Control of the mutagenicity of aromatic amines by protein kinases and phosphatases. I. The protein phosphatase inhibitors okadaic acid and ortho-vanadate drastically reduce the mutagenicity of aromatic amines.
The role of protein kinase C and protein phosphatases was examined in the control of mutagenic metabolites of aromatic amines. Various metabolic activating systems derived from rat liver were treated with: 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C modulator; okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatases (PP1 and PP2A); and ortho-vanadate (OV), an inhibitor of tyrosine phosphatases. TPA used over a wide concentration range (10(-9)-10(-6) M) did not affect the bacterial mutagenicity of the aromatic amines and of the aromatic amide investigated, 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene (2AAF). At the molecular level, TPA did not affect the function of cytochrome P450s 1A1 or 1A2, which are known key factors for the activation and inactivation of aromatic amines/amides. By contrast the OA and OV treatment of rat hepatocytes, rat liver homogenate, fraction S9 and the nuclear fraction drastically reduced (by > 80%) the mutagenicity of the aromatic amines/amide investigated. This is by far the most pronounced change in genotoxicity observed to date via modulation of phosphorylation. Whilst the mutagenicity of the primary toxication product 2-N-OH-acetylaminofluorene (2-N-OH-AAF) in the presence of exogenous activating systems (hepatocytes, S9-fraction, nuclear fraction) was also reduced by OV, OA had no influence. Thus the tyrosine protein phosphatase inhibitor and the serine/threonine protein phosphatase inhibitor influence the genotoxicity of aromatic amines/amides on different levels. Moreover, this shows that the drastic reduction in mutagenicity by OA was due to its influence on a step prior to the presence of the primary toxication product 2-N-OH-AAF. This reduction could be due to changes in the activity of cytochrome P4501A1 and/or 1A2. However, no incorporation of 32P-labelled phosphate from intracellularly prelabelled [32P]-ATP into cytochromes P450 1A1 or 1A2 nor any change in their catalytic activities was observed in the presence of OA. Furthermore, a phosphorylation dependent change in the function of P-glycoprotein (known for its role in the transport of diverse xenobiotic substances and their metabolites) was shown not to contribute to the observed decrease in mutagenicity. Our results reveal an important role for protein phosphatase 1 and/or 2A and tyrosine phosphatase(s) in the control of the genotoxicity of aromatic amines and amides. However, the present study does not distinguish between effects mediated by individual proteins affected by these protein phosphatases.
Oesch-Bartlomowicz
B
B
Institute of Toxicology, University of Mainz, Germany.
Arens
H J
HJ
Richter
B
B
Hengstler
J G
JG
Oesch
F
F
D015073:2-Acetylaminofluorene / Q000633:toxicity*; D000818:Animals; D000873:Anthracenes / Q000633:toxicity*; D001711:Biotransformation; D002273:Carcinogens / Q000633:toxicity; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005449:Fluorenes / Q000633:toxicity*; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity*; D019319:Okadaic Acid / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D054645:Protein Phosphatase 1; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000008:administration & dosage; D014638:Vanadates / Q000494:pharmacology*
D000873:Anthracenes; D002273:Carcinogens; D004791:Enzyme Inhibitors; D005449:Fluorenes; D009153:Mutagens; D019319:Okadaic Acid; C012177:2-aminofluorene; D014638:Vanadates; C006593:2-anthramine; D015073:2-Acetylaminofluorene; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D013755:Tetradecanoylphorbol Acetate