Genotoxic and mutagenic effects of vigabatrin, a γ-aminobutyric acid transaminase inhibitor, in Wistar rats submitted to rotarod task.

Vigabatrin (VGB) is an antiepileptic drug thatincreases brain γ-aminobutyric acid (GABA) levels through irreversible inhibition of GABA transaminase. The aim of this study was to evaluate neurotoxicological effects of VGB measuring motor activity and genotoxic and mutagenic effects after a single and repeated administration. Male Wistar rats received saline, VGB 50, 100, or 250 mg/kg by gavage for acute and subchronic (14 days) treatments and evaluated in the rotarod task. Genotoxicity was evaluated using the alkaline version of the comet assay in samples of blood, liver, hippocampus, and brain cortex after both treatments. Mutagenicity was evaluated using the micronucleus test in bone marrow of the same animals that received subchronic treatment. The groups treated with VGB showed similar performance in rotarod compared with the saline group. Regarding the acute treatment, it was observed that only higher VGB doses induced DNA damage in blood and hippocampus. After the subchronic treatment, VGB did not show genotoxic or mutagenic effects. In brief, VGB did not impair motor activities in rats after acute and subchronic treatments. It showed a repairable genotoxic potential in the central nervous system since genotoxicity was observed in the acute treatment group.

(-)-Linalool, a naturally occurring monoterpene compound, impairs memory acquisition in the object recognition task, inhibitory avoidance test and habituation to a novel environment in rats.

It is known that (-)-linalool is a competitive antagonist of NMDA receptors, which play a key role in the learning and memory processes; however, only a few studies have reported a possible interference of (-)-linalool in memory. The purpose of this study was to investigate the (-)-linalool effects on acquisition of short- and long-term memories through the objects recognition task, inhibitory avoidance test and habituation to a novel environment. Furthermore, the open field test was used to investigate the interference of (-)-linalool in motivation, locomotion and exploration by animals. Wistar male adult rats received an intraperitoneal injection (i.p.) of saline (NaCl 0.9%), tween 5% or (-)-linalool (50 or 100 mg/kg) before training in the tasks; MK-801 (0.1 mg/kg), a glutamate antagonist, was used as positive control. Short-term (STM) and long-term (LTM) memories were tested 1.5 and 24 h after training, respectively, in the inhibitory avoidance and recognition objects. The results suggested that (-)-linalool (as 50- and 100-mg/kg doses) impaired LTM acquisition, but not STM acquisition, in the object recognition task. In the inhibitory avoidance test, animals receiving linalool (both doses) showed impairment in acquisition of both memories measured. In the open field test, the animals that received (-)-linalool showed no significant difference in the crossings and latency to start the locomotion in any of the doses tested, although (-)-linalool 100 mg/kg reduced rearing behavior. When re-exposed to open field 24 h after training, the rats that received (-)-linalool 100mg/kg showed no habituation. Taken together, these data suggested that (-)-linalool was able to impair the acquisition of memory in rats, which can be associated to (-)-linalool antagonist capacity as regards NMDA glutamatergic receptors, since other glutamate antagonists also seem to affect memory.

DNA brain damage after stress in rats.

OBJECTIVE: The purpose of this study was to verify the presence of DNA brain lesion after acute stress in rats. METHOD: Adult male Wistar rats were divided into 3 groups according to the stressor (control, forced swimming or restraint), and sampled at 2 time points: immediately or 1week after stress. Trunk blood and the brain areas (prefrontal cortex, amygdala and hippocampus) were extracted for DNA analysis by the comet assay. The cells were classified according to the damage index and damage frequency based on the comet tail size. RESULTS: Immediately after the stress, DNA damage was detected in the amygdala area and in the hippocampus after restraint and forced swimming. In the prefrontal cortex, DNA was damaged after forced swimming. However, no alteration was seen in blood. Seven days after the stress, DNA damage was still identified in the hippocampus after forced swimming and restraint, whereas no alteration was detected in the other brain areas or in blood. CONCLUSION: One week after a single stressful event, a reversible DNA damage was identified in the prefrontal cortex and in the amygdala, whereas DNA damage in the hippocampus still remained.

Evaluation of the protective effects of quercetin in the hepatopulmonary syndrome.

The hepatopulmonary syndrome (HPS) occurs when intrapulmonary dilatation causes hypoxemia in cirrhosis. The free radicals may play a significant contributory role in the progression of HPS, and flavonoid agents could protect against deleterious effects of free radicals. The flavonoid quercetin was evaluated in an experimental model of biliary cirrhosis induced by bile duct ligation (BDL) in rats. Quercetin was administered at 50mg/kg for 14 days to cirrhotic and non-cirrhotic rats. Bone marrow was extracted from animals to analyze micronuclei. Lung, liver and blood were extracted to detect DNA damage using the comet assay. The results showed that the micronuclei and DNA damages to lung and liver were increased in BDL rats. Quercetin caused no damage to the DNA while decreasing the occurrence of micronucleated cells in bone marrow as well as DNA damage to lung and liver in cirrhotic rats. Quercetin showed antimutagenic activity against hydroperoxides as evaluated by the oxidative stress sensitive bacterial strains TA102 Salmonella typhimurium and IC203 Escherichia coli, suggesting protection by free radical scavenging. In Saccharomyces cerevisie yeast strains lacking mitochondrial or cytosolic superoxide dismutase, these results indicate that quercetin protects cells by induction of antioxidant enzymes. The present study is the first report of genotoxic/antigenotoxic effects of quercetin in a model of animal cirrhosis. In this model, quercetin was not able to induce genotoxicity and, conversely, it increased the genomic stability in the cirrhotic rats, suggesting beneficial effects, probably by its antioxidant properties.

Genotoxicity of stannous chloride in yeast and bacteria.

Stannous chloride was found genotoxic in microbial test systems of the yeast Saccharomyces cerevisiae, in one strain of Salmonella typhimurium and in the Mutoxitest of Escherichia coli. Five isogenic haploid yeast strains differing only in a particular repair-deficiency had the following ranking in Sn2+ -sensitivity: rad52delta>rad6delta>rad2delta>rad4delta>RAD, indicating a higher relevance of recombinogenic repair mechanisms than nucleotide excision in repair of Sn2+ -induced DNA damage. Sn2+ -treated cells formed aggregates that lead to gross overestimation of toxicity when not undone before diluting and plating. Reliable inactivation assays at exposure doses of 25-75 mM SnCl2 were achieved by de-clumping with either EDTA- or phosphate buffer. Sn2+ -induced reversion of the yeast his1-798, his1-208 and lys1-1 mutant alleles, in diploid and haploid cells, respectively, and putative frameshift mutagenesis (reversion of the hom3-10 allele) was observed. In diploid yeast, SnCl2 induced intra-genic mitotic recombination while inter-genic (reciprocal) recombination was very weak and not significant. Yeast cells of exponentially growing cultures were killed to about the same extend at 0.1% of SnCl2 than respective cells in stationary phase, suggesting a major involvement of physiological parameters of post-diauxic shift oxidative stress resistance in enhanced Sn2+ -tolerance. Superoxide dismutases, but not catalase, protected against SnCl2-induced reactive oxygen species as sod1delta had a three-fold higher sensitivity than the WT while the sod2delta mutant was only slightly more sensitive but conferred significant sensitivity increase in a sod1delta sod2delta double mutant. In the Salmonella reversion assay, SnCl2 did not induce mutations in strains TA97, TA98 or TA100, while a positive response was seen in strain TA102. SnCl2 induced a two-fold increase in mutation in the Mutoxitest strain IC203 (uvrA oxyR), but was less mutagenic in strain IC188 (uvrA). We propose that the mutagenicity of SnCl2 in yeast and bacteria occurs via error-prone repair of DNA damage that is produced by reactive oxygen species.

Genotoxic, neurotoxic and neuroprotective activities of apomorphine and its oxidized derivative 8-oxo-apomorphine.

Apomorphine is a dopamine receptor agonist proposed to be a neuroprotective agent in the treatment of patients with Parkinson's disease. Both in vivo and in vitro studies have shown that apomorphine displays both antioxidant and pro-oxidant actions, and might have either neuroprotective or neurotoxic effects on the central nervous system. Some of the neurotoxic effects of apomorphine are mediated by its oxidation derivatives. In the present review, we discuss recent studies from our laboratory in which the molecular, cellular and neurobehavioral effects of apomorphine and its oxidized derivative, 8-oxo-apomorphine-semiquinone (8-OASQ), were evaluated in different experimental models, i.e., in vitro genotoxicity in Salmonella/microsome assay and WP2 Mutoxitest, sensitivity assay in Saccharomyces cerevisiae, neurobehavioral procedures (inhibition avoidance task, open field behavior, and habituation) in rats, stereotyped behavior in mice, and Comet assay and oxidative stress analyses in mouse brain. Our results show that apomorphine and 8-OASQ induce differential mutagenic, neurochemical and neurobehavioral effects. 8-OASQ displays cytotoxic effects and oxidative and frameshift mutagenic activities, while apomorphine shows antimutagenic and antioxidant effects in vitro. 8-OASQ induces a significant increase of DNA damage in mouse brain tissue. Both apomorphine and 8-OASQ impair memory for aversive training in rats, although the two drugs showed a different dose-response pattern. 8-OASQ fails to induce stereotyped behaviors in mice. The implications of these findings are discussed in the light of evidence from studies by other groups. We propose that the neuroprotective and neurotoxic effects of dopamine agonists might be mediated, in part, by their oxidized metabolites.

Genotoxic, neurotoxic and neuroprotective activities of apomorphine and its oxidized derivative 8-oxo-apomorphine

Apomorphine is a dopamine receptor agonist proposed to be a neuroprotective agent in the treatment of patients with Parkinson's disease. Both in vivo and in vitro studies have shown that apomorphine displays both antioxidant and pro-oxidant actions, and might have either neuroprotective or neurotoxic effects on the central nervous system. Some of the neurotoxic effects of apomorphine are mediated by its oxidation derivatives. In the present review, we discuss recent studies from our laboratory in which the molecular, cellular and neurobehavioral effects of apomorphine and its oxidized derivative, 8-oxo-apomorphine-semiquinone (8-OASQ), were evaluated in different experimental models, i.e., in vitro genotoxicity in Salmonella/microsome assay and WP2 Mutoxitest, sensitivity assay in Saccharomyces cerevisiae, neurobehavioral procedures (inhibition avoidance task, open field behavior, and habituation) in rats, stereotyped behavior in mice, and Comet assay and oxidative stress analyses in mouse brain. Our results show that apomorphine and 8-OASQ induce differential mutagenic, neurochemical and neurobehavioral effects. 8-OASQ displays cytotoxic effects and oxidative and frameshift mutagenic activities, while apomorphine shows antimutagenic and antioxidant effects in vitro. 8-OASQ induces a significant increase of DNA damage in mouse brain tissue. Both apomorphine and 8-OASQ impair memory for aversive training in rats, although the two drugs showed a different dose-response pattern. 8-OASQ fails to induce stereotyped behaviors in mice. The implications of these findings are discussed in the light of evidence from studies by other groups. We propose that the neuroprotective and neurotoxic effects of dopamine agonists might be mediated, in part, by their oxidized metabolites.

Further phenotypic characterization of pso mutants of Saccharomyces cerevisiae with respect to DNA repair and response to oxidative stress

The sensitivity responses of seven pso mutants of Saccharomyces cerevisiae towards the mutagens N-nitrosodiethylamine (NDEA), 1,2:7,8-diepoxyoctane (DEO), and 8-hydroxyquinoline (8HQ) further substantiated their allocation into two distinct groups: genes PSO1 (allelic to REV3), PSO2 (SNM1), PSO4 (PRP19), and PSO5 (RAD16) constitute one group in that they are involved in repair of damaged DNA or in RNA processing whereas genes PSO6 (ERG3) and PSO7 (COX11) are related to metabolic steps protecting from oxidative stress and thus form a second group, not responsible for DNA repair. PSO3 has not yet been molecularly characterized but its pleiotropic phenotype would allow its integration into either group. The first three PSO genes of the DNA repair group and PSO3, apart from being sensitive to photo-activated psoralens, have another common phenotype: they are also involved in error-prone DNA repair. While all mutants of the DNA repair group and pso3 were sensitive to DEO and NDEA the pso6 mutant revealed WT or near WT resistance to these mutagens. As expected, the repair-proficient pso7-1 and cox11-Delta mutant alleles conferred high sensitivity to NDEA, a chemical known to be metabolized via redox cycling that yields hydroxylamine radicals and reactive oxygen species. All pso mutants exhibited some sensitivity to 8HQ and again pso7-1 and cox11-Delta conferred the highest sensitivity to this drug. Double mutant snm1-Delta cox11-Delta exhibited additivity of 8HQ and NDEA sensitivities of the single mutants, indicating that two different repair/recovery systems are involved in survival. DEO sensitivity of the double mutant was equal or less than that of the single snm1-Delta mutant. In order to determine if there was oxidative damage to nucleotide bases by these drugs we employed an established bacterial test with and without metabolic activation. After S9-mix biotransformation, NDEA and to a lesser extent 8HQ, lead to significantly higher mutagenesis in an Escherichia coli tester strain WP2-IC203 as compared to WP2, whereas DEO-induced mutagenicity remained unchanged

Dose finding in the Ames Salmonella assay.

Threshold dose/concentration values, such as the lowest effective dose, minimum effective dose or the lowest effective concentration (LED, MED or LEC, respectively) are in use as an alternative to the mutagen potency measures based on the 'rate' measurements (e.g., the slope of the initial part of the dose-response curve). In this respect, several statistical procedures for the corresponding so-called 'dose finding' were proposed during the last decades. However, most of them disregard the discrete nature of responses such as the plate colony count in the Ames Salmonella assay. When the plate counts agree with the Poisson assumption, two procedures considered here seem to be appropriate for the dose finding. One is based on the stepwise collapsing of the homogeneous control and dose counts; another consists of constructing the confidence limits for the mutation induction factor (MIF). When the dose and control counts are non-overlapping, the simple 'visual' non-parametric estimation of LED is possible. Applicability and validity of the methods is demonstrated with the two data sets on the mutagenicity of the beta-carboline alkaloid, harmine, and one of the oxidation products of apomorphine.

Glutathione, but not transcription factor Yap1, is required for carbon source-dependent resistance to oxidative stress in Saccharomyces cerevisiae.

Resistance of haploid yeast to hydrogen peroxide and to tert-butylhydroperoxide strongly increases when 4% glucose is replaced by glycerol or ethanol as the carbon source of the complex medium. Using a GSH1-promoter-lacZ-fusion reporter construct we could demonstrate that GSH1 is one of the genes that are up-regulated during the shift from fermentative to oxidative metabolism. A gsh1 mutant did not exhibit respiratory growth resistance to H2O2, whereas it was only slightly impaired in acquiring resistance against t-BOOH in the same experimental conditions. An isogenic deltayap1 mutant, although more sensitive to oxidative stress than the wild-type (WT), could increase resistance to both peroxides by a similar factor as observed for the WT when shifted from 4% glucose to a non-fermentable carbon source. This indicates that in this case induction of resistance to oxidative stress is independent from Yap1 and from the Yap1-mediated stress response via the STRE motif.

Deletogenic activity of 1,2:7,8-diepoxyoctane in the Salmonella typhimurium tester strain TA102.

1,2:7,8-Diepoxyoctane (DEO), whose deletogenic activity was first demonstrated in ad-3 system of Neurospora crassa and then in different species, has been tested in Salmonella typhimurium tester strain TA102 (hisG428(Ochre)). It was confirmed that it is a direct acting mutagen and was found that its activity is stimulated with the S9 mix. Obtained His(+) revertants were screened on their response to the histidine analog, N-(2-thiazolyl)-DL-alanine (ThiAla). Thirty-two percent of spontaneous and 52% of DEO-induced revertants were resistant to the analog while no resistance was observed among those induced with 4-nitroquinoline-N-oxide (4NQO). Resistance to ThiAla was interpreted as due to small deletions surrounding the target TAA codon in hisG428(Ochre). Thus, at least two simple test-systems, ad-3 of N. crassa and hisG428(Ochre) of S. typhimurium, gave compatible results and might be useful in searching of deletogens.

Genotoxic effects of structurally related beta-carboline alkaloids.

beta-Carboline alkaloids, found in medicinal plants, tobacco smoke and well-cooked foods, have shown a variety of actions in biological systems related to their interaction with DNA. Therefore, these alkaloids can be considered potentially mutagenic. In this work, the genotoxic, mutagenic, and cytotoxic activities of three aromatic beta-carboline alkaloids (harman, harmine, and harmol) and two dihydro-beta-carboline alkaloids (harmaline and harmalol) were evaluated by means of the Salmonella/microsome assay (Salmonella typhimurium TA98, TA97, TA100, and TA102) and SOS chromotest (Escherichia coli PQ37) with and without metabolic activation. Moreover, harman and harmine were analyzed by the micronucleus assay in vivo. It was shown that genotoxicity was inhibited by the addition of S9 mix for aromatic beta-carbolines harman and harmol in TA97. However, harmine showed signs of mutagenicity only in the presence of S9 mix in TA98 and TA97 frameshift strains. In the SOS chromotest, only harman induced SOS functions in the absence of S9 mix. Dihydro-beta-carbolines were not genotoxic in any of the microorganisms used. The negative responses obtained in the micronucleus assay indicated that harman and harmine were not able to induce chromosomal mutations.