The new EU regulation for chemicals, REACH, specifically requires the development of alternatives in order to reduce and eventually replace studies on vertebrates. At the moment the cell transformation assay performed on rodent cell lines (BALB/c 3T3 or C3H10T1/2) or primary cells from Syrian Hamster is regarded as the only possible in vitro alternative to animal testing for carcinogenesis studies. Cell transformation assays have been proposed as screening tests for the carcinogenic potential of compounds that have no evidence of genotoxicity (OECD, 2007) and are listed among the REACH methods, as reported in EU regulation EC 440/2008. For the last 20 years we have been tested many chemicals and complex mixtures by using BALB/c 3T3 A 31 cells in different experimental protocols (Colacci et al, 1990; Mascolo et al, 2010). Recently, several scientists proposed to switch to the BALB/c 3T3 A 31-1-1 cell clone to develop a validated protocol to fulfill REACH requirements. The present study was performed in the aim to compare the results obtained with the two different clones. Cells were treated with PAHs (3-MCA 2.5 µg/ml, B(a)P 2.5 µg/ml) and aloethanes (1,2-DBE 50 µg/ml). The induction of cytotoxicity and the onset of chemically transformed foci were evaluated by two different experimental protocols: i) the originally recommended protocol (Kakunaga, 1973; IARC/NCI/EPA Working Group, 1985), where cells were seeded at 10.000 cells/60 mm dish and exposed to chemicals in the culture medium for 72 h; ii) an improved protocol aiming at reducing the toxicity of the chemical treatment, where the number of seeded cells was increased from 10.000 to 30.000 per dish and the cell treatment started two days later and lasted 48 h instead of 72 h (Matthews et al, 1993). In both treatment schedules, at the end of the exposure, the treatment medium was replaced with complete medium and the cultures were maintained for 4–6 weeks to allow the expression of transformed foci. The two clones differed for the response to chemicals, probably because of the different metabolizing capacity. The A31-1-1 cells showed a higher inherent transformation rate after PAH treatment, but they were insensitive to 1,2-DBE. As DBE is bioactivated to reactive forms able to bind DNA mainly through the conjugation with intracellular glutathione (Guengerich, 2003), these results suggested a reduced activity of phase-2 enzymes involved in gluthatione conjugation in A31-1-1 cells. Our results seem to suggest that in vitro cell transformation protocols performed under REACH regulation should take in account the different sensitivity of BALB/c 3T3 clones to different classes of chemicals.

THE BALB/c 3T3 CELL TRANSFORMATION ASSAY TO ASSESS THE CARCINOGENIC ACTIVITY OF CHEMICALS

COLACCI, ANNAMARIA;MASCOLO, MARIA GRAZIA;PERDICHIZZI, STEFANIA;ROTONDO, FRANCESCA;MORANDI, ELENA;GUERRINI, ANGELA;SILINGARDI, PAOLA;GRILLI, SANDRO;VACCARI, MONICA
2010

Abstract

The new EU regulation for chemicals, REACH, specifically requires the development of alternatives in order to reduce and eventually replace studies on vertebrates. At the moment the cell transformation assay performed on rodent cell lines (BALB/c 3T3 or C3H10T1/2) or primary cells from Syrian Hamster is regarded as the only possible in vitro alternative to animal testing for carcinogenesis studies. Cell transformation assays have been proposed as screening tests for the carcinogenic potential of compounds that have no evidence of genotoxicity (OECD, 2007) and are listed among the REACH methods, as reported in EU regulation EC 440/2008. For the last 20 years we have been tested many chemicals and complex mixtures by using BALB/c 3T3 A 31 cells in different experimental protocols (Colacci et al, 1990; Mascolo et al, 2010). Recently, several scientists proposed to switch to the BALB/c 3T3 A 31-1-1 cell clone to develop a validated protocol to fulfill REACH requirements. The present study was performed in the aim to compare the results obtained with the two different clones. Cells were treated with PAHs (3-MCA 2.5 µg/ml, B(a)P 2.5 µg/ml) and aloethanes (1,2-DBE 50 µg/ml). The induction of cytotoxicity and the onset of chemically transformed foci were evaluated by two different experimental protocols: i) the originally recommended protocol (Kakunaga, 1973; IARC/NCI/EPA Working Group, 1985), where cells were seeded at 10.000 cells/60 mm dish and exposed to chemicals in the culture medium for 72 h; ii) an improved protocol aiming at reducing the toxicity of the chemical treatment, where the number of seeded cells was increased from 10.000 to 30.000 per dish and the cell treatment started two days later and lasted 48 h instead of 72 h (Matthews et al, 1993). In both treatment schedules, at the end of the exposure, the treatment medium was replaced with complete medium and the cultures were maintained for 4–6 weeks to allow the expression of transformed foci. The two clones differed for the response to chemicals, probably because of the different metabolizing capacity. The A31-1-1 cells showed a higher inherent transformation rate after PAH treatment, but they were insensitive to 1,2-DBE. As DBE is bioactivated to reactive forms able to bind DNA mainly through the conjugation with intracellular glutathione (Guengerich, 2003), these results suggested a reduced activity of phase-2 enzymes involved in gluthatione conjugation in A31-1-1 cells. Our results seem to suggest that in vitro cell transformation protocols performed under REACH regulation should take in account the different sensitivity of BALB/c 3T3 clones to different classes of chemicals.
ALTERNATIVES TO ANIMAL EXPERIMENTATION
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Colacci A.; Mascolo M.G.; Perdichizzi S.; Rotondo F.; Morandi E.; Guerrini A.; Gazzilli A.; Silingardi P.; Grilli S.; Vaccari M.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/102732
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