Testicular toxicity in mice exposed to terephthalic acid in utero and during lactation.

Terephthalic acid (TPA) is a worldwide aromatic compound widely used to manufacture resins and the raw material for the polymerization reaction with ethylene glycol to produce polyethylene terephthalate, known as PET. The use of TPA extends to the synthesis of phthalates, plasticizers used in various industrialized products such as toys and cosmetics. The present study aimed to evaluate the testicular toxicity of terephthalic acid on male mice exposed in utero and during lactation to TPA in different developmental windows. The animals were treated intragastric with TPA at stock dispersal dosages corresponding to 0.0014 g/ml and 0.56 g/ml of TPA in 0.5% v/v carboxymethylcellulose as well as the control dose, composed solely of dispersion of carboxymethylcellulose (0.5% v/v). Four experimental windows were established: group I-treatment in utero, in the fetal period (gestational day-GD 10.5-18.5), with euthanasia at GD 18.5; group II-treatment in utero, in the fetal period (GD 10.5-18.5) and the lactational period (postnatal day (PND-15)), with euthanasia at 15 days; group III-treatment in utero in the fetal period (DG 10.5-18.5) with euthanasia at 70 days (age of sexual maturity, PND 70); group IV-treatment in utero, in the fetal period (GD 10.5-18.5) and the lactational period (PND-15), with euthanasia at 70 days (PND70). The results indicate that TPA changes the reproductive parameters (testicular weight, GI, penis size, and anogenital index) only at the dose of 0.56 g/ml in the fetal period. Data on the volumetric ratio of the testis elements show that the dispersion with the highest concentration of TPA significantly altered the blood vessel/capillary, lymphatic vessel, and connective tissue percentages. Only at the dose of 0.56 g/ml TPA was it effective in decreasing the Leydig and Sertoli cell numbers of the euthanized animals at GD 18.5. In group II, TPA increased the diameter and lumen of seminiferous tubules, which indicates that TPA accelerated the maturation process of Sertoli cells without changing the number and the nuclear volume of these cells. The Sertoli and Leydig cell numbers of the 70-day animals exposed to TPA in the gestational and lactational period were similar to the control. Therefore, the present study is the first in the literature to show that TPA presents a testicular toxicity during fetal (DG18.5) and postnatal life (PND15), without repercussion in adulthood (70 days).

New Insights Into Blue Light Phototherapy in Experimental Trypanosoma cruzi Infection.

The search for an effective etiologic treatment to eliminate Trypanosoma cruzi, the causative agent of Chagas disease, has continued for decades and yielded controversial results. In the 1970s, nifurtimox and benznidazole were introduced for clinical assessment, but factors such as parasite resistance, high cellular toxicity, and efficacy in acute and chronic phases of the infection have been debated even today. This study proposes an innovative strategy to support the controlling of the T. cruzi using blue light phototherapy or blue light-emitting diode (LED) intervention. In in vitro assays, axenic cultures of Y and CL strains of T. cruzi were exposed to 460 nm and 40 µW/cm2 of blue light for 5 days (6 h/day), and parasite replication was evaluated daily. For in vivo experiments, C57BL6 mice were infected with the Y strain of T. cruzi and exposed to 460 nm and 7 µW/cm2 of blue light for 9 days (12 h/day). Parasite count in the blood and cardiac tissue was determined, and plasma interleukin (IL-6), tumoral necrosis factor (TNF), chemokine ligand 2 (CCL2), and IL-10 levels and the morphometry of the cardiac tissue were evaluated. Blue light induced a 50% reduction in T. cruzi (epimastigote forms) replication in vitro after 5 days of exposure. This blue light-mediated parasite control was also observed by the T. cruzi reduction in the blood (trypomastigote forms) and in the cardiac tissue (parasite DNA and amastigote nests) of infected mice. Phototherapy reduced plasma IL-6, TNF and IL-10, but not CCL2, levels in infected animals. This non-chemical therapy reduced the volume density of the heart stroma in the cardiac connective tissue but did not ameliorate the mouse myocarditis, maintaining a predominance of pericellular and perivascular mononuclear inflammatory infiltration with an increase in polymorphonuclear cells. Together, these data highlight, for the first time, the use of blue light therapy to control circulating and tissue forms of T. cruzi. Further investigation would demonstrate the application of this promising and potential complementary strategy for the treatment of Chagas disease.

Pyriproxyfen does not cause microcephaly or malformations in a preclinical mammalian model.

Pyriproxyfen is used in Brazil to combat epidemics of Dengue Fever, Chikungunya Fever, and Zika virus. This study assessed the effects of pyriproxyfen on reproductive performance, embryo-fetal development, head measurements, and DNA integrity in a preclinical model. Thirty pregnant mice were divided into three groups (n = 10): control (drinking water-0.1 ml/10 g (body weight-b.w., gavage) and treated with pyriproxyfen 0.0002 mg/kg and 0.0021 mg/kg (b.w., gavage) during the gestational period. Analysis of biometric, reproductive performance and embryo-fetal development parameters related to control presented no significant differences, suggesting no maternal or embryo-fetal toxicity. Head measurements showed no differences except an increase in anterior/posterior measurement and glabella/external occipital protuberance. Analysis of DNA integrity showed an increase in micronucleus only at 72 h for the lowest dose group. Thus, we infer that pyriproxyfen is not related to the occurrence of microcephaly, nor does it alter reproductive performance, embryo-fetal development or DNA integrity.

Effects of 3-Heptyl-3,4,6-trimethoxy-3H-isobenzofuran-1-one alone or/in association with cyclophosphamide on testicular function.

Chemotherapy for cancer treatment may result in a temporary or long-term gonadal damage resulting in subfertility or infertility. Cyclophosphamide (CY) is a cytotoxic alkylating agent that has been widely used in the treatment of cancer. Recent studies have shown that synthetic resorcinol lipid AMS35AA (3-Heptyl-3,4,6-trimethoxy-3H-isobenzofuran-1-one) may be an important adjuvant chemotherapy that potentiates mutagenic damage and increases apoptosis caused by CY. The present study investigates the action of AMS35AA alone or/in association with CY on testicular function. Animals were divided into four groups: (a) control group: received only water; (b) CY group: received 150 µg/g of CY b.w., i.p.; (c) AMS35AA group: received 10 µg/g of AMS35AA b.w., i.p; and (d) associated group: received 10 µg/g of AMS35AA + 150 µg/g of CY b.w., i.p. Four weeks after the treatment, the results showed that testes weight of CY and associated groups decreased. However, the number of Sertoli cell and Leydig cell per testis was similar in control and treated groups. Our findings provide strong evidence that the AMS35AA alone or in CY association is not toxic to spermatogenesis. The absence of toxicity of AMS35AA supports the view that the resorcinolic lipid could be used associated with CY chemotherapy without causing adverse effects to testes function.

Evaluation of the testis function of mice exposed in utero and during lactation to Pfaffia glomerata (Brazilian ginseng).

Pfaffia glomerata (Spreng.) Pedersen, popularly known as "Brazilian ginseng," is used as medicinal plant in Brazil to treat inflammatory diseases in general. Previous studies showed that its extract increases the nitric oxide (NO) levels. Knowing that NO downregulates steroidogenesis and that alterations in the action/production of androgens during perinatal life could alter testis development, the present studies sought to investigate the reproductive toxicity of Pfaffia glomerata on male mice exposed to hydroalcoholic extract in utero and during lactation. The present study shows that P. glomerata extract does not alter body weight, tubular diameter and testis function in male mice. Although a reduction in the testis weight was observed in the animals that received the highest dose directly in early post-natal life, our findings show clearly that P. glomerata may not act as an endocrine disruptor, and it is not an "antiandrogenic" compound that could lead to testicular dysgenesis syndrome.

Resorcinolic lipid 3-heptyl-3,4,6-trimethoxy-3H-isobenzofuran-1-one is a strategy for melanoma treatment.

AIMS: Previous studies performed by our research group indicated that cytosporone analogues are capable of prevent or repair DNA damages. This work presents the evaluation of the activity of AMS35AA for metastatic murine melanoma cells (B16F10) in experimental model in vitro and, in pre-clinic assay of metastatic melanoma in vivo, using mice lineage C57BL/6. MAIN METHODS: In vitro assays were performed: MTT and comet assay, flow cytometry evaluation, gene expression assay by RT-PCR, qualitative evaluation of cell death using B16F10 cells. In vivo assays: micronucleus and comet assay, splenic phagocytosis, melanoma murine model and histopathological analysis, using mice lineage C57BL/6 (n = 20). KEY FINDINGS: In vitro results performed by MTT assay showed that AMS35AA is cytotoxic for B16F10 cells (p < 0.05). Based on comet assay the genotoxicity of the IC50 was determined (95.83 µg/mL) (p < 0.05). These data were corroborated by flow cytometry analysis after the treatment with AMS35AA, which indicates the cellular death by apoptosis (p < 0.05) and increasing of ATR, p53, p21 and GADD45 gene expressions verified using RT-PCR. With respect to in vivo results, it was observed that AMS35AA did not show genotoxic activity. Data of tumor volume ex vivo indicate reduction of tumor for the treated animals with AMS35AA up to 15.84×, which is superior to Dacarbazina (50 mg/Kg, p.c.; i.p.). SIGNIFICANCE: In summary, the study showed that AMS35AA reveals relevant results regarding to cytotoxicity of B16F10 murine melanoma cells, inducing death by apoptosis via mitochondrial and/or mediated by DNA damages.

Evaluation of the effects of the larvicides temephos on reproductive performance, embryofetal development and DNA integrity of Swiss mice.

Temephos is considered the gold standard by the Ministry of Health for controlling the larvae of the mosquito Aedes aegypti. The present study evaluated the effects of Temephos larvicide on the reproductive performance, embryo-fetal development and DNA integrity of Swiss mice. This study used 30 pregnant female mice: 10 were controls treated with drinking water at a dosage of 0.1 mL/10 g (body weight - b.w., administered orally - a.o.), and 20 were treated with Temephos at doses of 0.0043 mg/kg and 0.043 mg/kg (b.w., a.o.) during the gestational period. Statistical analysis showed that Temephos did not alter the biometric or reproductive parameters. Comparing the weight of the fetus to the stage of pregnancy demonstrated that the 0.0043 mg/kg dosage increased the size of the fetuses. No external malformations were detected. However, the 0.043 mg/kg dosage induced changes in the sternum, with the main change being the center of the sternum, xiphoid processes and absence of the manubrium. The other skeletal and visceral alterations did not differ from the control group and are considered variants of normality. The analysis of head measurements showed an increase in the anterior/posterior measurements of the glabella, the external occipital protuberance and the biauricular plane. The circumference and area of the head did not present significant differences. The micronucleus test showed only a 0.043 mg/kg increase in 48 h. Thus, it is considered that Temephos has a low teratogenic and genotoxic risk.

The mixture of cashew nut shell liquid and castor oil results in an efficient larvicide against Aedes aegypti that does not alter embryo-fetal development, reproductive performance or DNA integrity.

Dengue fever, chikungunya fever and Zika virus are epidemics in Brazil that are transmitted by mosquitoes, such as Aedes aegypti or Aedes albopictus. The liquid from shells of cashew nuts is attractive for its important biological and therapeutic activities, which include toxicity to mosquitoes of the genus Aedes. The present study evaluated the effects of a mixture of surfactants from natural cashew nutshell liquid and castor oil (named TaLCC-20) on the mortality of larvae and on the reproductive performance, embryonic and fetal development and genetic stability of Swiss mice. A total of 400 Ae. aegypti larvae (third larval stage) were treated with TaLCC-20 concentrations of 0.05 mg/L, 0.5 mg/L, or 5 mg/L (ppm). Twenty pregnant female mice were also orally administered TaLCC-20 at doses of 5 mg/kg and 50 mg/kg body weight (b.w.), and 10 animals were given only drinking water at 0.1 mL/10 g b.w. (orally). The results of a larvicide test demonstrated that 5 mg/mL TaLCC-20 killed 100% of larvae within three hours, which is comparable to the gold standard indicated by the Ministry of Health. Overall, these results show that TaLCC-20 is an efficient larvicide that does not induce genetic damage. In addition, changes in reproductive performance and embryo-fetal development appear positive, and the formulation is cost effective. Therefore, TaLCC-20 is an important product in the exploration of natural larvicides and can assist in fighting mosquitos as vectors for dengue fever, chikungunya fever and Zika virus, which are emerging/re-emerging and require proper management to ensure minimal harm to the human population. Therefore, TaLCC-20 can be considered a key alternative to commercial products, which are effective yet toxigenic.

In vivo chemotherapeutic insight of a novel isocoumarin (3-hexyl-5,7-dimethoxy-isochromen-1-one): Genotoxicity, cell death induction, leukometry and phagocytic evaluation.

Chemotherapy is one of the major approaches for the treatment of cancer. Therefore, the development of new chemotherapy drugs is an important aspect of medicinal chemistry. Chemotherapeutic agents include isocoumarins, which are privileged structures with potential antitumoral activity. Herein, a new 3-substituted isocoumarin was synthesized from 2-iodo-3,5-dimethoxy-benzoic acid and oct-1-yne in a cross-coupling Sonogashira reaction followed by a copper iodide-catalyzed intramolecular cyclization as key step using MeOH/Et3N as the solvent system. The present study also evaluated the leukometry, phagocytic activity, genotoxic potential and cell death induction of three different doses (5 mg/kg, 10 mg/kg and 20 mg/kg) of this newly synthesized isocoumarin, alone and in combination with the commercial chemotherapeutic agents cyclophosphamide (100 mg/kg) and cisplatin (6 mg/kg) in male Swiss mice. The results suggest that the isocoumarin has genotoxicity and causes cell death. Noteworthy, this new compound can increase splenic phagocytosis and lymphocyte frequency, which are related to immunomodulatory activity. When combined with either cyclophosphamide or cisplatin, chemopreventive activity led to a reduction in the effects of both chemotherapeutic drugs. Thus, the new isocoumarin is not a candidate for chemotherapeutic adjuvant in treatments using cyclophosphamide or cisplatin. Nevertheless, the compound itself is an important prototype for the development of new antitumor drugs.

In vivo chemotherapeutic insight of a novel isocoumarin (3-hexyl-5,7-dimethoxy-isochromen-1-one): Genotoxicity, cell death induction, leukometry and phagocytic evaluation

Abstract Chemotherapy is one of the major approaches for the treatment of cancer. Therefore, the development of new chemotherapy drugs is an important aspect of medicinal chemistry. Chemotherapeutic agents include isocoumarins, which are privileged structures with potential antitumoral activity. Herein, a new 3-substituted isocoumarin was synthesized from 2-iodo-3,5-dimethoxy-benzoic acid and oct-1-yne in a cross-coupling Sonogashira reaction followed by a copper iodide-catalyzed intramolecular cyclization as key step using MeOH/Et3N as the solvent system. The present study also evaluated the leukometry, phagocytic activity, genotoxic potential and cell death induction of three different doses (5 mg/kg, 10 mg/kg and 20 mg/kg) of this newly synthesized isocoumarin, alone and in combination with the commercial chemotherapeutic agents cyclophosphamide (100 mg/kg) and cisplatin (6 mg/kg) in male Swiss mice. The results suggest that the isocoumarin has genotoxicity and causes cell death. Noteworthy, this new compound can increase splenic phagocytosis and lymphocyte frequency, which are related to immunomodulatory activity. When combined with either cyclophosphamide or cisplatin, chemopreventive activity led to a reduction in the effects of both chemotherapeutic drugs. Thus, the new isocoumarin is not a candidate for chemotherapeutic adjuvant in treatments using cyclophosphamide or cisplatin. Nevertheless, the compound itself is an important prototype for the development of new antitumor drugs.

Effects of dichloromethane and butanol fractions of Gochnatia polymorpha floccosa in maternal reproductive outcome, embryo-fetal development and DNA integrity in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Gochnatia polymorpha ssp. floccosa (Asteraceae) also known as ''Cambará'' is used as medicinal plant in Brazil to treat infections and inflammation. Previous studies showed that its ethanolic extract could be bioprospecting of a new anti-inflammatory phytotherapy for use during pregnancy. This work aimed to evaluate dichloromethane (DCM) and butanolic (BT) fractions from G. polymorpha on embryo-fetal development and DNA integrity in mice. MATERIALS AND METHODS: Female mice were treated with 50 and 20mg/kg of the DCM and BT fractions, respectively, during organogenesis and gestational period. RESULTS AND CONCLUSION: The present study shows that DCM and BT fractions from G. polymorpha possess mutagenic activity but are not teratogenic. Based on the fact that the reproductive indices are similar in control and treated animals, we may infer that the mutagenic effect was in somatic cell, at least in part, because the reabsorption number and reabsorption rates did not change in DCM and BT exposed groups.

Adulteration and Contamination of Commercial Sap of Hymenaea Species.

The Hymenaea stigonocarpa and Hymenaea martiana species, commonly known as "jatobá," produce a sap which is extracted by perforation of the trunk and is commonly used in folk medicine as a tonic. For this study, the authenticity of commercial samples of jatobá was verified by the identification of the main compounds and multivariate analysis and contamination by microbial presence analysis. The acute toxicity of the authentic jatobá sap was also evaluated. The metabolites composition and multivariate analysis revealed that none of the commercial samples were authentic. In the microbiological contamination analysis, five of the six commercial samples showed positive cultures within the range of 1,700-100,000 CFU/mL and the authentic sap produced no signs of toxicity, and from a histological point of view, there was the maintenance of tissue integrity. In brief, the commercial samples were deemed inappropriate for consumption and represent a danger to the population.

Gestational exposure to Byrsonima verbascifolia: teratogenicity, mutagenicity and immunomodulation evaluation in female Swiss mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Byrsonima verbascifolia is used in folk medicine to treat diarrhea, intestinal infections, chronic wounds, Chagas disease, inflammation and as a diuretic. However there is no investigation regarding the Byrsonima verbascifolia hydrometanolic extract (BVHME) used during gestation. MATERIALS AND METHODS: The pregnant females were randomly divided into 5 groups. Control group received saline plus DMSO (1%) in a volume of 0.1 mL/10 g (b.w.), via gavage, for at least 15 days prior to mating and throughout the gestational period. The Pre-treatment group received the BVHME, via gavage, at a dose of 50 mg/kg (b.w.) for at least 15 days prior to mating and up to the appearance of the vaginal plug. The Organogenesis group received the BVHME at a dose of 50 mg/kg (b.w.), via gavage, on the 5-15th gestational day. The Gestational group received the BVHME at a dose of 50 mg/kg (b.w.), via gavage, throughout the gestational period (from the 1st to the 18th day of pregnancy). The Pre+Gestational group received the BVHME at a dose of 50mg/kg (b.w.), via gavage, for at least 15 days prior to mating and up to throughout the gestational period. The clinical signals of maternal and fetuses toxicity were evaluated, as the mutagenicity and immunomodulation tests were performed. RESULTS AND CONCLUSIONS: The present investigation shows, for the first time, that the use of Byrsonima verbascifolia extract in pregnant Swiss mice, did not alter the female reproductive function, mutagenicity or immunostimulation as well as not interfere with embryofetal development at least in our experimental conditions.

Reproductive toxicity of Campomanesia xanthocarpa (Berg.) in female Wistar rats.

ETHNOPHARMACOLOGICAL RELEVANCE: There is no evidence in the literature that substantiates the safety of Campomanesia xanthocarpa (Berg.) use during pregnancy. MATERIALS AND METHODS: Thirty three female rats were randomly assigned to three groups. One group of animals received the Campomanesia xanthocarpa extract via gavage at a dose of 26.3mg/kg/day from 6 to 15 days of pregnancy (organogenic period, T1) and another group received the same extract throughout the gestational period (from the 1st to the 20th day of pregnancy, T2). Control groups received distilled water. Euthanasia was done on 20th day, when the liver, kidney, spleen ovaries, fetuses and their respective placentas were removed. Implantations, reabsorptions, live and dead fetuses were recorded. RESULTS AND CONCLUSIONS: Campomanesia xanthocarpa, in these experimental conditions, did not disturb the reproductive function of female rats and did not interrupt the progress of the embryofetal development. Moreover, our results provide further evidence that the Campomanesia xanthocarpa treatment reduces reabsorption sites, increases placenta weight and the number of live fetuses and may therefore have therapeutic applications.

Maternal exposure to Cochlospermum regium: a toxicological evaluation

Cochlospermum regium (Schrank) Pilg., Bixaceae, is a Brazilian plant widely used as a folk medicine in the southwestern of the Brazil to treat inflammation and infection diseases. However, the effects of C. regium hydroethanolic extract on pregnant rats have not been assessed. To evaluate the effects of the C. regium on pregnant rats during the organogenic period, the hydroethanolic extract was administered via gavage at a dose of 11.5 mg/kg/day to rats from 6th to 15th day of pregnancy. No clinical signs of maternal toxicity were observed. The placenta's and fetuses' weight were similar in control and treated animals. The term fetuses dis not present malformations or anomalies although the number of live fetuses and birth rate were significantly decreased. In conclusion, the C. regium hydroethanolic extract is nontoxicant to the pregnant rat although it would be likely to interfere in the progress of the embryofetal development.

Postnatal testis development, Sertoli cell proliferation and number of different spermatogonial types in C57BL/6J mice made transiently hypo- and hyperthyroidic during the neonatal period.

The role of thyroid hormones in testis structure and function has been fairly well studied in laboratory rodents. However, there are no comprehensive data in the literature for mice regarding the effects of transiently induced neonatal hypo- and hyperthyroidism on testis and spermatogonial cell development from birth to adulthood. Our goals were to evaluate the effects of propylthiouracil (PTU) and triidothyronine (T3) on Sertoli cell proliferation/differentiation and to correlate these events with the evolution of the spermatogenic process, tubular lumen formation, blood vessel volume density, and size and number of different spermatogonial types. Although Sertoli cell maturation was accelerated or delayed, respectively, in T3- and PTU-treated mice, the pace of the germ cell maturation was only slightly altered before puberty and the period of Sertoli cell proliferation was apparently not affected by the treatments. However, compared with controls, the total number of Sertoli cells per testis from 10 days of age to adulthood was significantly increased and decreased in PTU- and T3-treated mice, respectively. In comparison to all other spermatogonia, type A(2) was the largest cell in all ages and groups investigated. The PTU-treated mice had a significantly increased total number of undifferentiated spermatogonia as well as volume and percentage of vessels/capillaries, probably due to the higher number of Sertoli cells, particularly at 10 days of age. Taken together, our results suggest that neonatal hypothyroidism may be a valuable tool for studying spermatogonial biology as well as a means for providing more spermatogonial stem cells that could potentially be used for spermatogonial transplantation, thereby optimizing the efficiency of this technique when young mice are used as donors.