Non-pregnant Female Rats Research Articles

In vitro study of the tocolytic effect of oroxylin A from Scutellaria baicalensis root

Scutellariae Radix is one of the well-known tocolytic Chinese herbs. Oroxylin A is isolated from the root of Scutellaria baicalensis. The main syndrome of preterm birth is caused by uterus contractions from excitatory factors. Administration of tocolytic agents is a strategy to prevent the occurrence of preterm births. The aim of this study was to investigate the effects of oroxylin A on contractions of uterine strips isolated from non-pregnant female Wistar rats (250~350 g). Contractions of the uterus were induced with acetylcholine (Ach) (1 μM), PGF2α (0.1 μM), oxytocin (10-3 U/ml), KCl (56.3 mM), tetraethylammonium (TEA; 1 and 10 mM), 4-aminopyridine (4-AP; 5 mM), glipizide (30 μM), a nitric oxide synthase (NOS) inhibitor (LNNA; 10-3M), a β-receptor blocker (propranolol; 10 μM), and a cyclooxygenase inhibitor (indomethacin; 60 μM). The inhibitory effects of the amplitude and frequency of spontaneous contractions by oroxylin A were antagonized with Ach (IC50 22.85 μM), PGF2α (IC5027.28 μM), oxytocin (IC50 12.34 μM), TEA; 1 and 10 mM (IC50 52.73 and 76.43 μM), 4-AP (IC50 67.16 μM), and glipizide (IC5027.53 μM), but oroxylin A was not influenced by Ca2+-free medium, LNNA, propranolol, or indomethacin. Otherwise, oroxylin A-mediated relaxation of the rat uterus might occur through opening of uterine calcium-dependent potassium channels or adenosine triphosphate potassium channel activation. This suggests that oroxylin A is the tocolytic principle constituent of Scutellariae Radix, and oroxylin A may provide a lead compound for new tocolytic drug development in the future.

Uterotonic effect of Harpagophytum procumbens DC (Pedaliaceae) secondary root aqueous extract on rat isolated uterine horns

Some traditional health practitioners of South Africa have claimed that Harpagophytum procumbens DC (family: Pedaliaceae) secondary root is a useful obstetric remedy for induction or acceleration of labour, as well as for expelling retained placentas in pregnant women. In the present study, therefore, we investigated the effect of H. procumbens secondary root aqueous extract (HPE) on longitudinal, tubular uterine horn muscle strips taken from non-pregnant and pregnant, young adult, female rats. HPE (10-800 microg/ml) induced concentration-related and significant (P<0.05) increases in the baseline tone, and caused powerful rhythmic, myogenic contractions of, oestrogen-dominated rat longitudinal uterine horn muscle strips taken from stilboesterol-pretreated, non-pregnant female rats. Relatively low to high concentrations of HPE (10-800 microg/ml) also provoked concentration-dependent and significant (P<0.05-0.001) increases in the baseline tone of, and contracted, longitudinal, tubular uterine horn muscle strips taken from female rats in the early, middle and late stages of pregnancy. Moderate to high concentrations of HPE (200-1,000 microg/ml) always provoked powerful contractions of isolated longitudinal, tubular uterine horn muscle preparations of non-pregnant and pregnant rats. The results of this in vitro study indicate that H. procumbens secondary root aqueous extract possesses spasmogenic, uterotonic action on mammalian uterine muscles. These findings lend pharmacological credence to the suggested folkloric obstetric uses of the plant's secondary root for induction and/or acceleration of labour, as well as for expelling retained placentas in pregnant women.

Collaborative work on evaluation of ovarian toxicity 1) Effects of 2- or 4-week repeated-dose administration and fertility studies with medroxyprogesterone acetate in female rats

As a part of the collaborative study to evaluate the relationship between histopathological changes of the ovary and functional changes in female fertility, 2- and 4-week repeated-dose toxicity studies and a female fertility study were conducted using female Crl:CD(SD) rats using a synthetic progestagen of medroxyprogesterone acetate (MPA) as a test compound. MPA was administered to female rats by gavage at 0, 0.4, 2.0 and 10 mg/kg/day for 2 and 4 weeks to assess the histopathological changes of ovaries and to pregnant rats at the same doses from 2 weeks prior to mating until day 7 of gestation to examine female fertility. The number of non-pregnant female rats with irregular estrous cycle increased in number and there was a decrease in weight of ovaries was observed at doses > or = 2.0 mg/kg in the 2- and 4-week-treatment groups. The histopathological examination revealed an increased number of large atretic follicles and decreases in currently formed corpora lutea and previously-formed large or small ones were observed at the same doses in the 2- and 4-week treatment groups. In female fertility study, the number of animals with an irregular estrous cycle and elongation of mean estrous cycle increased at 0.4 mg/kg, with no changes in fertility. A decreased number of copulating animals and a decreased gestation rate with low preimplantation loss were observed in the 2.0 mg/kg-treatment group and no copulation was observed in the group treated with 10 mg/kg. Based on these results, changes in fertility induced by MPA correlated well with histopathological changes of ovaries after 2 and 4 weeks of treatment, which suggests that a 2 weeks administration period is sufficient to detect ovarian toxicity of MPA with repeated dosing

Synthetic FP-prostaglandin-induced contraction of rat uterus smooth muscle in vitro

Numerous synthetic FP-class prostaglandin (PG) analogs stimulated the contraction of isolated non-pregnant female rat uterus in a concentration-dependent manner with the following agonist potencies: bimatoprost acid (17-phenyl-trinor PGF 2 α ; EC 50=0.68±0.06 nM)=cloprostenol (EC 50=0.73±0.01 nM)>travoprost acid (EC 50=1.3±0.07 nM)>latanoprost acid (EC 50=2.7±0.08 nM)>PGF 2 α (EC 50=52±11 nM)>unoprostone (UF-021; EC 50=310±101 nM)>S-1033 (EC 50=610±4 nM)>bimatoprost (EC 50=1130±173 nM). The FP-receptor antagonist, AL-8810, antagonized the contractile effects of PGF 2 α ( K i=2.9±0.2 μM), travoprost acid ( K i=0.6±0.1 μM) and bimatoprost ( K i=0.2±0.02 μM). Agonist and antagonist potencies for rat uterus contraction by these PGs compared well with their potencies for inducing/blocking functional responses in other systems ( r=0.83–0.94) except with bovine iris sphincter ( r=0.2; p<0.7). In conclusion, the rat uterus contains functionally active FP-receptors whose activation by a variety of free acid and an amide forms of synthetic PGs leads to the contraction of this tissue and which can be pharmacologically blocked by an FP-receptor antagonist, AL-8810.

The comparison of the relaxant effects of propofol, thiopental, ketamine, and etomidate on isolated rat uterine smooth muscle

Background: Intravenous hypnotics are used in pregnancy, labor and delivery. The aim of the present study was to investigate and compare the relaxant effects of propofol, thiopental, ketamine, and etomidate on isolated rat uterine smooth muscles. Methods: Uterine smooth muscle preparations were obtained from non-pregnant female rats. The uterus of the rat was dissected and cut into 10 mm strips. The muscle strips were bathed in Krebs solution. After spontaneous uterine contractile activity had been accomplished, propofol, ketamine, thiopental, and etomidate in various concentrations were added cumulatively to the baths and resting tension, active tension, and frequency of contration were recorded at each concentration of agents. EC5, EC25, EC50, EC75, and EC95 of each drug on active tension and frequency of contraction were calculated using a probit model. Results: Propofol, thiopental, and etomidate reduced uterine contractions in a concentration-dependent manner. Ketamine concentrations of 10−7 to 10−5 M augmented uterine contractions but ketamine concentrations of 10−4 to 10−3 M attenuated uterine contractions. The EC50's of propofol, thiopental, ketamine, and etomidate on active tension were 1.56 × 10−5 M, 4.97 × 10−5 M, 3.52 × 10−4 M, and 2.73 × 10−5 M, respectively. Conclusions: All four intravenous hypnotics relaxed the uterine smooth muscle of rats except for ketamine in low concentrations (10−7 to 10−5 M). Propofol had the greatest relaxant effects on isolated rat uterine smooth muscle among these hypnotics. It seems that ketamine is a suitable obstetric hypnotic agent for hypovolemic parturients and propofol is a useful hypnotic agent for uterine relaxation during pregnancy.

Ovarian blood flow is reflexively regulated by mechanical stimulation of a hindlimb in nonpregnant anesthetized rats

Stimulation of various types of somatosensory receptors is known to modify visceral functions via autonomic nerves in anesthetized animals (Sato et al., 1997). Recent studies have shown that reproductive organs, such as uterus and ovaries are controlled by not only hormone but also autonomic nerves. It is important to know how these reproductive organs are reflexively regulated by somatosensory stimulations via autonomic nerves. In the present experiment, we investigated if the ovarian blood flow is reflexively regulated by somatosensory stimulation in anesthetized nonpregnant adult female rats. Ovarian blood flow at the left side was continuously measured using a laser Doppler flowmeter. A mechanical stimulus by pressing was delivered for 30 s to either a hindpaw or a hind leg, at the right or left side. Pressing stimulation of a hind leg at the right or left side produced a decrease in ovarian blood flow 8+2% accompanied with a decrease in blood pressure. Pressing of a hindpaw produced no effects on these two parameters measured. These responses of ovarian blood flow and blood pressure were abolished after severing the femoral and sciatic nerves at the same side in which stimulation was unilaterally delivered. After severance of ovarian sympathetic nerves, the blood pressure response was not influenced, while the decrease response in ovarian blood flow following pressing of a hind leg was slightly augmented. The activity of sympathetic nerves innervating the ovary was decreased during pressing of a hind leg of either side. Electrical stimulation of the distal part of the severed splanchnic nerve at the left side decreased the left ovarian blood flow, and this response was abolished by intravenous injection of phentolamine, the alpha-adrenoceptor antagonist. The present results demonstrate that ovarian blood flow decrease passively to response of a decrease in systemic arterial blood pressure following mechanical afferent stimulation of a hindlimb by pressing, and this passive response of ovarian blood flow is modulated by reflexive decrease of ovarian sympathetic vasoconstrictor nerves to attenuate blood pressure-dependent passive decrease in ovarian blood flow.

The effects of alcoholic extract of Arbutus andrachne on some biochemical parameters and visceral organs in rats

SummaryThe effect of the alcoholic extract of Arbutus andrachne on certain biochemical parameters and the histology of the visceral organs in nonpregnant adult female rats was studied.The lethal dose 50% (LD)50 was estimated, and three different doses (1/10th , 1/15th and 1/20th ) of the LD50 extracts were injected intra‐peritoneally for 14 consecutive days into three groups of animals (n = 5 in each group), with one group used as a control and injected with distilled water through the same route. Blood was collected after sacrificing the animals and the following biochemical parameters were detected: glutamate pyruvate transaminase (GPT), glutamate oxaloacetate transaminase, alkaline phosphatase, cholesterol, glucose, triglycerides, creatinine and bilirubin. In addition, the total white blood (WBC) and red blood cells (RBC) were estimated in the blood of all groups. The gross and microscopic findings were reported after the collection of specimens from the animals, and processed routinely for standard histological procedures. The results showed an increase in the number of WBC, which indicated a defence response; while the RBC were decreased because of damage in tissues or organs. The gross and the microscopic changes seemed to be dose‐dependent, as very little or no effects were noticed in the lowest treated group, and more obvious changes were detected in the highest‐dose group of animals. In addition, changes were observed in almost all biochemical parameters under investigation, with the exception of creatinine and GPT, which reflect the general effect of the material used on the animal health.

Distribution and characterization of estrogen receptor G protein-coupled receptor 30 in the rat central nervous system

The G protein-coupled receptor 30 (GPR 30) has been identified as the non-genomic estrogen receptor, and G-1, the specific ligand for GPR30. With the use of a polyclonal antiserum directed against the human C-terminus of GPR30, immunohistochemical studies revealed GPR30-immunoreactivity (irGPR30) in the brain of adult male and non-pregnant female rats. A high density of irGPR30 was noted in the Islands of Calleja and striatum. In the hypothalamus, irGPR30 was detected in the paraventricular nucleus and supraoptic nucleus. The anterior and posterior pituitary contained numerous irGPR30 cells and terminal-like endings. Cells in the hippocampal formation as well as the substantia nigra were irGPR30. In the brainstem, irGPR30 cells were noted in the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus; a cluster of cells were prominently labeled in the nucleus ambiguus. Tissue sections processed with pre-immune serum showed no irGPR30, affirming the specificity of the antiserum. G-1 (100 nM) caused a large increase of intracellular calcium concentrations [Ca(2+) ](i) in dissociated and cultured rat hypothalamic neurons, as assessed by microfluorometric Fura-2 imaging. The calcium response to a second application of G-1 showed a marked homologous desensitization. Our result shows a high expression of irGPR30 in the hypothalamic-pituitary axis, hippocampal formation, and brainstem autonomic nuclei; and the activation of GPR30 by G-1 is associated with a mobilization of calcium in dissociated and cultured rat hypothalamic neurons.

Distribution of androstenedione and its effects on total free fatty acids in pregnant rats

Androstenedione, an anabolic steroid used to enhance athletic performance, was administered in corn oil by gastric intubation once daily in the morning to nonpregnant female rats at a dose of 5 or 60 mg/kg/day, beginning two weeks before mating and continuing through gestation day (GD) 19. On GD 20, the distribution of androstenedione and other steroid metabolites was investigated in the maternal plasma and target organs, including brain and liver. The concentration of estradiol in plasma approached a statistically significant increase after treatment as compared with the controls, whereas the levels of androstenedione, testosterone and progesterone were not significantly different from the controls. In the liver, the concentrations of androstenedione and estradiol only were increased in a dose-related manner. None of these steroids was detectable in the brain. Androstenedione treatment also produced changes in the level of selected free fatty acids (FFAs) in the maternal blood, brain, liver and fetal brain. The concentrations of palmitic acid (16:0) and stearic acid (18:0) in the plasma were not significantly different between the controls and treated rats. However, oleic acid (18:1), linoleic acid (18:2) and docosahexaenoic acid (DHA, 22:6) were 17.94 +/- 2.06 microg/ml, 24.23 +/- 2.42 microg/ml and 4.08 +/- 0.53 microg/ml, respectively, in the controls, and none of these fatty acids was detectable in the treated plasma. On the other hand, palmitic, stearic, oleic, linoleic and DHA were present in both control and treated livers. Among the FFAs in liver, linoleic and DHA were increased 87% and 169%, respectively, over controls. Palmitic, stearic and oleic acids were not significantly affected by the 60 mg/kg treatment. These were present in both control maternal and fetal brains, whereas linoleic acid was found only in fetal brain control. DHA was present only in the control maternal brain (0.02 +/- 0.02 microg/mg protein) and fetal brain (0.24 +/- 0.15 microg/mg protein). The results indicated that androstenedione exhibits significantly different effects on the FFA composition among target organs during pregnancy.

Acute toxicity of sodium arsenite in a complex food matrix

Acute toxicity of a single oral dose of sodium arsenite (As), administered in half and half cream (HH), was assessed in male and non-pregnant female rats (0.41, 4.1, 41.0 and 410.0 mg/kg body weight) and pregnant rats (0.41, 4.1 and 41.0 mg/kg body weight). Control rats received deionized water alone, HH alone or 41.0 mg/kg As in deionized water (41 mg/kg As–water). Male and non-pregnant rats were monitored for 14 consecutive days post-dosing. Pregnant rats, dosed on gestation day 10 (GD-10), were monitored until fetuses were collected on GD 20. High mortality (100%) was observed in male and non-pregnant female rats exposed to 410.0 mg/kg As-HH. Low mortality (25%) was observed in non-pregnant female rats exposed to 41 mg/kg As–water. No mortality was observed in other control or treated groups. Reduced female fetal numbers were observed in the 41 mg/kg As–water group but not in the other control groups. Developmental effects were not observed in the controls or the As-HH treatment groups. In conclusion, As toxicity was not reduced when a high dose (410 mg/kg) was administered in HH however, at lower doses (41 mg/kg), HH reduced acute As oral toxicity in the female and developing fetus.

Metabotropic glutamate receptor/phospholipase C pathway is increased in rat brain at the end of pregnancy

Wistar pregnant rats were sacrificed at the end of pregnancy and the status of metabotropic glutamate receptors/phospholipase C (mGluR/PLC) pathway was studied in brain from pregnant and non-pregnant female rats. Pregnancy causes a significant increase in metabotropic glutamate receptors number, determined by radioligand binding assay, without significant changes on receptor affinity. Similar increase in mGluR 1 type was obtained by immunoblotting assay using specific anti-mGluR 1 antibody. However, no significant differences were observed in mGluR 5 type, suggesting that the increase detected by radioligand assays could be due to mGluR 1 up-regulation. On the other hand, a significant increase in the α subunit of G q protein was also detected in pregnant rats by immunoblotting assays. Real-time PCR experiments revealed a significant increase in gene expression of metabotropic glutamate receptors and G q proteins. Neither protein level nor gene expression of phospholipase C β 1 isoform was altered in pregnant rats. However, an increase in basal and agonist-stimulated phospholipase C activity was observed in membranes from pregnant rats. These results suggest that gestational period causes the up-regulation of both metabotropic glutamate receptors and coupled G q-protein and, in turn, an increase in phospholipase C activity.

Peroxisome Proliferator–Activated Receptor-α and Glucocorticoids Interactively Regulate Insulin Secretion During Pregnancy

We evaluated the impact of peroxisome proliferator-activated receptor (PPAR)alpha activation and dexamethasone treatment on islet adaptations to the distinct metabolic challenges of fasting and pregnancy, situations where lipid handling is modified to conserve glucose. PPARalpha activation (24 h) in vivo did not affect glucose-stimulated insulin secretion (GSIS) in nonpregnant female rats in the fasted state, although fasting suppressed GSIS. Dexamethasone treatment (5 days) of nonpregnant rats lowered the glucose threshold and augmented GSIS at high glucose; the former effect was selectively opposed by PPARalpha activation. Pregnancy-induced changes in GSIS were opposed by PPARalpha activation at day 19 of pregnancy. Dexamethasone treatment from day 14 to 19 of pregnancy did not modify the GSIS profile of perifused islets from 19-day pregnant rats but rendered the islet GSIS profile refractory to PPARalpha activation. During sustained hyperglycemia in vivo, dexamethasone treatment augmented GSIS in nonpregnant rats but limited further modification of GSIS by pregnancy. We propose that the effect of PPARalpha activation to oppose lowering of the glucose threshold for GSIS by glucocorticoids is important as part of the fasting adaptation, and modulation of the islet GSIS profile by glucocorticoids toward term facilitates the transition of maternal islet function from the metabolic demands of pregnancy to those imposed after parturition.

Relaxin Is Essential for Systemic Vasodilation and Increased Global Arterial Compliance during Early Pregnancy in Conscious Rats

During early pregnancy, there are marked increases in cardiac output (CO) and global arterial compliance (AC), as well as decreases in systemic vascular resistance (SVR). We recently reported that administration of recombinant human relaxin to nonpregnant female rats elicits changes in systemic hemodynamics and arterial mechanical properties similar to those observed during normal pregnancy. In the present study, we directly tested whether endogenous relaxin mediates the cardiovascular adaptations of pregnancy by neutralizing circulating relaxin with monoclonal antibodies during early gestation. Relaxin neutralizing antibodies were administered daily, beginning on d 8 of rat gestation, to block the functional effects of circulating relaxin. Systemic hemodynamics and arterial properties were assessed between gestational d 11 and 15 using techniques we have previously reported. Pregnant rats administered the neutralizing antibodies failed to exhibit the gestational increases in stroke volume, CO, and global AC or decreases in SVR that were observed in control pregnant rats administered an irrelevant antibody against fluorescein or PBS. In fact, in the pregnant rats administered the relaxin neutralizing antibodies, cardiovascular parameters were not statistically different from those in virgin rats. Interestingly, small renal and first-order mesenteric arteries isolated from midterm pregnant rats administered either relaxin-neutralizing or control antibodies did not exhibit any changes in passive mechanical properties compared with virgin rats. These findings indicate that circulating relaxin mediates the transition of the systemic circulation from the virgin to the pregnant state in the gravid rat model, suggesting a potential role for aberrant relaxin regulation in abnormal pregnancies wherein these cardiovascular adaptations are inadequate or excessive.

Suppression of Prolactin-Induced Signal Transducer and Activator of Transcription 5b Signaling and Induction of Suppressors of Cytokine Signaling Messenger Ribonucleic Acid in the Hypothalamic Arcuate Nucleus of the Rat during Late Pregnancy and Lactation

During late pregnancy and lactation, the tuberoinfundibular dopamine (TIDA) neurons that regulate prolactin secretion by negative feedback become less able to produce dopamine in response to prolactin, leading to hyperprolactinemia. Because prolactin-induced activation of dopamine synthesis in these neurons requires the Janus kinase/signal transducer and activator of transcription 5b (STAT5b) signaling pathway, we investigated whether prolactin-induced STAT5b signaling is reduced during lactation and whether induction of suppressors of cytokine signaling (SOCS) mRNAs occur at this time and in late pregnancy. During lactation, the ability of exogenous prolactin to induce STAT5 phosphorylation and STAT5b nuclear translocation was markedly reduced when compared with diestrous rats. In nonpregnant female rats, acute treatment with ovine prolactin markedly increased levels of SOCS-1 and -3 and cytokine-inducible SH2-containing protein mRNA in arcuate nucleus micropunches. On gestation d 22, SOCS-1 and SOCS-3 mRNA levels were 10-fold that on G20. SOCS-1 and -3 and cytokine-inducible SH2-containing protein mRNA levels were also elevated on lactation d 7. At these times, dopaminergic activity was decreased and the rats were hyperprolactinemic. The high levels of SOCS mRNA were prevented by bromocriptine pretreatment (gestation d 22) or pup removal (lactation d 7), which suppressed circulating prolactin to basal levels. These results demonstrate that around the end of pregnancy, prolactin loses the ability to activate STAT5b, associated with an increase in SOCS mRNAs. The loss of this stimulating pathway may underlie the reduced tuberoinfundibular dopamine neuron dopamine output and hyperprolactinemia that characterizes late pregnancy and lactation. The high maternal levels of SOCS mRNAs appear to be dependent on prolactin, presumably acting through an alternative signaling pathway to STAT5b.

Effects of oral androstenedione on phospholipid fatty acids, ATP, caspase-3, prostaglandin E 2 and C-reactive protein in serum and livers of pregnant and non-pregnant female rats

Androstenedione, a steroidal dietary supplement taken to enhance athletic performance, could affect serum and liver lipid metabolism, induce liver toxicity or alter inflammatory response depending on dose and duration of exposure. Pregnancy could further exaggerate these effects. To examine this, mature female rats were gavaged with 0, 5, 30 or 60 mg/kg/day androstenedione beginning two weeks prior to mating and continuing through gestation day 19. Non-pregnant female rats were gavaged over the same time frame with 0 or 60 mg/kg/day androstenedione. Serum was collected and livers were removed from dams on gestation day 20 and from non-pregnant rats after 5 weeks of treatment. Androstenedione had no effect on serum total cholesterol, triglycerides or HDL-cholesterol, but significantly decreased C-reactive protein in pregnant rats and prostaglandin E 2 in serum of both pregnant and non-pregnant rats. There were treatment related decreases in liver ATP and, to a lesser degree, caspase-3 and no change in alkaline phosphatase of pregnant female rats. Androstenedione decreased docosahexaenoic acid in both serum and liver phospholipids of pregnant female rats. In conclusion, oral androstenedione did not result in overt hepatotoxicity in pregnant female rats, but produced modest changes in lipid metabolism and may impair regeneration of injured hepatic cells or tissue.

Concentrations of N-methyl-2-pyrrolidone (NMP) and its metabolites in plasma and urine following oral administration of NMP to rats

The primary aims were to study the metabolism in rats and to determine the biological levels after one oral developmentally toxic dose of N-methyl-2-pyrrolidone (NMP), a widely used industrial chemical. Non-pregnant female Sprague-Dawley rats were given an oral single dose of either a non-toxic dose of 125 mg NMP/kg (group 1) by gavage or a developmentally toxic dose of 500 mg/kg (group 2). Blood plasma (7 rats per time point) and urine (10 rats per time point) were sampled up to 72 h after administration and analyzed using mass spectrometry. In both plasma and urine NMP, 5-hydroxy- N-methyl-2-pyrrolidone (5-HNMP), N-methylsuccinimide and 2-hydroxy- N-methylsuccinimide (2-HMSI) and 2-pyrrolidone (2-P) were identified. In urine 48% of the administered dose was recovered as 5-HNMP and 2–5% as 2-HMSI. The total recovery in urine was 53–59%. The peak concentrations for NMP in plasma were 1.2 and 6.9 mmol/l, 0.42 and 0.76 mmol/l for 5-HNMP, 0.07 and 0.31 mmol/l for MSI and for 2-HMSI the concentrations were 0.02 and 0.05 mmol/l for groups 1 and 2, respectively. In summary, the same metabolites were found in rats as in humans and the biological levels were reported for NMP and its metabolites after oral exposure to a developmentally toxic dose and one non-toxic dose of NMP.

Synthesis of Oxytocin Analogues with Replacement of Sulfur by Carbon Gives Potent Antagonists with Increased Stability

[Chemical reaction: See text] The neuropeptide oxytocin 1 controls mammary and uterine smooth muscle contraction. Atosiban 2, an oxytocin antagonist, is used for prevention of preterm labor and premature birth. However, the metabolic lifetimes of such peptide drugs are short because of in vivo degradation. Facile production of oxytocin analogues with varying ring sizes wherein sulfur is replaced by carbon (methylene or methine) could be achieved by standard solid-phase peptide synthesis using olefin-bearing amino acids followed by on-resin ring-closing metathesis (RCM). These were tested for agonistic and antagonistic uteronic activity using myometrial strips taken from nonpregnant female rats. Peptide 8 showed agonistic activity in vitro (EC50= 1.4 x 10(3) +/- 4.4 x 10(2) nM) as compared to 1 (EC50= 7.0 +/- 2.1 nM). Atosiban analogues 17 (pA2= 7.8 +/- 0.1) and 18 (pA2= 8.0 +/- 0.1) showed substantial activity compared to the parent oxytocin antagonist 2 (pA2= 9.9 +/- 0.3). Carba analogue 35 (pA2= 6.1 +/- 0.1) had an agonistic activity over 2 orders of magnitude less than its parent 3 (8.8 +/- 0.5). A comparison of biological stabilities of 1,6-carba analogues of both an agonist 8 and antagonist 18 versus parent peptides 1 and 2 was conducted. The half-lives of peptides 8 and 18 in rat placental tissue were shown (Table 2) to be greatly improved versus their parents oxytocin 1 and atosiban 2, respectively. These results suggest that peptides 8 and 18 and analogues thereof may be important leads into the development of a long-lasting, commercially available therapeutic for initiation of parturition and treatment of preterm labor.

Effects of oral androstenedione on steroid metabolism in liver of pregnant and non-pregnant female rats

It is unknown whether androstenedione, a steroidal dietary supplement taken to enhance athletic performance, can affect physiological hormone levels by altering liver enzyme activities that metabolize steroid hormones. Altered hormone levels could be especially devastating during pregnancy. Mature female rats were gavaged with 0, 5, 30 or 60 mg/kg/day androstenedione beginning two weeks prior to mating and continuing through gestation day 19. Non-pregnant female rats were gavaged over the same time frame with 0 or 60 mg/kg/day androstenedione. Livers were removed from dams on gestation day 20 and from non-pregnant rats after five weeks’ treatment. Liver microsomes were incubated with 200 μM testosterone, and the reaction products were isolated and analyzed by HPLC. In pregnant rats, formation of 6α-, 15β-, 7α-, 16β-, and 2β-hydroxytestosterone was increased significantly vs. control at the highest dose level only. Formation of 6β-hydroxytestosterone increased significantly at both the 30 and 60 mg/kg/day dose levels. In non-pregnant rats, 60 mg/kg/day androstenedione significantly increased formation of 15β-, 6β-, 16β-, and 2β-hydroxytestosterone. The data suggest that high oral doses of androstenedione can induce some female rat liver cytochromes P450 that metabolize steroid hormones and that the response to androstenedione does not differ between pregnant and non-pregnant female rats.

Effects of Pregnancy, Age and Sex in the Metabolism of Styrene in Rat Liver in Relation to the Regulation of Cytochrome P450 Enzymes

To elucidate the effect of maternal styrene exposure, which is due to various postnatal changes in the development and behavior of offspring, we investigated pregnancy-induced changes in the metabolism of styrene in rat liver in relation to the regulation of cytochrome P450 enzymes. We also examined age and sex-induced changes in the metabolism of styrene. Pregnancy appeared to exert a negative effect on cytochrome P450 content at the late stage, whereas microsomal protein content showed little change during pregnancy. Pregnancy significantly decreased the rate of formation of styrene glycol at the late stage. The percentage of remaining activity in microsomes exposed to anti-CYP2E1 was lower than that exposed to anti-CYP2C11/6 in pregnant and non-pregnant female rats and immature male rats, indicating that CYP2E1 contributes to the metabolism of styrene more than CYP2C11/6 in these rats. Although pregnancy seemed to decrease styrene metabolism, the contribution of CYP2E1 seemed to be slightly increasing. In conclusion, pregnancy clearly influences the metabolism of styrene as well as other characteristic factors such as age and sex. It is very important to elucidate the changes in specific P450 isozyme composition related to their characteristic modification and in their affinity for chemicals.

Relaxin-induced changes in renal sodium excretion in the anesthetized male rat

Pregnancy is associated with profound changes in renal hemodynamics and electrolyte handling. Relaxin, a hormone secreted by the corpus luteum, has been shown to induce pregnancy-like increases in renal blood flow and glomerular filtration rate (GFR) and alter osmoregulation in nonpregnant female and male rats. However, its effects on renal electrolyte handling are unknown. Accordingly, the influence of short (2 h)- and long-term (7 day) infusion of relaxin on renal function was determined in the male rat. Short term infusion of recombinant human relaxin (rhRLX) at 4 microg.h(-1).100 g body wt(-1) induced a significant increase in effective renal blood flow (ERBF) within 45 min, which peaked at 2 h of infusion (vehicle, n = 6, 2.1 +/- 0.4 vs. rhRLX, n = 7, 8.1 +/- 1.1 ml.min(-1).100 g body wt(-1), P < 0.01). GFR and urinary excretion of electrolytes were unaffected. After a 7-day infusion of rhRLX at 4 microg/h, ERBF (1.4 +/- 0.2 vs. 2.5 +/- 0.4 ml.min(-1).100 g body wt(-1), P < 0.05), urine flow rate (3.1 +/- 0.3 vs. 4.3 +/- 0.4 microl.min(-1).100 g body wt(-1), P < 0.05) and urinary sodium excretion (0.8 +/- 0.1 vs. 1.2 +/- 0.1 micromol.min(-1).100 g body wt(-1), P < 0.05) were significantly higher; plasma osmolality and sodium concentrations were lower in rhRLX-treated rats. These data show that long-term relaxin infusion induces a natriuresis and diuresis in the male rat. The mechanisms involved are unclear, but they do not involve changes in plasma aldosterone or atrial natriuretic peptide concentrations.