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Changes in 2-aminoisobutyric acid and cycloleucine uptake produced by 2,4-dichlorophenoxyacetic acid in chinese hamster ovary cells
Toxicology Letters, 36 (1987) 189 -196
189
Elsevier
TXL 01759
CHANGES IN 2-AMINOISOBUTYRIC ACID AND CYCLOLEUCINE UPTAKE PRODUCED BY 2,4-DICHLOROPHENOXYACETIC ACID IN CHINESE HAMSTER OVARY CELLS (2-Aminoisobutyric
acid; cycloleucine; uptake; Chinese hamster ovary cells)
JORGE
VIVIANA
R. BERGESSE,
A. RIVAROLA
and HECTOR
F. BALEGNO*
Departamento de Biologia Molecular, Universidad National de Rio Cuarto, 5800 - Rio Cuarto, Cdrdoba (Argentina) (Received
28 June
(Accepted
5 December
1986) 1986)
SUMMARY The effect
of dichlorophenoxyacetic
2-aminoisobutyric The herbicide contact
acid on the transport
acid (AIB) and cycloleucine
medium
restored
the influx
cell division.
The transport
indicate
2,4-dichlorophenoxyacetic
that
modification
in Chinese hamster
did not exert any direct effect on the AIB transport.
with the cells for 24 h an inhibition
the culture
of two non-metabolizable
(CL) was studied
kinetics
of the uptake
of the amino
showed
changes
However,
was observed.
a decrease
values
cells. was in
of the pesticide
maximum
in V,,,., but no variations
acid produces
acids,
(CHO)
when the pesticide
Removal
acids which reached
amino
ovary
from
1 h before
in K,,,. These results may
in the carrier
number
but without
of the affinity.
INTRODUCTION
It has been reported that the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) inhibits cell growth, DNA and protein synthesis of cultured Chinese hamster ovary cells (CHO) [l]. This effect was reversed when the biocide was removed from the culture medium. The same herbicide was also indicated as an inhibitor of glycine uptake into the gills of Mytifus californianus [2]. The 2,4-D analogue, 2,4,5-T was found to interfere with [14C]thymidine- and [)H]uridine-facilitated transport into * To whom
correspondence
Abbreviations:
CHO,
trichlorophenoxyacetic (cycloleucine);
037%4274/87/$
should
Chinese acid;
be addressed.
hamster
AIB,
ovary
cells;
2-aminoisobutyric
2,4-D, acid;
CL,
dichlorophenoxyacetic
HU, hydroxyurea.
03.50
0 Elsevier
Science
Publishers
acid;
I-aminocyclopentanecarboxylic
B.V. (Biomedical
Division)
2,4,5-T, acid
190
L-929 cells [3]. This diminution was considered the direct result of the action of the pesticide upon the cell membrane. Nevertheless, the same authors indicated that the drug did not affect the uptake of [14C]leucine into the cells, although protein synthesis was inhibited. Since we found that protein synthesis was profoundly inhibited by 2,4-D in CHO cells, we thought that it would be of interest to investigate whether the transport of two nonmetabolizable aminoacids; i.e., 2-aminoisobutyric acid (AIB) and cycloleucine (CL), would be affected by 2,4-D at the membrane level. MATERIAL
AND
METHODS
A CHO strain (provided by Petra Martinez of the Fundacion de Genetica Humana, Buenos Aires, Argentina), kept in liquid nitrogen, was used for this investigation. The cells were grown as a monolayer in a glass vial with a 4.12 cm’ surface base, employing McCoy’s culture medium [4] supplemented with 15% fetal bovine serum and 50 pg/ml gentamycin. 0.25% trypsin (1:250, Difco) in Hepes buffer, pH 7.2 [5] was used to release the cells from the vials. 2-Amino-[l-14C]isobutyric acid (AIB) (60 Ci/mol) and 1-aminocyclopentane-[ l-14C]carboxylic acid (CL) (59 Ci/mol) were obtained from the Radiochemical Centre (Amersham, England). The unlabelled amino acids were purchased from Sigma. All other chemicals used were of reagent grade. The radioactive amino acid solutions were prepared in a sodium medium containing Hank’s/Hepes minus glucose (pH 7.4) [6] with a 121 mM concentration of Na+ . The sodium salt of 2,4-D (pH 7.3) was prepared by treating 2,4-dichlorophenoxyacetic acid (Sigma) with NaOH. Assay for AIB and CL transport
The assays for AIB and CL uptake were carried out following the method of Kelley and Potter [7] with a slight modification. Briefly, the method was as follows: approximately 2.5 x lo5 cells in logarithmic growth were plated directly into the vials for the amino acid transport experiments. Two series of vials were run simultaneously, one for the determination of the amino acid uptake and the other for cell counting. Each container contained 1.8 ml of culture medium and was incubated for 24 h at 37°C. The culture medium of the cells grown as a monolayer was discarded, the cells washed twice with 2 ml of Hank’s/Hepes buffer (pH 7.3) at 37°C followed by incubation at 37°C for 15 min with 3 ml of the same buffer to allow the depletion of the internal amino acid pool. The buffer was removed and 1 ml of radioactive amino acid solution added and incubated at 37°C for 8 min and washed 3 times with 2 ml of cold Hank’s/Hepes buffer (pH 7.3) at 4°C. Finally, the cells were solubilized in 0.5 ml of 0.2 N NaOH. A 0.2-ml aliquot of the solubilized cell solution used to determine the radioactivity contained 2.5 ml of a toluene/Triton X-100 (2: 1) mixture with 0.02 g% of 1,4-bis-2-(5-phenyloxazolyl)-
191
benzene; 1,4-bis(2-(5phenyloxazolyl))benzene (POPOP) (Sigma) and 0.4% of 2,5diphenyloxazole (PPO, Amersham/Searle). Radioactivity was determined employing a Beckman liquid scintillation counter. The AIB and CL uptake was linear for at least 10 min and is expressed as pmol of aminoacid/106 cells per min. To determine the effects of hydroxyurea (HU; Sigma) and 2,4-D on cell transport, a monolayer of approximately 4-5 x 10’ cells in logarithmic growth plus 1.8 ml of culture medium per vial were used. Final concentrations of 1.2 mM HU or 1 mM 2,4-D were added to the vials and they were incubated at 37°C for 24 h. After this time the media containing the drugs were discarded, the monolayers rinsed twice with 2 ml PBS solution A [8] and the transport determined either immediately (t,,) or at different times (t,,) after the addition of 1.8 ml normal medium. The kinetics values (K, and Vmax)were determined by Lineweaver-Burke plots by examining the 8-min uptake of 0.125-8 mM AIB or CL and 0.0125-0.8 &i/ml of [14C]AIB or [14C]CL in sodium-containing Hank’s/Hepes medium. RESULTS
To determine whether the 2,4-D had any direct effect of AIB uptake, logarithmic growth cultures were treated with 1.2 mM HU for 24 h after which the culture medium was replaced by normal medium and the transport immediately tested in absence of 2,4-D (control) or with different concentrations of the herbicide. The results are shown in Table I where it can be seen that the biocide does not have a direct effect even at the highest concentration tested. Table II depicts the results when AIB and CL uptake was compared between logarithmic growth cells and those treated for 24 h with 2,4-D. There is no difference between the amino acid uptake of cells treated with HU for 24 h and cells growing in a logarithmic state, but there is a remarkable decrease in the amino acid influx in cells treated with 2,4-D for 24 h. This herbicide produces a growth inhibition of CHO cells which appears at concentration of 1 mM. However, when the cell culture in logarithmic growth is treated TABLE
I
DIFFERENT
2,4-CONCENTRATION
Cells in logarithmic eliminated mined.
growth
and the uptake
AIB concentration:
EFFECTS
were treated
ON AIB UPTAKE
for 24 h with
of AIB in the presence
1.2 mM hydroxyurea
of different
1 mM; [‘%Z]AIB: 0.1 &i/ml.
2,4-D concentrations
The results are the average
run by duplicate. 2,4-D mM
pmol of AIB/106
0 (control)
3.86
0.1
3.69
1
3.80
10
3.23
cells per min
(HU).
The HU was
immediately
deter-
of two experiments
192
TABLE
II
AIB AND CL UPTAKE
BY CHO CELLS
IN LOGARITHMIC
TO 1.2 mM HU OR 1 mM 2,4-D TREATMENT AIB and CL uptake growth)
was determined
that were treated
determined
in logarithmic
(log) growth
medium
containing
Amino
the drugs was removed.
acid
and in cells (also in log
acid uptake
was immediately
The results represent
pmol/106
AIB
4.35
I.2 Mm HU
AIB
4.13
1 mM 2,4-D
AIB
2.95
CL
4.80
1.2 mM HU
CL
5.54
1 mM 2,4-D
CL
3.19
cells
Log growth
SUBJECTION
the average
run in duplicate.
Treatment Log growth
OR AFTER
cells (controls)
for 24 h with 1.2 mM HU or 1 mM 2,4-D. Amino
after the culture
of two experiments
GROWTH
FOR 24 h
cells
cells per min
with this concentration followed by its elimination from the culture medium, cell growth is reestablished (unpublished data). Fig. 1 shows the effects of HU and 2,4-D on AIB uptake when it was determined in cells after they had attained logarithmic growth and which were treated for 24 h with 1 mM of the herbicide or 1.2 mM HU. This was followed by the elimination
0
2
4
6
8
10
12
14
16 hours
Fig. 1. Logarithmically
growing
with the drugs was replaced mined or at different HU. (0) AIB uptake the arrows
indicate
cells were treated
by a normal
culture
with 1.2 mM HU or 1 mM 2,4-D for 24 h. The medium medium
and the amino
acid uptake
immediately
times after the normal medium was added. (0) AIB uptake by cells treated by cells treated with 2,4-D. Each point is the average of three determinations the time that the cells divided.
AIB: 1 mM; [%]AIB:
0.1 &i/ml;
Na+:
deterwith and
121 mM.
193
TABLE III K,, AND V,,, VALUESOF Amino
acid
AIB AND CL INFLUX
Treatment
h
Km
(mM)
V,,, (pm011
VmaX
lo6 cells per min)
increase
AIB
HU
to
1.16
9.54
AIB
HU
ts
1.11
13.36
AIB
2,4-D
to
1.15
4.00
AIB
2,4-D
t15
1.10
9.90
CL
HU
to
0.40
8.06
CL
HU
t9
0.55
10.48
CL
2,4-D
to
0.55
4.54
CL
2,4-D
t1s
0.63
8.45
Logarithmic containing
growth
cells were treated
the drugs was replaced
+ 51%
+ 150%
f
32.7%
+ 96.8%
with 1.2 mM HU or I mM 2,4-D for 24 h. The culture
by a normal
one and the kinetic parameters
determined
medium
at zero time
(to) and at t9 for the cells treated with HU, and at tl5 for those treated with 2,4-D after changing culture
the
medium.
of the drugs by transfer to a normal culture medium. The recovery of the amino acid transport was determined immediately (to) and at different times (t,,) after the initiation of cell growth in the normal medium. We could observe that the uptake of AIB by cells treated with HU exhibits a slow increase as the cells traverse the S phase of the cell cycle. Similar results were obtained when the [‘Hlleucine, [3H]lysine and [‘Hltryptophane uptake was studied during the CHO cell cycle [9].
0
2
Fig. 2. Experimental
4
6
conditions
8
10
12
14
16 hours
were similar to those indicated
with HU. ( n ) CL uptake by cells treated with 2,4-D. CL: 1 mM: [r4C]CL: 0.1 &i/ml; Na+: 121 mM.
Arrows
in Fig. 1. indicate
(o)CL uptake by cells treated the time that the cells divided.
194
L
2
1
2-AMINOISOBUTYHIC
Fig. 3. Lineweaver-Burke with 1.2 mM HU. Transport Other cultures
were treated
and at 15 h, tts (
n
plots of AIB incorporation was determined
8 ACID
(mM)-l
in CHO
cells: cell cultures
immediately,
to (0)
with 1 mM 2,4-D for 24 h and uptake
) after the herbicide
were treated
and at 9 h, ts (0) was determined
for 24 h
after HU removal. immediately,
to ( q )
removal.
Ten hours after the HU elimination from the medium, the cells began to divide and the increment of the AIB uptake was 52% between to and t9. Similar to the cells treated with HU, the AIB transport increased in those cells subjected to the action of 2,4-D as the cells passed through the S phase. The cells divided at 16 h and the increment of the uptake was 150% from to to tl5. One hour before cell division the incorporation of AIB by cells treated with 2,4-D did not reach the uptake level of those treated with HU. Similar results were obtained when the CL uptake was determined under identical experimental conditions (Fig. 2). Table III shows the K,,, and I/maxvalues of logarithmic growth cells treated with HU or 2,4-D for 24 h, immediately after drug removal (to) and at t9 after HU elimination and 115 after 2,4-D elimination and the addition of normal culture medium (these parameters were calculated based upon the results of Figs. 3 and 4). The K,,, did not show variations, however, Vmaxincreased from the initial moments
195
1
Fig. 4. Lineweaver-Burke
8
2
plots of CL incorporation
in CHO cells: cell cultures
1.2 mM HU. The transport
was immediately
Other
with 1 mM 2,4-D for 24 h and the uptake
cultures
were treated
and at 15 h, trs ( n ), after
herbicide
determined,
to (0)
were treated
and at 9 h, t9 (0) determined
for 24 h with
after HU removal. immediately,
to ( q)
removal.
of the drug removal up until 1 h before cell division: t9 in the case of HU treatment and at tls when 2,4-D was employed. The increment of Vmaxwas considerably larger when the cells were treated with 2,4-D than if they were treated with HU. DISCUSSION
Based on the above results it may be concluded that 2,4-D does not have a direct effect on the AIB transport in CHO cells (Table I). However, when the cells were in contact with 2,4-D for 24 h the herbicide not only stopped cell growth by DNA and protein synthesis inhibition [l] but also inhibited the uptake of the two nonmetabolizable amino acids tested (Table II). The AIB and CL transport inhibition produced by 2,4-D may be a consequence of its interference in the synthesis of certain membrane proteins which are indispensable as carriers. As was previously demonstrated, 2,4-D produces a cell accumulation at the Gr/S boundary of the cell cycle with a simultaneous inhibition of protein synthesis [l]. The removal of the herbicide is accompanied by an initiation of protein and DNA synthesis. The inhibition of protein synthesis can in part be caused by a decrease
196
of the amino acid transport which is restored when the herbicide is eliminated from the culture medium and the cell advances into the S phase (Figs. 1 and 2) and reaches a maximum at 15 h. The cells are capable of division at this moment and their plasma membrane has probably been restored to normal with its corresponding amino acid carriers. The kinetic transport parameters (Table III) show a change only in the V,,, and not in the Km which may indicate that the 2,4-D produced a decrease in the number of carriers but with no change in the affinity. The recuperation period that follows the elimination of 2,4-D from the medium is accompanied by an increase in protein synthesis possibly including carrier proteins. It is not clear why 2,4-D produced an elongation of the S phase and perhaps also of the GZ phase. The possibility exists that one of the limiting factors for cell division would be the integrity of the cell membrane. Additional research should be carried out specifically on membrane protein synthesis to shed more light on this subject. ACKNOWLEDGEMENTS
We are grateful to Mrs. Donna E.M. de Balegno for her help in preparing the manuscript and to Mr. Adalberto Allione for his technical assistance. This investigation was supported by the Consejo National de Investigaciones Cientificas y Technicas (Argentina). REFERENCES 1 V.A. Rivarola, J.R. Bergesse and H.F. Balegno, DNA and protein synthesis inhibition in Chinese Hamster Ovary Cells by dichlorophenoxyacetic acid, Toxicol. Lett., 29 (1985) 137-144. 2 J.H. Swinehart and M.A. Cheney, 2,4-Dichlorophenoxyacetic acid (2,4-D) and paranitrophenol (PNP) interactions with gills of Anodonta culiforniensis and Mytilus californianus: uptake and effects on membrane fluxes, Pestic. Biochem. Physiol., 20 (I983) 49-56. 3 L. Dragsnes and K. Helgeland, Effects of 2,4,5-T on the incorporation of f’4C]-thymidine; [‘HIuridine and L-[3H]-Ieucine into L-929 cells, Acta Pharmacol. Toxicol., 35 (1974) 103-112. 4 R.G. Ham and W.L. McKeehan, Media and growth requirements, in W.B. Jakoby and I.H. Pastan (Eds.), Methods in Enzymology, Vol. LVIII, Academic Press, New York, 1979. 5 C. Shipman, Jr., Trypsin, A: mammalian tissues, in P.J. Kruse, Jr. and M.K. Patterson (Eds.), Tissue Culture Methods and Applications, Academic Press, New York, 1973. 6 M.A. Lieberman, D.M. Raben, B. Whittenberger and L. Glaser, Effect of plasma membranes on solute transport in 3T3 cells, .I. Biol. Chem., 254 (1979) 6357-6361. 7 D.S. Kelley and V.R. Potter, Regulation of amino acid transport systems by amino acid depletion and supplemen~tion in monolayer cultures of rat hepatocytes, 3. Biol. Chem., 253 (1978) 9009-9017. 8 R.L.P. Adams, in Cefl Culture for Biochemists, Elsevier/North-HolIand, Amsterdam, 1980, pp. 247. 9 O.C. Blair and J.L. Roti Roti, Incorporation of 3H-leucine IH-lysine and 3H-tryptophan during the cell cycle of Chinese Hamster Ovary cells, J. Histochem. Cytochem. 28 (1980) 487-492.
189
Elsevier
TXL 01759
CHANGES IN 2-AMINOISOBUTYRIC ACID AND CYCLOLEUCINE UPTAKE PRODUCED BY 2,4-DICHLOROPHENOXYACETIC ACID IN CHINESE HAMSTER OVARY CELLS (2-Aminoisobutyric
acid; cycloleucine; uptake; Chinese hamster ovary cells)
JORGE
VIVIANA
R. BERGESSE,
A. RIVAROLA
and HECTOR
F. BALEGNO*
Departamento de Biologia Molecular, Universidad National de Rio Cuarto, 5800 - Rio Cuarto, Cdrdoba (Argentina) (Received
28 June
(Accepted
5 December
1986) 1986)
SUMMARY The effect
of dichlorophenoxyacetic
2-aminoisobutyric The herbicide contact
acid on the transport
acid (AIB) and cycloleucine
medium
restored
the influx
cell division.
The transport
indicate
2,4-dichlorophenoxyacetic
that
modification
in Chinese hamster
did not exert any direct effect on the AIB transport.
with the cells for 24 h an inhibition
the culture
of two non-metabolizable
(CL) was studied
kinetics
of the uptake
of the amino
showed
changes
However,
was observed.
a decrease
values
cells. was in
of the pesticide
maximum
in V,,,., but no variations
acid produces
acids,
(CHO)
when the pesticide
Removal
acids which reached
amino
ovary
from
1 h before
in K,,,. These results may
in the carrier
number
but without
of the affinity.
INTRODUCTION
It has been reported that the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) inhibits cell growth, DNA and protein synthesis of cultured Chinese hamster ovary cells (CHO) [l]. This effect was reversed when the biocide was removed from the culture medium. The same herbicide was also indicated as an inhibitor of glycine uptake into the gills of Mytifus californianus [2]. The 2,4-D analogue, 2,4,5-T was found to interfere with [14C]thymidine- and [)H]uridine-facilitated transport into * To whom
correspondence
Abbreviations:
CHO,
trichlorophenoxyacetic (cycloleucine);
037%4274/87/$
should
Chinese acid;
be addressed.
hamster
AIB,
ovary
cells;
2-aminoisobutyric
2,4-D, acid;
CL,
dichlorophenoxyacetic
HU, hydroxyurea.
03.50
0 Elsevier
Science
Publishers
acid;
I-aminocyclopentanecarboxylic
B.V. (Biomedical
Division)
2,4,5-T, acid
190
L-929 cells [3]. This diminution was considered the direct result of the action of the pesticide upon the cell membrane. Nevertheless, the same authors indicated that the drug did not affect the uptake of [14C]leucine into the cells, although protein synthesis was inhibited. Since we found that protein synthesis was profoundly inhibited by 2,4-D in CHO cells, we thought that it would be of interest to investigate whether the transport of two nonmetabolizable aminoacids; i.e., 2-aminoisobutyric acid (AIB) and cycloleucine (CL), would be affected by 2,4-D at the membrane level. MATERIAL
AND
METHODS
A CHO strain (provided by Petra Martinez of the Fundacion de Genetica Humana, Buenos Aires, Argentina), kept in liquid nitrogen, was used for this investigation. The cells were grown as a monolayer in a glass vial with a 4.12 cm’ surface base, employing McCoy’s culture medium [4] supplemented with 15% fetal bovine serum and 50 pg/ml gentamycin. 0.25% trypsin (1:250, Difco) in Hepes buffer, pH 7.2 [5] was used to release the cells from the vials. 2-Amino-[l-14C]isobutyric acid (AIB) (60 Ci/mol) and 1-aminocyclopentane-[ l-14C]carboxylic acid (CL) (59 Ci/mol) were obtained from the Radiochemical Centre (Amersham, England). The unlabelled amino acids were purchased from Sigma. All other chemicals used were of reagent grade. The radioactive amino acid solutions were prepared in a sodium medium containing Hank’s/Hepes minus glucose (pH 7.4) [6] with a 121 mM concentration of Na+ . The sodium salt of 2,4-D (pH 7.3) was prepared by treating 2,4-dichlorophenoxyacetic acid (Sigma) with NaOH. Assay for AIB and CL transport
The assays for AIB and CL uptake were carried out following the method of Kelley and Potter [7] with a slight modification. Briefly, the method was as follows: approximately 2.5 x lo5 cells in logarithmic growth were plated directly into the vials for the amino acid transport experiments. Two series of vials were run simultaneously, one for the determination of the amino acid uptake and the other for cell counting. Each container contained 1.8 ml of culture medium and was incubated for 24 h at 37°C. The culture medium of the cells grown as a monolayer was discarded, the cells washed twice with 2 ml of Hank’s/Hepes buffer (pH 7.3) at 37°C followed by incubation at 37°C for 15 min with 3 ml of the same buffer to allow the depletion of the internal amino acid pool. The buffer was removed and 1 ml of radioactive amino acid solution added and incubated at 37°C for 8 min and washed 3 times with 2 ml of cold Hank’s/Hepes buffer (pH 7.3) at 4°C. Finally, the cells were solubilized in 0.5 ml of 0.2 N NaOH. A 0.2-ml aliquot of the solubilized cell solution used to determine the radioactivity contained 2.5 ml of a toluene/Triton X-100 (2: 1) mixture with 0.02 g% of 1,4-bis-2-(5-phenyloxazolyl)-
191
benzene; 1,4-bis(2-(5phenyloxazolyl))benzene (POPOP) (Sigma) and 0.4% of 2,5diphenyloxazole (PPO, Amersham/Searle). Radioactivity was determined employing a Beckman liquid scintillation counter. The AIB and CL uptake was linear for at least 10 min and is expressed as pmol of aminoacid/106 cells per min. To determine the effects of hydroxyurea (HU; Sigma) and 2,4-D on cell transport, a monolayer of approximately 4-5 x 10’ cells in logarithmic growth plus 1.8 ml of culture medium per vial were used. Final concentrations of 1.2 mM HU or 1 mM 2,4-D were added to the vials and they were incubated at 37°C for 24 h. After this time the media containing the drugs were discarded, the monolayers rinsed twice with 2 ml PBS solution A [8] and the transport determined either immediately (t,,) or at different times (t,,) after the addition of 1.8 ml normal medium. The kinetics values (K, and Vmax)were determined by Lineweaver-Burke plots by examining the 8-min uptake of 0.125-8 mM AIB or CL and 0.0125-0.8 &i/ml of [14C]AIB or [14C]CL in sodium-containing Hank’s/Hepes medium. RESULTS
To determine whether the 2,4-D had any direct effect of AIB uptake, logarithmic growth cultures were treated with 1.2 mM HU for 24 h after which the culture medium was replaced by normal medium and the transport immediately tested in absence of 2,4-D (control) or with different concentrations of the herbicide. The results are shown in Table I where it can be seen that the biocide does not have a direct effect even at the highest concentration tested. Table II depicts the results when AIB and CL uptake was compared between logarithmic growth cells and those treated for 24 h with 2,4-D. There is no difference between the amino acid uptake of cells treated with HU for 24 h and cells growing in a logarithmic state, but there is a remarkable decrease in the amino acid influx in cells treated with 2,4-D for 24 h. This herbicide produces a growth inhibition of CHO cells which appears at concentration of 1 mM. However, when the cell culture in logarithmic growth is treated TABLE
I
DIFFERENT
2,4-CONCENTRATION
Cells in logarithmic eliminated mined.
growth
and the uptake
AIB concentration:
EFFECTS
were treated
ON AIB UPTAKE
for 24 h with
of AIB in the presence
1.2 mM hydroxyurea
of different
1 mM; [‘%Z]AIB: 0.1 &i/ml.
2,4-D concentrations
The results are the average
run by duplicate. 2,4-D mM
pmol of AIB/106
0 (control)
3.86
0.1
3.69
1
3.80
10
3.23
cells per min
(HU).
The HU was
immediately
deter-
of two experiments
192
TABLE
II
AIB AND CL UPTAKE
BY CHO CELLS
IN LOGARITHMIC
TO 1.2 mM HU OR 1 mM 2,4-D TREATMENT AIB and CL uptake growth)
was determined
that were treated
determined
in logarithmic
(log) growth
medium
containing
Amino
the drugs was removed.
acid
and in cells (also in log
acid uptake
was immediately
The results represent
pmol/106
AIB
4.35
I.2 Mm HU
AIB
4.13
1 mM 2,4-D
AIB
2.95
CL
4.80
1.2 mM HU
CL
5.54
1 mM 2,4-D
CL
3.19
cells
Log growth
SUBJECTION
the average
run in duplicate.
Treatment Log growth
OR AFTER
cells (controls)
for 24 h with 1.2 mM HU or 1 mM 2,4-D. Amino
after the culture
of two experiments
GROWTH
FOR 24 h
cells
cells per min
with this concentration followed by its elimination from the culture medium, cell growth is reestablished (unpublished data). Fig. 1 shows the effects of HU and 2,4-D on AIB uptake when it was determined in cells after they had attained logarithmic growth and which were treated for 24 h with 1 mM of the herbicide or 1.2 mM HU. This was followed by the elimination
0
2
4
6
8
10
12
14
16 hours
Fig. 1. Logarithmically
growing
with the drugs was replaced mined or at different HU. (0) AIB uptake the arrows
indicate
cells were treated
by a normal
culture
with 1.2 mM HU or 1 mM 2,4-D for 24 h. The medium medium
and the amino
acid uptake
immediately
times after the normal medium was added. (0) AIB uptake by cells treated by cells treated with 2,4-D. Each point is the average of three determinations the time that the cells divided.
AIB: 1 mM; [%]AIB:
0.1 &i/ml;
Na+:
deterwith and
121 mM.
193
TABLE III K,, AND V,,, VALUESOF Amino
acid
AIB AND CL INFLUX
Treatment
h
Km
(mM)
V,,, (pm011
VmaX
lo6 cells per min)
increase
AIB
HU
to
1.16
9.54
AIB
HU
ts
1.11
13.36
AIB
2,4-D
to
1.15
4.00
AIB
2,4-D
t15
1.10
9.90
CL
HU
to
0.40
8.06
CL
HU
t9
0.55
10.48
CL
2,4-D
to
0.55
4.54
CL
2,4-D
t1s
0.63
8.45
Logarithmic containing
growth
cells were treated
the drugs was replaced
+ 51%
+ 150%
f
32.7%
+ 96.8%
with 1.2 mM HU or I mM 2,4-D for 24 h. The culture
by a normal
one and the kinetic parameters
determined
medium
at zero time
(to) and at t9 for the cells treated with HU, and at tl5 for those treated with 2,4-D after changing culture
the
medium.
of the drugs by transfer to a normal culture medium. The recovery of the amino acid transport was determined immediately (to) and at different times (t,,) after the initiation of cell growth in the normal medium. We could observe that the uptake of AIB by cells treated with HU exhibits a slow increase as the cells traverse the S phase of the cell cycle. Similar results were obtained when the [‘Hlleucine, [3H]lysine and [‘Hltryptophane uptake was studied during the CHO cell cycle [9].
0
2
Fig. 2. Experimental
4
6
conditions
8
10
12
14
16 hours
were similar to those indicated
with HU. ( n ) CL uptake by cells treated with 2,4-D. CL: 1 mM: [r4C]CL: 0.1 &i/ml; Na+: 121 mM.
Arrows
in Fig. 1. indicate
(o)CL uptake by cells treated the time that the cells divided.
194
L
2
1
2-AMINOISOBUTYHIC
Fig. 3. Lineweaver-Burke with 1.2 mM HU. Transport Other cultures
were treated
and at 15 h, tts (
n
plots of AIB incorporation was determined
8 ACID
(mM)-l
in CHO
cells: cell cultures
immediately,
to (0)
with 1 mM 2,4-D for 24 h and uptake
) after the herbicide
were treated
and at 9 h, ts (0) was determined
for 24 h
after HU removal. immediately,
to ( q )
removal.
Ten hours after the HU elimination from the medium, the cells began to divide and the increment of the AIB uptake was 52% between to and t9. Similar to the cells treated with HU, the AIB transport increased in those cells subjected to the action of 2,4-D as the cells passed through the S phase. The cells divided at 16 h and the increment of the uptake was 150% from to to tl5. One hour before cell division the incorporation of AIB by cells treated with 2,4-D did not reach the uptake level of those treated with HU. Similar results were obtained when the CL uptake was determined under identical experimental conditions (Fig. 2). Table III shows the K,,, and I/maxvalues of logarithmic growth cells treated with HU or 2,4-D for 24 h, immediately after drug removal (to) and at t9 after HU elimination and 115 after 2,4-D elimination and the addition of normal culture medium (these parameters were calculated based upon the results of Figs. 3 and 4). The K,,, did not show variations, however, Vmaxincreased from the initial moments
195
1
Fig. 4. Lineweaver-Burke
8
2
plots of CL incorporation
in CHO cells: cell cultures
1.2 mM HU. The transport
was immediately
Other
with 1 mM 2,4-D for 24 h and the uptake
cultures
were treated
and at 15 h, trs ( n ), after
herbicide
determined,
to (0)
were treated
and at 9 h, t9 (0) determined
for 24 h with
after HU removal. immediately,
to ( q)
removal.
of the drug removal up until 1 h before cell division: t9 in the case of HU treatment and at tls when 2,4-D was employed. The increment of Vmaxwas considerably larger when the cells were treated with 2,4-D than if they were treated with HU. DISCUSSION
Based on the above results it may be concluded that 2,4-D does not have a direct effect on the AIB transport in CHO cells (Table I). However, when the cells were in contact with 2,4-D for 24 h the herbicide not only stopped cell growth by DNA and protein synthesis inhibition [l] but also inhibited the uptake of the two nonmetabolizable amino acids tested (Table II). The AIB and CL transport inhibition produced by 2,4-D may be a consequence of its interference in the synthesis of certain membrane proteins which are indispensable as carriers. As was previously demonstrated, 2,4-D produces a cell accumulation at the Gr/S boundary of the cell cycle with a simultaneous inhibition of protein synthesis [l]. The removal of the herbicide is accompanied by an initiation of protein and DNA synthesis. The inhibition of protein synthesis can in part be caused by a decrease
196
of the amino acid transport which is restored when the herbicide is eliminated from the culture medium and the cell advances into the S phase (Figs. 1 and 2) and reaches a maximum at 15 h. The cells are capable of division at this moment and their plasma membrane has probably been restored to normal with its corresponding amino acid carriers. The kinetic transport parameters (Table III) show a change only in the V,,, and not in the Km which may indicate that the 2,4-D produced a decrease in the number of carriers but with no change in the affinity. The recuperation period that follows the elimination of 2,4-D from the medium is accompanied by an increase in protein synthesis possibly including carrier proteins. It is not clear why 2,4-D produced an elongation of the S phase and perhaps also of the GZ phase. The possibility exists that one of the limiting factors for cell division would be the integrity of the cell membrane. Additional research should be carried out specifically on membrane protein synthesis to shed more light on this subject. ACKNOWLEDGEMENTS
We are grateful to Mrs. Donna E.M. de Balegno for her help in preparing the manuscript and to Mr. Adalberto Allione for his technical assistance. This investigation was supported by the Consejo National de Investigaciones Cientificas y Technicas (Argentina). REFERENCES 1 V.A. Rivarola, J.R. Bergesse and H.F. Balegno, DNA and protein synthesis inhibition in Chinese Hamster Ovary Cells by dichlorophenoxyacetic acid, Toxicol. Lett., 29 (1985) 137-144. 2 J.H. Swinehart and M.A. Cheney, 2,4-Dichlorophenoxyacetic acid (2,4-D) and paranitrophenol (PNP) interactions with gills of Anodonta culiforniensis and Mytilus californianus: uptake and effects on membrane fluxes, Pestic. Biochem. Physiol., 20 (I983) 49-56. 3 L. Dragsnes and K. Helgeland, Effects of 2,4,5-T on the incorporation of f’4C]-thymidine; [‘HIuridine and L-[3H]-Ieucine into L-929 cells, Acta Pharmacol. Toxicol., 35 (1974) 103-112. 4 R.G. Ham and W.L. McKeehan, Media and growth requirements, in W.B. Jakoby and I.H. Pastan (Eds.), Methods in Enzymology, Vol. LVIII, Academic Press, New York, 1979. 5 C. Shipman, Jr., Trypsin, A: mammalian tissues, in P.J. Kruse, Jr. and M.K. Patterson (Eds.), Tissue Culture Methods and Applications, Academic Press, New York, 1973. 6 M.A. Lieberman, D.M. Raben, B. Whittenberger and L. Glaser, Effect of plasma membranes on solute transport in 3T3 cells, .I. Biol. Chem., 254 (1979) 6357-6361. 7 D.S. Kelley and V.R. Potter, Regulation of amino acid transport systems by amino acid depletion and supplemen~tion in monolayer cultures of rat hepatocytes, 3. Biol. Chem., 253 (1978) 9009-9017. 8 R.L.P. Adams, in Cefl Culture for Biochemists, Elsevier/North-HolIand, Amsterdam, 1980, pp. 247. 9 O.C. Blair and J.L. Roti Roti, Incorporation of 3H-leucine IH-lysine and 3H-tryptophan during the cell cycle of Chinese Hamster Ovary cells, J. Histochem. Cytochem. 28 (1980) 487-492.