Multilevel Approach for the Treatment of Giardiasis by Targeting Arginine Deiminase.

Giardiasis represents a latent problem in public health due to the exceptionally pathogenic strategies of the parasite Giardia lamblia for evading the human immune system. Strains resistant to first-line drugs are also a challenge. Therefore, new antigiardial therapies are urgently needed. Here, we tested giardial arginine deiminase (GlADI) as a target against giardiasis. GlADI belongs to an essential pathway in Giardia for the synthesis of ATP, which is absent in humans. In silico docking with six thiol-reactive compounds was performed; four of which are approved drugs for humans. Recombinant GlADI was used in enzyme inhibition assays, and computational in silico predictions and spectroscopic studies were applied to follow the enzyme's structural disturbance and identify possible effective drugs. Inhibition by modification of cysteines was corroborated using Ellman's method. The efficacy of these drugs on parasite viability was assayed on Giardia trophozoites, along with the inhibition of the endogenous GlADI. The most potent drug against GlADI was assayed on Giardia encystment. The tested drugs inhibited the recombinant GlADI by modifying its cysteines and, potentially, by altering its 3D structure. Only rabeprazole and omeprazole decreased trophozoite survival by inhibiting endogenous GlADI, while rabeprazole also decreased the Giardia encystment rate. These findings demonstrate the potential of GlADI as a target against giardiasis.

Fluoxetine-induced androgenic failure impairs the seminiferous tubules integrity and increases ubiquitin carboxyl-terminal hydrolase L1 (UCHL1): Possible androgenic control of UCHL1 in germ cell death?

The selective serotonin reuptake inhibitor fluoxetine has been used for the treatment of depression. Although sexual disorders have been reported in male patients, few studies have demonstrated the fluoxetine effect on the reproductive histophysiology, and the target of this antidepressant in testes is unknown. We evaluated the impact of short-term treatment with fluoxetine on the adult rat testes, focusing on steroidogenesis by Leydig cells (LC) and androgen-dependent testicular parameters, including Sertoli cells (SC) and peritubular myoid cells (PMC). Since UCHL1 (ubiquitincarboxyl-terminal hydrolase L1) seems to control spermatogenesis, the immunoexpression of this hydrolase was also analyzed. Adult male rats received 20 mg/kg BW of fluoxetine (FG) or saline (CG) for eleven days. In historesin-embedded testis sections, the seminiferous tubule (ST) and epithelial (Ep) areas, and the LC nuclear diameter (LCnu) were measured. The number of abnormal ST, androgen-dependent ST, SC and PMC was quantified. Testicular ß-tubulin levels and peritubular actin immunofluorescence were evaluated. Serum testosterone levels (STL) and steroidogenesis by 17ß-HSD6 immunofluorescence were analyzed, and either UCHL1-immunolabeled or TUNEL-positive germ cells were quantified. In FG, abnormal ST frequency increased whereas ST and Ep areas, androgen-dependent ST number, LCnu, 17ß-HSD6 activity and STL reduced significantly. TUNEL-positive PMC and SC was related to decreased number of these cells and reduction in peritubular actin and ß-tubulin levels. In FG, uncommon UCHL1-immunoexpression was found in spermatocytes and spermatids, and the number of UCHL1-immunolabeled and TUNEL-positive germ cells increased in this group. These findings indicate that LC may be a fluoxetine target in testes, impairing PMC-SC integrity and disturbing spermatogenesis. The increase of UCHL1 in the damaged tubules associated with high incidence of cell death confirms that this hydrolase regulates germ cell death and may be controlled by androgens. The fertility in association with the androgenic status of patients treated with fluoxetine should be carefully evaluated.

Alteraciones clínicas y bioquímicas en ratas tratadas con dosis altas de vitamina A / Clinical and biochemical alterations in rats treated with high doses of vitamin A

En el presente trabajo se estudió el efecto de la administración intramuscular de 30.000, 50.000 y 100.000 UI de palmitato de vitamina A/día, durante 7 días, respectivamente, sobre la actividad enzimática hepática en 45 ratas Wistar machos, de 12 semanas de edad, con pesos entre 180 y 200 gramos. El grupo control estuvo integrado por 15 ratas Wistar sanas, con género, edad y peso similares a los animales tratados. El consumo de alimentos y de agua, y el peso de las ratas se determinó al finalizar el período experimental. Las ratas se examinaron en busca de manifestaciones clínicas de toxicidad. Al final el estudio, las ratas se sacrificaron bajo anestesia con éter y se tomaron muestras de tejido hepático para la determinación de la actividad enzimática. La administración de vitamina A en exceso incrementó de manera significativa (p menor que 0,05) el contenido hepático del retinol, determinó diversos y variados signos clínicos (tales como: anorexia, pérdida de peso, alopecia, conjuntivitis, hemorragias internas y externas, alteraciones cutáneas y muerte de los animales) e incrementó (p menor que 0,05) la actividad de las siguientes enzimas: alanina aminotransferasa, aspartato aminotransferasa, maltasa ácida (alfa-1,4-glucosidasa ácida), proteasas ácidas, lactato dehidrogenasa y fosfatasa alcalina mientras que las actividades de la glucosa-6-fosfatasa, glucógeno fosforilasa, alfa-amilasa, colinesterasa y arginasa disminuyeron (p menor que 0,05) al comparar con los controles no tratados. Estos cambios son proporcionales a las dosis inyectadas de vitamina A. En conclusión, nuestros resultados proporcionan evidencias que la administración de dosis altas de vitamina A a corto plazo determina diversos y variados signos clínicos y produce una marcada alteración de la actividad enzimática hepática.

In the present work the effect of intramuscular administration of 30.000, 50.000 and 100.000 IU of vitamin A palmitate daily for seven days, respectively, on the liver enzyme activity in 45 white male Wistar rats, aged 12 weeks and weighing 180-200 g, have been studied. The group control was integrated by 15 healthy rats with similar characteristics (strain, gender, age and weight) to treated animals. Food and water consumption and body weights were recorded at the end of the experimental period. Rats were observed for clinical signs of toxicity. At the end of the study, rats were sacrificed under ether anesthesia. Liver samples were taken for the determination of enzyme activity. Administration of excess of vitamin A produced a significant (p menor 0.05) increase in the content of liver vitamin A, determined diverse and variable clinical signs (such as, anorexia, loss of body weight, alopecia, conjunctivitis, external and internal hemorrhages, skin abnormalities and death) and increased (p menor que 0.05) the activity of the following enzymes: alanine aminotransferase, aspartate aminotransferase, acid maltase (acid alfa-1,4-glucosidase), acid proteases, lactate dehydrogenase and alkaline phosphatase while glucose-6-phosphatase, glycogen phosphorylase, alfa-amylase, cholinesterase and arginase decreased (p menor que 0.05) as compared with untreated controls. These changes depend on the doses given of vitamin A. In conclusion, our results provide evidence that short-term administration of high doses of vitamin A determined diverse and variable clinical signs and produces a marked alteration of activity of liver enzymes.