Systemic L-Buthionine -S-R-Sulfoximine Treatment Increases Plasma NGF and Upregulates L-cys/L-cys2 Transporter and γ-Glutamylcysteine Ligase mRNAs Through the NGF/TrkA/Akt/Nrf2 Pathway in the Striatum.

Glutathione (GSH) is the most abundant intracellular antioxidant. GSH depletion leads to oxidative stress and neuronal damage in the central nervous system (CNS). In mice, the acute systemic inhibition of GSH synthesis by L-buthionine-S-R-sulfoximine (BSO) triggers a protective response and a subsequent increase in the CNS GSH content. This response might be modulated by a peripheral increment of circulating nerve growth factor (NGF). NGF is an important activator of antioxidant pathways mediated by tropomyosin-related kinase receptor A (TrkA). Here, we report that peripheral administration of BSO increased plasma NGF levels. Additionally, BSO increased NGF levels and activated the NGF/TrkA/Akt pathway in striatal neurons. Moreover, the response in the striatum included an increased transcription of nrf2, gclm, lat1, eaac1, and xct, all of which are involved in antioxidant responses, and L-cys/L-cys2 and glutamate transporters. Using antibody against NGF confirmed that peripheral NGF activated the NGF/TrkA/Akt/Nrf2 pathway in the striatum and subsequently increased the transcription of gclm, nrf2, lat1, eaac1, and xct. These results provide evidence that the reduction of peripheral GSH pools increases peripheral NGF circulation that orchestrates a neuroprotective response in the CNS, at least in the striatum, through the NGF/TrkA/Akt/Nrf2 pathway.

Dietary and pharmacological compounds altering intestinal calcium absorption in humans and animals.

The intestine is the only gate for the entry of Ca to the body in humans and mammals. The entrance of Ca occurs via paracellular and intracellular pathways. All steps of the latter pathway are regulated by calcitriol and by other hormones. Dietary and pharmacological compounds also modulate the intestinal Ca absorption process. Among them, dietary Ca and P are known to alter the lipid and protein composition of the brush-border and basolateral membranes and, consequently, Ca transport. Ca intakes are below the requirements recommended by health professionals in most countries, triggering important health problems. Chronic low Ca intake has been related to illness conditions such as osteoporosis, hypertension, renal lithiasis and incidences of human cancer. Carbohydrates, mainly lactose, and prebiotics have been described as positive modulators of intestinal Ca absorption. Apparently, high meat proteins increase intestinal Ca absorption while the effect of dietary lipids remains unclear. Pharmacological compounds such as menadione, dl-butionine-S,R-sulfoximine and ursodeoxycholic acid also modify intestinal Ca absorption as a consequence of altering the redox state of the epithelial cells. The paracellular pathway of intestinal Ca absorption is poorly known and is under present study in some laboratories. Another field that needs to be explored more intensively is the influence of the gene × diet interaction on intestinal Ca absorption. Health professionals should be aware of this knowledge in order to develop nutritional or medical strategies to stimulate the efficiency of intestinal Ca absorption and to prevent diseases.

CYP2E1 induction leads to oxidative stress and cytotoxicity in glutathione-depleted cerebellar granule neurons.

Increasing evidence suggests that brain cytochrome P450 (CYP) can contribute to the in situ metabolism of xenobiotics. In the liver, some xenobiotics can be metabolized by CYPs into more reactive products that can damage hepatocytes and induce cell death. In addition, normal CYP activity may produce reactive oxygen species (ROS) that contribute to cell damage through oxidative mechanisms. CYP2E1 is a CYP isoform that can generate ROS leading to cytotoxicity in multiple tissue types. The aim of this study was to determine whether CYP2E1 induction may lead to significant brain cell impairment. Immunological analysis revealed that exposure of primary cerebellar granule neuronal cultures to the CYP inducer isoniazid, increased CYP2E1 expression. In the presence of buthionine sulfoximine, an agent that reduces glutathione levels, isoniazid treatment also resulted in reactive oxygen species (ROS) production, DNA oxidation and cell death. These effects were attenuated by simultaneous exposure to diallyl sulfide, a CYP2E1 inhibitor, or to a mimetic of superoxide dismutase/catalase, (Euka). These results suggest that in cases of reduced antioxidant levels, the induction of brain CYP2E1 could represent a risk of in situ neuronal damage.

Antidepressant-like responses in the forced swimming test elicited by glutathione and redox modulation.

Glutathione (GSH) displays a broad range of functions, among them a role as a neuromodulator with some neuroprotective properties. Taking into account that oxidative stress has been associated with depressive disorders, this study investigated the possibility that GSH, a major cell antioxidant, elicits an antidepressant-like effect in mice. Thus, GSH was administered by i.c.v. route to mice that were tested in the forced swimming test and in the tail suspension test, two predictive tests for antidepressant drug activity. In addition, GSH metabolism and the redox environment were modulated in order to study the possible mechanisms underlying the effects of GSH in the forced swimming test. The administration of GSH decreased the immobility time in the forced swimming test (300-3000nmol/site) and tail suspension test (100-1000nmol/site), consistent with an antidepressant-like effect. GSH depletion elicited by l-buthionine sulfoximine (3.2µmol/site, i.c.v.) did not alter the antidepressant-like effect of GSH, whereas the inhibition of extracellular GSH catabolism by acivicin (100nmol/site, i.c.v.) prevented the antidepressant-like effect of GSH. Moreover, a sub-effective dose (0.01nmol/site, i.c.v.) of the oxidizing agent DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) potentiated the effect of GSH (100nmol/site, i.c.v.), while the pretreatment (25-100mg/kg, i.p.) with the reducing agent DTT (dl-dithiothreitol) prevented the antidepressant-like effect of GSH (300nmol/site, i.c.v.). DTNB (0.1nmol/site, i.c.v.), produced an antidepressant-like effect, per se, which was abolished by DTT (25mg/kg, i.p.). The results show, for the first time, that centrally administered GSH produces an antidepressant-like effect in mice, which can be modulated by the GSH metabolism and the thiol/disulfide reagents. The redox environment may constitute a new venue for future antidepressant-drug development.

Modulation of glutathione intracellular levels alters the spontaneous proliferation of lymphocyte from HTLV-1 infected patients.

The human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus associated with neoplasias and inflammatory diseases, such as adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1-infected individuals present a spontaneous T lymphocyte proliferation. This phenomenon is related to the HTLV-1-proviral load and the persistence of the infection. Viral proteins induce many cellular mediators, which can be associated with the abnormal cellular proliferation. The intracellular levels of glutathione (GSH) are important to modulate the cellular proliferation. The aim of this study was to investigate the correlation between the modulation of intracellular GSH levels and the spontaneous lymphocyte proliferation during the HTLV-1 infection. Intracellular GSH level can be modulated by using dl-buthionine-[S,R]-sulfoximine (BSO, GSH synthesis inhibitor) and N-acetylcysteine (NAC, peptide precursor). Our results demonstrated that BSO was capable of inducing a decrease in the spontaneous proliferation of PBMC derived from HTLV-1 carriers. On the other hand, the GSH precursor induces an increase in mitogen-stimulated cellular proliferation in infected and uninfected individuals. Similar results were observed by the inhibition of ABCC1/MRP1 protein, augmenting the mitogen-induced proliferation. This effect can be related with an increase in the GSH levels since ABCC1/MRP1 transports GSH to the extracellular medium. There was a significant difference on the expression of CD69 and CD25 molecules during the lymphocyte activation. We did not observe any alterations on CD25 expression induced by BSO or NAC. However, our results demonstrated that NAC treatment induced an increase in CD69 expression on unstimulated CD8(+) T lymphocytes obtained from HTLV-1 infected individuals, healthy donors and HTLV carriers. Therefore, our results suggest that the cellular proliferation promoted by the infection with HTLV-1 and the activation phenotype of CD8(+) T lymphocytes can be regulated by changing the intracellular GSH levels; suggesting the modulation of these intracellular levels as a new approach for the treatment of pathologies associated with the HTLV-1 infection.

Co-operative effect of glutathione depletion and selenium induced oxidative stress on API and NFkB expression in testicular cells in vitro: insights to regulation of spermatogenesis

Previous studies have shown that transcription factors, API and NFkB exert important roles in the process by which selenium regulates spermatogenesis. Glutathione, an intracellular thiol, acts as a source of reducing power and aids in maintenance of the cellular redox status. The activities of selenium are closely related to the availability of glutathione. Presently, mouse testicular cells were cultured in the presence of BSO, a known glutathione depletor, to generate oxidative stress. Selenium (Se) was added as sodium selenite to these cells at concentrations of 0.5 µM and 1.5 µM. It was observed that at 1.5 µM, Se acted as a pro-oxidant and significantly decreased the redox ratio. RT PCR analysis revealed that cjun, cfos expression increased in testicular cells cultured with Se compared to control. However, the major outcome was that the combined effect of Se supplementation and GSH depletion resulted in reduced expression of cjun and cfos while p65 expression increased. This suggests that selenium affects both these transcription factors differently. Our study indicates that though low levels of oxidative stress generated by moderate doses of selenium augments the expression of cjun and cfos, a robust increase in the ROS generation caused by the dual effect high levels of selenium and glutathione depletion leads to decrease in the expression of these genes. The present work substantiates our in vivo experiments and indicates the detrimental effect of excess selenium supplementation on male fertility.