Cancer Letters, 62 (1992) 159 - 165 Elsevier Scientific Publishers Ireland Ltd.
159
Morphological transformation mezerein A.S. Tu*, R.W. Tennant
of Syrian hamster embryo cells by
and J.W. Spalding
“Experimental Carcinogenesis and Mutagenesis Branch, National Toxicology mental Health Sciences, Research Triangle Park, North Carolina (U.S.A.)
Program,
National
Institute of Enuiron-
(Received 6 October 1991) (Revision received 8 November 1991) (Accepted 11 November 1991)
Summary Mezerein
(MEZ)
weak complete
has been
tumor promoter
described
as a but an effec-
tive stage !I promoter in the mouse skin initiation-promotion tumor model. In this study MEZ produced a strong transformation response when tested under code in the Syrian hamster embryo (SHE) clonal morphological transformation assay. Using a standard 7-day exposure protocol designed to detect complete carcinogens, MEZ was actioe at non-toxic concentrations, producing a linear response between 0.3- 10 ng/ml in a log-log plot of transformation activity uersus concentration. These concentrations were in the same range as those which had been shown to elicit promotion activity in several in uitro cell culture systems. Our data suggest that the SHE assay has the ability to detect some tumor promoters under the same conditions used to test for complete carcinogens. The possibility that MEZ may possess in vivo carcinogenic activity cannot be excluded.
Correspondence
genesis
to: J.W. Spalding, Experimental Carcinoand Mutagenesis Branch, NIEHS, P.O. Box 12233,
Research Triangle Park, NC 27709, U.S.A. ‘Present address: A.S. Tu, 112-D Anita Road, Burlingame, 94010, U.S.A.
0304-3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific in Ireland
CA
Publishers
Keywords: mezerein; SHE clonal transformation; tumor promoter Introduction The induction of morphological transformation in the Syrian hamster embryo (SHE) cells by chemical carcinogens was first reported by Berwald and Sachs [2,3]. Over the last two decades, a number of independent and collaborative efforts have been made to validate the SHE morphological transformation assay system for the testing of chemicals with carcinogenic potential [1,7,11,15,16,20,25]. In addition to its utility for detecting carcinogens, the SHE transformation system has been reported to detect tumor promoters [17,18]. Using a two-stage chemical exposure procedure mimicking that of the mouse skin epidermal papilloma tumor promotion model, SHE cells which were ‘initiated’ with a low concentration of carcinogen such as benzo[a]pyrene (BaP), then ‘promoted’ with a tumor promoter such as 12-O-tetradecanoylphorbol- 13-acetate (TPA) , were shown to have an enhanced transformation response above that of exposure to either the initiator or the promoter alone [19]. As part of a continuing program to evaluate the SHE transformation assay system for the Ireland Ltd
160
screening and characterizing of chemical carcinogens, we tested a series of chemicals under code in the SHE morphological transformation assay using a standard 7-day continuous exThis report presents the posure protocol. results of one of the chemicals, mezerein (MEZ), which produced a strong transformation response under the conditions of the assay. MEZ, a diterpene structurally related to the phorbol esters, is classified as a stage II tumor promoter in the mouse skin epidermal papilloma system [ZZ]. The activity of MEZ is discussed relative to the context in which it was evaluated in this study. Materials
and methods
Chemicals The positive control benzo[a]pyrene (BaP), and mezerein (MEZ), supplied as a coded chemical, were provided by the National Toxicology Program Chemical Repository (Radian Corporation, Austin, TX). The purity of the compounds were 98% and 99% for BaP and MEZ, respectively. The chemicals were stored desiccated in a - 20°C freezer. The solvent for BaP was dimethylsulfoxide (DMSO, spectrograde, Fisher Scientific). A 0.5% w/v pluronic acid F68 (Fluka Corporation, NY) in culture medium was used to dissolve MEZ. The final concentrations of DMSO and F68 in the test medium were 0.25% for both solvents. Cell culture
SHE cells were prepared from Syrian hamster embryos at days 12 - 14 of gestation and cryopreserved as described previously [24]. The clonal assay procedures for measuring cytotoxicity and transformation have also been described [24,25], with the exception that a reduced bicarbonate concentration was used in the culture medium. This medium, which had a pH of 6.7, was demonstrated by LeBoeuf and Kerchaert [ 13,141 to be optimal for the clonal growth and transformation of SHE cells.
SHE
clonal
cytotoxicity
and
transformation
assay
The procedures for conducting the cytotoxicity and transformation assays were identical with the exception that 5 dishes per test condition were used for the former and 15 dishes were used for the latter assay. Cryopreserved cells were thawed and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with low phenol red (5 mg/l) and reduced sodium bicarbonate (0.75 g/l) supplemented with 10% prescreened fetal bovine serum (FBS). The SHE cells were collected after 4 - 5 days, irradiated (5000 rad) and plated as feeder cells at 4 x lo4 cells/60 mm dish in 2 ml of medium containing 20% FBS. On the same day that the feeder cells were plated, another ampule of cryopreserved cells was thawed and cultured for use as target cells. The target cells were plated the following day onto dishes containing the feeder cells in a 2-ml aliquot at 200 cells/60 mm dish. Test chemicals were added the day after at twice the final concentration in a volume of 4 ml. The dishes were incubated undisturbed in a humidified 10% CO2 incubator. At the end of the 7-day exposure period, the dishes were rinsed in warmed (37OC) phosphate-buffered saline, fixed in methanol and stained with 10% Giemsa. To determine the cloning efficiency of SHE cells under the different test conditions, colonies from 5 representative dishes of the 15-dish sets were counted to calculate the mean colonies per dish. All the dishes were examined under a dissecting microscope and the colonies were classified as normal, morphologically altered (MA) or transformed (T), based on criteria previously established [25]. The MA colonies were recorded but not included in the calculation of transformation freas percent quency that was expressed transformation. Results The
cytotoxic
effect
of coded
chemicals
161
on the clonal growth of SHE cells was screened up to a concentration of 1 mg/ml. MEZ exhibited a shallow cytotoxicity profile over a four-log concentration range from 1 ng/ml10 pg/ml (Fig. 1). The LCsO (concentration which reduced cloning efficiency by 50%) for MEZ was estimated to be 0.15 pg/ml. The maximal toxic effect of MEZ was achieved at 10 pg/ml, resulting in a relative surviving fraction of approximately 30% for the SHE cells. There was no further reduction in cloning efficiency with higher concentrations of MEZ. This indicates that the solubility limit of MEZ in the test medium may have been reached. The clonal transformation assay data (Table I) showed that MEZ consistently induced a dose-dependent transformation response in three independent assays. The transformation of the negative controls ranged from no transformant scored in two of the assays, to a frequency of 0.30% and 0.55% for F68 and DMSO, respectively, in assay 2. These ranges are within the values we obtained for spon-
taneous transformation frequency of this assay performed in pH 6.7 medium. The level of induced transformation frequency varied substantially between assays. At 10 ng/ml of MEZ, the induced transformation activity in assay 1, 2 and 3 were 1.33%, 7.72% and 17.39% , respectively. Some variation of transformation activity between repeat experiments is not unusual for this assay which is based on a morphological endpoint and is sensitive to subtle differences in experimental conditions. In this particular study, however, a major source of variability is likely to be due to the limited solubility of MEZ. For example, when a lower concentration range was used in the third assay, the data showed that the toxicity level induced by 10 ng/ml of MEZ was similar to that induced by 100 ng/ml in assay 2; resulting in surviving fractions of 0.51 and 0.48, respectively. This suggests that the actual soluble concentrations in assay 1 and 2 might have been much lower than the calculated concentrations. In all assays performed, however, MEZ
+
0.12 lo+
lo-*
10-'
loo
MEZ Concentration
10'
102
lo3
b..LQ/ml)
Fig. 1. Cytotoxicity of MEZ on clonal growth of SHE cells. The SHE cells were exposed to various concentrations of MEZ for 7 days. The relative surviving fraction was determined from the mean number of colonies of 5 treated cultures divided by the mean number of colonies in 5 solvent control-treated cultures, the solvent control having a surviving fraction of 1.00. Each symbol represents results of an independent experiment.
162 Table 1. Morphological Assay
1
2
3
Chemical
transformation
Concentration
of Syrian hamster
embryo
Cytotoxicity
cells by MEZ. Transformation No. T
No. MA
% T
888 855 837 825 768 531
0 1 3 11 22 21
1 2 2 11 11 12
159
Morphological transformation mezerein A.S. Tu*, R.W. Tennant
of Syrian hamster embryo cells by
and J.W. Spalding
“Experimental Carcinogenesis and Mutagenesis Branch, National Toxicology mental Health Sciences, Research Triangle Park, North Carolina (U.S.A.)
Program,
National
Institute of Enuiron-
(Received 6 October 1991) (Revision received 8 November 1991) (Accepted 11 November 1991)
Summary Mezerein
(MEZ)
weak complete
has been
tumor promoter
described
as a but an effec-
tive stage !I promoter in the mouse skin initiation-promotion tumor model. In this study MEZ produced a strong transformation response when tested under code in the Syrian hamster embryo (SHE) clonal morphological transformation assay. Using a standard 7-day exposure protocol designed to detect complete carcinogens, MEZ was actioe at non-toxic concentrations, producing a linear response between 0.3- 10 ng/ml in a log-log plot of transformation activity uersus concentration. These concentrations were in the same range as those which had been shown to elicit promotion activity in several in uitro cell culture systems. Our data suggest that the SHE assay has the ability to detect some tumor promoters under the same conditions used to test for complete carcinogens. The possibility that MEZ may possess in vivo carcinogenic activity cannot be excluded.
Correspondence
genesis
to: J.W. Spalding, Experimental Carcinoand Mutagenesis Branch, NIEHS, P.O. Box 12233,
Research Triangle Park, NC 27709, U.S.A. ‘Present address: A.S. Tu, 112-D Anita Road, Burlingame, 94010, U.S.A.
0304-3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific in Ireland
CA
Publishers
Keywords: mezerein; SHE clonal transformation; tumor promoter Introduction The induction of morphological transformation in the Syrian hamster embryo (SHE) cells by chemical carcinogens was first reported by Berwald and Sachs [2,3]. Over the last two decades, a number of independent and collaborative efforts have been made to validate the SHE morphological transformation assay system for the testing of chemicals with carcinogenic potential [1,7,11,15,16,20,25]. In addition to its utility for detecting carcinogens, the SHE transformation system has been reported to detect tumor promoters [17,18]. Using a two-stage chemical exposure procedure mimicking that of the mouse skin epidermal papilloma tumor promotion model, SHE cells which were ‘initiated’ with a low concentration of carcinogen such as benzo[a]pyrene (BaP), then ‘promoted’ with a tumor promoter such as 12-O-tetradecanoylphorbol- 13-acetate (TPA) , were shown to have an enhanced transformation response above that of exposure to either the initiator or the promoter alone [19]. As part of a continuing program to evaluate the SHE transformation assay system for the Ireland Ltd
160
screening and characterizing of chemical carcinogens, we tested a series of chemicals under code in the SHE morphological transformation assay using a standard 7-day continuous exThis report presents the posure protocol. results of one of the chemicals, mezerein (MEZ), which produced a strong transformation response under the conditions of the assay. MEZ, a diterpene structurally related to the phorbol esters, is classified as a stage II tumor promoter in the mouse skin epidermal papilloma system [ZZ]. The activity of MEZ is discussed relative to the context in which it was evaluated in this study. Materials
and methods
Chemicals The positive control benzo[a]pyrene (BaP), and mezerein (MEZ), supplied as a coded chemical, were provided by the National Toxicology Program Chemical Repository (Radian Corporation, Austin, TX). The purity of the compounds were 98% and 99% for BaP and MEZ, respectively. The chemicals were stored desiccated in a - 20°C freezer. The solvent for BaP was dimethylsulfoxide (DMSO, spectrograde, Fisher Scientific). A 0.5% w/v pluronic acid F68 (Fluka Corporation, NY) in culture medium was used to dissolve MEZ. The final concentrations of DMSO and F68 in the test medium were 0.25% for both solvents. Cell culture
SHE cells were prepared from Syrian hamster embryos at days 12 - 14 of gestation and cryopreserved as described previously [24]. The clonal assay procedures for measuring cytotoxicity and transformation have also been described [24,25], with the exception that a reduced bicarbonate concentration was used in the culture medium. This medium, which had a pH of 6.7, was demonstrated by LeBoeuf and Kerchaert [ 13,141 to be optimal for the clonal growth and transformation of SHE cells.
SHE
clonal
cytotoxicity
and
transformation
assay
The procedures for conducting the cytotoxicity and transformation assays were identical with the exception that 5 dishes per test condition were used for the former and 15 dishes were used for the latter assay. Cryopreserved cells were thawed and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with low phenol red (5 mg/l) and reduced sodium bicarbonate (0.75 g/l) supplemented with 10% prescreened fetal bovine serum (FBS). The SHE cells were collected after 4 - 5 days, irradiated (5000 rad) and plated as feeder cells at 4 x lo4 cells/60 mm dish in 2 ml of medium containing 20% FBS. On the same day that the feeder cells were plated, another ampule of cryopreserved cells was thawed and cultured for use as target cells. The target cells were plated the following day onto dishes containing the feeder cells in a 2-ml aliquot at 200 cells/60 mm dish. Test chemicals were added the day after at twice the final concentration in a volume of 4 ml. The dishes were incubated undisturbed in a humidified 10% CO2 incubator. At the end of the 7-day exposure period, the dishes were rinsed in warmed (37OC) phosphate-buffered saline, fixed in methanol and stained with 10% Giemsa. To determine the cloning efficiency of SHE cells under the different test conditions, colonies from 5 representative dishes of the 15-dish sets were counted to calculate the mean colonies per dish. All the dishes were examined under a dissecting microscope and the colonies were classified as normal, morphologically altered (MA) or transformed (T), based on criteria previously established [25]. The MA colonies were recorded but not included in the calculation of transformation freas percent quency that was expressed transformation. Results The
cytotoxic
effect
of coded
chemicals
161
on the clonal growth of SHE cells was screened up to a concentration of 1 mg/ml. MEZ exhibited a shallow cytotoxicity profile over a four-log concentration range from 1 ng/ml10 pg/ml (Fig. 1). The LCsO (concentration which reduced cloning efficiency by 50%) for MEZ was estimated to be 0.15 pg/ml. The maximal toxic effect of MEZ was achieved at 10 pg/ml, resulting in a relative surviving fraction of approximately 30% for the SHE cells. There was no further reduction in cloning efficiency with higher concentrations of MEZ. This indicates that the solubility limit of MEZ in the test medium may have been reached. The clonal transformation assay data (Table I) showed that MEZ consistently induced a dose-dependent transformation response in three independent assays. The transformation of the negative controls ranged from no transformant scored in two of the assays, to a frequency of 0.30% and 0.55% for F68 and DMSO, respectively, in assay 2. These ranges are within the values we obtained for spon-
taneous transformation frequency of this assay performed in pH 6.7 medium. The level of induced transformation frequency varied substantially between assays. At 10 ng/ml of MEZ, the induced transformation activity in assay 1, 2 and 3 were 1.33%, 7.72% and 17.39% , respectively. Some variation of transformation activity between repeat experiments is not unusual for this assay which is based on a morphological endpoint and is sensitive to subtle differences in experimental conditions. In this particular study, however, a major source of variability is likely to be due to the limited solubility of MEZ. For example, when a lower concentration range was used in the third assay, the data showed that the toxicity level induced by 10 ng/ml of MEZ was similar to that induced by 100 ng/ml in assay 2; resulting in surviving fractions of 0.51 and 0.48, respectively. This suggests that the actual soluble concentrations in assay 1 and 2 might have been much lower than the calculated concentrations. In all assays performed, however, MEZ
+
0.12 lo+
lo-*
10-'
loo
MEZ Concentration
10'
102
lo3
b..LQ/ml)
Fig. 1. Cytotoxicity of MEZ on clonal growth of SHE cells. The SHE cells were exposed to various concentrations of MEZ for 7 days. The relative surviving fraction was determined from the mean number of colonies of 5 treated cultures divided by the mean number of colonies in 5 solvent control-treated cultures, the solvent control having a surviving fraction of 1.00. Each symbol represents results of an independent experiment.
162 Table 1. Morphological Assay
1
2
3
Chemical
transformation
Concentration
of Syrian hamster
embryo
Cytotoxicity
cells by MEZ. Transformation No. T
No. MA
% T
888 855 837 825 768 531
0 1 3 11 22 21
1 2 2 11 11 12
