Toxicology, 73 (1992) 71-79 Elsevier Scientific Publishers Ireland Ltd.
71
In vitro protein synthesis is affected by the herbicide 2,4-dichlorophenoxyacetic acid in Azospirillum
brasilense Viviana Rivarola, Adriana Fabra, Gladys Mori and Hector Balegno Facultad de Ciencias Exactas, Fisico-Quimica y Naturales, Universidad Nacional de Rio Cuarto, 5800, Rio Cuarto, C6rdoba (Argentina) (Received December 9th, 1991; accepted February 29th, 1992)
Summary The effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on growth and protein, D N A and R N A synthesis of Azospirillum brasilense Cd were studied. At a concentration of 1 mM, 2,4-D inhibited cell growth, an effect that was reversed either by transferring bacteria to a control (2,4-D-free) medium or to a 2,4-Dtreated medium supplemented with polyamines. The herbicide also affected in vitro protein synthesis, either when Azospirillum brasilense Cd's own cellular m R N A or an artificial m R N A was used. This effect was also reversed by the addition of polyamines to the 2,4-D-treated medium. Similar results were observed when D N A synthesis was studied in synchronous cultures. Taking into account the effects of this herbicide on animal cells (V.A. Rivarola and H.F. Balegno, Toxicology, 68 (1991) 109) we postulate that the mechanism of action of 2,4-D is similar on both procaryotic and eucaryotic cells, probably acting through the polyamine metabolism.
Key words." 2,4-Dichlorophenoxyacetic acid (2,4-D); Polyamines; Azospirillum brasilense; Protein synthesis
Introduction One of the most important ecotoxicological problems in agriculture is the intensive use of pesticides affecting non-target organisms, including soil microorganisms
[2]. Previous studies in our laboratory demonstrated that the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) can inhibit cell growth and DNA, RNA, protein and polyamine biosynthesis in cultured hamster ovary cells (CliO) [1,3-6]. These effects can be reversed when the pesticide is removed from the culture medium [3] or by adding polyamines to the medium [5].
Correspondence to." Hector Balegno, Facultad de Ciencias Exactas, Fisico-Quimica y Naturales, Universidad Nacional de Rio Cuarto, 5800, Rio Cuarto, C6rdoba, Argentina. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
72 The aim of this work was to study the effects of 2,4-D on bacteria. Azospirillum brasilense Cd was chosen as a model because of its ecological and agricultural importance. Bacteria of the genus Azospirillum are free living diazotrophs that can be readily isolated from the rhizosphere and roots of forage and grain grasses [7]. It has been postulated that biological nitrogen fixation by Azospirillum sp. in association with roots may contribute a significant amount of nitrogen to the plant, explaining beneficial effects on crop yield [8,9]. Other authors have suggested that the production of phytohormone-like substances by Azospirillum strains also contributes to the yield of various cereals [10]. On the basis of our previous results with C H O cells and in an attempt to compare the mechanism of action of 2,4-D in eucaryotic and procaryotic cells, we decided to study the effect of the herbicide on the growth of and on DNA, R N A and protein biosynthesis by A. brasilense Cd. Since polyamines are involved in the biosynthesis of protein and nucleic acids [11], their relation to these amines was investigated too. Materials and methods
Chemicals All the chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), while radiochemicals were purchased as follows: [3H]thymidine (58 Ci/mmol) and L-[U-14C]leucine (10 mCi/mmol) (Amersham); [3H]uridine (25.5 Ci/mmol); L-[U-14C]amino acid mixture (NEC. 445), [lac]phenyalalanine (450 mCi/mmol) (New England).
Bacteria and growth conditions Stock cultures of Azospirillum brasilense Cd were maintained in solid NfB medium, containing NH4CI 2.5 g 1-1, malic acid 5 g 1-I, K2HPO4 0.5 g I 1, MgSO 4" 7H20 0.2 g 1-j, NaC1 0.1 g 1 i, FeEDTA 0.066 g 1-1, K O H 4.5 g L -1, CaC12 0.02 g 1-1, Na2MO 4 • 2H20 0.4 mg 1 1 MnSO4' H20, 0.47 mg 1 i BO3H 3 0.56 mg 1-1, biotin, 10 #g 1-l, pyridoxal, 20 ~g 1-1 and agar 15 g 1-I [12]. Cultures were transferred monthly and stored at 4°C.
Growth studies NfB broth medium [12] was used in all the experiments. Growth experiments were conducted in 250-ml or 1000 -ml Erlenmeyer flasks which contained 50 ml or 250 ml broth, respectively. The concentrations of herbicide used were 0.5, 1 and 2 mM. The flasks were inoculated with 2 or 10 ml of a culture in early logarithmic phase (optical density (O.D.)620 nm: 0.3) and were incubated at 37 + 2°C on a rotary shaker. In some experiments, both polyamines (1 mM putrescine and 1 mM spermidine) were added to the medium at the initiation of the incubation or after 8 h of culture. Growth was followed by determining the absorbance at 620 nm. To determine if the effects of 2,4-D could be reversed by changing bacteria from 2,4-D-treated medium to control (2,4-D-free) or polyamine-supplemented control media, cells growing for 8 h in the 2,4-D-treated medium were harvested by centrifugation at 10 000 rev./min. The cellular pellets were resuspended in an appropriate volume of the corresponding fresh media, with the O.D. being the same as before harvesting. Growth was followed as explained above.
73
In vitro protein synthesis determination In vitro protein synthesis was determined according to the method of Kato et al.
[13]. Preparation of pH5 enzymes In order to obtain the enzymes and protein synthesis factors, a suspension called 'pH5 enzymes' was obtained in the following way: approximately 1 g of liver from a 200-g rat, fasted for 24 h, was homogenized in 3 ml of buffer (1 mM Tris-HCl pH 7, 4.5 mM MgCI2, 5 mM KC1 and 0.25 M sucrose) and centrifuged at 42 000 rev./min for 2 h. The supernatant was separated, taken to pH 5-5.2 with cold 1 N acetic acid and centrifuged at 2000 rev./min for 2 rain at 5°C. The resultant pellet was resuspended in cold distilled water and centrifuged at 2000 rev./min for 2 min. It was then washed with cold distilled water and resuspended in 1/3 of the original volume of the supernatant in the following buffer: 50 mM T r i s - H C l pH 7.6, 5 mM MgC12, 25 mM KC1, 0.25 M sucrose, 1 mM dithiothreitol and centrifuged at 10 000 rev./min for 10 rain. The supernatant containing the enzymes and protein synthesis factors was divided into aliquots and frozen at -20°C.
Preparation of the ribosomal fraction In vitro protein synthesis was determined by the following technique. Bacteria in logarithmic growth (8 h of culture) were centrifuged at 8000 rev./min for 10 rain and the spheroplasts were obtained using the lysozyme-EDTA method [14]. Spheroplasts were disrupted by homogenization in a Potter homogenizer in an appropriate volume of buffer (1 mM Tris-HC1, 5 mM MgCI2, 5 mM KCI, 0.25 M sucrose) and centrifuged at 10 000 rev./min for 10 min. The supernatant was separated and centrifuged at 45 000 rev./min for 2 h. The resultant pellet was resuspended in 0.5 ml of Tris buffer without sucrose. Protein concentration was determined by the method of Bradford [15].
Incorporation of 14C amino acid mixture in the ribosomal fraction To a ribosomal suspension containing 700/~g of proteins, 200 #g of pH5 protein enzymes were added. The following reagents in a total volume of 200/~1 were added: 10 mM phosphate buffer (pH 7.4), 1.25 mM ATP, 0.25 mM GTP, 43 mM sucrose, 5 mM MgC12, 50 mM glutathione, 20 mM phosphocreatine, 30 izl of a mixture of L-[U-14C]amino acids (radioactive), 80 tzl of a 3-mM mixture of non-radioactive amino acids (proline, cysteine, valine, tyrosine, aspartic acid, glutamic acid, methionine, threonine, serine, tryptophane) and 60 IU of phosphocreatine kinase. The mixture, with a final pH of 7.4, was incubated for 20 min at 37°C. The reaction was stopped with 0.4 M HCIO4, tubes were kept in ice for 10 rain and then centrifuged at 2000 rev./min for 5 min. The final pellet was dissolved in 1 ml of 0.1 N NaOH. Protein content was determined as previously described [15]. Scintillant (3 ml) consisting of 0.2 g of 1,4, bis(2,4-methylphenyloxozolyl) benzene and 4 g of 2,5-diphenyloxozole per litre of triton:toluene (1:3, v/v) was added and the radioactivity measured in a Beckman LS 100 scintillation counter.
[14C]Phenylalanine incorporation in the ribosomal fraction with artificial mRNA To a ribosomal suspension containing 700 #g of proteins, 200/~g of pH5 protein
74
enzymes were added. The same reagents used in the previous experiment (except the amino acid mixture) were also added in a total volume of 200 #1. The sample was placed in ice and the following mixture was added: creatine phosphokinase, 60 IU; 40 #mol of creatine phosphate, 0.085 /zmol of [lac]phenylalanine and 300 #g of polyuridylic acid. The tube was incubated at 37°C for 30 min and the reaction stopped by adding cold 0.4 N HC10 4. After centrifugation at 2000 rev./min for 10 min, the pellet was dissolved in 1 ml 0.1 N NaOH, the proteins were reprecipitated with 0.4 M HCIO4 and centrifuged. The final pellet was dissolved in 1 ml 1 N NaOH and the radioactivity determined in the resulting solution, as previously described.
[3H] Uridine and [14C]leucine uptake experiments Bacteria were grown in control or treated medium for 8 h, centrifuged and the pellets were resuspended in 6 ml of phosphate buffered saline (PBS), pH 7.4. [3H]Uridine or [14C]leucine (200 t~Ci/~mol) were added and the cells were maintained at 37 4- 2°C for 15 min. Incorporation was stopped by the addition of 15 ml cold PBS. After centrifugation at 10 000 rev./min, radioactivity was measured in (a) whole cells, (b) spheroplasts and (c) proteins and RNA isolated according to the method of Munro and Fleck [16].
[~H]Thymidine uptake in synchronous cultures Synchronization of bacterial cultures was performed by carbohydrate starvation as described by Evans [17]. When the absorbance of the bacteria in minimal medium was maintained constant (O.D.620 nm: 0.3), cells were transferred to control or treated media in a dilution of 1:25 and [laC]thymidine (200 ~zCi/#mol) was added.
Absor banoe (62Onto) 1.6 1.4 1"3
171
CI
~
~
~1
1.2 I
1.0---
I __1
-~.__~
0.8
I $
•
I
I
. . . .
0.6 0.4 0.2 O.C 0
I
I
I
I
1
6
10
16
20
25
30
Culture time (hours) Control
--4-- 1 mM 2,4-D
~
2 mM 2.4-D
~
Translerred 1
Fig. 1. 2,4-D effect on Azospirillum brasilense growth. ITransferred: the arrow indicates the moment bacteria, grown for 8 h in 2,4-D-treated medium, were transferred to fresh control (2,4-D free) medium, n = 3
75
When the optical density was doubled, the radioactive incorporation was stopped by the addition of 0.2 M trichloroacetic acid (TCA) and 0.4 M NaCI (final concentrations) [18]. The radioactivity was measured in the pellet after the addition of scintillation liquid.
Statistical analysis Means were compared using the Student t-test, at the 0.05 significance level. Results and Discussion
The addition of 1 or 2 mM 2,4-D to NfB broth, just before inoculation with
Azospirillum brasilense Cd, produced a decrease of the growth rate (Fig. 1). This effect was more pronounced when 2 mM 2,4-D was used. However, when the cells were transferred to a control medium after 8 h incubation in the presence of 1 mM 2,4-D, the effect of the herbicide was reversed (Fig. 1). The same observation was done when 1 mM putrescine and 1 mM spermidine were added to the culture medium (Fig. 2). These results suggest that the growth decrease produced by 2,4-D might be due to an effect of the herbicide on the polyamine biosynthesis because the same reversion effects were obtained either by transferring bacteria from treated to control (2,4-D-free) medium or to polyamine-supplemented-treated medium.
A b e o r l ~ n c e ( 0 2 0 nm)
1°f 1.4
Y.Z" ~
~
"
"
.
12
.
.
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0
";"
";"
: :i:-: ,5
10
Culture Control
-4-
":"
-"
"~
_:_
,¢
o:o F. 7
.
Control + P
1,5
time ~
,
,
20
26
30
(hours) 2,4-£) 1 mM
2,4-D + P
Rever810n
Fig. 2. Polyamine effect on bacteria growing with 2,4-D. Control + P, 1 mM putrescine and 1 mM spermidine were added to control medium before incubation; 2,4-D + P, 1 mM putrescine and 1 mM spermidine were added to I mM 2,4-D-treated medium before incubation; reversion, the arrow indicates the moment when 1 mM putrescine and 1 mM spermidine were added after 8 h of culture in a l-mM 2,4-Dtreated medium; n = 3.
76
300
opm x 10"3/ mg ribosomal prol.
250 200 150 100 ,50
OONTROL
2,4-D
Fig. 3. In vitro protein synthesis (cellular mRNA), in vitro protein synthesis was determined by the method of Kato et al. [13] (see Materials and methods); *P < 0.05; n = 5,
When polyamines were added to the 2,4-D-treated medium at the initiation of the incubation, bacteria grew similarly to control (Fig. 2). These data suggest that polyamines could protect cells from 2,4-D inhibitory effects. Because polyamines are involved in D N A and R N A as well as in protein biosynthesis [ 11 ], we therefore decided to investigate the effects of 2,4-D on these important processes. In vitro protein biosynthesis in bacteria grown for 8 h in the presence of the herbicide was decreased by 64%, (Fig. 3). In order to discern if the inhibition observed opm x lO'3/mg ribosomal Drot, 25
20
1,5
10
o
O
C +P
2,4-0
2,4-D ÷ P
2,4-D c.f.o.
Fig. 4. In vitro protein synthesis (artificial mRNA). C, cell-free extract from bacteria grown for 8 h in control medium; C + P, cell-free extract from bacteria grown for 8 h in control medium supplemented with polyamines; 2,4-D, cell-free extract from bacteria grown for 8 h in 2,4-D-treated medium; 2,4-D + P, cell-free extract from bacteria grown for 8 h in 2,4-D-treated medium supplemented with polyamines; 2,4-D c.f.e., 1 mM 2,4-D was added to a control cell-free extract (C); *P -< 0.01; n = 3-6.
77
0.008
nmole8 uridino/mg coil prof.
0.006
0.004
0.002
0.000
C (sph)
T (sph)
O (RNA)
T (RNA)
Fig. 5. [3H]Uridine uptake. Incorporation of [3H]uridine in control spheroplasts (C(sph)) and in treated spheroplasts (T(sph)) and in isolated RNA from control and treated groups (C(RNA)) and (T(RNA)); n = 3-6; *P < 0.0125.
was due to an alteration of the mRNA, the cellular mRNA was replaced by an artificial one. Under these conditions an inhibition in the protein biosynthesis was also found, being reversed by polyamine addition (Fig.4). These results suggest that the 2,4-D inhibition of protein biosynthesis is not mainly dependent upon mRNA. The addition of 2,4-D directly to the control cell ribosomes (in cell-free extract), did not produce alterations in the protein biosynthesis (Fig. 4), indicating that the herbicide must be in contact with the cells for at least 8 h to produce any effect. We nrnole8 leucine / rng cell prot.
v . v ~
0 (8ph)
T (sph)
0 (prot)
T (prol)
Fig. 6. [14C]Leucine uptake. Incorporation of [14C]leucine in control (C(sph)) and in treated spheroplasts (T(sph)) and in proteins from control and treated groups (C(prot)) and (T(prot)); n = 3-6; *P _~ 0.0005.
78
0.2(
nmolo8 /rag ooll prot.
0.1,'
0.1(
0.0~
O.OC 0
O+P
T
T+P
Fig. 7. [3H]Thymidine uptake in synchronized bacteria. Bacterial cultures were synchronized as in Materials and methods and [3H]thymidine uptake was determined in cells transferred to control medium (C), polyamine-supplemented control medium ( C + P ) , 2,4-D-treated medium (T), polyamine -supplemented-treated medium (T + P); n = 3; *P -< 0.05.
could speculate that 2,4-D is altering the polyamine metabolism and simultaneously affecting one of the components of the protein synthesis process. Results obtained from [3H]uridine uptake experiments are shown in Fig. 5. There is a reduction in the radioactive incorporation in treated spheroplasts, but in spite of a similar trend in isolated RNA, no statistically significant differences were observed. In Fig. 6 the results of [lac]leucine uptake are shown. A diminution was observed in both treated spheroplasts and in isolated proteins, being in agreement with the in vitro (cell-free extract) protein synthesis inhibition. With respect to DNA biosynthesis, a decrease in [3H]thymidine incorporation was observed that was reversed by the addition of polyamines to the culture medium (Fig. 7). In conclusion, these results suggest that the mechanism of action of 2,4-D may be similar in both procaryotic and eucaryotic cells [1,5], acting in some manner on the metabolism of polyamines and thus altering protein and nucleic acid synthesis and cell growth. We are currently investigating the effects of 2,4-D on the metabolism of polyamines.
Acknowledgements We are grateful to Mrs. Donna Balegno for her help in preparing the manuscript and to Mr. Miguel Bueno and Mr. Walter Giordano for their technical assistance. This investigation was supported by the Consejo Nacional de Investigaciones Cientificas y T6cnicas (Argentina).
79
References 1 2
3
4 5 6 7
8
9 10
11 12 13 14 15 16 17
18
V.A. Rivarola and H.F. Balegno, 2,4-Dichlorophenoxyacetic acid (2,4-D) effects on polyamine biosynthesis. Toxicology, 68 (1991) 109. D.M.S., Mano, A.C.M. Matos and T. Langebach, The effect ofdicofol on morphology growth and nitrogenase activity of Azospirillum lipoferum, in W. Klinmiiller (Ed.), Azospirillum IV: Genetics. Physiology, Ecology, Springer-Verlag, Berlin, Heidelberg, 1988, p. 159. A.M. Evangelista de Duffard, V. Rivarola and R. Duffard, Changes of glycolipids and gangliosides in 2,4-dichlorophenoxyacetic acid treated Chinese hamster ovary cells in monolayer culture. Toxicity Assess., 3 (1988) 117. V.A., Rivarola, J.R. Bergesse and H.F. Balegno, DNA and protein synthesis inhibition in Chinese hamster ovary cells by 2,4-dichlorophenoxyacetic acid. Toxicol. Lett., 29 (1985) 137. V., Rivarola, H. Balegno, Effects of 2,4-D on polyamine metabolism in Chinese hamster ovary cells (CHO). Toxicol. Lett., 56 (1991) 151. V.A. Rivarola and H.F. Balegno, 2,4-Dichlorophenoxyacetic acid (2,4-D) effects on in vitro protein synthesis and its relation to polyamines. Drug Chem. Toxicol., 15 (1992) 3. J. D6bereiner and J.M. Day, Associative symbiosis in tropical grasses; characterization of microorganisms and dinitrogen-fixing sites, in W.E. Newton and C.J. Nyman (Eds.), Proc. First int. Symp. Nitrogen Fixation, Washington State University Press, Pullman, 1976, p. 518. R.M. Boddey and J. D6bereiner, Association of Azospirillum and other diazotrophs with tropical graminae, 12th Int. Congr. Soil Sci., Symp. papers, Vol. 1, Int. Soc. Soil Sci., Food Agric. Organ., Rome, 1982, p. 28. C.A. Neyra and Dfbereiner, Nitrogen fixation in grasses. Adv. Agron., 29 (1977) 1. H., Levanony, Y. Bashan and Z. Kahana, Enzyme-linked immunosorbent assay for specific identification and enumeration of Azospirillum brasilense Cd in cereal roots. Appl. Environ. Microbiol., 53 (1987) 358. I.D. Algranati and S.H. Goldemberg, Polyamines and their role in protein synthesis. Trends Biochem. Sci., 2 (1977) 272. J. D6bereiner, Forage grasses and grain crops, in F.J. Bergesen (Ed.), Methods for Evaluating Biological Nitrogen Fixation, Wiley, 1980, p. 535. R., Kato, L. Loeb and H.V. Gelboin, Increased sensitivity of microsomes from phenobarbitaltreated rats to synthetic messenger RNA: lack of effect on ribosomes. Nature, (1965) 668. H.R. Kavack Bacterial Membranes, in S. Collowick and N.O. Kaplan (Eds.), Methods of Enzymology, Vol. XXII, 1971, p. 99. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem., 72 (1976) 243. H.N. Munro and A. Fleck, Recent development in the measurement of nucleic acid in biological material. Analyst, 91 (1966) 78. J.E. Evans, Techniques and their application for synchronization of populations of microorganisms, in A.I. Laskin and H.A. Lechevalier, (Eds.) Handbook of Microbiology, Vol. 4, CRC Press, Boca Ratfn, FL 1974, p. 813. H. Bremer and L. Chuang, The cell cycle in Escherichia coli B/r. J. Theor. Biol., 88 (1981) 47.
71
In vitro protein synthesis is affected by the herbicide 2,4-dichlorophenoxyacetic acid in Azospirillum
brasilense Viviana Rivarola, Adriana Fabra, Gladys Mori and Hector Balegno Facultad de Ciencias Exactas, Fisico-Quimica y Naturales, Universidad Nacional de Rio Cuarto, 5800, Rio Cuarto, C6rdoba (Argentina) (Received December 9th, 1991; accepted February 29th, 1992)
Summary The effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on growth and protein, D N A and R N A synthesis of Azospirillum brasilense Cd were studied. At a concentration of 1 mM, 2,4-D inhibited cell growth, an effect that was reversed either by transferring bacteria to a control (2,4-D-free) medium or to a 2,4-Dtreated medium supplemented with polyamines. The herbicide also affected in vitro protein synthesis, either when Azospirillum brasilense Cd's own cellular m R N A or an artificial m R N A was used. This effect was also reversed by the addition of polyamines to the 2,4-D-treated medium. Similar results were observed when D N A synthesis was studied in synchronous cultures. Taking into account the effects of this herbicide on animal cells (V.A. Rivarola and H.F. Balegno, Toxicology, 68 (1991) 109) we postulate that the mechanism of action of 2,4-D is similar on both procaryotic and eucaryotic cells, probably acting through the polyamine metabolism.
Key words." 2,4-Dichlorophenoxyacetic acid (2,4-D); Polyamines; Azospirillum brasilense; Protein synthesis
Introduction One of the most important ecotoxicological problems in agriculture is the intensive use of pesticides affecting non-target organisms, including soil microorganisms
[2]. Previous studies in our laboratory demonstrated that the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) can inhibit cell growth and DNA, RNA, protein and polyamine biosynthesis in cultured hamster ovary cells (CliO) [1,3-6]. These effects can be reversed when the pesticide is removed from the culture medium [3] or by adding polyamines to the medium [5].
Correspondence to." Hector Balegno, Facultad de Ciencias Exactas, Fisico-Quimica y Naturales, Universidad Nacional de Rio Cuarto, 5800, Rio Cuarto, C6rdoba, Argentina. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
72 The aim of this work was to study the effects of 2,4-D on bacteria. Azospirillum brasilense Cd was chosen as a model because of its ecological and agricultural importance. Bacteria of the genus Azospirillum are free living diazotrophs that can be readily isolated from the rhizosphere and roots of forage and grain grasses [7]. It has been postulated that biological nitrogen fixation by Azospirillum sp. in association with roots may contribute a significant amount of nitrogen to the plant, explaining beneficial effects on crop yield [8,9]. Other authors have suggested that the production of phytohormone-like substances by Azospirillum strains also contributes to the yield of various cereals [10]. On the basis of our previous results with C H O cells and in an attempt to compare the mechanism of action of 2,4-D in eucaryotic and procaryotic cells, we decided to study the effect of the herbicide on the growth of and on DNA, R N A and protein biosynthesis by A. brasilense Cd. Since polyamines are involved in the biosynthesis of protein and nucleic acids [11], their relation to these amines was investigated too. Materials and methods
Chemicals All the chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), while radiochemicals were purchased as follows: [3H]thymidine (58 Ci/mmol) and L-[U-14C]leucine (10 mCi/mmol) (Amersham); [3H]uridine (25.5 Ci/mmol); L-[U-14C]amino acid mixture (NEC. 445), [lac]phenyalalanine (450 mCi/mmol) (New England).
Bacteria and growth conditions Stock cultures of Azospirillum brasilense Cd were maintained in solid NfB medium, containing NH4CI 2.5 g 1-1, malic acid 5 g 1-I, K2HPO4 0.5 g I 1, MgSO 4" 7H20 0.2 g 1-j, NaC1 0.1 g 1 i, FeEDTA 0.066 g 1-1, K O H 4.5 g L -1, CaC12 0.02 g 1-1, Na2MO 4 • 2H20 0.4 mg 1 1 MnSO4' H20, 0.47 mg 1 i BO3H 3 0.56 mg 1-1, biotin, 10 #g 1-l, pyridoxal, 20 ~g 1-1 and agar 15 g 1-I [12]. Cultures were transferred monthly and stored at 4°C.
Growth studies NfB broth medium [12] was used in all the experiments. Growth experiments were conducted in 250-ml or 1000 -ml Erlenmeyer flasks which contained 50 ml or 250 ml broth, respectively. The concentrations of herbicide used were 0.5, 1 and 2 mM. The flasks were inoculated with 2 or 10 ml of a culture in early logarithmic phase (optical density (O.D.)620 nm: 0.3) and were incubated at 37 + 2°C on a rotary shaker. In some experiments, both polyamines (1 mM putrescine and 1 mM spermidine) were added to the medium at the initiation of the incubation or after 8 h of culture. Growth was followed by determining the absorbance at 620 nm. To determine if the effects of 2,4-D could be reversed by changing bacteria from 2,4-D-treated medium to control (2,4-D-free) or polyamine-supplemented control media, cells growing for 8 h in the 2,4-D-treated medium were harvested by centrifugation at 10 000 rev./min. The cellular pellets were resuspended in an appropriate volume of the corresponding fresh media, with the O.D. being the same as before harvesting. Growth was followed as explained above.
73
In vitro protein synthesis determination In vitro protein synthesis was determined according to the method of Kato et al.
[13]. Preparation of pH5 enzymes In order to obtain the enzymes and protein synthesis factors, a suspension called 'pH5 enzymes' was obtained in the following way: approximately 1 g of liver from a 200-g rat, fasted for 24 h, was homogenized in 3 ml of buffer (1 mM Tris-HCl pH 7, 4.5 mM MgCI2, 5 mM KC1 and 0.25 M sucrose) and centrifuged at 42 000 rev./min for 2 h. The supernatant was separated, taken to pH 5-5.2 with cold 1 N acetic acid and centrifuged at 2000 rev./min for 2 rain at 5°C. The resultant pellet was resuspended in cold distilled water and centrifuged at 2000 rev./min for 2 min. It was then washed with cold distilled water and resuspended in 1/3 of the original volume of the supernatant in the following buffer: 50 mM T r i s - H C l pH 7.6, 5 mM MgC12, 25 mM KC1, 0.25 M sucrose, 1 mM dithiothreitol and centrifuged at 10 000 rev./min for 10 rain. The supernatant containing the enzymes and protein synthesis factors was divided into aliquots and frozen at -20°C.
Preparation of the ribosomal fraction In vitro protein synthesis was determined by the following technique. Bacteria in logarithmic growth (8 h of culture) were centrifuged at 8000 rev./min for 10 rain and the spheroplasts were obtained using the lysozyme-EDTA method [14]. Spheroplasts were disrupted by homogenization in a Potter homogenizer in an appropriate volume of buffer (1 mM Tris-HC1, 5 mM MgCI2, 5 mM KCI, 0.25 M sucrose) and centrifuged at 10 000 rev./min for 10 min. The supernatant was separated and centrifuged at 45 000 rev./min for 2 h. The resultant pellet was resuspended in 0.5 ml of Tris buffer without sucrose. Protein concentration was determined by the method of Bradford [15].
Incorporation of 14C amino acid mixture in the ribosomal fraction To a ribosomal suspension containing 700/~g of proteins, 200 #g of pH5 protein enzymes were added. The following reagents in a total volume of 200/~1 were added: 10 mM phosphate buffer (pH 7.4), 1.25 mM ATP, 0.25 mM GTP, 43 mM sucrose, 5 mM MgC12, 50 mM glutathione, 20 mM phosphocreatine, 30 izl of a mixture of L-[U-14C]amino acids (radioactive), 80 tzl of a 3-mM mixture of non-radioactive amino acids (proline, cysteine, valine, tyrosine, aspartic acid, glutamic acid, methionine, threonine, serine, tryptophane) and 60 IU of phosphocreatine kinase. The mixture, with a final pH of 7.4, was incubated for 20 min at 37°C. The reaction was stopped with 0.4 M HCIO4, tubes were kept in ice for 10 rain and then centrifuged at 2000 rev./min for 5 min. The final pellet was dissolved in 1 ml of 0.1 N NaOH. Protein content was determined as previously described [15]. Scintillant (3 ml) consisting of 0.2 g of 1,4, bis(2,4-methylphenyloxozolyl) benzene and 4 g of 2,5-diphenyloxozole per litre of triton:toluene (1:3, v/v) was added and the radioactivity measured in a Beckman LS 100 scintillation counter.
[14C]Phenylalanine incorporation in the ribosomal fraction with artificial mRNA To a ribosomal suspension containing 700 #g of proteins, 200/~g of pH5 protein
74
enzymes were added. The same reagents used in the previous experiment (except the amino acid mixture) were also added in a total volume of 200 #1. The sample was placed in ice and the following mixture was added: creatine phosphokinase, 60 IU; 40 #mol of creatine phosphate, 0.085 /zmol of [lac]phenylalanine and 300 #g of polyuridylic acid. The tube was incubated at 37°C for 30 min and the reaction stopped by adding cold 0.4 N HC10 4. After centrifugation at 2000 rev./min for 10 min, the pellet was dissolved in 1 ml 0.1 N NaOH, the proteins were reprecipitated with 0.4 M HCIO4 and centrifuged. The final pellet was dissolved in 1 ml 1 N NaOH and the radioactivity determined in the resulting solution, as previously described.
[3H] Uridine and [14C]leucine uptake experiments Bacteria were grown in control or treated medium for 8 h, centrifuged and the pellets were resuspended in 6 ml of phosphate buffered saline (PBS), pH 7.4. [3H]Uridine or [14C]leucine (200 t~Ci/~mol) were added and the cells were maintained at 37 4- 2°C for 15 min. Incorporation was stopped by the addition of 15 ml cold PBS. After centrifugation at 10 000 rev./min, radioactivity was measured in (a) whole cells, (b) spheroplasts and (c) proteins and RNA isolated according to the method of Munro and Fleck [16].
[~H]Thymidine uptake in synchronous cultures Synchronization of bacterial cultures was performed by carbohydrate starvation as described by Evans [17]. When the absorbance of the bacteria in minimal medium was maintained constant (O.D.620 nm: 0.3), cells were transferred to control or treated media in a dilution of 1:25 and [laC]thymidine (200 ~zCi/#mol) was added.
Absor banoe (62Onto) 1.6 1.4 1"3
171
CI
~
~
~1
1.2 I
1.0---
I __1
-~.__~
0.8
I $
•
I
I
. . . .
0.6 0.4 0.2 O.C 0
I
I
I
I
1
6
10
16
20
25
30
Culture time (hours) Control
--4-- 1 mM 2,4-D
~
2 mM 2.4-D
~
Translerred 1
Fig. 1. 2,4-D effect on Azospirillum brasilense growth. ITransferred: the arrow indicates the moment bacteria, grown for 8 h in 2,4-D-treated medium, were transferred to fresh control (2,4-D free) medium, n = 3
75
When the optical density was doubled, the radioactive incorporation was stopped by the addition of 0.2 M trichloroacetic acid (TCA) and 0.4 M NaCI (final concentrations) [18]. The radioactivity was measured in the pellet after the addition of scintillation liquid.
Statistical analysis Means were compared using the Student t-test, at the 0.05 significance level. Results and Discussion
The addition of 1 or 2 mM 2,4-D to NfB broth, just before inoculation with
Azospirillum brasilense Cd, produced a decrease of the growth rate (Fig. 1). This effect was more pronounced when 2 mM 2,4-D was used. However, when the cells were transferred to a control medium after 8 h incubation in the presence of 1 mM 2,4-D, the effect of the herbicide was reversed (Fig. 1). The same observation was done when 1 mM putrescine and 1 mM spermidine were added to the culture medium (Fig. 2). These results suggest that the growth decrease produced by 2,4-D might be due to an effect of the herbicide on the polyamine biosynthesis because the same reversion effects were obtained either by transferring bacteria from treated to control (2,4-D-free) medium or to polyamine-supplemented-treated medium.
A b e o r l ~ n c e ( 0 2 0 nm)
1°f 1.4
Y.Z" ~
~
"
"
.
12
.
.
"~"~
1'0
0
";"
";"
: :i:-: ,5
10
Culture Control
-4-
":"
-"
"~
_:_
,¢
o:o F. 7
.
Control + P
1,5
time ~
,
,
20
26
30
(hours) 2,4-£) 1 mM
2,4-D + P
Rever810n
Fig. 2. Polyamine effect on bacteria growing with 2,4-D. Control + P, 1 mM putrescine and 1 mM spermidine were added to control medium before incubation; 2,4-D + P, 1 mM putrescine and 1 mM spermidine were added to I mM 2,4-D-treated medium before incubation; reversion, the arrow indicates the moment when 1 mM putrescine and 1 mM spermidine were added after 8 h of culture in a l-mM 2,4-Dtreated medium; n = 3.
76
300
opm x 10"3/ mg ribosomal prol.
250 200 150 100 ,50
OONTROL
2,4-D
Fig. 3. In vitro protein synthesis (cellular mRNA), in vitro protein synthesis was determined by the method of Kato et al. [13] (see Materials and methods); *P < 0.05; n = 5,
When polyamines were added to the 2,4-D-treated medium at the initiation of the incubation, bacteria grew similarly to control (Fig. 2). These data suggest that polyamines could protect cells from 2,4-D inhibitory effects. Because polyamines are involved in D N A and R N A as well as in protein biosynthesis [ 11 ], we therefore decided to investigate the effects of 2,4-D on these important processes. In vitro protein biosynthesis in bacteria grown for 8 h in the presence of the herbicide was decreased by 64%, (Fig. 3). In order to discern if the inhibition observed opm x lO'3/mg ribosomal Drot, 25
20
1,5
10
o
O
C +P
2,4-0
2,4-D ÷ P
2,4-D c.f.o.
Fig. 4. In vitro protein synthesis (artificial mRNA). C, cell-free extract from bacteria grown for 8 h in control medium; C + P, cell-free extract from bacteria grown for 8 h in control medium supplemented with polyamines; 2,4-D, cell-free extract from bacteria grown for 8 h in 2,4-D-treated medium; 2,4-D + P, cell-free extract from bacteria grown for 8 h in 2,4-D-treated medium supplemented with polyamines; 2,4-D c.f.e., 1 mM 2,4-D was added to a control cell-free extract (C); *P -< 0.01; n = 3-6.
77
0.008
nmole8 uridino/mg coil prof.
0.006
0.004
0.002
0.000
C (sph)
T (sph)
O (RNA)
T (RNA)
Fig. 5. [3H]Uridine uptake. Incorporation of [3H]uridine in control spheroplasts (C(sph)) and in treated spheroplasts (T(sph)) and in isolated RNA from control and treated groups (C(RNA)) and (T(RNA)); n = 3-6; *P < 0.0125.
was due to an alteration of the mRNA, the cellular mRNA was replaced by an artificial one. Under these conditions an inhibition in the protein biosynthesis was also found, being reversed by polyamine addition (Fig.4). These results suggest that the 2,4-D inhibition of protein biosynthesis is not mainly dependent upon mRNA. The addition of 2,4-D directly to the control cell ribosomes (in cell-free extract), did not produce alterations in the protein biosynthesis (Fig. 4), indicating that the herbicide must be in contact with the cells for at least 8 h to produce any effect. We nrnole8 leucine / rng cell prot.
v . v ~
0 (8ph)
T (sph)
0 (prot)
T (prol)
Fig. 6. [14C]Leucine uptake. Incorporation of [14C]leucine in control (C(sph)) and in treated spheroplasts (T(sph)) and in proteins from control and treated groups (C(prot)) and (T(prot)); n = 3-6; *P _~ 0.0005.
78
0.2(
nmolo8 /rag ooll prot.
0.1,'
0.1(
0.0~
O.OC 0
O+P
T
T+P
Fig. 7. [3H]Thymidine uptake in synchronized bacteria. Bacterial cultures were synchronized as in Materials and methods and [3H]thymidine uptake was determined in cells transferred to control medium (C), polyamine-supplemented control medium ( C + P ) , 2,4-D-treated medium (T), polyamine -supplemented-treated medium (T + P); n = 3; *P -< 0.05.
could speculate that 2,4-D is altering the polyamine metabolism and simultaneously affecting one of the components of the protein synthesis process. Results obtained from [3H]uridine uptake experiments are shown in Fig. 5. There is a reduction in the radioactive incorporation in treated spheroplasts, but in spite of a similar trend in isolated RNA, no statistically significant differences were observed. In Fig. 6 the results of [lac]leucine uptake are shown. A diminution was observed in both treated spheroplasts and in isolated proteins, being in agreement with the in vitro (cell-free extract) protein synthesis inhibition. With respect to DNA biosynthesis, a decrease in [3H]thymidine incorporation was observed that was reversed by the addition of polyamines to the culture medium (Fig. 7). In conclusion, these results suggest that the mechanism of action of 2,4-D may be similar in both procaryotic and eucaryotic cells [1,5], acting in some manner on the metabolism of polyamines and thus altering protein and nucleic acid synthesis and cell growth. We are currently investigating the effects of 2,4-D on the metabolism of polyamines.
Acknowledgements We are grateful to Mrs. Donna Balegno for her help in preparing the manuscript and to Mr. Miguel Bueno and Mr. Walter Giordano for their technical assistance. This investigation was supported by the Consejo Nacional de Investigaciones Cientificas y T6cnicas (Argentina).
79
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3
4 5 6 7
8
9 10
11 12 13 14 15 16 17
18
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