As our landslide frequency-magnitude analysis is based on data that were obtained during a 50-year period, they do not necessarily reflect the long-term change in denudation rate after human disturbances. More research is needed to get a comprehensive understanding of the impact of human activities on landslide-induced sediment fluxes on longer time-scales. Data collection and logistic support for this project was provided through the Belgian Science Policy, Research Program for Earth Observation Stereo II, contract SR/00/133, as part of the FOMO project (remote sensing of the forest transition and its ecosystem impacts in mountain

environments). M. Guns was funded through a PhD fellowship from the Fonds National de la Recherche Scientifique (FRS-FNRS, Belgium), and the Prize for Tropical find more Geography Yola Verhasselt of the Royal Academy for Overseas Sciences (Belgium). http://www.selleckchem.com/products/KU-55933.html The authors would like to thank Dr. A. Molina (University of Goettingen, Germany) and Dr. Vincent Balthazar for their precious help during fieldwork and Dr. Alain Demoulin for its advices. “
“Human modification of the surface of the Earth is now extensive. Clear and obvious

changes to the landscape, soils and biota are accompanied by pervasive and important changes to the atmosphere and oceans. These have led to the concept of the Anthropocene (Crutzen and Stoermer, 2000 and Crutzen, 2002), which is now undergoing examination as a potential addition to the Geological Time Scale (Zalasiewicz et al., 2008, Williams et al., 2011 and Waters et al., 2014). These changes are significant geologically, and have attracted wide interest because of the potential consequences, for human populations, of living in a world changed geologically by humans themselves. Humans have also had an impact on the

underlying rock structure of the Earth, for up to several kilometres below the planetary surface. Indirect effects of this activity, such as the carbon transfer from rock to atmosphere, are cumulatively of considerable importance. However, the extent and geological significance next of subsurface crustal modifications are commonly neglected: out of sight, out of mind. It is a realm that ranges from difficult to impossible to gain access to or to experience directly. However, any deep subsurface changes, being well beyond the reach of erosion, are permanent on any kind of human timescale, and of long duration even geologically. Hence, in imprinting signals on to the geological record, they are significant as regards the human impact on the geology of the Earth, and therefore as regards the stratigraphic characterization of the Anthropocene.

alba enzyme is most closely related to those from other Gammaproteobacteria (not shown), and the genes encoding it (including sdhDE) are found together in a possible operon. The three BOGUAY subunits selleck chemicals identified, on the other hand, are interior to three different contigs. Succinate dehydrogenase also plays a role in oxidative phosphorylation (see Section 3.4.2). The BOGUAY isocitrate dehydrogenase

(IcdA; Fig. S4D) likewise has a complex inferred history, being most closely related to sequences from hydrothermal vent gammaproteobacterial endosymbionts, the Chlorobium Chloroherpeton thalassium, and an uncultured archaeon (Thermoplasmatales archaeon SCGC AB-549-N05 Lloyd et al., 2013). Two enzymes are specific to the oxidative TCA cycle, citrate synthase and pyruvate dehydrogenase. Bacterial citrate synthases may be either Type I (homodimeric) or Type II (hexameric) (Nguyen et al., 2001). The B. alba and BgP genomes encode putative copies of both types (Table S5), but only Type I is found in the BOGUAY genome. It is closely related to the B. alba but not the BgP Type I sequence (Fig. S5A), and to sequences from other Gammaproteobacteria.

Unusually, it is also related to a sequence reportedly derived from sponge chromosomal DNA; no further information is available on this, but BLASTX matches to other regions of this scaffold (XP_003390620.1) are overwhelmingly Dabrafenib clinical trial bacterial (not shown). It may either be a contaminating sequence in the sponge genome, or recently acquired by lateral transfer. All three genomes possess putative pyruvate dehydrogenase genes (Table S5), whose phylogenies appear dissimilar, with the BOGUAY and BgP derived amino acid sequences more closely related to each other

and to sequences from a great diversity of other bacteria (Fig. S5B, C) than to the B. alba sequence. For BOGUAY, no gene for 2-oxoglutarate dehydrogenase (SucAB) was identified; however, this gap can be filled by the “reductive” KorAB in some bacteria (e.g., Baughn et al., 2009). No gene for the succinate dehydrogenase/fumarate reductase membrane anchor (SdhD) could be found, but the oxidative pathway is otherwise complete. Three enzymes are specific to the rTCA pathway: ATP citrate lyase (AclAB), 2-oxoglutarate ferredoxin oxidoreductase (KorAB), and pyruvate oxidoreductase (PorABCD). Of the three relatively complete Beggiatoaceae see more genomes, only orange Guaymas Beggiatoa possesses a complete set of these ( Fig. 5, Table S5), and their inferred phylogenies suggest histories of horizontal transfer via different routes. The putative BOGUAY AclA and AclB amino acid sequences (Fig. S6A, B) are both most closely related to sequences from a small cluster of other Gammaproteobacteria (Thioflavicoccus mobilis 8321, a tubeworm endosymbiont, and a hydrothermal vent environmental sequence), but beyond that to sequences from diverse proteobacteria. For the pyruvate:ferredoxin oxidoreductase KorAB (Fig.

Despite the recent decrease in total catch compared with 10 years ago, fish exports have increased constantly; this

increase seems to occur at the expense of local consumption and has caused significant increases in fish prices in local markets [44]. Artisanal fishing accounts for well over 90% of the total production [27]. The key fisheries resources, shown in Table 1, include pelagic fishery for tuna and tuna-like species and demersal fishery for fish, cuttlefish, shrimp, and lobster. Tuna and tuna-like species and cuttlefish are prevalent in the Gulf of Aden and the Arabian Sea, whereas demersal fish are more abundant in the Red Sea. Key pelagic species include yellowfin tuna, longtail tuna, little tuna, narrow-barred Spanish mackerel, Indian mackerel, anchovy, and sharks; key demersal fish species include emperors, groupers, snappers, and jacks [27] and [32]. Despite selleck chemicals the lack of comprehensive Forskolin ic50 stock assessment studies and reliable catch statistics, it is believed that most fish stocks, except small pelagic species for which there is no market demand, are either fully exploited or overexploited [37]; interviews with fishermen and

different stakeholders confirm these beliefs. Cuttlefish (Sepia pharaonis) has been harvested since 1967 by industrial fleets in the Gulf of Aden and the Arabian Sea region. The intensive trawling on their spawning aggregations has led to overfishing and a major decline of the fishery by 1982–1983 with reported annual landings falling from around 9000 to 1500 t. Landings of the rock lobster (Panulirus homarus) virtually collapsed to near zero in the late 1990s from peaks of around 400 t in the early part of the decade. This collapse was attributed to the widespread use of nets rather than traps to capture lobsters [37]. Large-scale harvest of sea cucumbers started in 2003 with the advent of air compressors, which facilitated diving; this process

led, a few years later, to the collapse of the fishery [45]. Many important demersal fish stocks and some pelagic species, such as Indian Tryptophan synthase mackerel [41], narrow-barred Spanish mackerel, and sharks [40] and [46], have experienced severe overfishing and their production levels have been beyond the maximum sustainable yields. The lack of FMPs, widespread IUU fishing, uncontrolled growth of fishing effort, and weak compliance and enforcement arrangements have led to significant economic losses associated with the suboptimal use of the resources, which has in turn resulted from weak and ineffective governance and subsequent overfishing. Small-scale fishermen typically use two types of fishing boats: small fiberglass boats called huris, 7–16 m long, with outboard engines and 2–6 crew members, and larger wooden boats called sambuks, 10–20 m long, with inboard or outboard engines and with a crew from 10 up to 25 or more [4] and [27]. Huris were traditionally used for single day trips in inshore waters, within 40 km of the shore [4].

Venom solution was prepared at the moment of use with 0.5 mg of lyophilized venom (provided by Instituto Butantan) in 1 mL of sterile phosphate buffered saline (PBS) (Na2HPO4·7H2O, 19.3 g/L; NaH2PO4·H2O, 3.9 g/L; NaCl, 8.77 g/L; pH 7.4), see more under mild mixing, for 10 min, at 4 °C and, then, centrifuged at 20,927×g (Cientec CT-14000 R), for 10 min at 4 °C. The pellet was discarded. The same lot of venom was used during this study. Lipoic acid (dl-alpha-lipoic acid) (Sigma, USA) was dissolved in absolute ethanol (50 mg/mL) at the moment of use. Immediately, this solution was diluted (1:5) in PBS (LA solution). The adopted

dose was 2 mg/20 g body mass, in a maximum volume of 0.2 mL, per oral (po). In order to evaluate its effects on AKI, this dose was administered 2 h after the envenomation. LA at

this dose and route was effective against nephrotoxicity induced by chloroquine, when compared with other doses (0.2 and 0.6 mg/20 g body A-1210477 order mass) ( Murugavel and Pari, 2004), and was also effective against certain nephrotoxic effects of C. d. terrificus envenomation ( Alegre et al., 2010). Intraperitoneal (ip) injection of LA at this same dose attenuated the ischaemia/reperfusion-induced increases in blood urea nitrogen, plasma concentrations of creatinine and fractional excretion of sodium ( Takaoka et al., 2002). Simvastatin (Novartis, Brasil) was dissolved in absolute ethanol (30 mg/mL) at the moment of use. Immediately, this solution was diluted (1:100) in PBS (SA solution). The adopted dose was 0.06 mg/20 g body mass,

in a maximum volume of 0.2 mL, po. In order to evaluate its effects on AKI, this dose was administered 2 h after the envenomation. SA at this dose and route was effective to mitigate uricosuria, renal oxidative stress and protein increase in C. d. terrificus envenomed mice ( Yamasaki et al., 2008). Adult male Swiss mice, weighing 18–20 g, provided by the Animal Facility of the Instituto Butantan, were maintained in polyethylene cages (inside length × width × height = 56 × 35 × 19 cm) with food and water ad libitum, in GNA12 a container with controlled temperature of 25 °C, relative humidity of 65.3 ± 0.9% and 12 h:12 h photoperiod light:dark (lights on at 6:00 am). Animals and research protocols used in this study are in agreement with the COBEA (Brazilian College of Animal Experimentation) and were approved by the Ethics Committee of Instituto Butantan (492/08). The venom was administered ip at a dose calculated on the basis of literature data. It is known that the LD50 ip of vBj in rats coincides with a minimum dose that causes renal damage ( Rezende et al., 1989). According to Ferreira et al. (2005b), the LD50 ip in mice is 2.

The acute effects of TBI (primary injuries) have been the focus of most biomarker studies, while sub-acute and long-term effects

of TBI (secondary injuries) have not been received as much attention. Secondary injuries due to mTBI are expected to be particularly subtle at the molecular level, posing a profound challenge for the discovery of clinically relevant biomarkers. Primary injuries are characterized by short-term increases in oxidative stress and decreases in selleck screening library motor function [[6], [7], [8] and [9]]. These initial events are followed by a poorly understood secondary response characterized by long-term effects associated with neuronal degeneration and functional and cognitive deficits, including deficits in memory, coordination, judgment, balance and

fine motor skills Nutlin-3 datasheet [7]. While the importance of investigating these long-term changes is becoming more appreciated due to strengthening links between TBI and multiple age-associated neurodegenerative diseases [[10], [11], [12], [13], [14] and [15]], few pre-clinical studies have examined the long-term functional and biochemical changes associated with mTBI [11,[16], [17], [18] and [19]]. The most sensitive (most true-positive) and specific (least false-positive) biomarkers are expected to be proteins. More than 24,000 genes are translated into an estimated 2 million protein isoforms in humans, encoding far more molecular diversity than the relatively static genome or transcriptome. Paradoxically, less than 100 proteins are routinely quantified in blood today [20,21]. Proteins must be measured directly due to the poor correlation between the transcriptome and proteome due to alternative splicing, post-translational

modifications, single nucleotide polymorphisms, limiting ribosomes available for translation, mRNA and protein stability, and other actors (e.g., microRNA). Central nervous system-specific proteins (CSPs), transported across the damaged blood–brain-barrier to cerebral spinal fluid (CSF) or blood, are attractive protein biomarkers for TBI because they are not expected at appreciable levels in the circulation of healthy Resminostat controls. However, amino acid sequence specific tandem mass spectrometry (MS/MS)-based proteomic analysis of low abundance CSPs can be confounded by masking effects due to high abundance proteins, particularly in CSF or blood where protein abundance can span up to 12 orders of magnitude. For these reasons and others, proteomic analysis of CSPs in brain tissue is a sound strategy for prioritizing putative protein biomarkers for future immunoassay (e.g., ELISA) measurements in CSF or blood. We hypothesized that changes in CSP expression might correlate to these long-term secondary effects. To test our hypothesis, we longitudinally assessed a closed-skull mTBI mouse model, vs. sham control, at 1, 7, 30, and 120 days post-injury.

Then, 20 μL of this suspension was injected into the HPLC (Shimadzu, C18 reverse

phase and UV detection at 280 nm) and eluted with a gradient of acetonitrile and 0.175 (g/100 mL) orthophosphoric acid ( Makkar et al., 1997). We use phorbol-12-myristate 13-acetate (Sigma) as standard. The soluble protein and reducing sugar contents were determined according to the colorimetric methods described by Bradford (1976) and Miller (1959). For this analysis, 10 g of the crushed mushrooms was placed in Erlenmeyer flasks (125 mL) containing 25 mL sodium citrate buffer PD0332991 (0.05 mol/L and pH 4.8). These flasks were kept in a shaker for 30 min at 150 rpm, and the extracts were filtered using Millipore membranes (Cavallazzi et al., 2004). This experiment was conducted using a completely randomized design with 5 replicates. The data were subjected to analysis

of variance, and the averages were compared by Tukey’s test (p < 0.05) using Saeg software (version 9.1, Federal University of Viçosa). The tannin concentrations Selleck Fasudil observed in the jatropha seed cake (Fig. 1) were similar to those found in the fruit peel of J. curcas ( Makkar, Aderibigbe, & Becker, 1998). The greatest concentration of this compound was observed in the substrate with coffee husk and eucalypt bark ( Fig. 1). This may have been due to the presence of tannins in the eucalypt bark ( Vázquez, González-Alvarez, Santos, Freire, & Antorrena, 2009) and in the coffee husk ( Barcelos, Paiva, Pérez, Santos, & Cardoso, 2001). The thermal treatment Methisazone of the substrates decreased the tannin concentration by 46%

(Fig. 1). This result was similar to that observed in vegetables after cooking or autoclaving at 121 °C and 128 °C for different periods of time (Rehman & Shah, 2005). Regardless of the substrate, tannin degradation by P. ostreatus Plo 6 increased as a function of the incubation time, and the highest rate was observed between 15 and 30 d in the substrate with coffee husk and eucalypt bark ( Fig. 1). The high degradation rate of this compound was also observed in Pleurotus sp. cultivated in coffee husk for 60 d ( Fan et al., 2006) and this tannin degradation may be related to tannase activity (tannin acyl hydrolase, EC 3.1.1.20). The activity of this enzyme in the polyphenols degradation has been reported in Aspergillus and Penicillium ( Batra & Saxena, 2005). Thus, P. ostreatus can degrade the tannins in J. curcas seed cake and the other tested substrates. The acid phytic content in the jatropha seed cake (Fig. 2A) was lower than the percentage this acid found in the seed of J. curcas by Makkar et al. (1998). This result shows that a percentage this acid was in the oil. Although phytic acid is considered to be heat-stable (Deshpande & Damodaran, 1990), the sterilization of the substrates decreased in 20% the content of phytic acid (Fig. 2A).

Os relatórios histológicos não dão aos clínicos e aos gastrenterologistas uma mensagem GSK1349572 research buy explícita de orientação daquele doente em concreto. O grau de atrofia e o tipo de metaplasia intestinal nem sempre são classificados. Como sabemos, a metaplasia intestinal pode ser de tipo entérico (completa, ou tipo I), enterocólica (incompleta, tipo II) ou colónica (incompleta, tipo III), sendo que este grau III tem sido tradicionalmente associado a uma maior gravidade, mas, na verdade, a extensão da atrofia e da metaplasia talvez seja o melhor marcador de pré‐malignidade, sendo a subtipagem da metaplasia, provavelmente de menor valor na prática clínica4. A causa mais

comum de metaplasia intestinal é a gastrite induzida pelo H. pylori, mas lembramos que a deteção da metaplasia intestinal em biopsias de rotina está sujeita a erros de amostragem e pode não ser o marcador desejável de risco aumentado de carcinoma gástrico 5. Tal como é referido no artigo «One day of upper gastrointestinal endoscopy in a southern European country», a extensão da metaplasia intestinal e da atrofia da mucosa ao corpo gástrico parece ter um papel relevante. Já há alguns anos, alguns AA advogavam que, com base na sua correlação com a metaplasia intestinal,

uma gastrite corporal pronunciada poderia ser considerada um marcador de cancro gástrico. Em comparação com a metaplasia intestinal este marcador de risco de cancro gástrico tem a vantagem de estar associado a uma menor variabilidade interobservadores p38 MAPK inhibitors clinical trials e, devido à sua apresentação difusa, a um menor risco de erros de amostragem6. Por outro lado, a localização das biopsias de rotina

não tem sido consensual, nomeadamente no que respeita às biopsias na incisura angularis, mas a sua realização nesta localização tem sido enfatizada em estudos recentes, dado que a incisura angularis está sujeita a um maior índice de gastrite atrófica severa, metaplasia e inflamação crónica why do que o corpo e o antro, pelo que é de considerar (ainda que não haja consenso) que estas biopsias devam ser rotineiramente incluídas nos protocolos 7. Parece óbvio que se torna importante estratificar os doentes de acordo com o risco de desenvolvimento de cancro gástrico, e os sistemas Operative Link for Gastritis Assessment (OLGA)8 e Operative Link on Gastric Intestinal Metaplasia (OLGIM) têm sido propostos com esse objetivo, sendo para isso necessária a real cooperação entre gastrenterologistas, na execução conveniente das biopsias, e anatomopatologistas, no uso destas escalas de valor analógico de classificação da atrofia gástrica e da metaplasia. Em termos de biopsias, a proposta do sistema OLGA consiste, basicamente, na realização de pelo menos 5 biopsias: na grande e pequena curvaturas do antro distal (A1 e A2); na pequena curvatura da incisura angularis (A3); e na parede anterior e posterior do corpo proximal (C1 e C2). Mas o número de biopsias continua a não ser consensual.

Thirdly, at high pH values (i.e. Complex III), dimers of GA or EGCG might be involved in the complexes. However, many of the spectra are derived from more than one complex, the spectra of which are not resolved from one another at RT, so it is inappropriate to discuss these details further. Characterisation of the EPR silent species that are formed at weakly acidic pH values is problematic. With the Cu/GA system, Ferreira Severino et al. [9] showed that the loss of signal was not the result of reduction of Cu(II) to Cu(I), and proposed

that the EPR silent species involved the formation of di- or polymeric complexes with coordination of the carboxyl group, as seen with simple carboxylic acids. However, coordination of carboxyl groups is not an option with EGCG, and any extended structure http://www.selleckchem.com/products/iox1.html with this polyphenol must be based on VE-822 mw coordination of pyrogallol groups. However, the similarity of the results with GA and EGCG indicates that the chemistry of the reactions with Cu(II) of both phenols is similar, and suggests that the EPR silent species involve extended structures in which the Cu is coordinated to the pyrogallol moiety. The complexation

chemistry of EGCG is further complicated by the presence of two pyrogallol groups in the same molecule. In previous work on the oxidation of EGCG [29], it was shown that the site of oxidation is dependent on the experimental conditions, and that the relative reactivities of the B and D rings is not always the same. The pH of the solution could be a factor in determining this, since it also determines the degree of proton dissociation. ADP ribosylation factor Thus it is possible that the products of reaction between Cu(II) and EGCG are not discrete molecules, but a group of closely related complexes. Nevertheless, the EPR results are consistent with those from the Cu(II)/GA system, and are consistent

with the formation of extended structures at acidic pH values, with the formation of mononuclear Cu(II) complexes gaining in importance at higher pH values and EGCG concentrations. Furthermore, as stated by Ferreira Severino et al. [9] for the Cu(II)/GA system, there is no convincing evidence for any redox reaction between Cu(II) and either of the polyphenols. The chemistry of the reactions of Cu(II) with the polyphenols EGCG and GA is similar, although the molecular mass of EGCG is four times that of GA with several more phenolic groups, but lacking any carboxyl group. With both polyphenols, EPR silent species are formed at weakly acidic pH values, and strong evidence is presented for these having extended structures rather than being the consequence of reduction of Cu(II) to Cu(I). The polyphenols, therefore, result in the removal of Cu from solution at an appreciably lower pH than is observed in the absence of the polyphenol (by ~ 2 pH units).

The commercialization of transgenic glyphosate-tolerant Talazoparib mw soybean in 1996 introduced a new pattern of use in which glyphosate can be applied to crops post-emergence to remove weeds without damage of crops. Since then, herbicide-tolerant crops have been quickly adopted by farmers. In 2012, herbicide tolerance, deployed in maize (Zea mays L.), Indian mustard (Brassica

juncea L.), Anemone vitifolia Buch.-Ham., soybean (Glycine max L.), sugar beet (Beta vulgaris L.), and erba medica (Medicago sativa L.) occupied 59% of 170.3 million hectares of transgenic crops planted globally [3]. Two basic strategies have been successfully used in glyphosate-tolerant crop development: expression of an insensitive form of the target enzyme EPSPS, and detoxification of the MAPK Inhibitor Library purchase glyphosate molecule. The first strategy has been used in most existing commercial glyphosate-tolerant crops. They were obtained by employing a mutated (TIPS) or a microbial (CP4) form of EPSPS that is not inhibited by glyphosate [4] and [5]. The theoretical disadvantage of this method is that glyphosate remains and accumulates in plant meristems, where it may hinder reproductive development

and lower crop yield [6]. The second approach avoids this limitation, because its functional mechanism is removal of herbicidal residue. N-acetylglyphosate is not herbicidal and does not inhibit EPSP synthase. Castle et al. [7] and [8] cloned glyphosate acetyltransferase (GLYAT) enzyme genes from Bacillus licheniformis. By stiripentol DNA shuffling, a Glyat gene was obtained that had catalytic efficiency appropriate for commercial levels of resistance to glyphosate in crops. The first trait, in which GLYAT is deployed in soybean and canola (Brassica campestris L.), is in advanced stages of development (Pioneer Hi-Bred Technical Update) [1]. In China, a key problem in herbicide-tolerance gene engineering is the

shortage of genes with higher glyphosate tolerance and independent intellectual property rights. Thus, it is of interest to seek new glyphosate-tolerance genes for developing glyphosate-tolerant crops that have high and stable heritability for glyphosate tolerance. Based on the biological diversity of microbial genetic resources in extremely polluted environments, a gat gene encoding N-acetyltransferase and a G2-aroA gene encoding EPSPS have been isolated by molecular biological methods [9] and [10]. G2-aroA showed enhanced glyphosate tolerance in transgenic crops [11]. In the present study, we simultaneously introduced the G2-aroA and gat genes into tobacco, Nicotiana tabacum L. Glyphosate tolerance analysis indicated that transgenic tobacco coexpressing G2-aroA and gat displayed higher tolerance to glyphosate than transgenic tobacco containing G2-aroA or gat alone.

Hepatocyte viability after isolation was determined by Trypan blue (0.16%) uptake, and initial cell viability in all experiments was more than 85%. Hepatocytes were suspended in Krebs-Henseleit buffer, pH 7.4, containing 12.5 mM Hepes and 0.1% albumin (BSA) and maintained at 4 °C. Cells (1 × 106/mL) were incubated in 25-mL Erlenmeyer flasks kept under constant agitation (30 rpm) at 37 °C AZD2281 cell line under a 95% O2 and 5% CO2 atmosphere. Reactions were initiated by the addition of MCT. Aliquots (0.5 mL) of the suspension were removed from the mixture at appropriate times for the determination of cell death and

biochemical parameters. In some experiments, cells were incubated with 20 mM fructose or 10 mM dithiotreitol (DTT) 15 min before the addition of selleck products MCT. Oxygen uptake by the isolated hepatocytes was monitored polarographically with an oxygraph equipped

with a Clark-type oxygen electrode (Strathkelvin Instruments Limited, Glasgow, Scotland, UK) at 37 °C. Respiration buffer contained 250 mM sucrose, 2 mM KH2PO4, 10 mM HEPES, pH 7.2, 0.5 mM EGTA, 0.5% bovine serum albumin, and 5 mM MgCl2. Cells were treated with 0.002% digitonin, and state 4 and state 3 mitochondrial respiration rates were measured in the presence of 1 μg/mL oligomycin and 2 mM ADP, respectively (Moreadith and Fisckum, 1984). MCT and DHM were added in the medium, immediately after the initiation of state 3 respirations by ADP. Cell viability was assessed by the leakage of alanine transaminase

(ALT) from hepatocytes. Cell suspensions were centrifuged (50 × g for 5 min). ALT in the supernatant was determined using an Alanine Transaminase Activity Assay Kit (Bioclin, Quibasa, Brazil) according to the manufacturer’s instructions. Absorbance was measured at 340 nm with a DU-800 spectrophotometer (Beckman Coulter Inc., Fullerton, CA, USA). Enzyme activity in the supernatant is expressed next as a percentage of the total activity, which was determined by lysing the cells with 0.5% Triton X-100. Cell ATP was determined by means of the firefly luciferin-luciferase assay system. The cell suspension was centrifuged at 50 × g for 5 min at 4 °C, and the pellet containing the hepatocytes was treated with 1 mL of ice-cold 1 M HClO4. After centrifugation at 2000 × g for 10 min at 4 °C, aliquots (100 μL) of the supernatant were neutralized with 70 μL of 2 M KOH, suspended in 100 mM Tris-HCl, pH 7.8 (1 mL final volume), and centrifuged again. Bioluminescence was measured in the supernatant with a Sigma-Aldrich assay kit according to the manufacturer’s instructions using a SIRIUS Luminometer (Berthold, Pforzheim, Germany). The levels of GSH were determined by a fluorimetric reaction with o-phthalaldialdehyde (OPT) (Hissin and Hilf, 1976). The cell suspension was treated with 0.2 mL of 30% TCA and centrifuged at 2000 × g for 6 min. Aliquots (100 μL) of the supernatant were mixed with 2 mL of 100 mM NaH2PO4 buffer, pH 8.0, containing 5 mM EGTA.