Cell response endpoints enhance sensitivity of the immature mouse uterotropic assay.

Outbred immature CD-1 mice were subcutaneously (s.c.) injected once on postnatal day 17 or on postnatal days 17, 18, and 19 with 17beta-estradiol, diethylstilbestrol, tamoxifen, 4-hydroxytamoxifen, methoxychlor, the methoxychlor metabolite HPTE, nonylphenol, o,p'-DDT, endosulfan, or kepone over a wide dose range (0.1 to 1,000,000 microg/kg). On the day following the last injection, uterine weight/body weight ratios were determined and uterine tissues processed for histologic examination. All compounds except endosulfan and kepone increased uterine wet weight compared to vehicle controls; however, the dose response curve and magnitude of response varied depending on the compound. Choosing the maximum wet weight dose for each compound, uterine tissue was evaluated for epithelial cell height, epithelial and stromal cell proliferation, endometrial gland number, and induction of estrogen-inducible proteins lactoferrin and complement C3. All compounds elicited estrogen-responsive changes in these endpoints that were individually more sensitive than uterine weight alone. We conclude that these endpoints enhance the sensitivity of the uterotropic bioassay.

Abolition of male sexual behaviors in mice lacking estrogen receptors alpha and beta (alpha beta ERKO).

Male mice with a knockout of the estrogen receptor (ER)-alpha gene, a ligand-activated transcription factor, showed reduced levels of intromissions and no ejaculations whereas simple mounting behavior was not affected. In contrast, all components of sexual behaviors were intact in male mice lacking the novel ER-beta gene. Here we measure the extent of phenotype in mice that lack both ER-alpha and ER-beta genes (alphabetaERKO). alphabetaERKO male mice did not show any components of sexual behaviors, including simple mounting behavior. Nor did they show ultrasonic vocalizations during behavioral tests with receptive female mice. On the other hand, reduced aggressive behaviors of alphabetaERKO mice mimicked those of single knockout mice of ER-alpha gene (alphaERKO). They showed reduced levels of lunge and bite aggression, but rarely showed offensive attacks. Thus, either one of the ERs is sufficient for the expression of simple mounting in male mice, indicating a redundancy in function. Offensive attacks, on the other hand, depend specifically on the ER-alpha gene. Different patterns of natural behaviors require different patterns of functions by ER genes.

Postnatal sex reversal of the ovaries in mice lacking estrogen receptors alpha and beta.

Mice lacking estrogen receptors alpha and beta were generated to clarify the roles of each receptor in the physiology of estrogen target tissues. Both sexes of alphabeta estrogen receptor knockout (alphabetaERKO) mutants exhibit normal reproductive tract development but are infertile. Ovaries of adult alphabetaERKO females exhibit follicle transdifferentiation to structures resembling seminiferous tubules of the testis, including Sertoli-like cells and expression of Müllerian inhibiting substance, sulfated glycoprotein-2, and Sox9. Therefore, loss of both receptors leads to an ovarian phenotype that is distinct from that of the individual ERKO mutants, which indicates that both receptors are required for the maintenance of germ and somatic cells in the postnatal ovary.

Protection against atherosclerosis by estrogen is independent of plasma cholesterol levels in LDL receptor-deficient mice.

Low density lipoprotein (LDL) receptor-deficient (LDLR-/-) mice consuming a high fat diet were used to assess the effect of endogenous and exogenous estradiol (E2) on atherosclerosis. Sexually mature female mice were ovariectomized (OVX) and implanted with subcutaneous, slow-release pellets designed to release 6 microg/day of exogenous 17beta-estradiol (17beta-E2 ), 17alpha-estradiol (17alpha-E2 ), or placebo (E2- deficient). Sham-operated control female (endogenous E2 ) and male mice were studied as controls. Aortic atherosclerotic lesion area was reduced by physiologic amounts of both endogenous and exogenous E2 compared to E2-deficient female mice. Although plasma cholesterol levels were reduced by exogenous E2 despite the absence of the LDL receptor, endogenous E2 was not associated with any cholesterol changes. In contrast, only 17alpha-E2 was associated with decreased fasting triglyceride. In subgroup analyses matched for time-averaged plasma total cholesterol, aortic lesion area was reduced by the presence of estradiol (E2 ). E2 protected LDLR-/- female mice from atherosclerosis and this protection was independent of changes in plasma cholesterol levels.

Intestinal absorption of trace amounts of aluminium in rats studied with 26aluminium and accelerator mass spectrometry.

1. Until recently studies of intestinal aluminium absorption used pharmacological amounts of stable 27Al. 2. To examine the intestinal absorption of trace amounts of different chemical compounds of aluminium, in the present study we have employed the long half-life isotope of aluminium, 26Al, and accelerator mass spectrometry. Trace amounts of 26Al (2.7-12.1 ng) as the hydroxide, citrate, citrate plus 1 mmol/kg sodium citrate, or maltolate respectively, were administered to four groups of rats (n = 9 per group) by gavage. Blood and urine samples were collected for 5 h and the 26Al content (as a percentage of the administered dose) determined by accelerator mass spectrometry. 3. The 5 h urinary 26Al excretion amounted to 0.1 +/- 0.02, 0.7 +/- 0.2, 5.1 +/- 1.5 and 0.1 +/- 0.1% of administered dose in the four groups respectively. There was a strong positive correlation between peak plasma 26Al (r = 0.98) and urinary 26Al excretion in individual animals (P < 0.001). 4. We conclude that the fractional intestinal absorption of trace oral doses of aluminium hydroxide is at least 0.1% (compared with the previous estimate of 0.01% using large 27Al oral loads). Absorption of aluminium citrate given alone is significantly greater (0.7%) and is further increased to 5% by the accompanying sodium citrate, consistent with an enhancing effect of added citrate upon mucosal aluminium permeability. Aluminium maltolate absorption approximates that of aluminium hydroxide (0.1%).

Induction of novel Grp75 isoforms by 2-deoxyglucose in human and murine fibroblasts.

Grp75 is a stress-inducible mitochondrial chaperone which has a high homology to senescence-related protein, p66mot mortalin. In human cells the mortalin gene assigns to the locus of a putative tumor suppressor gene for myeloid malignancies. In order to study expression and localization of Grp75 and p66mot in human and murine fibroblast lines, polyclonal antibodies were raised to conserved portions of each sequence. HT1080 and C3H10T1/2 cells were treated with various Grp-inducing agents. A single 75 kDa band was detected by Western blot of cytoplasmic proteins which was not greatly altered after thermal stress or treatment with L-azetidine-2-carboxylic acid or nonactin. However, glucose deprivation by 2-deoxyglucose treatment induced five novel isoforms at 74-75 kDa mass. Mortalin at 66 kDa could not be detected under these treatment conditions.

Semiautomated method for the quantitation of plasma or sera androstenedione, testosterone, and dihydrotestosterone in population studies.

A method is presented that analyzes quantitatively and reproducibly the androgens testosterone, androstenedione, and dihydrotestosterone from human sera or plasma. The chromatographic separation step generates an unattended throughput of one preparative separation per hour. Controls are built into the method to account for changing chromatographic conditions that otherwise result in shifts in retention characteristics. Separation factors for the three androgens are as follows (mean +/- standard deviation): alpha = 1.23 +/- 0.011 between androstenedione and testosterone and alpha = 1.38 +/- 0.025 between testosterone and dihydrotestosterone. Sensitivities of the method are androstenedione 5 pg, testosterone 3 pg, and dihydrotestosterone 14 pg. A study of procedural losses associated with initial sample processing, a validation, and application to two sample sets which demonstrates the methods utility for the analysis of hypoandrogenic populations (postmenopausal women) and hyperandrogenic groups (prostate cancer patients) is also reported. The precision for replicate aliquots of control plasma is androstenedione and testosterone = 5-11% CV and dihydrotestosterone = 10-20% CV.

Tissue disposition of 26aluminum in rats measured by accelerator mass spectrometry.

A trace quantity of 26aluminum (26Al) was administered intravenously to 1 normal and 1 uremic rat. After a 3-week period, the animals were sacrificed and samples of bone, muscle, kidney, liver, heart, and brain were analyzed for their 26Al content. In the normal and uremic rats, most of the tissue 26Al was found in bone amounting to 0.9% and 2.0%, respectively, of administered dose/g dry weight of tissue. Much smaller amounts of isotope were found in the other tissues in both animals. In the normal rat, the descending order of 26Al content in other tissues was: kidney, 0.2% > liver, 0.06% > heart, 0.03%, > brain and muscle, 0.02%. In the uremic rat, the same order of tissue 26Al content was found with kidney, 0.37% > liver, 0.06% > heart, 0.02% > brain and muscle, 0.01% per g dry weight of tissue. When expressed per g wet weight of tissue in the 2 animals, a similar order of tissue 26Al content was found. In comparing the amount of 26Al in the bone of the 2 rats, the uremic animal was found to have more than twice that found in the bone of the normal rat when expressed either per g dry or wet weight of bone. However, 26Al content of other tissues was similar in the 2 animals. This suggests that uremic bone may have a greater affinity for aluminum than normal bone, but kidney, liver, brain, heart, and muscle appear to behave similarly in uremic and normal rats in regard to incorporation of a single trace dose of isotope in the 3-week time frame of the present study.

Enteric and mild hyperoxaluria.

Enteric hyperoxaluria complicates extensive disease or resection of the small intestine in the presence of an intact colon, and is associated with calcium oxalate nephrolithiasis. In addition to hyperoxaluria these patients have a low urine volume, low urinary ionic strength and hypocitraturia. Many forms of treatment have been recommended, but none has been subjected to a prospective clinical trial. Mild idiopathic hyperoxaluria is reported in 8-50% of idiopathic calcium oxalate stoneformers. Several pathophysiological mechanisms have been proposed, including low dietary calcium and possible oxalate transport defects in the gut and/or the kidney. Mild hyperoxaluria, or a high oxalate:calcium ratio in the urine, may be particularly important risk factors for calcium oxalate stone formation; an approach to the correction of these abnormalities is proposed.

Mechanism of polyuria after cisplatin therapy.

Cisplatin is an antineoplastic agent. Several nephrotoxic effects are associated with its use including chronic and acute renal failure, renal magnesium wasting, and polyuria. We have investigated polyuria in groups of rats treated with cisplatin at doses of 2.5 and 5 mg/kg body weight given once weekly for 3 weeks to determine possible mechanisms of this impairment. After cisplatin administration, glomerular filtration rate was reduced and significant increases in sodium and water loss were also seen. These changes were associated with decreases in urinary cAMP. Inner medullary collecting duct (IMCD) cells were removed from these animals and were stimulated with graded doses of vasopressin. Cells from cisplatin-treated rats showed an impaired response in cAMP generation to vasopressin stimulation as compared to cells from normal animals. To determine more precisely the site of impairment, the adenylate cyclase complex of the IMCD cells was further studied with forskolin and NaF. Forskolin was used to probe the catalytic unit activating adenylate cyclase, and NaF the guanine nucleotide regulatory protein (G protein). In response to forskolin, cells from cisplatin-treated rats and normal rats responded similarly in generating cAMP. However, following NaF, the cAMP response was blunted in the cells from the cisplatin rats. These results suggested that the catalytic unit was not injured by cisplatin (forskolin study) but the G protein was (NaF). In conclusion, the present study suggests that the polyuria seen following cisplatin administration is associated with an end-organ resistance to vasopressin manifested by reduced cAMP generation, secondary in part or whole to a defect at the level of the G protein.

Plasma and urinary oxalate and glycolate in healthy subjects.

High-performance ion chromatography (HPIC) has been widely used for oxalate analysis and, more recently, for glycolate analysis. We describe a procedure for sample preparation in which the plasma ultrafiltrate is acidified during harvesting with a cation-exchange resin, and the chloride is removed before the ion chromatography, which is performed with a newly developed AS10 column. The same ultrafiltrate sample is analyzed for glycolate. For plasma oxalate, the mean recovery of sample in eluted fractions was 95-96%, and intraassay CV was 6.2-8.1%. The reference interval (mean +/- 2 SD) for men was 0.8-3.2 mumol/L and for women, 1.0-2.6 mumol/L. For urinary oxalate, the reference interval for men was 175-560 mumol/day and for women, 107-432 mumol/day. For plasma glycolate, the mean analytical recovery was 96-98%, and the intra-assay CV was 2.4-6.2%. The reference interval for men was 1.9-7.5 mumol/L and for women, 1.4-7.4 mumol/L. For urinary glycolate, the reference interval for men was 0-1400 mumol/day and for women, 91-1001 mumol/day.

Accelerator mass spectrometry: application to study of aluminum kinetics in the rat.

The advent of accelerator mass spectrometry (AMS) now permits the ultrasensitive detection of extremely long-lived isotopes, including 14C, 26Al, and 41Ca. Until now, tracer studies of aluminum kinetics have not been possible because aluminum has only two isotopes, with half-lives of 6.5 min (29Al) and 7 x 10(5) yr (26Al), neither of which is suitable for conventional studies. In a novel experiment we have employed AMS to study aluminum kinetics in a normal rat and a 5/6-nephrectomized rat over a 3-wk period of intravenous injection of a tracer dose of 26Al. Kinetics were similar in the two animals; approximately 75% of intravenously injected tracer 26Al was excreted in the urine in the first 24 h as was approximately 80% after 3 wk. Renal clearance of 26Al was approximately 0.75 ml.min-1.kg body wt-1 in both rats. The results clearly demonstrate the potential of this technique for isotope tracer studies in animals as well as in humans.

Chronic hypomagnesemia caused by cisplatin: effect of calcitriol.

A group of six patients with hypomagnesemia (serum magnesium less than or equal to 0.5 mmol/L), previously given treatment with cisplatin for ovarian or testicular cancer, received calcitriol at a dose of 0.5 to 1.0 microgram/day for a period of 4 weeks to determine whether treatment with this vitamin D metabolite could improve their hypomagnesemia. In response to treatment, the serum magnesium concentration fell progressively in association with a rise in serum and urinary calcium levels and a decrease in parathyroid hormone level. In a single previous report, active vitamin D metabolites markedly improved renal magnesium wasting. However, in the present study, increases in serum and urinary calcium levels and suppression of parathyroid hormone, factors known to decrease magnesium reabsorption, presumably overwhelmed any direct effect calcitriol may have had to enhance magnesium reabsorption, so that the net effect was a marked exacerbation of the renal magnesium wasting.

Bone resorption and hypercalciuria in calcium stoneformers.

The circadian rhythm of urinary total hydroxyproline (THP) excretion was determined in matched groups of ten male idiopathic calcium stoneformers and ten normal subjects in order to determine whether enhanced resorption of bone might contribute to hypercalciuria in these patients. THP increased progressively in normal subjects in successive eight-hour urine collections from period 1 (8 AM to 4 PM) to period 3 (12 midnight to 8 AM), the nocturnal high level in period 3 being significantly greater than in period 1 (P less than 0.01) and in period 2 (P less than 0.05). By contrast, no significant circadian rhythm was observed in THP excretion in the stoneformers. Their THP excretion was similar to that of normal subjects in period 3, but was significantly higher than that of normal subjects in period 1 (THP/creatinine ratio(mg/mg): 0.026 +/- 0.003 v 0.017 +/- 0.001; P less than 0.05. Indices of parathyroid hormone activity were not significantly different between stoneformers and normal subjects; mean serum 1,25(OH)2 vitamin D levels were higher in the stoneformers than the normal subjects (44 v 37 pg/mL) but the difference was not significant (P greater than 0.05). These studies suggest that increased bone turnover may contribute to hypercalciuria in these calcium stoneformers.

Urinary adenosine cyclic 3',5'-monophosphate in idiopathic calcium stone-formers: response to an oral calcium load.

Idiopathic calcium stone-formers with hypercalciuria during fasting have significantly lower urinary cyclic AMP levels (nmol/dl of glomerular filtrate) than fasting normocalciuric stone-formers. Female subjects, including both normal subjects and idiopathic calcium stone-formers, have higher urinary cyclic AMP levels than their male counterparts, and this difference is significant when urinary cyclic AMP is expressed in the units mumol/g of creatinine. Expressing urinary cyclic AMP in nmol/dl of glomerular filtrate reduces this difference but does not abolish it. Thus, in comparing urinary cyclic AMP levels in various subgroups of the calcium stone-formers and in normal subjects, both sex differences and the units of urinary cyclic AMP expression must be taken into consideration. The magnitude of the change in urinary cyclic AMP in response to an oral calcium load appears to depend on the antecedent urinary cyclic AMP excretion rate, whereby those individuals (either normal subjects or calcium stone-formers) having the highest urinary cyclic AMP levels demonstrate the greatest fall in urinary cyclic AMP after a calcium load.

Responses to hydrochlorothiazide and acetazolamide in patients with calcium stones. Evidence suggesting a defect in renal tubular function.

Urinary excretion of sodium, calcium, and magnesium has been measured after single oral doses of hydrochlorothiazide (100 mg) and acetazolamide (500 mg) in unselected patients with calcareous renal stone formation and in normal control subjects. With hydrochlorothiazide, 36 stone formers had significantly greater increments in sodium (P less than 0.01), calcium (P less than 0.05), and magnesium (P less than 0.05) excretion than 20 normal subjects. With acetazolamide, 13 stone formers had a smaller increment in sodium excretion (P less than 0.05) than 10 normal subjects. The abnormal responses to both diuretics were most marked in the patients with hypercalciuria during fasting. These data suggest that the tubular handling of sodium, magnesium, and calcium may be abnormal in patients with calcareous renal stones and are consistent with the presence of a defect in proximal-tubular reabsorption of fluid and electrolytes that may be partly offset by increased reabsorption in the distal nephron.

Cortical thickness measurements and photon absorptiometry for determination of bone quantity.

Reproducibility of estimates of cortical bone quantity in the appendicular skeleton by two methods was studied in healthy individuals and patients undergoing long-term hemodialysis. Repeated measurements of cortical thickness (CT) at the midpoint of the second metacarpal were taken from single radiographs of both hands by two independent observers. Repeated measurements by the same observer were more reproducible and the degree of reproducibility was far greater in healthy subjects than in dialysis patients. Repeated measurements were made of bone mineral content (BMC) and bone width (W) of the distal radius by photon absorptiometry. Repeated BMC/W determinations were highly reproducible in both healthy subjects and dialysis patients. High correlation was found between BMC and cross-sectional cortical area and between both simple cortical thickness and cortical area/width. Thus the photon absorptiometric technique is superior for the serial monitoring of bone quantity, particularly in patients with uremic osteodystrophy, but results obtained by the two methods in group studies should be comparable.

Measurement of bone mineral content in vivo using photon absorptiometry.

Progress in evaluating treatment of systemic bone disease has been hampered in the past by lack of precise in vivo quantitative techniques. Recently a method has been developed for measurement of bone mineral content (BMC), based on bone absorption of low-energy monochromatic radiation. This paper discusses a technique of photon absorptiometry using (125)l as a collimated point source. The technique is simple, with accuracy and precision within 2%.BMC and bone width (W) were measured in the distal radius of 359 normal subjects ranging in age from 5 to 82 years. A "normal" curve of BMC/W with age as the independent variable was then obtained from this population and was constructed for each sex. A positive correlation of BMC/W with height and body weight was found in a group of normal males.A series of patients with osteoporosis or malabsorption, or undergoing hemodialysis or steroid treatment, was then assessed in order to demonstrate changes in BMC/W that may occur secondary to disease or disturbances in calcium metabolism. Many of these patients were found to have a BMC/W below the normal mean value for their age and sex.