Effect of particle size on the biodistribution of lipid nanocapsules: Comparison between nuclear and fluorescence imaging and counting

In vivo biodistribution of nanoparticles depends on several physicochemical parameters such as size. After intravenous injection of 25, 50 and 100 nm lipid nanocapsules (LNC) in nude mice bearing HEK293(β₃) tumour xenografts, biodistribution was evaluated by γ-scintigraphy and by γ-counting. The small LNC 25 nm disappeared faster than the larger LNC 50 and 100 nm from the blood circulation due to faster elimination and wider tissue distribution. At 24 h, biodistribution profiles of all these LNC were similar. Low LNC quantities were found in this weak EPR (enhanced permeability and retention) tumour regardless the particle size. Co-injected 50 nm fluorescent DiD-LNC and ^99mTc-LNC allowed direct comparison of biodistribution as evaluated by the two methods. Optical imaging underestimated LNC quantity especially in dark-colored organs that were observed to capture extensive quantities of the particles by γ-counting (i.e. liver, spleen, and kidney).     

Developmental toxic potential of 1,3-dichloro-2-propanol in Sprague–Dawley rats

This study investigated the potential adverse effects of 1,3-dichloro-2-propanol (1,3-DCP) on pregnant dams and the embryo-fetal development after maternal exposure on gestational days (GD) 6 through 19 in Sprague–Dawley rats. The test chemical was administered to pregnant rats by gavage at dose levels of 0, 10, 30, and 90 mg/kg per day (n = 10 for each group). All dams underwent Caesarean sections on GD 20, and their fetuses were examined for morphological abnormalities. Maternal toxicity was noted at 90 mg/kg/day. Manifestations of toxicity included clinical signs of illness, lower body weight gain, decreased food intake, and increases in the weight of the adrenal glands and the liver. Developmental toxic effects including decreases in fetal body weight and increases in visceral and skeletal variations also occurred at the highest dose. At 30 mg/kg, only a minimal maternal toxicity, including a decrease in maternal food intake and an increase in the liver weight, was observed. No adverse maternal or developmental effects were observed at 10 mg/kg/day. These results revealed that a 14-day repeated oral dose of 1,3-DCP was minimally embryotoxic but not teratogenic at a maternal toxic dose (90 mg/kg/day), and was not embryotoxic at a minimally maternal toxic dose (30 mg/kg/day) in rats. Because the developmental toxicity of 1,3-DCP was observed only in the presence of maternal toxicity, it is concluded that the developmental findings observed in the present study are secondary effects to maternal toxicity. Under these experimental conditions, the no-observed-adverse-effect level of 1,3-DCP is considered to be 10 mg/kg/day for dams and 30 mg/kg/day for embryo-fetal development.     

Maternal fenvalerate exposure during pregnancy persistently impairs testicular development and spermatogenesis in male offspring

Fenvalerate, a widely used pyrethroid insecticide, has been associated with poor semen quality. As yet, little is known about the effects of prenatal fenvalerate exposure on testicular development. The present study investigated the effects of prenatal fenvalerate exposure on testicular development and spermatogenesis. The pregnant mice were administered fenvalerate (30 mg/kg) by gavage daily from gestational day (gd) 13 to gd 18. The weights of testes and epididymides were significantly decreased in mice whose mothers were exposed to fenvalerate during pregnancy. Importantly, maternal fenvalerate exposure during pregnancy markedly decreased the number of mature seminiferous tubules (stages VII and VIII) in testes of adult male offspring. In addition, maternal fenvalerate exposure during pregnancy significantly reduced the number of epididymal spermatozoa in adult male offspring. Additional experiments showed that the level of serum testosterone (T) was significantly decreased in male fetuses whose mothers were exposed to fenvalerate during pregnancy. Correspondingly, mRNA and protein levels of P450_17α, a T synthetic enzyme, were significantly decreased in fetal testes. Moreover, the disruptive effect of prenatal fenvalerate exposure on testicular T synthesis was irreversible. In conclusion, prenatal fenvalerate exposure irreversibly impairs testicular development and spermatogenesis at least into early adulthood.     

Particle size-dependent and surface charge-dependent biodistribution of gold nanoparticles after intravenous administration

Gold nanoparticles (GNP) provide many opportunities in imaging, diagnostics, and therapies of nanomedicine. Hence, their biokinetics in the body are prerequisites for specific tailoring of nanomedicinal applications and for a comprehensive risk assessment.We administered ¹⁹⁸Au-radio-labelled monodisperse, negatively charged GNP of five different sizes (1.4, 5, 18, 80, and 200 nm) and 2.8 nm GNP with opposite surface charges by intravenous injection into rats. After 24 h, the biodistribution of the GNP was quantitatively measured by gamma-spectrometry.The size and surface charge of GNP strongly determine the biodistribution. Most GNP accumulated in the liver increased from 50% of 1.4 nm GNP to >99% of 200 nm GNP. In contrast, there was little size-dependent accumulation of 18-200 nm GNP in most other organs. However, for GNP between 1.4 nm and 5 nm, the accumulation increased sharply with decreasing size; i.e. a linear increase with the volumetric specific surface area. The differently charged 2.8 nm GNP led to significantly different accumulations in several organs.We conclude that the alterations of accumulation in the various organs and tissues, depending on GNP size and surface charge, are mediated by dynamic protein binding and exchange. A better understanding of these mechanisms will improve drug delivery and dose estimates used in risk assessment.     

In vivo evaluation of poly-l-asparagine nanocapsules as carriers for anti-cancer drug delivery

Here, we report the in vivo proof of-concept of a novel nanocarrier, poly-l-asparagine (PASN) nanocapsules, as an anticancer targeted drug delivery system. The nanocapsules were loaded with the fluorescent marker DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate) and also with the model drug docetaxel to evaluate the biodistribution and efficacy profiles in healthy and glioma-bearing mice, respectively. Regardless of their cargo, the nanocapsules presented a size close to 180 nm, a surface charge around −40 mV and an encapsulation efficiency of 75–90%. The biodistribution study in healthy mice showed that PASN nanocapsules led to a two- and three-fold increment in the mean residence time (MRT) and area under the curve (AUC) values, respectively, compared to those of a non-polymeric nanoemulsion. Finally, the efficacy/toxicity study indicated that the encapsulated drug was as efficacious as the commercial formulation (Taxotere^®), with the additional advantage of being considerably less toxic. Overall, these results suggest the potential of PASN nanocapsules as drug nanocarriers in anticancer therapy.     

Acute hyperglycemia in uterine arteries from pregnant, but not non-pregnant mice, enhances endothelium-dependent relaxation

Poorly controlled diabetes in pregnancy, characterized by hypo- and hyperglycemia, is associated with adverse outcomes. We hypothesized that aberrant glucose levels affect vascular function in pregnancy. The effects of glucose concentration on constriction and endothelium-dependent relaxation in uterine arteries of normal C57BL/6 mice were examined. Ex-vivo arteries from 18 pregnant and 14 non-pregnant mice were mounted on a wire myograph, constricted with phenylephrine and relaxed with incremental doses of acetylcholine, in physiological saline solution containing 5 mmol/L glucose. Arteries were then exposed to solutions with 2, 5, 8 or 12 mmol/L glucose for 30 min and constriction/relaxation repeated. On altering glucose concentrations to 2, 8 or 12 mmol/L, maximal constriction was increased in arteries from pregnant but not from non-pregnant mice (paired t-test, p<0.05). Endothelium-dependent relaxation was enhanced at 12 mmol/L glucose in arteries from pregnant (two-way ANOVA, p<0.01), but not from non-pregnant mice. Endothelium-dependent relaxation in the uterine artery was pre-dominantly mediated by a non-nitric oxide/non-prostanoid mechanism, with a smaller contribution from nitric oxide, and no prostanoid-mediated relaxation. In summary, acute changes in glucose concentration alter both constriction and endothelium-dependent relaxation in uterine arteries of normal pregnant mice; these effects are unique to pregnancy.     

Effects of maternal exposure to di(2-ethylhexyl)phthalate (DEHP) during pregnancy on susceptibility to neonatal asthma

Di(2-ethylhexyl) phthalate (DEHP) is used as a plasticizer and is widely dispersed in the environment. In this study, we investigated the effects of maternal exposure to DEHP during pregnancy on neonatal asthma susceptibility using a murine model of asthma induced by ovalbumin (OVA). Pregnant BALB/c mice received DEHP from gestation day 13 to lactation day 21. Their offspring were sensitized on postnatal days (PNDs) 9 and 15 by intraperitoneal injection of 0.5 μg OVA with 200 μg aluminum hydroxide. On PNDs 22, 23 and 24, live pups received an airway challenge of OVA for 30 min. Offspring from pregnant mice that received DEHP showed reductions in inflammatory cell count, interleukin (IL)-4, IL-13, and eotaxin in their bronchoalveolar lavage fluid and in total immunoglobulin E and OVA-specific IgE in their plasma compared with offspring from pregnant mice that did not receive DEHP treatment. These results were consistent with histological analysis and immunoblotting. Maternal exposure to DEHP reduces airway inflammation and mucus production in offspring, with a decrease in inducible nitric oxide synthase (iNOS) in the lung tissue. This study suggests that maternal exposure to DEHP during pregnancy reduces asthmatic responses induced by OVA challenge in offspring. These effects were considered to be closely related to the suppression of Th2 immune responses and iNOS expression.     

DNA damage and apoptosis of endometrial cells cause loss of the early embryo in mice exposed to carbon disulfide

Carbon disulfide (CS₂) may lead to spontaneous abortion and very early pregnancy loss in women exposed in the workplace, but the mechanism remains unclear. We designed an animal model in which gestating Kunming strain mice were exposed to CS₂ via i.p. on gestational day 4 (GD4). We found that the number of implanted blastocysts on GD8 was significantly reduced by each dose of 0.1 LD₅₀ (157.85 mg/kg), 0.2 LD₅₀ (315.7 mg/kg) and 0.4 LD₅₀ (631.4 mg/kg). In addition, both the level of DNA damage and apoptosis rates of endometrial cells on GD4.5 were increased, showed definite dose–response relationships, and inversely related to the number of implanted blastocysts. The expressions of mRNA and protein for the Bax and caspase-3 genes in the uterine tissues on GD4.5 were up-regulated, while the expressions of mRNA and protein for the Bcl-2 gene were dose-dependently down-regulated. Our results indicated that DNA damage and apoptosis of endometrial cells were important reasons for the loss of implanted blastocysts induced by CS₂.     

Renal Toxicity after Oral Administration of Lead Acetate during Pre- and Post-Implantation Periods: Effects on Trace Metal Composition,Metallo-Enzymes and Glutathione

Abstract: The present study was undertaken to compare the effects of 10–50 mg/kg b.wt. of Pb acetate after chronic treatment through oral gavage on: (a) the distribution of trace elements such as Fe, Cu, Zn, and Mn, (b) enzyme activity of δ-amino levulinic acid dehydratase (δ-ALAD) and alkaline phosphatase, and (c) glutathione (GSH) in kidney and (d) δ-ALAD in blood of pregnant and non-pregnant mice. Treatment with Pb acetate was given on every alternate day for 4 weeks prior to mating and for 3–4 weeks until pregnancy became apparent and confirmed by laporatomy. Lead administration reduced the rate of reproduction as assessed by number of living viable embryos. During normal pregnancy renal Cu, Fe and GSH tended to decline although non-significantly and continued to do so after lead administration. Mn was considerably and significantly elevated, whereas activity of δ–ALAD (non-activated) was quite low in pregnant mice. Following administration of Pb acetate, kidneys of pregnant and non-pregnant dams accumulated Pb in a dose-dependent manner, but as compared to non-pregnant mice, Pb increase in pregnant dams was significantly lower. Pb toxicity was associated with the loss of δ-ALAD in blood and kidney, but unlike the non-activated form of δ-ALAD, the dithiothreitol-activated form of δ-ALAD declined in a significant amount. The residual activity showed a high degree of negative correlation with endogenous Pb as well as with Zn/Pb ratio. Pb toxicity did not modify renal Fe, Cu and Zn in the nonpregnant state, but reduced renal Fe during pregnancy. On the other hand, Mn decline is a sensitive indicator of Pb toxicity and correlated with the Pb content during pregnancy. While the increase in renal alkaline phosphatase activity in pregnancy persisted even after Pb administration up to 25 mg/kg b.wt. Pb acetate, the activity of alkaline phosphatase in non-pregnant mice did not change after lead treatment. The level of alkaline phosphatase did not correlate with the Zn content but correlated with endogenous levels of Pb, Mn or Zn/Mn ratio. Increase in GSH during the pregnant and nonpregnant state after Pb exposure was apparent. It appears that the body attempts to regulate the Pb toxicity by promoting self-defence by enhanced production of thiol compounds such as glutathione. It is concluded that pregnancy modifies renal lead retention and lead toxicity in terms of trace metal composition and some metalloenzymes.     

Role of transporter-mediated efflux in the placental biodisposition of bupropion and its metabolite, OH-bupropion

Cigarette smoking during pregnancy is a preventable risk factor associated with maternal and fetal complications. Bupropion is an antidepressant used successfully for smoking cessation in non-pregnant patients. Our goal is to determine whether it could benefit the pregnant patient seeking smoking cessation. The aim of this investigation was to determine the role of human placenta in the disposition of bupropion and its major hepatic metabolite, OH-bupropion. The expression of efflux transporters P-gp and BCRP was determined in placental brush border membrane (n = 200) and revealed a positive correlation (p < 0.05). Bupropion was transported by BCRP (K_t 3 μM, V_max 30 pmol/mg protein/min) and P-gp (K_t 0.5 μM, V_max 6 pmol/mg protein*min) in placental inside-out vesicles (IOVs). OH-bupropion crossed the dually-perfused human placental lobule without undergoing further metabolism, nor was it an efflux substrate of P-gp or BCRP. In conclusion, our data indicate that human placenta actively regulates the disposition of bupropion (via metabolism, active transport), but not its major hepatic metabolite, OH-bupropion.     

Mannosylated gelatin nanoparticles bearing isoniazid for effective management of tuberculosis

The mannosylated gelatin nanoparticles (Mn-GNPs) were prepared for the selective delivery of an antitubercular drug, isoniazid (INH), to the alveolar macrophages. The gelatin nanoparticles (GNPs) were prepared by using a two-step desolvation method and efficiently conjugated with mannose. Various parameters such as particle size, polydispersity index, zeta potential, % entrapment efficiency, in vitro drug release, macrophage uptake, in vivo biodistribution, antitubercular activity and hepatotoxicity of plain and Mn-GNPs were determined. The size of nanoparticles (both plain and Mn-GNPs) was found to be in range of 260–380?nm, and maximum drug payload was found to be 40–55%. Average particle size of Mn-GNPs was more, whereas drug entrapment was lesser compared to plain GNPs. The organ distribution studies demonstrated the efficiency of Mn-GNPs for spatial delivery of INH to alveolar tissues. Intravenous administration of INH loaded Mn-GNPs (I-Mn-GNPs) resulted in significant reduction in bacterial counts in the lungs and spleen of tuberculosis-infected (TB-infected) mice and also reduction in the hepatotoxicity of the drug. This study revealed that mannose conjugated GNPs may be explored as potential carrier for safer and efficient management of TB through targeted delivery of INH when compared to plain GNPs and free drug.     

Protein binding modulates the cellular uptake of silver nanoparticles into human cells: Implications for in vitro to in vivo extrapolations?

Nanoparticles (NP) absorbed in the body will come in contact with blood proteins and form NP/protein complexes termed protein coronas, which may modulate NP cellular uptake. This study quantitated human epidermal keratinocyte (HEK) uptake of silver (Ag) NP complexed to different human serum proteins. Prior to HEK dosing, AgNP (20 nm and 110 nm citrate BioPure™; 40 nm and 120 nm silica-coated) were preincubated for 2 h at 37 °C without (control) or with physiological levels of albumin (44 mg/ml), IgG (14.5 mg/ml) or transferrin (3 mg/ml) to form protein-complexed NP. HEK were exposed to the protein incubated AgNP for 3 h, rinsed and incubated for 24 h, rinsed in buffer and lysed. Ag was assayed by inductively-coupled plasma optical emission spectrometry. Uptake of Ag in HEK was <4.1% of applied dose with proteins suppressing citrate, but not silica coated Ag uptake. IgG exposure dramatically reduced 110 nm citrate AgNP uptake. In contrast, greatest uptake of 20 nm silica AgNP was seen with IgG, while 110 nm silica AgNP showed minimal protein effects. Electron microscopy confirmed cellular uptake of all NP but showed differences in the appearance and agglomeration state of the NP within HEK vacuoles. This work suggests that NP association with different serum proteins, purportedly forming different protein coronas, significantly modulates Ag uptake into HEK compared to native NP uptake, suggesting caution in extrapolating in vitro uptake data to predict behavior in vivo where the nature of the protein corona may determine patterns of cellular uptake, and thus biodistribution, biological activity and toxicity.     

Effects of pregnancy on the metabolism of drugs in the rat and rabbit

In rats 19–20 days pregnant, liver weight is increased by 40 per cent, cytochrome P-450 concentration is decreased by 25 per cent and the specific activities of 4-methylumbelliferone glucuronyl transferase and biphenyl-4-hydroxylase are reduced by 25 and 30 per cent, respectively; biphenyl-2-hydroxylase and p-nitrobenzoic acid reductase are not changed. In rats, 15–16 days pregnant, liver weight is increased by 33 per cent but the concentration of cytochrome P-450 and the specific activities of the drug microsomal enzymes are unchanged. Expressed as total amounts per whole liver, there is an increase in microsomal protein and nitro-reductase in both 15–16 and 19–20 day pregnant animals but no changes occur in cytochrome P-450, glucuronyl transferase or biphenyl hydroxylases.Hexobarbital administered to rats at doses related to pregnant body weight increases the sleeping-time from 50 min in non-pregnant animals to 110 min at full-term, but when administered on the basis of the non-pregnant body weight the duration of anaesthesia remains unchanged.Pretreatment of pregnant (19–20 days) and non-pregnant rats with phenobarbital leads to similar increases in microsomal protein (25 per cent) and nitroreductase activity (40 per cent); cytochrome P-450 is increased in non-pregnant animals (30 per cent) but not in the pregnant, although biphenyl-4-hydroxylase is increased in both to such extents as to annul the inhibitory effect of pregnancy. Pretreatment with methylcholanthrene gives rise to similar increases in cytochrome P-450 (30 per cent) and biphenyl-2-hydroxylase (10-fold increase) in both pregnant and non-pregnant rats and again increases biphenyl-4-hydroxylase so as to annul the effect of pregnancy.With rabbits, no change occurs in liver weight, microsomal protein, nitro-reductase, cytochrome P-450, or biphenyl-4-hydroxylase at full-term pregnancy, but glucuronyl transferase is reduced by 20 per cent, and coumarin-7-hydroxylase by 60 per cent. Pretreatment of rabbits with phenobarbital increases microsomal protein (15, 25 per cent), nitro-reductase (70, 80 per cent), cytochrome P-450 (130, 90 per cent), biphenyl-4- hydroxylase (50, 60 per cent), coumarin-7-hydroxylase (40, 150 per cent), and glucuronyl transferase (65, 15 per cent) in both non-pregnant and pregnant animals, respectively.The decrease during pregnancy of hepatic glucuronyl transferase is attributed to competitive inhibition by high levels of endogenous estrogenic and progestational steroids, but the decrease in the activities of the microsomal hydroxylating enzymes is attributed to the decrease in P-450, which may result from high levels of growth factors.     

Comparison of gene expression profiles in mice liver following intravenous injection of 4 and 100 nm-sized PEG-coated gold nanoparticles

Gold nanoparticles (AuNPs) have been widely used in various biomedical applications for photothermal therapy, imaging and drug delivery. Although AuNPs have been recognized as a biologically safe material, very little is known about their molecular and cellular effects. To evaluate the gene expression profile and mechanism of the molecular level of polyethylene glycol (PEG)-coated AuNPs and the effect of particle size, we applied an expression profiling approach. PEG-coated AuNPs of different particle sizes, 4 and 100 nm, were intravenously administered to BALB/c mice (4.26 mg/kg, body weight). Thirty minutes after injection of AuNPs, the mice were sacrificed and liver tissues were removed. Then, pathological examination and microarray analysis were performed on the liver tissues. Histology of the liver tissues did not indicate any pathological changes in all treatment groups. Only 0.38% (170 genes) and 0.50% (224 genes) of the total genes (45,000 genes) were significantly induced by the treatment of 4 or 100 nm AuNPs, respectively. In addition, the 4 and 100 nm AuNPs treatment groups shared 67.1% and 50.9% of the significantly changed genes, respectively. Commonly expressed genes by a single intravenous injection of 4 or 100 nm AuNPs were categorized as apoptosis, cell cycle, inflammation, and metabolic process. In the specifically expressed genes of 4 or 100 nm AuNPs, although the genes were different each other, 4 and 100 nm AuNPs showed similar gene categories such as cell cycle, response to stress, signal transduction, and metabolic process. Therefore, we can conclude that 4 and 100 nm AuNPs showed similar biological effects on liver tissues of mice.     

Maternal high fat feeding and gestational dietary restriction: Effects on offspring body weight, food intake and hypothalamic gene expression over three generations in mice

Excessive gestational weight gain and maternal obesity have both been associated with increased incidence of obesity and metabolic disorder in offspring in both humans and animal models. The objectives of this study were to determine (1) whether mild gestational food restriction during the third trimester (GFR) would alter food intake and growth parameters of offspring, (2) whether effects of GFR depended on diet (high fat [HF] vs chow), (3) whether effects of excessive gestational weight gain (WG) would become magnified across generations, and (4) whether diet and GFR would alter hypothalamic gene expression in adult offspring. Three generations of female C57BL/6 mice were fed chow or HF diet, mated at 11 weeks of age and assigned to ad libitum feeding or 25% GFR. Offspring were fed the same diet as their mothers. Results showed (1) maternal gestational WG was positively correlated with offspring WG. (2) HF offspring weighed less (p < 0.01) at weaning (WWT) but gained more during the 8 weeks after weaning than chow-fed offspring (p < 0.05), resulting in higher final body weights (BW) (p < 0.01). (3) HF males from GFR mothers had higher WWT (p < 0.05), but subsequent WG and final BW were less (p < 0.05) compared to males from ad lib mothers. (4) In the HF group, GFR also resulted in decreased FI (p < 0.05) and FE (p < 0.07) in offspring, compared to offspring from ad lib mothers. (5) In generation 3, hypothalamic expression of tyrosine hydroxylase was lower in HF males from GFR mothers compared to HF males from ad lib mothers (p < 0.05). In conclusion, gender and maternal GFR had independent effects on growth and FI, and hypothalamic gene expression was dependent on both gender and maternal GFR in HF offspring. Even mild food restriction of obese mothers during pregnancy may have beneficial effects in reducing the risk or degree of obesity in offspring.     

Effects of maternal cadmium exposure during late pregnant period on testicular steroidogenesis in male offspring

Cadmium (Cd) is a testicular toxicant and endocrine disruptor. In the present study, we investigated the effects of maternal Cd exposure during the late pregnant period on testicular development and steroidogenesis in male offspring. Pregnant mice were injected intraperitoneally with CdCl₂ (0.5 mg/kg) daily from gestational day (gd) 13 to gd 17. As expected, fetal weight and crown length were significantly decreased in pups whose mothers were exposed to Cd. Importantly, absolute and relative weights of testes were significantly decreased in male fetuses. In addition, maternal Cd exposure during pregnancy markedly reduced serum T level and downregulated the expression of steroidogenic acute regulatory (StAR) protein, P450scc, P45017α and 17β-HSD in testes of male fetuses. Interestingly, the level of serum and testicular T at adulthood remained decreased in male offspring of Cd-exposed mice. Correspondingly, the expression of testicular P450scc was downregulated in male adult offspring whose mothers were exposed to Cd during pregnancy. Fertility analysis found that the number of live fetuses per litter in F2 generation was significantly decreased in Cd-treated group. Additional experiment showed that placental Cd level was increased about 750 folds in dams injected with Cd. However, only traces of blood Cd was measured in fetuses whose mothers were exposed to Cd during the late pregnant period. Taken together, these results suggest that placenta could deter most of Cd from passing from dams to fetuses. The impairments on testicular steroidogenesis in male offspring could not be attributed to a direct action of Cd on fetal testes.     

THE EFFECT OF PREGNANCY ON MINERALO- AND GLUCO-CORTICOID SECRETION IN THE SHEEP

1. The peripheral blood concentrations of aldosterone, corticosterone and cortisol were measured during pregnancy in conscious, undisturbed sheep. 2. Aldosterone levels did not change during pregnancy and the mean pregnant value, 1·2 s.d. 1·4 ng/100 ml (n= 12) was not significantly different from the non-pregnant value, 2·1 s.d. 1·7 (n= 16). 3. Cortisol levels likewise were unchanged by pregnancy–non-pregnant values were 0·56 s.d. 0·50 μg/100 mi (n= 12) compared with 0·46 s.d. 0·40 μg/100 ml (n= 16) in pregnant sheep. 4. Sheep of 110–140 days gestation had a 400 mmol greater total exchangeable sodium than non-pregnant sheep. Plasma volume and plasma renin concentration tended to be elevated near to term. 5. Very high aldosterone secretion rates and peripheral blood levels could be produced in pregnant sheep by stress, intravenous ACTH or angiotensin II infusions, and by sodium deficiency. It is suggested that the pregnant sheep may show increased sensitivity in contrast to non-pregnant sheep to these stimuli and the enlarged size of their adrenals may be a contributing factor.