Nephrotoxicity evaluation of 3-monochloropropane-1,2-diol exposure in Sprague-Dawley rats using data-independent acquisition-based quantitative proteomics analysis.

3-Monochloropropane-1,2-diol (3-MCPD), as a heat-induced food process contaminant, possesses strongly toxic effect on kidney. The present study focuses on characterizing the proteome and clarifying the underlying molecular regulatory mechanisms in a model of kidney injury in rats treated with 3-MCPD. Data-independent acquisition (DIA)-mass spectrometry (MS) based proteomics was used to identify dysregulated proteins in kidney tissues of Sprague-Dawley (SD) rats treated with 30 mg/kg/day 3-MCPD by gavage for 28 days. It was found that a total of 975 proteins were deregulated after 3-MCPD treatment. Bioinformatic analyses revealed that several enzymes related to the metabolisms of amino acid, lipid and carbohydrate in endogenous metabolism were altered in response to 3-MCPD treatment. Moreover, some proteins involved in these pathways were also changed, mainly including oxidative stress, oxidative phosphorylation, apoptosis and autophagy. Our study unravels the vital roles of loss of mitochondrial homeostasis and function and cell death pathways in the development of renal damage induced by 3-MCPD, which provides further valuable insights into the initiation and resolution of 3-MCPD nephrotoxicity. The proposed DIA-MS workflow not only provides a choice for proteomic analysis in toxicological research, but also provides a more comprehensive understanding of the molecular mechanisms of nephrotoxicity induced by toxins.

Formulation, physical, in vitro and ex vivo evaluation of nanomedicine-based chemosterilant for non-surgical castration of male animals.

The overpopulation of free-roaming companion animals has become the global crisis. The development and application of a suitable, effective, non-surgical approach for animal sterilization would have an enormous advantage over the current surgical method. The main purpose of this study was to develop and evaluate a novel nanomedicine-based chemosterilant for non-surgical castration of male companion animals. In this study, we first sought to investigate the testicular toxicity of different apoptosis-inducing agents. We next synthesized and characterized nano-sized particles which encapsulated the most potent testicular toxicants and evaluated in vitro sterilant properties. Our result showed that doxorubicin exhibited the highest cytotoxic activity against mouse spermatogenic cells. We therefore synthesized and characterized doxorubicin-encapsulated nanoemulsion. The negatively charged particle of doxorubicin-encapsulated nanoemulsion exhibited the anti-proliferative activity towards spermatogetic cells. Apoptosis studies revealed activation of Caspases 3 and 7 as well as annexin V expression. In addition, doxorubicin-encapsulated nanoemulsion exhibited anti-inflammatory activity in lipopolysaccharide-stimulated macrophages. Cell death was observed following treatment of isolated and cultured rat seminiferous tubules with doxorubicin-encapsulated nanoemulsion. In conclusion, nanoemulsion can be a potential carrier for prolonged release and to enhance activity of doxorubicin that may have utility in non-surgical castration of male animals.

Post-operative outcomes of surgical and chemical castration with zinc gluconate in dogs presenting to veterinary field clinics.

The objective of this study was to characterize post-operative outcomes of chemical castration as compared to surgical castration performed by existing municipal field clinics. Fifty-four healthy adult male dogs underwent chemical castration with zinc gluconate solution and 55 healthy adult male dogs underwent surgical castration in veterinary field clinics. Dogs in each group were evaluated for swelling, inflammation, and ulceration (chemical castration) or dehiscence (surgical castration) at Days 3, 7, and 14 following castration. More surgically castrated dogs required medical intervention than chemically castrated dogs (P=0.0328); the number of dogs requiring surgical repair within each group did not differ (P=0.3421). Seven chemically castrated dogs and 22 surgically castrated dogs experienced swelling, inflammation, and/or ulceration; all were managed medically. Two chemically castrated dogs experienced scrotal ulceration requiring surgical castration at Days 3 and 7. One surgically castrated dog experienced partial incisional dehiscence requiring surgical repair at Day 3. Our results suggest that chemical castration of dogs in field clinics is a feasible alternative to surgical castration, but proper follow-up care should be ensured for at least 7days post-procedurally.

Examination of the use of intraovarian administration of CaCl2 and zinc gluconate as potential chemosterilants in Bos indicus heifers.

OBJECTIVE: To investigate if administration of potential chemosterilants by transvaginal, ultrasound-guided intraovarian injection is an alternative to surgical sterilisation. DESIGN: Randomised control study. METHODS: Bos indicus heifers were treated with intraovarian injections of saline (n = 10), CaCl2 (n = 10), zinc gluconate (ZG; n = 10) or a combination of CaCl2 and ZG (CaCl2 + ZG; n = 10). Heifers were exposed to a bull from 82 to 84 days after treatment and slaughtered between 364 and 396 days after treatment. RESULTS: After treatment administration, the concentrations of haptoglobin and anti-Müllerian hormone and the total oocyte counts did not differ (P > 0.150) between treatments. Pain responses were observed in heifers treated with ZG and CaCl2 + ZG but not in heifers treated with saline or CaCl2 . Total ovarian mass at slaughter was less in heifers treated with CaCl2 compared with the other treatments (P < 0.05). Complete regression of one ovary was observed in 40% (4/10) of the heifers treated with CaCl2 . Pregnancies were recorded in ≥ 70% of heifers administered each treatment. CONCLUSION: Treatments used in this study were not able to sterilise most of the B. indicus heifers, but treatment with CaCl2 has the potential to cause complete ovarian atrophy without causing detectable pain.

Effects of surgical and chemical sterilization on the behavior of free-roaming male dogs in Puerto Natales, Chile.

Population management of free-roaming domestic dogs (Canis lupus familiaris) is of interest due to the threat these animals pose to people, other animals and the environment. Current sterilization procedures for male dogs include surgical and chemical methods. However, little is known about how these procedures affect their behavior. The primary objective of this study was to investigate changes in selected behaviors following chemical and surgical sterilization in a male free-roaming dog (FRD) population in southern Chile. We also examined the association between serum testosterone levels and behaviors thought to be influenced by circulating androgens. A total of 174 dogs were randomly assigned to either a surgical or chemical sterilization group, or a control group. At the onset of the intervention period, 119 dogs remained and 102 dogs successfully completed the study. Each dog was monitored pre- and post-intervention using video recordings, GPS collars, and blood samples for the measurement of testosterone. Analysis of behavior revealed that surgically castrated dogs showed no reduction of sexual activity or aggression when compared to their pre-intervention behavior. Chemically sterilized dogs showed a statistically significant increase in dog-directed aggression, but no change in sexual activity. There was no change in home range size in any groups between the pre- and post-intervention measurement. We found no consistent association between levels of serum testosterone concentration and behavioral changes in any of the groups. This study presents the first detailed behavioral observations following surgical and chemical sterilization in male FRDs. The information generated is highly relevant to communities struggling with the control of FRDs. Complementary studies to further our understanding of the effects of male sterilization on the behavioral and reproductive dynamics of FRD populations are needed.

Estabelecimento de protocolo para micropropagação de Lippia gracilis Schauer / Establishment of protocol for Lippia gracilis Schauer micropropagation

A espécie Lippia gracilis SCHAUER (Verbenaceae) é nativa do Nordeste brasileiro e se destaca pela capacidade de acumular nos tricomas glandulares óleos essenciais com atividade antimicrobiana. Tendo em vista que não constam trabalhos na literatura sobre o estabelecimento in vitro dessa espécie, este trabalho teve como objetivo estabelecer protocolo para micropropagação de L. gracilis. Para tanto, ramos contendo folhas foram coletados de plantas matrizes no habitat natural para a confecção de estacas. Em laboratório, os explantes provenientes do processo de estaquia foram assepticamente tratados e inoculados em meio MS, acrescido de fitorreguladores, com o intuito de se estabelecer a melhor dose para o desenvolvimento dos explantes. Em decorrência de altos níveis de contaminação, avaliou-se o efeito da cefalexina. No entanto, apesar do antibiótico ter apresentado diminuição na contaminação bacteriana, a porcentagem de oxidação foi elevada. Portanto, testou-se o carvão ativado, ácido ascórbico, ácido cítrico e metade dos sais de MS quanto a eficiência no controle da oxidação. Concluiu-se que, o antibiótico na concentração utilizada provocou a oxidação dos explantes e os fitorreguladores, bem como os métodos antioxidantes testados, não apresentaram resultados consistentes para o melhor desenvolvimento dos explantes e controle da oxidação, respectivamente.

The species Lippia gracilis Schauer (Verbenaceae) is native to Northeastern Brazil and has been important for its ability to accumulate essential oils with antimicrobial activity in the glandular trichomes. Since there are no reports in the literature on the micropropagation of this species, the present work aimed to establish a protocol for L. gracilis micropropagation. Thus, branches containing leaves were collected from plant matrices in their natural habitat to prepare cuttings. In the laboratory, explants from cutting were aseptically treated and inoculated onto MS medium plus plant growth regulators in order to establish the best dose for the development of explants. Due to high levels of contamination, the effect of cephalexin was evaluated. Although the antibiotic decreased the bacterial contamination, the percentage of oxidation was high. Then, activated charcoal, ascorbic acid, citric acid and half the salts of MS were tested for their effectiveness to control oxidation. In conclusion, the used antibiotic concentration resulted in oxidation of explants. Furthermore, plant growth regulators and antioxidant methods did not show consistent results for a better development of explants and control of oxidation, respectively.

Evaluation of single intratesticular injection of calcium chloride for nonsurgical sterilization in adult albino rats.

OBJECTIVE: To investigate a method of chemical sterilization and its efficacy in adult albino rats. METHOD: Evaluation was conducted 3 weeks after a single bilateral intratesticular injection of calcium chloride (CaCl2) at the dose of 5, 10, 15 or 20 mg per testis per 100 g body weight. RESULTS: The significant graded diminution in relative sex organ weights, testicular androgenic enzymes like Delta5,3beta-hydroxysteroid dehydrogenase (Delta5,3beta-HSD) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD), glutathione peroxidase (GPx), glutathione S-transferase (GST) and superoxide dismutase (SOD) activities, testicular content of reduced glutathione (GSH), plasma and intratesticular concentrations of testosterone, epididymal sperm count as well as significant elevation in plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testicular content of malondialdehyde (MDA) were noted in all the treated groups with respect to vehicle control. There was no chronic general stress in experimental animals as indicated by insignificant changes in plasma concentrations of corticosterone, prolactin, total protein, blood urea nitrogen and fasting blood sugar level. Dose-dependent responses on testicular histopathology were recorded by noting multinucleated giant cells in seminiferous tubules, derangement of tubular architecture along with infiltration of leucocytes and appearance of fibrous tissue throughout the testicular sections. The fertility efficacy of the 10, 15 or 20 mg CaCl2-treated males was nil, proven after mating with fertile, virgin healthy females, as there were no implantation sites in each uterine horns noted by laparotomy. CONCLUSION: Intratesticular CaCl2 injection at a specific dose might serve as a way of sterilization and may be considered as an alternative to surgical castration in male animals.

Antispermatogenic and antifertility effects of 20,25-diazacholesterol dihydrochloride in mice.

The effect of intraperitoneal administration for 28 days of 10, 20, and 30 mg/kg body weight per day of 20,25-diazacholesterol dihydrochloride (SC 12937), a hypocholesterolemic agent, on the testis of Parkes (P) strain mice was investigated. Histologically, testes in mice treated with 10 or 20 mg/kg body weight of SC 12937 showed non-uniform degenerative changes in the seminiferous tubules as both affected and normal tubules were observed in the same section; the affected tubules showed intraepithelial vacuolation, occurrence of giant cells, exfoliation of germ cells, and marginal condensation of chromatin in round spermatids. In both dosage groups, only 11-12% of the seminiferous tubules were affected, and no significant differences were found in the frequency of affected tubules between the two groups. By contrast, testes in mice treated with 30 mg/kg body weight of the drug exhibited a degenerated appearance of germ cells in all seminiferous tubules. The treatment also had adverse effects on motility, viability, morphology, and number of spermatozoa in the cauda epididymidis, and on fertility. Even 56 days after drug withdrawal, the above parameters remained markedly affected. Our results thus suggest that SC 12937 treatment causes antispermatogenic and antifertility effects in P mice and that the effects are not reversible up to 56 days after drug withdrawal. This compound may prove useful in the control of rodent populations.

[Study on the absorption, distribution and excretion of 3-chloro-1,2-propandiol in rats].

OBJECTIVE: To explore the absorption, distribution and excretion of 3-Chloro-1,2-propandiol (3-MCPD) in healthy male SD rats after oral administration. METHODS: 3-MCPD was administrated with a single oral dosage of 75 mg/kg BW to each rat. Samples of blood, tissues (including liver, kidney, brain and testicle) and excreta were then collected, and analyzed by the GC-MS method to determine 3-MCPD concentrations. The reported value is the mean value of three rats. RESULTS: At 2 h after the administration, 3-MCPD concentrations in blood, testicle and kidney were (67.46 +/- 7.72), (78.37 +/- 5.15) and (56.21 +/- 3.64) microg/g, respectively. At 24 h, however, the corresponding values changed to (1.07 +/- 0.97) microg/g, (49.43 +/- 28.18) microg/g and (11.41 +/- 2.55) microg/g. During the 24-hour period, 9.74 +/- 3.05% of the given parent compound was excreted in urine, whereas 0.56 +/- 0.22% and 0.28 +/- 0.03% were excreted in feces and bile, respectively, which implies that kidney is a major organ for excretion 3-MCPD. CONCLUSIONS: 3-MCPD was quickly absorbed through the alimentary tract and quickly distributed into a number of tissues, and then accumulated in the target organs, especially in the testicle. The excretion of the parent compound was largely through the kidney. It was inferred that 3-MCPD was mainly metabolized in the liver.

Dose-dependent response to an intratesticular injection of calcium chloride for induction of chemosterilization in adult albino rats.

This study concerned the minimum and optimum effective doses of calcium chloride needed for induction of chemosterilization in male albino rats, 30 days after a single intratesticular injection of calcium chloride (CaCl2.2H2O) solution at 2.5, 5, 10 or 20 mg per 100 g body weight per testis. There was a significant diminution in the relative wet weight of the sex organs (p<0.01), epididymal sperm count (p<0.001), plasma concentration of testosterone (p<0.01), testicular activities of delta5,3beta-hydroxy steroid dehydrogenase (delta5,3beta-HSD), 17beta-hydroxy steroid dehydrogenase (17beta-HSD) (p<0.01), glutathione S-transferase (GST) (p<0.01), superoxide dismutase (SOD) (p<0.01), and peroxidase (p<0.01), significant elevations in testicular content of malondialdehyde (MDA) and conjugated dienes (p<0.01), along with derangement of seminiferous tubular architecture and degeneration of the Leydig cells in the testis and elevations in the concentrations in the plasma of LH and FSH (p<0.01), commencing at a dose of 5 mg, with the greatest effects at a dose of 20 mg. No significant alterations in these factors occurred at the dose of 2.5 mg in comparison to the control that received only the vehicle. There was no significant alteration in the plasma concentrations of prolactin (p>0.05), corticosterone (p>0.05) or fasting blood glucose or in the rectal temperature (p>0.05) at any of the doses relative to the control group, suggesting that this chemosterilizing procedure did not exert any chronic stress on the experimental animals. From these observations, it may be suggested that 5 mg should be considered as the minimum dose, and 10 mg or 20 mg as the optimum dose, whereas 2.5 mg was ineffective for induction of chemosterilization. There would seem to be little point in using more than 20 mg of calcium chloride for this purpose. Intratesticular injection of calcium chloride at an effective dose may be considered as an alternative to surgical castration.

Genotoxicity of a crude leaf extract of neem in male germ cells of mice.

The oral administration of a soxhlated crude ethanolic extract of leaves of neem (Azadirachta indica Ajuss; family Meliaceae) to adult male mice for 6 weeks (one spermatogenic duration) at the rate of 0.5, 1.0 or 2.0 g/kg body weight per day increased the incidences of structural changes and synaptic-disturbances in meiotic chromosomes and also caused more disruptions of meiosis. The extract reduced the sperm count and increased the frequency of spermatozoa with abnormal head morphology. It is suggested that at least one of the constituents of the extract may have interfered with the DNA. The result was chromosome strand breakages, or spindle disturbances, and the regulation of genes responsible for sperm shaping was affected.

Avaliaçäo da utilizaçäo dos métodos de esterilizaçäo em ortodontia / Evaluation of the use of sterilization methods in orthodontics

Os principais métodos de esterilizaçäo empregados em odontologia foram revisados, especialmente os físicos, pelo uso do calor seco ou forno de Pasteur-estufa e do calor úmido-autoclave, e a esterilizaçäo química, feita pelo emprego de glutaraldeído a 2 por cento por 10 horas ou formaldeído a 38 por cento por 18 horas. Este trabalho também teve como objetivo a avaliaçäo do emprego dos métodos de esterilizaçäo química e física pelos ortodontistas da cidade de Curitiba. Os resultados mostraram que 83 por cento dos ortodontistas entrevistados empregavam tanto os métodos físicos como os químicos para esterilizaçäo, 11 por cento empregavam somente os métodos físicos e 6 por cento somente os métodos químicos. Concluiu-se que 27,7 por cento dos ortodontistas empregavam tempo de esterilizaçäo inferior a 10 horas para o glutaraldeído e para o formaldeído, tempo este inferior ao recomendado para a esterilizaçäo. O álcool etílico a 70 por cento era utilizado por 2,4 por cento dos profissionais como agente esterilizante químico, mesmo sendo este um agente desinfetante de grau intermediário. O monitoramento da temperatura interna da estufa era feito pelo termömetro próprio do aparelho (termostato) por 56,5 por cento dos ortodontistas, com tempo de exposiçäo inferior a uma hora, o que näo garante a esterilizaçäo em todo o interior da estufa. Mais de 40 por cento dos ortodontistas empregavam desinfecçäo para o instrumental e o material, embora estes admitissem somente esterilizaçäo, o que pode ser um fator de infecçäo cruzada

Sperm motion predicts fertility in male hamsters treated with alpha-chlorohydrin.

An understanding of the relationship between altered sperm motion and sperm function (fertility) is important when interpreting the biological significance of toxicant-induced changes in sperm velocity in rodent test species. Previous studies showed that a brief (4-day) exposure of male hamsters to the model chemical alpha-chlorohydrin (ACH) results in significant deficits in epididymal and uterine sperm velocity, which are associated with both a delay and a failure of fertilization in vivo. To characterize this effect in terms of fertility, similarly treated male hamsters were bred to untreated females and pups were counted the day before parturition. ACH treatment resulted in a dose-dependent decline in the percentage of sperm-positive females that were pregnant at the end of gestation (100, 78, 67, 22, and 0 where males were treated with 0, 33, 49, 66, and 83 mg ACH/kg/day, respectively). Cauda epididymal sperm from the same males were assayed for motion characteristics using computer-assisted sperm analysis (CASA), and for fertilizing ability in vitro. While the percentage of motile sperm was unaffected by ACH treatment, sperm velocity declined in a dose-dependent manner at all ACH treatment levels. Furthermore, the velocity of sperm from infertile males was shifted downward consistently across the entire velocity distribution. Since treated males tended to either be infertile (no pups) or have near normal litter size, the correlation between sperm velocity and litter size was nonlinear. Therefore, logistic regression models using velocity cut-off values were the most useful models for predicting fertility. These results support the contention that fertility relies on there being a sufficient number of sperm that exceed a velocity threshold. Sperm from treated males were also less likely to support in vitro fertilization (IVF), providing further evidence of impaired sperm function associated with acute exposure to ACH.