Biotransformation of the polycyclic aromatic hydrocarbon pyrene in the marine polychaete Nereis virens

Anne Jørgensen

Studenteropgave: Speciale


#The marine polychaete Nereis virens is an important species in estuarine sediments, which are often impacted by anthropogenic persistent compounds including polycyclic aromatic hydrocarbons (PAHs). Therefore, infaunal deposit-feeding invertebrates like Nereis virens can be extensively exposed to PAHs due to feeding activity and surface contact. The degree of systemic accumulation of PAHs depends on intake but also on efficiency of biotransformation by the organism. N. virens is known to extensively biotransform PAHs. In this study, in vivo and in vitro biotransformation studies were conducted using the model PAH pyrene. Assays were designed to characterise activities of phase I and II enzymes isolated from gut tissue by measuring the production of 1-hydroxypyrene, pyrene-1-glucuronide, pyrene-1-sulfate and pyrene-1-glucoside by high-pressure liquid chromatography (HPLC). Formation of metabolites was used to estimate the activity of pyrene hydroxylase, glucuronosyl transferase, and sulfotransferase. Pyrene hydroxylase activity was determined to be cytochrome P450 (CYP) mediated and the prominent phase II pathway in this organism was glucuronidation. Pyrene hydroxylase activity was significantly induced after pre-exposure to sediment associated pyrene (10 μg/g dry wt) whereas none of the phase II enzymes appeared inducible after pre-exposure to pyrene and B(a)A in the chosen concentrations. Apparent kinetic parameters were determined for all enzymatic reactions and glucuronidation was confirmed as being the prominent phase II reaction, based on its high apparent maximum velocity (Vmax(a)). However, sulfotransferase enzymes had the lowest apparent Michaelis constant (Km(a)), indicating high specificity of this enzymatic reaction. Apparent kinetic parameters for pyrene hydroxylase activity were changed after induction with pyrene. Induced worms showed increased Vmax(a) and decreased Km(a) compared to noninduced worms, indicating that the relative amount of the CYP enzyme(s) responsible for pyrene hydroxylation was increased. Since CYP enzymes catalyse the initial step in biotransformation of PAHs and the kinetic results indicate that this might be the rate-limiting step in the overall biotransformation, further characterisation could be interesting. Therefore, complete cDNA sequences of two novel CYP genes were isolated from N. virens gut tissue. One was named CYP4BB1 and the other was named CYP342A1 by the Cytochrome P450 nomenclature committee. The cDNA sequence of both genes was heterologously expressed and the proteins purified. Both CYP enzymes had monooxygenase activity and catalysed hydroxylation of pyrene to 1-hydroxypyrene. Furthermore, site directed mutagenesis replacing the conserved cysteine residue of the heme binding domain with a serine residue resulted in complete loss of monooxygenase activity of both CYP enzymes. This indicates that the cysteine residue is essential for monooxygenase activity of invertebrate CYP enzymes, as has been well documented in vertebrates. The data on specific CYP enzymes in N. virens presented here contribute to the understanding of PAH biotransformation in this species, which is important in attempting to understand the differences in sensitivity towards PAHs among marine invertebrates.

UddannelserMiljøbiologi, (Bachelor/kandidatuddannelse) KandidatMolekylærbiologi, (Bachelor/kandidatuddannelse) Kandidat
Udgivelsesdato1 jun. 2005


  • CYP genes
  • phase II enzymes
  • Biotransformation
  • PAH
  • CYP enzymes
  • Nereis virens