Germination,Viability and Clearance of Stachybotrys chartarum in the Lungs of Infant Rats

Observing that the conidia of Stachybotrys chartarum can germinate in the lung of infant rats, it became important to ascertain whether an infection can ensue. Viable conidia of S. chartarum were instilled into the lungs of 4 and 14 day-old rat pups. Germination was observed frequently in the lungs of 4 day-old but rarely in the 14 day-old pups. In the 4 day-old pups, pulmonary inflammation with hemorrhagic exudates was observed and resulted in about 15% mortality rate compared to 0% for the controls instilled with phosphate buffered saline. Acute neutrophilic inflammation and intense interstitial pneumonia with poorly formed granulomas observed three days following exposure were associated with fungal hyphae and conidia. The surviving experimental pups showed significantly slower weight gain for seven days. Dilution plating and quantitative PCR analysis were used to follow total fungal load in the rat pups lung homogenates. In the 4 day-old rat pups viable fungi decreased rapidly and were less than 1% by day seven. Similarly, fungal DNA decreased exponentially and was only 0.03% by fourteen days after exposure. However, 14 day-old rat pups showed neither the lethal effects of exposures to viable conidia of S. chartarum nor the slower weight gain, and the fungal load decreased even more rapidly. We conclude that S. chartarum conidia can initially germinate and form hyphae but even in the immature rat pups do not establish an effective infection, although a very limited persistence cannot be excluded.This revised version was published online in October 2005 with corrections to the Cover Date.     

Cerebrospinal fluid monoamine and metabolite concentrations and aggression in rats

In humans and other primates low cerebrospinal fluid (CSF) levels of the major serotonin (5-HT) metabolite 5-hydroxyindoleacetic acid (5-HIAA) have been correlated to high aggressiveness. This finding forms the basis of the 5-HT deficiency hypothesis of aggression. Surprisingly, this correlation has not been confirmed in rodents so far, while manipulation studies aimed to investigate the link between 5-HT and aggressive behaviour are mostly carried out in rodents. In this study the relation between aggression and CSF monoamine and metabolite concentrations was investigated in male Wildtype Groningen rats. In sharp contrast to the hypothesis and our expectation, a clear positive correlation was found between the individual level of trait-like aggressiveness and CSF concentrations of 5-HT, 5-HIAA, norepinephrine (NE), dopamine (DA), and 3,4-dihydroxyphenylacetic acid (DOPAC). Shortly after the acute display of aggressive behaviour (as a state-like phenomenon), decreased 5-HT levels and an increase in 5-HIAA/5-HT ratio and NE concentrations were found. Surprisingly, pharmacological challenges known to influence 5-HT transmission and aggressive behaviour did not affect CSF 5-HT and 5-HIAA concentrations, only the NE level was increased. Lesioning 5-HT terminals by 5,7-dihydroxytryptamine (5,7-DHT) administration caused a decrease in CSF 5-HT and 5-HIAA, but without affecting aggressive behaviour. The observed positive correlation between CSF 5-HIAA and trait aggressiveness makes it questionable whether a direct extrapolation of neurobiological mechanisms of aggression between species is justified. Interpretation of CSF metabolite levels in terms of activity of neural substrates requires a far more detailed knowledge of the dynamics and kinetics of a neurotransmitter after its release.     

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。     

慢性镉负荷雄性大鼠的睾丸及生殖内分泌功能活动

选择健康SD雄性成年大鼠60只,随机分成对照组（C组）、镉负荷中剂量组（M组）和镉负荷高剂量组（H组）,每天分别饲喂含镉0,5,10mg/kg的大鼠全价饲料,连续6周,研究了镉负荷对大鼠睾丸及生殖内分泌功能活动的影响。结果显示：在整个实验期内,M和H组大鼠睾丸组织中的镉含量极明显上升,锌含量销有下降,与对照组差异不显著;血浆镉、锌含量虽分别表现稍有升高和下降,但与对照组比较无明显差异;H组睾丸精子头计数和每日精子生成量在镉负荷第3周极显著下降,第6周时,M和H组均极明显低于对照组;在整个实验期内,H组大鼠ALP活明显低于C组;LDH－X活性在M和H组大鼠均极明显低于C组;M和H组血浆T水平下降,均低于或显著低于C组;3组间的FSH和LH水平无明显差异。结果提示：慢性镉负荷在睾丸组织中逐步蓄积可引起睾丸一些酶活性改变、精子生成减少及内分泌功能活动低下。     

Chlorogenic acid modifies plasma and liver concentrations of: cholesterol,triacylglycerol, and minerals in (fa/fa) Zucker rats

Chlorogenic acid, a phenolic compound found ubiquitously in plants, is an in vitro antioxidant and metal chelator. Some derivatives of chlorogenic acid are hypoglycemic agents and may affect lipid metabolism. Concentrations of cholesterol and triacylglycerols are of interest due to their association with diseases such as non-insulin-dependent-diabetes- mellitus and obese insulin resistance. As little is known about the effects of chlorogenic acid in vivo, studies using obese, hyperlipidemic, and insulin resistant (fa/fa) Zucker rats were conducted to test the effect of chlorogenic acid on fasting plasma glucose, plasma and liver triacylglycerols and cholesterol concentrations. Aditionally, the effects of chlorogenic acid on selected mineral concentrations in plasma, spleen, and liver were determined. Rats were implanted with jugular vein catheters. Chlorogenic acid was infused (5 mg/Kg body weight/day) for 3 weeks via intravenous infusion. Chlorogenic acid did not promote sustained hypoglycemia and significantly lowered the postprandial peak response to a glucose challenge when compared to the same group of rats before Chlorogenic acid treatment. In Chlorogenic acid-treated rats, fasting plasma cholesterol and triacylglycerols concentrations significantly decreased by 44% and 58% respectively, as did in liver triacylglycerols concentrations (24%). We did not find differences (p > 0.05) in adipose triacylglycerols concentration. Significant differences (p < 0.05) in the plasma, liver, and spleen concentration of selected minerals were found in chlorogenic acid-treated rats. In vivo, chlorogenic acid was found to improve glucose tolerance, decreased some plasma and liver lipids, and improve mineral pool distribution under the conditions of this study.