Multilevel assessment of carbamazepine effects: An integrative approach using zebrafish early-life stages.

Carbamazepine (CBZ) is the most commonly used drug in epilepsy treatment, and its metabolites are commonly detected among persistent pharmaceuticals in the aquatic environment. This study aimed to investigate CBZ effects on early-life-stage zebrafish (Danio rerio) (from 2 to 168 hpf) by employing of an integrative approach linking endpoints from molecular to individual level: (i) development; (ii) locomotor activity; (iii) biochemical markers (lactate dehydrogenase, glutathione-S-transferase, acetylcholinesterase and catalase) and (iv) transcriptome analysis using microarray. A 168 h - LC50 of 73.4 mg L-1 and a 72 h - EC50 of 66.8 mg L-1 for hatching were calculated while developmental effects (oedemas and tail deformities) were observed at CBZ concentrations above 37.3 mg L-1. At the biochemical level, AChE activity proved to be the most sensitive parameter, as evidenced by its decrease across all concentrations tested (∼25% maximum reduction, LOEC (lowest observed effect concentration) < 0.6 µg L-1). Locomotor behaviour seemed to be depressed by CBZ although this effect was only evident at the highest concentration tested (50 mg L-1). Molecular analysis revealed a dose-dependent effect of CBZ on gene expression. Although only 25 genes were deregulated in organisms exposed to CBZ when compared to controls, both 0.6 and 2812 µg L-1 treatments impaired gene expression related to development (e.g. crygmxl1, org, klf2a, otos, stx16 and tob2) and the nervous system (e.g. Rtn3, Gdf10, Rtn3), while activated genes were associated with behavioural response (e.g. prlbr and taar). Altogether, our results indicate that environmentally relevant CBZ concentrations might affect biochemical and genetic traits of fish. Thus, the environmental risk of CBZ cannot be neglected, especially in a realistic scenario of constant input of domestic effluents into aquatic systems.

ZDHHC9 X-linked intellectual disability: Clinical and molecular characterization.

The ZDHHC9 gene encodes the Zinc Finger DHHC-Type Containing 9 protein that functions as a palmitoyltransferase. Variants in this gene have been reported as the cause of Raymond-type X-linked intellectual disability with only 16 families described in the literature. This study reviews molecular and clinical data from previously reported patients and reports the case of a 13-year-old patient with a splicing variant in ZDHHC9 presenting intellectual disability, developmental delay, facial dysmorphisms, and skeletal defects. Although intellectual disability and developmental delay with severe speech delay have been reported in all cases with available clinical data, the remaining clinical signs differ significantly between patients. Missense, nonsense, frameshift, and splicing variants, in addition to large exonic deletions, have been described suggesting a loss of function mechanism. Though variants are distributed in almost all exons, most missense and nonsense variants affect arginine residues located in the cytoplasmic domains of this transmembrane protein, suggesting possible mutational hotspots.

Effects of incubation temperature on development, morphology, and thermal physiology of the emerging Neotropical lizard model organism Tropidurus torquatus.

Incubation temperature is among the main phenotypic trait variation drivers studied since the developmental trajectory of oviparous animals is directly affected by environmental conditions. In the last decades, global warming predictions have aroused interest in understanding its impacts on biodiversity. It is predicted that the effects of direct warming will be exacerbated by other anthropogenic factors, such as microclimatic edge effects. Although the Brazilian Cerrado biome is one of the most affected by these issues, little is known about the aforementioned effects on its biodiversity. Therefore, the aim of our study is to investigate the influence of incubation temperature on developmental parameters, morphology and thermal physiology traits of the collared lizard (Tropidurus torquatus). Furthermore, we discuss our findings regarding lizard developmental biology and the climate change paradigm. Therefore, we incubated T. torquatus eggs under five temperature regimes ranging from artificial nest temperature (28.7 °C) to 35.0 °C. We found that elevated incubation temperatures affect several investigated traits: egg mass gain is positively affected, without any influence in newborn mass; incubation period is broadly reduced with temperature increase; survival rate is negatively affected by temperature, constant 35.0 °C regime is confirmed as a lethal incubation temperature, and the sex ratio is affected at 30.0 °C, with a prevailing outbreak of females. Increased incubation temperature also affects body and head size but has no effect on limb size. Newborn thermoregulation and the critical thermal maximum (CTmax) are not affected by incubation temperature. On the other hand, basal body temperature (Tbb) and the critical thermal minimum (CTmin) were positively affected. Thermal physiology was also affected by age, with newborns differing from adults for all analyzed thermal traits. Our findings indicate that future modifications in incubation temperature regimes at nesting sites caused by warming may affect several features of the development, morphology, and thermal physiology of newborns of this species. Laboratory experiments have pointed to possible drastic effects of warming on lizard survival rates, also affecting aspects of its natural history and population distribution. Moreover, in addition to being more vulnerable than adults in aspects such as predation and feeding, T. torquatus newborns are also more vulnerable regarding thermal physiological traits.

Characterization of Associated Nonclassical Phenotypes in Patients with Deletion in the WAGR Region Identified by Chromosomal Microarray: New Insights and Literature Review.

WAGR syndrome (Wilms' tumor, aniridia, genitourinary changes, and intellectual disability) is a contiguous gene deletion syndrome characterized by the joint deletion of PAX6 and WT1 genes, located in the short arm of chromosome 11. However, most deletions include other genes, leading to multiple associated phenotypes. Therefore, understanding how genes deleted together can contribute to other clinical phenotypes is still considered a challenge. In order to establish genotype-phenotype correlation in patients with interstitial deletions of the short arm of chromosome 11, we selected 17 patients with deletions identified by chromosomal microarray analysis: 4 new subjects and 13 subjects previously described in the literature with detailed clinical data. Through the analysis of deleted regions and the phenotypic changes, it was possible to suggest the contribution of specific genes to several nonclassical phenotypes, contributing to the accuracy of clinical characterization of the syndrome and emphasizing the broad phenotypic spectrum found in the patients. This study reports the first patient with a PAX6 partial deletion who does not present any eye anomaly thus opening a new set of questions about the functional activity of PAX6.

SHORT syndrome in an adult Brazilian patient.

We report an individual from Brazil with SHORT syndrome. The term SHORT stands for its common characteristics: short stature (S), hyperextensibility of joints, and/or inguinal hernia (H), ocular depression (O), Rieger anomaly (R), and teething delay (T). In addition to most of the clinical signs previously described in SHORT syndrome, the patient presented here also shows microcephaly and intellectual disability. Diagnosis was confirmed by exome sequencing revealing a novel heterozygous variant c.1456G>A (p.Ala486Thr) at PIK3R1. Human recombinant growth hormone (r-hGH) therapy was administered prior to diagnosis; however, the use of r-hGH may have had a role in anticipating and worsening the glucose metabolic profile in the patient, as previously described. This article contributes to providing a better understanding of the SHORT syndrome genotype and its correlation with the phenotype, by comparing with it other reported cases.

Insights into how environment shapes post-mortem RNA transcription in mouse brain.

Most biological features that occur on the body after death were already deciphered by traditional medicine. However, the molecular mechanisms triggered in the cellular microenvironment are not fully comprehended yet. Previous studies reported gene expression alterations in the post-mortem condition, but little is known about how the environment could influence RNA degradation and transcriptional regulation. In this work, we analysed the transcriptome of mouse brain after death under three concealment simulations (air exposed, buried, and submerged). Our analyses identified 2,103 genes differentially expressed in all tested groups 48 h after death. Moreover, we identified 111 commonly upregulated and 497 commonly downregulated genes in mice from the concealment simulations. The gene functions shared by the individuals from the tested environments were associated with RNA homeostasis, inflammation, developmental processes, cell communication, cell proliferation, and lipid metabolism. Regarding the altered biological processes, we identified that the macroautophagy process was enriched in the upregulated genes and lipid metabolism was enriched in the downregulated genes. On the other hand, we also described a list of biomarkers associated with the submerged and buried groups, indicating that these environments can influence the post-mortem RNA abundance in its particular way.

Toxicity of ayahuasca after 28 days daily exposure and effects on monoamines and brain-derived neurotrophic factor (BDNF) in brain of Wistar rats.

Ayahuasca is a hallucinogenic beverage that affects the serotonergic system and have therapeutic potential for many diseases and disorders, including depression and drug addiction. The objectives of this study were to evaluate the potential toxic effects of ayahuasca on rats after chronic exposure, and the levels of monoamines, their metabolites and the brain-derived neurotrophic factor (BDNF) in the brain. Female and male rats were treated orally for 28 days with H2O (control), fluoxetine (FLX), a selective serotonin reuptake inhibitor antidepressant, or ayahuasca (Aya) at doses of 0.5X, 1X and 2X the ritualistic dose (7 to 10 animals/group). Clinical, hematological and macroscopic results showed that ayahuasca was safe to the rats. Behavior tests conducted one hour after the last treatment showed that male rats from the Aya1 group explored the open field central area less than the control group, and the number of entries in the central area compared to total locomotion was also significantly lower in this group and in the FLX group. The hippocampus was removed for BDNF analysis and the remaining brain was used for monoamine analysis by HPLC-FL. Serotonin levels were significantly higher than control only in the Aya2 female group, while a significant reduction of its metabolite 5-HIAA was observed in the FLX group. Dopamine levels were similar among the experimental groups, but the levels of its metabolite DOPAC increased significantly in the Aya1 and Aya2 groups compared to controls, especially in females, and the DOPAC/dopamine turnover was significantly higher in Aya2 group. The levels of HVA, another dopamine metabolite, did not change with the treatments compared to controls, but HVA/DOPAC ratio was significantly lower in all ayahuasca male groups. Norepinephrine was not detected in any brain sample, and the levels of its metabolite MHPG did not change significantly among the groups. BDNF levels in the hippocampus were significantly higher in the FLX and Aya2 female groups compared to controls when expressed in relation to the total brain weight. The mechanisms involved in the increase in serotonin, dopamine turnover and BDNF levels observed in ayahuasca treated animals should be further investigated in specific brain areas.

Genetic and genomics in congenital heart disease: a clinical review.

OBJECTIVE: Discuss evidence referring to the genetic role in congenital heart diseases, whether chromosomic alterations or monogenic diseases. DATA SOURCE: LILACS, PubMed, MEDLINE, SciELO, Google Scholar, and references of the articles found. Review articles, case reports, book chapters, master's theses, and doctoral dissertations were included. SUMMARY OF FINDINGS: Congenital heart diseases are among the most common type of birth defects, afflicting up to 1% of the liveborn. Traditionally, the etiology was defined as a multifactorial model, with both genetic and external contribution, and the genetic role was less recognized. Recently, however, as the natural evolution and epidemiology of congenital heart diseases change, the identification of genetic factors has an expanding significance in the clinical and surgical management of syndromic or non-syndromic heart defects, providing tools for the understanding of heart development. CONCLUSIONS: Concrete knowledge of congenital heart disease etiology and recognition of the genetic alterations may be helpful in the bedside management, defining prognosis and anticipating complications.

Effects of the hallucinogenic beverage ayahuasca on voluntary ethanol intake by rats and on cFos expression in brain areas relevant to drug addiction.

Ayahuasca is a hallucinogenic infusion used in religious rituals that has serotoninergic properties and may be a potential therapeutic option for drug addiction. In this study, Wistar rats had intermittent access to ethanol for 8 weeks, receiving water (control), naltrexone (NTX, 2 mg/kg body weight [bw] intraperitoneally [i.p.]) or ayahuasca (Aya) at 0.5x, 1x, or 2x the ritual dose in the final 5 days. A naïve group had access only to water. Ethanol intake was estimated throughout the experiment, and cFos expression was evaluated in medial orbital cortex (MO), ventral orbital cortex (VO), lateral orbital cortex (LO), nucleus accumbens (NAc), and striatum. Treatment with either NTX or Aya (oral) did not decrease ethanol intake compared to the baseline level (5th to 7th week), but the NTX group intake was significantly lower than controls (p < 0.05). Ethanol significantly increased cFos expression in the MO region for control (p < 0.0001), NTX (p < 0.05), Aya1 (p < 0.001), and Aya2 (p < 0.0001) groups. This increase was also observed in the VO for the Aya1 group (p = 0.035), in the LO for the Aya2 group (p < 0.01), and in NAc for NTX and ayahuasca groups (p < 0.005). Furthermore, NTX and Aya0.5 treatment decreased cFos expression compared to controls in the MO region (p < 0.05 and p < 0.01, respectively), but only the ayahuasca group reached levels not significantly different from the naïve group. Studies using other protocols and dose regime are necessary to better investigate the impact of ayahuasca on alcohol intake by rats to support the observations in humans. Additionally, the role of ayahuasca in mediating cFos expression in other selected brain regions and its relationship with the serotoninergic/dopaminergic systems and drug addiction need further investigation.

Defining the Critical Region for Intellectual Disability and Brain Malformations in 6q27 Microdeletions.

Terminal microdeletions of the long arm of chromosome 6 are associated with a phenotype that includes multiple brain malformations, intellectual disability, and epilepsy. A 1.7-Mb region has been proposed to contain a gene responsible for the brain anomalies. Here, we present the case of a 12-year-old girl with multiple brain alterations and moderate intellectual disability with a 18-kb deletion in chromosome 6q27, which is smaller than the microdeletions previously described by microarray analysis. We refined the smallest region of overlap possibly associated with the phenotype of brain malformations and intellectual disability to a segment of 325 kb, comprising the DLL1, PSMB1, TBP, and PDCD2 genes since these genes were structurally and/or functionally lost in the smaller deletions described to date. We hypothesize that DLL1 is responsible for brain malformations and possibly interacts with other adjacent genes. The TBP gene encodes a transcription factor which is potentially related to cognitive development. TBP is linked to PSMB1 and PDCD2 in a conserved manner among mammals, suggesting a potential interaction between these genes. In conclusion, the 6q27 microdeletion is a complex syndrome with variable expressivity of brain malformations and intellectual disability phenotypes which are possibly triggered by the 4 genes described and adjacent genes susceptible to gene regulation changes.

A Balanced Reciprocal Translocation t(2;9)(p25;q13) Disrupting the LINC00299 Gene in a Patient with Intellectual Disability.

Long intergenic noncoding RNAs (lincRNAs) are a class of noncoding RNAs implicated in several biological processes. LincRNA 299 (LINC00299) maps to 2p25.1 and its function is still unknown. However, this gene has been proposed as a candidate for intellectual disability (ID) in a patient with a balanced translocation where the breakpoint disrupted its ORF. Here, we describe a new case of LINC00299 disruption associated with ID. The individual, a 42-year-old woman, was referred to the clinical geneticist because of her son who had severe syndromic ID. G-banding and chromosomal microarray analysis were performed. Karyotyping of the boy revealed an extranumerary derivative chromosome identified as an unbalanced translocation between chromosomes 2 and 9 of maternal origin. The mother's karyotype showed a balanced translocation 46,XX,t(2;9)(p25;q13). Chromosomal microarray indicated a disruption of LINC00299. These data corroborate the role of LINC00299 as a causative gene for ID and broadens the spectrum of LINC00299-related phenotypes.

Wound Healing Effect of Essential Oil Extracted from Eugenia dysenterica DC (Myrtaceae) Leaves.

The use of natural oils in topical pharmaceutical preparations has usually presented safe agents for the improvement of human health. Based on research into the immense potential of wound management and healing, we aimed to validate the use of topical natural products by studying the ability of the essential oil of Eugenia dysenterica DC leaves (oEd) to stimulate in vitro skin cell migration. Skin cytotoxicity was evaluated using a fibroblast cell line (L929) by MTT assay. The oil chemical profile was investigated by GC-MS. Moreover, the inhibition of lipopolysaccharide (LPS) induced nitric oxide (NO) production in the macrophage cell line (RAW 264.7) tested. The Chick Chorioallantoic Membrane (CAM) assay was used to evaluate the angiogenic activity and irritating potential of the oil. The oEd induces skin cell migration in a scratch assay at a concentration of 542.2 µg/mL. α-humulene and ß-caryophyllene, the major compounds of this oil, as determined by GC-MS, may partly explain the migration effect. The inhibition of nitric oxide by oEd and α-humulene suggested an anti-inflammatory effect. The CAM assay showed that treatment with oEd ≤ 292 µg/mL did not cause skin injury, and that it can promote angiogenesis in vivo. Hence, these results indicate the feasibility of the essential oil of Eugenia dysenterica DC leaves to developed dermatological products capable of helping the body to repair damaged tissue.

Syndromic hearing loss molecular diagnosis: Application of massive parallel sequencing.

Syndromic hearing loss accounts for approximately 30% of all cases of hearing loss due to genetic causes. Mutation screening in known genes is important because it potentially sheds light on the genetic etiology of hearing loss and helps in genetic counseling of families. In this study, we describe a customized Ion AmpliSeq Panel, specifically designed for the investigation of syndromic hearing loss. The Ion AmpliSeq Panel was customized to cover the coding sequences of 52 genes. Twenty-four patients were recruited: 17 patients with a clinical diagnosis of a known syndrome, and seven whose clinical signs did not allow identification of a syndrome. Of 24 patients sequenced, potentially causative mutations were found in nine, all of which belonged to the group with a previous clinical diagnostic and none in the group not clinically diagnosed. We were able to provide conclusive molecular diagnosis to six patients, constituting a diagnostic rate of 25% (6/24). In the group of patients with a suspected clinical diagnosis, the diagnostic rate was 35% (6/17). Of the nine different mutations identified, three are novel, and were found in patients with Waardenburg, Treacher Collins and CHARGE syndromes. Since all patients with a conclusive molecular diagnosis through this panel had a previous suspected clinical diagnosis, our results suggest that this panel was more effective in diagnosing this group of patients. Therefore, the panel demonstrated effectiveness in molecular diagnosis when compared to others in the literature, especially for patients with a defined clinical diagnosis.

Exposure to ayahuasca induces developmental and behavioral alterations on early life stages of zebrafish.

Ayahuasca is a psychoactive concoction prepared from the plants Banisteriopsis caapi and Psychotria viridis which are used ancestrally by Amazonian Indian populations and more recently, by Christian religious groups in Brazil and other countries. The aims of the present study were to identify the effects of ayahuasca on zebrafish embryo development and neurobehavior. Toxicity and developmental endpoints for zebrafish embryos were assessed from 0 to 1000 mg/L over 96 h of exposure. The effects on locomotor activity of zebrafish larvae were assessed using a video tracking system (ZebraBox) from 0 to 20 mg/L and after 120 and 144 h of exposure. The LC50 of ayahuasca in zebrafish was determined as 236.3 mg/L. Ayahuasca exposure caused significant developmental anomalies in zebrafish embryos, mainly at the highest concentration tested, including hatching delay, loss of equilibrium, edema and the accumulation of red blood cells. Embryo behavior was also significantly affected, with decreased locomotor activity at the highest concentration tested. These results are in accordance with data obtained in mammal studies highlighting the possible risks of uncontrolled use of ayahuasca. Further research employing more specific behavior analysis could provide additional data on both therapeutic benefits and possible toxicological risk of ayahuasca.

Maternal and developmental toxicity of the hallucinogenic plant-based beverage ayahuasca in rats.

Rats were treated orally with ayahuasca (AYA) on gestation days (GD) 6-20 at doses corresponding to one-(1X) to eight-fold (8X) the average dose taken by a human adult in a religious ritual, and the pregnancy outcome evaluated on GD21. Rats treated with 4X and 8X doses died during the treatment period (44 and 52%), and those that survived showed kidney injury. Rats surviving the 8X dose showed neuronal loss in hippocampal regions and in the raphe nuclei, and those from the 2X dose neuronal loss in CA1. Delayed intrauterine growth, induced embryo deaths and increased occurrence of foetal anomalies were observed at the 8X dose. At non-lethal doses, AYA enhanced embryolethality and the incidence of foetal soft-tissue and skeleton anomalies. This study suggested that AYA is developmentally toxic and that its daily use by pregnant women may pose risks for the conceptus.

High Frequency of Copy-Neutral Loss of Heterozygosity in Patients with Myelofibrosis.

Myelofibrosis is the rarest and most severe type of Philadelphia-negative classical myeloproliferative neoplasms. Although mutually exclusive driver mutations in JAK2, MPL, or CALR that activate JAK-STAT pathway have been related to the pathogenesis of the disease, chromosome abnormalities have also been associated with the phenotype and prognosis of the disease. Here, we report the use of a chromosomal microarray platform consisting of both oligo and SNP probes to improve the detection of chromosome abnormalities in patients with myelofibrosis. Sixteen patients with myelofibrosis were tested, and the results were compared to karyotype analysis. Driver mutations in JAK2, MPL, or CALR were investigated by PCR and MLPA. Conventional cytogenetics revealed chromosome abnormalities in 3 out of 16 cases (18.7%), while chromosomal microarray analysis detected copy-number variations (CNV) or copy-neutral loss of heterozygosity (CN-LOH) alterations in 11 out of 16 (68.7%) patients. These included 43 CN-LOH, 14 deletions, 1 trisomy, and 1 duplication. Ten patients showed multiple chromosomal abnormalities, varying from 2 to 13 CNVs or CN-LOHs. Mutational status for JAK2, CALR, and MPL by MLPA revealed a total of 3/16 (18.7%) patients positive for the JAK2 V617F mutation, 9 with CALR deletion or insertion and 1 positive for MPL mutation. Considering that most of the CNVs identified were smaller than the karyotype resolution and the high frequency of CN-LOHs in our study, we propose that chromosomal microarray platforms that combine oligos and SNP should be used as a first-tier genetic test in patients with myelofibrosis.

Expanding the spectrum of TBL1XR1 deletion: Report of a patient with brain and cardiac malformations.

The TBL1XR1 gene product is a nuclear protein ubiquitously produced. The protein is a component of SMRT/N-CoR co-repressor complexes and participates in the molecular switch of specific gene transcription. Deletions of the TBL1XR1 gene have been described in two families to date, both presenting intellectual disability and dysmorphisms. Rare recurrent chromosomal micro-rearrangements, particularly those involving single genes, represent a challenge for clinicians to ensure correlation with phenotype due to the paucity of previously described cases. Here we present a patient harbouring a TBL1XR1 gene deletion detected by chromosome microarray analysis. In addition to intellectual disability, the patient presents dysmorphic features and multiple cardiac malformations, together with brain malformation, thus contributing to the phenotypic characterization of this rare microdeletion and to the TBL1XR1 gene function.

Integrated assessment of toxic effects of maghemite (γ-Fe2O3) nanoparticles in zebrafish.

The increasing use of nanotechnology in the last decade has raised concerns about the impact of nanoparticles in the environment. In particular, the potential harmful effects of iron oxide nanoparticles (IONPs) in aquatic organisms have been poorly addressed. We analyze here the toxic effects induced by IONPs in zebrafish using a combination of classical (genotoxicity, oxidative stress) and molecular (transcriptomic) methodologies. Adult animals were exposed for 96h to five sub-lethal IONP concentrations, ranging from 4.7 to 74.4mg/L. Comet and micronucleus assays revealed a significant number of DNA lesions induced by IONPs at all concentrations tested. Conversely, the thiobarbituric acid reactive substances (TBARS) test detected only a mild oxidative damage in liver cells (∼1.5-fold increase of malondialdehyde concentrations) and only at the two higher IONP concentrations tested. Microarray analysis of liver samples identified 953 transcripts (927 unique genes) differentially expressed between controls and IONP-exposed samples. Subsequent functional analysis identified genes related to cation/metal ion binding, membrane formation, and morphogenesis among the transcripts overrepresented upon IONP treatments, whereas mRNAs encompassing genes associated with RNA biogenesis, translation, ribosomes, and several metabolic processes became underrepresented in treated samples. Taken together, these results indicate considerable genotoxic effects of IONPs combined with general negative effect on cell growth and on the ability of the cell produce new proteins. On the contrary, IONPs showed only a limited capacity to induce oxidative stress. To our knowledge, this is the first study on IONPs toxicity using such an integrative approach in an aquatic organism.

Reproductive effects of the psychoactive beverage ayahuasca in male Wistar rats after chronic exposure

Abstract Ayahuasca is a psychoactive beverage used ancestrally by indigenous Amazonian tribes and, more recently, by Christian religions in Brazil and other countries. This study aimed to investigate the reproductive effects of this beverage in male Wistar rats after chronic exposure. The rats were treated by gavage every other day for 70 days at 0 (control), 1×, 2×, 4× and 8× the dose used in a religious ritual (12 animals per group), and animals euthanized on the 71st day. Compared to controls, there was a significant decrease in food consumption and body weight gain in rats from the 4× and 8× groups, and a significant increase in the brain and stomach relative weight at the 8× group. There was a significant increase in total serum testosterone, and a decrease in spermatic transit time and spermatic reserves in the epididymis caudae in the 4× group, but not in the highest dose group. No significant changes were found in the other reproductive endpoints (spermatozoid motility and morphology, total spermatozoid count and daily sperm production), and histology of testis and epididymis. This study identified a no-observed-adverse-effect-level for chronic and reproductive effects of ayahuasca in male Wistar rats at 2× the ritualistic dose, which corresponds in this study to 0.62 mg/kg bw N, N-dimethyltryptamine, 6.6 mg/kg bw harmine and 0.52 mg/kg bw harmaline. A potential toxic effect of ayahuasca in male rats was observed at the 4× dose, with a non-monotonic dose-response. Studies investigating the role of ayahuasca components in regulating testosterone levels are needed to better understand this action.