Chem. -Biol. Zntemctions, 24 (1979) 137-l 51 o Elsevier/North-Holland Scientific PutiIishers Ltd.
DIFFERENTIAL CYTOTOXIC ACTIVITY OF POTASSIUM DICHROMATE ON NUCLEOSIDE UPTAKE IN BHK FIBROBLASTS
137
*
V. BIANCHI, A.G. LEVIS and D. SAGGIORO Institute of Animal Biology,
Via Loredan 10, University of Padova, 35100 Padova (Italy)
(Received March 21st, 3.978) (Revision received August 5th, 1978) (Accepted August 26th. 1978)
SUMMARY
In cultures of hamster fibroblasts (BHK cell line) treated with potassium dichromate (K&qO,) nucleic acid and protein syntheses are differentially inhibited, and nucleoside uptake into the intracellular pool is characterized by a sthnulation phase followed by an inhibition phase. Different patterrs are observed for the uptake of each ribo- and deoxyribonucleoside, pyrimidine nucleoside (particularly deoxycytidine) uptake reaching the highest stimulation level. Kinetics of thymidine and deoxycytidine initial uptake at different exogenous nucleoside concentrations show that K&r207 affects both simple and facilitated diffusion of nucleosides. The time course of thymidine and deoxycytidine pool saturation suggests however that the effects of KZCrlO, on plasma *membrane permeability are partially counterbalanced by modifications of pool size deriving from the concomitant alteration of steps of nucleoside metabolism separate from nucleoside uptake.
INTRODUCTION
Studies on the cytogenetic effects of environmental contaminants contaming metal compounds have been recently stimulated by observations suggesting a relation between their carcinogenic and mutagenic actinn [ 1,2]. As for chromium compounds, lung tumor incidence is increased up to 20 times in workers of chromate-producing plants [3-5-j ; moreover several chromates, * Supported by a grant from the National Research Council of Italy (Consiglio Nazionale delle Ricerche, Programma Finalizzato: “Promozione della Qualita de11’Ambiente”). Abbreviations: t3H]dAdo, [6-3H]deoxyadenosine; t3H]Ado, [6-3H]riboadenosine; BSS, Hank’s balanced salt solution; [ 3H]dCyd, [ 5-3 Hldeoxycytidine; [ 3H]Cyd, [ 5-3H]ribocytidine; [3H]dGuo, [ 8-3H]deoxyguanosine; [ 3H]Guo, [ 8-3H]riboguanosine; [ jHlLeu, L-[4,5-3H]1eucine; MEM, Eagle’s minimal essential medium supplemented with 10% calf serum; PCA, perchloric acid; [jH]dThd, [ 6-3H]thymidine; [3H]Wrd, [ 5-3Hluridine.
138 namely Ca-, Zn-, Sr- and Pb-chromate, are also capable of inducing tumors in experimental animals [ 5-71. Chromates and dichromates have been shown lid induce point mutations in bacteria [1,8-lo] and yeasts [ll], infidelity of DNA replication in vitro [ 21, chromosome aberrations in mammalian cells [12,13], and cell transformation in vitro [13,14]. Citotoxic, mutagenic and carcinogenic effects of chromium have been attributed to the oxidizing action of hexavalent chromium [8-10,14,X], namely to the formation of aldehydes and epoxyaldehydes via ,the oxidation of cell metabolites [16]. However, some trivalent chromium compounds which are not mutagenic in bacteria [ 1,8-lo] are carcinogenic in test animals [ 57,171, induce chromosome aberrations in ‘VicQ faba [18] and cultured mouse cells [19] and interact with nucleic acids both in purified form in vitro [20-223 and in cultured hamster cells [23], modifying their physico-chemical and biological properties. Furthermore, only the trivalent oxidation state is present inside the cell, even after treatment with hexavalent chromium compounds [X,24]. Therefore, the actual chromium oxidation state responsible for the above biological effects, especially for the ~~ractions with the genetic material, is still uncertain [2,15,25]. By treating cultures of a hamster fibroblast line (BHK) with potassium dichromate (K&r&), a strong oxidizing agent,, it has been reported that nucleic acid and protein syntheses are differentially affected and that aminoacid uptake into the intracellular pool is always inhibited, whereas thymidine and uridine uptake can be markedly stimulated [2f?]. The latter effect is related to the action of hexavalent chromium on the plasma membrane as it does not take place after treatment with trivalent chromium [ 271. In order better to understand the mechanisms of nucleoside uptake stimulation the cytotoxic activity of K2CrZ0, on other ribo- and deoxyribonucleoside uptake and on thymidine and deoxycytidine simple and facilitated diffusion was studied. MATERIALS
AND METHODS
Cells
Cultures of the established BHK21 hamster fibroblast line, clone 12, are routinely grown in our laboratory at 37°C as monolayers, in Eagle’s minimal medium supplemented with 10% calf serum (MEM). Potassium dichromate (K2Cr207, Ma~inc~~t Inc., St. Louis, MO.U.S.A.) was solubilized in steriIe twiced~~led water at concent~tions of 10” to 10e3 M imm~?dia~ly before use and, afterwards, it was diluted 100 times in prewarmed MEM or BSS to final concent~tions of 10m3 to 10q5 M. Experimental treatments were carried out in MEM or BSS on exponential cultures at 37°C in a climatized room; prewarmed solutions were used to avoid any thermic shock. After different lengths of treatment, the cultures were rinsed twice with BSS, and the medium containing K&&O, was replaced with
139
normal growth medium. At different intervals after exposure to KZCr2G,, the cultures were incubated with tritiated nucleic acid and protein precursors (The Radiochemi~~ Centre, Amen&am, England). [3H]dTd (2 Ci~mmol), f3H]dAdo (14.5 Cijmmol, i3H] dCyd (2 Ci/mmol) j3H]dGuo (3.1 Cifmmol, [jH]Urd (2 Cijmmol), t3H]Ado (6.4 Cilmmol), i3H]Cyd (2.8 Cijmmol), 13H] Guo (7.7 Ci/mmol) and [3H] Leu (0.5-l Ci/mmol) are used at the con centration of 1 &i/ml, unless otherwise specified. At the end of the incubation, tritiated precursor uptake was blocked by rinsing the cultures twice with ice-cold BSS. Extraction procedures and analytical methods From labelled cultures, nucleotides of the intracellular pool, RNA, DNA and proteins were differentially extracted with PCA and KOH and measured by UV absorption as detailed elsewhere [26,27]. Radioa~ti~ty counting of liquid samples (0.5 ml) of the different fractions was carried out by a Packard Tri-Carb 2425 scintillation counter, with 10 ml Bray’s solution. The radioactivity in the different fractions of a culture were normalized by dividing them by the DNA concentration of the same culture, giving values referred to as normalized (radio) activities. In the treated cultures, normalized activities were expressed as percentages of control values. Since KZCr#& affects the uptake of labelled precursors into the intracellular pool, changing their relative concentrations, the original percent values of nucleic acid and protein normalized activities were divided by the corresponding percentage of normalized activities of intracellular precursors. Such values are assumed to express the actual rates of precursor incorporation into macromolecular compounds and represent the net levels of RNA, DNA and protein synthesis after treatment with K,CrzO, [26,27]. RESULTS
Precursor uptake and macromolecule synthesis in BHK cells treated for l-10 h with lo-$ to 10v3 M KZCr207 in MEM or BSS are shown in Fig. 1. For the specific (radio)activities of nucleic acid and protein precursors in the intracellular pool, the following observations can be made: (1) treatment with lows and 10m4 M KzCrzO, in MEM sharply stimulates uridine uptake (Fig. lB), whereas it immediately inhibits leucine uptake (Fig. 1C). Thymidine uptake is stimulated over the whole observation period by treatment with 10e5 and 10m4 M KzCrzO, (Fig. lA), whereas it is stimulated only by treatments shorter than 30 min when 10e3 M K&&O, is used 2261; (2) the kinetics of uridine and thymidine uptake in MEM are characterized by an initial period where the uptake is more stimulated the longer the exposure to KZCr20,, followed by a period of pro~essively reduced stimuiation (Fig. IA: 10e4 M K2Cr20,; Fig. 1B: 3.0d4 and 10e3 M K2Cr20,). With 10e3 M K2Cr207, the stimulation of thymidine uptake diminishes very early and switches to a marked inhibition when treatment is prolonged over 1 h [26] ; (3) the onset of the inhibition phase of nucleoside uptake is particularly evident when the
(SlOJ.NlO3
40
141 reagent with lo-’ to W3 M K&r& is made in Bs,I fFig. lA, 3.31,and a&3 feucine uptake is more inhibited in BSS than in MEM (Fig. 1C). Data in Fig. 123-F show that KzCr207 a&o exerts a differential ~bibit~~ action on DNA, RNA and protein synthesis: the primary effect of dichramate hes in the blockage of DNA replication, whereas RNA t~sc~pti#n and protein sy~~~s~s are secondarily i~ibi~~ ~xpe~rnen~ have been also carried out where BHK cultures were repeatedly treated with 1W4 M K&rzO, for 1 h in MEM, at 24 h interns (Fig. 2). The effect on ~ymid~e uptake is net ex~~ish~ within 24 h, but the maximum st~u~~un achieved after each new treatment remainsc~n~t~t; the increment in the uptake st~rn~a~u~ is therefore ~~~~~ve~y reduced. As for the libation uf EMA lynxes, it ceases by S h after the fZrsttreatment, whereas it is mu& more marked after the 2md tr~tme~t and recovery shifts towards the 24 h. Following the 3rd treatment, the ~n~b~ti~n lasts throughout the peziod of crbservationand no significant recovery is detected.
tiQQfS
aftlN tf&lS@Qt
In order to verify the stability of the cytotoxic activity of K2Cr20, solutions, BHK cultures were treated with K2Crz0, preincubated for different lengths of time at 37°C in BSS or MEM (Fig. 3). The cytotoxic activity was similar regardless of the preincubation time even in MEM, which contains several compounds (e.g. aminoacids, vitamins, serum proteins and nucleotides) capable of reducing Cf”. As previously observed (Fig. l), the stimulalOO--
D
K
60-.
’
0 r-g-
0
i
_~__~~~~O
l_-l 0
20
--a--
0
E
2
8 II
24
36
II
Preincubation (hours)
Fig. 3. Stability of KzCrz07 action on precursor uptake and macromolecular syntheses in BHK cultures. Normalized activities of [3H]dThd (A), r3H]Urd (B) and [3H]Leu (C) in the intracellular pool and normalized rates of DNA (D), RNA (E) and protein (F) syn-
theses were detzinmined in BHK cultures at the end of 1 h treatment with 10d4 M K$2rt07 in MEM (0) or BSS (0).The solutions of K&r207 in MEM and BSS were preincubated at 37OC for different times up to 48 h, before use.
143 tion of nucleoside uptake is less pronounced and the inhibition of leucine uptake and of macromolecular syntheses is more marked when treatment is made in BSS rather than iv MEM. Cfl? reduction in MEM and BSS solutions was determined by the colored reaction complex with diphenylcarbazide [27]. It has been observed that, following the initial reduction of a fractioia of CP to Cr3+occurring at the solubilizaticm of K&O, in MEM [ 27 1, the CP level remains constant up to 48 h in BSS as well as in MEM when such solutions are incubated for different times at 37°C in the absence of cells (data not shown). ..
K
IO0
i
ZOO
1
o_.-
-0---------
0
,(, y
DIFFERENTIAL CYTOTOXIC ACTIVITY OF POTASSIUM DICHROMATE ON NUCLEOSIDE UPTAKE IN BHK FIBROBLASTS
137
*
V. BIANCHI, A.G. LEVIS and D. SAGGIORO Institute of Animal Biology,
Via Loredan 10, University of Padova, 35100 Padova (Italy)
(Received March 21st, 3.978) (Revision received August 5th, 1978) (Accepted August 26th. 1978)
SUMMARY
In cultures of hamster fibroblasts (BHK cell line) treated with potassium dichromate (K&qO,) nucleic acid and protein syntheses are differentially inhibited, and nucleoside uptake into the intracellular pool is characterized by a sthnulation phase followed by an inhibition phase. Different patterrs are observed for the uptake of each ribo- and deoxyribonucleoside, pyrimidine nucleoside (particularly deoxycytidine) uptake reaching the highest stimulation level. Kinetics of thymidine and deoxycytidine initial uptake at different exogenous nucleoside concentrations show that K&r207 affects both simple and facilitated diffusion of nucleosides. The time course of thymidine and deoxycytidine pool saturation suggests however that the effects of KZCrlO, on plasma *membrane permeability are partially counterbalanced by modifications of pool size deriving from the concomitant alteration of steps of nucleoside metabolism separate from nucleoside uptake.
INTRODUCTION
Studies on the cytogenetic effects of environmental contaminants contaming metal compounds have been recently stimulated by observations suggesting a relation between their carcinogenic and mutagenic actinn [ 1,2]. As for chromium compounds, lung tumor incidence is increased up to 20 times in workers of chromate-producing plants [3-5-j ; moreover several chromates, * Supported by a grant from the National Research Council of Italy (Consiglio Nazionale delle Ricerche, Programma Finalizzato: “Promozione della Qualita de11’Ambiente”). Abbreviations: t3H]dAdo, [6-3H]deoxyadenosine; t3H]Ado, [6-3H]riboadenosine; BSS, Hank’s balanced salt solution; [ 3H]dCyd, [ 5-3 Hldeoxycytidine; [ 3H]Cyd, [ 5-3H]ribocytidine; [3H]dGuo, [ 8-3H]deoxyguanosine; [ 3H]Guo, [ 8-3H]riboguanosine; [ jHlLeu, L-[4,5-3H]1eucine; MEM, Eagle’s minimal essential medium supplemented with 10% calf serum; PCA, perchloric acid; [jH]dThd, [ 6-3H]thymidine; [3H]Wrd, [ 5-3Hluridine.
138 namely Ca-, Zn-, Sr- and Pb-chromate, are also capable of inducing tumors in experimental animals [ 5-71. Chromates and dichromates have been shown lid induce point mutations in bacteria [1,8-lo] and yeasts [ll], infidelity of DNA replication in vitro [ 21, chromosome aberrations in mammalian cells [12,13], and cell transformation in vitro [13,14]. Citotoxic, mutagenic and carcinogenic effects of chromium have been attributed to the oxidizing action of hexavalent chromium [8-10,14,X], namely to the formation of aldehydes and epoxyaldehydes via ,the oxidation of cell metabolites [16]. However, some trivalent chromium compounds which are not mutagenic in bacteria [ 1,8-lo] are carcinogenic in test animals [ 57,171, induce chromosome aberrations in ‘VicQ faba [18] and cultured mouse cells [19] and interact with nucleic acids both in purified form in vitro [20-223 and in cultured hamster cells [23], modifying their physico-chemical and biological properties. Furthermore, only the trivalent oxidation state is present inside the cell, even after treatment with hexavalent chromium compounds [X,24]. Therefore, the actual chromium oxidation state responsible for the above biological effects, especially for the ~~ractions with the genetic material, is still uncertain [2,15,25]. By treating cultures of a hamster fibroblast line (BHK) with potassium dichromate (K&r&), a strong oxidizing agent,, it has been reported that nucleic acid and protein syntheses are differentially affected and that aminoacid uptake into the intracellular pool is always inhibited, whereas thymidine and uridine uptake can be markedly stimulated [2f?]. The latter effect is related to the action of hexavalent chromium on the plasma membrane as it does not take place after treatment with trivalent chromium [ 271. In order better to understand the mechanisms of nucleoside uptake stimulation the cytotoxic activity of K2CrZ0, on other ribo- and deoxyribonucleoside uptake and on thymidine and deoxycytidine simple and facilitated diffusion was studied. MATERIALS
AND METHODS
Cells
Cultures of the established BHK21 hamster fibroblast line, clone 12, are routinely grown in our laboratory at 37°C as monolayers, in Eagle’s minimal medium supplemented with 10% calf serum (MEM). Potassium dichromate (K2Cr207, Ma~inc~~t Inc., St. Louis, MO.U.S.A.) was solubilized in steriIe twiced~~led water at concent~tions of 10” to 10e3 M imm~?dia~ly before use and, afterwards, it was diluted 100 times in prewarmed MEM or BSS to final concent~tions of 10m3 to 10q5 M. Experimental treatments were carried out in MEM or BSS on exponential cultures at 37°C in a climatized room; prewarmed solutions were used to avoid any thermic shock. After different lengths of treatment, the cultures were rinsed twice with BSS, and the medium containing K&&O, was replaced with
139
normal growth medium. At different intervals after exposure to KZCr2G,, the cultures were incubated with tritiated nucleic acid and protein precursors (The Radiochemi~~ Centre, Amen&am, England). [3H]dTd (2 Ci~mmol), f3H]dAdo (14.5 Cijmmol, i3H] dCyd (2 Ci/mmol) j3H]dGuo (3.1 Cifmmol, [jH]Urd (2 Cijmmol), t3H]Ado (6.4 Cilmmol), i3H]Cyd (2.8 Cijmmol), 13H] Guo (7.7 Ci/mmol) and [3H] Leu (0.5-l Ci/mmol) are used at the con centration of 1 &i/ml, unless otherwise specified. At the end of the incubation, tritiated precursor uptake was blocked by rinsing the cultures twice with ice-cold BSS. Extraction procedures and analytical methods From labelled cultures, nucleotides of the intracellular pool, RNA, DNA and proteins were differentially extracted with PCA and KOH and measured by UV absorption as detailed elsewhere [26,27]. Radioa~ti~ty counting of liquid samples (0.5 ml) of the different fractions was carried out by a Packard Tri-Carb 2425 scintillation counter, with 10 ml Bray’s solution. The radioactivity in the different fractions of a culture were normalized by dividing them by the DNA concentration of the same culture, giving values referred to as normalized (radio) activities. In the treated cultures, normalized activities were expressed as percentages of control values. Since KZCr#& affects the uptake of labelled precursors into the intracellular pool, changing their relative concentrations, the original percent values of nucleic acid and protein normalized activities were divided by the corresponding percentage of normalized activities of intracellular precursors. Such values are assumed to express the actual rates of precursor incorporation into macromolecular compounds and represent the net levels of RNA, DNA and protein synthesis after treatment with K,CrzO, [26,27]. RESULTS
Precursor uptake and macromolecule synthesis in BHK cells treated for l-10 h with lo-$ to 10v3 M KZCr207 in MEM or BSS are shown in Fig. 1. For the specific (radio)activities of nucleic acid and protein precursors in the intracellular pool, the following observations can be made: (1) treatment with lows and 10m4 M KzCrzO, in MEM sharply stimulates uridine uptake (Fig. lB), whereas it immediately inhibits leucine uptake (Fig. 1C). Thymidine uptake is stimulated over the whole observation period by treatment with 10e5 and 10m4 M KzCrzO, (Fig. lA), whereas it is stimulated only by treatments shorter than 30 min when 10e3 M K&&O, is used 2261; (2) the kinetics of uridine and thymidine uptake in MEM are characterized by an initial period where the uptake is more stimulated the longer the exposure to KZCr20,, followed by a period of pro~essively reduced stimuiation (Fig. IA: 10e4 M K2Cr20,; Fig. 1B: 3.0d4 and 10e3 M K2Cr20,). With 10e3 M K2Cr207, the stimulation of thymidine uptake diminishes very early and switches to a marked inhibition when treatment is prolonged over 1 h [26] ; (3) the onset of the inhibition phase of nucleoside uptake is particularly evident when the
(SlOJ.NlO3
40
141 reagent with lo-’ to W3 M K&r& is made in Bs,I fFig. lA, 3.31,and a&3 feucine uptake is more inhibited in BSS than in MEM (Fig. 1C). Data in Fig. 123-F show that KzCr207 a&o exerts a differential ~bibit~~ action on DNA, RNA and protein synthesis: the primary effect of dichramate hes in the blockage of DNA replication, whereas RNA t~sc~pti#n and protein sy~~~s~s are secondarily i~ibi~~ ~xpe~rnen~ have been also carried out where BHK cultures were repeatedly treated with 1W4 M K&rzO, for 1 h in MEM, at 24 h interns (Fig. 2). The effect on ~ymid~e uptake is net ex~~ish~ within 24 h, but the maximum st~u~~un achieved after each new treatment remainsc~n~t~t; the increment in the uptake st~rn~a~u~ is therefore ~~~~~ve~y reduced. As for the libation uf EMA lynxes, it ceases by S h after the fZrsttreatment, whereas it is mu& more marked after the 2md tr~tme~t and recovery shifts towards the 24 h. Following the 3rd treatment, the ~n~b~ti~n lasts throughout the peziod of crbservationand no significant recovery is detected.
tiQQfS
aftlN tf&lS@Qt
In order to verify the stability of the cytotoxic activity of K2Cr20, solutions, BHK cultures were treated with K2Crz0, preincubated for different lengths of time at 37°C in BSS or MEM (Fig. 3). The cytotoxic activity was similar regardless of the preincubation time even in MEM, which contains several compounds (e.g. aminoacids, vitamins, serum proteins and nucleotides) capable of reducing Cf”. As previously observed (Fig. l), the stimulalOO--
D
K
60-.
’
0 r-g-
0
i
_~__~~~~O
l_-l 0
20
--a--
0
E
2
8 II
24
36
II
Preincubation (hours)
Fig. 3. Stability of KzCrz07 action on precursor uptake and macromolecular syntheses in BHK cultures. Normalized activities of [3H]dThd (A), r3H]Urd (B) and [3H]Leu (C) in the intracellular pool and normalized rates of DNA (D), RNA (E) and protein (F) syn-
theses were detzinmined in BHK cultures at the end of 1 h treatment with 10d4 M K$2rt07 in MEM (0) or BSS (0).The solutions of K&r207 in MEM and BSS were preincubated at 37OC for different times up to 48 h, before use.
143 tion of nucleoside uptake is less pronounced and the inhibition of leucine uptake and of macromolecular syntheses is more marked when treatment is made in BSS rather than iv MEM. Cfl? reduction in MEM and BSS solutions was determined by the colored reaction complex with diphenylcarbazide [27]. It has been observed that, following the initial reduction of a fractioia of CP to Cr3+occurring at the solubilizaticm of K&O, in MEM [ 27 1, the CP level remains constant up to 48 h in BSS as well as in MEM when such solutions are incubated for different times at 37°C in the absence of cells (data not shown). ..
K
IO0
i
ZOO
1
o_.-
-0---------
0
,(, y
