Modeling the metabolic competency of glutathione S-transferases using genetically modified cell lines (original) (raw)
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Toxicological Sciences, 2007
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-Stransferase 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 -7,8-diol). Expression of hGSTM1 in the presence of hCYP1A1 conferred significant 8.5fold 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.
Cancer Research
The role of glutathione-S-transferase (GST) in alkylator drug resistance has been studied in Malli rat mammary carcinoma cells. A series of GST transfectant cell lines was established by using an expression vector con taining the complementary DNA for the rat GST )'<• gene under regulation of the SV40 early region promoter and the antibiotic resistance plasmid pSV2neo. Transfectant cell lines expressing up to 4-fold higher total GST activity than in the parental wild type cell line were identified. Southern blot analysis confirmed a DNA fragment corresponding in size to the transfected GST Yc complementary DNA. Wild type Mallt cells contain very low levels of Yc protein, whereas the Yc* clones showed greatly increased amounts of the Yc subunit. The effect of increased GST Yc activity on the sensitivity of the transfected clones to various cytotoxic agents was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival assay. The clones expressing recombi nant GST Yc were more resistant to melphalan (6-to 12-fold), mechlorethamine (10-to 16-fold), and chlorambucil (7-to 30-fold). In late passage populations of the GST Yc* clones that had been grown over a period of 14 months under continuous selection in G418, GST activity was de creased and it was paralleled by a decrease in Yc protein. These late passage clones with diminished GST Yc content also demonstrate a partial reversion toward the wild type phenotype as determined by cytotoxicity assays using melphalan, mustargen, and chlorambucil. Interstrand DNA cross-links induced by mechlorethamine were significantly lower at 0, 2, and 20 h posttreatment in one of the GST Yc* clones when compared to wild type Mall! cells. These studies indicate that GST Yc overexpression can confer resistance to alkylating agents and that this correlates with inhibition of DNA cross-link formation.
GLUTATHIONE S-TRANSFERASES: A BRIEF ON CLASSIFICATION AND GSTM1-T1 ACTIVITY
The glutathione S-transferase (GST) isoenzyme superfamilies detoxify a wide-range of toxic chemicals and environmental substances are extensively expressed in mammalian tissues. Liver and pancreas are the sites where cytosolic Phase I and phase II biotransformation GSTs enzymes have characteristic expression. GSTs play a key role in the deactivation of reactive oxygen species (ROS) and the metabolism of lipids, chemotherapeutic agents. GSTs are mainly involved in conjugation of reduced glutathione (GSH) with diverse substrates specificity and it is possible that genetic variations in these enzymes will influence cellular response to the environmental agents. GSTs are overexpressed in response to a chemical or oxidative stress as an adaptive physiology and upregulated in cancerous state of organ or tissue. GSTs are essentially involved in susceptibility to various forms of cancer as they are vital in detoxification mechanism to metabolize the environmental carcinogens. GSTM1 encodes for a class mu GST isoenzyme involved in polycyclic aromatic hydrocarbons (PAHs) detoxification. The substrates of GSTM1 include benzo(a)pyrene, benzo(c)phenanthrene, benzo(g)chrysene and other carcinogens. They can catalyze in-vitro GSH conjugation with several potent carcinogenic epoxides including aflatoxin B1(AFB1)8,9-epoxide and electrophilic metabolites of PAHs present in tobacco smoke. Ethylene dibromide, p-nitrobenzyl chloride, p-nitrophenetyl bromide, methyl chloride, and methyl iodide, are known substrates for GSTT1 or GST Theta (θ). GST Theta is most primitive among other known GSTs and widely expressed in nature.
Mutation Research-fundamental and Molecular Mechanisms of Mutagenesis, 2007
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 (IC50 = 2.7 and 0.7 nM, respectively) than in V79MZh1B1 (IC50 = 6.0 and 4.8 nM, 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.
Scientific Reports, 2013
The gene for glutathione-S-transferase (GST) M1 (GSTM1), a member of the GST-superfamily, is widely studied in cancer risk with regard to the homozygous deletion of the gene (GSTM1 null), leading to a lack of corresponding enzymatic activity. Many of these studies have reported inconsistent findings regarding its association with cancer risk. Therefore, we employed in silico, in vitro, and in vivo approaches to investigate whether the absence of a functional GSTM1 enzyme in a null variant can be compensated for by other family members. Through the in silico approach, we identified maximum structural homology between GSTM1 and GSTM2. Total plasma GST enzymatic activity was similar in recruited individuals, irrespective of their GSTM1 genotype (positive/null). Furthermore, expression profiling using real-time PCR, western blotting, and GSTM2 overexpression following transient knockdown of GSTM1 in HeLa cells confirmed that the absence of GSTM1 activity can be compensated for by the overexpression of GSTM2. G lutathione-S-transferases (GSTs) belong to a superfamily of ubiquitous, multifunctional dimeric cytosolic enzymes that play a very important role in the Phase II detoxification (or biotransformation) pathway in humans and confer protection against a wide array of toxic insults 1,2. Several GST isoforms have been identified and characterised, forming seven distinct classes: a, m, p, o, t, k, and f 3,4. Functionally, most GSTs catalyse the conjugation of the nucleophilic tripeptide glutathione to a wide range of electrophilic substrates for detoxification. However, the conjugation reaction can occasionally lead to the formation of compounds that are far more toxic than the initial substrate, thereby leading to disease outcomes 1,5,6. Interestingly, a null variant is encountered for two members, GSTT1 and GSTM1, whereby the entire gene is homozygously deleted in a considerable proportion of different populations, resulting in the complete absence of the corresponding enzyme activity 7,8. The GSTM1 gene is highly polymorphic and is located on chromosome 1p13.3. A wide range of variation in GSTM1 homozygous deletion polymorphism (approximately 20-67%) has been observed globally with regard to various ethnicities 9-12. It is often hypothesised that, due to the lack of functional GSTT1 and/or GSTM1, the null phenotype is unable to efficiently perform the conjugation reaction (biotransformation) and the subsequent elimination of toxic products via urine and bile. The null variant of GSTM1 is of particular interest, as a plethora of studies have demonstrated the difference in susceptibility, exposure to environmental toxicants, resistance to chemotherapy treatment, variability in drug response, manifestation of several diseases, and, most importantly, cancerous outcomes. The four other members of the GSTm subfamily, i.e., GSTM2, GSTM3, GSTM4, and GSTM5, exhibit high levels of sequence homology and substrate specificity with GSTM1 13. Among these genes, GSTM1 has largely been studied due to its null genotype. Although a large number of studies have attempted to associate the GSTM1-null genotype with cancer risk, the results are inconclusive. Several studies have attempted to identify the association of GSTM1 null with cancer risk through meta-analysis using the existing literature; however, these analyses failed to show a significant association of GSTM1 with cancer 14-20. These observations prompted us to search for the functional relevance of this ''well known gene'' with other family members that are relatively less studied. A possible explanation of the apparently inconsistent results could be that other members of the GST family compensate for the absence of a functional GSTM1 enzyme. In this study, we attempted to ascertain whether
Archives of Biochemistry and Biophysics, 1997
forms toward anti-CDE was investigated by molecular modeling of the two proteins with GSH conjuga-The kinetics of the conjugation of glutathione tion products in their active sites. These studies re-(GSH) with anti-1,2-dihydroxy-3,4-oxy-1,2,3,4-tetravealed that the enantioselectivity of hGSTP1-1 for hydrochrysene (anti-CDE), the activated form of the (/)-anti-CDE and the differential catalytic efficiencwidespread environmental pollutant chrysene, cataies of the V104 and I104 forms of hGSTP1-1 in the lyzed by two naturally occurring polymorphic forms GSH conjugation of (/)-anti-CDE were due to the difof the pi class human GSH S-transferase (hGSTP1ferences in the active-site architecture of the two 1), has been investigated. The polymorphic forms of proteins. The results of the present study, for the hGSTP1-1, which differ in their primary structure by first time, provide evidence for the toxicological relea single amino acid in position 104, exhibited prefervance of GSTP1-1 polymorphism in humans and sugence for the GSH conjugation of (/)-anti-CDE, which gest that the population polymorphism of hGSTP1-1 is a far more potent carcinogen than (0)-anti-CDE. variants with disparate enzyme activities may, at When concentration of anti-CDE was varied (5-200 least in part, account for the differential susceptibil-mM) and the GSH concentration was kept constant ity of individuals to environmental carcinogens such at 2 mM, both hGSTP1-1(I104) and hGSTP1-1(V104) as anti-CDE and possibly other similar carcinogens. obeyed Michaelis-Menten kinetics. However, the ᭧ 1997 Academic Press V max of GSH conjugation of anti-CDE was approxi-Key Words: Chrysene; carcinogenesis; glutathione Smately 5.3-fold higher for the V104 variant than for transferase P1-1; polymorphism; detoxification. the I104 form. Calculation of catalytic efficiency (k cat / K m) thus resulted in a value for hGSTP1-1(V104), 28 mM 01 s 01 , that was 7.0-fold higher than that for hGSTP1-1(I104), 4 mM 01 s 01. The mechanism of the GSTs 3 belong to a superfamily of multifunctional isodifferences in the kinetic properties of hGSTP1-1 isoenzymes which can detoxify a wide variety of electrophilic xenobiotics primarily by catalyzing their conju-1 This investigation was supported, in part, by United States Public Health Service Grants CA 55589 (S.V.S.), CA 63660 (S.A.), ES 07804 (P.Z.), and GM 32304 (Y.C.A.) and by the National Cancer 3 Abbreviations used: (/)-anti-CDE, chrysene-1R,2S-diol 3S,4R-Institute, Department of Health and Human Services, under contract with ABL (X.J.). The contents of this publication do not necessarily oxide; (0)-anti-CDE, chrysene-1S,2R-diol 3R,4S-oxide; (/)-anti-BPDE, benzo(a)pyrene-7R,8S-diol 9S,10R-oxide; CDNB, 1-chloro-reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or 2,4-dinitrobenzene; GSH, glutathione; GST, glutathione S-transferase; H-site, electrophilic xenobiotic binding site; PAHs, polycyclic organizations imply endorsement by the United States Government. 2 To whom correspondence may be addressed. aromatic hydrocarbons.
Mobilization of Pentachlorophenol by GlutathioneS-Transferase μ Increases Cellular Toxicity
Pesticide Biochemistry and Physiology, 1996
Glutathione S-transferases (GSTs) are a class of proteins that have intracellular binding and sequestration, as well as catalytic, capabilities. This study investigated the role of GSTs in the intracellular mobilization of pentachlorophenol (PCP). A single mouse GST isoform, GST , specifically and competitively bound PCP with a k i ס 7 M with respect to the substrate 1-chloro-2,4-dinitrobenzene (CDNB), yet did not metabolize PCP. Instead, the binding of GST to PCP resulted in the partitioning of PCP from lipid to aqueous compartments. The physiological significance of the mobilization was then investigated using two mouse hepatocyte cell lines that differ significantly in their expression of GST. These cells were treated with butylated hydroxyanisole (BHA) to induce GST and provided several levels of GST expression. The toxicity of PCP to these cells based on trypan blue dye exclusion was assessed, which demonstrated a significant correlation between GST levels within the cell and PCP toxicity. The BHA-treated, high GST expressor cells were approximately 40% more sensitive to PCP toxicity than were the untreated low GST expressors, suggesting that GST was acting to make PCP more bioavailable to elicit toxicity. Furthermore, the increase in toxicity was not due to a difference in PCP accumulation or in GST metabolism by the cells. These results suggest that GST may increase the cellular toxicity of PCP by mobilizing this lipophilic compound within the cell.
Hypochlorous Acid is a Potent Inhibitor of GST P1-1
Chemico-Biological …, 2001
Glutathione S-transferase is a phase II detoxification enzyme that can be inactivated by hydrogen peroxide (H2O2). During oxidative stress various other reactive oxygen species are generated that are more reactive than the relatively stable H2O2. Hypochlorous acid (HOCl) is a powerful oxidant which is highly reactive towards a range of biological substrates. We studied the influence of HOCl on the activity of GST P1-1. HOCl inhibits purified glutathione S-transferase P1-1 in a concentration dependent manner with an IC50-value of 0.6 microM, which is more than 1000 times as low as IC50 reported for H2O2. HOCl lowered the Vmax value, but did not affect the Michaelis Menten constant (Km) for CDNB. Our results show that HOCl is a potent, non-competitive inhibitor of GST P1-1. The relevance of this effect is discussed.
PLOS ONE
Environmental and endogenous electrophiles cause tissue damage through their high reactivity with endogenous nucleophiles such as DNA, proteins, and lipids. Protection against damage is mediated by glutathione (GSH) conjugation, which can occur spontaneously or be facilitated by the glutathione S-transferase (GST) enzymes. To determine the role of GST enzymes in protection against electrophiles as well as the role of specific GST families in mediating this protection, we exposed mutant mouse lines lacking the GSTP, GSTM, and/or GSTT enzyme families to the model electrophile acrylamide, a ubiquitous dietary contaminant known to cause adverse effects in humans. An analysis of urinary metabolites after acute acrylamide exposure identified the GSTM family as the primary mediator of GSH conjugation to acrylamide. However, surprisingly, mice lacking only this enzyme family did not show increased toxicity after an acute acrylamide exposure. Therefore, GSH conjugation is not the sole mechanism by which GSTs protect against the toxicity of this substrate. Given the prevalence of null GST polymorphisms in the human population (approximately 50% for GSTM1 and 20-50% for GSTT1), a substantial portion of the population may also have impaired acrylamide metabolism. However, our study also defines a role for GSTP and/or GSTT in protection against acrylamide mediated toxicity. Thus, while the canonical detoxification function of GSTs may be impaired in GSTM null individuals, disease risk secondary to acrylamide exposure may be mitigated through non-canonical pathways involving members of the GSTP and/or GSTT families.