The milk protein promoter is a useful tool for developing a rat with tolerance to a human protein

Biopharmaceuticals intended for humans are immunogenic in animals. Antibodies associated with their administration make it difficult to perform repeated-dose pharmacology and toxicology studies in animals. Despite suggestions to solve this problem with transgenic animal technology, an effective strategy has not yet been reported. The objective of the present study was to provide an efficient strategy to develop rats tolerant to biopharmaceuticals such as human gene-based proteins. The present study used transgenic rat lines (lines 311-6, 308-5, and 305-1) carrying a fusion gene designed to express the human growth hormone (hGH) gene under the control of the bovine S1 casein gene promoter. Three lactating females with the transgene, produced approximately 4mg/ml, 300g/ml, and 10ng/ml in their milk. Male 8-week-old rats from these three lines were immunized with hGH three times (week 0, 1, and 3 ) and the production of antibodies against hGH in their sera were examined at week 4. While the hGH serum antibody titers increased over 1000-fold in wild-type control rats, there was no detectable antibody against hGH in the sera of these three transgenic lines. Human growth hormone in their sera was undetectable (lines 308-5 and 305-1) or much lower than the endogenous biologic level of rat growth hormone (line 311-6). Importantly, lines 308-5 and 305-1 developed tolerance to hGH without detectable hGH in their sera and these lines will be very useful for the repeated dose pharmacology and toxicology studies. These results suggest that a milk protein promoter can be a useful tool to develop transgenic rats that are tolerant to biopharmaceuticals intended for humans.     

Extrapolation of Rodent Studies on Amniotic Fluid Contaminatnts to Human Populations

If endocrine active chemicals (EACs) adversely affect human development, then there must be evidence of effects in animal models at properly scaled levels of exposure during pertinent sensitive periods as derived from quantified exposures of the human fetus. Our recent studies attempt to address both effects and exposures. First Study: Dams were gavaged from Gestation Day (GD) 14 through weaning on Post-Natal Day (PND) 21 with either corn oil alone (unexposed controls) or Low DES (0.5?mg/kg BW); High DES (5.0?mg/kg BW); GEN (15?mg/kg BW); GEN + DES (GEN at 15?mg/kg BW and DES at 0.5?mg/kg BW). No treatments affected duration of gestation, litter size or birth anogenital distance / birth body weights ((bAGD/bBW) or ratios of bAGD/cube root of bBW of pups of either sex. The ratio of weaning AGD to weaning body weight (wAGD/wBW) differed significantly between the control group and each of the estrogenic treatments in both sexes with larger wAGD/wBW values associated with each of the estrogenic treatments. Males exposed to High DES and GEN alone exhibited earlier onset of puberty. Only females in the low DES group showed an earlier onset of puberty. At 50 to 70 days of age, the ratios of male reproductive organ weights/body weight were unaffected by estrogen treatment in all groups except high DES which increased testicle weight and decreased epididymis, seminal vesicle, and prostate weights. Initial vaginal cycle lengths were affected only in the high DES group. Thus low doses of DES and GEN at levels comparable to the upper range of human exposure affect some but not all markers of sexual development. Second Study: Amniotic fluid samples obtained at routine amniocentesis between 15 and 23 weeks of gestation were assayed by gas chromatographic/mass spectrometric (GC/MS) analysis. The first group of amniotic fluid samples (n = 53) from 51 women were analysed for several xenobiotic EACs. Alpha-hexachlorocyclohexane, with a mean (± SD) concentration of 0.15 ± 0.06 (ng/ml), and p,p′-DDE, with a mean (± SD) concentration of 0.21 ± 0.18?ng/ ml, were detected in several specimens. Overall one in three amniotic fluid samples tested positive for at least one xenobiotic EAC. Another group of amniotic fluid samples (n = 62) from 56 women were analysed for phytoestrogenic EACs. The mean (± SD) concentration of daidzein and genistein in amniotic fluid were 1.14 ± 1.04 and 1.37 ± 1.00?ng/ml with maximum levels of 5.52 and 4.86?ng/ml, respectively. Overall, 26 and 34 of the samples had quantifiable levels of daidzein and genistein, respectively. Conclusions: One in three human fetuses were exposed to xenobiotic EACs and two of three human fetuses were exposed to phytoestrogenic EACs in utero. Our demonstrations that EACs have developmental effects in an animal model at levels of exposure that mimic those found in humans in North America during sensitive time-frames sustains concerns about potential adverse health effects of developmental exposures to EACs for the human fetus/neonate.     

Liver Lipid Composition and Antioxidant Enzyme Activities of Spontaneously Hypertensive Rats After Ingestion of Dietary Fats (Fish, Olive and High-Oleic Sunflower Oils)

Hypertension is associated with greater than normal lipoperoxidation and an imbalance in antioxidant status, suggesting that oxidative stress is important in the pathogenesis of this disease. Although many studies have examined the effect of antioxidants in the diet on hypertensión and other disorders, less attention has been given to the evaluation of the role of specific dietary lipids in modulating endogenous antioxidant enzyme status. Previously, we have described that liver antioxidant enzyme activities may be modulated by consumption of different oils in normotensive rats. The purpose of the present study was to examine the effects of feeding different lipidic diets (olive oil, OO, high-oleic-acid sunflower oil, HOSO, and fish oil, FO) on liver antioxidant enzyme activities of spontaneously hypertensive rats (SHR). Plasma and liver lipid composition was also studied. Total triacylglycerol concentration increases in plasma and liver of animals fed on the HOSO and OO diets and decreases in those fed on the FO diet, relative to rats fed the control diet. The animals fed on the oil-enriched diet show similar hepatic cholesterol and phospholipid contents, which are higher than the control group. Consumption of the FO diet results in a decrease in the total cholesterol and phospholipid concentration in plasma, compared with the high-oleic-acid diets. In liver, the FO group show higher levels of polyunsaturated fatty acids (PUFA) of the (n-3) series, in relation to the animals fed on the diets enriched in oleic acid. Livers of FO-fed rats, compared with those of OO- and HOSO-fed rats showed: (i) significantly higher activities of catalase, glutathione peroxidase and Cu/Zn superoxide dismutase; (ii) no differences in the NADPH-cytochrome c reductase activity. The HOSO diet had a similar effect on liver antioxidant enzyme activities as the OO diet. In conclusion, it appears that changes in the liver fatty acid composition due mainly to n-3 lipids may enhance the efficiency of the antioxidant defence system and may yield a benefit in the hypertension status. The two monounsaturated fatty acids oils studied (OO and HOSO), with the same high content of oleic acid, but different content of natural antioxidants, had similar effects on the antioxidant enzyme activities studied.     

Effects of oral aluminum on essential trace elements metabolism during pregnancy

The present study was conducted to assess in rats the effects of oral aluminum (Al) exposure on calcium (Ca), magnesium (Mg), manganese (Mn), copper (Cu), zinc (Zn), and iron (Fe) accumulation and urinary excretion. Three groups of plug-positive Sprague-Dawley (SD) rats were given by gavage 0, 200, and 400 mg/kg/d of Al(OH)₃ on gestational days 1–20. Three groups of nonpregnant female SD rats of the same age received Al(OH)₃ by gavage at the same doses for 20 consecutive days. At the end of the treatment period, 24-h urine samples were collected for analysis of Al and essential elements. Subsequently, all animals were sacrificed and samples of liver, bone, spleen, kidneys, and brain were removed for metal analyses. With some exceptions, the urinary amounts of Al, Mn, and Cu excreted by pregnant animals as well as the urinary levels of Al excreted by nonpregnant rats were higher in the Al-treated groups than in the respective control groups. Although higher Al levels were found in the liver of pregnant rats, the concentrations of Al in the brain of these animals were lower than those found in the same tissues of nonpregnant rats. With regard to the essential elements, tissue accumulation was most affected in pregnant than in nonpregnant animals. In pregnant rats, the hepatic and renal concentrations of Ca, Mg, Mn, Cu, Zn, and Fe, as well as the levels of Ca in bone, and the concentrations of Cu in brain were significantly higher in the Al-exposed groups than in the control group. According to the current results, oral Al exposure during pregnancy can produce significant changes in the tissue distribution of a number of essential elements.     

Investigation of the mechanisms affecting Cu and Fe bioavailability from legumes

Chemical composition and content in polyphenols, phytic acid, and dietary fiber of whole cooked common bean (Phaseolus vulgaris L.) and faba bean (Vicia faba L.) and of soluble and insoluble fractions separated from them were determined. Simultaneous determination of Cu, Fe, and protein bioavailability in the small intestine of rat was carried out in single-dose, short-term (1 h) experiments. After cooking, about 80% of seed components (on a weight basis) of either legume was recovered in the precipitate (insoluble fraction) after extraction with water. Protein, lipid, starch, dietary fiber, and polyphenols underwent the most severe insolubilization, together with more than 70% of total Cu and Fe. Cu, Fe, and protein bioavailability showed a similar trend (i.e., the lower the protein, the lower the Cu and Fe availability). Availability of proteins, Cu, and Fe in the insoluble fractions were the lowest, but Cu bioavailability was higher than that of Fe in all fractions. The results provide evidence that the heat-induced insolubilization process adversely affects not only protein but also Cu and Fe bioavailability from legumes and that polyphenols are likely to be a major inhibitor on absorption.     

电刺激下丘脑外侧区对大鼠胃缺血-再灌注损伤的影响

采用夹闭大鼠腹腔动脉30min,松开动脉夹血流复灌60min的胃缺血－再灌注损伤(gastric ischemia reperfusion injury,GI-RI)模型,用电和化学刺激,电损毁的方法观察了下丘脑外侧区（lateral hypothalamic area,LHA)对GI－RI的影响,并对其机制进行了初步分析,结果表明：（1）以0．2,0．4,0．6mA的电流强度刺激LHA,GI－RI均显著加重,且有强度－效应依赖关系,LHA内注射L－谷氨酸（L－Glu)后,对LI－RI的效应与电刺激相似,电损毁双侧LHA,GI－RI面积较电刺激组明显减小;（2）损毁双侧背侧迷走复合体（dorsal vagal complex,DVC)或切损毁是LHA,GI－RI面积较电刺激组明显减小;（2）损 侧背侧迷走复合体（dorsal vagal complex,DVC)或切断膈下迷走神经均能取消电刺激LHA加重GI－RI的作用。（3）电刺激LHA使缺血－再灌注（ischemia-reperfusion,I-R)的胃粘膜丙二醛（MDA）含量升高,超氧化物歧化酶（SOD）活性降低;（4）电刺激LHA使I－R的胃液量和总酸排出量增多,而酸度,胃蛋白酶活性和胃壁结合粘液量等无明显改变,结果提示;LHA是对GI－RI具有加重作用的中枢部位,其作用是通过DVC及迷走神经下传的,电刺激LHA加重GI－RI的作用与胃粘膜MDA含量增加,SOD活性降低,胃液量和总酸排出量增加等因素有关,而似与酸度,胃蛋白酶活性,胃壁结合粘液量等因素无关。     

Involvement of Accessory Neurosecretory Nuclei of Hypothalamus in the Formation of Hypothalamohypohysial System during Prenatal and Postnatal Development in Rats

We studied the development of direct axonal connections of the accessory neurosecretory hypothalamic nuclei with the posterior pituitary lobe on the fixed rat brain from day 15 of embryogenesis until day 10 of postnatal development using the retrograde diffusion method of the lipophilic fluorescent carbocyanine dye 1,1"-dioctadecyl-3,3,3",3"-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The marker was applied onto the posterior pituitary lobe and, after incubation in a fixative, fluorescing bodies of nerve cells were visualized in the hypothalamus. Neuronal axons of the retrochiasmatic nucleus were the first of the accessory nuclei to ingrow in the posterior pituitary lobe (on days 16–17 of embryogenesis). Neurons of the circular and dorsolateral nuclei and the nuclei of the median bundle of the forebrain sent their axons to the posterior pituitary lobe starting from the first days of postnatal development. No direct connections of the anterior commissure and perifornical accessory nuclei with the posterior pituitary lobe were found in perinatal development. These facts are discussed in the light of concepts about the different functional role of accessory peptidergic hypothalamic nuclei in rats.     

Involvement of 5-HT(2A/2B/2C) receptors on memory formation: simple agonism,antagonism, or inverse agonism?

1. The 5-HT₂ receptors subdivision into the 5-HT_2A/2B/2C subtypes along with the advent of the selective antagonists has allowed a more detailed investigation on the role and therapeutic significance of these subtypes in cognitive functions. The present study further analyzed the 5-HT₂ receptors role on memory consolidation.2. The SB-200646 (a selective 5-HT_2B/2C receptor antagonist) and LY215840 (a nonselective 5-HT_2/7 receptor antagonist) posttraining administration had no effect on an autoshaped memory consolidation. However, both drugs significantly and differentially antagonized the memory impairments induced by 1-(3-chlorophenyl)piperazine (mCPP), 1-naphtyl-piperazine (1-NP), mesulergine, or N-(3-trifluoromethylphenyl) piperazine (TFMPP).3. In contrast, SB-200646 failed to modify the facilitatory procognitive effect produced by (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) or ketanserin, which were sensitive to MDL100907 (a selective 5-HT_2A receptor antagonist) and to a LY215840 high dose.4. Finally, SB-200646 reversed the learning deficit induced by dizocilpine, but not that by scopolamine; while SB-200646 and MDL100907 coadministration reversed memory deficits induced by both drugs.5. It is suggested that 5-HT_2B/2C receptors might be involved on memory formation probably mediating a suppressive or constraining action. Whether the drug-induced memory impairments in this study are explained by simple agonism, antagonism, or inverse agonism at 5-HT₂ receptors remains unclear at this time.6. Notably, the 5-HT₂ receptor subtypes blockade may provide some benefit to reverse poor memory consolidation conditions associated with decreased cholinergic, glutamatergic, and/or serotonergic neurotransmission.     

Infant animal model of pulmonary mycotoxicosis induced by Stachybotrys chartarum

In recent years cases of often fatal pulmonary hemorrhage in infants have been associated with water damaged homes and the toxigenic fungusStachybotrys chartarum. The fungal spores contain mycotoxins which could be injurious to the rapidly developing lung. In order to understand the developmental pathophysiology of this disease we developed an infant rat model of stachybotrytoxicosis describing the effects of fungal spores on survival, growth, histopathology of the lung and respiration. Conidia ofS. chartarum were instilled intratracheally (1.0–8.0 × 10⁵/gm wt.) in 4-dold Sprague-Dawley rat pups. Two control groups received either sterile PBS or a suspension of spores extensively extracted with ethanol to remove toxins. Lethal dose response was determined (LD₅₀ = 2.7 × 10⁵ spores/gm wt.). All dead pups had extensively hemorrhagic lungs. Growth of surviving animals was impaired in a dose-dependent manner. Changes of pulmonary function parameters in rats treated with 1.1 × 10⁵ spores/g were consistent with an increased respiratory resistance. Histology of lungs revealed fresh hemorrhage, sparse hemosiderin-laden macrophages, and evidence of inflammation including thickened alveolar septa infiltrated by lymphocytes and mononuclear cells and intra-alveolar macrophages. Significant increases (p = 0.001) in numbers of macrophages (2-fold), lymphocytes (5-fold) and neutrophils (7-fold) were found in BAL fluid. Hemoglobin was elevated 2-fold (p = 0.004). Proinflammatory mediator IL-1β increased more than 6-fold and TNF-α30-fold (p = 0.001). Extracted spores had a minimal effect on all examined parameters in BAL fluid indicating that mycotoxins are primarily responsible for the hemorrhagic and inflammatory response. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of Nigella sativa L. and Urtica dioica L. on selected mineral status and hematological values in CCl4-treated rats

This study was designed to investigate the effects of Nigella sativa L. (NS), known as black seed, or/and Urtica dioica L. (UD), known as stinging nettle root, treatments on serum Na, K, Cl, and Ca levels and some hematological values of CCl₄-treated rats. Sixty healthy male Sprague-Dawley rats, weighing 250–300 g, were randomly allotted into 1 of 4 experimental groups: A (CCl₄-only treated), B (CCl₄+UD treated), C (CCl₄+NS treated), and D (CCl₄+UD+NS treated), each containing 15 animals. All groups received CCl₄ (0.8 mL/kg of body weight, subcutaneously, twice a week for 90 d starting d 1). In addition, B, C, and D groups also received the daily ip injection of 0.2 mL/kg NS and/or 2 mL/kg UD oils for 45 d starting d 46. Group A, on the other hand, received only 2 mL/kg normal saline solution for 45 d starting d 46. Blood samples for the biochemical analysis were taken by cardiac puncture from five randomly chosen rats in each treatment group at the beginning, d 45, and d 90 of the experiment. The CCl₄ treatment for 45 d significantly (p<0.05) increased the serum K and Ca and decreased (p<0.05) the red blood cell count (RBC), white blood cell count (WBC), packed cell volume (PCV), and Hb levels without changing (p>0.05) the serum Na and Cl levels. NS or UD treatments (alone or combination) for 45 d starting d 46 significantly (p<0.05) decreased the elevated serum K and Ca levels and also increased (p<0.05) the reduced RBC, WBC, PCV, and Hb levels. It is concluded that NS and/or UD treatments might ameliorate the CCl₄-induced disturbances of anemia, some minerals, and body’s defense mechanism in CCl₄-treated rats.