Role of multidrug-resistance protein 2 in glutathione S-transferase P1-1-mediated resistance to 4-nitroquinoline 1-oxide toxicities in HepG2 cells

Previous studies in our laboratory have shown that the phase III efflux transporter multidrug-resistance protein (MRP)1 can act synergistically with the phase II conjugating glutathione S-transferases (GST) to confer resistance to the toxicities of some electrophilic drugs and carcinogens. To determine whether the distinct efflux transporter MRP2 could also potentiate GST-mediated protection from electrophilic toxins, we examined the effect of regulatable GSTP1-1 expression in MRP2-rich HepG2 cells on 4-nitroquinoline 1-oxide (4NQO)-induced cytotoxicity and genotoxicity (nucleic-acid adduct formation). Expression of GSTP1-1 was associated with a fourfold to tenfold protection from 4NQO-induced cytotoxicity. Inhibition of MRP2-mediated efflux activity by sulfinpyrazone or cyclosporin A completely reversed GSTP1-1-associated resistance-a result indicating that GSTP1-1-mediated cytoprotection is absolutely dependent on MRP2 efflux activity. Moreover, MRP2 efflux activity also augmented GSTP1-1-mediated protection from 4NQO-induced nucleic-acid adduct formation. We conclude that MRP2-mediated efflux of the glutathione conjugate of 4NQO and/or another toxic derivative of 4NQO is required to support GSTP1-1-associated protection from 4NQO toxicities in HepG2 cells.     

Nitroreductases and glutathione transferases that act on 4-nitroquinoline 1-oxide and their differential induction by butylated hydroxyanisole in mice.

These studies concern the initial steps in 4-nitroquinoline 1-oxide (4NQO) metabolism in relation to mechanisms of anticarcinogenesis. Butylated hydroxyanisole (BHA) administration by a protocol known to inhibit the pulmonary tumorigenicity of 4NQO in A/HeJ mice enhanced hepatic and pulmonary activities for 4NQO metabolism by two major pathways, conjugative detoxification and nitroreductive activation. High-performance liquid chromatography analysis showed approximate doubling of two types of glutathione transferase subunits with 4NQO-conjugating activity in livers of BHA-treated mice. Similar increases were observed in hepatic 4NQO-conjugating activity and in Vmax, while Km for 4NQO was 39 to 43 microM. Pulmonary 4NQO-glutathione transferase activity increased 24 to 29%. DT diaphorase activity toward 4NQO was elevated 3.3-fold in livers and 2.7-fold in lungs of BHA-treated mice. However, the predominant 4NQO reductase of liver and lung was dicumarol resistant, had a strong preference for NADH, and showed little if any response to BHA. This Mr 200,000 enzyme, partially purified from livers of Swiss mice, exhibited the stoichiometry of 2-NADH/4NQO expected for reduction of 4NQO to 4-hydroxyaminoquinoline 1-oxide. Its high affinity for 4NQO (Km, 15 microM) signified a much greater influence on 4NQO metabolism than DT diaphorase (Km, 208 microM). The dicumarol-resistant 4NQO reductase differed from several known cytosolic nitroreductases. The results suggest that protection by BHA may result from alteration of the balance between 4NQO activation and conjugation.     

Overcoming glutathione S-transferase P1-related cisplatin resistance in osteosarcoma

Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most used drugs for osteosarcoma chemotherapy. By using a series of CDDP-resistant variants, which were established from the U-2OS and Saos-2 human osteosarcoma cell lines, we found that CDDP resistance was mainly associated with the increase of both the intracellular level and enzymatic activity of glutathione S-transferase P1 (GSTP1). On the basis of these findings, we evaluated the clinical effect of GSTP1 in a series of 34 high-grade osteosarcoma patients and we found that the increased expression of GSTP1 gene was associated with a significantly higher relapse rate and a worse clinical outcome. These indications prompted us to assess the in vitro effectiveness of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), a promising new anticancer agent that is a highly efficient inhibitor of GSTP1. NBDHEX was tested on a panel of 10 human osteosarcoma cell lines and 20 variants of the U-2OS or Saos-2 cell lines that were resistant to CDDP, doxorubicin, or methotrexate. NBDHEX proved to be very active on the vast majority of these cell lines, including those with higher GSTP1 levels and enzymatic activity. Drug combination studies showed that NBDHEX can be used in association with CDDP and provided useful information about the best modality of their combined administration. In conclusion, our findings show that GSTP1 has a relevant effect for both CDDP resistance and clinical outcome of high-grade osteosarcoma and that targeting GSTP1 with NBDHEX may be considered a promising new therapeutic possibility for osteosarcoma patients who fail to respond to conventional chemotherapy.     

Identification of the glutathione conjugate of 4-nitroquinoline 1-oxide formed in the reaction catalyzed by murine glutathione transferases

The product of the enzyme-catalyzed conjugation of glutathioneand 4-nitroquinoline 1-oxide was isolated and its structuredetermined by MS and NMR. The results indicate that the cysteinesulfur of glutathione replaces the nitro group of 4-nitroquinoline1-oxide in the reaction with the formation of 4-(glutathion-S-yI)-quinoline1-oxide. No evidence was found for the binding of glutathioneto any other position of 4-nitroquinoline 1-oxide or throughany group other than the cysteine sulfur.     

Fpg protein protects Escherichia coli K-12from mutation induction by the carcinogen 4-nitroquinoline 1-oxide

This paper investigates the role of the fpg gene product inprotecting Escherichia coli cells against the lethal and mutageniceffects of 4-nitroquinoline 1-oxide (4NQO). To this end, thearaD81 mutation which make the cells sensitive to L-arabinosewas combined with an fpg-1::Kn^r allele, in either an uvrA⁺ oruvrA^–, umuC⁺ or umuC^–, genetic background. Mutationinduction was monitored by selecting forward mutations to L-arabinoseresistance (Ara^r). The formamidopyrimidine-DNA glycosylase (Fpgprotein) protected bacteria from 4NQO-induced mutagenesis sinceFpg^– defective cells showed greater Ara^r mutation inductionthan fpg⁺ bacteria did. This was confirmed since the increasedsensitivity of the fpg^– cells to mutagenesis by 4NQO wassuppressed when the Fpg protein was overproduced by placingthe fpg gene in a multicopy plasmid vector. The fpg^– mutationhad no detectable influence on 4NQO mutagenesis in a uvrA^–genetic background, but its effect was magnified in umuC^–cells. No influence on cell survival was observed after 4NQOtreatment. Our data suggest that 8-hydroxyguanine, a non-lethal,non-bulky and directly miscoding lesion, might be responsiblefor the detected influence of Fpg protein expression on mutationinduction by 4NQO. This is in agreement with the reported invivo formation of 8-hydroxyguanine in cellular DNA after 4NQOexposure. The increased 4NQO-induction of GC to TA transversionson fpg^– bacteria further support such a possibility. Thiswork reinforces the role of Fpg protein in the bacterial defenseagainst the mutagenicity by genotoxic agents.     

Differential activity of human, rat, mouse and bacteria glutathione transferase isoenzymes towards 4-nitroquinoline 1-oxide

The conjugation capacity of 4-nitroquinoline 1-oxide (4-NQO) with GSH by a number of human, rat, mouse and bacteria glutathione transferases (GSTs) was investigated. Pi and mu classes GSTs exhibited maximum conjugation capacity. Alpha class glutathione transferases as well as bacteria glutathione transferases were found to be unable to conjugate GSH to 4-NQO. The Km values as well as the catalytic efficiency (Kcat/Km) for most of the GSTs investigated were also determined. Mouse liver GST MIII (class mu) was the most efficient of the various isoenzymes tested. Its Kcat/Km value was 162 times higher than that of mouse liver GST MI (class alpha). The relatively high catalytic efficiency exhibited by GST-alpha (class pi) is prevalently due to its low affinity for 4-NQO.     

Polyclonal antibodies to DNA modified with 4-nitroquinoline 1-oxide: application for the detection of 4-nitroquinoline 1-oxide-DNA adducts in vivo

Antibodies against 4-nitroquinoline 1-oxide (4NQO) adducts were elicited in rabbits immunized with 4NQO-modified DNA complexed with methylated bovine serum albumin. In enzyme-linked immunosorbent assay (ELISA), the antibodies could recognize either denatured or native 4NQO-modified DNA, but not unmodified DNA, DNA modified with other carcinogens or free 4NQO derivative. Modification levels as low as 5 mumol of adduct per one mole DNA nucleotide (5 adducts/10(6) nucleotides) can be easily detected by the competitive ELISA. Indirect immunofluorescence staining by anti 4NQO-DNA antibody indicated that the antibodies bound specifically to the nuclei of normal human skin fibroblast cells treated with 4NQO. The intensity of fluorescence was proportional to the dose of 4NQO used to treat the cells, and the fluorescence-positive cells could be detected after treatment with 0.25 microM 4NQO (which resulted in the formation of 10(4) adducts per cell). Applying the competitive ELISA to the quantitation of DNA-adducts in rats treated with 4NQO, it was confirmed that the sensitivity of immunochemical assays was equivalent to that of isotopic assays. These methods should be helpful in studies on the formation of adducts and their removal in cells and tissues.     

Separation and identification of N4-(guanosin-7-yl)-4-aminoquinoline 1-oxide, a novel nucleic acid adduct of carcinogen 4-nitroquinoline 1-oxide

A novel nucleic acid adduct of the carcinogen 4-nitroquinoline 1-oxide, N4-(guanosin-7-yl)-4-aminoquinoline 1-oxide, was separated and identified after RNA was treated with activated 4-hydroxyaminoquinoline 1-oxide. This is the first report of a 7-arylaminated guanine adduct produced by arylaminating carcinogens.     

Genetic characterization of adenine-3 mutants induced by 4-nitroquinoline 1-oxide and 4-hydroxyaminoquinoline 1-oxide in Neurospora crassa.

Specific locus mutations induced by the chemical carcinogens, 4-nitroquinoline 1-oxide (4NQO) and 4-hydroxyaminoquinoline 1-oxide (4HAQO), have been characterized to obtain a presumptive identification of the genetic alterations at the molecular level. One hundred eighty-four 4NQO-induced and 219 4HAQO-induced ad-3 mutants of Neurospora crassa obtained in previous studies were studied with a series of genetic tests that permits determination of their genotype and the frequencies of point mutations and multilocus deletions. These tests have shown that the spectrum of ad-3 mutations among 4NQO-induced mutants is similar to that of 4HAQO-induced mutants. None of the 4NQO- or 4HAQO-induced mutants is a multilocus deletion mutant. The ratio of ad-3A to ad-3B mutants is the same in the two samples, as well as the frequencies of complementing ad-3B mutants. These data suggest, then, that the mechanism of mutation induction by 4NQO in N. crassa is identical to that of 4HAQO. It is not clear, however, whether 4NQO is mutagenic per se or reduction of 4NQO to 4HAQO is the first step involved in the mutagenesis of this compound in Neurospora. The heterotaryon tests have shown that the relatively high frequencies of 4NQO- or 4HAQO-induced ad-3B mutants show allelic complementation and that most of the complementing ad-3B mutants (74% of 4NQO induced and 71% of 4HAQO induced) have nonpolarized complementation patterns. From this we conclude that both agents induce predominantly base-pair substitution mutations in N. crassa. The results are in agreement with our other studies which show that potent chemical carcinogens induce predominantly base-pair substitution mutations in N. crassa.     

Expression of glutathione S-transferase alpha, P1-1 and T1-1 in the human gastrointestinal tract.

Glutathione S-transferases (GSTs) form a family of enzymes, which play an important role in the prevention of cancer by detoxifying numerous potentially carcinogenic compounds. GSTs catalyze the conjugation of glutathione to such harmful molecules, and enable their secretion. Human GSTs can be divided into five main classes. The theta class of isoenzymes was only recently identified and limited (immunohistochemical) data on these enzymes are available. In the present study, paraffin-embedded sections of different gastrointestinal tissues were analyzed immunohistochemically for GSTalpha, GSTP1-1 and GSTT1-1 expression using specific antibodies. GSTalpha, GSTP1-1 and GSTT1-1 were highly expressed in all gastrointestinal tissues examined, with a unique cellular distribution. GSTT1-1 is the first GST isoenzyme demonstrated in duodenal Paneth cells and glands of Brunner. The common expression of GSTalpha, GSTT1-1 and GSTP1-1 in many cell types along the human gastrointestinal tract suggests an important role in the protection against carcinogens and other xenobiotics.     

Breakage of a DNA-protein complex induced by 4-nitroquinoline 1-oxide, 4-nitropyridine 1-oxide, and their derivatives in cultured mouse fibroblasts.

The effects of a number of 4-nitroquinoline 1-oxide and 4-nitropyridine 1-oxide derivatives, with varying carcinogenic potencies, on the scission of proteins linking DNA were studied in cultured mouse fibroblasts, strain L-P-3. With twenty-two 4-nitroquinoline 1-oxide derivatives and twelve-4-nitroquinoline 1-oxide derivatives tested, an excellent correlation was found between the scission effect of each compound and its carcinogenicity. All carcinogens, whether strong or weak, showed positive results in the scission test. Strong carcinogens such as 4-nitroquinoline 1-oxide, 2-methyl-4-nitroquinoline 1-oxide, 6-methyl-4-nitroquinoline 1-oxide, 6-chloro-4-nitroquinoline 1-oxide,and 4-hydroxyaminoquinoline 1-oxide induced the scission at a low concentration of 1 x 10-5 M, while weak carcinogens such as 3-methyl-4-nitroquinoline 2-oxide, 6-n-butyl-4-nitroquinoline 1-oxide, 6-tert-butyl-4-nitroquinoline 1-oxide, 6-n-hexyl-4-nitroquinoline 1-oxide, and 6-carboxy-4-nitroquinoline 1-oxide only produced the same effect a dose levels higher than 5 x10-5 M. On the other hand, some noncarcinogenic derivatives such as 8-nitroquinoline 1-oxide, 4-hydoxy-quinoline 1-oxide, 4-aminoquinoline 1-oxide, and 6-nitroquinoline could not induce the scission, while other noncarcinogens such as 3-nitroquinoline 1-oxide, 5-nitroquinoline 1-oxide, and 5-nitroquinoline did induce scission at concentrations higher than 1 x 10-4 M. Throughout these tests the effective concentrations of active compounds were generally much lower than the concentration at which the compounds were cytotoxic. The implication of the results and the feasibility of the present method of analysis as a screening procedure for potential carcinogens and muagens are discussed.     

Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2-ASK1 signals

Human glutathione S-transferase P1-1 (GSTP1-1) is an ubiquitously expressed protein that plays an important role in the detoxification and xenobiotics metabolism. It has been shown that GSTP1-1 interacts with c-Jun NH(2)-terminal kinase (JNK) and suppresses its activity. Here, we report a novel function of GSTP1-1 in regulating tumor necrosis factor-alpha (TNF-alpha)-triggered signaling. The present experiments showed that GSTP1-1 physically associated with tumor necrosis factor receptor-associated factor 2 (TRAF2) in vivo and in vitro. Overexpression of GSTP1-1 inhibited TRAF2-induced activation of both JNK and p38 but not of nuclear factor-kappaB (NF-kappaB). Glutathione S-transferase P1-1 also attenuated TRAF2-enhanced apoptosis signal-regulating kinase 1 (ASK1) autophosphorylation and inhibited TRAF2-ASK1-induced cell apoptosis by suppressing the interaction of TRAF2 and ASK1. Conversely, silencing of GSTP1-1 expression through RNA interference (RNAi) resulted in increase of TNF-alpha-dependent TRAF2-ASK1 association followed by hyper-activation of ASK1 and JNK. A mutant GSTP1-1 lacking TRAF domain-binding motif exhibited a significant decline of capacity to bind TRAF2 and block TRAF2-ASK1 signaling compared with the wild type of GSTP1-1. Moreover, the glutathione-conjugating activity of GSTP1-1 was not involved in the regulation of TRAF2 signaling. These findings indicate that GSTP1-1 plays an important regulatory role in TNF-alpha-induced signaling by forming ligand-binding interactions with TRAF2, which provides a new insight for analysing the protective effects of GSTP1-1 in tumor cells.     

细胞色素P450 2E1和谷胱甘肽转硫酶P1基因与食管癌易患性

目的研究与致癌物亚硝胺代谢激活有关的细胞色素Ｐ４５０２Ｅ１基因（ＣＹＰ２Ｅ１）,和与致癌物代谢解毒有关的谷胱甘肽转硫酶Ｐ１基因（ＧＳＴＰ１）多型性与食管癌易患性的关系。方法采用病例－对照分子流行病学方法。以ＰＣＲ－ＲＦＬＰ方法分析食管癌、食管上皮重度增生病例,和与其年龄性别配对的正常对照者（各４５例）ＣＹＰ２Ｅ１和ＧＳＴＰ１的基因型。结果ＧＳＴＰ１基因型在病例和对照者中的分布无显著差别,但ＲｓａＩ识别的ＣＹＰ２Ｅ１基因型,在食管癌、食管上皮重度增生病例及其正常对照者中的分布差别显著。ＣＹＰ２Ｅ１突变型基因频率在正常对照组中为５５．６％,显著高于食管上皮重度增生病例（１７．８％）和食管癌病例（２０．０％;χ２＝２０．８,Ｐ＜０．００１）;携带野生型ＣＹＰ２Ｅ１的个体,发生食管上皮重度增生和食管癌的危险性,比携带变异型ＣＹＰ２Ｅ１的个体各高５倍。结论ＣＹＰ２Ｅ１基因是涉及食管癌变早期过程的遗传易患性因素。     

Phase I study of TLK286 (glutathione S-transferase P1-1 activated glutathione analogue) in advanced refractory solid malignancies.

PURPOSE: The purpose of this study was to determine the dose-limiting toxicities (DLTs), the maximum tolerated dose, and the pharmacokinetics of the novel glutathione analog TLK286 administered by i.v. infusion. EXPERIMENTAL DESIGN: Patients with advanced malignancies received i.v. TLK286 administered as a 30-min constant rate infusion once every 3 weeks in escalating doses from 60 to 1280 mg/m(2). Patients underwent tumor assessment on day 43 and continued on treatment until disease progression or unacceptable toxicity. RESULTS: A total of 35 patients were treated with 109 cycles of TLK286. At 1280 mg/m(2), 3 of 5 patients developed one of two observed dose limiting toxicities (DLTs). The DLTs were: mild pancreatitis (1 of 5) and bladder symptoms (2 of 5) consisting of hematuria, dysuria, and urinary frequency. All of the patients with DLTs continued on TLK286 treatment at 960 mg/m(2) (one dose below maximum tolerated dose) without recurrence of DLTs. DLTs were transient, resolved without sequelae, and noncumulative. TLK286-related toxicities included grade 1-2 nausea, vomiting, fatigue, transient microscopic hematuria, and anemia. Of 31 evaluable patients, 10 patients continued therapy (median six cycles; range, four to nine cycles). Pharmacokinetic studies of TLK286 on cycle 1 revealed a mean elimination half-life of 18 min (95% confidence interval, 16.1-19.9). Dose-proportional increases in both maximum blood concentrations and area under the blood-concentration-time curve were observed over the dose range of 60-960 mg/m(2). CONCLUSION: TLK286 was well tolerated in this study. TLK286 safety and pharmacokinetics support disease-specific evaluations of TLK286 at doses <1280 mg/m(2) administered once every three weeks in the treatment of patients with advanced malignancies.