Purification and characterization of a benzo[a]pyrene hydroxylase from Pleurotus pulmonarius.

Cytochrome P450 has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by white-rot fungi. We have purified and reconstituted a benzo[a]pyrene hydroxylating cytochrome P450 (P450) from microsomal fractions of the white rot fungus Pleurotus pulmonarius. The microsomal P450 was recovered using a combination of n-aminooctyl agarose and hydroxyapatite chromatography and had an apparent molecular mass of 55 kDa. The purified protein exhibited moderate affinity for benzo[a]pyrene with a K(s) of 66 microM calculated from the Type I substrate binding spectra produced. Reconstitution of activity was achieved and a turnover of 0.75 nmol 3-hydroxybenzo[a]pyrene product/min/nmol P450 was observed, comparable to levels of metabolism observed by animal cytochromes P450 involved in xenobiotic detoxification.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on benzo(a)pyrene hydroxylase activity in embryos of the Japanese medaka (Oryzias latipes).

When Japanese medaka embryos were exposed to 12 ng/l 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) beginning on the day of fertilization (day 0), benzo(a)pyrene hydroxylase (B(a)PH) activity was induced in the whole embryo 105,000 g fraction by day 5 of development, which coincided with liver development. The induction of B(a)PH activity also coincided with the appearance of 2,3,7,8-TCDD induced hemorrhagic and edematous lesions. B(A)PH induction only occurred in embryos exposed to toxic concentrations (greater than 10 ng/l) of 2,3,7,8-TCDD. B(a)PH induction also occurred in embryos after exposure to 10 ng/l 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 50 micrograms/l 1,2,7,8-TCDD. Both 2,3,7,8-TCDF and 1,2,7,8-TCDD are toxic to Japanese medaka embryos at concentrations that resulted in the induction of B(a)PH activity. B(a)PH activity was not induced by the non-toxic congener 1,3,6,8-TCDD at concentrations as high as 50 micrograms/l. The structure activity relationship for B(a)PH induction in Japanese medaka embryos was similar to that which is observed in other species and biological systems, suggesting that the biological activities of these compounds may also be mediated through the putative Ah receptor in these fish embryos. At 50 micrograms/l, beta-naphthoflavone (BNF) induced B(a)PH activity in Japanese medaka embryos to similar levels as 2,3,7,8-TCDD did at toxic concentrations. However, at 50 micrograms/l, BNF was not toxic to Japanese medaka embryos. Therefore, the induction of B(a)PH activity probably did not directly result in the toxicity observed in these fish embryos after exposure to 2,3,7,8-TCDD.