In vitro response of the brown bullhead catfish (BB) and rainbow trout (RTG-2) cell lines to benzo[a]pyrene.
Established cell lines from rainbow trout (RTG-2) and brown bullhead catfish (BB) were evaluated as bioindicators of benzo[a]pyrene (B[a]P) toxicity with 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and neutral red (NR) uptake assays. Cytochrome P450 1A1 (CYP1A1) enzymatic activity was also evaluated, and taken as a biological indicator of the B[a]P induction power by ethoxyresorufin O-deethylase (EROD) and ethoxycoumarin O-deethylase (ECOD) assays. The BB and RTG-2 cells were compared after 1 and 6 days of exposure to B[a]P. The photoactivation of the compound (B[a]PUV) was another parameter taken into consideration. Cytotoxicity was not observed after 1 day of incubation with B[a]P in both cell lines, although the enzymatic activities of ECOD and EROD presented an induction. Apparently, after 1 day, cells did not metabolise sufficient amounts of B[a]P to cytotoxic metabolites. After 6 days of exposure to this compound a significant reduction in cell viability was observed, this reduction being superior to 50% at the highest B[a]P concentrations for the RTG-2 cell line. These results are in agreement with the values observed for the ECOD and EROD induction. The B[a]P cytotoxicity determined in both cell lines could be ascribed to the significant increase of EROD activity by 6 days of exposure. The photoactivation of B[a]P showed marked differences in both cytotoxic assays and CYP1A1 enzymatic activities, for both cell lines. After 1 day of exposure there was a significant reduction in cell viability, superior to 50% for the RTG-2 cell line. However, it was observed that no induction occurred but rather a decrease in ECOD and EROD activities. Six days of incubation with B[a]PUV showed a decrease in cell viability at the highest concentrations for the BB cells and at the lowest concentrations for the RTG-2 cell line, and the CYP1A1 enzymatic activity presented a significant induction. These results and those observed after 1 day of exposure suggest that B[a]PUV acts as a direct-acting toxicant as well as a metabolism-mediated toxicant-like B[a]P. The RTG-2 cells were more sensitive to B[a]P and its toxic metabolites as well as to the photoactivation of the compound, in both exposure times tested. The finding that the cell lines responded to the CYP1A1 induction in a very efficient way gives proof of the applicability of this system to environmental biomonitoring and toxicology.
Araújo
C S
CS
Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
Marques
S A
SA
Carrondo
M J
MJ
Gonçalves
L M
LM
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000818:Animals; D001564:Benzo(a)pyrene / Q000633:toxicity*; D002397:Catfishes; D002460:Cell Line; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D004784:Environmental Monitoring; D009499:Neutral Red; D017686:Oncorhynchus mykiss; D013778:Tetrazolium Salts; D013844:Thiazoles; D014874:Water Pollutants, Chemical / Q000633:toxicity*
D013778:Tetrazolium Salts; D013844:Thiazoles; D014874:Water Pollutants, Chemical; D009499:Neutral Red; D001564:Benzo(a)pyrene; D015660:7-Alkoxycoumarin O-Dealkylase; D019363:Cytochrome P-450 CYP1A1; C022616:thiazolyl blue