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Effects of antibiotics on the growth and differentiation in dissociated brain cell cultures
Neuroscience Vol. 3. pp.465-468. Pergamon PressLtd. 1978. Printedin GreatBritain. 0 IBRO
0306.4522/78/0501-0465102.00/O
EFFECTS OF ANTIBIOTICS ON THE GROWTH AND DIFFERENTIATION IN DISSOCIATED BRAIN CELL CULTURES F. AMONN,U. BAUMANN,U. N. WIESMANN,’
K. HOFMANNand N. HERSCHKOWITZ Department of Pediatrics, University of Bern, 3010 Bern, Switzerland Abstract-Penicillin,
streptomycin or gentamicin in medium concentrations routinely used in cell culture interfere with the levels of total protein and DNA and with the synthesis of sulfatide (a component of the myelin membrane and indicator of cell differentiation) in brain cell cultures of newborn mice. At 6, 9 and 12 days in culture, the combination of 200 I.U. penicillin and 5Opg streptomycin/ml as well as 5Opg streptomycin alone depresses total protein, DNA and sulfatide synthesis. Up to 300 I.U. penicillin or 50 pg gentamicin/ml shows no effect on protein and DNA while there is a dose-dependent interference with sulfatide synthesis. Less than 100 I.U. penicillin or 6 pg gentamicin/ml do not affect sulfatide synthesis. Since the antimicrobial activity is preserved even at lower concentrations, the doses of antibiotics routinely used in cell culture should be reduced to levels no longer affecting apparent cell metabolism. The cultures could, however, be used to study the effects of drugs other than antibiotics on cellular growth and differentiation.
ANTIBIOTICSare generally used to maintain sterility in organ and cell cultures, because complete sterility during the removal of organs such as brains from
cultures were incubated undisturbed without change of the medium at 37°C for 4 days. From day 4 the cultures were gassed by a flow-through system with 5% CO,, 40% O2 and 55% Nr. 10% (v/v) fetal calf serum was present in non-sterile animals is often difficult to achieve. Penithe culture media. cillin and streptomycin are most frequently used for At 6, 9 and 12 days in culture the cells were labelled this purpose. The recommended concentrations vary for 6 h with 10 ml medium containing 40 @/ml carrierbetween 100 and 300 I.U. penicillin and 10 and 100 pg free [35S04], then harvested by scraping with a rubber streptomycin/ml medium (RUDIN, 1970; TRACHTEN- policeman, washed, centrifuged and homogenized by ultraBERG, 1972; WIESMANN, HOFMANN, BURKART & sonication. Total protein was determined by the method HERSCHKOWITZ, 1975; WILSON, SHRIER, FARBER, of LOWRY, RO~EBROUGH, FARR & RANDALL (1951) and DNA according to CERIOTTI(1952). Total [35S0,]-incorTHOMPSON, ROSENBERG,BLUME & NIRENBERG, 1972). poration into the homogenate and the synthesis of sulfatide For these doses no adverse short- or long-term effects ([35S]-activity in the lipid extract prepared according to on growth, cell division and differentiation of brain HERSCHKOWITZ, MCKHANN, SAXENA & SHOOTER, 1968) cells have been reported. We have examined in this were measured as described by WIESMANNet al. (1975). paper whether penicillin and streptomycin as well as Sulfatide (cerebroside sulfate) had been identified by thingentamicin may exert an adverse effect on primary layer chromatography as the only [3sS0,]-labelled lipid cultures of dissociated mouse brain cells with respect in the cell homogenate. to DNA and protein content and the synthesis of sulThe basic culture medium contained no antibiotics. The fatide as an indicator of cell differentiation (WIES- antibiotics were added to the medium of the test cultures MANN f?t al., 1975). for the entire culture period as well as during the 6-h labelling with [3sS04] in concentrations specified under results. Penicillin was purchased from Mycofarm, Delft, NetherEXPERIMENTAL PROCEDURES lands, streptomycin-sulfate from Novo Industry, Kopenhagen, Denmark, and gentamicin-sulfate from Shering Dissociated brain cells were prepared from newborn mice (Jack River Albino) according to a method described Corporation U.S.A., Bloomfield, N.J. previously (WIESMANNet al., 1975), except that no trypsin was used to dissociate the cells. An aliquot of cell suspension equivalent to one whole brain was seeded into RESULTS 1OOmm culture dishes (Falcon Plastics) but not coated Protein DNA, total [3sS04]-incorporation and sulwith collagen, containing 10 ml Dulbecco’s modified Eagle fatide synthesis in table 1 are expressed as mean + medium buffered to pH 7.4 by 5% Cot-bicarbonate. The S.E.M.of the values in cultures grown with and without antibiotics. ’ Requests for reprints should be sent to Dr. Wiesmann, The combination of penicillin and streptomycin in Department of Pediatrics, Section of Metabolic Disorders, Freiburgstrasse 23, 3010 Bern, Switzerland. concentrations commonly used in tissue culture 465
50 pg
200 I.U.
50 pg
6 9 12
6 9 12
6 9 12
DIC 6 9 12
Values are expressed as the mean Days in cultures at which the cultures between the data for test and control
Gentamicin
Penicillin
Streptomycin
Penicillin 200 I.U. Streptomycin 50 wcg
Antibiotics (ml of medium)
0.26 * 0.03 0.46 k 0.07
3.0 f 0.4 3.6 f 0.8 4.1 f 1.0
1.9 f 0.1 2.0 + 0.1 2.3 + 0.2
0.1 I + O.OI
I.0 + I.0
0.15 + 0.01 0.14 f 0.01 0.19 f 0.03
0.26 _+ 0.03 0.20 f 0.03 0.33 + 0.03
* 0.01
2.0 f 0.2 3.0 + 0.4
C 0.30 f 0.03 0.51 * 0.12
0.12
T 0.01
0.12 + 0.03 0.14 _+ 0.01 0.18 f 0.02
0.19 f 0.04 0.19 + 0.02 0.26 + 0.02
0.32 + 0.05
0.08 + O.Ol O./X * 0.03
0.136 * 0.03 0.193 + O./Z
0.10 f
3309 * 93 4596 _+ 139 4835 + 103
1208 _+ 43 1301 + 182 1402 f 155
550 * 79 1245 f 228 1487 5 204
C 691 f 21 1750 + 49 1846 f 64
5344 * 550 18972 f 41 I 26221 * 411
1875 + I07 23x0 + 618 8002 + 870
523 f 70 1939 f 356 315x * 686
C 500 * 77 1432 + I44 1950 * 171
2565 * 256 10814 & 823 13373 + 1266
600 * 152 I095 + 309 2961 * 698
2135 * 387
S;rl + I54
345 * 62
T 282 + 29 378 * 54 560 f 206
[35S0,]-sulfatide (d.p.m./culture)
T, Test cultures with antibiotics. DIC, analysis (Student’s r test) was performed
2548 + 146 4320 f 109 4110 f 235
1401 + 94 1540 f 137 1416 + 86
397 f 71 813 + 155 x44 f I31
T 530 + 27 I236 f 51 1651 f. 102
[35S0,]-incorporation (d.p.m. x 10-2/culture)
+ standard error of the mean (s.E.M.) (n = 3 cultures). C, Control cultures without antibiotics. were labelled for 6 h with 10 ml medium containing 40 ntci carrier-free [35S04] per ml. Statistical cultures. Values that demonstrate a statistically significant difference (P < 0.05) are in italics.
3.3 + 0.3 3.3 f 0.5 3.7 * 0.5
1.8 f 0.2 1.9 + 0.2 2.5 + 0.2
1.4 f 0.2 2.4 f 0.2 3.9 + 0.4
C 1.7 + 0.03 2.7 + 0.3 4.7 f 0.1
DNA (mg/culture)
DOSES OF ANTIBIOTICS ON DISSOCIATED BRAIN CELL CULTURES
T 1.5 * 0.1 2.1 f 0.1 4.1 + 0.4
1. EFFECT OF STANDARD
Protein (mg/culture)
TABLE
Metabolic effects of antibiotics on brain cell cultures (200 I.U.
and 50pg/ml) moderately reduced the growth of brain cell cultures and markedly inhibited the sulfatide synthesis. Two hundred International Units of penicillin per ml had no effect on the protein or DNA content of cultures nor on total [35SOJincorporation but significantly inhibited sulfatide synthesis. Streptomycin (50 pg./ml) moderately depressed the accumulation of protein and DNA and [35S0,]incorporation per culture and markedly reduced sulfatide synthesis. Unlike streptomycin, gentamicin
ld Control o--o IO0I.U. /m\ +~200LU./m 0-4 300I.UJm
Penicillin
t S.E.M., n =3-6 100% =2689 d.p.m.
za -
400-
?
6
12
9 Doys
in culture
.d e--c
Control
6.5pg/ml b--r) 13 pg/ml Q--Q5Opg/ml
Gentomicin
?:S&M., n=3 100% = 5344 d.p.m. s ;
400
-
; k 2 \ 4 ._ + 0
300-
200 -
Z : IO0 A z 6
Days
I
I
9
12
in culture
FIGS 1 and 2. Effect of various concentrations of penicillin and gentamicin on sulfatide synthesis in dissociated brain cell cultures. At 6, 9 and 12 days in culture (DIG) cultures were labelled for 6 h with 10 ml medium containing 40 &i carrier-free [?SO,]/ml. Values (d.p.m./culture) obtained at 6 DIC were taken as lOO?<.
467
(SO,ng/ml) only interfered with the synthesis of sulfatide. Figures 1 and 2 show the effect of various concennations of penicillin and gentamicin on sulfatide synthesis. For both drugs a dose-de~ndent depression is evident.
DISCUSSION Streptomycin is concentrated in the lysosomes of cultured cells (DE Duvn, DE BARSY,POOLE,TROUET, TULKENS& VAN HOOF, 1974) and interferes with protein synthesis (WEINSTEIN,1965). Penicillin is known to compete with muraminic acid in cell wall synthesis of bacteria (WEINSTEIN, 1965). However, little is known about the effect of these drugs on mammalian cells. All of the antibiotics tested can produce neurotoxic symptoms in patients when given above safe limits (WEINSTEIN,1965; WALTER& HEILMEYER, 1969). In our culture system the combination of penicillin and streptomycin affected the growth (accumulation of protein and DNA) of dissociated brain cells whereas penicillin or gentamicin alone did not. An increase of osmolality in the medium of less than 7% (measured by freezing point depression) cannot explain these effects by the drugs. The synthesis of.sulfatide is a specific characteristic of cultured brain cells and parallels their development in uivo (WI~SMANN et af., 1975). Inhibition of the synthesis of sulfatide can be observed for all antibiotics tested. The effects of penicillin and gentamicin are closely dose-dependent. Since our medium contained 200mg MgSO,/l, the sulfate added by streptomycinand gentamycin-sulfate accounted for less than 5% of the nonradioactive SO,-pool and could not explain a reduction of more than 511/in the [35S0,J-labelling experiments. The antibiotics may therefore directly inhibit the synthesis of sulfatide or selectively suppress the proliferation or differentiation of sulfatide synthesizing cells. The difference in the metabolic effect on brain cell cultures of the structurally similar molecules streptomycin and gentamicin is noteworthy. It corresponds well with observations by SHAFER, BASCALE,SHIMONASKI & CAME(1972) on the effect of antibiotics on the morphology of cultured cells. The addition of antibiotics to the medium of cell cultures introduces one more factor to the experimental system and may unpredictably alter the results of experiments. Since the use of antibiotics in brain cell cultures cannot always be completely avoided, their doses should be reduced to apparent non-toxic levels, especially because the antimicrobial activity is preserved even at much lower concentrations than the ones recommended for use in tissue culture (WEINSTEIN,1965; WALTER& HEILMEYER, 1969). Due to their extraordinary sensitivity to antibiotics, primary cultures of dissociated brain cells could be used to study the effects of antibiotics and of other
468
F. AMONN,U. BAUMANN,U. N. WIESMANN,K. HOFMANNand N. HERSCHKOWITZ
drugs on growth and differentiation vitro. This might help to understand side effects in viva
of brain cells in drug action and
Acknowledgements-This study was supported by a Grant from Hoffmann-La Roche (Emil Barell-Stiftung) and by the Swiss National Science Foundation No. 3.768.76 and 3.660.75
REFERENCES CERIO~TIG. (1952) A microchemical determination of desoxyribonucleic acid. J. biol. Chem. 198, 297-303. DE DUVE C., DE BARSYT., POOLEB., TROUET A., TULKENS P. & VAN Hook F. (1974) Lysosomotropic agents. Biochem. Pharmac. 23, 2495-2541.
HERSCHKOWITZ N., MCKHANN G. M., SAXENAA. & SHOOTERE. M. (1968) Studies of water soluble lipoproteins in rat brain. J. Neurochem. 15, 161-168. LOWRY0. H., RO~EBROUGH N. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. RUDIN A. (1970) Antibacterial activity of gentamicin sulfate in tissue culture. Appl. Microbial. 20, 989-990. SCHAFERT. W., BAXCALEA., SHIMONASKIG. & CAME P. (1972) Evaluation of gentamicin for use in virology and tissue culture. Appl. Microbial. 23, 565-570. TRACHTENHERG M. C. (1972) Biophysical properties of cultured human glial cells. Brain Res. 38. 279-298. WALTERA. M. & HEILMEYER L. (1969) Antibiotikafibel. 3rd Edn. Thieme, Stuttgart. WEINSTEINL. (1965) Antibiotics. The Pharmacological Basis of Therapeutics (eds GOODMAN L. S. & GILMANA.). Revised Edn. Macmillan, New York. WIESMANNU. N., HOFMANN,K., BURKARTT. & HERSCHKOWITZ N. (1975) Dissociated cultures of newborn mouse brain--l. Metabolism of sulfatid lipids and mucopolysaccharides. Neurobiology 5, 305-315. WILSONS. H., SCHRIERB. K., FARBERJ. L., THOMPSONE. J., ROSENBERG R. N., BLUMEA. J. & NWENBERGM. W. (1972) Markers for gene expression in cultured cells from the nervous system. J. biol. Chem. 247, 3159-3169. (Accepted 8 December 1977)
0306.4522/78/0501-0465102.00/O
EFFECTS OF ANTIBIOTICS ON THE GROWTH AND DIFFERENTIATION IN DISSOCIATED BRAIN CELL CULTURES F. AMONN,U. BAUMANN,U. N. WIESMANN,’
K. HOFMANNand N. HERSCHKOWITZ Department of Pediatrics, University of Bern, 3010 Bern, Switzerland Abstract-Penicillin,
streptomycin or gentamicin in medium concentrations routinely used in cell culture interfere with the levels of total protein and DNA and with the synthesis of sulfatide (a component of the myelin membrane and indicator of cell differentiation) in brain cell cultures of newborn mice. At 6, 9 and 12 days in culture, the combination of 200 I.U. penicillin and 5Opg streptomycin/ml as well as 5Opg streptomycin alone depresses total protein, DNA and sulfatide synthesis. Up to 300 I.U. penicillin or 50 pg gentamicin/ml shows no effect on protein and DNA while there is a dose-dependent interference with sulfatide synthesis. Less than 100 I.U. penicillin or 6 pg gentamicin/ml do not affect sulfatide synthesis. Since the antimicrobial activity is preserved even at lower concentrations, the doses of antibiotics routinely used in cell culture should be reduced to levels no longer affecting apparent cell metabolism. The cultures could, however, be used to study the effects of drugs other than antibiotics on cellular growth and differentiation.
ANTIBIOTICSare generally used to maintain sterility in organ and cell cultures, because complete sterility during the removal of organs such as brains from
cultures were incubated undisturbed without change of the medium at 37°C for 4 days. From day 4 the cultures were gassed by a flow-through system with 5% CO,, 40% O2 and 55% Nr. 10% (v/v) fetal calf serum was present in non-sterile animals is often difficult to achieve. Penithe culture media. cillin and streptomycin are most frequently used for At 6, 9 and 12 days in culture the cells were labelled this purpose. The recommended concentrations vary for 6 h with 10 ml medium containing 40 @/ml carrierbetween 100 and 300 I.U. penicillin and 10 and 100 pg free [35S04], then harvested by scraping with a rubber streptomycin/ml medium (RUDIN, 1970; TRACHTEN- policeman, washed, centrifuged and homogenized by ultraBERG, 1972; WIESMANN, HOFMANN, BURKART & sonication. Total protein was determined by the method HERSCHKOWITZ, 1975; WILSON, SHRIER, FARBER, of LOWRY, RO~EBROUGH, FARR & RANDALL (1951) and DNA according to CERIOTTI(1952). Total [35S0,]-incorTHOMPSON, ROSENBERG,BLUME & NIRENBERG, 1972). poration into the homogenate and the synthesis of sulfatide For these doses no adverse short- or long-term effects ([35S]-activity in the lipid extract prepared according to on growth, cell division and differentiation of brain HERSCHKOWITZ, MCKHANN, SAXENA & SHOOTER, 1968) cells have been reported. We have examined in this were measured as described by WIESMANNet al. (1975). paper whether penicillin and streptomycin as well as Sulfatide (cerebroside sulfate) had been identified by thingentamicin may exert an adverse effect on primary layer chromatography as the only [3sS0,]-labelled lipid cultures of dissociated mouse brain cells with respect in the cell homogenate. to DNA and protein content and the synthesis of sulThe basic culture medium contained no antibiotics. The fatide as an indicator of cell differentiation (WIES- antibiotics were added to the medium of the test cultures MANN f?t al., 1975). for the entire culture period as well as during the 6-h labelling with [3sS04] in concentrations specified under results. Penicillin was purchased from Mycofarm, Delft, NetherEXPERIMENTAL PROCEDURES lands, streptomycin-sulfate from Novo Industry, Kopenhagen, Denmark, and gentamicin-sulfate from Shering Dissociated brain cells were prepared from newborn mice (Jack River Albino) according to a method described Corporation U.S.A., Bloomfield, N.J. previously (WIESMANNet al., 1975), except that no trypsin was used to dissociate the cells. An aliquot of cell suspension equivalent to one whole brain was seeded into RESULTS 1OOmm culture dishes (Falcon Plastics) but not coated Protein DNA, total [3sS04]-incorporation and sulwith collagen, containing 10 ml Dulbecco’s modified Eagle fatide synthesis in table 1 are expressed as mean + medium buffered to pH 7.4 by 5% Cot-bicarbonate. The S.E.M.of the values in cultures grown with and without antibiotics. ’ Requests for reprints should be sent to Dr. Wiesmann, The combination of penicillin and streptomycin in Department of Pediatrics, Section of Metabolic Disorders, Freiburgstrasse 23, 3010 Bern, Switzerland. concentrations commonly used in tissue culture 465
50 pg
200 I.U.
50 pg
6 9 12
6 9 12
6 9 12
DIC 6 9 12
Values are expressed as the mean Days in cultures at which the cultures between the data for test and control
Gentamicin
Penicillin
Streptomycin
Penicillin 200 I.U. Streptomycin 50 wcg
Antibiotics (ml of medium)
0.26 * 0.03 0.46 k 0.07
3.0 f 0.4 3.6 f 0.8 4.1 f 1.0
1.9 f 0.1 2.0 + 0.1 2.3 + 0.2
0.1 I + O.OI
I.0 + I.0
0.15 + 0.01 0.14 f 0.01 0.19 f 0.03
0.26 _+ 0.03 0.20 f 0.03 0.33 + 0.03
* 0.01
2.0 f 0.2 3.0 + 0.4
C 0.30 f 0.03 0.51 * 0.12
0.12
T 0.01
0.12 + 0.03 0.14 _+ 0.01 0.18 f 0.02
0.19 f 0.04 0.19 + 0.02 0.26 + 0.02
0.32 + 0.05
0.08 + O.Ol O./X * 0.03
0.136 * 0.03 0.193 + O./Z
0.10 f
3309 * 93 4596 _+ 139 4835 + 103
1208 _+ 43 1301 + 182 1402 f 155
550 * 79 1245 f 228 1487 5 204
C 691 f 21 1750 + 49 1846 f 64
5344 * 550 18972 f 41 I 26221 * 411
1875 + I07 23x0 + 618 8002 + 870
523 f 70 1939 f 356 315x * 686
C 500 * 77 1432 + I44 1950 * 171
2565 * 256 10814 & 823 13373 + 1266
600 * 152 I095 + 309 2961 * 698
2135 * 387
S;rl + I54
345 * 62
T 282 + 29 378 * 54 560 f 206
[35S0,]-sulfatide (d.p.m./culture)
T, Test cultures with antibiotics. DIC, analysis (Student’s r test) was performed
2548 + 146 4320 f 109 4110 f 235
1401 + 94 1540 f 137 1416 + 86
397 f 71 813 + 155 x44 f I31
T 530 + 27 I236 f 51 1651 f. 102
[35S0,]-incorporation (d.p.m. x 10-2/culture)
+ standard error of the mean (s.E.M.) (n = 3 cultures). C, Control cultures without antibiotics. were labelled for 6 h with 10 ml medium containing 40 ntci carrier-free [35S04] per ml. Statistical cultures. Values that demonstrate a statistically significant difference (P < 0.05) are in italics.
3.3 + 0.3 3.3 f 0.5 3.7 * 0.5
1.8 f 0.2 1.9 + 0.2 2.5 + 0.2
1.4 f 0.2 2.4 f 0.2 3.9 + 0.4
C 1.7 + 0.03 2.7 + 0.3 4.7 f 0.1
DNA (mg/culture)
DOSES OF ANTIBIOTICS ON DISSOCIATED BRAIN CELL CULTURES
T 1.5 * 0.1 2.1 f 0.1 4.1 + 0.4
1. EFFECT OF STANDARD
Protein (mg/culture)
TABLE
Metabolic effects of antibiotics on brain cell cultures (200 I.U.
and 50pg/ml) moderately reduced the growth of brain cell cultures and markedly inhibited the sulfatide synthesis. Two hundred International Units of penicillin per ml had no effect on the protein or DNA content of cultures nor on total [35SOJincorporation but significantly inhibited sulfatide synthesis. Streptomycin (50 pg./ml) moderately depressed the accumulation of protein and DNA and [35S0,]incorporation per culture and markedly reduced sulfatide synthesis. Unlike streptomycin, gentamicin
ld Control o--o IO0I.U. /m\ +~200LU./m 0-4 300I.UJm
Penicillin
t S.E.M., n =3-6 100% =2689 d.p.m.
za -
400-
?
6
12
9 Doys
in culture
.d e--c
Control
6.5pg/ml b--r) 13 pg/ml Q--Q5Opg/ml
Gentomicin
?:S&M., n=3 100% = 5344 d.p.m. s ;
400
-
; k 2 \ 4 ._ + 0
300-
200 -
Z : IO0 A z 6
Days
I
I
9
12
in culture
FIGS 1 and 2. Effect of various concentrations of penicillin and gentamicin on sulfatide synthesis in dissociated brain cell cultures. At 6, 9 and 12 days in culture (DIG) cultures were labelled for 6 h with 10 ml medium containing 40 &i carrier-free [?SO,]/ml. Values (d.p.m./culture) obtained at 6 DIC were taken as lOO?<.
467
(SO,ng/ml) only interfered with the synthesis of sulfatide. Figures 1 and 2 show the effect of various concennations of penicillin and gentamicin on sulfatide synthesis. For both drugs a dose-de~ndent depression is evident.
DISCUSSION Streptomycin is concentrated in the lysosomes of cultured cells (DE Duvn, DE BARSY,POOLE,TROUET, TULKENS& VAN HOOF, 1974) and interferes with protein synthesis (WEINSTEIN,1965). Penicillin is known to compete with muraminic acid in cell wall synthesis of bacteria (WEINSTEIN, 1965). However, little is known about the effect of these drugs on mammalian cells. All of the antibiotics tested can produce neurotoxic symptoms in patients when given above safe limits (WEINSTEIN,1965; WALTER& HEILMEYER, 1969). In our culture system the combination of penicillin and streptomycin affected the growth (accumulation of protein and DNA) of dissociated brain cells whereas penicillin or gentamicin alone did not. An increase of osmolality in the medium of less than 7% (measured by freezing point depression) cannot explain these effects by the drugs. The synthesis of.sulfatide is a specific characteristic of cultured brain cells and parallels their development in uivo (WI~SMANN et af., 1975). Inhibition of the synthesis of sulfatide can be observed for all antibiotics tested. The effects of penicillin and gentamicin are closely dose-dependent. Since our medium contained 200mg MgSO,/l, the sulfate added by streptomycinand gentamycin-sulfate accounted for less than 5% of the nonradioactive SO,-pool and could not explain a reduction of more than 511/in the [35S0,J-labelling experiments. The antibiotics may therefore directly inhibit the synthesis of sulfatide or selectively suppress the proliferation or differentiation of sulfatide synthesizing cells. The difference in the metabolic effect on brain cell cultures of the structurally similar molecules streptomycin and gentamicin is noteworthy. It corresponds well with observations by SHAFER, BASCALE,SHIMONASKI & CAME(1972) on the effect of antibiotics on the morphology of cultured cells. The addition of antibiotics to the medium of cell cultures introduces one more factor to the experimental system and may unpredictably alter the results of experiments. Since the use of antibiotics in brain cell cultures cannot always be completely avoided, their doses should be reduced to apparent non-toxic levels, especially because the antimicrobial activity is preserved even at much lower concentrations than the ones recommended for use in tissue culture (WEINSTEIN,1965; WALTER& HEILMEYER, 1969). Due to their extraordinary sensitivity to antibiotics, primary cultures of dissociated brain cells could be used to study the effects of antibiotics and of other
468
F. AMONN,U. BAUMANN,U. N. WIESMANN,K. HOFMANNand N. HERSCHKOWITZ
drugs on growth and differentiation vitro. This might help to understand side effects in viva
of brain cells in drug action and
Acknowledgements-This study was supported by a Grant from Hoffmann-La Roche (Emil Barell-Stiftung) and by the Swiss National Science Foundation No. 3.768.76 and 3.660.75
REFERENCES CERIO~TIG. (1952) A microchemical determination of desoxyribonucleic acid. J. biol. Chem. 198, 297-303. DE DUVE C., DE BARSYT., POOLEB., TROUET A., TULKENS P. & VAN Hook F. (1974) Lysosomotropic agents. Biochem. Pharmac. 23, 2495-2541.
HERSCHKOWITZ N., MCKHANN G. M., SAXENAA. & SHOOTERE. M. (1968) Studies of water soluble lipoproteins in rat brain. J. Neurochem. 15, 161-168. LOWRY0. H., RO~EBROUGH N. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. RUDIN A. (1970) Antibacterial activity of gentamicin sulfate in tissue culture. Appl. Microbial. 20, 989-990. SCHAFERT. W., BAXCALEA., SHIMONASKIG. & CAME P. (1972) Evaluation of gentamicin for use in virology and tissue culture. Appl. Microbial. 23, 565-570. TRACHTENHERG M. C. (1972) Biophysical properties of cultured human glial cells. Brain Res. 38. 279-298. WALTERA. M. & HEILMEYER L. (1969) Antibiotikafibel. 3rd Edn. Thieme, Stuttgart. WEINSTEINL. (1965) Antibiotics. The Pharmacological Basis of Therapeutics (eds GOODMAN L. S. & GILMANA.). Revised Edn. Macmillan, New York. WIESMANNU. N., HOFMANN,K., BURKARTT. & HERSCHKOWITZ N. (1975) Dissociated cultures of newborn mouse brain--l. Metabolism of sulfatid lipids and mucopolysaccharides. Neurobiology 5, 305-315. WILSONS. H., SCHRIERB. K., FARBERJ. L., THOMPSONE. J., ROSENBERG R. N., BLUMEA. J. & NWENBERGM. W. (1972) Markers for gene expression in cultured cells from the nervous system. J. biol. Chem. 247, 3159-3169. (Accepted 8 December 1977)