The role of CB2 cannabinoid receptors (CBR) in global brain lesions induced by hypoxia-ischemia (HI) insult is still unresolved. The aim of this study was to evaluate the involvement of CBR in the behavioural and biochemical underpinnings related to brain damage induced by HI in adult mice, and the mechanisms involved. CBR knockout (KO) mice and wild-type littermates (WT) underwent permanent ligation of the left common carotid artery and hypoxia. Behavioural measurements in the rotarod, beam walking, object recognition, open field, and Irwin tests were carried out 24h, 72h and 7 days. In KO mice, more extensive brain injury was observed. Behavioural deficits in the Irwin test were observed in both genotypes; while WT mice showed progressive recovery by day 7, KO mice did not. Only KO mice showed alterations in motor learning, coordination and balance, and did not recover over time. A higher expression of microglia and astrocytes was observed in several brain areas of lesioned WT and KO mice. The possible alteration of the inflammatory-related factors HIF-1α and TIM-3 was evaluated in these animals. In both genotypes, HIF-1α and TIM-3 expression was observed in lesioned areas associated with activated microglia. However, the expression levels of these proteins were exacerbated in KO mice in several lesioned and non-lesioned brain structures. Our results indicate that CBR may have a crucial neuroprotective role following HI insult through the modulation of the inflammatory-related HIF-1α/TIM-3 signalling pathway in microglia.

Inorganic arsenic is nongenotoxic in the Drosophila melanogaster wing somatic mutation and recombination test (SMART). Recent evidence in mammalian systems indicates that methylated metabolites of arsenic are more genotoxic than inorganic arsenic. Thus, we hypothesized that inorganic arsenic is nongenotoxic in Drosophila because they are unable to biotransform arsenic to methylated forms. In the present study, we fed trivalent and pentavalent inorganic arsenic to Drosophila larvae and adults and measured the production of methylated derivatives. No biomethylated arsenic species were found in the organisms or in the growth medium, which suggests that Drosophila are unable to biomethylate inorganic arsenic. Exposure of Drosophila to the methylated arsenic derivative dimethylarsinic acid (DMA(V)) resulted in incorporation of this organoarsenic compound without demethylation. In addition, we used the SMART wing spot assay, which measures loss of heterozygosity (LOH) resulting from gene mutation, chromosomal rearrangement, chromosome breakage, and chromosome loss, to evaluate the genotoxicity of DMA. DMA by itself induced significant increases in the frequency of total spots, small spots, and large single spots. These results are consistent with the important role of arsenic biomethylation as a determinant of the genotoxicity of arsenic compounds. The absence of biomethylation in Drosophila could explain the lack of genotoxicity for inorganic arsenic and the genotoxicity of methylated arsenic species in the SMART wing spot assay.

The PPAR-α agonist, oleoylethanolamide (OEA) has neuroprotective properties in stroke models. However, its rapid degradation represents a limitation for an effective therapeutic approach. In this study, we evaluated the effects of a stable OEA-modeled compound, octadecylpropyl sulfamide (SUL) on the cognitive, behavioral, cellular and molecular alterations associated with hypoxia-ischemia (HI) in mice. Mice subjected to HI were treated with the PPAR-α antagonist GW6471 (GW) (1 mg/kg) followed 15 min later by SUL (3 and 10 mg/kg). Behavioral, motor, and cognitive tests were carried out 24 h and 7 days after the HI. The levels of microglia, reactive astrocytes and neuronal nuclei were studied using immunofluorescence, and the expression of genes related to the N-acyl-ethanolamides/endocannabinoid signaling systems was determined by qRT-PCR at the end of the experimental sequence. HI induced brain damage in the ipsilateral hippocampus and cortex, which lead to severe memory impairments, and motor coordination deficits. Significant neuronal loss, increased microglia and reactive astrocytes, and compensatory changes in genes associated with the inflammation/immune and endocannabinoid systems were observed in these brain structures of lesioned mice. SUL reversed the memory and motor deficits, decreased the overexpression of microglia and astrocytes, and reduced neurodegeneration induced by HI. Cnr1 and Cnr2 gene expression was modulated by SUL in both sham and HI mice, while Pparα and Faah expression was regulated in HI mice. GW completely blocked the beneficial actions of SUL. These findings suggest that treatment with SUL reduces brain damage and the associated motor and memory deficits induced by HI probably by normalizing the changes in neuroinflammation/immune system mediators.

Chronic alcohol intake has been shown to disrupt gut microbiota homeostasis, but whether microbiota modulation could prevent behavioral alterations associated with chronic alcohol intake remains unknown. We investigated the effects of synbiotic dietary supplementation on the development of alcohol-related addictive behavior in female and male mice and evaluated whether these effects were associated with changes in bacterial species abundance, short-chain fatty acids, tryptophan metabolism, and neurotransmitter levels in the prefrontal cortex and hippocampus. Chronic intermittent exposure to alcohol during 20 days induced escalation of intake in both female and male mice. Following alcohol deprivation, relapse-like behavior was observed in both sexes, but anxiogenic and cognitive deficits were present only in females. Synbiotic treatment reduced escalation and relapse to alcohol intake in females and males. In addition, the anxiogenic-like state and cognitive deficits observed in females following alcohol deprivation were abolished in mice exposed to synbiotic. Alcohol-induced differential alterations in microbial diversity and abundance in both sexes. In females, synbiotic exposure abrogated the alterations provoked by alcohol in Prevotellaceae UCG-001 and Ruminococcaceae UCG-014 abundance. In males, synbiotic exposure restored the changes induced by alcohol in Akkermansia and Muribaculum uncultured bacterium abundance. Following alcohol withdrawal, tryptophan metabolites, noradrenaline, dopamine, and γ-aminobutyric acid concentrations in the prefrontal cortex and the hippocampus were correlated with bacterial abundance and behavioral alterations in a sex-dependent manner. These results suggested that a dietary intervention with a synbiotic to reduce gut dysbiosis during chronic alcohol intake may impact differently the gut-brain-axis in females and males.

The wing spot test in Drosophila melanogaster was used to investigate the genotoxicity of arsenic and its effects on the action of two clearly genotoxic agents: potassium dichromate (PDC) and ethyl methanesulfonate (EMS). This assay is based on the principle that the loss of heterozygosity of the suitable recessive markers multiple wing hairs (mwh) and flare-3 (flr(3)) can lead to the formation of mutant clones of larval cells, which are then expressed as spots on the wings of adult flies. These spots can be attributed to different genotoxic events: either mitotic recombination or mutation (deletion, point mutation, and specific types of translocation). Pretreatments and chronic cotreatments were comparatively used for combined treatments. From the results obtained it is evident that sodium arsenite (SA) does not increase the frequency of any of the three categories of spots recorded (small, large, and twin spots) at the concentrations tested. The effects of SA in combination with PDC, in both cotreatments and pretreatments, indicate that SA almost suppressed the clones induced by PDC. Nevertheless, no effects of arsenic were observed with respect to the pre- and cotreatments with EMS. Thus, SA does not modify the frequencies of mutant clones induced by EMS.

No abstract

The genotoxic activity of cadmium chloride (CC) has been evaluated in the somatic mutation and recombination test (SMART) in Drosophila melanogaster. In addition, its possible modulating effect on the genotoxicity of two known mutagenic agents, potassium dichromate (PDC) and ethyl methanesulfonate (EMS), was investigated. Three different types of combined treatments of CC with the two genotoxins were performed: pretreatment, cotreatment, and posttreatment. The SMART assay is based on the principle that loss of heterozygosity for the recessive markers, multiple wing hairs (mwh) and flare-3 (flr(3)), leads to the formation of mutant clones in the imaginal disks of larvae, which are expressed as mutant spots on the wings of adult flies. Thus, after adult emergence, the wings of the adult flies were scored for the presence of single and/or twin spots. Our results show that CC alone was not effective in increasing the frequency of any of the three categories of spots (small, large, and twin). In the cotreatment experiments, CC increased the genotoxicity of PDC but it decreased the genotoxicity of EMS. No effects of CC were observed in the pretreatment or posttreatment experiments; however, only low concentrations of CC, PDC, and EMS were tested in the pretreatment assays due to the high toxicity of the treatment. Although our results with PDC are consistent with the hypothesis that cadmium can interfere with repair mechanisms, the EMS data suggest that other modulating mechanisms are also involved in the genotoxicity of this metal.