota) was dissolved in 20 ml of n-heptane and added to 100 ml of Eagle’s minimum essential medium (MEM) containing 3 g of fatty acid-free bovine albumin, fraction V (Miles Laboratories Inc., Elkhart, Indiana). The mixture was shaken gently at room temperature for 48 hr, centrifuged, and the heptane layer was removed by thorough aspiration. Any residual dissolved heptane was removed by evaporation under a stream of nitrogen, The pH was adjusted to 7.0 with 0.1 M NaOH. The solution was then sterilized by filtration through a 0.22-pm membrane filter (Millipore Corp., Bedford, Massachusetts) and stored under a nitrogen atmosphere. Fatty acid-free albumin solutions were prepared in the same manner by incubating with n-heptane without added fatty acid for use in control experiments. No detectable differences on cellular growth, viability, or morphology were observed in cultures receiving either unincubated albumin solutions or n-heptaneincubated albumin solutions. Previously, Goodman (1958) and Spector et al. (1969) reported no apparent physical or chemical alterations in albumin similarly treated with heptane. The prepared solutions were analyzed for FFA concentration by the method of Novak ( 1965). Albumin content was determined by a modified Lowry procedure (Oyama and Eagle, 1956). A molar ratio (Y) of FFA to albumin of approximately 8:l was routinely obtained in the test solutions with an average FFA concentration of 3 X 10e3M. Tissue Culture Studi& Strain L (Earle) mouse fibroblasts were grown in suspension tissue culture in 250-ml Ehrlenmeyer flasks agitated on a rotary shaker at 37°C (Kuchler and Merchant, 1956). Each flask contained 80 ml of culture medium composed of Eagle’s MEM supplemented with 10% horse serum, penicillin (100 units/ml), and streptomycin (100 pg/ml). An appropriate number of cells from stock cultures in logarithmic growth were used to obtain an initial inoculum in the experimental cultures of approximately 250,000 cells/ml. The pH was adjusted to approximately 7.0 by gassingwith filtered 8% CO:! in air. For each experiment, replicate cultures were established and growth curves were determined by daily cell counts and cellular protein determinations. The cell counts were performed using a standard hemocytometer as described previously (King et al., 1959), and cell protein content was determined by the modified Lowry method of Oyama and Eagle (1956). After 24 hr of incuba-
264
GERALD
B. GORDON
tion with the cultures in the logarithmic growth phase, 8 ml of culture medium was removed from each experimental culture and replaced with 8 ml of Eagle’s MEM containing the appropriate albumin-bound long-chain free fatty acid (3 x lo-” hl). Control cultures received 8 ml of medium containing fatty acidfree albumin. The actual media concentration of FFA and protein was estimated by mixing the supernatant removed from each culture with the test FFA solution in the exact proportion used in each experiment. The average media molar ratio (Y) of FFA to albumin in the experimental cultures was determined to be 3.0 -C 0.2 as compared to 0.3 * 0.1 in the controls. In different experiments, cultures were allowed to incubate with the added long-chain free fatty acid for varying intervals: 1, 2, 4, 12, 24, and 48 hr. After incubation, the cells were counted and cell viability was estimated by trypan blue dye exclusion ( McLimans et nl., 1957). From each culture, aliquots of 5 ml for protein determination and 50 ml for lipid studies were taken. The remainder was prepared for electron microscopy. The experiments with each fatty acid were done in triplicate and repeated twice so that the reported data on ultrastructure, cell growth, and lipid analysis each represents the findings of six separate cultures. Statistical analyses for significance were performed by using Student’s t test. I,? .A; ” 4 ‘L, %J Suspension fixation of the L cells was performed as described previously (Gordon et al., 1963). Cold 2% glutaraldehyde in 0.5 M cacodylate buffer, pH 7.2, was added immediately in equal amounts to the aliquots of the cultures taken for electron microscopy. After 10 min at 5”C, the cells were centrifuged at 4OOg for 3 min, and the supernatant was discarded. Additional glutaraldehyde fixative was gently layered over the cell buttons, which were allowed to fix overnight in the cold. The cell buttons were then washed in 0.1 M cacodylate buffer containing 0.2 M sucrose, sliced into l-mm” blocks and postfixed in veronalbuffered 1% osmium tetroxide (Palade, 1952). Dehydration through alcohol and propylene oxide was followed by embedding in Araldite (Luft, 1961). Sections were cut with an LKB ultramicrotome and stained with uranyl acetate and lead citrate prior to examination in a Philips EM 300 electron microscope.
Electron
Microscopy
Lipid Analyses
4 .
