2‐aminofluorene bioactivation by human term placental peroxidase

Abstract

Earlier investigations from our laboratory demonstrated that human term placental peroxidase (HTPP) is capable of metabolism of xenobiotics and endogenous compounds. In this study, purified HTPP was found to bioactivate 2-aminofluorene (2-AF) in the presence of H2O2. 2-AF oxidation was studied spectrophotometrically while radiometry was employed to assess the bioactivation. The rate of oxidation and covalent binding to protein and DNA was dependent upon the pH of the reaction medium and the concentration of 2-AF, the enzyme, and H2O2. To observe maximal enzyme velocity of oxidation, the presence of 16.5 microM H2O2, 100 microM 2-AF, 37 micrograms of the enzyme protein/ml, and pH 7.2 was required. Under optimal assay conditions, the range of specific activity between 130 and 165 nmol of 2-AF oxidized/min/mg HTPP was observed. Using similar assay conditions, the magnitude of covalent binding of [3H]-2-AF to protein (BSA) and calf thymus DNA was found to be about 508 pmol bound/min/mg HTPP/mg BSA and 84 pmol bound/min/mg HTPP/mg DNA, respectively. Potassium cyanide and sodium azide, the known inhibitors of different peroxidases, significantly blocked both the oxidation and covalent binding of 2-AF in a dose dependent manner. These results strongly suggest that peroxidase may be one of the important pathways responsible for the bioactivation of arylamines in human term placenta.

Role of Biotransformation in Conceptal Toxicity of Drugs and Other Chemicals

“…The importance of these findings is heightened by the observations that the same or similar enzyme capable of xenobiotic oxidation also occurs in human intrauterine tissues during the organogenesis ( 4-6 weeks of gestation) [213] and early fetal (10 weeks of gestation) [98] periods. Quantitatively, it is noteworthy that the partially 10 Wk Fetal [98] 3,3'-Dimethoxybenzidine Term placenta [209] 4-6 Wk Conceptus [213] Pyrogallol Term placenta [209] 4-6 Wk Conceptus [213] Tetramethyl phenylenediamine Term placenta [209] 4-6 Wk Conceptus [213] 10 Wk Fetal [98] Tetramethylbenzidine Term placenta [209] 4-6 Wk Conceptus [213] ABTS** Term placenta [209] 4-6 Wk Conceptus [213] Benzidine Term placenta [209] 4-6 Wk Conceptus [213] 10 Wk Fetal [98] Epinephrine Term placenta [209] 4-6 Wk Conceptus [213] Bilirubin Term placenta [209] 4-6 Wk Conceptus [213] p-Phenylenediamne Term placenta [209] 4-6 Wk Conceptus [213] 2-Aminofluorene Term placenta [208] 4-6 Wk Conceptus [213] 8 Wk Conceptus [214] purified conceptal peroxidase can catalyze oxidation of model substrates with several fold greater efficiency than the term placental enzyme [98,213]. Purified human fetal tissue peroxidase can also bioactivate the teratogen, 2-AF [214].…”

“…Nelson and Kulkarni,[206] noted that in human term placenta, the highest concentration of the enzyme occurs in mitochondria while substantial amounts of the activity also resides in microsomes and nuclei as well. The methods to extract and purify the membrane-bound enzyme to the electrophoretic homogeneity and free of hemoglobin are now available [206][207][208]. Besides endogenous chemicals such as bilirubin and epinephrine, the purified term placental peroxidase can oxidize several hydrogen donors (Table 5) such as guaiacol, pyrogallol, arylamines namely benzidine, 3,3',5,5'-tetramethyl benzidine, dimethoxy benzidine, ρ-phenylenediamine, tetramethylphenylenediamine, and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid [209].…”

“…Thiobenzamide, a model hepatotoxicant, serves as an excellent substrate for placental peroxidase and undergoes sulfoxidation [207]. Further studies have revealed that the term placental enzyme can activate animal teratogens or developmental toxicants such as BP-diol [210], 2-AF [208], as well as various phenothiazines [211] and eugenol (Table 5) [212]. The importance of these findings is heightened by the observations that the same or similar enzyme capable of xenobiotic oxidation also occurs in human intrauterine tissues during the organogenesis ( 4-6 weeks of gestation) [213] and early fetal (10 weeks of gestation) [98] periods.…”