R. Van Bree

Transplacental transfer of anthracyclines, vinblastine, and 4-hydroxy-cyclophosphamide in a baboon model

OBJECTIVE The paucity of data on the fetal effects of prenatal exposure to chemotherapy prompted us to study transplacental transport of chemotherapeutic agents. METHODS Fluorouracil-epirubicin-cyclophosphamide (FEC) and doxorubicin-bleomycin-vinblastine-dacarbazine (ABVD) were administered to pregnant baboons. At predefined time points over the first 25 h after drug administration, fetal and maternal blood samples, amniotic fluid (AF), urine, fetal and maternal tissues, and cerebrospinal fluid (CSF) were collected. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) were used for bioanalysis of doxorubicin, epirubicin, vinblastine, and cyclophosphamide. RESULTS In nine baboons, at a median gestational age of 139 days (range, 93-169), FEC 100% (n = 2), FEC 200% (n=1), ABVD 100% (n = 5), and ABVD 200% (n = 1) were administered. The obtained ratios of fetal/maternal drug concentration in the different simultaneously collected samples were used as a measure for transplacental transfer. Fetal plasma concentrations of doxorubicin and epirubicin averaged 7.5 ± 3.2% (n = 6) and 4.0 ± 1.6% (n = 8) of maternal concentrations, respectively. Fetal tissues contained 6.3 ± 7.9% and 8.7 ± 8.1% of maternal tissue concentrations for doxorubicin and epirubicin, respectively. Vinblastine concentrations in fetal plasma averaged 18.5 ± 15.5% (n=9) of maternal concentrations. Anthracyclines and vinblastine were neither detectable in maternal nor in fetal brain/CSF. 4-Hydroxy-cyclophosphamide concentrations in fetal plasma and CSF averaged 25.1 ± 6.3% (n = 3) and 63.0% (n = 1) of the maternal concentrations, respectively. CONCLUSION This study shows limited fetal exposure after maternal administration of doxorubicin, epirubicin, vinblastine, and 4-hydroxy-cyclophosphamide.

Effects of exercise and disuse on bone remodeling, bone mass, and biomechanical competence in spontaneously diabetic female rats

Diabetes is associated with low bone formation. In this study we investigate the effect of additional or reduced mechanical loading on indices of bone formation and resorption, bone mass, and biomechanical properties in spontaneously diabetic BB rats. Female diabetic (mean age 13 weeks) and age-matched control rats were each allocated to three experimental groups: no-intervention; supervised running exercise program (Ex); and unloading induced by unilateral sciatic neurectomy (USN). The study period was 8 weeks. We measured biochemical parameters of bone formation (plasma osteocalcin) and resorption (urinary deoxypyridinoline [Dpd]); bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA) at middiaphyseal and metaphyseal regions of the femur; histomorphometry of the proximal tibial metaphysis (PTM); and biomechanical properties of the femur (neck, diaphysis, and metaphysis) and lumbar vertebra (L-5). In nondiabetic rats, Ex did not affect parameters of bone formation/resorption and BMD, and had little effect on biomechanical properties. USN increased Dpd excretion, whereas there was a decreased trabecular bone formation rate (BFR) on morphometry of PTM in both paralyzed and intact limbs. Compared with intact limbs, paralyzed limbs of USN rats showed decreased trabecular bone volume at the PTM, and decreased BMD and biomechanical properties at the distal femoral metaphysis (DFM) and, to a lesser extent, femoral neck. Diabetic rats of the three experimental groups had low plasma osteocalcin levels and Dpd excretion, as well as low BFR on morphometry. The BMD and biomechanical properties of both femur and L-5 were unchanged in diabetic rats. Diabetic Ex rats, however, showed a lower maximum load and stress at DFM than control Ex rats. Diabetic USN rats showed no increase in Dpd excretion; their paralyzed limbs showed decreased maximum load at DFM, but there was no significant decrease in trabecular bone volume at PTM or BMD at DFM. Thus, the running exercise does not affect low bone formation in diabetic rats; however, trabecular bone loss caused by disuse is less pronounced in diabetic rats, probably as a result of low bone resorption.

In Vivo Glucose Utilization by Individual Tissues in Virgin and Pregnant Offspring of Severely Diabetic Rats

Adult offspring of diabetic rats or SDF rats are characterized by insulin resistance in the liver and extrahepatic tissues; this insulin resistance does not worsen during pregnancy. In this study, we determined the glucose metabolic index in tissues of anesthetized virgin and pregnant control and SDF rats in basal conditions and during a euglycemic hyperinsulinemic clamp. Tissues comprised insulin-sensitive tissues (five skeletal muscles, diaphragm, and periovarian white adipose tissue) and control tissues (duodenum and cerebrum). In addition, this study measured the GMI of placenta and fetuses. In basal conditions, SDF rats showed a slight decrease (9–29%) in the GMI of skeletal muscles compared with control rats; it was not altered by pregnancy in any of the tissues. During physiological hyperinsulinemia, virgin SDF rats exhibited a 25–70% decrease in the GMI of skeletal muscles compared with control rats; this decrease was not observed in diaphragm, or in adipose tissue in which the GMI was found to be increased. During pregnancy, SDF rats did not show an additional drop in the GMI of skeletal muscles, whereas the GMI of both skeletal muscles and adipose tissue was clearly diminished (25–60%) in control rats. The GMI of skeletal muscles was therefore comparable in pregnant control rats and SDF rats. The placental, but not fetal, GMI was increased by 24% during hyperinsulinemia in control rats; the placental and fetal GMIs, in basal and hyperinsulinemic conditions, were similar in control rats and SDF rats. In conclusion, skeletal muscles, but not white adipose tissue, are involved in the peripheral insulin resistance of the SDF rats. However, pregnancy does not induce a further decrease in glucose utilization by skeletal muscles in SDF rats.

Plasma amino acids in diabetic pregnant rats and in their fetal and adult offspring

Amino acid profiles and total amino-acid concentrations are established in nonfasting plasma of pregnant control, mildly diabetic and severely diabetic rats, and of their fetal and adult offspring. In pregnant rats at day 20 of gestation plasma amino acids can be regarded as normal in mildly diabetic mothers, but are significantly decreased in severely diabetic mothers. In fetuses of control rats, amino acid levels are twice as high as in the mother (fetomaternal ratio 2.0); in the fetuses of mildly diabetic mothers they are significantly lower than normal (fetomaternal ratio 1.3); in the fetuses of severely diabetic mothers they are also significantly lower than normal but with a normal fetomaternal ratio (fetomaternal ratio 2.0). In adult offspring of mildly diabetic mothers the concentration of almost all amino acids as well as that of total amino acid pool is significantly lower than in the controls; in the offspring of severely diabetic mothers they can be regarded as normal. No specific amino acid or group of amino acids can be held responsible for any of these changes, since all differences with control values display an overall effect, involving all or almost all amino acids.

Effects of long-term diabetes and/or high-dose 17β-estradiol on bone formation, bone mineral density, and strenght in ovariectomized rats

Abstract Long-term diabetes in female rats preserves the bone mineral density (BMD) but impairs the strength of the femur. In this study, we have compared the effects of diabetes and high-dose 17β-estradiol (E 2 ), two conditions of low bone formation, in ovariectomized (ovx) rats. Spontaneously diabetic BB rats were ovx 0–3 days after onset, and nondiabetic ovx littermates were used as controls; the rats were either untreated or treated with E 2 (30 μg/day), subcutaenously), for 6 or 12 weeks (n = 9 in each of the eight groups). Analysis included: plasma 1,25-dihydroxyvitamin D 3 , insulin-like growth factor-I (IGF-I), and osteocalcin concentrations; histomorphometry of the proximal tibial metaphysis (PTM); and DXA and biomechanical testing of the femur. Both E 2 treatment and diabetes markedly lowered plasma IGF-I and osteocalcin concentrations, as well as dynamic morphometric parameters of bone formation in the PTM. Plasma IGF-I and osteocalcin were correlated ( R 2 = 0.55; p 2 treatment in both control and diabetic ovx rats increased the trabecular bone volume in the PTM and the BMD in the metaphysis of the distal femur; there was no difference between control and diabetic rats, however. The diaphyseal area and BMC were decreased in E 2 -treated or/and diabetic ovx rats, but the diaphyseal BMD remained unchanged compared with untreated ovx rats. The biomechanical properties of the whole femur (strength, angular deformation, and stiffness) were decreased in E 2 -treated and diabetic E 2 -treated ovx rats after 12 weeks. The data indicate that in situations of chronic low bone formation, whole bone strenght does not reflect total BMD but correlates better with bone size and bone mineral content measurements.

Maternal Apelin Physiology during Rat Pregnancy: The Role of the Placenta

OBJECTIVE Apelin is a multifunctional peptide which is catabolized by the angiotensin-converting enzyme-related carboxypeptidase-2 (ACE2). The peptide is well known for its hemodynamic effects and its role in energy and fluid homeostasis. Pregnancy is a state of dramatically altered maternal hemodynamics and metabolism, but the role of apelin is unknown. To gain further insight in apelin physiology, we investigated relative tissue expression, plasma clearance and metabolic pathways of apelin in pregnant rats. METHODS We measured maternal plasma apelin levels throughout normal rat gestation and examined relative apelin gene expression in several tissues, including the placenta. We documented apelin clearance using radiolabeled apelin and assessed maternal plasma levels in rats that underwent surgical reduction of the fetoplacental mass, thereby further examining the role of the placenta in apelin clearance. Finally, we localized apelin and ACE2 in the placenta and mesometrial triangle using immunohistochemistry. RESULTS Maternal apelin plasma concentrations dropped by 50% between mid- and late gestation. Apelin expression was comparable between non-pregnant and late-pregnant rats in non-reproductive tissues. The placenta showed low apelin gene expression compared to brain tissue. Apelin clearance was enhanced in term gestation as evidenced by a steeper decline of the slow phase of the elimination curve of radiolabeled apelin. Compared to sham-operated dams, maternal plasma apelin was raised by 23% in late-pregnant rats in which half of the fetoplacental units were removed at day 16 of gestation. ACE2 mRNA expression was detectable in late- but not mid-pregnancy placental tissue; immunohistochemically, ACE2 was primarily localized in the smooth muscle layer of fetal arterioles in the labyrinth. CONCLUSION Maternal circulating apelin drops considerably between mid- and late- pregnancy owing to faster clearance. The current data suggest a role for placental ACE2 in the accelerated apelin metabolism.

Pathogenesis of Fetal Hypomineralization in Diabetic Rats: Evidence for Delayed Bone Maturation

There is some evidence that fetuses of diabetic rats (FDR) are hypomineralized. To explore the pathogenic role of decreased maternal duodenal Ca absorption, fetal hypotrophy, and decreased placental calbindin-D9K, respectively, spontaneously diabetic rats fed a 1.0% Ca diet were compared with diabetic rats treated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (15 ng/100 g) during week 3 of pregnancy, which restored duodenal calbindin-D9K concentrations to normal; with nondiabetic rats semistarved during week 3, which resulted in similar fetal hypertrophy; and with nondiabetic rats fed high cation diets (1.5% Ca-1.5% Sr and 1.5% Ca-3.5% Sr) during week 3, the latter of which repressed duodenal and placental calbindin-D9K toward concentrations measured in diabetic rats. In addition, fetal tibiae were studied histologically. Ca content was lower in 21.5-d-old FDR than in control fetuses. FDR had lower plasma osteocalcin (OC) levels and, on histomorphometry, increased hypertrophic cartilage width, indicating retarded bone maturation. Maternal 1,25(OH)2D3 treatment did not change Ca content and hypertrophic cartilage width in FDR. Fetuses of semistarved rats had plasma OC levels and hypertrophic cartilage width comparable to those of control fetuses. Fetuses of rats fed the 1.5% Ca-3.5% Sr diet were more severely hypomineralized than FDR but had higher plasma OC than both FDR and control fetuses, compatible with fetal Ca deficiency. Whereas diabetic placentas showed weak but homogeneous staining of calbindin-D9K in the labyrinth on immunohistology, degenerative zones were present in placentas of rats fed the 1.5% Ca-3.5% Sr diet. Thus, there is no mineralization defect in FDR caused by disturbed maternal duodenal Ca absorption or transplacental Ca transport, but a delay in bone maturation that is unexplained by their lower body weight.

Influence of a vitamin E-deficient diet on prostacyclin production by mesometrial triangles and aortic rings from nondiabetic and diabetic pregnant rats

We investigated the effect of a vitamin E-deficient diet on the synthesis of prostacyclin in nondiabetic and diabetic pregnant rats. In both groups the diet reduced fetal growth and prostacyclin production by mesometrial triangles; it also increased serum lipid peroxides, which are known to inhibit prostacyclin synthesis. Independently of the diet, mesometrial triangles of diabetic rats produced more prostacyclin than those of control rats, whereas an opposite tendency was found in the aortic rings. Our model seems useful for fundamental and pharmacologic studies of the vascular pathology of pregnancy.

Differential Inhibition of Vascular Prostacyclin and Platelet Thromboxane Synthesis by Different Doses Aspirin and by the Thromboxane Inhibitor Ridogrel (R 68070)

The present study has examined the effect of different doses of aspirin and of ridogrel, a compound combining specific TXA2-synthetase and TXA2-prostaglandin endoperoxide receptor blockade in one molecule, on prostacyclin (PGI2) and thromboxane A2 (TXA2)-production in a rat model. Prostaglandin-production by incubated vascular rings and in serum were evaluated. The equivalent of a daily 60 mg aspirin-dose in an adult person, significantly inhibited both serum PGI2-concentration (-44 %) and direct vascular PGI2-production (-50 %). No significant effect on PGI2-formation was seen with a 20 mg aspirin equivalent. There was on the other hand no significant difference in the degree of TXA2-inhibition obtained with the higher (76 %) and lower dose (62 %), so that a more interesting PGI2-TXA2 ratio was obtained with the latter. With ridogrel however a nearly complete (99 %) TXA2-inhibition was acquired with no effect on PGI2. The differences in PGI2-TXA2 balance after intraperitoneally or intramuscularly adminis...

Influence of pregnancy, prostacyclin and labetalol on the vascular-response to angiotensin-ii in the rat

The mean arterial blood pressure and the vascular response to angiotensin II were studied in chronically catheterized and unanesthetized rats.Pregnancy significantly decreased the pressor response. This effect could be mimicked by prostacyclin infusions in non pregnant rats, whereas labetalol had an opposite effect.The model seems suitable for investigations into the control of vascular reactivity, and reinforces the hypothesis that the relative vascular refractoriness to angiotensin II during pregnancy could result, at least in part, from the action of vasodilator prostaglandins, in this instance prostacyclin