Tanniniferous Dichrostachys cinerea fruits do not require detoxification for goat nutrition: in sacco and in vivo evaluations

This study investigated the potential of Dichrostachys cinerea fruits as a protein supplement in semi-arid areas of Zimbabwe. The tanniniferous fruits were treated with aqueous solutions of polyethylene glycol (PEG) or sodium hydroxide (NaOH). Both treatments increased the soluble fraction, rate of degradation and effective degradability (ED) of nitrogen (N) in sacco. The PEG effects were higher than the NaOH effects (e.g. a 25% vs. 6% increase in effective N degradabilities, respectively). Five treatments were evaluated in a N-balance trial using Matebele goats: ground, PEG- or NaOH-treated D. cinerea fruits, a commercial protein supplement (CPS) and no supplement. Animals offered ground fruits or CPS retained most N (3.7 or 4.1 g N/day, respectively), while those offered NaOH- or PEG-treated fruits retained significantly less N (2.7 or 1.0 g/day, respectively). Unsupplemented animals were in negative N balance (-2.4 g/day). PEG treatment deactivated the tannins more than the NaOH treatment. PEG treatment resulted in excessive protein degradation in the rumen leading to high urine N loss. It is concluded that the D. cinerea fruits were beneficial for goat N-nutrition and that the tannins did not require inactivation. D. cinerea fruits can, therefore, replace the expensive commercial protein supplement. It is also suggested that the collection and grinding of fruits could be used as a management tool to control bush encroachment. (C) 2004 Elsevier B.V. All rights reserved.

Modulation of detoxification enzymes by watercress: in vitro and in vivo investigations in human peripheral blood cells

Epidemiological studies indicate that consumption of cruciferous vegetables (CV) can reduce the risk of cancer. Supposed mechanisms are partly the inhibition of phase I and the induction of phase II enzymes. The aim of this study was to investigate in vitro and in vivo effects of watercress (WC), a member of the CV family, on chemopreventive parameters using human peripheral blood mononuclear cells (PBMC) as surrogate cells. We investigated the hypothesis that WC reduces cancer risk by inducing detoxification enzymes in a genotype-dependent manner. In vitro gene expression and enzyme activity experiments used PBMC incubated with a crude extract from fresh watercress (WCE, 0.1-10 mu L/mL with 8.2 g WC per 1 mL extract) or with one main key compound phenethyl isothiocyanate (PEITC, 1-10 mu M). From an in vivo perspective, gene expression and glutathione S-transferase (GST) polymorphisms were determined in PBMC obtained from a human intervention study in which subjects consumed 85 g WC per day for 8 weeks. The influence of WC consumption on gene expression was determined for detoxification enzymes such as superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1), whilst the SOD and GPX activities in red blood cells were also analysed with respect to GST genotypes. In vitro exposure of PBMC to WCE or PEITC (24 h) increased gene expression for both detoxification enzymes GPX1 (5.5-fold, 1 mu L/mL WCE, 3.7-fold 1 mu M PEITC) and SOD2 (12.1-fold, 10 mu L/mL WCE, 7.3-fold, 10 mu M PEITC), and increased SOD2 activity (1.9-fold, 10 mu L/mL WCE). The WC intervention had no significant effect on in vivo PBMC gene expression, as high individual variations were observed. However, a small but significant increase in GPX (p = 0.025) and SOD enzyme activity (p = 0.054) in red blood cells was observed in GSTM1*0, but not in GSTM1*1 individuals, whilst the GSTT1 genotype had no impact. The results indicate that WC is able to modulate the enzymes SOD and GPX in blood cells in vitro and in vivo, and suggest that the capacity of moderate intake of CV to induce detoxification is dependent in part on the GSTM1 genotype.

Sulforaphane protects cortical neurons against 5-S-cysteinyl-dopamine-induced toxicity through the activation of ERK1/2, Nrf-2 and the upregulation of detoxification enzymes

The degeneration of dopaminergic neurons in the substantia nigra has been linked to the formation of the endogenous neurotoxin 5-S-cysteinyl-dopamine. Sulforaphane (SFN), an isothiocyanate derived from the corresponding precursor glucosinolate found in cruciferous vegetables has been observed to exert a range of biological activities in various cell populations. In this study, we show that SFN protects primary cortical neurons against 5-S-cysteinyl-dopamine induced neuronal injury. Pre-treatment of cortical neurons with SFN (0.01-1 microM) resulted in protection against 5-S-cysteinyl-dopamine-induced neurotoxicity, which peaked at 100 nM. This protection was observed to be mediated by the ability of SFN to modulate the extracellular signal-regulated kinase 1 and 2 and the activation of Kelch-like ECH-associated protein 1/NF-E2-related factor-2 leading to the increased expression and activity of glutathione-S-transferase (M1, M3 and M5), glutathione reductase, thioredoxin reductase and NAD(P)H oxidoreductase 1. These data suggest that SFN stimulates the NF-E2-related factor-2 pathway of antioxidant gene expression in neurons and may protect against neuronal injury relevant to the aetiology of Parkinson's disease.

Exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to triclosan induces a species-specific response, including drug detoxification

Objectives: The use of triclosan within various environments has been linked to the development of multiple drug resistance (MDR) through the increased expression of efflux pumps such as AcrAB-ToIC. In this work, we investigate the effect of triclosan exposure in order to ascertain the response of two species to the presence of this widely used biocide. Methods: The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli K-12 MG1655 after exposure to the MIC of triclosan (0.12 mg/L) were determined in microarray experiments. Phenotypic validation of the transcriptomic data included RT-PCR, ability to form a biofilm and motility assays. Results: Despite important differences in the triclosan-dependent transcriptomes of the two species, increased expression of efflux pump component genes was seen in both. Increased expression of soxS was observed in Salmonella Typhimurium, however, within E. coli, decreased expression was seen. Expression of fabBAGI in Salmonella Typhimurium was decreased, whereas in E. coli expression of fabABFH was increased. Increased expression of ompR and genes within this regulon (e.g. ompC, csgD and ssrA) was seen in the transcriptome of Salmonella Typhimurium. An unexpected response of E. coli was the differential expression of genes within operons involved in iron homeostasis; these included fhu, fep and ent. Conclusions: These data indicate that whilst a core response to triclosan exposure exists, the differential transcriptome of each species was different. This suggests that E. coli K-12 should not be considered the paradigm for the Enterobacteriaceae when exploring the effects of antimicrobial agents.

Dichrostachys cinerea and Acacia nilotica fruits as dry season feed supplements for goats in a semi-arid environment: Summary of a DFID funded project in Zimbabwe

Indehiscent fruits of six tree species, common in Matabeleland were examined in in vitro and in vivo trials. The results for two of them, Acacia nilotica and Dichrostachys cinerea are presented here. Acacia nilotica-contained more total phenolics than D. cinerea, but less nitrogen (N) and fibre (ADF and NDF). After 48 h incubation, in vitro OMD of both species was increased by PEG and NaOH or wood ash treatment, except when NaOH or wood ash were used in combination with PEG with D. cinerea fruits. DM intake, DMD were lowest and N-retention negative in goats fed A. nilotica as supplement. However when fed a supplement of D. cinerea, untreated or treated with PEG or NaOH, digestibility and N-retention were highest, and similar to a commercial goat meal, with the untreated fruit. In a trial in which milking does were supplemented with D. cinerea fruits, for 65 before and 65 days after kidding, kid birthweight and weaning weight were increased by supplementation. Deaths of twin-born kids were lowest in the supplemented but unmilked group. Supplementation with D. cinerea fruit resulted in improved goat performance. The only treatment applied of practical significance, wood ash, is currently being tested in an in vivo study. More research is required on detoxification of tannins, especially with A. nilotica. (c) 2005 Elsevier B.V. All rights reserved.

Bacterial iron homeostasis

Iron is essential to virtually all organisms, but poses problems of toxicity and poor solubility. Bacteria have evolved various mechanisms to counter the problems imposed by their iron dependence, allowing them to achieve effective iron homeostasis under a range of iron regimes. Highly efficient iron acquisition systems are used to scavenge iron from the environment under iron-restricted conditions. In many cases, this involves the secretion and internalisation of extracellular ferric chelators called siderophores. Ferrous iron can also be directly imported by the G protein-like transporter, FcoB. For pathogens, host-iron complexes (transferrin, lactoferrin, haem, haemoglobin) are directly used as iron sources. Bacterial iron storage proteins (ferritin, bacterioferritin) provide intracellular iron reserves for use when external supplies are restricted, and iron detoxification proteins (Dps) are employed to protect the chromosome from iron-induced free radical damage. There is evidence that bacteria control their iron requirements in response to iron availability by downregulating the expression of iron proteins during iron-restricted growth. And finally, the expression of the iron homeostatic machinery is subject to iron-dependent global control ensuring that iron acquisition, storage and consumption are geared to iron availability and that intracellular levels of free iron do not reach toxic levels. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Aldose reductase and the importance of experimental design

Kinetic studies on the AR (aldose reductase) protein have shown that it does not behave as a classical enzyme in relation to ring aldose sugars. As with non-enzymatic glycation reactions, there is probably a free radical element involved derived from monosaccharide autoxidation. in the case of AR, there is free radical oxidation of NADPH by autoxidizing monosaccharides, which is enhanced in the presence of the NADPH-binding protein. Thus any assay for AR based on the oxidation of NADPH in the presence of autoxidizing monosaccharides is invalid, and tissue AR measurements based on this method are also invalid, and should be reassessed. AR exhibits broad specificity for both hydrophilic and hydrophobic aldehydes that suggests that the protein may be involved in detoxification. The last thing we would want to do is to inhibit it. ARIs (AR inhibitors) have a number of actions in the cell which are not specific, and which do not involve them binding to AR. These include peroxy-radical scavenging and effects of metal ion chelation. The AR/ARI story emphasizes the importance of correct experimental design in all biocatalytic experiments. Developing the use of Bayesian utility functions, we have used a systematic method to identify the optimum experimental designs for a number of kinetic model data sets. This has led to the identification of trends between kinetic model types, sets of design rules and the key conclusion that such designs should be based on some prior knowledge of K-m and/or the kinetic model. We suggest an optimal and iterative method for selecting features of the design such as the substrate range, number of measurements and choice of intermediate points. The final design collects data suitable for accurate modelling and analysis and minimizes the error in the parameters estimated, and is suitable for simple or complex steady-state models.

Genistein blocks homocysteine-induced alterations in the proteome of human endothelial cells

Dietary isoflavones from soy are suggested to protect endothelial cells from damaging effects of endothelial stressors and thereby to prevent atherosclerosis. In search of the molecular targets of isoflavone action, we analyzed the effects of the major soy isoflavone, genistein, on changes in protein expression levels induced by the endothelial stressor homocysteine (Hcy) in EA.hy 926 endothelial cells. Proteins from cells exposed for 24 h to 25 mu M Hcy alone or in combination with 2.5 mu M genistein were separated by two-dimensional gel electrophoresis and those with altered spot intensities were identified by peptide mass fingerprinting, Genistein reversed Hcy-induced changes of proteins involved in metabolism, detoxification, and gene regulation: and some of those effects can be linked functionally to the antiatherosclerotic properties of the soy isoflavone. Alterations of steady-state levels of cytoskeletal proteins by genistein suggested an effect oil apoptosis. As a matter of fact genistein caused inhibition of Hcy-mediated apoptotic cell death as indicated by inhibition of DNA fragmentation and chromatin condensation. In conclusion, proteome analysis allows the rapid identification of cellular target proteins of genistein action in endothelial cells exposed to the endothelial stressor Hcy and therefore enables the identification of molecular pathways of its antiatherosclerotic action

Intracellular metabolism and bioactivity of quercetin and its in vivo metabolites

Understanding the cellular effects of flavonoid metabolites is important for predicting which dietary flavonoids might be most beneficial in vivo. Here we investigate the bioactivity in dermal fibroblasts of the major reported in vivo metabolites of quercetin, i.e. 3'-O-methyl quercetin, 4'-O-methyl quercetin and quercetin 7-O-beta-D-glucuronide, relative to that of quercetin, in terms of their further metabolism and their resulting cytotoxic and/or cytoprotective effects in the absence and presence of oxidative stress. Uptake experiments indicate that exposure to quercetin led to the generation of two novel cellular metabolites, one characterized as a 2'-glutathionyl quercetin conjugate and another product with similar spectral characteristics but 1 mass unit lower, putatively a quinone/quinone methide. A similar product was identified in cells exposed to 3'-O-methyl quercetin, but not in the lysates of those exposed to its 4'-O-methyl counterpart, suggesting that its formation is related to oxidative metabolism. There was no uptake or metabolism of quercetin 7-O-beta-D-glucuronide by fibroblasts. Formation of oxidative metabolites may explain the observed concentration-dependent toxicity of quercetin and 3'-O-methyl quercetin, whereas the formation of a 2'-glutathionyl quercetin conjugate is interpreted as a detoxification step. Both O -methylated metabolites conferred less protection than quercetin against peroxide-induced damage, and quercetin glucuronide was ineffective. The ability to modulate cellular toxicity paralleled the ability of the compounds to decrease the level of peroxide-induced caspase-3 activation. Our data suggest that the actions of quercetin and its metabolites in vivo are mediated by intracellular metabolites.

The Gly482Ser genotype at the PPARGC1A gene and elevated blood pressure: a meta-analysis involving 13,949 individuals.

The protein encoded by the PPARGC1A gene is expressed at high levels in metabolically active tissues and is involved in the control of oxidative stress via reactive oxygen species detoxification. Several recent reports suggest that the PPARGC1A Gly482Ser (rs8192678) missense polymorphism may relate inversely with blood pressure. We used conventional meta-analysis methods to assess the association between Gly482Ser and systolic (SBP) or diastolic blood pressures (DBP) or hypertension in 13,949 individuals from 17 studies, of which 6,042 were previously unpublished observations. The studies comprised cohorts of white European, Asian, and American Indian adults, and adolescents from South America. Stratified analyses were conducted to control for population stratification. Pooled genotype frequencies were 0.47 (Gly482Gly), 0.42 (Gly482Ser), and 0.11 (Ser482Ser). We found no evidence of association between Gly482Ser and SBP [Gly482Gly: mean = 131.0 mmHg, 95% confidence interval (CI) = 130.5-131.5 mmHg; Gly482Ser mean = 133.1 mmHg, 95% CI = 132.6-133.6 mmHg; Ser482Ser: mean = 133.5 mmHg, 95% CI = 132.5-134.5 mmHg; P = 0.409] or DBP (Gly482Gly: mean = 80.3 mmHg, 95% CI = 80.0-80.6 mmHg; Gly482Ser mean = 81.5 mmHg, 95% CI = 81.2-81.8 mmHg; Ser482Ser: mean = 82.1 mmHg, 95% CI = 81.5-82.7 mmHg; P = 0.651). Contrary to previous reports, we did not observe significant effect modification by sex (SBP, P = 0.966; DBP, P = 0.715). We were also unable to confirm the previously reported association between the Ser482 allele and hypertension [odds ratio: 0.97, 95% CI = 0.87-1.08, P = 0.585]. These results were materially unchanged when analyses were focused on whites only. However, statistical evidence of gene-age interaction was apparent for DBP [Gly482Gly: 73.5 (72.8, 74.2), Gly482Ser: 77.0 (76.2, 77.8), Ser482Ser: 79.1 (77.4, 80.9), P = 4.20 x 10(-12)] and SBP [Gly482Gly: 121.4 (120.4, 122.5), Gly482Ser: 125.9 (124.6, 127.1), Ser482Ser: 129.2 (126.5, 131.9), P = 7.20 x 10(-12)] in individuals <50 yr (n = 2,511); these genetic effects were absent in those older than 50 yr (n = 5,088) (SBP, P = 0.41; DBP, P = 0.51). Our findings suggest that the PPARGC1A Ser482 allele may be associated with higher blood pressure, but this is only apparent in younger adults.

Chemotherapeutic agents and gene expression in cardiac myocytes

It is becoming apparent that anti-cancer chemotherapies are increasingly associated with cardiac dysfunction or even congestive heart failure (Minotti et al., 2004; Eliott, 2006; Suter et al., 2004; Ren, 2005). Our data suggest that one of the contributing factors to the cardiotoxicitiy of these drugs may be the activation of the AhR-response (including the increased expression of Cyp1a1) and/or other detoxification program in cardiac myocytes themselves. The induction of such responses may have secondary effects (e.g. to increase the level of intracellular oxidative stress), which may influence the contractility or even survival of cardiac myocytes. Furthermore, the specific response of cardiac myocytes, both with respect to the metabolizing enzymes and the export channels, potentially differs from other cells (e.g. we failed to detect any increase in expression of other “classical” AhR-responsive genes, Ugt1a1 and Ugt1a6). This could account for, for example, the observation that doxoribicinol (the 13-hydroxy form of doxorubicin) accumulates in cardiac myocytes but not in hepatocytes (Del Tacca et al., 1985; Olson et al., 1988). Given the vulnerability of the heart and the almost irreparable damage that can be done by severe oxidative stress, further studies would seem to be merited specifically on the effects of chemotherapeutic agents on cardiac myocytes.

Proanthocyanidins inhibit Ascaris suum glutathione-S-transferase activity and increase susceptibility of larvae to levamisole and ivermectin in vitro

Proanthocyanidins (PAC) are a class of plant secondary metabolites commonly found in the diet that have shown potential to control gastrointestinal nematode infections. The anti-parasitic mechanism(s) of PAC remain obscure, however the protein-binding properties of PAC suggest that disturbance of key enzyme functions may be a potential mode of action. Glutathione-S-transferases (GSTs) are essential for parasite detoxification and have been investigated as drug and vaccine targets. Here, we show that purified PAC strongly inhibit the activity of both recombinant and native GSTs from the parasitic nematode Ascaris suum. As GSTs are involved in detoxifying xenobiotic substances within the parasite, we hypothesised that this inhibition may render parasites hyper-susceptible to anthelmintic drugs. Migration inhibition assays with A. suum larvae demonstrated that the potency of levamisole (LEV) and ivermectin (IVM) were significantly increased in the presence of PAC purified from pine bark (4.6-fold and 3.2-fold reduction in IC50 value for LEV and IVM, respectively). Synergy analysis revealed that the relationship between PAC and LEV appeared to be synergistic in nature, suggesting a specific enhancement of LEV activity, whilst the relationship between PAC and IVM was additive rather than synergistic, suggesting independent actions. Our results demonstrate that these common dietary compounds may increase the efficacy of synthetic anthelmintic drugs in vitro, and also suggest one possible mechanism for their well-known anti-parasitic activity.