Ochratoxin A-induced teratogenesis in rats: partial protection by phenylalanine.

Ochratoxin A (OA), an important foodborne mycotoxin, is a potent teratogenic and nephrotoxic agent produced by several species of Aspergillus and Penicillium. OA is a known inhibitor of protein synthesis via competition with phenylalanine (Phe) in the phenylalanyl-tRNA synthetase-catalyzed reaction. It also has been reported that a variety of toxic effects of OA can be prevented by Phe. This study was designed to determine whether Phe could prevent or diminish the teratogenic effects of OA in rats. Pregnant Sprague-Dawley rats were injected with a single individual dose of OA (1.75 mg/kg) alone or in combination with a single dose of Phe (20 mg/kg) or in combination with either a single or daily dose of Phe (25 mg/kg). OA dissolved in 5% sodium bicarbonate and Phe dissolved in normal saline were administered subcutaneously on gestation day 7 to rats. The incidences of OA-induced fetal malformations (gross and skeletal) were significantly diminished in the presence of added Phe. These results indicate that coadministered Phe provides partial prenatal protection from the teratogenic effects of OA.     

Ochratoxin A-induced DNA damage in human fibroblast: protective effect of cyanidin 3-O-beta-d-glucoside

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus ochraceus and other moulds, has recently received growing attention because of its carcinogenic, teratogenic and nephrotoxic properties in both humans and farm animals. Nevertheless, with regard to the mechanism of toxicity, the data in the literature are inconclusive. The aim of our work was to verify in human fibroblasts treated with different OTA dosages the involvement of oxidative pathway in the damage mechanism of this mycotoxin and the possible protective effect exerted by cyanidin 3-O-beta-D-glucoside (C3G), an anthocyanin present in pigmented oranges, red wines, fruits and vegetables. The addition of OTA at 25 and 50 microM concentrations for 48 h determined only a slight but significant (P<.05) increase in radical oxygen species, whereas a substantial increase in their production was observed at longer exposure, in particular, when the fibroblasts were treated with 50 microM OTA for 72 h. Under the same experimental conditions, our data showed a significant (P<.05) increase in the rupture of cellular membrane and high damage to genomic DNA, evaluated by single-cell gel electrophoresis (comet assay), thus confirming the involvement of oxidative stress in the OTA genotoxicity in agreement with other studies. Diversely, mitochondrial functionality does not appear influenced by OTA treatment. C3G (0.125, 0.250 mM) added to the cells treated with 50 microM OTA significantly reduced free radical species production and prevented genomic DNA damage.     

Experimental hyperphenylalaninemia in the pregnant guinea pig: possible phenylalanine teratogenesis and p-chlorophenylalanine embryotoxicity

Maternal hyperphenylalaninemia (HPH) due to deficient phenylalanine (Phe) hydroxylation is a recognized human teratogen associated with an increased incidence of intrauterine growth retardation, microcephaly, congenital heart disease, and mental retardation. There are no previous reports of experimental HPH during organogenesis. Sustained HPH was produced in pregnant guinea pigs by adding 3.5% Phe and 1.0% parachlorophenylalanine (pCPA), an inhibitor of Phe hydroxylase, to standard guinea pig chow. Animals consumed the supplemented test diets from gestation day 1 until killed on gestation day 17. Examination of day 17 embryos revealed that embryonic mortality was associated only with maternal pCPA administration and was independent of the degree of maternal HPH. Embryonic malformation was associated with maternal HPH as well as maternal pCPA administration. Both maternal HPH and pCPA administration were associated with embryonic growth retardation. There was no association between maternal food intake or plasma tyrosine levels and embryonic abnormality or mortality. Both Phe and tyrosine were found to be concentrated in gestation day 17 yolk sac fluid when compared to maternal plasma Phe and tyrosine. The association of embryonic malformation and maternal HPH is consistent with human data. The embryotoxicity of pCPA requires further study and highlights the necessity of appropriate controls in models of experimental HPH.     

Acetazolamide teratogenesis in Wistar rats: potentiation and antagonism by adrenergic agents

Acetazolamide, a carbonic anhydrase inhibitor, induced right forelimb ectrodactyly in rat fetuses when the mothers were treated on late day 10 and early day 11 of gestation. Coadministration of the selective alpha-1-adrenergic agonist phenylephrine significantly increased the incidence of acetazolamide-induced right forelimb ectrodactyly while failing to induce the lesion when administered alone. Pretreatment with the alpha-adrenergic antagonists phenoxybenzamine and prazosin prevented the phenylephrine-induced increase in right forelimb ectrodactyly. In addition, treatment with either phenoxybenzamine or prazosin in the absence of stimulation with phenylephrine significantly decreased the incidence of acetazolamide-induced ectrodactyly. The results suggest an adrenergic component in acetazolamide teratogenesis. Alterations in uterine blood flow are discussed as a plausible mechanism for the modification of the incidence of ectrodactyly by these adrenergic agents.     

Mycotoxin Ochratoxin A-induced cell death and changes in oxidative metabolism of Arabidopsis thaliana

We evaluated the phytotoxicity of mycotoxin ochratoxin A (OTA) from Aspergillus and Penicillium strains on Arabidopsis thaliana. The results demonstrate that the growth of Arabidopsis thaliana on media containing OTA was inhibited significantly. Moreover, OTA induced necrotic lesions in detached leaves, which are reminiscent of hypersensitive response lesions that are activated during plant–pathogen interactions and other abiotic stress factors. From our study, we can see that OTA exposure stimulated a biphasic oxidative burst in the leaves, resulting in the generation of hydrogen peroxide (H₂O₂) and superoxide anion radicals (O₂^·−) and in the concomitant down-regulation of antioxidant enzyme defense responses and up-regulation of lipid peroxidation. These results suggested that OTA damage might result from reactive oxygen species pathways. Our experiments provide a useful model plant system for research on OTA-induced plant cell death.     

Maternal metabolism and teratogenesis in zinc-deficient rats

Embryos removed at 11.5 days gestation from pregnant rats allowed a zinc-deficient diet from the time of mating showed a high frequency of malformations of all organ systems. There were, however, large differences between litters of individual dams. Comparison of the daily food intake of zinc-deficient dams with the appearance of the embryos suggested that fluctuations in the maternal serum zinc levels induced by feeding or fasting influenced the availability of zinc to the embryos. By cyclically feeding zinc-deficient dams to a predetermined schedule, low maternal serum zinc levels were induced at selected stages of development. This was accompanied by specific malformations of the organs developing at that time.     

Radiologic diagnosis of diethylstilbestrol induced teratogenesis in rats

Radiographs were used for the assessment of diethylstilbestrol-induced teratogenesis of the urogenital system of rats. Lesions such as female hypospadias were demonstrable with a 4:1 magnification of radiographs that were not observed using subgross dissection and histologic step sections.     

Hydrocortisone attenuates cyclosporin A-induced nephrotoxicity in rats

Cyclosporin A (CsA) is the prototype of immunosuppressant drugs that have revolutionized the management of all transplantation and autoimmune diseases. Side effects of CsA mainly affecting the kidney but also observed in liver and heart, limit the therapeutic use of this drug after organ transplantation. The renal toxicity of CsA is attributed to reduced renal blood flow which leads to hypoxia-reoxygenation injury accompanied by excessive generation of oxygen-derived free radicals. In several therapeutic protocols, CsA is used in association with corticosteroids to obtain better therapeutic results. Recently, our studies showed that hydrocortisone (HY) has a protective effect on CsA-induced cardiotoxicity. In fact our previous results demonstrated that in rat cardiomyocytes, CsA toxicity is due to a calcium overload, which in turn induce lipid peroxidation and determines oxidative stress-induced cell injury. Treatment with HY effectively inhibits CsA-induced toxicity, decreasing lipid peroxidation as well as calcium intracellular concentration. In this study we evaluated in vivo the effects of CsA, used alone or in association with HY, on some parameters of renal dysfunction (blood urea nitrogen; BUN, creatinine, and cholesterol), malondialdheyde (MDA) levels, antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and apoptosis. CsA administration for 24 days resulted in a marked renal oxidative stress, which significantly deranged the renal functions. Treatment with CsA in association with HY significantly improved the renal dysfunction and renal oxidative status. This study clearly suggests the role of oxidative stress in the pathogenesis of CsA-induced nephrotoxicity.     

Non-confirmation of thalidomide induced teratogenesis in rats and mice

It has been reported that thalidomide, dissolved in a 1:3 mixture of Tween 20 and physiological saline and administered intraperitoneally to pregnant mice and rats induces a variety of malformations, including limb deformaties, characteristic of the primate syndrome. The studies reported herein attempted to confirm these findings without success although the rodent strains used were not the same. A low level of non-specific malformations was observed in the fetuses of both species at dose levels reported to cause a 47 percent and 92 percent rate of malformation in mice and rats respectively. One possible source of difference was Tween 20 which was toxic to the point of lethality in these studies at dose levels reported to be non-toxic in the earlier studies.     

Preventive effect of piperonyl butoxide on cyclophosphamide-induced teratogenesis in rats

BACKGROUND: Cyclophosphamide induces fetal defects through metabolic activation by cytochrome P-450 monooxygenases (CYP). The effects of piperonyl butoxide (PBO), a CYP inhibitor, on the fetal development and external, visceral, and skeletal abnormalities induced by cyclophosphamide were investigated in rats. METHODS: Pregnant rats were daily administered PBO (400 mg/kg) by gavage for 7 days (the 6th to 12th day of gestation), and intraperitoneally administered with cyclophosphamide (12 mg/kg) 4 h after the final treatment. On the 20th day of gestation, maternal and fetal abnormalities were determined by Cesarean section. RESULTS: Cyclophosphamide reduced fetal body weights by 30–40% without increasing resorption or death. In addition, it induced malformations in live fetuses: 100, 98, and 98.2% of the external (head and limb defects), visceral (cerebroventricular dilatation, cleft palate, and renal pelvic/ureteric dilatation), and skeletal (acrania, vertebral/costal malformations, and delayed ossification) abnormalities, respectively. The pre-treatment of PBO greatly decreased mRNA expression and activity of hepatic CYP2B, which metabolizes cyclophosphamide into teratogenic acrolein and cytotoxic phosphoramide mustard. Moreover, PBO remarkably attenuated cyclophosphamide-induced body weight loss and abnormalities of fetuses; score 3.57 versus 1.87 for exencephaly, 75.5% versus 42.5% for limb defects, 65.3% versus 22% for cerebroventricular dilatation, 59.2% versus 5.1% for cleft palate, score 1.28 versus 0.93 for renal pelvic/ureteric dilatation, 71.9–82.5% versus 23–45.9% for vertebral/costal malformations, and 84.2% versus 57.4% for delayed ossification in cyclophosphamide alone and PBO co-administration groups. CONCLUSIONS: These results suggest that repeated treatment with PBO may improve cyclophosphamide-induced body weight loss and malformations of fetuses by down-regulating CYP2B. Birth Defects Res (Part B) 86:402–408, 2009. © 2009 Wiley-Liss, Inc.     

Ochratoxin A-induced renal cortex fibrosis and epithelial-to-mesenchymal transition: molecular mechanisms of ochratoxin A-injury and potential effects of red wine

We characterized the effect of chronic ochratoxin A (OTA) on rat kidney cortex, analyzing collagen content and collagen turnover and the major markers of epithelial-to-mesenchymal transition (EMT), such as alpha-smooth muscle actin (alphaSMA), cadherins, and MMP-9. Because OTA nephrotoxicity is mediated by free radicals, we also investigated whether antioxidants in red wine provided protection for the kidney and attenuated OTA-induced EMT. Collagen content, determined by computerized analysis of Sirius red-stained kidney sections, increased in OTA, OTA-wine, and OTA-EtOH treated rats. In kidney cortex homogenates, COL-I and COL-III mRNA levels tended to rise in OTA treated rats, but were similar to CT after OTA-wine and OTA-EtOH administration. TIMP-1 gene expression was up-regulated in OTA, OTA-wine, and OTA-EtOH treated rats. LH2b mRNA/COL-I mRNA was significantly up-regulated in OTA-wine and OTA-EtOH treated rats, compared with CT and OTA alone. TGF-beta1 signaling tended to dominate after OTA, OTA-wine, and OTA-EtOH. MMP-1 protein levels were not affected. OTA induced proMMP-9 and alphaSMA overexpression, decreases of E-cadherin and N-cadherin, and DSC-2 up-regulation. OTA-wine caused a further, unexpected decrease of E- and N-cadherins and further up-regulation of OTA-induced DSC-2, while strongly reducing the OTA-induced increases of alphaSMA and proMMP-9. Posttranslational collagen modifications, such as decreased collagen degradation through MMP inhibition and increased collagen cross-links, seem to be key mechanisms leading to OTA-induced kidney cortex fibrosis. This mechanism was not affected by red wine in these conditions. Red wine seems to have some protective role against OTA-induced EMT, although without completely blocking the process and determining a condition in which abundant cells display an intermediate translational phenotype, but there are no alphaSMA or epithelial markers.     

Lycopene protects against cyclosporine A-induced testicular toxicity in rats

Cyclosporine A (CsA)-induced direct failures in hypothalamic-pituitary-gonadal axis and Sertoli cell phagocytic function have been considered for testicular toxicity so far. It has clearly been reported that oxidative stress leads to damage in sperm functions and structure of the testis. Therefore, this study was conducted to demonstrate whether CsA causes testicular and spermatozoal toxicity associated with the oxidative stress, and to investigate the possible protective effect of lycopene against CsA-induced damages in all reproductive organs and sperm characteristics in male rats. While the daily administration of CsA at the dose 15 mg/kg for 21 days significantly decreased the seminal vesicles weight, epididymal sperm concentration, motility, testicular tissue glutathione (GSH), glutathione peroxidase (GSH-Px) and catalase (CAT), diameter of seminiferous tubules and germinal cell thickness, it increased malondialdehyde (MDA) level and abnormal sperm rates along with degeneration, necrosis, desquamative germ cells in testicular tissue. However, the CsA along with simultaneous administration of lycopene at the dose of 10mg/kg markedly ameliorated the CsA-induced all the negative changes observed in the testicular tissue, sperm parameters and oxidant/antioxidant balance. In conclusion, CsA-induced oxidative stress leads to the structural and functional damages in the testicular tissue and sperm quality of rats and, lycopene has a potential protective effect on these damages.     

Endogenous corticosteroids modulate Clostridium difficile toxin A-induced enteritis in rats

We examined the role of glucocorticoids in acute inflammatory diarrhea mediated by Clostridium difficile toxin A. Toxin A (5 microg) or buffer was injected in rat ileal loops, and intestinal responses were measured after 30 min to 4 h. Ileal toxin A administration increased plasma glucocorticoids after 1 h, at which time the toxin-stimulated secretion was not significant. Administration of the glucocorticoid analog dexamethasone inhibited toxin A-induced intestinal secretion and inflammation and downregulated toxin A-mediated increase of macrophage inflammatory protein-2. Adrenalectomy followed by replacement with glucocorticoids at various doses suggested that intestinal responses to toxin A were related to circulating levels of glucocorticoids. Administration of the glucocorticoid receptor antagonist RU-486 enhanced toxin A-mediated intestinal secretion and inflammation. We conclude that C. difficile toxin A causes increased secretion of endogenous glucocorticoids, which diminish the intestinal secretory and inflammatory effects of toxin A.     

An in vivo function of glucagon-lnduced phenylalanine: Pyruvate transaminase in p-chlorophenylalanine-treated rats

The effect of glucagon-induced phenylalanine:pyruvate transaminase on the urinary excretion of the unconjugated metabolites of phenylalanine transamination was studied in rats. Chronic injection of glucagon induced an 18-fold increase in hepatic phenylalanine:pyruvate transaminase activity. Treatment with p-chlorophenylalanine (PCPA) blocked phenylalanine hydroxylase and caused an elevation of plasma phenylalanine following administration of an intraperitoneal loading dose of this amino acid. Gasliquid Chromatographic analysis demonstrated the presence of phenylpyruvate, phenyllactate, and O-hydroxyphenylacetate in the urine of PCPA- and PCPA-glucagontreated rats, but not control or glucagon-treated animals. Combined PCPA-glucagon treatment caused twofold increase in phenylpyruvate and phenyllactate concentrations and a fivefold increase in O-hydroxyphenylacetate concentration, when compared to urinary metabolite levels from rats receiving only PCPA treatment. A decrease in plasma phenylalanine was found together with the elevated urinary levels of the phenylalanine transamination metabolites. The results provide the first evidence that the unconjugated transamination metabolite concentrations increase when concurrent treatment with glucagon causes high-level induction of hepatic phenylalanine:pyruvate transaminase.     

Lectin teratogenesis: defects produced by concanavalin A in fetal rabbits.

Concanavalin A (con A) is teratogenic to rabbit embryos during gestational days 12--15. Intracoelomic injections of 40 microliter con A solution (4 microgram/microliter) were performed on rabbit embryos during gestational days 10--15. Control embryos received either 40 microliter of saline, sham injection or no treatment. Con A caused increased fetal resorptions on days 10 and 11, but malformation levels did not differ from controls. On days 12--15, con A produced craniofacial, trunk and limb anomalies. The highest percentage of malformation occurred on day 14. The defects were classified into four groups: (1) malformations of limbs including paw and digital dysplasias as well as fusions of the limbs to the head or body wall; (2) "closure" defects such as umbilical hernia, encephalocoele, exencephaly or ectopia cordis; (3) "contracture" defects such as club paws, extended knees, or clenched digits, which exhibited normal osseous and cartilaginous skeletons; and (4) miscellaneous, non-specific anomalies including fused or dysplastic sternebrae or ribs. Histologic analysis of selected 12-day embryos 4 to 18 hours post-injection was performed to ascertain potential sites of teratogenic action. At 12 hours ectodermal necrosis was observed in the limb buds adjacent to the apical ectodermal ridge. By 18 hours, the ectoderm had eroded, exposing the basal lamina to the amniotic fluid. Focal areas of mesenchymal necrosis were observed in association with the ectodermal erosion. The potential roles of amniocentesis and limb bud repair in the genesis of the malformations are discussed.