Author(s): Sundberg K, Dreij K, Seidel A, Jernstrm B
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Abstract Mammalian V79 cells stably expressing human glutathione transferase (GST) A1-1, M1-1, and P1-1 (the allelic variant with Val105 and Ala114) have been constructed and characterized. The cells have been used to study the capacity of individual GST isoenzymes in conjunction with GSH to detoxify diol epoxides from dibenzo[a,l]pyrene (DBPDE), the most carcinogenic polycyclic aromatic hydrocarbon (PAH) identified so far, and diol epoxides from benzo[a]pyrene (BPDE). The relationship between GSH-conjugation and DNA adduct-formation has been investigated as well as factors governing the accessibility of lipophilic diol epoxide substrates for the soluble GSTs in the cells. Relative to control cells, those expressing GSTA1-1 showed the highest rate (about 50-fold increase) to perform GSH-conjugation of (-)-anti-DBPDE (R-absolute configuration at the benzylic oxirane carbon in the fjord-region) followed by GSTM1-1 (25-fold increase) and GSTP1-1 (10-fold increase). GSTA1-1 was found to be strongly inhibited when expressed in cells (10\% of fully functional protein). Taking this factor into account, the rates of conjugation found in the cells fairly well reflected the order of catalytic efficiencies (k(cat)/K(m)) obtained with the pure enzymes. Increased GSH conjugation of (-)-anti-DBPDE was associated with a reduction in DNA adduct formation. GSTA1-1 inhibited the formation of adducts more than 6-fold and GSTM1-1 and GSTP1-1 about 2-fold. With (+)-anti-BPDE, GSTP1-1-expressing cells demonstrated a substantially higher rate of GSH-conjugate formation than cells with GSTA1-1 and GSTM1-1 cells (33- and 10-fold increase, respectively). Relative to control cells, GSTM1-1 was found to inhibit DNA adduct formation of (+)-anti-BPDE most effectively followed by GSTP1-1 and GSTA1-1 (12-, 4-, and 3-fold, respectively). Values of k(cat)/K(m) and estimated oil/water partition coefficients of DBPDE and BPDE were used to calculate the concentration of free diol epoxides in solution and expected rates of GSH conjugate formation in cells, and these theoretical results were compared with the observed ones. With the highly reactive (+)-anti-BPDE, 1-2\% of the expected activity was observed, whereas the corresponding values for the less reactive (-)-anti-DBPDE were up to 13\%. The most obvious explanations for the low observed rate with (+)-anti-BPDE are rapid and competing reactions such as hydrolysis and/or more unspecific chemical and physical reactions with cellular constituents (proteins, lipids, nucleic acids, etc.). In addition, the difference between the theoretical and observed rates may also reflect participation of factors such as macromolecular crowding and reduced rates of diffusion, factors expected to further restrict the accessibility of GST and the diol epoxides in the intact cell.
This article was published in Chem Res Toxicol
and referenced in Journal of Analytical & Bioanalytical Techniques