Vanillic acid and vitamin C attenuated di-2-ethylhexyl phthalate-induced testicular toxicity in adult male rats.

Abstract: Di-2-ethylhexyl phthalate (DEHP) is an extensively used plasticizer which has raised some concerns about its safety on human health. This study aimed at evaluating the effects of vanillic acid (VA) and vitamin C (VC) supplementation on DEHP-induced testicular toxicity. Thirty-five adult male Wistar rats were randomly divided into 7 groups (A-G) (n = 5) receiving distilled water; 250 mg/kg bw of DEHP only; 30 mg/kg bw of VA and 250 mg/kg bw of DEHP; 30 mg/kg bw of VC and 250 mg/kg bw of DEHP; 30 mg/kg bw of DEHP plus 30 mg/kg bw of VA and 30 mg/kg bw of VC; 30 mg/kg bw of VA only; and 30 mg/kg bw of VC only, respectively. At the end of the experiment, blood was taken from the heart via cardiac puncture and stored, semen was collected from the caudal epididymis for immediate sperm analysis, while the testes were excised and preserved for histological examination and biochemical analysis. The results showed a significant decrease (P < 0.05) in body weights, sperm motility, sperm volume, sperm viability and count, antioxidant levels, and reproductive hormonal levels, with a significant increase (P < 0.05) in sperm morphological defect and lipid peroxidation level in DEHP-only group compared with the control but was ameliorated after VA and VC administration compared to the DEHP-only treated animals. VA and VC supplementation attenuated the toxic effects of DEHP on the testicular functions, morphology, and semen characterization of the experimental adult male Wistar rats. Lay summary: Male infertility is considered when identifiable female causes of infertility are excluded and semen quantity and quality fail to fulfil World Health Organization criteria. From conception through to adulthood, people are exposed to limitless environmental toxicants among which di-2-ethylhexyl phthalate (DEHP) commonly found in personal care products, cosmetics, and medical devices is prevalent. The present study elaborated on the importance of taking antioxidant-rich foods containing vitamin C and vanillic acid, such as those found in various fruits, olives, whole wheat, and cereal grains, in combating infertility caused by environmental toxicants. An experiment was carried out on rats to see the effect of vanillic acid and vitamin C supplementation on preventing DEHP-induced testicular toxicity. The testicles and semen were analyzed from five rats in each treated and control groups. The data led us to conclude that vanillic acid and vitamin C supplementation do have attenuating effects on DEHP-induced testicular toxicity, due to their high antioxidant and anti-inflammatory properties.

Rat age-related benign prostate hyperplasia is concomitant with an increase in the secretion of low ramified α-glycosydic polysaccharides.

This work analysed the expression of prostate polysaccharides in rats with age-related benign prostatic hyperplasia (BPH) for a better understanding of the possible relationship between prostate polysaccharides secretion and BPH onset. For this, prostatic glands from 1 month-old, 3 months-old, 6 months-old and 12 months-old Sprague-Dawley rats were processed in order to identify their overall polysaccharide content. Additionally, serum testosterone was also determined. One-month old rats showed significantly (P < 0.05) lower testosterone levels (0.77 ng/mL±0.12 ng/mL) compared with the other groups, which showed no significant difference among them. PAS staining showed positive polysaccharides markings in both the prostatic lumen and inside of luminal prostatic cells in all groups. Semiquantitative analysis of intraluminal PAS showed that one month-old rats had significantly (P < 0.005) lower PAS intensity when compared with all other groups (100.0 ± 0.5, arbitrary units vs. 107.3 ± 0.6, arbitrary units in 3 months-old ones), whereas 12 months-old ones showed significantly (P < 0.005) higher values when compared with all other groups (133.6 ± 3.5, arbitrary units in 12 months-old rats vs. 108.6 ± 1.4, arbitrary units in 6 months-old ones). The PAS + content practically disappeared when tissues were pre-incubated with either α-amylase or amyloglucosidase, regardless of a previous incubation with proteinase K. Incubation of prostate extracts from 12 months-old rats for 2 h with α-amylase yielded a significantly higher amount of free glucose (1.47 nmol/mg protein±0.23 nmol/mg protein vs. 0.32 nmol/mg protein±0.01 nmol/mg protein in untreated extracts). Similar results were obtained when extracts were pre-incubated with amyloglucosidase. Contrarily, pre-incubation with N-glycosidase induced a significantly (P < 0.05), much lower increase of free glucose. Pre-treatment with proteinase K did not significantly modify these results, which indicate that BPH is related to an increase in the secretion of low ramified ductal α-glycosydic polysaccharides that were not protected against lysis by any type of protein protective core. These changes seem to not be related with concomitant variations in serum testosterone levels.

Protective effects of methanolic leaf extracts of Monanthotaxis caffra against aflatoxin B1-induced hepatotoxicity in rats.

Aflatoxins are potent hepatotoxic and carcinogenic secondary metabolites produced by toxigenic fungi. The present study investigated the protective effect of methanolic leaf extracts of Monanthotaxis caffra (MLEMC) against aflatoxin B1-induced toxicity in male Sprague-Dawley rats. The rats were randomly divided into 6 groups of 8 animals each. Five groups were administered orally for seven days with three different concentrations of MLEMC (100 mg/kg, 200 mg/kg and 300 mg/kg), curcumin (10 mg/kg) or vehicle (25% propylene glycol). The following day, these groups were administered 1 mg/kg b.w. of aflatoxin B1 (AFB1). The experiment was terminated three days after administration of AFB1. Group 6 represented untreated healthy control. Serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, creatinine and liver histopathology were evaluated. Methanolic leaf extracts of M. caffra decreased the levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase and creatinine in the sera of rats as compared with the AFB1 intoxicated group. Co-administration of MLEMC improved the histological characteristics of the hepatocytes in contrast to the AFB1 treated group, which had mild to severe hepatocellular injuries including bile duct proliferation, bile duct hyperplasia, lymphoplasmacytic infiltrate and fibrosis. Extracts of M. caffra were beneficial in mitigating the hepatotoxic effects of AFB1 in rats by reducing the levels of liver enzymes and preventing hepatic injury.

Effect of tetra-hydroxylated bile acid on size and insulin sensitivity of subcutaneous adipocytes in healthy lean cats.

Obesity leads to insulin resistance and is a major risk factor for the development of diabetes mellitus in cats. Prevention of obesity and obesity-induced insulin resistance is difficult, and reliable long-term strategies are currently lacking. Retinoid-related orphan receptor gamma (RORγ) was recently identified as an important transcription factor in the development of large insulin-resistant adipocytes in mice and humans. RORγ negatively affects adipocyte differentiation through expression of its target gene matrix metalloproteinase 3 (MMP3) and promotes the development of large insulin-resistant adipocytes. Preliminary studies in mice showed that RORγ can be inhibited by its ligand tetra-hydroxylated bile acid (THBA). In the present study, serum THBA levels were determined in healthy and diabetic cats. Moreover, potential side effects and the effects of THBA supplementation on adipocyte size, mRNA expression of RORγ, MMP3, interleukin 6, tumor necrosis factor α, adiponectin and leptin in feline subcutaneous adipocytes and insulin sensitivity were investigated in healthy normal weight cats. Thirteen healthy and 13 diabetic cats were used for determination of serum THBA level, and six healthy normal-weight cats were included in a feeding trial. Similar THBA levels were determined in serum of healthy and diabetic cats. Supplementation of 5 mg/kg THBA for 8 wk did not cause any negative effect on feeding behavior, general condition and blood parameters of tested cats. It significantly reduced adipocyte size and mRNA expression of MMP3, interleukin 6, and tumor necrosis factor α in adipocytes, while mRNA expression of adiponectin significantly increased and mRNA expression of RORγ and leptin remained unchanged. Administration of THBA did not influence fasting blood glucose levels or the response of cats to acute insulin administration. Based on these results, THBA is palatable and is considered safe for use in cats. It reduces expression of MMP3 and promotes the development of small adipocytes with increased expression of adiponectin and reduced expression of interleukin 6 and tumor necrosis factor α. Further studies are recommended to evaluate the effect of THBA on adipocyte size and insulin sensitivity in obese cats.

Ulcerative Dermatitis in C57BL/6NCrl Mice on a Low-Fat or High-Fat Diet With or Without a Mineralized Red-Algae Supplement.

Ulcerative dermatitis (UD) is a spontaneous idiopathic disease that often affects C57BL/6 mice or mice on a C57BL/6 background. UD is characterized by intense pruritus and lesion formation, most commonly on the head or dorsal thorax. Self-trauma likely contributes to wound severity and delayed wound healing. Histologically, changes are nonspecific, consisting of ulceration with neutrophilic and mastocytic infiltration and epithelial hyperplasia and hyperkeratosis. Diet appears to have a profound effect on the development and progression of UD lesions. We investigated the incidence and severity of UD in C57BL/6NCrl mice on a high-fat western-style diet (HFWD) compared with a standard rodent chow. In addition, we examined the protective effects of dietary supplementation with a multimineral-rich product derived from marine red algae on UD in these 2 diet groups. HFWD-fed mice had an increased incidence of UD. In addition, mice on a HFWD had significantly more severe clinical and histologic lesions. Dietary mineral supplementation in mice on a HFWD decreased the histologic severity of lesions and reduced the incidence of UD in female mice in both diets. In conclusion, a high-fat western-style diet may potentiate UD in C57BL/6NCrl mice. Insufficient mineral supply and mineral imbalance may contribute to disease development. Mineral supplementation may be beneficial in the treatment of UD.

Ulcerative dermatitis in C57BL/6 mice lacking stearoyl CoA desaturase 1.

Ulcerative dermatitis (UD) is a common cause of morbidity and euthanasia in mice with a C57BL/6 (B6) background. The purposes of the current study were to determine whether UD lesions could be reliably produced in B6 mice lacking stearoyl-CoA desaturase 1 (SCD1(-/-) mice), to ascertain whether the UD lesions in SCD1(-/-) mice were similar to those found in other B6 mice, and to characterize the cell invasion phenotype of Staphlococcus xylosus cultured from the lesions. S. xylosus isolates from the environment and human skin were used as controls. SCD1(-/-) (n = 8 per group) and nontransgenic B6 control mice (n = 22 mice pooled from 3 groups that received different concentrations of conjugated linoleic acid) were fed standard rodent chow or a semipurified diet (NIH AIN76A) for 4 wk. Samples from other B6 mice with UD (field cases; n = 7) also were submitted for histology and culture. All of the SCD1(-/-) mice developed UD lesions by 4 wk on NIH AIN76A. None of SCD1(-/-) fed standard rodent chow and none of the wildtype B6 mice fed NIH AIN76A developed UD. Supplementation with conjugated linoleic acid did not affect ulcerogenesis. UD lesions in SCD1(-/-) mice and field cases were grossly and histologically similar. S. xylosus was isolated from SCD1(-/-) mice with UD (71%) and field cases of UD (43%). These isolates were the most cell-invasive, followed by the environmental isolate, and finally the human skin isolate. Our results provide a basis for further pathologic and clinical study of UD.

Melatonin reduces the severity of experimental amoebiasis.

BACKGROUND: Melatonin has immunomodulatory effects but very little is known about its influence in protozoan infections, such as Entamoeba histolytica, which causes amoebiasis, a disease with significant morbidity and mortality. In this study, we evaluated the effects of exogenous melatonin interference in experimental amoebiasis and on interactions between human blood cells and E. histolytica trophozoites. METHODS: The effect of melatonin was investigated in models of experimental amoebiasis in hamsters and rats by evaluating the area of necrosis induced by E. histolytica. The activity of melatonin on the interactions between leukocytes and amoebae was determined by examining leukophagocytosis. For in vitro tests, polymorphonuclear and mononuclear human blood leucocytes were incubated with E. histolytica trophozoites. RESULTS: The areas of amoebic necrosis were significantly reduced in animals treated with melatonin. Melatonin treatment increased leukophagocytosis but was associated with a greater number of dead amoebae. CONCLUSIONS: These results suggest that melatonin may play a beneficial role in the control of amoebic lesions, raising the possibility that this drug may be used as an adjuvant in anti-amoebic therapy.

Treatment of pododermatitis in the guinea pig.

Pododermatitis is a painful inflammation of the footpads. This column describes appropriate treatment for pododermatitis in guinea pigs.

Vitamin E as a treatment for ulcerative dermatitis in C57BL/6 mice and strains with a C57BL/6 background.

In this study, we fed a standard NIH-31 diet fortified with vitamin E to C57BL/6 mice and strains of mice with a C57BL/6 background that had spontaneously developed ulcerative dermatitis (UD). In addition to the therapeutic response to increased levels of vitamin E, we also defined the occurrence of UD within our facility in terms of age, sex, coat color, and lesion location on the body. Mice with spontaneous UD were fed a vitamin E-fortified diet (3000 IU/kg) for a period of 8 weeks and entered the study without regard to vendor source, age, sex, coat color, or the site or number of UD lesions. We found that lesions occurred most commonly on the dorsal cervical and scapular regions and spared the ventral abdomen and thorax. No sex or coat color predilection was noted for the development of UD, however males were older than females at the time of lesion development. Of 71 mice, 32 (45%) had complete lesion re-epithelialization with hair regrowth. Complete lesion repair was not influenced by sex, age, or coat color. The average time to complete lesion repair ranged from 2 to 5 weeks, and there was no correlation with sex or coat color. The positive response to vitamin E suggests that protection from oxidative injury may play a role in the resolution of UD lesions and offers veterinarians and investigators a new treatment option with ease of compliance.

Diet and kidney diseases in rats.

Diet-associated kidney diseases of rats includes nephropathy in both sexes and nephrocalcinosis in females. High protein content of diets appears to be the major cause for severe nephropathy and changing the source of protein to one such as soy protein, restricting caloric intake, or modifying the diet to decrease protein consumption could decrease the severity of nephropathy. The NTP-2000 diet with lower protein content than most diets decreases the severity of nephropathy and increases the survival of Fischer 344 rats without substantial changes in growth patterns and body weights. Nephrocalcinosis, characterized by mineralization of renal tubules at the corticomedullary junction, has been reported in young and adult female rats of most strains and stocks suggesting a major contribution of female sex hormones to the development of this lesion. Calcium (Ca), phosphorous (P), magnesium (Mg), and chloride (Cl) imbalances, especially a Ca:P ratio of less than 1.0 in diet, are considered to be associated with this lesion. Most commercial diets commonly used for toxicology studies have a Ca:P molar ratio of less than 1.0. Increasing the Ca:P molar ratio to more than 1.0 and closer to 1.3 in the AIN-93 purified diet and NTP-2000 nonpurified diet prevents the development of this lesion. Genetics will predispose rats to some diseases and environmental factors will influence the severity of these diseases. Diet is one of the most important environmental factors. Diets balanced for nutrients without excesses could markedly improve the health of rats used in chronic studies leading to substantial increases in survival and thereby accomplish the objective of chronic toxicity and carcinogenicity studies.

Rat hypoplastic kidney (hpk/hpk) induces renal anemia, hyperparathyroidism, and osteodystrophy at the end stage of renal failure.

In rats with genetically hypoplastic kidneys (hpk/hpk) and associated hypogonadism (hgn/hgn), their kidneys contain only one-quarter the number of nephrons that are found in those of normal rats [26]. Not surprisingly, therefore, renal excretive function has been shown to be depressed in hpk/hpk rats [26]. In the study presented here, we have examined the process of the progression of renal failure and the development of renal secondary disease in hpk/hpk rats. The plasma concentrations of urea-nitrogen and creatinine were significantly higher in adult hpk/hpk rats than in normal rats. These values elevated gradually and the degree of renal histological damage also progressed with advancing age in the hpk/hpk rats. In addition, renal anemia appeared at 140 days of age or later in these rats, and hyperplasia of the parathyroid glands was visible macroscopically at 280 days of age. In the hpk/hpk rats plasma levels of calcium and phosphorus were significantly lower and higher than in normal rats, respectively, at 280 days of age. Pathologically, the left femora of hpk/hpk rats exhibited fibrous osteodystrophy at 280 days of age and the calcium content of the right femora (as a percentage of the dry weight of bone) was significantly lower than in normal rats at both 210 and 280 days of age. These results indicate that the reduced nephrogenesis of the hpk/hpk rats causes progressive renal failure, secondarily inducing anemia, hyperparathyroidism, and osteodystrophy.

Familial dysmyelination in a Long Evans rat mutant.

Tremors were observed in 15 Long Evans rats beginning at 10 to 12 days of age. These were followed by progressively worsening ataxia, hind limb paresis, episodes of immobility, and seizures by 5 to 14 weeks. Gross lesions were not observed at necropsy in rats euthanized and perfused at 4 to 16 weeks of age. Neurohistologic examination revealed dysmyelination in the central nervous system. Astrogliosis in the white matter with marked increase of expression of the glial fibrillary acid protein marker was accompanied by diffuse microgliosis. Scattered glial cells, interpreted to be oligodendrocytes, contained minute periodic acid-Schiff-positive cytoplasmic granules. Large mineralized periodic acid-Schiff-positive and laminated structures were observed in the cerebellar white matter, midbrain, and thalamus of rats over 6 weeks old. Neuronal degeneration and loss was evident in the cortex, hippocampus, and midbrain. Large axonal spheroids were found in the ventral and lateral funiculi of the spinal cord. An ultrastructural study of four affected rats revealed an almost complete absence of myelinated axons and normal sheaths, and degeneration and necrosis of oligodendrocytes. The Long Evans shaker rat represents a novel myelin mutant with a remarkable survival period and appears to have an autosomal recessive mode of inheritance.

Calcification of the urinary bladder in the wild cotton rat (Sigmodon hispidus).

Calcification of the urinary bladder epithelium was observed in 19 of 30 and 18 of 30 wild cotton rats from control and petrochemical-contaminated sites, respectively. The rats in the two sites did not differ significantly in respect of serum calcium and phosphorus concentrations. The calcification was considered to be dystrophic in nature. An unidentified factor common to both control and petrochemical-contaminated sites was considered to be responsible for this syndrome.

Effects of humoral hypercalcemia of malignancy on the parathyroid gland in nude mice.

The ultrastructure of parathyroid chief cells was examined from four groups of nude mice (NIH:Swiss) with different serum calcium concentrations. The groups consisted of eight male mice with hypercalcemia induced by transplantable canine adenocarcinoma (CAC-8), eight female mice with hypercalcemia induced by infusion of parathyroid hormone-related protein, ten male control mice, and six male mice fed a low calcium (0.01%) diet. Hypercalcemia induced by malignancy or parathyroid hormone-related protein infusion was associated with low serum phosphorus concentration, a decrease in the number of secretory and prosecretory granules in the parathyroid chief cells, and an increase in the cytoplasmic area of chief cells. Prominent myelinlike membranous whorls were present in the cytoplasm of chief cells of tumor-bearing and parathyroid hormone-related protein-infused hypercalcemic mice. Mice fed a low calcium diet had decreases in the number of secretory granules and cell area but increases in the number of prosecretory granules compared with control mice. The number of mitochondria and the nuclear area of chief cells were similar in all four groups. The prominent membranous whorls and increased cytoplasmic area of chief cells from these hypercalcemic mice mark these cells as distinctly different from the parathyroid chief cells of other species with hypercalcemia.

Corneal dystrophy in Fischer 344 rats.

A spontaneous degenerative lesion of the cornea resembling calcific band keratopathy in man has been observed in 10-15% of the F-344 rats (aged 35-300 days) purchased from a private vendor's closed breeding colony. The lesion appears clinically as punctuate to linear superficial corneal opacities located in the interpalpebral fissure of one or both eyes. Occasional roughening, bleb formation, or pitting of the corneal surface resembling superficial ulcers may be observed. The lesion occurs in both sexes. It is rarely associated with inflammation or irritation. Histologically, it consists of mineral deposits along the epithelial basement membrane and Bowman's space, some of which are large enough to disrupt or destroy portions of the basilar epithelium. Energy dispersive X-ray analysis of the deposits proved them to be composed of calcium and phosphorus. Electron microscopic examination revealed a variety of extracellular laminated and crystalline arrays similar to those seen in humans with band keratopathy. The etiology of the lesion is as yet undetermined. A genetic-associated susceptibility due to hypercalcemia may be involved.

Vitamin E and selenium status of guinea pigs with myocardial necrosis.

Myocardial necrosis and mineralization has been identified in a colony of guinea pigs which were subsequently tested for vitamin E and selenium deficiency. Serum vitamin E and whole blood selenium levels were within normal ranges. The erythrocyte glutathione peroxidase test has potential as a predictor of whole blood selenium levels in the guinea pig. The red blood cell hemolysis test used in this study did not correlate consistently with the serum vitamin E levels. We suspect that myocardial necrosis and mineralization may have resulted from inbreeding guinea pigs within the closed colony.

Paresis of peristalsis and ileus lead to death in lactating mice.

Based on the examination of 45 dead and 5 moribund female mice during a 2-year period, we are able to describe a new disease entity: ileus of the small intestine in lactating mice caused by a paresis of peristalsis. Diarrhoea was not observed and inflammation and infectious agents were not found. Females were affected during the 2nd week of their first lactation. The condition may have a mortality rate as high as 40%. It is assumed that exhaustion (calcium, glucose, etc.) is the cause of this condition. Consequently, the development of a dietary supplement or of a special diet for lactating mice may prove beneficial in preventing this disease. Endogenic (Clostridia) or exogenic toxic components may also play a role.

Effect of vitamin A deficiency on mammary gland development and susceptibility to mastitis through intramammary infusion with Staphylococcus aureus in mice.

Weanling mice were fed 0 or 150 micrograms retinol equivalent/kg of diet for 5 weeks, were bred, and allowed to complete gestation. On day 3 of lactation, all mice were separated from their litters for 1 hour and were then anesthetized. The 4th right or left mammary gland was inoculated with 0.1 ml of S Aureus (10(10) colony-forming units/0.1 ml). Exactly 24 hours after inoculation, the mice were euthanatized and the mammary glands were removed and fixed for histologic evaluations. Vitamin A-deficient dams had smaller litter size and lower liver stores of vitamin A; however, deficiency was not severe enough to produce external signs of vitamin A deficiency in the dams. Morphologic studies showed large areas of adipose tissue, greatly reduced ductal and lobule-alveolar development, and decreased total secretory activity in mammary glands from vitamin A-deficient females. On the other hand, mammary glands from vitamin A-supplemented mice had extensive lobule-alveolar development and highly distended alveoli. Extensive necrosis of alveolar tissue was observed in staphylococcus-infused mammary glands of all mice. Large numbers of leukocytes and cell debris were present in the lumen of alveoli and ducts. However, mammary glands from vitamin A-deficient females had more extensive pathologic damage compared with corresponding glands from vitamin A-supplemented mice. Results indicted that vitamin A-deficient mice had reduced mammary development and increased pathologic damage to the mammary gland after intramammary challenge with staphylococcus.