María I. Conti

Failure of Polycythemia-Induced Increase in Arterial Oxygen Content to Suppress the Anorexic Effect of Simulated High Altitude in the Adult Rat

The anorexic effect of exposure to high altitude may be related to the reduction in the arterial oxygen content (Ca(O2)) induced by hypoxemia and possibly the associated decreased convective oxygen transport (COT). This study was then performed to evaluate the effects of either transfusion-induced polycythemia or previous acclimation to hypobaria with endogenously induced polycythemia on the anorexic effect of simulated high altitude (SHA) in adult female rats. Food consumption, expressed in g/d/100 g body weight, was reduced by 40% in rats exposed to 506 mbar for 4 d, as compared to control rats maintained in room air. Transfusion polycythemia, which significantly increased hematocrit, hemoglobin concentration, Ca(O2), and COT, did not change the anorexic response to the exposure to hypobaric air. Depression of food intake during exposure to SHA also occurred in rats fasted during 31 h before exposure and allowed to eat ad libitum for 2 h during exposure. Body mass loss was similar in 48-h fasted rats that were either hypoxic or normoxic. Body mass loss was similar in normoxic and hypoxic rats, the former eating the amount of food freely eaten by the latter. Hypoxia-acclimated rats with endogenously induced polycythemia taken to SHA again had diminished food intake and lost body mass at rates that were very close to those found in nonacclimated ones. Exposure to SHA also led to a decrease in food consumption, body weight, and plasma leptin in adult female mice. Analysis of data suggest that body mass loss that accompanies SHA-induced hypoxia is due to hypophagia and that experimental manipulation of the blood oxygen transport capacity cannot ameliorate it. Leptin does not appear to be an inducer of the anorexic response to hypoxia, at least in mice and rats.

Plasma disappearance of exogenous erythropoietin in mice under different experimental conditions

Erythropoietin (EPO) is a glycoprotein hormone produced primarily in the kidneys and to a lesser extent in the liver that regulates red cell production. Most of the studies conducted in experimental animals to assess the role of EPO in the regulation of erythropoiesis were performed in mouse models. However, little is known about the in vivo metabolism of the hormone in this species. The present study was thus undertaken to measure the plasma t 1/2 of radiolabeled recombinant human EPO (rh-EPO) in normal mice as well as in mice with altered erythrocyte production rates (EPR), plasma EPO (pEPO) titer, marrow responsiveness, red cell volume, or liver function. Adult CF-1 mice of both sexes were used throughout. For the EPO life-span studies, 30 mice in each experiment were intravenously injected with 600,000 cpm of 125I-rh-EPO and bled by cardiac puncture in groups of five every hour for 6 h. Trichloroacetic acid (TCA) was added to each plasma sample and the radioactivity in the precipitate measured in a γ-counter. EPO, pEPO, marrow responsiveness, or red cell volume were altered by either injections of rh-EPO, 5-fluorouracil, or phenylhydrazine, or by bleeding, or red cell transfusion. Liver function was altered by Cl4C administration. In the normal groups of mice, the estimated t 1/2 was 182.75 ± 14.4 (SEM) min. The estimated t 1/2 of the other experimental groups was not significantly different from normal. These results, therefore, strongly suggest that the clearance rate of EPO in mice is not subjected to physiologic regulation and that pEPO titer can be really taken as the reflection of the EPO production rate, at least in the experimental conditions reported here.

Alveolar bone loss associated to periodontal disease in lead intoxicated rats under environmental hypoxia

Previously reported studies from this laboratory revealed that rats chronically intoxicated with lead (Pb) under hypoxic conditions (HX) impaired growth parameters and induced damages on femoral and mandibular bones predisposing to fractures. We also described periodontal inflammatory processes under such experimental conditions. Periodontitis is characterised by inflammation of supporting tissues of the teeth that result in alveolar bone loss. The existence of populations living at high altitudes and exposed to lead contamination aimed us to establish the macroscopic, biochemical and histological parameters consistent with a periodontal disease in the same rat model with or without experimental periodontitis (EP). Sixty female rats were divided into: Control; Pb (1000ppm of lead acetate in drinking water); HX (506mbar) and PbHX (both treatments simultaneously). EP was induced by placing ligatures around the molars of half of the rats during the 14 days previous to the autopsy. Hemi-mandibles were extracted to evaluate bone loss by histomorphometrical techniques. TNFα plasmatic concentration was greater (p<0.01) in Pb and HX animals. TBA-RS content was significantly higher in gums of rats with or without EP only by means of Pb. The SMG PGE2 content increased by Pb or HX was higher in PbHX rats (p<0.01). Pb and HX increased EP induced alveolar bone loss, while Pb showed spontaneous bone loss also. In conclusion, these results show that lead intoxication under hypoxic environment enhanced not only alveolar bone loss but also systemic and oral tissues inflammatory parameters, which could aggravate the physiopathological alterations produced by periodontal disease.

Enhanced Erythropoietin Response to Acute Hypoxemia in Mice with Pharmacological Depression of Erythropoiesis

The recent report of a depression of stimulated production of erythropoietin (EPO) in mice with enhanced erythropoiesis suggests that unknown mechanism (s) other than hypoxia may be involved in the regulation of EPO formation. The present study was thus designed to investigate EPO production during acute hypoxemia in a mouse model in which the oxygen-carrying capacity of blood, the plasma EPO level, and the plasma EPO half-life were within normal values in spite of a marked depression of the red cell production rate (RCPR) induced by cyclophosphamide (CP) administration. Injection of 100 mg/Kg of the drug into adult female CF-1 mice that previously received 0.4 ml of packed red cells depressed markedly the 24-hour RBC 59Fe uptake without affecting the plasma immunoreactive EPO level and the plasma disappearance of 1251-labeled recombinant human EPO. The EPO production rate, calculated from the change in plasma EPO levels and the estimated EPO clearance rate, after 4 h of exposure to hypobaric air was about 2.8 times higher in mice with CP-induced inhibition of the RCPR than in mice with normal RCPR. The results support the hypothesis that the EPO production rate in mammals is not only related to the oxygen supply to the tissues relative to their oxygen needs (main stimulus) but also to the erythroid activity of the marrow (modulatory action).

Correlation between lead and prolactin in males exposed and unexposed to lead in Buenos Aires (Argentina) area

Lead is an important factor in environmental pollution due to its intensive industrial use and in Argentina it is perhaps the main industrial contaminant. In most cases lead toxicity is related to alterations in heme synthesis. It is also responsible for changes in the central nervous system. Researchers have reported the action of lead on the dopaminergic transmission mechanism in the central nervous system of rats. The control of prolactin levels by an agent whose identity is still uncertain is known to involve undoubtedly a dopaminergic mechanism by which an increase in dopamine depresses plasma prolactin values. The purpose of this work was to determine the correlation between blood lead and plasma prolactin levels in humans. Normal plasma prolactin and blood lead values in healthy individuals exposed to lead at work had already been obtained in males and ranged from 1 to 17 ng/ml for prolactin and up to 26 ug/100 ml for lead.

Chronic lead poisoning magnifies bone detrimental effects in an ovariectomized rat model of postmenopausal osteoporosis.

Lead (Pb) is a persistent environmental contaminant that is mainly stored in bones being an important source of endogenous lead exposure during periods of increased bone resorption as occurs in menopause. As no evidence exists of which bone biomechanical properties are impaired in those elderly women who had been exposed to Pb during their lifetime, the aim of the present study is to discern whether chronic lead poisoning magnifies the deterioration of bone biology that occurs in later stages of life. We investigated the effect of Pb in the femora of ovariectomized (OVX) female Wistar rats who had been intoxicated with 1000 ppm of Pb acetate in drinking water for 8 months. Structural properties were determined using a three-point bending mechanical test, and geometrical and material properties were evaluated after obtaining the load/deformation curve. Areal Bone Mineral Density (BMD) was estimated using a bone densitometer. Femoral histomorphometry was carried out on slices dyed with H&E (Hematoxylin and Eosin). Pb and OVX decreased all structural properties with a higher effect when both treatments were applied together. Medullar and cortical area of femurs under OVX increased, allowing the bone to accommodate its architecture, which was not observed under Pb intoxication. Pb and OVX significantly decreased BMD, showing lead treated ovariectomized rats (PbOVX) animals the lowest BMD levels. Trabecular bone volume per total volume (BV/TV%) was decreased in OVX and PbOVX animals in 54% compared to the control animals (p<0.001). Pb femurs also showed 28% less trabeculae than the control (p<0.05). We demonstrated that Pb intoxication magnifies the impairment in bone biomechanics of OVX rats with a consequent enhancement of the risk of fracture. These results enable the discussion of the detrimental effects of lead intoxication in bone biology in elderly women.

Permanent reduction of mandibular size and bone stiffness induced in post-weaning rats by cyclophosphamide

It has been previously reported that several doses of cyclophosphamide (CPA) reduce body weight gain, diaphyseal torsional strength and longitudinal femoral growth in the growing rat. The present study was thus designed to estimate both the initial and the possible long-term effects of CPA treatment, by analyzing mandibular dimensions and biomechanical performance of the bone in adulthood in rats treated with the drug around weaning. Female Sprague-Dawley rats (N=20), 26 d of age, received 100mg/kg of CPA by the intraperitoneal route during days 0, 7 and 21 of the experimental period. Controls (C) received saline. Groups of rats were sacrificed at day 28 to estimate initial changes induced by the drug and on day 126 in order to determine long-term effects. The dimensions of the excised mandibles were measured directly between anatomical points; the geometry and material biomechanical quality of mandibular bone were assessed using a three-point bending mechanical test in an Instron Universal Testing Machine model 4442. CPA reduced body weight, body length and mandibular size (posterior part of the bone) significantly, when the parameters were measured at day 28. They did not recover with time, which means that catch-up growth did not occur and that the overall growth of the body was permanently affected by the drug. CPA treatment was also associated with a marked depression of the natural increase in the mandibular bone mass (cross-sectional area). The bending cross-sectional moment of inertia of the fracture sections (xCSMI) was also negatively affected by treatment. Significant decreases of both ultimate load and stiffness were also observed. The above structural parameters did not recover enough with time to attain control values at the end of the study. The intrinsic stiffness (E) of the mandibular bone was not affected by treatment. These findings suggest that CPA treatment during early postnatal life causes permanent changes in mandibular morphology and affects the adaptation of mandibular bone architecture to body growth, thus not allowing complete compensation at the end of the study because of an inadequate distribution of the resistive material through its cross-section rather than a qualitative impairment of cortical bone.

Enhanced hypoxia-stimulated erythropoietin production in mice with depression of erythropoiesis induced by hyperoxia.

Current evidence suggests that a modulatory action on O(2)-dependent EPO secretion is exerted by the erythroid/precursor cell population in the erythropoietic organs through a negative feedback system. The hypothesis is based on studies of stimulated-EPO secretion performed in mice in whom the erythropoietic rates were either enhanced or depressed in the presence of normal plasma EPO half-lives. Since erythropoietic depression was elicited by cyclophosphamide administration, which could have altered EPO production directly, the aim of the present investigation was to estimate hypoxia-stimulated EPO secretion in a mouse model of functional depressed erythropoiesis induced by exposure to normobaric hyperoxia. Females CF#1 mice aged 70 d were divided into control (C) and experimental (E) groups. The former was maintained in plastic cages in a normal environment, while the latter was placed in an environment of 60% O(2)/40% N(2) in an 85-dm(3) atmospheric chamber with air flow of 1 L/min. Erythropoiesis was evaluated by either 24-h RBC-(59)Fe uptake or iron kinetics performed 3 h after IV injection of a tracer dose of (59)Fe. Both indexes of the red cell production rate were significantly depressed in E mice. Plasma disappearance of exogenous EPO in C mice, as well as in E mice exposed to hyperoxia for 4 d, was estimated by injecting (125)I-rHuEPO intravenously. Linear regression analysis indicated that neither the differences between the slopes of both curves nor the Y-intercepts were significant. Hypobaric hypoxemia was used as stimulus for EPO production. Plasma immuno-EPO titer after a 4-h exposure to hypobaric air was 73% higher in mice with hyperoxia-induced hypoerythropoiesis than in control mice with normal erythropoiesis. Data support the concept that the rate of erythropoiesis, perhaps through the number of the erythroid progenitor/precursor cell population, modulates O(2)-dependent EPO secretion.

Erythropoietin-Hypersecretory State Without Kidney Hypertrophy in Testosterone-Treated Hypophysectomised Rats

Abstract: An erythropoietin hypersecretory state (EPO-HS) has been defined as a condition evidenced in hypertransfused-polycythaemic rats and mice as a consequence of several treatments imposed before transfusion in which stimulated secretion of EPO is higher than in non-treated hypertransfused-polycythaemic controls at equal levels of polycythaemia. We have recently reported that sustained administration of testosterone induced the appearance of an EPO-HS in female mice, which was accompanied by kidney hypertrophy. Since the erythropoietic effect of androgens has been associated with the latter effect, the present study was performed to test the hypothesis that sustained administration of testosterone could induce an EPO-HS in the hypophysectomised rat model without concurrently increasing renal mass. Normal and hypophysectomised rats were injected with 5 mg of testosterone propionate, given three times per week by the subcutaneous route. Treatment started 90 days after hypophysectomy (performed when rats were 30 days old) to allow the erythropoietic system to reach steady-state conditions in the hypophysectomised animals before androgenic treatment began. The erythropoietic effect of testosterone in the hypophysectomised rats was demonstrated by normalisation of the circulating red cell volume at the end of the treatment period, whilst the undosed hypophysectomised controls gave values of 30% less than normal control rats. Testosterone administration produced a significant increase in renal weight in non-hypophysectomised rats, an effect that was negligible in the hypophysectomised animals. Some of the rats from all groups were hypertransfused at the end of the dosing period. Plasma EPO (pEPO) levels were not significantly different between undosed polycythaemic hypophysectomised and polycythaemic non-hypophysectomised rats exposed to hypobaria for 6 h. However, pEPO was significantly higher in response to exposure to hypobaria in both testosterone-treated hypophysectomised and non-hypophysectomised rats than in their corresponding untreated controls. Thus, data confirmed the hypothesis and suggest that the EPO-HS induced by testosterone should be considered non-specific and independent of kidney hypertrophy.

Effects of chronic lead exposure on bone mineral properties in femurs of growing rats

Lead exposure has been associated with several defective skeletal growth processes and bone mineral alterations. The aim of the present study is to make a more detailed description of the toxic effects of lead intoxication on bone intrinsic material properties as mineral composition, morphology and microstructural characteristics. For this purpose, Wistar rats were exposed (n=12) to 1000ppm lead acetate in drinking water for 90days while control group (n=8) were treated with sodium acetate. Femurs were examined using inductively coupled plasma optical emission spectrometry (ICP-OES), Attenuated Total Reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray diffraction (XRD), and micro-Computed Tomography (μCT). Results showed that femur from the lead-exposed rats had higher carbonate content in bone mineral and (Ca2++Mg2++ Na+)/P ratio values, although no variations were observed in crystal maturity and crystallite size. From morphological analyses, lead exposure rats showed a decreased in trabecular bone surface and distribution while trabecular thickness and cortical area increased. These overall effects indicate a similar mechanism of bone maturation normally associated to age-related processes. These responses are correlated with the adverse actions induced by lead on the processes regulating bone turnover mechanism. This information may explain the osteoporosis diseases associated to lead intoxication as well as the risk of fracture observed in populations exposed to this toxicant.