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c 3T3 cell clones in the response to carcinogens
Toxicology in Vitro 25 (2011) 1183–1190
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Different sensitivity of BALB/c 3T3 cell clones in the response to carcinogens Annamaria Colacci a, Maria Grazia Mascolo a, Stefania Perdichizzi a, Daniele Quercioli a, Antonio Gazzilli a, Francesca Rotondo b, Elena Morandi b, Angela Guerrini c, Paola Silingardi a, Sandro Grilli c, Monica Vaccari a,⇑ a Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency – Emilia-Romagna Region (ER-EPA), Viale Filopanti 22, 40126 Bologna, Italy b Interdepartmental Center for Cancer Research ‘‘G. Prodi’’, University of Bologna, Italy c Department of Experimental Pathology, Cancer Research Section, University of Bologna, Italy
a r t i c l e
i n f o
Article history: Available online 6 June 2011 Keywords: Cell transformation assay Alternative methods Carcinogens Metabolic activation REACH
a b s t r a c t Cell transformation assays (CTAs) are currently regarded as the only possible in vitro alternative to animal testing for carcinogenesis studies. CTAs have been proposed as screening tests for the carcinogenic potential of compounds that have no evidence of genotoxicity but present structural alerts for carcinogenicity. We have extensively used the BALB/c 3T3 model based on the A31 cell clone to test single chemicals, complex mixtures and environmental pollutants. In the prevalidation study carried out by ECVAM, the improved protocol is based on BALB/c 3T3 A31-1-1 cells, a clone derived by A31 cells, that is very sensitive to PAH-induced transformation. The present study was performed in the aim to compare the results obtained with the two different clones exposed to different classes of carcinogens. Cells were treated with PAHs (3-methylcholanthrene, benzo(a)pyrene), alkylating agents (melphalan) and aloethanes (1,2-dibromoethane). The induction of cytotoxicity and the onset of chemically transformed foci were evaluated by two experimental protocols, differing for cell seeding density and chemical treatment duration. The A311-1 cells showed higher inherent transformation rate after PAHs treatment, but they were insensitive to 1,2-dibromoethane at concentrations that usually induced transformation in A31 cells. As 1,2-dibromoethane is bioactivated to reactive forms able to bind DNA mainly through the conjugation with intracellular glutathione, these results suggested a reduced activity of phase-2 enzymes involved in glutathione conjugation in A31-1-1 cells. Our results give evidence that inherent metabolic capacity of cells may play a critical role in in vitro cell transformation, cautioning against possible misclassification of chemicals. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Chemical carcinogenesis is a complex multistage and multifactorial process that is usually studied in animal assays. Among in vitro testing methods, cell transformation assay appears to be one of the most suitable tools to predict the carcinogenic properties of chemicals (Lilienblum et al., 2008). CTAs closely model the various stages of in vivo carcinogenesis, involving cellular and molecular changes similar to those detected in the process of in vivo tumor formation (Combes et al., 1999; LeBoeuf et al., 1999; Sakai, 2007; OECD, 2007). At the present, CTAs are regarded as the only possible in vitro alternative to animal testing for the screening of potential genotoxic and nongenotoxic carcinogens
Abbreviations: ACE, absolute clonal efficiency; B(a)P, benzo(a)pyrene; 1,2-DBE, 1,2 dibromoethane; CTA, cell transformation assay; GSH, glutathione; 3-MCA, 3methylcholanthrene; MEL, melphalan; PAHs, polycyclic aromatic hydrocarbons; RCE, relative clonal efficiency; TF, transformation frequency. ⇑ Corresponding author. Tel.: +39 51 2094790. E-mail addresses: [email protected], [email protected] (M. Vaccari). 0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2011.05.032
(Combes et al., 1999; OECD, 2007; Sakai, 2007; Lilienblum et al., 2008; Wells and Williams, 2009). For this reason CTAs are listed in the accepted methods for REACH (Reg. EC 440/2008). One of the most commonly used CTAs is based on the immortalized embryonic mouse fibroblasts BALB/c 3T3. These cells retain contact-inhibition, form a monolayer culture and stop growing at confluence. The treatment with carcinogenic compounds leads to the release from contact-inhibition, followed by the onset of morphologically transformed foci (Kakunaga, 1973; Sakai, 2007). In our 20 year-experience in the use of the BALB/c 3T3 model (Colacci et al., 1990; Perocco et al., 1993; Vaccari et al., 1999), we tested many classes of chemicals and complex mixtures, including also environmental pollutants, following the original protocol (Kakunaga, 1973; IARC/NCI/EPA Working Group, 1985). Recently, we confirmed the suitability of the BALB/c 3T3 A31 CTA by using a modified protocol, which aimed at reducing the toxicity of the chemical treatment (Matthews et al., 1993a; Vaccari et al., 2009; Mascolo et al., 2010). The two protocols mainly differ in the number of cells at seeding and the schedule of chemical exposure (Fig. 1).
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PROTOCOL 1 CYTOTOXICITY TEST 0
Day 4
Day 1
Day 10
carcinogen seeding 250 cells
fixing & staining
TRANSFORMATION TEST 0
Day 4
Day 1
Day 28-35
carcinogen seeding 1x104 cells
medium changes
fixing & staining
PROTOCOL 2 CYTOTOXICITY TEST Day 4
Day 2
0
Day 10
carcinogen seeding 250 cells
fixing & staining
TRANSFORMATION TEST 0
Day 4
Day 2
Day 28-35
carcinogen carcinogen seeding 3x104 cells
medium changes
fixing & staining
Fig. 1. Scheme of the experimental protocols.
To answer the need of a suitable alternative method to carcinogenicity bioassay, ECVAM coordinated a prevalidation study, which developed an experimental protocol based on the BALB/c 3T3 A311-1 cells (OECD, 2007). This clone was originally selected for its susceptibility to chemicals and ultraviolet light as well as its resistance to spontaneous transformation (Kakunaga and Crow, 2008). The prevalidation study demonstrated that the BALB/c 3T3 cell transformation assay is valuable to detect rodent carcinogens, even if its robustness should be further confirmed (Vanparys et al., 2011). In the present study we report the results obtained in comparative experiments to assess cytotoxicity and transforming properties of chemicals in the two BALB/c 3T3 clones, namely A31 and A31-1-1 cells. Cells were treated with PAHs (3-MCA, B(a)P), alkylating agents (MEL) and aloethanes (1,2-DBE), in order to evaluate possible differences in the response to chemicals. The effects on cloning efficiency and the onset of chemically transformed foci were evaluated in both original and modified protocols. Our results highlight some differences in the susceptibility of the 3T3 subclones to chemicals such as 1,2-DBE, which are mainly bioactivated through GSH conjugation (Guengerich, 2003). Among all variables we considered, only the use of subclones seemed to affect the final outcome, warning about possible misclassification due to different sensitivity of target cells. 2. Material and methods
>95%) were purchased from Sigma–Aldrich, St Louis, MO, USA. All these chemicals were dissolved in dimethylsulfoxide (DMSO). The stock solutions of 3-MCA (0.5 mg/ml), (B(a)P (0.5 mg/ml), 1,2-DBE (10 mg/ml) and MEL (1.2 mg/ml) were delivered to the culture medium to obtain suitable working solutions. The final concentration of DMSO in the experimental medium was adjusted, if needed, to 0.5%. 2.2. Cells The original stock of BALB/c 3T3 A31 cells was obtained from the American Type Culture Collection (Manassas, VA, USA). Working cultures were expanded from the original cryopreserved stock. Cells were grown in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with 10% Newborn Calf Serum (NCS). Cells were frozen in a 10% DMSO and 90% NCS solution and stored in liquid nitrogen and used in the CTAs at passage 79-80. The BALB/c 3T3 A31-1-1 clone was obtained from the Health Science Research Resource Bank (Osaka, Japan). The cells were grown in Minimum Essential Medium (MEM) with 10% Fetal Bovine Serum (FBS). Cells were cryoconserved in MEM 10% FBS solution and were used for the CTAs at passage 3-5 from the arrival. Cultures were routinely maintained in a humidified incubator with an atmosphere of 5% CO2 in air at 37 °C. For the transformation assays, only sub-confluent cells (about 70% confluence) were used. The target cells were not maintained beyond the third passage after thawing.
2.1. Chemicals 2.3. Cytotoxicity test Methylcholanthrene (3-MCA, 56-49-5, purity 98%), benzo(a)pyrene (B(a)P, 50-32-8, purity 96%), 1,2 dibromoethane (1,2-DBE, 106-93-4, purity 99%) and melphalan (MEL, 148-82-3, purity
The cytotoxicity assay was performed by seeding exponentially growing cells at 250 cells/60 mm dish (BD Falcon, BD Biosciences,
A. Colacci et al. / Toxicology in Vitro 25 (2011) 1183–1190
CA), in five replicates for each treatment. Plates were incubated at 37 °C in a 5% CO2 humidified atmosphere. Cells were exposed to chemicals according to the original protocol (Protocol 1) (IARC/ NCI/EPA Working Group, 1985) or the modified protocol (Protocol 2) (Matthews et al., 1993a) (Fig. 1). At the end of the chemical treatment, plates were washed with phosphate-buffered saline (PBS) then fresh culture medium was added. Cells were maintained in culture for ten days, then fixed with methanol, stained with 10% aqueous Giemsa and scored for colony formation. Only colonies containing more than 50 cells were counted (IARC/NCI/EPA Working Group, 1985; Franken et al., 2006). Untreated BALB/c 3T3 cells and vehicle-treated cells were used as negative controls. Results were expressed as: (i) mean number of colonies/ plate ± standard error (SE); (ii) absolute clonal efficiency (ACE), i.e. the fraction of cells which survived to the chemical treatment with respect to the number of seeded cells; (iii) relative clonal efficiency (RCE), which estimates the per cent reduction of cell clonal efficiency in treated groups as compared to that of the relative control (vehicle-treated cells).
2.4. Cell transformation assay The transformation assay was performed according to two different experimental protocols: (i) the originally recommended protocol (Kakunaga, 1973; IARC/NCI/EPA Working Group, 1985) (Protocol 1); (ii) the modified protocol (Matthews et al., 1993a; Mascolo et al., 2010) (Protocol 2) (Fig. 1). At the end of the chemical treatment, cells were maintained in culture for 4–6 weeks and the medium was changed twice a week for the duration of the assay. Then cells were fixed with methanol, stained with 10% aqueous Giemsa and scored for foci formation. For each experimental point, ten replicates were carried out, unless otherwise specified. Untreated BALB/c 3T3 cells and solvent-treated cells were used as negative controls. In order to calculate the number of cells surviving the chemical treatment, the colony-forming efficiency assay was also performed in parallel with the transformation test.
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specifically transforms the substitution products obtained with GSH into a chromophoric thione with a maximal absorbance at 400 nm. After 0, 24 and 48 h of exposure, the cell pellets of exponentially growing BALB/c 3T3 A31 cells were collected, suspended in methaphosphoric acid 5% (MPA) and homogenized by a Teflon pestle. The cell homogenates were then centrifuged (3000g, 10 min, 4 °C) and aliquots of the resulting supernatants were assayed for GSH content. Color development was measured at 405 nm in a microplate reader (Multiskan Ascent) after 1 min at room temperature. The concentration of reduced thiols was determined by comparing the absorbance of the sample with a standard curve obtained by dilutions of the standard GSH solution. The results were expressed as nM GSH/106cells.
2.7. Statistical analysis The difference between the mean colony numbers in the treated group compared to the control group was evaluated by the Student t test. Significant differences in the ACE of cells exposed to the chemical treatments were tested by the z test for comparison of two proportions. The RCE was analyzed by the Chi-square test of significance in 2 2 contingency tables. The significant percentage of plates with foci with respect to scored plates was calculated according to the Fisher–Yates test of significance in 2 2 contingency tables. Statistical analysis of foci distribution was performed by the Mann–Whitney unpaired t test. The TF was analyzed by the comparison of Poisson rates, after verifying that TF values fit a Poisson distribution. Positive relationship between the employed doses and the TF in the cell transformation assay and the clonal efficiency in the cytotoxicity assay was analyzed using linear regression analysis and by the Cochran-Armitage test for positive trend.
3. Results
2.5. Scoring of foci
3.1. Evaluation of toxicity
The scoring of foci was carried out according to the recommended guidelines. Only foci considered as positive (type III) (Schechtman, 1985a; IARC/NCI/EPA Working Group, 1985), showing deeply basophilic, dense multilayering of cells, random cell orientation at all parts of the focus edge, invasion into the surrounding contact-inhibited monolayer and domination of spindle-shaped cells, were counted. Foci of less than 1 mm in diameter were not scored (Kakunaga, 1985; IARC/NCI/EPA Working Group, 1985). Data was reported as: (i) number of positive plates (plates with foci/scored plates); (ii) mean number foci/plate ± standard error (SE); (iii) transformation frequency (TF), calculated on the cells that survived after chemical exposure. TF is expressed as a function of the total number of foci per treatment divided by the number of surviving cells estimated from the clonal efficiency observed in the cytotoxicity assay performed in parallel with the transformation test (Schechtman, 1985a).
In Table 1 the cytotoxicity induced in both BALB/c 3T3 A31-1-1 cells and the A31 parental clone by carcinogens with different mechanisms of action was evaluated according to the modified protocol routinely used in our laboratory (Protocol 2). The clonal efficiency of BALB/c 3T3 A31-1-1 cells was drastically reduced after the exposure to the alkylating agent MEL (0.075–0.3 lg/ml) or to B(a)P (0.5–2.5 lg/ml). The effects were strictly related to the dose. Unexpectedly, 1,2-DBE did not exert any toxic effect in A31-1-1 cells, when it was administered at doses which were effective in A31 cells (10–50 lg/ml). Also the cell clones response to the cytotoxic effects of PAHs was different. In A31 cells, a slight even if significant cell toxicity was observed only when high doses of B(a)P (>2.5 lg/ml) were used. In Fig. 2, the toxic effects induced in both cell clones by the positive control 3-MCA were represented. Data confirmed the high sensitivity of A31-1-1 cells to PAHs. To understand better the cells response to chemicals in relation to the treatment duration, A31-1-1 cells were exposed to the highest concentration of the chemicals, in both standard and modified protocols (Table 2). The strong susceptibility of A31-1-1 cells to the cytotoxicity induced by MCA (2.5 lg/ml) and B(a)P (2.5 lg/ml) was always confirmed regardless of the adopted experimental procedure. 1,2-DBE (50 lg/ml) was toxic only when cells were treated for 72 h. MEL (0.3 lg/ml) also induced a significant decrease of the relative cloning efficiency. The MEL effect was more evident by treating cells according to the modified protocol.
2.6. Determination of GSH levels Intracellular GSH was measured using the Glutathione Assay Kit, according to the manufacturer suggestions (CalBiochem). The assay involves a two-step chemical reaction. The first step leads to the formation of thioethers from mercaptans that are present in the sample. The second step is a b-elimination reaction, which
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Table 1 Clonal efficiency of BALB/c 3T3 A31-1-1 and A31 cells exposed to chemicalsa. Treatment
BALB/c A31-1-1
MEL [lg/ml] 0 (DMSO 0.5%) 0.075 0.15 0.3 0.45 0.6 UC 1,2-DBE [lg/ml] 0 (DMSO 0.5%) 10 25 50 94 188 UC B(a)P [lg/ml] 0 (DMSO 0.5%) 0.13 0.5 1 2.5 10 UC
BALB/c A31
Mean of colonies ± SE
ACE
RCE (%)
Mean of colonies ± SE
ACE
RCE (%)
96.60 ± 3.66 74.00 ± 2.65c 57.00 ± 4.32c 16.80 ± 4.26c nd nd 114.80 ± 9.25c
0.386 0.296d 0.228d 0.067d nd nd 0.459d
100 77e 59e 17e nd nd 119e
60.40 ± 1.03b 59.50 ± 1.98b 48.60 ± 1.08b,c 47.60 ± 0.93b,c 41.60 ± 1.29b,c 19.40 ± 1.21b,c 70.20 ± 0.66b,c
0.242b 0.238b 0.194b,d 0.190b,d 0.166b,d 0.078b,d 0.281b,f
100b 98b 80b,e 79b,e 69b,e 32b,e 116b,e
96.60 ± 3.66 113.80 ± 3.22c 118.60 ± 4.48c 97.00 ± 5.82c nd nd 114.80 ± 9.25c
0.386 0.455d 0.474d 0.388d nd nd 0.459d
100 118e 123e 100e nd nd 119e
39.50 ± 2.25 nd 32.75 ± 2.25 30.20 ± 2.33h 24.60 ± 1.29c 20.00 ± 1.08c 40.50 ± 1.06
0.158 nd 0.131 0.121f 0.098d 0.080d 0.130
nd 83 76g 62e 51e 103
96.60 ± 3.66 nd 8.60 ± 1.17c 3.80 ± 1.11c 1.00 ± 0.32c nd 114.80 ± 9.25c
0.386 nd 0.034d 0.015d 0.004d nd 0.459
100 nd 9e 4e 1e nd 119e
55.80 ± 2.15 51.40 ± 4.78 nd nd 42.80 ± 0.86c 36.20 ± 1.20c 54.00 ± 3.62
0.223 0.206 nd nd 0.171d 0.145d 0.216
100 92 nd nd 77e 65e 97
ACE = absolute clonal efficiency; RCE = relative clonal efficiency; UC = untreated cells. Positive relationship between the employed dose and absolute clonal efficiency has been analyzed by the linear regression analysis (MEL p < 0.01, A31 and A31-1-1; 1,2-DBE p < 0.05, A31) and by the Cochran-Armitage test for positive trend (B(a)P, MEL p < 0.01, A31-1-1). a The cells were seeded at 250 cells/60 mm plate and the chemical treatment lasted for 48 h. Data are reported as a mean of five replicates. b From Mascolo et al. (2010). c Significantly different (p < 0.01) from controls (solvent-treated cells) at the Student t test. d Significantly different (p < 0.01) from controls (solvent-treated cells) at the z test. e Significantly different (p < 0.01) from controls (solvent-treated cells) at the Chi-square test of significance in 2 2 contingency tables. f Significantly different (p < 0.05) from controls (solvent-treated cells) at the z test. g Significantly different (p < 0.05) from controls (solvent-treated cells) at the Chi-square test of significance in 2 2 contingency tables. h Significantly different (p < 0.05) from controls (solvent-treated cells) at the Student t test.
150
RCE (%)
A 100
50
0 1 µg/ ml
2.5 µg/ ml
3-MCA 150
RCE (%)
B 100
50
0 1 µg/ml
2.5 µg/ml
3-MCA Fig. 2. Box-plot of the RCE of 3-MCA-treated cells. A = BALB/c 3T3 A31 cells (2.5 lg/ ml, n = 10; 1 lg/ml, n = 7); B = BALB/c A31-1-1 cells (2.5 lg/ml, n = 4; 1 lg/ml, n = 4). All the experiments are performed by Protocol 2.
3.2. Evaluation of cell transformation The ability of the standard as well as the modified protocol to classify correctly chemicals for their transforming potential was
evaluated. As previously described (Mascolo et al., 2010), positive chemicals should fulfill the following criteria: (i) increase of the mean number of transformed foci/plate in the treatment groups that would be statistically significant at the 99% confidence level for just one of the tested doses or at the 95% confidence level, when two or more doses induced positive effects; (ii) increase of the transformation frequency in the treatment groups that would be twofold higher than that of the solvent control. Chemicals that meet only the first criterion but not the second one would be considered equivocal. Negative chemicals were those that did not induce any statistically significant increase of transformed foci at any concentration. As we routinely use 1,2-DBE-treated cells as positive controls in the BALB/c 3T3 A31 transformation assay, we checked the transforming activity of this compound in the modified protocol, comparing the results with the data previously obtained with the standard procedure (Perocco et al., 1991) (Table 3). Based on the results in A31 cells, 1,2-DBE was judged as positive in the transformation assay. The increment in TF and foci number/plate was significant, regardless of the experimental protocol. Then BALB/c 3T3 A31-1-1 cells were exposed to the well-known carcinogens 3-MCA and 1,2-DBE to assess the occurrence of transformed foci. In BALB/c 3T3 A31-1-1 cells (Table 4), 3-MCA induced a dose-related increase of cell transformation as determined by linear regression analysis (p < 0.05 standard protocol; p < 0.01 modified protocol). 1,2-DBE failed to induce transformation in the A31-1-1 cell subclone and did not affect the TF as well. As a consequence 1,2-DBE was classified as negative in this transformation system.
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A. Colacci et al. / Toxicology in Vitro 25 (2011) 1183–1190 Table 2 Clonal efficiency of BALB/c 3T3 A31-1-1 cells exposed to chemicalsa. Protocol 1b
Treatment
UC DMSO 0.5% 3-MCA [lg/ml] 2.5 B(a)P[lg/ml] 2.5 1,2-DBE [lg/ml] 50 MEL [lg/ml] 0.3
Protocol 2c
Mean of colonies ± SE
ACE
RCE (%)
Mean of colonies ± SE
ACE
RCE (%)
133.00 ± 9.17 126.00 ± 12.66
0.532 0.505
105 100
90.20 ± 5.03 80.00 ± 3.27
0.361d 0.320
113e 100
33.40 ± 3.04f
0.134g
27h
29.20 ± 2.82f
0.117g
37h
0.80 ± 0.58f
0.003g
1h
0.20 ± 0.20f
0.001g
0h
95.40 ± 3.66i
0.382g
76h
92.40 ± 6.19
0.370d
116h
94.20 ± 5.95
0.377g
75h
25.20 ± 9.2f
0.101g
32h
ACE = absolute clonal efficiency; RCE = relative clonal efficiency; UC = untreated cells. a Data are reported as a mean of five replicates. b The cells were seeded at 250 cells/60 mm plate and the chemical treatment lasted for 72 h. c The cells were seeded at 250 cells/60 mm plate and the chemical treatment lasted for 48 h. d Significantly different (p < 0.05) from controls (solvent-treated cells) at the z test. e Significantly different (p < 0.05) from controls (solvent-treated cells) at the Chi-square test of significance in 2 2 contingency tables. f Significantly different (p < 0.01) from controls (solvent-treated cells) at the Student t test. g Significantly different (p < 0.01) from controls (solvent-treated cells) at the z test. h Significantly different (p < 0.01) from controls (solvent-treated cells) at the Chi-square test of significance in 2 2 contingency tables. i Significantly different (p < 0.05) from controls (solvent-treated cells) at the Student t test.
Table 3 Effects of the treatment with 1,2-DBE on the transformation rate of BALB/c 3T3 A31 cells. Treatment
UC DMSO 0.5% 1,2-DBE [lg/ml] 24 48 94 188
Protocol 1a
Protocol 2b
Plates with foci/ plates scored
Foci/plate ± SE
RCE (%)
TF ( 10
6/9 5/8
0.77 ± 0.22 0.75 ± 0.25
120c 100
7/7 9/9 9/9 8/8
4.57 ± 0.78 3.66 ± 0.50 4.66 ± 0.52 3.50 ± 0.62
109 107 90 57c
4
)
Plates with foci/ plates scored
Foci/plate ± SE
RCE(%)
TF ( 10
2.5 2.9
0/10 0/10
0.00 ± 0.00 0.00 ± 0.00
103 100
0.00 0.00
16.3d 13.3d 20.2d 24.6d
0/10 7/10e 8/10e 8/10e
0.00 ± 0.00 1.30 ± 0.40f 1.40 ± 0.37f 1.20 ± 0.29f
83 76c 62c 51c
0.00 3.59e 4.74e 5.00e
4
)
RCE = relative clonal efficiency ; UC = untreated cells. Positive relationship between the employed dose and transformation frequency (TF) has been analyzed by the linear regression analysis and by the Cochran–Armitage test for positive trend (1,2-DBE: Protocol 1, p < 0.01; Protocol 2, p < 0.01). a The cells were seeded at 1 104 cell/60 mm plate and the chemical treatment lasted for 72 h (data from Perocco et al., 1993). b The cells were seeded at 3 104 cell/60 mm plate and the chemical treatment lasted for 48 h. c Significantly different (p < 0.01) from controls (solvent-treated cells) at the Chi-square test of significance in 2 2 contingency tables. d Significantly different (p < 0.01) from controls (solvent-treated cells) at the Poisson test. e Significantly different from control (solvent-treated plates) at the Fisher–Yates test of significance in 2 2 contingency tables (p < 0.01). f Significantly different (p < 0.01) from controls (solvent-treated cells) at the Mann–Whitney unpaired t test.
3.3. GSH intracellular levels The basal endogenous level of GSH was determined in BALB/c 3T3 A31 cells (Fig. 3). GSH was measured at low but detectable levels, which were not modified by the incubation time. The treatment with the antioxidant alpha-lipoic acid (ALA, 0.5 mM in NaOH 1 N) significantly increased the content of intra-cellular GSH. The enhancement was affected by ALA treatment duration. 3.4. Morphological observations In Fig. 4, plates of BALB/c 3T3 A31 cell transformation in the modified protocol are shown. The small basophilic structures, which could be observed in the untreated BALB/c 3T3 A31 plates, are formed by not overlapping cells showing higher grow density (>cell number/cm2). Isolated cells from these dense structures did not show tumorigenic or invasive properties, whereas the chemical transformed foci induced by carcinogens were both tumorigenic and metastatic in nude mice (Melchiori et al., 1992; Colacci et al., 1993). The monolayer of untreated A31-1-1 cells is more homogeneous and, as a result, the scoring of foci is easier (Fig. 5). The spontaneous
cell transformation frequency is lower and the chemical treatment induces the onset of large and numerous transformed foci. 4. Discussion New EU Regulations (REACH, cosmetics, biocides) strongly recommend the developing of alternative experimental assays to screen the toxicological profile of chemicals, including the assessment of carcinogenic properties. The reduction of animal testing for carcinogenicity would be an important goal to reach before 2013, when the evaluation and registration of chemicals under REACH is going to affect the small-medium enterprise production. Several protocols and experimental conditions have been proposed for setting up a suitable CTA to predict carcinogenic properties in vitro. We have extensively used the BALB/c 3T3 cell line by following both the original and the modified protocols (Colacci et al., 1990; Mascolo et al., 2010). The original protocol (Protocol 1, Fig. 1) implies seeding cells at a density of 1 104 cells/60 mm dish, a chemical treatment after 24 h prolonged for 72 h, and may or may not require the addition of an exogenous metabolic system (Kakunaga, 1973; IARC/NCI/EPA Working Group, 1985). The modified protocol (Matthews et al., 1993a, b) involves an
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Table 4 Effects of the treatment with 3-MCA or 1,2-DBE on the transformation rate of BALB/c 3T3 A31-1-1 cells. Protocol 1a
Treatment
Protocol 2b
Plates with foci/plates scored UC 0/6 DMSO 0.5% 0/6 3-MCA [lg/ml] 0.5 3/8 1 6/6e 2.5 7/7e 1,2-DBE [lg/ml] 10 1/9 25 1/8 50 0/8
Foci/plate ± SE
RCE (%)
TF (x 10
0.00 ± 0.00 0.00 ± 0.00
93 100
0.38 ± 0.18 1.33 ± 0.21f 2.29 ± 0.36f 0.11 ± 0.11 0.13 ± 0.13 0.00 ± 0.00
4
)
Plates with foci/plates scored
Foci/plate ± SE
RCE (%)
TF(x 10
0.00 0.00
0/7 2/10
0.00 ± 0.00 0.20 ± 0.13
98 100
0.00 0.17
29 27g 23g
3.18c 12.46c 25.12c
3/8 7/7e 8/8e
0.75 ± 0.41 2.00 ± 0.22f 4.25 ± 0.37f
51 36g 27g
1.26d 4.76c 13.75c
91 77 65
0.30 0.41 0.00
0/7 0/7 1/10
0.00 ± 0.00 0.00 ± 0.00 0.10 ± 0.10
108 110 102
0.00 0.00 0.08
4
)
RCE = relative clonal efficiency ; UC = untreated cells. Positive relationship between the employed dose and transformation frequency (TF) has been analyzed by the linear regression analysis (3-MCA: protocol 1, p < 0.05; protocol 2, p < 0.01) and by the Cochran-Armitage test for positive trend (3-MCA: protocol 1, p < 0.01; protocol 2, p < 0.01). a The cells were seeded at 1 104 cell/60 mm plate and the chemical treatment lasted for 72 h. b The cells were seeded at 3 104 cell/60 mm plate and the chemical treatment lasted for 48 h. c Significantly different (p < 0.01) from controls (solvent-treated cells) at the Poisson test. d Significantly different (p < 0.05) from controls (solvent-treated cells) at the Poisson test. e Significantly different from control (solvent-treated plates) at the Fisher–Yates test of significance in 2 2 contingency tables (p < 0.01). f Significantly different (p < 0.01) from controls (solvent-treated cells) at the Mann–Whitney unpaired t test. g Significantly different (p < 0.01) from controls (solvent-treated cells) at the Chi-square test of significance in 2 2 contingency tables.
6
GSH (nM) / 10 cells
30
UC 0.5 mM ALA
20
10
0 0
10
20
30
40
50
Fig. 4. Giemsa-stained plates from a cell transformation experiment performed by using BALB/c 3T3 A31 cells. A = DMSO-treated A31 cells; B = 3-MCA-treated A31cells; C = transformed foci induced by 3-MCA (100).
Time (h) Fig. 3. Basal and ALA-induced GSH levels in BALB/c 3T3 A31 cells.
increase in the number of plated cells up to 3 104 cells/60 mm dish, a delayed treatment at the day 2 after seeding and an exposure to the chemical for only 48 h (Protocol 2, Fig. 1). It also does not require any exogenous metabolic activation. In a previous work, we compared the standard and the modified protocol with regard to the cellular density at seeding. The modified protocol improved the specificity of the method by reducing the cytotoxic effects induced by the chemical administration (Vaccari et al., 1999). As the transformation rate is linked to the number of surviving cells after the chemical treatment, a strong toxic effect can lead to overestimate the chemical transforming potential (Schechtman, 1985a). Recently, some technical modifications aiming at optimize reproducibility and transferability of the original protocol were proposed (Hayashi et al., 2008). It has also been reported the relevance for regulatory purpose of the application of the improved BALB/c 3T3 A31-1-1 cell transformation assay to detect the initiating and promoting activities of chemicals (Tsuchiya et al., 2010). The prevalidation study coordinated by ECVAM aiming at assessing the predictivity and reproducibility of in vitro CTAs in carcinogenicity testing of chemicals was performed by using the A31-1-1 subclone (OECD, 2007; Vanparys et al., 2011). In the present study, the higher inherent clonal efficiency of the A31-1-1 clone, with respect to that of the parental A31 cell line, represents a useful property in assaying chemicals which are expected to reduce significantly cell growth and survival. The flat and regular monolayer and the low rate of spontaneous TF in un-
Fig. 5. Giemsa-stained plates from a cell transformation experiment performed by using BALB/c 3T3 A31-1-1 cells. A = DMSO-treated A31-1-1 cells; B = 3-MCAtreated A31-1-1 cells; C = transformed foci induced by 3-MCA (40).
treated or solvent-treated cells made the scoring of foci, which is a complex and critical step in the evaluation of results from CTAs, faster and easier (Figs. 4 and 5). The A31-1-1 cells were also very responsive to 3-MCA and B(a)P, as demonstrated by the dose-dependent inhibition of colony formation (Table 1 and Fig. 2). The induction of cell transformation by 3-MCA, evaluated by the increase in both foci number and TF, was demonstrated when the A31-1-1 cells were seeded at low density and treated according to the OECD proposed protocol (OECD, 2007). However, we are still able to classify 3-MCA as a carcinogen, even when it is tested in the CTA by the modified protocol (Table 4). Target BALB/c 3T3 cells exhibit enough inherent metabolizing capacity to convert the polycyclic aromatic hydrocarbon B[a]P to hydrophilic intermediates. However, different subclones of 3T3 cells (A31-714 and A31-1) have been found to differ in their B(a)P metabolizing capacity by as much as threefold (Schechtman, 1985b). Recent reports
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(Hayashi et al., 2008; Tsuchiya et al., 2010) on the improved BALB/c 3T3 assay protocol did not include the use of exogenous metabolizing systems, like rat liver S9 fractions, suggesting that the A31-1-1 cells could efficiently metabolize chemicals. This is particularly true for B(a)P and 3-MCA that were able to modulate the expression of several genes involved in xenobiotic defense, including CYP 1A1 and 1B1, in A31-1-1 cells (Rohrbeck et al., 2010). Our data confirm that, regardless of the utilized protocol, the dose-related transforming ability of 3-MCA was easily identified. Significant differences were observed in the response of A31 and A31-1-1 cells to 1,2-DBE (Tables 3 and 4). 1,2-DBE is classified as probably carcinogenic to humans (IARC, 1999; Cancer, 1999). The cancer risk assessment from long term exposure to 1,2-DBE had caused its withdrawal from the market (Sun, 1984; Guengerich, 2003). As most dihaloalkanes, 1,2-DBE is a bifunctional electrophile, which can be activated by either cytochrome P450-mediated oxidation or GSH conjugation by GSH transferases (Guengerich, 2003). N7-guanyl adduct represents the major DNA adduct formed in liver or kidney of rats treated with 1,2-DBE (Inskeep et al., 1986). In previous published results, we reported 1,2-DBE acting as both an initiating and promoting compound in in vitro cell transformation (Perocco et al., 1991; Colacci et al., 1995, 1996). Our studies also seemed to indicate basal levels of intracellular glutathione in BALB/c 3T3 A31 cells (Fig. 3), that were enhanced by the treatment with the potent antioxidant ALA. Thus, we have extensively used 1,2-DBE as a positive control in CTAs when analyzing compounds whose activity could be possibly affected by phase II enzymes (Mascolo et al., 2010). As the positive control, 1,2-DBE is used in the transformation test at the dose of 50 lg/ml (50 ppm). This concentration was chosen as the reference dose in vitro, on the basis of the previous dose–response studies in A31 cells (Perocco et al., 1991), since it is comparable to the dose that was effective in inducing tumors in animals (mice) after oral administration (60 ppm), without enhancing the mortality rate (NIOSH, 1977). In the present study, A31-1-1 cells failed to identify the transforming activity of 1,2-DBE (Table 4) at the reference dose (Table 3). As the A31 CTA judged 1,2-DBE as positive by using both the standard and the modified protocols, we can speculate that the classification of 1,2-DBE as positive or negative is strictly related more to the cell clone used in the test than to cell seeding density or treatment duration. The experimental data seems to imply that chemicals which are bioactivated also by phase II enzymes could represent a critical task for CTAs based on BALB/c 3T3 A31-1-1. When it was established, this cell clone was selected for its high susceptibility to transformation induced by carcinogens, as PAHs, activated by phase I enzymes, but other important metabolic pathways could not be expressed at the same optimal level in this cell clone. Despite the long-time use in toxicity and carcinogenicity testing, little is known about inherent metabolic activity of both A31 and A31 -1-1 BALB/c 3T3 cell clones. Since the crucial importance of bioactivation in deciding the fate of carcinogens, the in vitro cell transformation protocols performed under REACH regulation should consider the different sensitivity of BALB/c 3T3 clones to different classes of chemicals. Acknowledgement The authors would like to thank Mr. James Klimeczak for the critical revision of the manuscript. References Colacci, A., Perocco, P., Vaccari, M., Mazzullo, M., Albini, A., Parodi, S., Taningher, M., Grilli, S., 1990. In vitro transformation of BALB/c 3T3 cells by 1,1,2,2tetrachloroethane. Japanese Journal of Cancer Research 81, 786–792.
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Colacci, A., Albini, A., Melchiori, A., Nanni, P., Nicoletti, G., Noonan, D., Parodi, S., Grilli, S., 1993. Induction of a malignant phenotype in BALB/c 3T3 cells by 1,1,2,2-tetrachloroethane. International Journal of Oncology 2, 937–945. Colacci, A., Perocco, P., Vaccari, M., Da Vià, C., Silingardi, P., Manzini, E., Horn, W., Bartoli, S., Grilli, S., 1995. 1,2-dibromoethane as initiating agent for cell transformation. Japanese Journal of Cancer Research 86, 168–173. Colacci, A., Vaccari, M., Perocco, P., Da Vià, C., Silingardi, P., Manzini, E., Horn, W., Grilli, S., 1996. Enhancement of BALB/c 3T3 cells transformation by 1,2dibromoethane promoting effect. Carcinogenesis 17 (2), 225–231. Combes, R., Balls, M., Curren, R., Fishback, M., Fusenig, N., Kirkland, D., Lasne, C., Landlph, J., LeBoeuf, R., Marquardt, H., McCormik, J., Muller, L., Rivedal, E., Sabbioni, E., Tanaka, N., Vasseur, P., Yamasaki, H., 1999. Cell transformation assays as predictors of human carcinogenicity. Alternatives to laboratory animals 27, 745–767. Franken, N.A., Rodermond, H.M., Stap, J., Haveman, J., Van Bree, C., 2006. Clonogenic assay of cells in vitro. Nature Protocols 1, 2315–2319. Guengerich, F.P., 2003. Activation of dihaloalkanes by thiol-dependent mechanisms. Journal of Biochemistry and Molecular Biology 36, 20–27. Hayashi, K., Sasaki, K., Asada, S., Tsuchiya, T., Hayashi, M., Yoshimura, I., Tanaka, N., Umeda, M., 2008. Technical modification of the Balb/c 3T3 Cell transformation assay: the use of serum-reduced medium to optimise the practicability of the protocol. ATLA 36, 653–665. International Agency for Research on Cancer, 1999. Re-evaluation of some organic chemicals hydrazine and hydrogen peroxide. IARC Monographs 71, 661. IARC/NCI/EPA Working Group, 1985. Cellular and molecular mechanisms of cell transformation and standardization of transformation assays of established cell lines for the prediction of carcinogenic chemicals: overview and recommended protocols. Cancer Research 45, 2395–2399. Inskeep, P.B., Koga, N., Cmarik, J.L., Guengerich, F.P., 1986. Covalent binding of 1,2dihaloalkanes to DNA and stability of the major DNA adduct, S-[2-(N7guanyl)ethyl]glutathione. Cancer Research 46, 2839–2844. Kakunaga, T., 1973. Quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/c 3T3. International Journal of Cancer 12, 463–473. Kakunaga, T., 1985. Critical review of the use of established cell lines for in vitro cell transformation. In: Kakunaga, T., Yamasaki, H. (Eds.), Transformation Assay of Established Cell Lines: Mechanisms and Application. IARC Scientific Publications 67, Lyon, pp. 55–69. Kakunaga, T., Crow, J.D., 2008. Cell variants showing differential susceptibility to ultraviolet light-induced transformation. Science 209, 505–507. LeBoeuf, R., Kerckaert, K.A., Aardema, M.J., Isfort, R.J., 1999. Use of Syrian hamster embryo and BALB/c 3T3 cell transformation for assessing the carcinogenic potential of chemicals. In: McGregor, D.B., Rice, J.M., Venitt, S. (Eds.), The Use of Short and Medium Term Tests for Carcinogens and Data on Genetic Effects in Carcinogenic Hazard Evaluation. IARC Scientific Publications 67, Lyon, pp. 409– 425. Lilienblum, W., Dekant, W., Foth, H., Gebel, T., Hengstler, J.G., Kahl, R., Kramer, P.J., Schweinfurth, H., Wollin, K-M., 2008. Alternative methods to safety studies in experimental animals: role in the risk assessment of chemicals under the new European chemicals legislation (REACH). Archives of Toxicology 82, 211–236. Mascolo, M.G., Perdichizzi, S., Rotondo, F., Morandi, E., Guerrini, A., Silingardi, P., Vaccari, M., Grilli, S., Colacci, A., 2010. BALB/c 3T3 transformation assay for the prediction of carcinogenic potential of chemicals and environmental mixtures. Toxicology In vitro 24, 1292–1300. Matthews, E.J., Spalding, J.W., Tennant, R.W., 1993a. Transformation of BALB/c 3T3 cells: IV. Rank ordered potency of 24 chemical responses detected in a sensitive new procedure. Environmental Health Perspectives 101 (2), 319–345. Matthews, E.J., Spalding, J.W., Tennant, R.W., 1993b. Transformation of BALB/c 3T3 cells: V. Transformation responses of 168 chemicals compared with mutagenicity in Salmonella and carcinogenicity in rodent bioassays. Environmental Health Perspectives 101 (2), 347–482. Melchiori, A., Colacci, A., Lollini, P-L, De Giovanni, C., Carlone, S., Grilli, S., Parodi, S., Albini, A., 1992. Induction of invasive and experimental metastasis potential in BALB/c 3T3 cells by benzo(a)pyrene transformation. Invasion and Metastasis 12, 1–11. NIOSH, Criteria for a recommended standard. Occupational exposure to ethylene dibromide. NIOSH Publication N 77, 1977, pp. 221. OECD, Detailed review paper on cell transformation assay for detection of chemicals carcinogens, Environment, Health and Safety Publications Series on Testing and Assessment 31 (2007) 1–164. Perocco, P., Colacci, A., Santucci, M.A., Vaccari, M., Grilli, S., 1991. Transforming activity of ethylene dibromide in BALB/c 3T3. Research Communication on Chemical Pathology and Pharmacology 73, 159–172. Perocco, P., Colacci, A., Grilli, S., 1993. In vitro cytotoxic and cell transforming activities exerted by the pesticides Cyanizine, Dithianon, Diflubenzuron, Procymidone, and Vinclozolin on BALB/c 3T3 cells. Environmental and Molecular Mutagenesis 21, 81–86. Regulation (EC) 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH). Official Journal of the European Union, 31 May 2008, vol. 51, L 142/ 1 – L 142/739. 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Sakai, A., 2007. BALB/c 3T3 cell transformation assay for the assessment of chemical carcinogenicity. Alternatives to Animal Testing and Experimentation 14 (Special Issue), 367–373. Schechtman, L.M., 1985a. BALB/c 3T3 cell transformation: protocols, problems and improvements. In: Kakunaga, T., Yamasaki, H. (Eds.), Transformation Assay of Established Cell Lines: Mechanisms and Application. IARC Scientific Publications 67, Lyon, pp. 165–181. Schechtman, L.M., 1985b. Metabolic activation of procarcinogens by subcellular enzyme fractions in the C3H 10T1/2 and BALB/c 3T3 cell transformation systems. In: Kakunaga, T., Yamasaki, H. (Eds.), Transformation Assay of Established Cell Lines: Mechanisms and Application. IARC Scientific Publications 67, Lyon, pp. 137–162. Sun, M., 1984. EDB contamination kindles federal action. Science 223, 464–466. Tsuchiya, T., Umeda, M., Tanaka, N., Sakai, A., Nishiyama, H., Yoshimura, I., Ajimi, S., Asada, S., Asakura, M., Baba, H., Dewa, Y., Ebe, Y., Fushiwaki, Y., Hagiwara, Y., Hamada, S., Hamamura, T., Iwase, Y., Kajiwara, Y., Kasahara, Y., Kato, Y., Kawabata, M., Kitada, E., Kaneko, K., Kizaki, Y., Kubo, K., Miura, D., Mashiko, K., Mizuhashi, F., Muramatsu, D., Nakajima, M., et al., 2010. Application of the
improved BALB/c 3T3 cell transformation assay to the examination of the initiating and promoting activities of chemicals: the second interlaboratory collaborative study by the non-genotoxic carcinogen study group of Japan. ATLA 38, 11–27. Vaccari, M., Argnani, A., Horn, W., Silingardi, P., Giungi, M., Mascolo, M.G., Bartoli, S., Grilli, S., Colacci, A., 1999. Effects of the protease inhibitor antipain on cell malignant transformation. Anticancer Research 19, 589–596. Vaccari, M., Mascolo, M.G., Rotondo, F., Silingardi, P., Morandi, E., Perdichizzi, S., Guerrini, A., Grilli, S., Colacci, A., 2009. The in vitro BALB/c 3T3 transformation assay to profile the carcinogenic activity of environmental mixtures. Alternatives to Animal Experimentation 26, 106. Vanparys, P., Corvi, R., Aardema, M., Gribaldo, L., Hayashi, M., Hoffmann, S., Schechtman, L., 2011. ECVAM prevalidation of three cell transformation assays. ALTEX 28, 56–59. Wells, M.Y., Williams, E.S., 2009. The transgenic mouse assay as an alternative test method for regulatory carcinogenicity studies – Implications for REACH. Regulatory Toxicology and Pharmacology 53, 150–155.
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Different sensitivity of BALB/c 3T3 cell clones in the response to carcinogens Annamaria Colacci a, Maria Grazia Mascolo a, Stefania Perdichizzi a, Daniele Quercioli a, Antonio Gazzilli a, Francesca Rotondo b, Elena Morandi b, Angela Guerrini c, Paola Silingardi a, Sandro Grilli c, Monica Vaccari a,⇑ a Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency – Emilia-Romagna Region (ER-EPA), Viale Filopanti 22, 40126 Bologna, Italy b Interdepartmental Center for Cancer Research ‘‘G. Prodi’’, University of Bologna, Italy c Department of Experimental Pathology, Cancer Research Section, University of Bologna, Italy
a r t i c l e
i n f o
Article history: Available online 6 June 2011 Keywords: Cell transformation assay Alternative methods Carcinogens Metabolic activation REACH
a b s t r a c t Cell transformation assays (CTAs) are currently regarded as the only possible in vitro alternative to animal testing for carcinogenesis studies. CTAs have been proposed as screening tests for the carcinogenic potential of compounds that have no evidence of genotoxicity but present structural alerts for carcinogenicity. We have extensively used the BALB/c 3T3 model based on the A31 cell clone to test single chemicals, complex mixtures and environmental pollutants. In the prevalidation study carried out by ECVAM, the improved protocol is based on BALB/c 3T3 A31-1-1 cells, a clone derived by A31 cells, that is very sensitive to PAH-induced transformation. The present study was performed in the aim to compare the results obtained with the two different clones exposed to different classes of carcinogens. Cells were treated with PAHs (3-methylcholanthrene, benzo(a)pyrene), alkylating agents (melphalan) and aloethanes (1,2-dibromoethane). The induction of cytotoxicity and the onset of chemically transformed foci were evaluated by two experimental protocols, differing for cell seeding density and chemical treatment duration. The A311-1 cells showed higher inherent transformation rate after PAHs treatment, but they were insensitive to 1,2-dibromoethane at concentrations that usually induced transformation in A31 cells. As 1,2-dibromoethane is bioactivated to reactive forms able to bind DNA mainly through the conjugation with intracellular glutathione, these results suggested a reduced activity of phase-2 enzymes involved in glutathione conjugation in A31-1-1 cells. Our results give evidence that inherent metabolic capacity of cells may play a critical role in in vitro cell transformation, cautioning against possible misclassification of chemicals. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Chemical carcinogenesis is a complex multistage and multifactorial process that is usually studied in animal assays. Among in vitro testing methods, cell transformation assay appears to be one of the most suitable tools to predict the carcinogenic properties of chemicals (Lilienblum et al., 2008). CTAs closely model the various stages of in vivo carcinogenesis, involving cellular and molecular changes similar to those detected in the process of in vivo tumor formation (Combes et al., 1999; LeBoeuf et al., 1999; Sakai, 2007; OECD, 2007). At the present, CTAs are regarded as the only possible in vitro alternative to animal testing for the screening of potential genotoxic and nongenotoxic carcinogens
Abbreviations: ACE, absolute clonal efficiency; B(a)P, benzo(a)pyrene; 1,2-DBE, 1,2 dibromoethane; CTA, cell transformation assay; GSH, glutathione; 3-MCA, 3methylcholanthrene; MEL, melphalan; PAHs, polycyclic aromatic hydrocarbons; RCE, relative clonal efficiency; TF, transformation frequency. ⇑ Corresponding author. Tel.: +39 51 2094790. E-mail addresses: [email protected], [email protected] (M. Vaccari). 0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2011.05.032
(Combes et al., 1999; OECD, 2007; Sakai, 2007; Lilienblum et al., 2008; Wells and Williams, 2009). For this reason CTAs are listed in the accepted methods for REACH (Reg. EC 440/2008). One of the most commonly used CTAs is based on the immortalized embryonic mouse fibroblasts BALB/c 3T3. These cells retain contact-inhibition, form a monolayer culture and stop growing at confluence. The treatment with carcinogenic compounds leads to the release from contact-inhibition, followed by the onset of morphologically transformed foci (Kakunaga, 1973; Sakai, 2007). In our 20 year-experience in the use of the BALB/c 3T3 model (Colacci et al., 1990; Perocco et al., 1993; Vaccari et al., 1999), we tested many classes of chemicals and complex mixtures, including also environmental pollutants, following the original protocol (Kakunaga, 1973; IARC/NCI/EPA Working Group, 1985). Recently, we confirmed the suitability of the BALB/c 3T3 A31 CTA by using a modified protocol, which aimed at reducing the toxicity of the chemical treatment (Matthews et al., 1993a; Vaccari et al., 2009; Mascolo et al., 2010). The two protocols mainly differ in the number of cells at seeding and the schedule of chemical exposure (Fig. 1).
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PROTOCOL 1 CYTOTOXICITY TEST 0
Day 4
Day 1
Day 10
carcinogen seeding 250 cells
fixing & staining
TRANSFORMATION TEST 0
Day 4
Day 1
Day 28-35
carcinogen seeding 1x104 cells
medium changes
fixing & staining
PROTOCOL 2 CYTOTOXICITY TEST Day 4
Day 2
0
Day 10
carcinogen seeding 250 cells
fixing & staining
TRANSFORMATION TEST 0
Day 4
Day 2
Day 28-35
carcinogen carcinogen seeding 3x104 cells
medium changes
fixing & staining
Fig. 1. Scheme of the experimental protocols.
To answer the need of a suitable alternative method to carcinogenicity bioassay, ECVAM coordinated a prevalidation study, which developed an experimental protocol based on the BALB/c 3T3 A311-1 cells (OECD, 2007). This clone was originally selected for its susceptibility to chemicals and ultraviolet light as well as its resistance to spontaneous transformation (Kakunaga and Crow, 2008). The prevalidation study demonstrated that the BALB/c 3T3 cell transformation assay is valuable to detect rodent carcinogens, even if its robustness should be further confirmed (Vanparys et al., 2011). In the present study we report the results obtained in comparative experiments to assess cytotoxicity and transforming properties of chemicals in the two BALB/c 3T3 clones, namely A31 and A31-1-1 cells. Cells were treated with PAHs (3-MCA, B(a)P), alkylating agents (MEL) and aloethanes (1,2-DBE), in order to evaluate possible differences in the response to chemicals. The effects on cloning efficiency and the onset of chemically transformed foci were evaluated in both original and modified protocols. Our results highlight some differences in the susceptibility of the 3T3 subclones to chemicals such as 1,2-DBE, which are mainly bioactivated through GSH conjugation (Guengerich, 2003). Among all variables we considered, only the use of subclones seemed to affect the final outcome, warning about possible misclassification due to different sensitivity of target cells. 2. Material and methods
>95%) were purchased from Sigma–Aldrich, St Louis, MO, USA. All these chemicals were dissolved in dimethylsulfoxide (DMSO). The stock solutions of 3-MCA (0.5 mg/ml), (B(a)P (0.5 mg/ml), 1,2-DBE (10 mg/ml) and MEL (1.2 mg/ml) were delivered to the culture medium to obtain suitable working solutions. The final concentration of DMSO in the experimental medium was adjusted, if needed, to 0.5%. 2.2. Cells The original stock of BALB/c 3T3 A31 cells was obtained from the American Type Culture Collection (Manassas, VA, USA). Working cultures were expanded from the original cryopreserved stock. Cells were grown in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with 10% Newborn Calf Serum (NCS). Cells were frozen in a 10% DMSO and 90% NCS solution and stored in liquid nitrogen and used in the CTAs at passage 79-80. The BALB/c 3T3 A31-1-1 clone was obtained from the Health Science Research Resource Bank (Osaka, Japan). The cells were grown in Minimum Essential Medium (MEM) with 10% Fetal Bovine Serum (FBS). Cells were cryoconserved in MEM 10% FBS solution and were used for the CTAs at passage 3-5 from the arrival. Cultures were routinely maintained in a humidified incubator with an atmosphere of 5% CO2 in air at 37 °C. For the transformation assays, only sub-confluent cells (about 70% confluence) were used. The target cells were not maintained beyond the third passage after thawing.
2.1. Chemicals 2.3. Cytotoxicity test Methylcholanthrene (3-MCA, 56-49-5, purity 98%), benzo(a)pyrene (B(a)P, 50-32-8, purity 96%), 1,2 dibromoethane (1,2-DBE, 106-93-4, purity 99%) and melphalan (MEL, 148-82-3, purity
The cytotoxicity assay was performed by seeding exponentially growing cells at 250 cells/60 mm dish (BD Falcon, BD Biosciences,
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CA), in five replicates for each treatment. Plates were incubated at 37 °C in a 5% CO2 humidified atmosphere. Cells were exposed to chemicals according to the original protocol (Protocol 1) (IARC/ NCI/EPA Working Group, 1985) or the modified protocol (Protocol 2) (Matthews et al., 1993a) (Fig. 1). At the end of the chemical treatment, plates were washed with phosphate-buffered saline (PBS) then fresh culture medium was added. Cells were maintained in culture for ten days, then fixed with methanol, stained with 10% aqueous Giemsa and scored for colony formation. Only colonies containing more than 50 cells were counted (IARC/NCI/EPA Working Group, 1985; Franken et al., 2006). Untreated BALB/c 3T3 cells and vehicle-treated cells were used as negative controls. Results were expressed as: (i) mean number of colonies/ plate ± standard error (SE); (ii) absolute clonal efficiency (ACE), i.e. the fraction of cells which survived to the chemical treatment with respect to the number of seeded cells; (iii) relative clonal efficiency (RCE), which estimates the per cent reduction of cell clonal efficiency in treated groups as compared to that of the relative control (vehicle-treated cells).
2.4. Cell transformation assay The transformation assay was performed according to two different experimental protocols: (i) the originally recommended protocol (Kakunaga, 1973; IARC/NCI/EPA Working Group, 1985) (Protocol 1); (ii) the modified protocol (Matthews et al., 1993a; Mascolo et al., 2010) (Protocol 2) (Fig. 1). At the end of the chemical treatment, cells were maintained in culture for 4–6 weeks and the medium was changed twice a week for the duration of the assay. Then cells were fixed with methanol, stained with 10% aqueous Giemsa and scored for foci formation. For each experimental point, ten replicates were carried out, unless otherwise specified. Untreated BALB/c 3T3 cells and solvent-treated cells were used as negative controls. In order to calculate the number of cells surviving the chemical treatment, the colony-forming efficiency assay was also performed in parallel with the transformation test.
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specifically transforms the substitution products obtained with GSH into a chromophoric thione with a maximal absorbance at 400 nm. After 0, 24 and 48 h of exposure, the cell pellets of exponentially growing BALB/c 3T3 A31 cells were collected, suspended in methaphosphoric acid 5% (MPA) and homogenized by a Teflon pestle. The cell homogenates were then centrifuged (3000g, 10 min, 4 °C) and aliquots of the resulting supernatants were assayed for GSH content. Color development was measured at 405 nm in a microplate reader (Multiskan Ascent) after 1 min at room temperature. The concentration of reduced thiols was determined by comparing the absorbance of the sample with a standard curve obtained by dilutions of the standard GSH solution. The results were expressed as nM GSH/106cells.
2.7. Statistical analysis The difference between the mean colony numbers in the treated group compared to the control group was evaluated by the Student t test. Significant differences in the ACE of cells exposed to the chemical treatments were tested by the z test for comparison of two proportions. The RCE was analyzed by the Chi-square test of significance in 2 2 contingency tables. The significant percentage of plates with foci with respect to scored plates was calculated according to the Fisher–Yates test of significance in 2 2 contingency tables. Statistical analysis of foci distribution was performed by the Mann–Whitney unpaired t test. The TF was analyzed by the comparison of Poisson rates, after verifying that TF values fit a Poisson distribution. Positive relationship between the employed doses and the TF in the cell transformation assay and the clonal efficiency in the cytotoxicity assay was analyzed using linear regression analysis and by the Cochran-Armitage test for positive trend.
3. Results
2.5. Scoring of foci
3.1. Evaluation of toxicity
The scoring of foci was carried out according to the recommended guidelines. Only foci considered as positive (type III) (Schechtman, 1985a; IARC/NCI/EPA Working Group, 1985), showing deeply basophilic, dense multilayering of cells, random cell orientation at all parts of the focus edge, invasion into the surrounding contact-inhibited monolayer and domination of spindle-shaped cells, were counted. Foci of less than 1 mm in diameter were not scored (Kakunaga, 1985; IARC/NCI/EPA Working Group, 1985). Data was reported as: (i) number of positive plates (plates with foci/scored plates); (ii) mean number foci/plate ± standard error (SE); (iii) transformation frequency (TF), calculated on the cells that survived after chemical exposure. TF is expressed as a function of the total number of foci per treatment divided by the number of surviving cells estimated from the clonal efficiency observed in the cytotoxicity assay performed in parallel with the transformation test (Schechtman, 1985a).
In Table 1 the cytotoxicity induced in both BALB/c 3T3 A31-1-1 cells and the A31 parental clone by carcinogens with different mechanisms of action was evaluated according to the modified protocol routinely used in our laboratory (Protocol 2). The clonal efficiency of BALB/c 3T3 A31-1-1 cells was drastically reduced after the exposure to the alkylating agent MEL (0.075–0.3 lg/ml) or to B(a)P (0.5–2.5 lg/ml). The effects were strictly related to the dose. Unexpectedly, 1,2-DBE did not exert any toxic effect in A31-1-1 cells, when it was administered at doses which were effective in A31 cells (10–50 lg/ml). Also the cell clones response to the cytotoxic effects of PAHs was different. In A31 cells, a slight even if significant cell toxicity was observed only when high doses of B(a)P (>2.5 lg/ml) were used. In Fig. 2, the toxic effects induced in both cell clones by the positive control 3-MCA were represented. Data confirmed the high sensitivity of A31-1-1 cells to PAHs. To understand better the cells response to chemicals in relation to the treatment duration, A31-1-1 cells were exposed to the highest concentration of the chemicals, in both standard and modified protocols (Table 2). The strong susceptibility of A31-1-1 cells to the cytotoxicity induced by MCA (2.5 lg/ml) and B(a)P (2.5 lg/ml) was always confirmed regardless of the adopted experimental procedure. 1,2-DBE (50 lg/ml) was toxic only when cells were treated for 72 h. MEL (0.3 lg/ml) also induced a significant decrease of the relative cloning efficiency. The MEL effect was more evident by treating cells according to the modified protocol.
2.6. Determination of GSH levels Intracellular GSH was measured using the Glutathione Assay Kit, according to the manufacturer suggestions (CalBiochem). The assay involves a two-step chemical reaction. The first step leads to the formation of thioethers from mercaptans that are present in the sample. The second step is a b-elimination reaction, which
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Table 1 Clonal efficiency of BALB/c 3T3 A31-1-1 and A31 cells exposed to chemicalsa. Treatment
BALB/c A31-1-1
MEL [lg/ml] 0 (DMSO 0.5%) 0.075 0.15 0.3 0.45 0.6 UC 1,2-DBE [lg/ml] 0 (DMSO 0.5%) 10 25 50 94 188 UC B(a)P [lg/ml] 0 (DMSO 0.5%) 0.13 0.5 1 2.5 10 UC
BALB/c A31
Mean of colonies ± SE
ACE
RCE (%)
Mean of colonies ± SE
ACE
RCE (%)
96.60 ± 3.66 74.00 ± 2.65c 57.00 ± 4.32c 16.80 ± 4.26c nd nd 114.80 ± 9.25c
0.386 0.296d 0.228d 0.067d nd nd 0.459d
100 77e 59e 17e nd nd 119e
60.40 ± 1.03b 59.50 ± 1.98b 48.60 ± 1.08b,c 47.60 ± 0.93b,c 41.60 ± 1.29b,c 19.40 ± 1.21b,c 70.20 ± 0.66b,c
0.242b 0.238b 0.194b,d 0.190b,d 0.166b,d 0.078b,d 0.281b,f
100b 98b 80b,e 79b,e 69b,e 32b,e 116b,e
96.60 ± 3.66 113.80 ± 3.22c 118.60 ± 4.48c 97.00 ± 5.82c nd nd 114.80 ± 9.25c
0.386 0.455d 0.474d 0.388d nd nd 0.459d
100 118e 123e 100e nd nd 119e
39.50 ± 2.25 nd 32.75 ± 2.25 30.20 ± 2.33h 24.60 ± 1.29c 20.00 ± 1.08c 40.50 ± 1.06
0.158 nd 0.131 0.121f 0.098d 0.080d 0.130
nd 83 76g 62e 51e 103
96.60 ± 3.66 nd 8.60 ± 1.17c 3.80 ± 1.11c 1.00 ± 0.32c nd 114.80 ± 9.25c
0.386 nd 0.034d 0.015d 0.004d nd 0.459
100 nd 9e 4e 1e nd 119e
55.80 ± 2.15 51.40 ± 4.78 nd nd 42.80 ± 0.86c 36.20 ± 1.20c 54.00 ± 3.62
0.223 0.206 nd nd 0.171d 0.145d 0.216
100 92 nd nd 77e 65e 97
ACE = absolute clonal efficiency; RCE = relative clonal efficiency; UC = untreated cells. Positive relationship between the employed dose and absolute clonal efficiency has been analyzed by the linear regression analysis (MEL p < 0.01, A31 and A31-1-1; 1,2-DBE p < 0.05, A31) and by the Cochran-Armitage test for positive trend (B(a)P, MEL p < 0.01, A31-1-1). a The cells were seeded at 250 cells/60 mm plate and the chemical treatment lasted for 48 h. Data are reported as a mean of five replicates. b From Mascolo et al. (2010). c Significantly different (p < 0.01) from controls (solvent-treated cells) at the Student t test. d Significantly different (p < 0.01) from controls (solvent-treated cells) at the z test. e Significantly different (p < 0.01) from controls (solvent-treated cells) at the Chi-square test of significance in 2 2 contingency tables. f Significantly different (p < 0.05) from controls (solvent-treated cells) at the z test. g Significantly different (p < 0.05) from controls (solvent-treated cells) at the Chi-square test of significance in 2 2 contingency tables. h Significantly different (p < 0.05) from controls (solvent-treated cells) at the Student t test.
150
RCE (%)
A 100
50
0 1 µg/ ml
2.5 µg/ ml
3-MCA 150
RCE (%)
B 100
50
0 1 µg/ml
2.5 µg/ml
3-MCA Fig. 2. Box-plot of the RCE of 3-MCA-treated cells. A = BALB/c 3T3 A31 cells (2.5 lg/ ml, n = 10; 1 lg/ml, n = 7); B = BALB/c A31-1-1 cells (2.5 lg/ml, n = 4; 1 lg/ml, n = 4). All the experiments are performed by Protocol 2.
3.2. Evaluation of cell transformation The ability of the standard as well as the modified protocol to classify correctly chemicals for their transforming potential was
evaluated. As previously described (Mascolo et al., 2010), positive chemicals should fulfill the following criteria: (i) increase of the mean number of transformed foci/plate in the treatment groups that would be statistically significant at the 99% confidence level for just one of the tested doses or at the 95% confidence level, when two or more doses induced positive effects; (ii) increase of the transformation frequency in the treatment groups that would be twofold higher than that of the solvent control. Chemicals that meet only the first criterion but not the second one would be considered equivocal. Negative chemicals were those that did not induce any statistically significant increase of transformed foci at any concentration. As we routinely use 1,2-DBE-treated cells as positive controls in the BALB/c 3T3 A31 transformation assay, we checked the transforming activity of this compound in the modified protocol, comparing the results with the data previously obtained with the standard procedure (Perocco et al., 1991) (Table 3). Based on the results in A31 cells, 1,2-DBE was judged as positive in the transformation assay. The increment in TF and foci number/plate was significant, regardless of the experimental protocol. Then BALB/c 3T3 A31-1-1 cells were exposed to the well-known carcinogens 3-MCA and 1,2-DBE to assess the occurrence of transformed foci. In BALB/c 3T3 A31-1-1 cells (Table 4), 3-MCA induced a dose-related increase of cell transformation as determined by linear regression analysis (p < 0.05 standard protocol; p < 0.01 modified protocol). 1,2-DBE failed to induce transformation in the A31-1-1 cell subclone and did not affect the TF as well. As a consequence 1,2-DBE was classified as negative in this transformation system.
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A. Colacci et al. / Toxicology in Vitro 25 (2011) 1183–1190 Table 2 Clonal efficiency of BALB/c 3T3 A31-1-1 cells exposed to chemicalsa. Protocol 1b
Treatment
UC DMSO 0.5% 3-MCA [lg/ml] 2.5 B(a)P[lg/ml] 2.5 1,2-DBE [lg/ml] 50 MEL [lg/ml] 0.3
Protocol 2c
Mean of colonies ± SE
ACE
RCE (%)
Mean of colonies ± SE
ACE
RCE (%)
133.00 ± 9.17 126.00 ± 12.66
0.532 0.505
105 100
90.20 ± 5.03 80.00 ± 3.27
0.361d 0.320
113e 100
33.40 ± 3.04f
0.134g
27h
29.20 ± 2.82f
0.117g
37h
0.80 ± 0.58f
0.003g
1h
0.20 ± 0.20f
0.001g
0h
95.40 ± 3.66i
0.382g
76h
92.40 ± 6.19
0.370d
116h
94.20 ± 5.95
0.377g
75h
25.20 ± 9.2f
0.101g
32h
ACE = absolute clonal efficiency; RCE = relative clonal efficiency; UC = untreated cells. a Data are reported as a mean of five replicates. b The cells were seeded at 250 cells/60 mm plate and the chemical treatment lasted for 72 h. c The cells were seeded at 250 cells/60 mm plate and the chemical treatment lasted for 48 h. d Significantly different (p < 0.05) from controls (solvent-treated cells) at the z test. e Significantly different (p < 0.05) from controls (solvent-treated cells) at the Chi-square test of significance in 2 2 contingency tables. f Significantly different (p < 0.01) from controls (solvent-treated cells) at the Student t test. g Significantly different (p < 0.01) from controls (solvent-treated cells) at the z test. h Significantly different (p < 0.01) from controls (solvent-treated cells) at the Chi-square test of significance in 2 2 contingency tables. i Significantly different (p < 0.05) from controls (solvent-treated cells) at the Student t test.
Table 3 Effects of the treatment with 1,2-DBE on the transformation rate of BALB/c 3T3 A31 cells. Treatment
UC DMSO 0.5% 1,2-DBE [lg/ml] 24 48 94 188
Protocol 1a
Protocol 2b
Plates with foci/ plates scored
Foci/plate ± SE
RCE (%)
TF ( 10
6/9 5/8
0.77 ± 0.22 0.75 ± 0.25
120c 100
7/7 9/9 9/9 8/8
4.57 ± 0.78 3.66 ± 0.50 4.66 ± 0.52 3.50 ± 0.62
109 107 90 57c
4
)
Plates with foci/ plates scored
Foci/plate ± SE
RCE(%)
TF ( 10
2.5 2.9
0/10 0/10
0.00 ± 0.00 0.00 ± 0.00
103 100
0.00 0.00
16.3d 13.3d 20.2d 24.6d
0/10 7/10e 8/10e 8/10e
0.00 ± 0.00 1.30 ± 0.40f 1.40 ± 0.37f 1.20 ± 0.29f
83 76c 62c 51c
0.00 3.59e 4.74e 5.00e
4
)
RCE = relative clonal efficiency ; UC = untreated cells. Positive relationship between the employed dose and transformation frequency (TF) has been analyzed by the linear regression analysis and by the Cochran–Armitage test for positive trend (1,2-DBE: Protocol 1, p < 0.01; Protocol 2, p < 0.01). a The cells were seeded at 1 104 cell/60 mm plate and the chemical treatment lasted for 72 h (data from Perocco et al., 1993). b The cells were seeded at 3 104 cell/60 mm plate and the chemical treatment lasted for 48 h. c Significantly different (p < 0.01) from controls (solvent-treated cells) at the Chi-square test of significance in 2 2 contingency tables. d Significantly different (p < 0.01) from controls (solvent-treated cells) at the Poisson test. e Significantly different from control (solvent-treated plates) at the Fisher–Yates test of significance in 2 2 contingency tables (p < 0.01). f Significantly different (p < 0.01) from controls (solvent-treated cells) at the Mann–Whitney unpaired t test.
3.3. GSH intracellular levels The basal endogenous level of GSH was determined in BALB/c 3T3 A31 cells (Fig. 3). GSH was measured at low but detectable levels, which were not modified by the incubation time. The treatment with the antioxidant alpha-lipoic acid (ALA, 0.5 mM in NaOH 1 N) significantly increased the content of intra-cellular GSH. The enhancement was affected by ALA treatment duration. 3.4. Morphological observations In Fig. 4, plates of BALB/c 3T3 A31 cell transformation in the modified protocol are shown. The small basophilic structures, which could be observed in the untreated BALB/c 3T3 A31 plates, are formed by not overlapping cells showing higher grow density (>cell number/cm2). Isolated cells from these dense structures did not show tumorigenic or invasive properties, whereas the chemical transformed foci induced by carcinogens were both tumorigenic and metastatic in nude mice (Melchiori et al., 1992; Colacci et al., 1993). The monolayer of untreated A31-1-1 cells is more homogeneous and, as a result, the scoring of foci is easier (Fig. 5). The spontaneous
cell transformation frequency is lower and the chemical treatment induces the onset of large and numerous transformed foci. 4. Discussion New EU Regulations (REACH, cosmetics, biocides) strongly recommend the developing of alternative experimental assays to screen the toxicological profile of chemicals, including the assessment of carcinogenic properties. The reduction of animal testing for carcinogenicity would be an important goal to reach before 2013, when the evaluation and registration of chemicals under REACH is going to affect the small-medium enterprise production. Several protocols and experimental conditions have been proposed for setting up a suitable CTA to predict carcinogenic properties in vitro. We have extensively used the BALB/c 3T3 cell line by following both the original and the modified protocols (Colacci et al., 1990; Mascolo et al., 2010). The original protocol (Protocol 1, Fig. 1) implies seeding cells at a density of 1 104 cells/60 mm dish, a chemical treatment after 24 h prolonged for 72 h, and may or may not require the addition of an exogenous metabolic system (Kakunaga, 1973; IARC/NCI/EPA Working Group, 1985). The modified protocol (Matthews et al., 1993a, b) involves an
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Table 4 Effects of the treatment with 3-MCA or 1,2-DBE on the transformation rate of BALB/c 3T3 A31-1-1 cells. Protocol 1a
Treatment
Protocol 2b
Plates with foci/plates scored UC 0/6 DMSO 0.5% 0/6 3-MCA [lg/ml] 0.5 3/8 1 6/6e 2.5 7/7e 1,2-DBE [lg/ml] 10 1/9 25 1/8 50 0/8
Foci/plate ± SE
RCE (%)
TF (x 10
0.00 ± 0.00 0.00 ± 0.00
93 100
0.38 ± 0.18 1.33 ± 0.21f 2.29 ± 0.36f 0.11 ± 0.11 0.13 ± 0.13 0.00 ± 0.00
4
)
Plates with foci/plates scored
Foci/plate ± SE
RCE (%)
TF(x 10
0.00 0.00
0/7 2/10
0.00 ± 0.00 0.20 ± 0.13
98 100
0.00 0.17
29 27g 23g
3.18c 12.46c 25.12c
3/8 7/7e 8/8e
0.75 ± 0.41 2.00 ± 0.22f 4.25 ± 0.37f
51 36g 27g
1.26d 4.76c 13.75c
91 77 65
0.30 0.41 0.00
0/7 0/7 1/10
0.00 ± 0.00 0.00 ± 0.00 0.10 ± 0.10
108 110 102
0.00 0.00 0.08
4
)
RCE = relative clonal efficiency ; UC = untreated cells. Positive relationship between the employed dose and transformation frequency (TF) has been analyzed by the linear regression analysis (3-MCA: protocol 1, p < 0.05; protocol 2, p < 0.01) and by the Cochran-Armitage test for positive trend (3-MCA: protocol 1, p < 0.01; protocol 2, p < 0.01). a The cells were seeded at 1 104 cell/60 mm plate and the chemical treatment lasted for 72 h. b The cells were seeded at 3 104 cell/60 mm plate and the chemical treatment lasted for 48 h. c Significantly different (p < 0.01) from controls (solvent-treated cells) at the Poisson test. d Significantly different (p < 0.05) from controls (solvent-treated cells) at the Poisson test. e Significantly different from control (solvent-treated plates) at the Fisher–Yates test of significance in 2 2 contingency tables (p < 0.01). f Significantly different (p < 0.01) from controls (solvent-treated cells) at the Mann–Whitney unpaired t test. g Significantly different (p < 0.01) from controls (solvent-treated cells) at the Chi-square test of significance in 2 2 contingency tables.
6
GSH (nM) / 10 cells
30
UC 0.5 mM ALA
20
10
0 0
10
20
30
40
50
Fig. 4. Giemsa-stained plates from a cell transformation experiment performed by using BALB/c 3T3 A31 cells. A = DMSO-treated A31 cells; B = 3-MCA-treated A31cells; C = transformed foci induced by 3-MCA (100).
Time (h) Fig. 3. Basal and ALA-induced GSH levels in BALB/c 3T3 A31 cells.
increase in the number of plated cells up to 3 104 cells/60 mm dish, a delayed treatment at the day 2 after seeding and an exposure to the chemical for only 48 h (Protocol 2, Fig. 1). It also does not require any exogenous metabolic activation. In a previous work, we compared the standard and the modified protocol with regard to the cellular density at seeding. The modified protocol improved the specificity of the method by reducing the cytotoxic effects induced by the chemical administration (Vaccari et al., 1999). As the transformation rate is linked to the number of surviving cells after the chemical treatment, a strong toxic effect can lead to overestimate the chemical transforming potential (Schechtman, 1985a). Recently, some technical modifications aiming at optimize reproducibility and transferability of the original protocol were proposed (Hayashi et al., 2008). It has also been reported the relevance for regulatory purpose of the application of the improved BALB/c 3T3 A31-1-1 cell transformation assay to detect the initiating and promoting activities of chemicals (Tsuchiya et al., 2010). The prevalidation study coordinated by ECVAM aiming at assessing the predictivity and reproducibility of in vitro CTAs in carcinogenicity testing of chemicals was performed by using the A31-1-1 subclone (OECD, 2007; Vanparys et al., 2011). In the present study, the higher inherent clonal efficiency of the A31-1-1 clone, with respect to that of the parental A31 cell line, represents a useful property in assaying chemicals which are expected to reduce significantly cell growth and survival. The flat and regular monolayer and the low rate of spontaneous TF in un-
Fig. 5. Giemsa-stained plates from a cell transformation experiment performed by using BALB/c 3T3 A31-1-1 cells. A = DMSO-treated A31-1-1 cells; B = 3-MCAtreated A31-1-1 cells; C = transformed foci induced by 3-MCA (40).
treated or solvent-treated cells made the scoring of foci, which is a complex and critical step in the evaluation of results from CTAs, faster and easier (Figs. 4 and 5). The A31-1-1 cells were also very responsive to 3-MCA and B(a)P, as demonstrated by the dose-dependent inhibition of colony formation (Table 1 and Fig. 2). The induction of cell transformation by 3-MCA, evaluated by the increase in both foci number and TF, was demonstrated when the A31-1-1 cells were seeded at low density and treated according to the OECD proposed protocol (OECD, 2007). However, we are still able to classify 3-MCA as a carcinogen, even when it is tested in the CTA by the modified protocol (Table 4). Target BALB/c 3T3 cells exhibit enough inherent metabolizing capacity to convert the polycyclic aromatic hydrocarbon B[a]P to hydrophilic intermediates. However, different subclones of 3T3 cells (A31-714 and A31-1) have been found to differ in their B(a)P metabolizing capacity by as much as threefold (Schechtman, 1985b). Recent reports
A. Colacci et al. / Toxicology in Vitro 25 (2011) 1183–1190
(Hayashi et al., 2008; Tsuchiya et al., 2010) on the improved BALB/c 3T3 assay protocol did not include the use of exogenous metabolizing systems, like rat liver S9 fractions, suggesting that the A31-1-1 cells could efficiently metabolize chemicals. This is particularly true for B(a)P and 3-MCA that were able to modulate the expression of several genes involved in xenobiotic defense, including CYP 1A1 and 1B1, in A31-1-1 cells (Rohrbeck et al., 2010). Our data confirm that, regardless of the utilized protocol, the dose-related transforming ability of 3-MCA was easily identified. Significant differences were observed in the response of A31 and A31-1-1 cells to 1,2-DBE (Tables 3 and 4). 1,2-DBE is classified as probably carcinogenic to humans (IARC, 1999; Cancer, 1999). The cancer risk assessment from long term exposure to 1,2-DBE had caused its withdrawal from the market (Sun, 1984; Guengerich, 2003). As most dihaloalkanes, 1,2-DBE is a bifunctional electrophile, which can be activated by either cytochrome P450-mediated oxidation or GSH conjugation by GSH transferases (Guengerich, 2003). N7-guanyl adduct represents the major DNA adduct formed in liver or kidney of rats treated with 1,2-DBE (Inskeep et al., 1986). In previous published results, we reported 1,2-DBE acting as both an initiating and promoting compound in in vitro cell transformation (Perocco et al., 1991; Colacci et al., 1995, 1996). Our studies also seemed to indicate basal levels of intracellular glutathione in BALB/c 3T3 A31 cells (Fig. 3), that were enhanced by the treatment with the potent antioxidant ALA. Thus, we have extensively used 1,2-DBE as a positive control in CTAs when analyzing compounds whose activity could be possibly affected by phase II enzymes (Mascolo et al., 2010). As the positive control, 1,2-DBE is used in the transformation test at the dose of 50 lg/ml (50 ppm). This concentration was chosen as the reference dose in vitro, on the basis of the previous dose–response studies in A31 cells (Perocco et al., 1991), since it is comparable to the dose that was effective in inducing tumors in animals (mice) after oral administration (60 ppm), without enhancing the mortality rate (NIOSH, 1977). In the present study, A31-1-1 cells failed to identify the transforming activity of 1,2-DBE (Table 4) at the reference dose (Table 3). As the A31 CTA judged 1,2-DBE as positive by using both the standard and the modified protocols, we can speculate that the classification of 1,2-DBE as positive or negative is strictly related more to the cell clone used in the test than to cell seeding density or treatment duration. The experimental data seems to imply that chemicals which are bioactivated also by phase II enzymes could represent a critical task for CTAs based on BALB/c 3T3 A31-1-1. When it was established, this cell clone was selected for its high susceptibility to transformation induced by carcinogens, as PAHs, activated by phase I enzymes, but other important metabolic pathways could not be expressed at the same optimal level in this cell clone. Despite the long-time use in toxicity and carcinogenicity testing, little is known about inherent metabolic activity of both A31 and A31 -1-1 BALB/c 3T3 cell clones. Since the crucial importance of bioactivation in deciding the fate of carcinogens, the in vitro cell transformation protocols performed under REACH regulation should consider the different sensitivity of BALB/c 3T3 clones to different classes of chemicals. Acknowledgement The authors would like to thank Mr. James Klimeczak for the critical revision of the manuscript. References Colacci, A., Perocco, P., Vaccari, M., Mazzullo, M., Albini, A., Parodi, S., Taningher, M., Grilli, S., 1990. In vitro transformation of BALB/c 3T3 cells by 1,1,2,2tetrachloroethane. Japanese Journal of Cancer Research 81, 786–792.
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