Amparo Urios

Contribution of hyperammonemia and inflammatory factors to cognitive impairment in minimal hepatic encephalopathy

To assess the contribution of hyperammonemia and inflammation to induction of mild cognitive impairment (or MHE). We analyzed the presence of mild cognitive impairment (CI) by using the PHES battery of psychometric tests and measured the levels of ammonia and of the inflammatory cytokines IL-6 and IL-18 in blood of patients with different types of liver or dermatological diseases resulting in different grades of hyperammonemia and/or inflammation. The study included patients with 1) liver cirrhosis, showing hyperammonemia and inflammation; 2) non-alcoholic fatty liver disease (NAFLD) showing inflammation but not hyperammonemia; 3) non-alcoholic steatohepatitis (NASH) showing inflammation and very mild hyperammonemia; 4) psoriasis, showing inflammation but not hyperammonemia; 5) keloids, showing both inflammation and hyperammonemia and 6) controls without inflammation or hyperammonemia. The data reported show that in patients with liver diseases, cognitive impairment may appear before progression to cirrhosis if hyperammonemia and inflammation are high enough. Five out of 11 patients with NASH, without liver cirrhosis, showed cognitive impairment associated with hyperammonemia and inflammation. Patients with keloids showed cognitive impairment associated with hyperammonemia and inflammation, in the absence of liver disease. Hyperammonemia or inflammation alone did not induce CI but the combination of certain levels of hyperammonemia and inflammation is enough to induce CI, even without liver disease.

Mutagenicity of 80 chemicals in Escherichia coli tester strains IC203, deficient in OxyR, and its oxyR(+) parent WP2 uvrA/pKM101: detection of 31 oxidative mutagens.

Strain IC203, deficient in OxyR, and its oxyR(+) parent WP2 uvrA/pKM101 (denoted IC188) are the basis of a new bacterial reversion assay, the WP2 Mutoxitest, which has been used in the evaluation of 80 chemicals for oxidative mutagenicity. The following 31 oxidative mutagens were recognized by their greater mutagenic response in IC203 than in IC188: (1) peroxides: hydrogen peroxide (HP), t-butyl hydroperoxide (BOOH) and cumene hydroperoxide (COOH); (2) benzoquinones (BQ): 2-methyl-1,4-BQ, 2,6-dimethyl-1,4-BQ and 2,3, 5,6-tetramethyl-1,4-BQ; (3) naphthoquinones (NQ): 1,4-NQ, 2-methyl-1, 4-NQ and 2-hydroxy-1,4-NQ; (4) phenol derivatives: catechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, t-butylhydroquinone, gallic acid and 4-aminophenol; (5) catecholamines: DL- and L-dopa, DL- and L-epinephrine, dopamine and L-norepinephrine; (6) thiols: L-cysteine methyl ester, L-cysteine ethyl ester, L-penicillamine and dithiothreitol; (7) diverse: 3,4-dihydroxyphenylacetic acid, hypoxanthine and xanthine, both in the presence of xanthine oxidase, L-ascorbic acid plus copper (II) and phenazine methosulfate. Among these oxidative mutagens, 25 were found to be uniquely positive in IC203. With the exception of BOOH and COOH, mutagenesis by all oxidative mutagens was inhibited by catalase present in rat liver S9, indicating that it is mediated by HP generation, probably in autoxidation reactions. These catalase-sensitive oxidative mutagens were poor inducers of mutations derived from 8-oxoguanine lesions, whereas such mutations were efficiently induced by organic hydroperoxides. The results support the usefulness of incorporating IC203 in the bacterial battery for testing of chemicals. The well-characterized oxidative mutagens available with the use of the WP2 Mutoxitest may serve as a reference in studies on the genotoxicity of oxidative stress.

New Escherichia coli WP2 tester strains highly sensitive to reversion by oxidative mutagens.

New Escherichia coli strains have been added to the WP2 mutagenicity test for the specific detection of oxidative mutagens. Strain IC203 derives from WP2 uvrA/pKM101 and is highly sensitive to oxidative stress due to a deficiency in the OxyR function. Following exposure to t-butyl hydroperoxide (BuOOH) or menadione (MD), but not to 4-nitroquinoline 1-oxide (4NQO), strain IC203 (oxyR) shows increased mutability with respect to the oxyR+ parent. The advantage that the OxyR deficiency confers on IC203 strain in detecting oxidative mutagens is not obtained with strains deficient in either katG or ahpCF, two OxyR-regulated genes. Strain IC206, a derivative of WP2 uvrA carrying a deletion of the umuDC genes and deficient in the MutY glycosylase, has also been added to the WP2 test for the detection of SOS-independent mutations promoted by 8-oxoguanine lesions. Induction of these mutations was observed after treatment with BuOOH, but not after MD or 4NQO exposure. The two new strains, IC203 and IC206, can be useful for the screening of mutations resulting from oxidative stress as well as in studies on antioxidants preventing mutagenesis.

Patients with minimal hepatic encephalopathy show impaired mismatch negativity correlating with reduced performance in attention tests.

Attention deficit is an early event in the cognitive impairment of patients with minimal hepatic encephalopathy (MHE). The underlying mechanisms remain unclear. Mismatch negativity (MMN) is an auditory event‐related potential that reflects an attentional trigger. Patients with schizophrenia show impaired attention and cognitive function, which are reflected in altered MMN. We hypothesized that patients with MHE, similarly to those with schizophrenia, should show MMN alterations related with attention deficits. The aims of this work were to assess whether (1) MMN is altered in cirrhotic patients with MHE, compared to those without MHE, (2) MMN changes in parallel with performance in attention tests and/or MHE in a longitudinal study, and (3) MMN predicts performance in attention tests and/or in the Psychometric Hepatic Encephalopathy Score (PHES). We performed MMN analysis as well as attention and coordination tests in 34 control subjects and in 37 patients with liver cirrhosis without MHE and 23 with MHE. Patients with MHE show reduced performance in selective and sustained attention tests and in visuomotor and bimanual coordination tests. The MMN wave area was reduced in patients with MHE, but not in those without MHE. In the longitudinal study, MMN area improved in parallel with performance in attention tests and PHES in 4 patients and worsened in parallel in another 4. Logistic regression analyses showed that MMN area predicts performance in attention tests and in PHES, but not in other tests or critical flicker frequency. Receiver operating characteristic curve analyses showed that MMN area predicts attention deficits in the number connection tests A and B, Stroop tasks, and MHE, with sensitivities of 75%‐90% and specificities of 76%‐83%. Conclusion: MMN area is useful to diagnose attention deficits and MHE in patients with liver cirrhosis. (HEPATOLOGY 2012;)

3-Nitro-Tyrosine as a Peripheral Biomarker of Minimal Hepatic Encephalopathy in Patients With Liver Cirrhosis

OBJECTIVES:Between 30 and 50% of the cirrhotic patients who do not show symptoms of clinical hepatic encephalopathy (HE) present minimal hepatic encephalopathy (MHE), with mild cognitive impairment. MHE impairs the quality of life, increases the risk of suffering accidents, predicts the appearance of clinical HE, and is associated with shortened lifespan. Early detection of MHE would be very useful. The “gold standard” for MHE diagnosis is the psychometric hepatic encephalopathy score (PHES). However, it is time consuming and needs adjusting for age and educational level. It would be very useful to have some blood biomarker reflecting the presence of MHE in cirrhotic patients. The aim of this work was to identify serum molecules useful as biomarkers for MHE.METHODS:We measured in 63 controls, 43 cirrhotic patients without MHE, and 44 patients with MHE, from Hospital Clinico de Valencia, serum levels of different amino acids, cyclic guanosine monophosphate (cGMP), nitrites+nitrates, and 3-nitrotyrosine. We analyzed for each parameter its diagnostic accuracy as an indicator of MHE, as assessed using the PHES.RESULTS:These studies supported that 3-nitro-tyrosine is a good marker for MHE. To validate its utility as a biomarker for MHE, we analyzed in a second cohort of 44 cirrhotic patients without MHE and 18 patients with MHE, from Hospital Arnau de Vilanova, serum levels of 3-nitro-tyrosine, methionine, and citrulline. Citrulline (173±17%), methionine (173±16%), and 3-nitro-tyrosine (857±92%) were increased in sera from patients with MHE when compared with those without MHE. The receiver operating characteristic (ROC) curve analysis of 3-nitro-tyrosine for the diagnosis of MHE in the first cohort showed an area under the curve (AUC) value of 0.96 (95% confidence interval 0.93–0.99). At the cutoff of 14 nM, the specificity was 93%, sensitivity 89%, and positive and negative predictive values were both 91%. When the same cutoff was applied to the second cohort, the specificity was 83% and sensitivity was 94%. The positive and negative predictive values were 70 and 97%, respectively.CONCLUSIONS:This pilot study, to be validated in a larger cohort, shows that determination of 3-nitro-tyrosine in serum, which is easy and not time consuming, is useful to identify patients with MHE, with good sensitivity, specificity, and positive and negative predictive values.

Specificity of spontaneous and t-butyl hydroperoxide-induced mutations in ΔoxyR strains of Escherichia coli differing with respect to the SOS mutagenesis proficiency and to the MutY and MutM functions

Mutations induced by oxidative DNA damage appear to occur by two pathways, differing in their dependence on SOS mutagenesis. We have analysed the specificity of mutations produced by each pathway. Base substitutions generating extragenic suppressors were characterized in Trp+ revertants of Escherichia coli strains carrying the trpE65 ochre mutation, which were hypersensitive to oxidative mutagenesis due to a deletion of the oxyR gene. In strain IC3821, containing MucA/B proteins and therefore proficient for SOS mutagenesis, the more frequently scored base substitutions, either spontaneous or induced by t-butyl hydroperoxide (BuOOH), were T:A-A:T transversions, followed by G:C-A:T transitions, while the frequency of G:C-T:A transversions was lower. This SOS-dependent mutability could be promoted by abasic sites. In strains IC3894 (mutY) and IC3981 (mutY mutM), lacking mutagenesis proteins, SOS-independent revertants arose almost exclusively via G:C-T:A transversions probably derived from oxidatively damaged 8-oxoguanine/adenine mispairs. Formation of these mispairs in IC3894 and IC3981 would be enhanced by BuOOH treatment since it caused a significant increase in the revertant number. Strains IC3894 and IC3981 could have a complementary role to that of IC3821 to analyse the mutagenicity and the mutational specificity of oxidants.

Screening for metabolites from Penicillium novae-zeelandiae displaying radical-scavenging activity and oxidative mutagenicity: isolation of gentisyl alcohol

In the search for new natural products with anti-oxidant activity, we have combined the cell-free assay based on the scavenging of the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), with a bioassay that detects oxidative mutagens. This bioassay uses a new Escherichia coli tester strain, IC203, specifically sensitive to oxidative stress due to a deficiency in the OxyR function. OxyR is a redox-sensitive transcriptional activator of genes encoding anti-oxidant enzymes such as catalase and peroxiredoxin alkyl hydroperoxide reductase. The positive response observed in E. coli IC203 with several known anti-oxidants, including cysteine, catechol and ascorbic acid, suggested to us the usefulness of the mutagenicity assay for a rapid screening of anti-oxidant compounds. The extract from Penicillium novae-zeelandiae was found to scavenge the DPPH radical. Subsequently, guided by the DPPH-scavenging assay and the oxidative mutagenesis assay, we isolated and identified three compounds in fractions from that active extract: patulin (1). 3-hydroxybenzyl alcohol (2). and gentisyl alcohol (2,5-dihydroxybenzyl alcohol) (3). Of these, gentisyl alcohol showed both DPPH-scavenging activity and oxidative mutagenicity. This compound also gave rise to intracellular formation of superoxide, evaluated by monitoring the oxidation of dihydroethidium, and was able to inhibit mutagenesis induced by the model oxidant t-butyl hydroperoxide (t-BuOOH).

Increased mutability by oxidative stress in OxyR-deficient Escherichia coli and Salmonella typhimurium cells: clonal occurrence of the mutants during growth on nonselective media

Escherichia coli and Salmonella typhimurium strains deficient in the OxyR-regulated adaptive response to oxidative stress were used to study the mode in which spontaneous SOS-dependent mutations are generated in a distressed bacterial population. When assayed on supplemented selective medium, the E. coli strain IC3821 (trpE65), carrying the delta oxyR30 mutation and containing the plasmid pRW144 (mucA/B), showed a frequency of spontaneous Trp+ revertants similar to that of the oxyR+ control. Instead, the IC3821 strain exhibited an enhancement in the clonal occurrence of spontaneous revertants arising at random during growth on a nonselective medium. A similar enhancement was observed for the S. typhimurium strain TA4125 (hisG428 delta oxyR2). The mutator effect observed in oxyR- cells would be induced by an increased background of reactive oxygen species; it provides a model for studying the mutability of a cell population constantly exposed to mutation-inducing agents. In the IC3821 strain, revertants were induced by t-butyl hydroperoxide with higher efficiency than in oxyR+. We suggest that strain IC3821 could be useful for the detection of SOS-dependent mutagenesis induced by chemical oxidants.

Expression of the recA gene is reduced in Escherichia coli topoisomerase I mutants

We studied the influence of DNA topological changes on Escherichia coli recA gene expression. This was monitored by measuring beta-galactosidase activity in cells containing a recA-lacZ fusion. To modulate DNA supercoiling we used mutations in the genes encoding for topoisomerase I and DNA gyrase. After either UV irradiation or treatment with the gyrase inhibitor ciprofloxacin, induction of the recA gene was reduced in topA10 mutants, this reduction being alleviated when gyrA or gyrB mutations causing DNA relaxation were present. A reduced induction of recA was also observed after incubation of cells carrying the recA441 mutation at 42 degrees C in the presence of adenine. Using bacteria deficient in the LexA repressor, we have demonstrated that the topA10 mutation reduces the constitutive expression of the recA gene. We suggest that the increase in negative supercoiling resulting from topoisomerase I deficiency interferes with transcription from the recA promoter. The reduction in the expression of the recA gene in topA10 bacteria could determine their increased UV sensitivity as well as their partial defectiveness in SOS mutability.

Mutagenicity of nitric oxide-releasing compounds in Escherichia coli: effect of superoxide generation and evidence for two mutagenic mechanisms

The mutagenicity of three nitric oxide (NO) donors, 3-morpholinosydnonimine (SIN-1), a compound generating the precursors of peroxynitrite NO and superoxide, diethylamine/NO (DEA/NO) and spermine/NO (SPER/NO), both releasing authentic NO was analyzed using Escherichia coli tester strains IC203, carrying a deletion of the oxyR gene, and its oxyR(+) parent IC188 (the alternative name of WP2 uvrA/pKM101). The OxyR protein is a redox-sensitive transcriptional activator of genes encoding antioxidant enzymes. Strains IC203 and IC188 contain error-prone DNA polymerases polV, encoded by the chromosomal umuDC genes, and polRI, encoded by mucAB genes carried by pKM101. SIN-1 was determined to be an oxidative mutagen giving a positive response only in IC203, whereas DEA/NO and SPER/NO induced similar positive responses in IC203 and IC188 and were considered as non-oxidative mutagens. The spectrum of ochre suppressors in Trp(+) revertants induced by SIN-1 in IC203 was characterized by a higher number of TA-->AT transversions and GC-->AT transitions, and a lower number of GC-->TA transversions, with respect to the untreated control. The mutagenicity of SIN-1 in IC203, probably induced by peroxynitrite through reactive derivatives, was enhanced in the presence of plumbagin (PLB), a superoxide generator. Superoxide generation by PLB, as well as formation of peroxynitrite in cells treated with SIN-1, evaluated by monitoring the oxidation, respectively, of dihydroethidium and dihydrorhodamine 123, were greater in IC203 than in IC188. Formation of peroxynitrite in IC203 treated with SIN-1 was stimulated by PLB. After treatment with DEA/NO and SPER/NO the number of revertants scored in IC188 was higher than in strains IC187, containing only polV, and IC204, deficient in both polV and polRI. For these compounds, induced suppressor revertants in IC187 and IC204 were almost exclusively GC-->AT transitions, whereas in IC188 significant levels of GC-->TA and TA-->AT transversions were also induced. Mutagenesis by both DEA/NO and SPER/NO was partially inhibited in the presence of PLB. The results show the usefulness of the new tester strain IC203 to differentiate NO-promoted mutagenic mechanisms that involve or do not involve oxygen radicals.