The in vitro porcine brain tubulin assembly assay: effects of a genotoxic carcinogen (aflatoxin B1), eight tumor promoters and nine miscellaneous substances.
Aflatoxin B1 (AFB1) had a reversible inhibitory effect on the assembly of porcine brain tubulin in vitro. The 30%-inhibition concentration was 0.3 mM AFB1. The 8 tumor promoters showed different effects. Five of them, anthralin, cholic acid, gamma-hexachlorocyclohexane (lindane, gamma-HCH), lithocholic acid and phenobarbital (PB), enhanced the in vitro assembly. The effect was reversible in the case of PB and anthralin, only partially reversible in the case of cholic acid and gamma-HCH, whereas the stimulating effects of lithocholic acid led to an irreversible modification of the tubulin structure, as shown by the insolubility of the microtubules at 0 degrees C. This could be confirmed by an electron microscopic study. The doses necessary for a 30% enhancement of the steady-state level were 3 mM (PB), 0.2 mM (anthralin), 6 mM (cholic acid), 0.7 mM (gamma-HCH) and less than 0.2 mM (lithocholic acid). The other 3 tumor promoters tested - diethylstilbestrol (DES), 4,4'-dichloro-diphenyl-trichloro-ethane (DDT) and saccharin - inhibited the assembly. The concentrations necessary for a 30% inhibition varied within a wide range: 0.025 mM, 0.4 mM and 7.5 mM for DES, DDT and saccharin, respectively. Five of the 9 miscellaneous compounds, namely asbestos (crocidolite), bavistan, colchicine, chloropropham and ethylacetate, showed inhibitory effects, whereas Fe2+ (a constituent of asbestos) and 5-azacytidine did not influence the assembly process. The 30%-inhibition concentrations for colchicine, ethylacetate and asbestos were 10 microM, 0.153 M and 0.19 mM, respectively. For bavistan and chloropropham the 30%-inhibition values were 0.7 mM and 2.0 mM, respectively. The inhibitory effects of chloropropham and asbestos were reversible. For colchicine and bavistan the reversibility of the effects was not assayed. In agreement with published data, dimethylsulfoxide (DMSO) and acetone enhanced the in vitro assembly of porcine brain tubulin. The doses needed for a 30% enhancement by DMSO and acetone were 0.4 mM and 0.136 M, respectively. The effect of DMSO was irreversible whereas acetone led to a reversible stimulation. Some compounds were tested for their influence on preformed microtubules (interaction with the equilibrium between assembly and disassembly). Anthralin, cholic acid, PB and DMSO showed no effect on the steady-state plateau. A slight reduction was induced by DDT and bavistan, whereas DES, colchicine and chloropropham led to a pronounced reduction.
Albertini
S
S
Institute of Toxicology, Swiss Federal Institute of Technology, Schwerzenbach.
Friederich
U
U
Holderegger
C
C
Würgler
F E
FE
D000348:Aflatoxins / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology*; D066298:In Vitro Techniques; D008870:Microtubules / Q000187:drug effects; D013552:Swine; D014404:Tubulin / Q000096:biosynthesis*
D000348:Aflatoxins; D002273:Carcinogens; D014404:Tubulin