Relationship of glutathione S-transferase genotypes with side-effects of pulsed cyclophsphamide therapy in patients with systemic lupus erythematosus

Aims
Cyclophosphamide (CTX) is an established treatment of severe systemic lupus erythematosus (SLE). Cytotoxic CTX metabolites are mainly detoxified by multiple glutathione S-transferases (GSTs). However, data are lacking on the relationship between the short-term side-effects of CTX therapy and GST genotypes. In the present study, the effects of common GSTM1, GSTT1, and GSTP1 genetic mutations on the severity of myelosuppression, gastrointestinal (GI) toxicity, and infection incidences induced by pulsed CTX therapy were evaluated in patients SLE.
Methods
DNA was extracted from peripheral leucocytes in patients with confirmed SLE diagnosis (n = 102). GSTM1 and GSTT1 null mutations were analyzed by a polymerase chain reaction (PCR)-multiplex procedure, whereas the GSTP1 codon 105 polymorphism (Ile→Val) was analyzed by a PCR-restriction fragment length polymorphism (RFLP) assay.
Results
Our study demonstrated that SLE patients carrying the genotypes with GSTP1 codon 105 mutation [GSTP1*-105I/V (heterozygote) and GSTP1*-105 V/V (homozygote)] had an increased risk of myelotoxicity when treated with pulsed high-dose CTX therapy (Odds ratio (OR) 5.00, 95% confidence interval (CI) 1.96, 12.76); especially in patients younger than 30 years (OR 7.50, 95% CI 2.14, 26.24), or in patients treated with a total CTX dose greater than 1.0 g (OR 12.88, 95% CI 3.16, 52.57). Similarly, patients with these genotypes (GSTP1*I/V and GSTP1*V/V) also had an increased risk of GI toxicity when treated with an initial pulsed high-dose CTX regimen (OR 3.33, 95% CI 1.03, 10.79). However, GSTM1 and GSTT1 null mutations did not significantly alter the risks of these short-term side-effects of pulsed high-dose CTX therapy in SLE patients.
Conclusions
The GSTP1 codon 105 polymorphism, but not GSTM1 or GSTT1 null mutations, significantly increased the risks of short-term side-effects of pulsed high-dose CTX therapy in SLE patients. Because of the lack of selective substrates for a GST enzyme phenotyping study, timely detection of this mutation on codon 105 may assist in optimizing pulsed high-dose CTX therapy in SLE patients.

Upregulation of glutathione-related genes and enzyme activities in cultured human cells by sublethgal concentrations of inorganic arsenic

Inorganic arsenic (jAs), a known human carcinogen, acts as a tumor promoter in part by inducing a rapid burst of reactive oxygen species (ROS) in mammalian cells. This causes oxidative stress and a subsequent increase in the level of cellular glutathione (GSH). Glutathione, a ubiquitous reducing sulfhydryl tripeptide, is involved in ROS detoxification and its increase may be part of an adaptive response to the oxidative stress. Glutathione related enzymes including glutathione reductase (GR) and glutathione S-transferase (GST) also play key roles in these processes. In this study the regulatory effects of inorganic arsenite (As111) on the activities of GSH-related enzymes were investigated in cultured human keratinocytes. Substantial increases in GR enzyme activity and mRNA levels were shown in keratinocytes and other human cell lines after exposure to low, subtoxic, micromolar concentrations of As111 for 24 h. Upregulation of GSH synthesis paralleled the upregulation of GR as shown by increases in glutamatecysteine lyase (GeL) enzyme activity and mRNA levels, cystine uptake, and intracellular GSH levels. Glutathione S-transferase activity was also shown to increase slightly in keratinocytes, but not in fibroblasts or breast tumor cells. Overall the results show that sublethal arsenic induces a multicomponent response in human keratinocytes that involves upregulation of parts, but not all of the GSH system and counteracts the acute toxic effects of jAs. The upregulation of GR has not previously been shown to be an integral part of this response, although GR is critical for maintaining levels of reduced GSH.

In vitro effect of arsenical compounds on glutathione-related enzymes

The mechanism of arsenic toxicity is believed to be due to the ability of arsenite (AsIII) to bind protein thiols. Glutathione (GSH) is the most abundant cellular thiol, and both GSH and GSH-related enzymes are important antioxidants that play an important role in the detoxification of arsenic and other carcinogens. The effect of arsenic on the activity of a variety of enzymes that use GSH has been determined using purified preparations of glutathione reductase (GR) from yeast and bovine glutathione peroxidase (GPx) and equine glutathione S-transferase (GST). The effect on enzyme activity of increasing concentrations (from 1 μM to 100 mM) of commercial sodium arsenite (AsIII) and sodium arsenate (AsV) and a prepared arsenic(III)−glutathione complex [AsIII(GS)3] and methylarsenous diiodide (CH3AsIII) has been examined.

Relationship between genotype and enzyme activity of glutathione S-transferases M1 and P1 in Chinese

Glutathione S-transferases (GSTs) are the major detoxifying Phase II enzyme for eliminating electrophilic compounds. Mutations in GSTM1, GSTP1 and GSTT1 in Caucasian and GSTA1 in Chinese have been found to reduce enzyme activity. However, data on the impact of common genetic polymorphisms of GSTM1 and GSTP1 on enzyme activity in Chinese is lacking. This study aimed to investigate the effect of common GSTP1 and GSTM1 polymorphisms on erythrocyte GST activity in healthy Chinese (n = 196). GSTM1 null mutation (GSTM1*0) was analyzed by a PCR-Multiplex procedure, whereas GSTP1 313A → G polymorphism (resulting in Ile105Val at codon 105) was analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis. Erythrocyte GST activity was measured using 1-chloro-2,4-dinitro-bezene (CDNB) as the model substrate. The frequency of GSTM1 null genotype was 54.3% and the frequency of GSTP1-Ile/Ile, -Ile/Val, and -Val/Val genotype was 60.7%, 35.2% and 4.1%, respectively, with a frequency of 21.7% for the 105 valine allele. Age, gender and smoking did not significantly affect the erythrocyte GST activities. The mean erythrocyte GST enzyme activity for GSTP1*-Ile/Val genotype group (3.53 ± 0.63 U/g Hb) was significantly lower than that for subjects with GSTP1-Ile/Ile genotype (4.25 ± 1.07 U/g Hb, P = 0.004), while subjects with the GSTP1-Val/Val genotype had the lowest enzyme activity (2.44 ± 0.67 U/g Hb). In addition, the GST activity in carriers of GSTM1*0/GSTP1-Ile/Ile was significantly higher than that of subjects inherited GSTM1*0/GSTP1-Ile/Val or GSTM1*0/GSTP1-Val/Val. However, there is no association between GSTM1 null mutation and reduced enzyme activity. GSTP1 codon 105 mutation led to reduced erythrocyte GST activity in Chinese. A combined GSTP1 and GSTM1 null mutations also resulted in significantly reduced GST activity. Further studies are needed to explore the clinical implications of GSTM1 and GSTP1 polymorphisms.

Genetic polymorphisms in glutathione-S-transferases are associated with anxiety and mood disorders in nicotine dependence

Nicotine dependence is associated with an increased risk of mood and anxiety disorders and suicide. The primary hypothesis of this study was to identify whether the polymorphisms of two glutathione-S-transferase enzymes (GSTM1 and GSTT1 genes) predict an increased risk of mood and anxiety disorders in smokers with nicotine dependence.

Alteration of GSH level, gene expression and cell transformation in NIH3T3 cells by chronic exposure to low dose of arsenic

It is well established that arsenic toxicity is postulated to be primarily due to the binding of As(III) to sulfhydryl-containing enzymes. However, the mechanism of carcinogenesis induced by arsenic is still unclear. The interaction of arsenic with GSH and related enzymes seems a very important issue regarding mechanism of arsenical induced toxicity or carcinogenesis. The purpose of this work is to investigate the effect of chronic exposure to low dose of As(III) on GSH level, gene expression and cell transformation in NIH3T3 cells. The results showed that long-term, low dose arsenic treatment makes 3T3 cell more resistant to acute arsenic treatment. There were morphology changes after long-term arsenic treatment. First, partially immortalized 3T3 cell became immortalized. In addition, the cells were doubling more quickly than the control cells and attained higher density than the control cells at confluence. Second, cells treated with 0.1 µ.M As(III) exhibited anchorage-independent growth. Arsenic could enhance GSH level at 0.5 -10 µM dose of arsenic in 24 h treatment and decrease it at 25 µM and above. In long-term treatment with low dose of arsenic, GSH levels were decreased. As(I1I) can increase both glutathione S-transferase (GST) and glutathione reductase (GR) activities at low dose (0.5-10 M), but decreased GST and GR activities at 25 M and higher dose of arsenic, while in long-term As(III) treatment, GST and GR activities are increased. Both long-term and short-term treatments with As(III) can induce GR gene expression. GPx mRNA levels were decreased both in acute and chronic arsenic treated cells. Chronic treatment with As(III) also decreased the p53 mRNA level. Taken together, our results suggest that As(III) can alter GST, GR enzyme activities as well as GSH level and related gene expression both in long-term and short-term treatment but in a different manner in different doses. Alteration of cellular GSH level by As(III) might play all important role in gene expression and arsenic induced cell transformation.

Regulation of redox and DNA repair genes by arsenic : low dose protection against oxidative stress?

We have evaluated the molecular responses of human epithelial cells to low dose arsenic to ascertain how target cells may respond to physiologically relevant concentrations of arsenic. Data gathered in numerous experiments in different cell types all point to the same conclusion: low dose arsenic induces what appears to be a protective response against subsequent exposure to oxidative stress or DNA damage, whereas higher doses often provoke synergistic toxicity. In particular, exposure to low, sub-toxic doses of arsenite, As(III), causes coordinate up-regulation of multiple redox and redox-related genes including thioredoxin (Trx) and glutathione reductase (GR). Glutathione peroxidase (GPx) is down-regulated in fibroblasts, but up-regulated in keratinocytes, as is glutathione S-transferase (GST). The maximum effect on these redox genes occurs after 24 h exposure to 5–10 mM As(III). This is 10-fold higher than the maximum As(III) concentrations required for induction of DNA repair genes, but within the dose region where DNA repair genes are co-ordinately down-regulated. These changes in gene regulation are brought about in part by changes in DNA binding activity of the transcription factors activating protein-1 (AP-1), nuclear factor kappa-B, and cAMP response element binding protein (CREB). Although sub-acute exposure to micromolar As(III) up-regulates transcription factor binding, chronic exposure to submicromolar As(III) causes persistent down-regulation of this response. Similar long-term exposure to micromolar concentrations of arsenate in drinking water results in a decrease in skin tumour formation in dimethylbenzanthracene (DMBA)/phorbol 12-tetradecanoate 13-acetate (TPA) treated mice. Altered response patterns after long exposure to As(III) may play a significant role in As(III) toxicology in ways that may not be predicted by experimental protocols using short-term exposures.

Effects of low dosage pyrene pollution on biochemical characters of earthworm (Eisenia fetida) in soil

By the method of artificial soil pollution, an exposure experiment with different concentrations of pyrene (0, 60, 120, 240, 480, 960 microg x kg(-1)) was conducted to determine the cytochrome P450 and MDA contents and the glutathione-S-transferase (GST), superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities in earthworm gut after exposure for 1, 3, 7 and 14 days. The results indicated that within the range of test pyrene concentrations, all the biochemical indices tested differed in their sensitivity to pyrene toxicity, among which, P450 content and GST and SOD activities were most sensitive, followed by POD and CAT activities, while MDA content did not show any obvious response. Exposure duration had stronger effects than exposure dosage. In diagnosing the ecotoxicity of soil pollutant, it could be necessary to use a combined multi-time and multi-index diagnostic method to enhance the sensitivity and effectiveness of the indices adopted.

Insights into exazaphosphorine resistance and possible approaches to its circumvention

The oxazaphosphorines cyclophosphamide, ifosfamide and trofosfamide remain a clinically useful class of anticancer drugs with substantial antitumour activity against a variety of solid tumors and hematological malignancies. A major limitation to their use is tumour resistance, which is due to multiple mechanisms that include increased DNA repair, increased cellular thiol levels, glutathione S-transferase and aldehyde dehydrogenase activities, and altered cell-death response to DNA damage. These mechanisms have been recently re-examined with the aid of sensitive analytical techniques, high-throughput proteomic and genomic approaches, and powerful pharmacogenetic tools. Oxazaphosphorine resistance, together with dose-limiting toxicity (mainly neutropenia and neurotoxicity), significantly hinders chemotherapy in patients, and hence, there is compelling need to find ways to overcome it. Four major approaches are currently being explored in preclinical models, some also in patients: combination with agents that modulate cellular response and disposition of oxazaphosphorines; antisense oligonucleotides directed against specific target genes; introduction of an activating gene (CYP3A4) into tumor tissue; and modification of dosing regimens. Of these approaches, antisense oligonucleotides and gene therapy are perhaps more speculative, requiring detailed safety and efficacy studies in preclinical models and in patients. A fifth approach is the design of novel oxazaphosphorines that have favourable pharmacokinetic and pharmacodynamic properties and are less vulnerable to resistance. Oxazaphosphorines not requiring hepatic CYP-mediated activation (for example, NSC 613060 and mafosfamide) or having additional targets (for example, glufosfamide that also targets glucose transport) have been synthesized and are being evaluated for safety and efficacy. Characterization of the molecular targets associated with oxazaphosphorine resistance may lead to a deeper understanding of the factors critical to the optimal use of these agents in chemotherapy and may allow the development of strategies to overcome resistance.

Ets2 maintains hTERT gene expression and breast cancer cell proliferation by interacting with c-Myc

Human telomerase reverse transcriptase (hTERT) underlies cancer cell immortalization, and the expression of hTERT is regulated strictly at the gene transcription. Here, we report that transcription factor Ets2 is required for hTERT gene expression and breast cancer cell proliferation. Silencing Ets2 induces a decrease of hTERT gene expression and increase in human breast cancer cell death. Reconstitution with recombinant hTERT rescues the apoptosis induced by Ets2 depression. In vitro and in vivo analyses show that Ets2 binds to the EtsA and EtsB DNA motifs on the hTERT gene promoter. Mutation of either Ets2 binding site reduces the hTERT promoter transcriptional activity. Moreover, Ets2 forms a complex with c-Myc as demonstrated by co-immunoprecipitation and glutathione S-transferase pulldown assays. Immunological depletion of Ets2, or mutation of the EtsA DNA motif, disables c-Myc binding to the E-box, whereas removal of c-Myc or mutation of the E-box also compromises Ets2 binding to EtsA. Thus, hTERT gene expression is maintained by a mechanism involving Ets2 interactions with the c-Myc transcription factor and the hTERT gene promoter, a protein-DNA complex critical for hTERT gene expression and breast cancer cell proliferation.

Comparative effects of the blue green algae Nodularia spumigena and a lysed extract on detoxification and antioxidant enzymes in the green lipped mussel (Perna viridis)

Nodularia spumigena periodically proliferates to cause toxic algal blooms with some aquatic animals enduring and consuming high densities of the blue green algae or toxic lysis. N. spumigena contains toxic compounds such as nodularin and lipopolysaccharides. This current work investigates physiological effects of exposure from bloom conditions of N. spumigena cells and a post-bloom lysis. Biochemical and antioxidative biomarkers were comparatively studied over an acute 3-day exposure. In general, a post-bloom N. spumigena lysis caused opposite physiological responses to bloom densities of N. spumigena. Specifically, increases in glutathione (GSH) and glutathione peroxidase (GPx) and decreases in glutathione S-transferase (GST) were observed from the N. spumigena lysis. In contrast, N. spumigena cell densities decreased GSH and increased GST and lipid peroxidation (LPO) in mussels. Findings also suggest that at different stages of a toxic bloom, exposure may result in toxic stress to specific organs in the mussel.

Steroid acyl transferase activity in the mammalian brain

This thesis investigates steroid fatty acid esters (which may have a possible role in myelination of the central nervous system) in mammalian brain. While these conjugates are abundant in brain tissue, little is known about their biological role. When measuring steroid acyl transferase activity, the optimum substrate concentration was found to vary significantly between the sheep and rat brain. Results also indicated that different enzymes may act to esterify different steroid substrates.