Nitric oxide storage and transport in cells are mediated by glutathione S-transferase P1-1 and multidrug resistance protein 1 via dinitrosyl iron complexes.

Nitrogen monoxide (NO) plays a role in the cytotoxic mechanisms of activated macrophages against tumor cells by inducing iron release. We showed that NO-mediated iron efflux from cells required glutathione (GSH) (Watts, R. N., and Richardson, D. R. (2001) J. Biol. Chem. 276, 4724-4732) and that the GSH-conjugate transporter, multidrug resistance-associated protein 1 (MRP1), mediates this release potentially as a dinitrosyl-dithiol iron complex (DNIC; Watts, R. N., Hawkins, C., Ponka, P., and Richardson, D. R. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7670-7675). Recently, glutathione S-transferase P1-1 (GST P1-1) was shown to bind DNICs as dinitrosyl-diglutathionyl iron complexes. Considering this and that GSTs and MRP1 form an integrated detoxification unit with chemotherapeutics, we assessed whether these proteins coordinately regulate storage and transport of DNICs as long lived NO intermediates. Cells transfected with GSTP1 (but not GSTA1 or GSTM1) significantly decreased NO-mediated 59Fe release from cells. This NO-mediated 59Fe efflux and the effect of GST P1-1 on preventing this were observed with NO-generating agents and also in cells transfected with inducible nitric oxide synthase. Notably, 59Fe accumulated in cells within GST P1-1-containing fractions, indicating an alteration in intracellular 59Fe distribution. Furthermore, electron paramagnetic resonance studies showed that MCF7-VP cells transfected with GSTP1 contain significantly greater levels of a unique DNIC signal. These investigations indicate that GST P1-1 acts to sequester NO as DNICs, reducing their transport out of the cell by MRP1. Cell proliferation studies demonstrated the importance of the combined effect of GST P1-1 and MRP1 in protecting cells from the cytotoxic effects of NO. Thus, the DNIC storage function of GST P1-1 and ability of MRP1 to efflux DNICs are vital in protection against NO cytotoxicity.

Enhanced glutathione depletion, protein adduct formation, and cytotoxicity following exposure to 4-hydroxy-2-nonenal (HNE) in cells expressing human multidrug resistance protein-1 (MRP1) together with human glutathione S-transferase-M1 (GSTM1).

4-Hydroxy-2-nonenal (HNE) is one of the most reactive products of lipid peroxidation and has both cytotoxic and genotoxic effects in cells. Several enzymatic pathways have been reported to detoxify HNE, including conjugation by glutathione-S-transferases (GSTs). Removal of the resulting HNE-glutathione conjugate (HNE-SG) by an efflux transporter may be required for complete detoxification. We investigated the effect of expression of GSTM1 and/or the ABC efflux transporter protein, multidrug-resistance protein-1 (MRP1), on HNE-induced cellular toxicity. Stably transfected MCF7 cell lines were used to examine the effect of GSTM1 and/or MRP1 expression on HNE-induced cytotoxicity, GSH depletion, and HNE-protein adduct formation. Co-expression in the MCF7 cell line of GSTM1 with MRP1 resulted in a 2.3-fold sensitization to HNE cytotoxicity (0.44-fold IC(50) value relative to control) rather than the expected protection. Expression of either GSTM1 or MRP1 alone also resulted in slight sensitization to HNE cytotoxicity (0.79-fold and 0.71-fold decreases in IC(50) values, respectively). Co-expression of GSTM1 and MRP1 strongly enhanced the formation of HNE-protein adducts relative to the non-expressing control cell line, whereas expression of either MRP1 alone or GSTM1 alone yielded similarly low levels of HNE-protein adducts to that of the control cell line. Glutathione (GSH) levels were reduced by 10-20% in either the control cell line or the MCF7/GSTM1 cell line with the same HNE exposure for 60min. However, HNE induced >80% depletion of GSH in cells expressing MRP1 alone. Co-expression of both MRP1 and GSTM1 caused slightly greater GSH depletion, consistent with the greater protein adduct formation and cytotoxicity in this cell line. Since expression of GSTM1 or MRP1 alone did not strongly sensitize cells to HNE, or result in greater HNE-protein adducts than in the control cell line, these results indicate that MRP1 and GSTM1 collaborate to enhance HNE-protein adduct formation and HNE cytotoxicity, facilitated by GSH depletion mediated by both MRP1 and GSTM1.

Nitroalkene fatty acids mediate activation of Nrf2/ARE-dependent and PPARγ-dependent transcription by distinct signaling pathways and with significantly different potencies.

Naturally occurring nitroalkene fatty acids (NAs) derived from oleic (NO(2)-OA) and linoleic (NO(2)-LA) acids mediate a variety of cellular responses. We examined the signaling pathways involved in NA activation of Nrf2/ARE-dependent versus PPARγ/PPRE-dependent transcription in human MCF7 breast cancer cells. Additionally, we compared the relative potencies of NO(2)-OA and NO(2)-LA in activating these two transcriptional programs. Here it is demonstrated that, in addition to the direct adduct formation of NA with the Nrf2 inhibitory protein, Keap1, shown by others, NA activation of Nrf2/ARE-mediated transcription results from increased nuclear Nrf2 levels and depends upon activation of the PI3K/AKT and PKC, but not ERK and JNK MAPK, signaling pathways. Examination of the relationship between NA stimulation of the Nrf2/ARE versus PPARγ/PPRE transcriptional programs revealed concentration-dependent activation of distinct signaling pathways that were readily distinguished by selective attenuation of Nrf2/ARE-dependent, but not PPARγ-dependent, transcription by inhibitors of PI3K and PKC. Moreover, measurable, statistically significant activation of PPARγ/PPRE-dependent transcription occurred at nanomolar concentrations of NAs-the 12-NO(2) isomer of NO(2)-LA showing the most potent activity-whereas significant activation of Nrf2/ARE-dependent transcription occurred at much higher NA concentrations (≥3 µM) with the NO(2)-OA isomers the most potent. These findings have implications for the physiological roles of NAs, suggesting that, at concentrations likely to be encountered in vivo, their direct activation of PPARγ transcription will dominate over their electrophilic activation of Nrf2 antioxidant/protective responses.

Lung tumor promotion by curcumin.

Curcumin exhibits anti-inflammatory and antitumor activity and is being tested in clinical trials as a chemopreventive agent for colon cancer. Curcumin's chemopreventive activity was tested in a transgenic mouse model of lung cancer that expresses the human Ki-ras(G12C) allele in a doxycycline (DOX) inducible and lung-specific manner. The effects of curcumin were compared with the lung tumor promoter, butylated hydroxytoluene (BHT), and the lung cancer chemopreventive agent, sulindac. Treatment of DOX-induced mice with dietary curcumin increased tumor multiplicity (36.3 +/- 0.9 versus 24.3 +/- 0.2) and progression to later stage lesions, results which were similar to animals that were co-treated with DOX/BHT. Microscopic examination showed that the percentage of lung lesions that were adenomas and adenocarcinomas increased to 66% in DOX/BHT, 66% in DOX/curcumin and 49% in DOX/BHT/curcumin-treated groups relative to DOX only treated mice (19%). Immunohistochemical analysis also showed increased evidence of inflammation in DOX/BHT, DOX/curcumin and DOX/BHT/curcumin mice relative to DOX only treated mice. In contrast, co-treatment of DOX/BHT mice with 200 p.p.m. [DOSAGE ERROR CORRECTED] of sulindac inhibited the progression of lung lesions and reduced the inflammation. Lung tissue from DOX/curcumin-treated mice demonstrated a significant increase (33%; P = 0.01) in oxidative damage, as assessed by the levels of carbonyl protein formation, relative to DOX-treated control mice after 1 week on the curcumin diet. These results suggest that curcumin may exhibit organ-specific effects to enhance reactive oxygen species formation in the damaged lung epithelium of smokers and ex-smokers. Ongoing clinical trials thus may need to exclude smokers and ex-smokers in chemopreventive trials of curcumin.

Noncatalytic interactions between glutathione S-transferases and nitroalkene fatty acids modulate nitroalkene-mediated activation of peroxisomal proliferator-activated receptor gamma.

The naturally occurring nitroalkenes, nitrolinoleic (NO(2)-LA) and nitrooleic (NO(2)-OA) acids, are among the most potent endogenous ligand activators of PPARgamma-dependent transcription. In order to understand mechanisms that regulate cellular response to these nitroalkenes, we previously demonstrated that glutathione conjugation of NO(2)-LA and MRP1-mediated efflux of the conjugates were associated with significant attenuation of PPARgamma activation by this nitroalkene [(2006) Biochemistry 45, 7889-7896]. Here we show that NO(2)-OA activation of PPARgamma is similarly affected by nonenzymatic conjugation and MRP1-mediated efflux. Moreover, the roles of glutathione S-transferases (GSTs) in the glutathione conjugation and bioactivities of NO(2)-LA and NO(2)-OA were investigated. While none of the GST isozymes tested (GSTA1-1, A4-4, M1a-1a, and P1a-1a) enhanced the rate of glutathione conjugation, expression of GSTA1-1, M1a-1a, or P1a-1a in MCF7 cells significantly reduced the magnitude of PPARgamma-dependent reporter gene transcription in response to NO(2)-LA and NO(2)-OA treatment, with GSTP1a-1a expression mediating the most potent inhibition of PPARgamma. Although these GSTs failed to catalyze nitroalkene conjugation with glutathione, the nitroalkenes were found to associate avidly with all four GST isozymes as indicated by their ability to inhibit GST activity with K(i)'s in the nanomolar range. Treatment of purified GSTP1a-1a with excess NO(2)-LA and NO(2)-OA resulted in the formation of covalent adducts between GSTP1a monomers and nitroalkenes, although separate experiments indicated that such covalent bond formation was not necessary for avid GST-nitroalkene interactions. These results suggest that GSTs can inhibit the activation of transcription by nitroalkenes via noncatalytic sequestration of these ligands, and their glutathione conjugates, away from their nuclear target, PPARgamma.

Differential protection by human glutathione S-transferase P1 against cytotoxicity of benzo[a]pyrene, dibenzo[a,l]pyrene, or their dihydrodiol metabolites, in bi-transgenic cell lines that co-express rat versus human cytochrome P4501A1.

Polycyclic aromatic hydrocarbons (PAHs) are activated by cytochrome P450 (CYP) isozymes, and a subset of the reactive metabolites generated is detoxified via conjugation with glutathione (GSH) by specific glutathione S-transferases (GSTs). We have used V79MZ cells stably transfected with either human or rat cytochrome P4501A1 (CYP1A1), alone or in combination with human GSTP1 (hGSTP1), to examine the dynamics of activation versus detoxification of benzo[a]pyrene (B[a]P), dibenzo[a,l]pyrene (DB[a,l]P), and their dihydrodiol metabolites. The cytotoxicity of B[a]P or DB[a,l]P was 9-11-fold greater in cells expressing human, as compared to rat CYP1A1, despite similar enzymatic activities. Co-expression of the hGSTP1 with the hCYP1A1 conferred 16-fold resistance to B[a]P cytotoxicity, compared to only 2.5-fold resistance when hGSTP1 was co-expressed with rat CYP1A1. The lower B[a]P cytotoxicity in the cells expressing rat CYP1A1, and weaker protection by hGSTP1 co-expression in these cells, were attributable to the much lower fraction of B[a]P metabolism via formation of the 7,8-dihydrodiol intermediate by the rat CYP1A1 compared to hCYP1A1. Resistance to the DB[a,l]P cytotoxicity conferred by hGSTP1 expression was also greater in cells co-expressing hCYP1A1 (7-fold) as compared to cells co-expressing rCYP1A1 (<2-fold). Resistance to B[a]P conferred by hGSTP1 was closely correlated with the activity level in two clonal transfectant lines with a 3-fold difference in hGSTP1-1 specific activity. Depletion of GSH to 20% of control levels via pretreatment with the de novo GSH biosynthesis inhibitor buthionine sulfoximine reduced the protection against B[a]P cytotoxicity by hGSTP1 from 16-fold to 5-fold, indicating that catalysis of conjugation with GSH, rather than binding or other effects, is responsible for the resistance. The cytotoxicity of the dihydrodiol intermediates of B[a]P or DB[a,l]P was much greater, and similar in cell lines expressing either human or rat CYP1A1. Again, however, the protection conferred by hGSTP1 co-expression was 2-5-fold greater in cells with hCYP1A1 than with rCYP1A1 expression. These results indicate that GST expression can effectively limit cytotoxicity following activation of B[a]P by human or rat CYP1A1, but is less effective as a defense against exposure of cells to the intermediate metabolite B[a]P-7,8-dihydrodiol.

Cytotoxicity and mutagenicity of 5-methylchrysene and its 1,2-dihydrodiol in V79MZ cells modified to express human CYP1A1 or CYP1B1, in the presence or absence of human GSTP1 coexpression.

The environmental carcinogen 5-methylchrysene (5MC) can be activated to mutagenic metabolites by several isozymes of cytochrome P-450 (CYP). The resulting reactive diol-epoxides can be detoxified via conjugation by glutathione S-transferases (GST). We investigated whether expression of human glutathione S-transferase P1 (hGSTP1) would differentially protect cells against the cytotoxicity or mutagenicity of 5MC or its 1,2-dihydrodiol intermediate (5MC-1,2-diol) in V79MZ cells with activation via stably transfected human CYP1B1 (hCYP1B1) as compared to activation by human CYP1A1 (hCYP1A1). The parent compound 5MC was only 2-fold more cytotoxic in the CYP-expressing cell lines than in the V79MZ parental cell line, while 5MC-1,2-dihydrodiol was more than 30-fold more cytotoxic in CYP-transfected cells compared to V79MZ cells. Cells co-expressing either hCYP1B1 or hCYP1A1 together with hGSTP1 were 2-fold less sensitive to 5MC or 5MC-1,2-diol cytotoxicity than their CYP-only parent lines. The 5MC was highly mutagenic with similar potency in both hCYP-transfected cell lines, while 5MC-1,2-diol was 2-fold more mutagenic in hCYP1B1-transfected cells as compared to hCYP1A1 cells. Coexpression of hGSTP1 with either hCYP reduced 5MC or 5MC-1,2-diol mutagenicity by 1.4-4.5-fold compared to the corresponding hCYP-only expressing cell lines. The greater protection against mutagenicity of 5MC is in contrast to our previous studies in which we found greater protection by hGSTP1 against cytotoxicity than mutagenicity of benzo[a]pyrene in cells co-expressing hCYP1A1. Protection against mutagenicity by hGSTP1 was greater with activation of either compound by hCYP1B1 than with hCYP1A1 activation. These studies show that the relative efficacy of protection by hGSTP1 against mutagenicity of 5MC or 5MC-1,2-diol is in part determined by the specific CYP pathway that catalyzes activation to the toxic or mutagenic metabolites.

Role of glutathione S-transferase P1-1 in the cellular detoxification of cisplatin.

Cells expressing elevated levels of allelic variants of human glutathione S-transferase P1 (GSTP1) and/or efflux transporters, MRP1 or MRP2, were used to evaluate the role of GSTP1-1 in cisplatin resistance. These studies revealed that GSTP1-1 confers low-level resistance (1.4- to 1.7-fold) to cisplatin-induced cytotoxicity in MCF7 cells. However, expression of MRP1 (MCF7 cells) or MRP2 (HepG2 cells) failed to augment or potentiate GSTP1-1-mediated resistance in either cell line. To understand the mechanism by which variants of GSTP1-1 confer resistance to cisplatin, their relative abilities to catalyze conjugation of cisplatin with glutathione were examined. Enzymes encoded by all three alleles tested, GSTP1a (I(104)A(113)), GSTP1b (V(104)A(113)), and GSTP1c (V(104)V(113)), increased the formation rate of the mono-platinum/glutathione derivative of cisplatin with relative catalytic activities of 1.0 (GSTP1a-1a variant) and 1.8 to 1.9 (GSTP1b-1b and GSTP1c-1c variants). Although these data are consistent with the idea that very low level resistance to cisplatin may be conferred by GSTP1-1-mediated cisplatin/glutathione conjugation, two observations indicate that such catalysis plays a minor role in the protection from cisplatin toxicity. First, the rates of GSTP1-1-mediated conjugation are extremely slow (1.7-2.6 h(-1) at 25 degrees C). Second, despite an 80% to 90% increase in catalysis of cisplatin conjugation by GSTP1b-1b or GSTP1c-1c over GSTP1a-1a, we observed no discernable differences in relative resistances conferred by these alternative variants when expressed in MCF7 cells. We conclude that high-level cisplatin resistance attributed to GSTP1-1 in other studies is not likely due to catalysis of cisplatin conjugation but rather must be explained by other mechanisms, which may include GSTP1-mediated modulation of signaling pathways.

Expression of MRP1 and GSTP1-1 modulate the acute cellular response to treatment with the chemopreventive isothiocyanate, sulforaphane.

A major component of the anticarcinogenic activity of the dietary chemopreventive agent sulforaphane (SFN) is attributed to its ability to induce expression of phase II detoxification genes containing the antioxidant response element (ARE) within their promoters. Because SFN is a reactive electrophile--readily forming conjugates with glutathione (GSH)--we asked whether expression of glutathione S-transferase (GST) P1-1 and the GSH conjugate efflux pump, multidrug resistance or resistance-associated protein (MRP) 1, would significantly modify the cellular response to SFN exposure. This was investigated using GST- and MRP1-poor parental MCF7 cells and transgenic derivatives expressing GSTP1-1 and/or MRP1. Compared with parental cells, expression of GSTP1-1 alone enhanced the rate of intracellular accumulation of SFN and its glutathione conjugate, SFN-SG--an effect that was associated with increased ARE-containing reporter gene induction. Expression of MRP1 greatly reduced SFN/SFN-SG accumulation and resulted in significant attenuation of SFN-mediated induction of ARE-containing reporter and endogenous gene expression. Coexpression of GSTP1-1 with MRP1 further reduced the level of induction. Depletion of GSH prior to SFN treatment or the substitution of tert-butylhydroquinone for SFN abolished the effects of MRP1/GSTP1-1 on ARE-containing gene induction-indicating that these effects are GSH dependent. Lastly, analysis of NF-E2-related factor 2 (Nrf2)--a transcription factor operating via binding to the ARE--showed that the increased levels of Nrf2 following SFN treatment were considerably less sustained in MRP1-expressing, especially those coexpressing GSTP1-1, than in MRP1-poor cells. These results suggest that the regulating effects of MRP1 and GSTP1-1 expression on SFN-dependent induction of phase II genes are ultimately mediated by altering nuclear Nrf2 levels.

Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (+/-)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1.

We have used V79MZ hamster lung fibroblasts stably transfected with human cytochrome P450-1A1 (hCYP1A1; cell line designated V79MZh1A1) or P450-1B1 (hCYP1B1; cell line designated V79MZh1B1) alone, or in combination with human glutathione-S-transferase (GST) alpha-1 (hGSTA1), in order to examine GST protection against cytotoxicity and mutagenicity of dibenzo[a,l]pyrene (DBP) and the intermediate dihydrodiol metabolite (+/-)-DBP-11,12-dihydrodiol (DBPD). At comparable expression levels of hCYP1A1 and hCYP1B1, both DBP and DBPD were more cytotoxic in V79MZ1A1 (IC(50)=2.7 and 0.7nM, respectively) than in V79MZh1B1 (IC(50)=6.0 and 4.8nM, respectively). In contrast, both DBP and DBPD were two- to four-fold more mutagenic in V79MZh1B1 than in V79MZ1A1. Co-expression of hGSTA1 with hCYP1A1 decreased DBP cytotoxicity two-fold compared to V79MZh1A1 with hCYP1A1 alone, and provided a small, yet still statistically significant, 1.3-fold protection against DBPD. Protection against mutagenicity of these compounds was comparable to that for cytotoxicity in cells expressing hCYP1A1. In V79MZh1B1 cells, co-expression of hGSTA1 conferred up to five-fold protection against DBP cytotoxicity, and up to nine-fold protection against the (+/-)-DBP-dihydrodiol cytotoxicity relative to the cells expressing hCYP1B1 alone. Co-expression of hGSTA1 also reduced mutagenicity of DBP or its dihydrodiol to a lesser extent (1.3-1.8-fold) than the protection against cytotoxicity in cells expressing hCYP1B1. These findings demonstrate that the protective efficacy of hGSTA1 against DBP and DBPD toxicity is variable, depending on the compound or metabolite present, the specific cytochrome P450 isozyme expressed, and the specific cellular damage endpoint examined.

Protective efficacy of hGSTM1-1 against B[a]P and (+)- or (-)-B[a]P-7,8-dihydrodiol cytotoxicity, mutagenicity, and macromolecular adducts in V79 cells coexpressing hCYP1A1.

Transgenic cell lines were constructed to study the dynamics of competition between activation versus detoxification of benzo[a]pyrene (B[a]P) or B[a]P-7,8-dihydrodiol metabolites. Stably transfected V79MZ cells expressing human cytochrome P4501A1 (hCYP1A1) alone or in combination with human glutathione-S-transferase M1 (hGSTM1) were used to determine how effectively this GST isozyme protects against cytotoxic, genotoxic, and mutagenic effects of B[a]P or the enantiomeric dihydrodiol metabolites (+)-benzo[a]pyrene-7,8-dihydrodiol ((+)-B[a]P-7,8-diol) and (-)-benzo[a]pyrene-7,8-dihydrodiol ((-)-B[a]P-7,8-diol). Expression of hGSTM1 in the presence of hCYP1A1 conferred significant 8.5-fold protection against B[a]P-induced cytotoxicity, but protection against cytotoxicity of either B[a]P-7,8-diol enantiomer was not significant. Mutagenicity of B[a]P at the hprt locus was dose and time dependent in cells that expressed hCYP1A1. Mutagenicity of B[a]P was reduced by 21-32% and mutagenicity induced by the B[a]P-7,8-diols was reduced 20-58% in cells further modified to coexpress hGSTM1-1 compared to cells expressing hCYP1A1 alone. Expression of hGSTM1-1 reduced adducts in total cellular macromolecules by twofold, in good correlation with the reduction in B[a]P mutagenicity. These results indicate that while hGSTM1-1 effectively protects against hCYP1A1-mediated cytotoxicity of B[a]P, a significant fraction of the mutagenicity that results from activation of B[a]P and its 7,8-dihydrodiol metabolites by hCYP1A1 is derived from B[a]P metabolites that are not detoxified by hGSTM1.

Expression of human glutathione S-transferase P1 confers resistance to benzo[a]pyrene or benzo[a]pyrene-7,8-dihydrodiol mutagenesis, macromolecular alkylation and formation of stable N2-Gua-BPDE adducts in stably transfected V79MZ cells co-expressing hCYP1A1.

Transgenic cell lines were constructed to study dynamic competition between activation versus detoxification of benzo[a]pyrene (B[a]P) and its metabolites. Transfected V79MZ cells expressing human cytochrome P4501A1 (hCYP1A1) alone, or expressing hCYP1A1 in combination with human glutathione S-transferase P1 (hGSTP1), were used to determine how effectively GST protects against macromolecular damage or mutagenicity of B[a]P or its enantiomeric dihydrodiol metabolites (+)-benzo[a]pyrene-7,8-dihydrodiol [(+)B[a]P-7,8-diol] and (-)-benzo[a]pyrene-7,8-dihydrodiol [(-)-B[a]P-7,8-diol]. Mutagenicity of B[a]P at the hprt locus was dose- and time-dependent in cells that expressed hCYP1A1. Mutagenicity was reduced in cells further modified to co-express hGSTP1. Dose-response and time-course studies indicated that mutagenicity was reduced up to 3-fold by hGSTP1 expression, compared with cells expressing hCYP1A1 alone. Mutagenicity induced by the B[a]P 7,8-dihydrodiols was also dose-dependent, and was reduced 2- to 5-fold by hGSTP1. Expression of hGSTP1 reduced B[a]P adducts in total cellular macromolecules by 3.8-fold, which correlated with the reduction in B[a]P mutagenicity and with reduction in the formation of the proximate metabolite B[a]P 7,8-dihydrodiols from B[a]P. However, measurement of total B[a]P metabolites bound to DNA isolated from cells incubated with [3H]-B[a]P revealed only 12, 33 and 24% reduction at 12, 24 and 48 h, respectively, by GSTP1 expression. Nevertheless, (32)P-post-labeling analysis demonstrated nearly total prevention of the known B[a]P-DNA adduct, N2-guanine-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), in cells co-expressing hGSTP1. This adduct, thought to be the most mutagenic of the stable B[a]P adducts, accounts for 15% or less of the total DNA adducts observed. These results indicate that the reduction in hCYP1A1-mediated B[a]P mutagenesis by hGSTP1 is probably largely due to prevention of the N2-guanine-BPDE adduct. However, the significant fraction (30-40%) of this mutagenesis and the majority of the total DNA binding that are not prevented together suggest formation by hCYP1A1 of a subset of mutagenic metabolites of B[a]P that are not effectively detoxified by hGSTP1.

Induction of DNA-protein crosslinks by dichloromethane in a V79 cell line transfected with the murine glutathione-S-transferase theta 1 gene.

Dichloromethane (DCM) is considered a probable human carcinogen. Laboratory studies have shown an increased incidence of lung and liver cancer in mice but not in rats or hamsters. Despite the correlation between metabolism of DCM by the glutathione-S-transferase (GST) pathway and the occurrence of tumors in different species, the mechanism of tumor induction by DCM metabolites produced through the GST pathway remains unclear. In this study a V79 cell line stably transfected with the murine GST theta 1 gene (mGSTT1) was compared to the parent cell line (MZ) to determine how the construct affects DCM metabolism and the sensitivity of the cell line to DNA damage and cytotoxicity. V79 cells were treated with DCM (2.5-10mM) or formaldehyde (150-600muM) for 2h. Also, formaldehyde produced by V79 cytosol metabolism of DCM was measured spectrophotometrically. DNA damage and DNA-protein crosslinks were measured by the standard and proteinase K-modified alkaline single cell gel electrophoresis (SCG) assays. Cytotoxicity was assessed by trypan blue stain exclusion, the Live/Dead((R)) cell viability/cytotoxicity kit for animal cells, and the neutral red assay. After DCM treatment a significant concentration-dependent increase in tail moment in the V79 MZ cells was observed compared to a significant concentration-dependent decrease in tail moment in the V79 mGSTT1 cells. Post-incubation with proteinase K significantly increased DNA migrations in DCM-treated V79 mGSTT1 cells. DCM formed significantly higher levels of formaldehyde in the cytosol of the V79 mGSTT1 cells than in the cytosol of the V79 MZ cells. Results using the cytotoxicity assays were comparable using the trypan blue and Live/Dead((R)) assays, neither showing a difference in response between the two cell lines when exposed to either formaldehyde or DCM. These results indicate that V79 mGSTT1 can metabolize DCM to a genotoxic and cytotoxic metabolite, which is likely formaldehyde. This is the first time that the magnitude of the GSTT1 effect can be observed in mammalian cells without confounding caused by using cells with different genetic backgrounds.

Expression of glutathione S-transferases in fetal lung and liver tissue from parental strains and F1 crosses between C57BL/6 and BALB/c F1 mice following in utero exposure to 3-methylcholanthrene.

GST isoforms have been extensively studied in adult tissues but little is known about the composition and levels of these enzymes in fetal tissues. As part of our ongoing studies to determine the potential role of metabolic enzymes in mediating the differential susceptibility of different strains of mice to lung tumorigenesis following in utero exposure to 3-methylcholanthrene (MC), we screened for GST enzyme activity and for expression of the individual GSTalpha, pi, mu, and theta isoforms in murine fetal lung and liver tissues isolated from the parental strains and F1 crosses between C57BL/6 (B6) and BALB/c (C) mice. Using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, we found that treatment with MC had no effect on the levels of GST enzyme activity in either the fetal lung or liver in either of the two parental strains or their F1 crosses. Low levels of expression of each of the four enzymes were detected by Western blotting in both fetal lung and liver tissues in all four strains. A statistically significant 3.5-fold induction was observed only for GSTmu in the fetal lung of the parental strain of BALB/c mice 48 h after exposure to MC. None of the other enzymes showed any significant differences in the levels of expression following exposure to MC. Although strain-specific differences in the expression of the GSTs that were independent of MC treatment were observed, they could not account for the differences previously observed in either the Ki-ras mutational spectrum or lung tumor incidence in the different strains of mice. Similar results were obtained when the maternal metabolism of MC was assayed in liver microsomal preparations. The results are consistent with previous studies showing low levels and poor inducibility of phase II enzymes during gestation, and demonstrate for the first time that all four of the major GST enzymes are expressed in fetal tissues. While the high inducibility of activating enzymes, such as Cyp1a1, and low, uninducible levels of phase II conjugating enzymes probably account for the high susceptibility of the fetus to transplacentally induced tumor formation, the results also suggest that factors other than metabolism may account for the strain-specific differences in susceptibility to carcinogen-mediated lung tumor induction following in utero exposure to chemical carcinogens.

Multidrug resistance protein 1 (MRP1, ABCC1) mediates resistance to mitoxantrone via glutathione-dependent drug efflux.

Based upon several previous reports, no consistent relationship between multidrug resistance protein 1 (MRP1, ABCC1) expression and cellular sensitivity to mitoxantrone (MX) toxicity can be ascertained; thus, the role of MRP1 in MX resistance remains controversial. The present study, using paired parental, MRP1-poor, and transduced MRP1-overexpressing MCF7 cells, unequivocally demonstrates that MRP1 confers resistance to MX cytotoxicity and that resistance is associated with reduced cellular accumulation of MX. This MRP1-associated reduced accumulation of MX was partially reversed by treatment of cells with 50 microM MK571 [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]-an MRP inhibitor that increased MX accumulation in MRP1-expressing MCF7 cells but had no effect on MRP-poor MCF7 cells. Moreover, in vitro experiments using inside-out membrane vesicles show that MRP1 supports ATP-dependent, osmotically sensitive uptake of MX. Unlike ABCG2 (breast cancer resistance protein, mitoxantrone-resistant protein), MRP1-mediated MX transport is dependent upon the presence of glutathione or its S-methyl analog. In addition, MX stimulates transport of [3H]glutathione. Together, these data are consistent with the interpretation that MX efflux by MRP1 involves cotransport of MX and glutathione. The results suggest that MRP1-like the alternative MX transporters ABCG2 and ABCB1 (MDR1, P-glycoprotein)-can significantly influence tumor cell sensitivity to and pharmacological disposition of MX.

Cancer chemotherapy and drug metabolism.

Drug-metabolizing enzymes and drug transporters are key determinants of the pharmacokinetics and pharmacodynamics of many antineoplastic agents. Metabolism and transport influence the cytotoxic effects of antineoplastic agents in target tumor cells and normal host tissues. This article summarizes several state-of-the-art approaches to enhancing the effectiveness and safety of cancer therapy based on recent developments in our understanding of antineoplastic drug metabolism and transport. Advances in four interrelated research areas presented at a recent symposium sponsored by the Division for Drug Metabolism of the American Society for Pharmacology and Experimental Therapeutics (Experimental Biology 2004; Washington D.C., April 17-21, 2004) are discussed: 1) interactions of anthracyclines with drug-metabolizing enzymes; 2) use of hypoxia-selective gene-directed enzyme prodrug therapy (GDEPT) in combination with bioreductive prodrugs; 3) synergy between glutathione conjugation and conjugate efflux in conferring resistance to electrophilic toxins; and 4) use of cytochromes P450 as prodrug-activating enzymes in GDEPT strategies. A clear theme emerged from this symposium: drug metabolism and transport processes can be modulated and exploited in ways that may offer distinct therapeutic advantages in the management of patients with cancer.

Dynamics of glutathione conjugation and conjugate efflux in detoxification of the carcinogen, 4-nitroquinoline 1-oxide: contributions of glutathione, glutathione S-transferase, and MRP1.

4-Nitroquinoline 1-oxide (NQO) is a reactive electrophile with potent cytotoxic as well as genotoxic activities. NQO forms a conjugate, QO-SG, with glutathione, which greatly reduces its chemical reactivity. Previous studies demonstrated that glutathione S-transferase (GST) P1a-1a and multidrug resistance protein (MRP) 1/2 act in synergy to confer resistance to both cyto- and genotoxicities of NQO, whereas protection afforded by GSTP1a-1a or MRP alone was much less. To better understand the role of glutathione, GSTP1a-1a, and MRP1 in NQO detoxification, we have characterized the kinetics and cofactor requirements of MRP1-mediated transport of QO-SG and NQO. Additionally, using recombinant GSTP1a-1a and physiological conditions, we have examined the enzymatic and nonenzymatic formation of QO-SG. Results show that MRP1 supports efficient transport of QO-SG with a K(m) of 9.5 microM and a V(max) comparable to other good MRP1 substrates. Glutathione or its S-methyl analogue enhanced the rate of (3)H-QO-SG transport, whereas QO-SG inhibited the rate of (3)H-glutathione transport. These data favor a mechanism for glutathione-enhanced, MRP1-mediated QO-SG transport that does not involve cotransport of glutathione. NQO was not transported by MRP1 either alone or in the presence of S-methyl glutathione. Transport of (3)H-NQO was observed in the presence of glutathione, but uptake into MRP1-containing vesicles was entirely attributable to its conjugate, QO-SG, formed nonenzymatically. While the nonenzymatic rate was readily measurable, enzyme catalysis was overwhelmingly dominant in the presence of GSTP1a-1a (rate enhancement factor, (k(cat)/K(m))/k(2), approximately 3 x 10(6)). We conclude that MRP1 supports detoxification of NQO via efficient, glutathione-stimulated efflux of QO-SG. While nonenzymatic QO-SG formation and MRP1-mediated conjugate efflux result in low-level protection from cyto- and genotoxicities, this protection is greatly enhanced by coexpression of GSTP1-1 with MRP1. This result emphasizes the quantitative importance of enzyme-catalyzed conjugate formation, a crucial determinant of high-level, MRP-dependent protection of cells from NQO toxicity.

Glutathione S-transferases (GSTs) inhibit transcriptional activation by the peroxisomal proliferator-activated receptor gamma (PPAR gamma) ligand, 15-deoxy-delta 12,14prostaglandin J2 (15-d-PGJ2).

15-Deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), a terminal metabolite of the J-series cyclopentenone prostaglandins, influences a variety of cellular processes including gene expression, differentiation, growth, and apoptosis. As a ligand of peroxisomal proliferator-activated receptor gamma (PPAR gamma), 15-d-PGJ(2) can transactivate PPAR gamma-responsive promoters. Previously, we showed that multidrug resistance proteins MRP1 and MRP3 attenuate cytotoxic and transactivating activities of 15-d-PGJ(2) in MCF7 breast cancer cells. Attenuation was glutathione-dependent and was associated with formation of the glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, and its active efflux by MRP. Here we have investigated whether the glutathione S-transferases (GST) can influence biological activities of 15-d-PGJ(2). MCF7 cells were stably transduced with human cytosolic GST isozymes M1a, A1, or P1a. These GSTs had no effect on 15-d-PGJ(2) cytotoxicity when expressed either alone or in combination with MRP1. However, expression of any of the three GSTs significantly inhibited 15-d-PGJ(2)-dependent transactivation of a PPAR gamma-responsive reporter gene. The degree of inhibition correlated with the level of GST expressed. Under physiologic conditions, the nonenzymatic rate of 15-d-PGJ(2) conjugation with glutathione was significant. Of the three GST isozymes, only GSTM1a-1a further stimulated the rate of 15-d-PGJ(2)-SG formation. Moreover, GSTM1a-1a rate enhancement was only a transient burst that was complete within 15 s. Hence, catalysis plays little, if any, role in GST inhibition of 15-d-PGJ(2)-dependent transactivation. In contrast, inhibition of transactivation was associated with strong GST/15-d-PGJ(2) interactions. Potent inhibition by 15-d-PGJ(2) and 15-d-PGJ(2)-SG of GST activity was observed with K(i) in the 0.15-2.0 microM range for the three GST isozymes, results suggesting avid associations between GST and 15-d-PGJ(2) or 15-d-PGJ(2)-SG. Electrospray ionization mass spectrometry (ESI/MS) studies revealed no stable adducts of GST and 15-d-PGJ(2) indicating that GST/15-d-PGJ(2) interactions are primarily noncovalent. These results are consistent with a mechanism of GST-mediated inhibition of transactivation in which GST binds 15-d-PGJ(2) and 15-d-PGJ(2)-SG thereby sequestering the ligands in the cytosol away from their nuclear target, PPAR gamma.

Role of multidrug resistance protein 2 (MRP2, ABCC2) in alkylating agent detoxification: MRP2 potentiates glutathione S-transferase A1-1-mediated resistance to chlorambucil cytotoxicity.

Our previous studies have shown that the glutathione S-transferases (GSTs) can operate in synergy with the efflux transporter multidrug resistance protein 1 (MRP1, ABCC1) to confer resistance to the cyto- and genotoxicities of some anticancer drugs and carcinogens. The current study was designed to determine whether the alternative efflux transporter, MRP2 (ABCC2), can also potentiate GST-mediated detoxifications in HepG2 cells. HepG2 cells, which express high-level MRP2 but not MRP1, were stably transduced with GST expression vectors under tetracycline-repressible transcriptional control. MRP2 was able to support GSTA1-1-mediated resistance to chlorambucil (CHB) cytotoxicity in HepG2 cells. Resistance was GST isozyme-specific in that GSTP1a-1a and GSTM1a-1a failed to confer protection from CHB toxicity. Moreover, inhibition of MRP2 with sulfinpyrazone completely reversed GSTA1-1-associated resistance, indicating that MRP2-efflux function is required to potentiate GSTA1-1-mediated resistance. Relative transport by MRP1 versus MRP2 of monoglutathionyl-CHB (CHB-SG) was examined using inside-out plasma membrane vesicles derived from MCF7 cells transduced with MRP1 or MRP2 expression vectors. Both MRP1 and MRP2 transported CHB-SG efficiently, at the levels of protein expressed, with similar Vmax and with Km of 0.39 and 10 microM, respectively. We conclude that detoxification of CHB by GSTA1-1 requires the removal of the glutathione conjugate formed and that either MRP1 or MRP2 can serve this efflux function. These findings have implications for the role of MRP2 in detoxification of alkylating agents in the apical epithelium of liver and kidney where it is highly expressed as well as the role of MRP2 in the emergence of alkylating drug resistance in cancer cells.

Multidrug resistance protein (MRP) 1 and MRP3 attenuate cytotoxic and transactivating effects of the cyclopentenone prostaglandin, 15-deoxy-Delta(12,14)prostaglandin J2 in MCF7 breast cancer cells.

One of the most potent cyclopentenone prostaglandins, 15-deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), has been shown to be cytotoxic in some tumor cells and, as a ligand of peroxisome proliferator activated receptor gamma (PPARgamma), to influence the transcriptional regulation of several genes. We examined whether a glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, is formed and if the glutathione conjugate efflux pumps, MRP1 and MRP3, could transport this conjugate, thereby attenuating the cytotoxicity and transactivating activity of 15-d-PGJ(2) in MCF7 breast cancer cells. Formation of 15-d-PGJ(2)-SG was demonstrated both in vitro and in cells, and its structure was determined by ESI/MS and NMR. Expression of MRP1 and MRP3 was achieved by stable transduction of parental MCF7 cells. Membrane vesicles derived from these cells supported efficient, ATP-dependent transport of 15-d-PGJ(2)-SG (K(M) 1.4 and 2.9 microM for MRP1 and MRP3, respectively). When compared with parental, MRP-minus MCF7 cells, expression of MRP1 and MRP3 conferred approximately 2-fold protection from 15-d-PGJ(2) cytotoxicity. 15-d-PGJ(2)-mediated transcriptional activation was evaluated in cells transiently transfected with a reporter gene under the transcriptional control of a PPAR responsive element. Treatment of parental MCF7 cells with 15-d-PGJ(2) resulted in a time-dependent induction of reporter gene activity-induction that was measurable with concentrations of added 15-d-PGJ(2) as low as 100 nM. In contrast, expression of MRP1 or MRP3 abolished 15-d-PGJ(2)-dependent reporter gene induction. Depletion of intracellular glutathione reversed MRP1- and MRP3-mediated attenuation of 15-d-PGJ(2) cytotoxicity and transactivation. These data indicate that MRP1 and MRP3 can modulate the biological effects of 15-d-PGJ(2), and likely other cyclopentenone prostaglandins, in a glutathione-dependent manner. The results are consistent with a mechanism for the attenuation of the biological activities of 15-d-PGJ(2) that involves the formation and active efflux of its glutathione conjugate, 15-d-PGJ(2)-SG.