Fd Chem. Toxic. Vol. 30, No. 8, pp. 669-672, 1992 Printed in Great Britain. All rights reserved
0278-6915/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
Research Section GENOTOXICITY AND CELL TRANSFORMATION STUDIES WITH SORBATES IN SYRIAN HAMSTER EMBRYO FIBROBLASTS D . SCHIFFMANN* a n d J. SCHLATTER~"
Institute of Toxicology, University of Wiirzburg, W-8700 Wiirzburg, Germany and tDivision of Food Science, Federal Office of Public Health, c/o Institute of Toxicology, CH 8603, Schwerzenbach, Switzerland (Accepted 26 March 1992)
Abstract--Sorbic acid, sodium sorbate and potassium sorbate were tested for their genotoxic potential in the Syrian hamster embryo (SHE) fibroblast micronucleus assay and the SHE cell transformation test in vitro. Sorbic acid and potassium sorbate showed no activity in either test system. When freshly prepared sodium sorbate solutions were used, no genotoxic or cell-transforming activity was detected. However, sodium sorbate as stored solution, which previously had been heated and sonicated to facilitate solubilization, yielded a positive response in both test systems. It is concluded that oxidation products of sodium sorbate that possess genotoxic and cell-transforming properties are formed under conditions of heating, sonication and storage.
INTRODUCTION Sorbic acid and sorbic acid salts have been used for food conservation on a wide scale (Liick, 1986). More recently, evidence has accumulated about the potential genotoxic hazards of sodium sorbate. Chromosomal as well as gene mutations have been described in mammalian cells (Hasegawa et al., 1984). In addition, clastogenicity of stored sodium sorbate has been reported in vivo (Munzner et al., 1990). In attempts to elucidate the possible mechanisms of the genotoxicity of stored sodium sorbate, an epoxide (the 4,5epoxy-2-hexenoic acid) was detected when crystalline sodium sorbate was stored or heated (personal communication, Hoechst AG, Frankfurt, Germany). In this paper the results are reported o f micronucleus formation and morphological transformation in the Syrian hamster embryo (SHE) cell system induced by heated, sonicated and stored sodium sorbate in comparison to the effects of freshly prepared solutions of sorbic acid and sorbic acid salts. MATERIALS AND METHODS
Chemicals. Sorbic acid and potassium sorbate (99% pure) were purchased from Serva, Heidelberg, Germany. F o r the preparation of sodium sorbate, 1 g sorbic acid was dissolved in 40 ml bidistilled water by adding 10 N-NaOH (at room temperature). In order
*To whom correspondence should be addressed. Abbreviations: FCS = foetal calf serum; 4-NQO = 4-nitro-
quinoline-N-oxide; PBS = Dulbeeco's phosphate buffered saline; SHE = Syrian hamster embryo.
to obtain a clear stock solution it was necessary to heat the mixture (80°C) and to sonicate it (after cooling) in a Branson sonifier, model B-12, at 80 W for 5 min. Subsequently the solution was stored at room temperature (48 hr) before use. Alternatively, sodium sorbate was prepared as above, with the omission of heating and sonication (Hoechst AG, Hoechst Germany). The solution obtained was kept under helium until use in order to avoid oxidation. Cell culture. Primary cultures of SHE fibroblasts were prepared from 13-day-old hamster foetuses and cryopreserved in liquid nitrogen as described previously (Schiffmann et al., 1988). Mass cultures were grown in IBR-modified Dulbecco's Eagle's reinforced medium (Gibco, Karlsruhe, Germany), supplemented with 15% foetal calf serum (FCS) at 37°C in an atmosphere containing 12% CO:. Ampoules of cryopreserved cells were used as stock cultures in the assay for micronucleus induction and in the transformation assay. Micronucleus assay. This test was carried out according to the protocol of Schmuck et al. (1988) with some minor modifications. Briefly, tertiary cultures of SHE cells (1 x 105 cells per 35-mm dish) were first incubated for 24 hr at 37°C. The test compounds, dissolved in Dulbecco's phosphate buffered saline (PBS), were added and after an incubation period of 5 hr the test substances were removed by changing the medium and the cells were incubated for another 18 hr. Subsequently they were fixed with methanol (10min) and stained with 10% aqueous Giemsa (Merck AG, Darmstadt, Germany). F o r each concentration of the test compounds, a total of 2000 cells
669
D. SCHIFFMANNand J, SCHLATTER
670
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30
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25
25'
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8 8 20
B
15 0 ::1 C o
_u
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10
0
Co.
a
b
e
d
u0.
a
D
e
a
Fig. 1. Induction of micronuclei in SHE ceils by (A) sodium sorbate (heated, sonicated and stored) and (B) sorbic acid at concentrations of(a) 120, (b) 300, (c) 600 and (d) 1200 #g/ml. Each data point represents the mean of three treated cultures from one experiment. The experiments were repeated four times with
consistent results. Co = control.
was scored microscopically for the presence of micronuclei. Cell transformation assay. The cell transformation assay with SHE cells was carried out as previously described in detail (Schiffmann et al., 1988). A feeder layer of 2 x 104 lethally X-irradiated SHE cells (50 Gy) was seeded in 3 ml complete medium (IBR supplemented with 20% FCS) on 60-mm petri dishes (Falcon Plastics, Oxnard, USA). The next day, 150-200 target cells suspended in 1 ml medium were added and, after 24 hr, various amounts of the test compounds dissolved in PBS. Following a 48-hr incubation period in a humidified incubator at 12% CO2 and 37°C, the medium was removed and the cells were washed with PBS and fed with fresh culture medium. Eight days later, the cells were fixed with methanol, stained with 10% aqueous Giemsa (Merck) and scored for cloning efficiency and morphological transformation according to the criteria (altered colony morphology, consisting of crisscrossing, piling up of cells and reduced cytoplasm/ nucleus ratio) described previously (DiPaolo et al., 1971; Pienta et al., 1977). RESULTS
In the first set of experiments, heated, sonicated and stored sodium sorbate and regularly dissolved sorbic acid were assayed for induction of micronuclei and morphological transformation in the SHE cell system. Sorbic acid showed neither genotoxic (Fig. 1B) nor cell-transforming activity (Table 1) whereas sodium sorbate prepared and stored under the conditions described yielded a positive response in the micronucleus test (Fig. 1A) and the cell transformation assay (Table 1). In a second set of exper-
iments, freshly prepared sodium sorbate (Hoechst), without further treatment for solubilization, and potassium sorbate, also freshly prepared, were assayed for induction of micronuclei and cell transformation. Prepared in this way, neither sodium sorbate (Fig. 2A; Table 2) nor potassium sorbate (Fig. 2B; Table 2) showed any effect in either test system. DISCUSSION
Heated, sonicated and stored sodium sorbate yielded positive results in the SHE micronucleus and morphological transformation tests. These data are in Table 1. Morphological transformation of SHE cells Transformation frequency Concentration Sodium sorbate PBS 1% (control) 4-NQO (10 -s M) Sodium sorbate (#g/ml) 120 300 600 1200 H20 (control) 4-NQO (10 -s M) Sorbic acid (gg/ml) 120 300 600 1200
Ratio
(%)
Cloning efficiency CE (%)
(heated, sonicated and stored) 0/1621 0.0 100 5/1615 0.31 99.6 1/1617 2/1600 3/1607 5/1352 Sorbic acid 0/1274 4/1157
0.06 0.13 0.19 0.37
99.8 98.7 99.1 83.4
0.0 0.38
100 95.4
0/1285 0/1259 0/1241 0/l 117
0.0 0.0 0.0 0,0
100 98.8 97.4 87.7
The transformation frequency (%) is calculated from the ratio of the number of transformed cells to the number of colonies surviving. 4-Nitroquinoline-N-oxide (4-NQO, 10-8 M) was used as a positive control. All experiments were repeated four times with consistent results. The data presented represent the results of one experiment.
Genotoxicity of sorbates
671
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9
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Co.
a
b
c
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Co.
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c
d
Fig. 2. Induction of micronuclei in SHE cells by (A) sodium sorbate (freshly prepared) and (B) potassium sorbate at concentrations of (a) 120, (b) 300, (c) 600 and (d) 1200 #g/ml. Each data point represents the mean of three treated cultures from one experiment. The experiments were repeated four times with consistent results. Co = control.
agreement with the results of Munzner et al. (1990), who described clastogenic events and formation of micronuclei in vivo. The results reported here were obtained at rather high dose ranges (~
0278-6915/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
Research Section GENOTOXICITY AND CELL TRANSFORMATION STUDIES WITH SORBATES IN SYRIAN HAMSTER EMBRYO FIBROBLASTS D . SCHIFFMANN* a n d J. SCHLATTER~"
Institute of Toxicology, University of Wiirzburg, W-8700 Wiirzburg, Germany and tDivision of Food Science, Federal Office of Public Health, c/o Institute of Toxicology, CH 8603, Schwerzenbach, Switzerland (Accepted 26 March 1992)
Abstract--Sorbic acid, sodium sorbate and potassium sorbate were tested for their genotoxic potential in the Syrian hamster embryo (SHE) fibroblast micronucleus assay and the SHE cell transformation test in vitro. Sorbic acid and potassium sorbate showed no activity in either test system. When freshly prepared sodium sorbate solutions were used, no genotoxic or cell-transforming activity was detected. However, sodium sorbate as stored solution, which previously had been heated and sonicated to facilitate solubilization, yielded a positive response in both test systems. It is concluded that oxidation products of sodium sorbate that possess genotoxic and cell-transforming properties are formed under conditions of heating, sonication and storage.
INTRODUCTION Sorbic acid and sorbic acid salts have been used for food conservation on a wide scale (Liick, 1986). More recently, evidence has accumulated about the potential genotoxic hazards of sodium sorbate. Chromosomal as well as gene mutations have been described in mammalian cells (Hasegawa et al., 1984). In addition, clastogenicity of stored sodium sorbate has been reported in vivo (Munzner et al., 1990). In attempts to elucidate the possible mechanisms of the genotoxicity of stored sodium sorbate, an epoxide (the 4,5epoxy-2-hexenoic acid) was detected when crystalline sodium sorbate was stored or heated (personal communication, Hoechst AG, Frankfurt, Germany). In this paper the results are reported o f micronucleus formation and morphological transformation in the Syrian hamster embryo (SHE) cell system induced by heated, sonicated and stored sodium sorbate in comparison to the effects of freshly prepared solutions of sorbic acid and sorbic acid salts. MATERIALS AND METHODS
Chemicals. Sorbic acid and potassium sorbate (99% pure) were purchased from Serva, Heidelberg, Germany. F o r the preparation of sodium sorbate, 1 g sorbic acid was dissolved in 40 ml bidistilled water by adding 10 N-NaOH (at room temperature). In order
*To whom correspondence should be addressed. Abbreviations: FCS = foetal calf serum; 4-NQO = 4-nitro-
quinoline-N-oxide; PBS = Dulbeeco's phosphate buffered saline; SHE = Syrian hamster embryo.
to obtain a clear stock solution it was necessary to heat the mixture (80°C) and to sonicate it (after cooling) in a Branson sonifier, model B-12, at 80 W for 5 min. Subsequently the solution was stored at room temperature (48 hr) before use. Alternatively, sodium sorbate was prepared as above, with the omission of heating and sonication (Hoechst AG, Hoechst Germany). The solution obtained was kept under helium until use in order to avoid oxidation. Cell culture. Primary cultures of SHE fibroblasts were prepared from 13-day-old hamster foetuses and cryopreserved in liquid nitrogen as described previously (Schiffmann et al., 1988). Mass cultures were grown in IBR-modified Dulbecco's Eagle's reinforced medium (Gibco, Karlsruhe, Germany), supplemented with 15% foetal calf serum (FCS) at 37°C in an atmosphere containing 12% CO:. Ampoules of cryopreserved cells were used as stock cultures in the assay for micronucleus induction and in the transformation assay. Micronucleus assay. This test was carried out according to the protocol of Schmuck et al. (1988) with some minor modifications. Briefly, tertiary cultures of SHE cells (1 x 105 cells per 35-mm dish) were first incubated for 24 hr at 37°C. The test compounds, dissolved in Dulbecco's phosphate buffered saline (PBS), were added and after an incubation period of 5 hr the test substances were removed by changing the medium and the cells were incubated for another 18 hr. Subsequently they were fixed with methanol (10min) and stained with 10% aqueous Giemsa (Merck AG, Darmstadt, Germany). F o r each concentration of the test compounds, a total of 2000 cells
669
D. SCHIFFMANNand J, SCHLATTER
670
O
30
3O
25
25'
2O
8 8 20
B
15 0 ::1 C o
_u
:E
10
0
Co.
a
b
e
d
u0.
a
D
e
a
Fig. 1. Induction of micronuclei in SHE ceils by (A) sodium sorbate (heated, sonicated and stored) and (B) sorbic acid at concentrations of(a) 120, (b) 300, (c) 600 and (d) 1200 #g/ml. Each data point represents the mean of three treated cultures from one experiment. The experiments were repeated four times with
consistent results. Co = control.
was scored microscopically for the presence of micronuclei. Cell transformation assay. The cell transformation assay with SHE cells was carried out as previously described in detail (Schiffmann et al., 1988). A feeder layer of 2 x 104 lethally X-irradiated SHE cells (50 Gy) was seeded in 3 ml complete medium (IBR supplemented with 20% FCS) on 60-mm petri dishes (Falcon Plastics, Oxnard, USA). The next day, 150-200 target cells suspended in 1 ml medium were added and, after 24 hr, various amounts of the test compounds dissolved in PBS. Following a 48-hr incubation period in a humidified incubator at 12% CO2 and 37°C, the medium was removed and the cells were washed with PBS and fed with fresh culture medium. Eight days later, the cells were fixed with methanol, stained with 10% aqueous Giemsa (Merck) and scored for cloning efficiency and morphological transformation according to the criteria (altered colony morphology, consisting of crisscrossing, piling up of cells and reduced cytoplasm/ nucleus ratio) described previously (DiPaolo et al., 1971; Pienta et al., 1977). RESULTS
In the first set of experiments, heated, sonicated and stored sodium sorbate and regularly dissolved sorbic acid were assayed for induction of micronuclei and morphological transformation in the SHE cell system. Sorbic acid showed neither genotoxic (Fig. 1B) nor cell-transforming activity (Table 1) whereas sodium sorbate prepared and stored under the conditions described yielded a positive response in the micronucleus test (Fig. 1A) and the cell transformation assay (Table 1). In a second set of exper-
iments, freshly prepared sodium sorbate (Hoechst), without further treatment for solubilization, and potassium sorbate, also freshly prepared, were assayed for induction of micronuclei and cell transformation. Prepared in this way, neither sodium sorbate (Fig. 2A; Table 2) nor potassium sorbate (Fig. 2B; Table 2) showed any effect in either test system. DISCUSSION
Heated, sonicated and stored sodium sorbate yielded positive results in the SHE micronucleus and morphological transformation tests. These data are in Table 1. Morphological transformation of SHE cells Transformation frequency Concentration Sodium sorbate PBS 1% (control) 4-NQO (10 -s M) Sodium sorbate (#g/ml) 120 300 600 1200 H20 (control) 4-NQO (10 -s M) Sorbic acid (gg/ml) 120 300 600 1200
Ratio
(%)
Cloning efficiency CE (%)
(heated, sonicated and stored) 0/1621 0.0 100 5/1615 0.31 99.6 1/1617 2/1600 3/1607 5/1352 Sorbic acid 0/1274 4/1157
0.06 0.13 0.19 0.37
99.8 98.7 99.1 83.4
0.0 0.38
100 95.4
0/1285 0/1259 0/1241 0/l 117
0.0 0.0 0.0 0,0
100 98.8 97.4 87.7
The transformation frequency (%) is calculated from the ratio of the number of transformed cells to the number of colonies surviving. 4-Nitroquinoline-N-oxide (4-NQO, 10-8 M) was used as a positive control. All experiments were repeated four times with consistent results. The data presented represent the results of one experiment.
Genotoxicity of sorbates
671
30B
25in
g
l
~
15
-! C
..i
r,
15"
9
+o
U
10
IE 5
0
5
Co.
a
b
c
d
0
Co.
ii
b
c
d
Fig. 2. Induction of micronuclei in SHE cells by (A) sodium sorbate (freshly prepared) and (B) potassium sorbate at concentrations of (a) 120, (b) 300, (c) 600 and (d) 1200 #g/ml. Each data point represents the mean of three treated cultures from one experiment. The experiments were repeated four times with consistent results. Co = control.
agreement with the results of Munzner et al. (1990), who described clastogenic events and formation of micronuclei in vivo. The results reported here were obtained at rather high dose ranges (~
