Glutathione Conjugation and DNA Adduct Formation of Dibenzo[a,l]pyrene and Benzo[a]pyrene Diol Epoxides in V79 Cells Stably Expressing Different Human Glutathione Transferases

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.

DNA adduct burden and tobacco carcinogenesis

“…The authors caution against extrapolating catalytic information from pure enzymes to the complex situation within the intact cell. Notably, they found only about 1 -2% of the expected rate of BPDE conjugation to be observed in vivo in cells, which the authors postulate may be a result of the reduced concentrations of lipophilic substrates available for conjugation by the soluble GSTs (Sundberg et al, 2002).…”

“…A re-examination of GSTP1 genotype -phenotype relationships (Sundberg et al, 2002) has suggested that the GSTP1 enzyme containing valine at position 105 may actually have less than, or at most equal activity in reducing, PAH -DNA adduct formation compared with the wildtype GSTP1 Ile105/Ala114 isoform. The authors caution against extrapolating catalytic information from pure enzymes to the complex situation within the intact cell.…”

Ultra-performance liquid chromatography–tandem mass spectrometry for rapid and highly sensitive analysis of stereoisomers of benzo[a]pyrene diol epoxide–DNA adducts

“…The metabolic intermediates of benzo[a]pyrene, 7,8-epoxide and trans -7,8-diol, as well as the two stereoisomeric diol epoxides (syn-and anti-BPDE) are all optically active. Glutathione conjugates (for detoxification) also display optical activity [12]. The frequency and ratio of stereoisomeric BPDE-DNA adducts, the endpoint of metabolic intermediates, can be used as a stereochemical indicator of these optically active intermediates if the developed analytical method has adequate sensitivity.…”

Integrating Field Analyses with Laboratory Exposures to Assess Ecosystems Health

“…GSTs encoded by polymorphic members of the mu (GSTM1), pi (GSTP1) and theta (GSTT1) gene families play important roles in the detoxication of a variety of reactive toxic and carcinogenic compounds, including PAH epoxides and diol epoxides (Hecht, 2002a). Isoforms involved in the formation of glutathionyl conjugates with PAH diol epoxides have been assigned (Jernström et al, 1996;Sundberg et al, 1998Sundberg et al, , 2002 and their rank order of importance based on catalytic efficiency (catalytic constant (k cat )/Michaelis constant (K m )) varies by PAH diol epoxide and individual stereochemistry. For example, for the conjugation of the (-)-anti-diol epoxide dibenzo [a,l]pyrene (R-configuration at the benzylic oxirane carbon in the fjord-region), the preference is GSTA1-1 > GSTM1-1 > GSTP1-1.…”