Beta-amyloid peptides(1-42) and (1-40) in the cerebrospinal fluid during pregnancy: a prospective observational study.

To evaluate changes in concentrations of selected biomarkers, neurotrophic factors, and growth factors in the cerebrospinal fluid during pregnancy. A prospective observational study was conducted in 32 pregnant women undergoing gynecological and obstetrical surgery under spinal anesthesia in a university hospital. Beta-amyloid(1-42) and beta-amyloid(1-40) peptides, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and vascular endothelial growth factor were analyzed in cerebrospinal fluid using an enzyme-linked immunosorbent assay. Eight women in second trimester pregnancy who underwent spinal anesthesia for gynecological or obstetrical surgery were compared with 24 matched women in third trimester pregnancies. CSF concentrations of beta-amyloid(1-42) were significantly higher in third trimester pregnancies (p = 0.025). During third trimester, the beta-amyloid ratio correlated with the vascular endothelial growth factor (rs = 0.657; p = 0.008). Higher concentrations of beta-amyloid(1-42) in cerebrospinal fluid of third trimester pregnancies and correlations between the beta-amyloid ratio and the vascular endothelial growth factor support the hypothesis that beta-amyloid peptides are involved in complex adaptive brain alterations during pregnancy.

Effects of high doses of vitamin D3 and 1,25-dihydroxyvitamin D3 in lactating rats on milk composition and calcium homeostasis of the suckling pups.

Changes in serum Ca and phosphorus and in kidney Ca were determined in lactating rats and their suckling pups after the mothers received high doses of vitamin D3 or 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. High dietary vitamin D3 intake (300 IU/g diet) or daily oral doses of vitamin D3 (1 microgram/g BW) to vitamin I)-replete (+D) lactating rats for 8 or 12 days caused significant increases in serum Ca in the mothers (1-2 mg/dl) and in their suckling pups (1.5 mg/dl). Daily oral doses of 1,25-(OH)2D3 (2 ng/g BW) to +D lactating rats caused a similar increase in serum Ca in the mothers, but did not affect the serum Ca of the pups. The administration of a high dose of 1,25-(OH)2D3 to vitamin D-deficient lactating rats or high doses of vitamin D3 to +D rats, caused no change in milk Ca, Mg, or phosphorus. Milk from +D rats given high doses of [3H]vitamin D3 (1 microgram/g BW) contained mostly [3H]vitamin D3 (85%) and a small amount of [3H]25-hydroxyvitamin D3 (6%). The results indicate that high doses of vitamin D3, but not 1,25-(OH)2D3, given to +D lactating rats can cause hypercalcemia in the suckling pups. The hypercalcemic effect on the pups observed after vitamin D3 treatment of the mother is probably a result of transport of toxic amounts of primarily vitamin D3 into the milk and is not due to altered mineral composition of the milk.

A short formal synthesis of squalamine from a microbial metabolite.

A short formal synthesis of squalamine is described, utilizing the biotransformation product 2, which is available in one step from commercially available 3-keto-23,24-bisnorchol-4-en-22-ol (1). Regioselective C-22 oxidation and C-24 sulfation of the corresponding alcohols in the presence of a free C-7 alcohol make for an efficient preparation of squalamine intermediate 11.

Repeated analysis of semen parameters in beagle dogs during a 2-year study with the HMG-CoA reductase inhibitor, atorvastatin.

Sperm analyses are often incorporated into reproductive toxicity studies in rats. Due to the relative ease of collecting multiple samples throughout a study, semen analysis in non-rodents such as dogs offers the opportunity to assess potential development of functional effects of compounds on male reproduction over time. In the present study, semen parameters were evaluated in beagle dogs during and at termination of a chronic toxicity study with the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, atorvastatin. Male dogs received 0, 10, 40, or 120 mg/kg orally in gelatin capsules for up to 104 weeks (n = 10/group). After 52 weeks of dosing, 3 dogs/group were euthanized, and 2/group were withdrawn from treatment for a 12-week reversal period and euthanized at Week 64. The remaining 5/group continued treatment until Week 104. Semen was collected from all animals for 3 consecutive weeks prior to termination of the 52-week animals (Weeks 50, 51, 52) for analysis of sperm parameters, using manual methods of evaluation. Semen was collected from the remaining animals at Weeks 64, 78, 91, and 104, and was analyzed. At necropsy, testes, epididymides, and prostates were weighed and evaluated histologically, and epididymal sperm counts were determined. Serum cholesterol was decreased 25--60% at all doses during the study. There were no drug-related differences in semen volume and color, total sperm count, and sperm concentration, morphology, progressiveness, and percent motility during treatment with atorvastatin. There were also no effects on reproductive organ weights or histopathology, and no effects on epididymal sperm count. Thus, incorporation of semen analyses into this study allowed the evaluation of potential male reproductive effects in dogs at multiple time points during the study. Statistical power calculations demonstrated acceptable statistical power (> 80%) for semen sperm count, concentration, morphology, and motility with group sizes of 8--10 animals, and for semen sperm count and concentration or epididymal sperm count with group sizes of 3--5 animals, using the methodology described in this paper.

Female reproductive and developmental toxicology: overview and current approaches.

In recent years, concern about possible female reproductive and developmental toxicity due to environmental contaminants, such as PCBs, has been growing. Because this area of toxicology had not been emphasized prior to this time, there are many gaps in current knowledge about female developmental and reproductive toxicology and only a limited number of validated tests to assay effects of toxicants on various parts of the reproductive and developmental cycle. This article reviews the current state of knowledge on this topic and also explores a variety of techniques for assessing female reproductive and developmental toxicity. These include an assay of the state of intercellular communication among the embryo, fetus and placenta; protocols for assessing toxicity in early pregnancy; and techniques for evaluating the role of glutathione in protecting the conceptus from xenobiotics.

Determination of diphenhydramine in rat milk and plasma and its effects on milk composition and mammary gland nucleic acids.

To study the excretion of diphenhydramine into rat milk, milk and plasma concentrations of diphenhydramine were determined in lactating rats after single or multiple oral doses. Four hours after a single dose of 40 or 100 mg/kg of diphenhydramine, milk concentrations of the drug averaged 0.30 and 2.2 micrograms/mL, respectively, in two experiments, and the milk:plasma ratios ranged from 4.4 to 7.5. Multiple doses did not significantly affect the plasma or milk concentrations or the milk:plasma ratios, which were similar to the theoretical milk:plasma ratio based on pH partitioning for this compound (i.e., 4.0). Although the concentration of diphenhydramine was higher in milk than in plasma, the estimated dose received by the pups (0.057 mg/kg/d) based on the milk concentrations was much lower than that given to the mother. Oral diphenhydramine treatment at doses which significantly reduced maternal food consumption had no effect on milk solid, lipid, protein, or lactose concentrations, nor on mammary gland RNA or DNA content, indicating that diphenhydramine did not adversely affect lactation.

Sperm motion parameters in vas deferens and cauda epididymal rat sperm.

Motion parameters were compared in rat sperm isolated from the distal vas deferens and the cauda epididymidis. Motion parameters were also compared in 20 microns and 50 microns deep muCellTM chambers using vas deferens sperm. Video recorded samples were analyzed manually for motility, and analyzed by a computer automated sperm analysis (CASA) system for motility, curvilinear velocity, linearity, mean and maximum amplitude of lateral head displacement (ALH), and beat/cross frequency using two versions of CellSoftTM (Series 3,000 and Series 4,000). Motility, linearity, and beat/cross frequency were not significantly different between sperm from vas deferens and cauda epididymidis, while velocity and ALH values were slightly greater in sperm from vas deferens than from cauda epididymidis. Sperm motility and linearity were not significantly different when analyzed in 20 microns and 50 microns mu CellTM chambers. Velocity and ALH values were slightly greater in 20 microns than in 50 microns chambers, and beat/cross frequency was slightly lower in 20 microns than in 50 microns chambers. Sperm motility was significantly greater when determined manually than when determined with the Series 3,000 but manually determined sperm motility was only slightly greater than motility determined with the Series 4,000. Several sperm motion parameters differed significantly between the Series 3,000 and Series 4,000 (curvilinear velocity, mean and maximum ALH, linearity, and beat/cross frequency) but the relative variability of the systems was comparable. Compared with manual determinations, the Series 3,000 overestimated and the Series 4,000 underestimated the number of cells analyzed for motility. Therefore, differences existed between manual and CellSoft (Series 3,000 and 4,000) analysis of sperm motility and number of cells, and between CellSoft systems in the analysis of sperm motion parameters. However, only minimal differences in sperm motion parameters were observed between the vas deferens and cauda epididymidis, and between 20 microns and 50 microns deep muCell chambers.

Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and dog: a consensus report. ILSI Risk Science Institute Expert Working Group on Sperm Evaluation.

Reproductive toxicity studies are increasingly including assessments of sperm parameters including motility, morphology, and counts. While these assessments can provide valuable information for the determination of potential reproductive toxicity, the methods for conducting the assessments have not been well developed in all laboratories and are continually evolving. The use of different methods in different laboratories makes comparison of data among laboratories difficult. To address the differences in methods, a working group was convened to discuss methods currently in use, share data, and try to reach consensus about optimal methods for assessing sperm parameters in rats, rabbits, and dogs. This article presents the consensus report, as well as future research needs, with the hope that optimized common methods will aid in the detection of reproductive effects and enhance interlaboratory comparisons.

Biotransformations of tocopherols by Streptomyces catenulae.

Streptomyces catenulae catalyzed the oxidation of alpha-tocopherol to alpha-tocopherolquinone. Nitrotocopherols isolated from S. catenulae grown in defined culture medium containing delta- and gamma-tocopherols are formed by a combination of enzymatic nitrate reduction to nitrite, and subsequent nonenzymatic acid-catalyzed nitration. The incorporation of 15N18O3-into nitrated tocopherols confirmed the origin of the nitrating species. Structures of chromatographically purified products obtained from S. catenulae transformations of tocopherols were deduced by spectral (mass spectrometry, 1H-, and 13C-nuclear magnetic resonance) analyses.

Renal calcification in suckling rats after high doses of calcitriol (1,25-dihydroxycholecalciferol).

Light and electron microscopic characteristics of renal calcification caused by high doses of calcitriol (1,25-dihydroxycholecalciferol) were examined in suckling rats. Four daily doses of calcitriol caused greater hypercalcemia and kidney calcification in 2-week-old than in 3-week-old rats. Calcium deposits, as localized with glyoxal bis(2-hydroxyanil), von Kossa's, or alizarin red S stains, were found primarily in the renal cortex. Glomeruli and tubules were calcified in younger pups, whereas only tubules were affected in older pups. Electron-dense deposits were found primarily in proximal tubules and consisted of needlelike crystals, large mitochondrial granules, and lamellar deposits along basal laminae. The location and appearance of the deposits were similar to those described in vitamin D-treated adult rats. The deposits probably resulted from the hypercalcemia and not from a direct toxic effect of calcitriol on the kidney.

Developmental toxicity study in rats treated with the anticonvulsant, ralitoline.

The developmental toxicity of the anticonvulsant compound, ralitoline, was investigated in Sprague-Dawley rats administered oral doses of 0, 15, 60, 120, 180, or 240 mg/kg on days 6 through 15 of gestation. An untreated control group and a vehicle control group pair-fed to the high dose group were included. Maternal and fetal parameters were evaluated on day 21 of gestation. Fetuses were examined for external, visceral, and skeletal malformations and variations. Maternal death occurred at 180 and 240 mg/kg. Dose-dependent decreases in body weight, food consumption, and water consumption were observed at 60 mg/kg and above. Body weight gain during treatment was similar in the pair-fed and 240 mg/kg groups. Dose-related CNS signs (hypoactivity, ataxia, prostration, and/or convulsions) were observed at 60 mg/kg and above. Decreased numbers of live fetuses and increased postimplantation loss were observed in a dose-related manner at 120, 180, and 240 mg/kg while no changes occurred in pair-fed controls. Fetal body weights and placental weights were decreased in pair-fed controls and in the 120, 180, and 240 mg/kg groups. The percent fetuses per litter, and the percent litters with external/visceral malformations, were significantly increased at 120, 180, and 240 mg/kg compared with vehicle and pair-fed controls. Dose-related increases in cardiovascular malformations, specifically of the aortic arch (interrupted, stenotic, extra vessel), were apparent at 120 mg/kg and above. The incidence of skeletal variations was increased at 120 mg/kg and above.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of vitamin D3 on duodenal calcium absorption in vivo during early development.

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and vitamin D deficiency on duodenal calcium absorption in suckling and weaned rats was determined by an in situ loop technique. In vitamin D-replete (+D) rats, the linear, or nonsaturable, component of calcium absorption was very efficient in 14-day-old pups and decreased with age until 35 days. The saturable component, which was undetectable in 14-day-old pups, became detectable by 18 days of age and increased until 26 days of age. Calcium absorption was not reduced in vitamin D-deficient (-D) 14-day-old pups as compared with +D pups. A high dose of 1,25(OH)2D3 increased the plasma calcium level of +D suckling rats but had no effect on calcium absorption even with milk present in the loop. Weaned -D rats had a reduced saturable component of absorption (P less than 0.01) compared with +D rats. A high dose of 1,25(OH)2D3 significantly increased calcium absorption and plasma calcium levels of -D rats. Our results indicate that during suckling calcium absorption occurs by a process that is insensitive to vitamin D. After weaning both saturable and nonsaturable processes appear to contribute to calcium absorption, and the saturable component can be influenced by vitamin D status or a high dose of 1,25(OH)2D3.

Calcium absorption during development: experimental studies of the rat small intestine.

During the period of natural weaning in the rat (beginning during the third week) the mechanisms of calcium absorption from the duodenum change from an efficient nonsaturable process, which is insensitive to vitamin D, to a combination of a less efficient nonsaturable process and a saturable vitamin D-dependent component. The stimulatory effect of vitamin D on active calcium transport requires the development of mucosal receptors for 1,25-(OH)2D3 and increasing circulating levels of 1,25-(OH)2D3. Concurrent increases in the concentration of the cytosolic calcium-binding protein (CaBP) support the suggestion that the CaBP mediates, in part, the effect of 1,25-(OH)2D3 on active calcium transport. The decline in the efficiency of nonsaturable calcium absorption during the third week coincides with, but is probably not due to, the loss of pinocytotic capacity of the small intestine. The time of appearance of the receptors for 1,25-(OH)2D3 and the decline in nonsaturable calcium absorption are under glucocorticoid control as are several other maturational events in the intestine at this time.

Mechanisms of ferulic acid conversions to vanillic acid and guaiacol by Rhodotorula rubra.

Resting cells of Rhodotorula rubra converted transferulic acid (1) to vanillic acid (2), then to guaiacol (3) and protocatechuic acid (4), under aerobic conditions. In an argon atmosphere, R. rubra transformed ferulic acid to vanillic acid and 4-hydroxy-3-methoxystyrene (5). Metabolites were isolated by solid-phase extraction and characterized by mass spectrometry, 1H, and 13C-nuclear magnetic resonance spectroscopy (NMR). The biotransformation of ferulic acid to vanillic acid by R. rubra cell-free extracts required CoA, ATP, and NAD+. Mass spectrometry and 13C-NMR were used to demonstrate the incorporation of oxygen from H2(18)O during the conversion of ferulic acid to vanillic acid. The results suggest a parallel between this bioconversion reaction and the beta-oxidation of fatty acids. Proton-carbon correlation NMR spectroscopy was used to demonstrate the specific incorporation of deuterium from D2O into guaiacol obtained from vanillic acid. The incorporation of deuterium implicates the involvement of a quinoid vanillic acid tautomer as an intermediate in the decarboxylation reaction.

Aeruginol [2-(2'-hydroxyphenyl)-4-hydroxymethylthiazole], a new secondary metabolite from Pseudomonas aeruginosa.

The CHCl3 extract of Pseudomonas aeruginosa UI 29791 cultures afforded a novel fluorescent compound, aeruginol [1]. The structure of 1 was elucidated by spectroscopic methods, including uv, eims, cims, hreims, and 1H nmr. Aeruginol appears to be biosynthetically related to aeruginoic acid.

Stereospecificity of microbial hydrations of oleic Acid to 10-hydroxystearic Acid.

We recently described a simple method for ascertaining the stereochemical purities of hydroxy fatty acids (S. H. El-Sharkawy, W. Yang, L. Dostal, and J. P. N. Rosazza, Appl. Environ. Microbiol. 58:2116-2122, 1992) based on the H-nuclear magnetic resonance spectral analysis of diastereomeric S-(+)-O-acetylmandelate esters of hydroxystearates. This report describes the stereochemistries of microbial hydrations of oleic acid to 10-hydroxystearic acid by Nocardia aurantia (also known as Rhodococcus rhodochrous) ATCC 12674, Nocardia restrictus ATCC 14887, Mycobacterium fortuitum UI-53387, Pseudomonas species strain NRRL-2994, Pseudomonas species strain NRRL B-3266, and baker's yeast. 10(R)-hydroxystearic acid isolated from Pseudomonas species strain NRRL-2994 was the standard for use in the H-nuclear magnetic resonance spectral technique to permit simple assignments of the absolute configurations of 10-hydroxystearic acid produced by different microorganisms. While the R. rhodochrous ATCC 12674-mediated hydration of oleic acid gave mixtures of enantiomers 10(R)-hydroxystearic acid and 10(S)-hydroxystearic acid, Pseudomonas species strain NRRL-B-3266 produced optically pure 10(R)-hydroxystearic acid. The remaining microorganisms stereoselectively hydrated oleic acid to 10(R)-hydroxystearic acid containing between 2 and 18% of the contaminating 10(S)-hydroxystearic acid.

Developmental toxicity of the HMG-CoA reductase inhibitor, atorvastatin, in rats and rabbits.

The developmental toxicity of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, atorvastatin, was investigated in pregnant rats and rabbits given daily oral doses during organogenesis. Rats received 0, 10, 100, or 300 mg/kg on days 6-15 of gestation, and rabbits received 0, 10, 50, or 100 mg/kg on days 6-18 of gestation. Maternal and fetal parameters were evaluated on day 20 (rats) or 29 (rabbits) of gestation. Live fetuses were examined for external, visceral, and skeletal malformations and variations. At 300 mg/kg in rats, 1 treatment-related death occurred on day 12 of gestation, and maternal body weight gain and food consumption were decreased during treatment (43% and 23%, respectively). In addition, 1 animal at 300 mg/kg had total litter resorption. Increased postimplantation loss (not statistically significant) and slightly decreased fetal body weight (statistically significant only in males) were also observed at 300 mg/kg. There were no significant differences between treated and control groups in the incidence of fetal malformations or variations. No maternal or developmental toxicity was observed in rats at 10 or 100 mg/kg. In rabbits, marked maternal toxicity (7 deaths, body weight loss during and after treatment, and decreased food consumption) and abortion occurred at 100 mg/kg. At 50 mg/kg, maternal toxicity (2 deaths and 72% body weight gain suppression) and abortion also occurred. There were no treatment-related effects on live litter size or sex ratio. At 50 and 100 mg/kg, nonstatistically significant increases in postimplantation loss and decreases in gravid uterine weight were observed, and at 100 mg/kg, decreases in fetal body weight were observed relative to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic peroxisome proliferation and hypolipidemic effects of di(2-ethylhexyl)phthalate in neonatal and adult rats.

To determine the relative sensitivity of suckling rats as compared to adults to the effects of di(2-ethylhexyl) phthalate (DEHP), five daily oral doses of 0, 10, 100, 1000, or 2000 mg DEHP/kg body weight were given to male Sprague-Dawley rats beginning at 6, 14, 16, 21, 42, and 86 days of age. Twenty-four hours after the last dose, rats were sacrificed and plasma cholesterol and triglyceride levels and the activities of the hepatic peroxisomal enzymes, palmitoyl CoA oxidase and carnitine acetyltransferase, were determined. Suckling rats (1-3 weeks of age) suffered severe growth retardation at doses of 1000 mg/kg and death at 2000 mg/kg while older rats only showed decreased weight gain at 2000 mg/kg. Of particular interest was the lethality at doses of 1000 mg/kg at 14 days of age but not at 16 days or at other ages. Increases in relative liver weight and hepatic peroxisomal enzyme activities were similar in all age groups except the 14-day old group in which the increases were greater. Relative kidney weight was increased in 21-, 42-, and 86-day-old rats at the highest doses but not in younger rats. Hypolipidemia was observed only in 21-, 42-, and 86-day-old rats at doses of 1000 and 2000 mg/kg, while elevated plasma cholesterol levels were observed in 6- and 14-day-old rats at the 1000 mg/kg dose, possibly due to the dietary differences between suckling and weaned rats. The results suggest that neonatal and suckling rats are more sensitive to the lethal and growth retardation effects of DEHP than are adult rats, but the hepatic peroxisome proliferation is similar at all ages with the exception of a greater increase at 14 days of age.

Transfer of di(2-ethylhexyl) phthalate through rat milk and effects on milk composition and the mammary gland.

Five daily oral doses of di(2-ethylhexyl) phthalate (DEHP) (2 g/kg) given to rats on Days 2-6, 6-10, or 14-18 of lactation caused significant decreases in body weight and increases in hepatic peroxisomal enzymes palmitoyl CoA oxidase and carnitine acetyltransferase in the dams and their suckling pups. Plasma cholesterol and triglyceride levels were decreased in the lactating dams. Decreased food consumption, as indicated by pair-fed rats, accounted for the decreased body weight in the pups but not the increases in enzyme activities. To determine whether DEHP and mono(2-ethylhexyl) phthalate (MEHP) were transferred through the milk, milk and plasma were collected from lactating rats 6 hr after the third dose of DEHP. The milk contained 216 +/- 23 micrograms/ml DEHP and 25 +/- 6 micrograms/ml MEHP (mean +/- SE), while the plasma contained less than 0.5 micrograms/ml DEHP and 75 +/- 12 micrograms/ml MEHP. The high milk/plasma ratio for DEHP (greater than 200) indicates efficient extraction of DEHP from the plasma into the milk. DEHP dosing during lactation also caused a decrease in mammary gland weight and a decrease in mammary gland RNA content which reflects synthetic activity. The water content of the milk was reduced, which probably accounted for the increase in lipid in the milk. Milk lactose was decreased in DEHP-treated and pair-fed rats, consistent with the decrease in milk production. The results show that exposure to high doses of DEHP during lactation in rats can result in changes in milk quality and quantity and can lead to DEHP and MEHP exposure in the suckling rat pups.