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 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.

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.

Role of glutathione in schizophrenia? The effect of brain glutathione depletion on prepulse inhibition (PPI) in rats and mice

Reductions in brain glutathione (GSH) levels have been reported in schizophrenia. We investigated the effects of brain GSH depletion on prepulse inhibition (PPI), a model of sensorimotor gating which is disrupted in individuals with schizophrenia. It was hypothesized that GSH depletion would lead to disruption of PPI similar to that seen in schizophrenia and enhance the effect of increased dopamine release by amphetamine. Sprague-Dawley rats and C57Bl/6 mice were treated with saline or 2-cyclohexene-1-one (CHX, 75 mg/kg and 120 mg/kg respectively) to deplete brain GSH. 225 minutes later the animals were injected with amphetamine (2.5 mg/kg in rats and 25 mg/kg in mice). Total brain GSH levels were measured using an enzymatic recycling assay. Surprisingly, in rats CHX treatment prevented the disruption of PPI by amphetamine. Thus, while there was the expected disruption of PPI caused by amphetamine on its own (average %PPI reduced from 58 ± 5 to 44 ± 4), in combination with CHX, amphetamine had no significant effect (67 ± 4 vs. 63 ± 3, respectively). In contrast to rats, in mice CHX had no effect on PPI. Thus, amphetamine similarly disrupted PPI after saline (41 ± 5 vs. 28 ± 5) and CHX pretreatment (45 ± 6 vs. 26 ± 5). There were significant 40-63% depletions of GSH in frontal cortex and striatum of CHX-treated rats and mice. These data show that GSH depletion in the brain by CHX treatment did not induce the expected decrease in PPI. Because the levels of GSH depletion in this study were similar to those found in schizophrenia, these results cast doubt on a direct interaction between brain GSH levels and PPI disruption in this illness. In rats, CHX treatment prevented the disruption of PPI caused by amphetamine. We have observed that resting levels of GSH are lower in rats than in mice. It is plausible that some oxidative damage may occur after amphetamine treatment alone, which induces marked release of the electroactive species, dopamine. In mice with their higher levels of GSH (either with or without CHX treatment) and in control rats, this does not cause functional effects. However, in CHX-treated rats GSH levels are reduced to a point where amphetamine-induced dopamine release may cause increased metabolism and lipid peroxidation inducing a decrease in postsynaptic dopamine receptor function and consequently leading to an apparent inhibition of the disruption of PPI. In conclusion, while individuals with schizophrenia show disruption of PPI and reduced brain GSH levels, in rats and mice brain GSH depletion alone does not impact on PPI. In combination with a hyperdopaminergic state, functional effects on PPI regulation were found. These effects warrant further investigation.