A novel micronucleus in vitro assay utilizing human hematopoietic stem cells.

The induction of micronucleated reticulocytes in the bone marrow is a sensitive indicator of chromosomal damage. Therefore, the micronucleus assay in rodents is widely used in genotoxicity and carcinogenicity testing. A test system based on cultured human primary cells could potentially provide better prediction compared to animal tests, increasing patient safety while also implementing the 3Rs principle, i.e. replace, reduce and refine. Hereby, we describe the development of an in vitro micronucleus assay based on animal-free ex vivo culture of human red blood cells from hematopoietic stem cells. To validate the method, five clastogens with direct action, three clastogens requiring metabolic activation, four aneugenic and three non-genotoxic compounds have been tested. Also, different metabolic systems have been applied. Flow cytometry was used for detection and enumeration of micronuclei. Altogether, the results were in agreement with the published data and indicated that a sensitive and cost effective in vitro assay to assess genotoxicity with a potential to high-throughput screening has been developed.

The effects of a primary-treated municipal effluent on the immune system of rainbow trout (Oncorhynchus mykiss): exposure duration and contribution of suspended particles.

Municipal sewage effluents are complex mixtures of contaminants known to disrupt both immune and endocrine functions in aquatic organisms. The present study sought to determine the impacts of municipal effluent on the immune systems of juvenile rainbow trout (Oncorhynchus mykiss), by exposing specimens to low concentrations (0.01%, 0.1%, 1% or 10%) of sewage effluent for periods of 28 or 90 days. The soluble and insoluble fractions of the effluent were also studied to assess the contribution of fractions rich in microorganisms and particles on fish immune systems. To this end, the trout were also exposed to soluble and insoluble fractions of the effluent for a period of 28 days. Immunocompetence was assessed by the following three parameters: phagocytosis, natural cytotoxic cells (NCC) and blastogenesis of lymphocytes under mitogen stimulation. Fish exposed to the 1% sewage effluent concentration for 28 days had enhanced phagocytic activity; at 90 days, phagocytic activity was reduced. T and B lymphocyte proliferation in fish from both groups was similarly stimulated. Phagocytosis and NCC activities were influenced more by the insoluble fraction than the soluble fraction of the effluent. Conversely, mitogen-stimulated T lymphocyte proliferation was enhanced in cells of fish exposed to the soluble fraction of the effluents, with a dampening effect on the insoluble (particulate) fraction of the effluent. In conclusion, the effects of the effluent and its fractions were higher at the cellular-mediated immunity level than at the acquired immunity level. Immunotoxicity data on the soluble fraction of the effluent were more closely associated to data on the unfractionated effluent, but the contribution of the particulate fraction could not be completely ignored for phagocytosis and B lymphocyte proliferation.

Effects of ozone, ultraviolet and peracetic acid disinfection of a primary-treated municipal effluent on the immune system of rainbow trout (Oncorhynchus mykiss).

Municipal sewage effluents are complex mixtures that are known to compromise the health condition of aquatic organisms. The aim of this study was to evaluate the impacts of various wastewater disinfection processes on the immune system of juvenile rainbow trout (Oncorhynchus mykiss). The trout were exposed to a primary-treated effluent for 28 days before and after one of each of the following treatments: ultraviolet (UV) radiation, ozonation and peracetic acid. Immune function was characterized in leucocytes from the anterior head kidney by the following three parameters: phagocytosis activity, natural cytotoxic cells (NCC) function and lymphocyte (B and T) proliferation assays. The results show that the fish mass to length ratio was significantly decreased for the primary-treated and all three disinfection processes. Exposure to the primary-treated effluent led to a significant increase in macrophage-related phagocytosis; the addition of a disinfection step was effective in removing this effect. Both unstimulated and mitogen-stimulated T lymphocyte proliferation in fish decreased dramatically in fish exposed to the ozonated effluent compared to fish exposed to either the primary-treated effluent or to aquarium water. Stimulation of T lymphocytes proliferation was observed with the peracetic acid treatment group. In conclusion, the disinfection strategy used can modify the immune system in fish at the level of T lymphocyte proliferation but was effective to remove the effects on phagocytosis activity.

Spanning or looping? The order and conformation of bipolar phospholipids in lipid membranes using 2H NMR spectroscopy.

Solid-state 2H NMR spectroscopy was used to study and characterize the conformation and order of bolaform lipid membranes. A series of 2H-labeled bolaform phosphatidylcholines has been synthesized and their properties compared to a [D4]dimyristoylphosphatidylcholine (DMPC) and a [D8]-32 macrocyclic phosphatidylcholine. 31P NMR measurements establish that the aqueous dispersions of these lipids adopt lamellar phases. Computational dePakeing was used to extract the spectrum of the oriented system from spectra consisting of a superposition of randomly oriented domains in an unoriented sample. A large (> 90 %) and constant value for the normalized segmental order parameter (Smol) was observed for all positions along the diacyl chain of the bolaform lipids and only a small population (< 10%) of a less ordered conformer was observed. The less ordered conformer is assigned to the looping conformation on the basis of comparison with the deuterated macrocyclic phospholipid which has an enforced loop in its diacyl chain. The predominant population(> 90%) of the bolaform lipids is assigned to a highly ordered, spanning conformer.

Solid-phase synthesis of a library of functionalized aminodiol scaffolds.

A combinatorial library motif has been developed based on orthogonally protected aminodiol scaffolds. Amine functionality was derivatized by commercially available electrophiles including carboxylic acids, sulfonyl chlorides, isocyanates, and aldehydes. A hydroxyl moiety was converted to a carbamate linkage, allowing a variety of amines to be incorporated. The scaffold was anchored to TentaGel at the second hydroxyl via a succinyl linker, which was hydrolyzed by mild aqueous basic conditions. The method was used to make a library of about 17,000 different members in mixtures of 5 per sample.

Deconvolution of combinatorial libraries for drug discovery: experimental comparison of pooling strategies.

An experimental evaluation of several different pooling strategies for combinatorial libraries was conducted using a library of 810 compounds and an enzyme inhibition assay (phospholipase A2). The library contained compounds with varying degrees of activity as well as inactive compounds. The compounds were synthesized in groups of three and pooled together in various formats to realize different pooling strategies. With one exception, all iterative deconvolution strategies and position scanning resulted in identification of the same compound. The results are in good agreement with the predicted outcome from theoretical and computational methods. These data support the tenet that active compounds for pharmaceutically relevant targets can be successfully identified from combinatorial libraries organized in mixtures.

Pharmacokinetic profile of recombinant human (rh) inhibin A and activin A in the immature rat. I. Serum profile of rh-inhibin A and rh-activin A in the immature female rat.

The serum pharmacokinetics of recombinant human inhibin A (rh-inhibin A) and rh-activin A were examined in immature female Sprague Dawley-derived rats after iv and sc injection of the drugs. After iv administration of rh-inhibin A (120 micrograms/kg), the serum concentrations were described by a biexponential equation. The weight-normalized clearance was 21.3 ml/min.kg, and the initial (t1/2 alpha) and terminal (t1/2 beta) half-lives were 2.9 min and 37.9 min, respectively. Subcutaneous administration of 120 micrograms/kg rh-inhibin A resulted in a peak serum concentration of 10.6 ng/ml at 30.8 min after injection. Approximately 24% of the sc administered material was absorbed. Serum concentrations of rh-activin A also declined biexponentially after iv injection of the drug (120 micrograms/kg). The clearance of rh-activin A was 5.1 ml/min.kg, the t1/2 alpha was 6.1 min, and the t1/2 beta was 46.3 min. The peak serum concentration of rh-activin A (104.7 ng/ml) was achieved 24.7 min after sc delivery of the drug. The bioavailability of the sc dose was 38%. Iodinated rh-inhibin A and rh-activin A were used to examine the serum forms and metabolites of the drugs. [125I]rh-inhibin A and [125I]rh-activin A associated with two serum-binding proteins. Within 2 min of iv injection, the labeled hormones bound follistatin and alpha-2-macroglobulin. Even though rh-inhibin A and rh-activin A are structurally similar and appear to bind to the same serum proteins, their disposition in the immature rat differ.

Restoration of lactation in bromocriptine-treated rats by prolactin replacement: comparison of constant versus pulsatile infusion and intrahepatic versus intrajugular routes of delivery.

The effectiveness of pulsed vs constant infusion of ovine(o) prolactin (PRL), given by different schedules, at restoring lactation in PRL-suppressed rats was compared, and the possibility that the liver participates in the restorative effects of the infused hormone was investigated. Lactating dams were given subcutaneous injections of bromocriptine (BC) between days 7 and 12 postpartum to suppress endogenous PRL secretion. Osmotic minipumps were used to infuse the oPRL into either the jugular vein or the hepatic portal vein. The latter route would expose the liver to higher concentrations of PRL than would intrajugular infusion. Constant infusion of oPRL in different doses was, overall, more effective at restoring lactation (i.e. litter weight gain) than was giving pulses, regardless of the site of delivery. Infusion of the PRL at 100 micrograms/rat/day in pulses of 1h duration was ineffective at frequencies of either 4 or 8/day, whereas pulses of 2h duration were effective at both of these frequencies. Infusing that dose of oPRL was equally effective whether it was given in 4 or 8 pulses/day of 2 h duration. Intrahepatic infusion of oPRL was not more effective than intrajugular delivery regardless of the schedule of administration. These results indicate that pulse duration is a more important determinant of the effectiveness of the galactopoietic action of PRL in the lactating rat than is pulse frequency. No evidence was obtained that the liver participates in the galactopoietic effects of PRL.

On the possible role of the liver in the galactopoietic action of prolactin in the rat.

The possibility that the liver contributes to the galactopoietic effects of PRL was assessed in lactating rats in which endogenous PRL secretion was suppressed by injections of bromocriptine. Pup weight gain over a 5-day period (i.e. days 7-12 of lactation) was used as an index of lactational performance in dams. Osmotic minipumps were used to infuse different doses of ovine (o) PRL into either the external jugular vein (JV) or the hepatic portal vein of the dams at a constant rate. The latter route of delivery would directly expose the liver to a higher concentration of PRL than would the former one. Twice daily sc injections of bromocriptine (1.5 mg/kg.injection) in corn oil into the dams completely suppressed litter weight gain. Infusion of oPRL into the JV at a dose of 2.0 mg/rat.day restored lactation to normal in the drug-treated mothers. Electrophoretic analysis indicated that about 95% of the oPRL remained in the intact monomeric form when incubated in the infusion solvent in the minipump at 37 C for 2 days, but by 4 and 6 days of incubation the amounts of that form decreased by about 25% and 50%, respectively. Measurement of serum oPRL levels by RIA showed that they were fairly constant, and after 5 days of infusion, the final concentration was directly related to the dose infused. Continuous infusion of oPRL into the JV was equally effective at restoring pup weight gain as was infusion into the hepatic portal vein over a wide range of doses. Hence, a physiological role of the liver in the maintenance of lactation by PRL is not supported by these experiments.

Interactions among prolactin, epidermal growth factor, and proinsulin on the growth and morphology of the pigeon crop-sac mucosal epithelium in vivo.

The local pigeon crop-sac assay was used to test the direct effects of epidermal growth factor (EGF) and several other growth factors and hormones on the growth of mucosal epithelial cells in vivo. Insulin, relaxin, multiplication-stimulating activity, proinsulin, and platelet-derived or fibroblast growth factors had no direct stimulatory activity by themselves. Human insulin-like growth factor I and EGF caused dose-related stimulation, but were less effective than ovine (o) PRL. Insulin, platelet-derived growth factor, fibroblast growth factor, and multiplication-stimulating activity did not affect the proliferative response to oPRL when injected along with the hormone. Proinsulin augmented the direct mitogenic action of oPRL, but not that of EGF. When EGF was injected locally with PRL no interaction occurred even though both hormones were independently mitogenic. A single sc injection of a high dose of oPRL (0.5 mg) given at a site distant from the crop-sac had no effect on the mucosal epithelial cells, but it caused a significant increase in their response to the direct action of oPRL. However, the systemically acting PRL did not affect the direct local effect of EGF. The gross pattern of mucosal cell proliferation induced by PRL (parallel ridges resembling gastric rugae) differed from that produced by EGF (usually irregular patches), and PRL, but not EGF, promoted the accumulation of lipid droplets in the stimulated mucosal epithelial cells. Furthermore, the crop mucosal cells in the midline of the organ are unresponsive to PRL, but were highly responsive to EGF. These results indicate that although EGF and PRL are both mitogenic to crop mucosal epithelial cells, the former does not mimic the latter. They produce a different growth pattern, and EGF fails to promote differentiation of the resultant daughter cells. Moreover, EGF is a less specific mitogen than is PRL, and the two hormones do not interact in their mitogenic effects.

Secretion of prolactin-synergizing activity (synlactin) by the liver of ectothermic vertebrates in vitro.

Recent work in our laboratory indicated that the liver of rats and pigeons secretes a prolactin synergist (synlactin) in vitro. We have investigated the secretion of this activity by the liver of nine species of ectothermic vertebrates. Liver from three teleosts (goby, salmon, and tilapia), four amphibians (Ambystoma, Necturus, bullfrog, and grass frog) and two reptiles (turtle and anole) was diced, washed, and incubated for 3 hr in isotonic medium. After dialysis, the liver incubation media (LIM) were tested with and without prolactin (PRL) in the local pigeon crop-sac bioassay. The LIM for turtle, larval bullfrog, freshwater salmon, and 5% seawater-adapted goby significantly augmented the local crop-sac response to PRL, but the LIM from anole, adult bullfrog, grass frog, fasted larval Ambystoma, Necturus, 100% seawater-adapted goby, and tilapia did not contain synergizing activity. We conclude that synlactin is secreted by the liver of several species representing three of the major ectothermic classes of vertebrates. It is significant that in two cases, larval bullfrog and 5% seawater-adapted goby, the presence of synlactin occurs in physiological states in which PRL is active. In the opposite cases (adult frog and 100% seawater-adapted goby) the activity was not detectable. We also found that the liver of larval and adult bullfrogs and tilapia released a factor in vitro that had proliferative activity in the crop-sac. This activity appears to be distinct from synlactin.

Lactogenic hormones stimulate the liver to secrete a factor that acts synergistically with prolactin to promote growth of the pigeon crop-sac mucosal epithelium in vivo.

We have investigated whether the liver is a source of a PRL-synergizing activity (i.e. synlactin) and we have obtained some information on endocrine control of its secretion. Livers were removed from 3-month-old Long-Evans rats (male, virgin, pregnant, or lactating female) or from virgin females that were injected with saline, bovine GH, or ovine PRL for 7 days, and hepatic slices were prepared for in vitro incubation in medium 199. The media were tested for synlactin activity by determining whether they could augment the pigeon crop-sac response to locally injected ovine PRL. Only the media containing factors from the liver of pregnant or lactating females and PRL-injected virgins had significant synlactin activity. They augmented the crop-sac mucosal growth response by 130%, 140%, and 103%, respectively. Medium in which slices of kidney from virgin or pregnant rats were incubated did not have detectable synlactin activity. The medium samples that had synlactin activity were also tested for the presence of bioactive (crop-sac assay) and immunoreactive (RIA) rat PRL, and none had detectable amounts of the hormone. Hence, the augmenting effect is not due to PRL that is sequestered in and released by the liver. The levels of insulin-like growth factor I/somatomedin-C in medium samples that did or did not have synlactin activity (from pregnant and virgin females, respectively) were measured by RIA and were found to be equivalent. Hence, synlactin activity is probably not due to insulin-like growth factor I. Overall, our results indicate that lactogenic hormones (i.e. pituitary PRL, and presumably placental lactogens in the pregnant rats) stimulate the liver to secrete synlactin activity. The hepatic PRL receptors which increase in number in pregnant females may be involved in the secretion of synlactin, which could then act in concert with ovarian steroids and GH and/or PRL to promote mammary growth.

Flexible valinomycin electrodes for on-line determination of intravascular and myocardial K+.

Flexible electrode systems capable of monitoring in vivo changes in venous and myocardial extracellular potassium activity were constructed using valinomycin-polyvinyl chloride matrix membrane and polyvinyl chloride tubing. Electrode impedance was 1--30 Momega, time constant 10--200 ms, drift less than 1 mV/h, and shelf life approximately 3 days (intramyocardial electrode) and approximately 6 wk (venous electrode). In vitro and in vivo accuracy were determined in 5 dogs and 18 pigs, anesthetized with sodium pentobarbital (35 mg/kg iv), with normal and elevated K+ levels. Serum and venous K+ concentration, determined using a K+ electrode with tip diameter of 1.5 mm, correlated well to serum K+ values determined by flame photometry (r = 0.997). Steady-state myocardial extracellular K+ concentration determined using double-barrel electrodes with total tip diameter of 0.6 mm also correlated well to the serum K+ concentration (r = 0.992). These electrode assemblies permit on-line, virtually instantaneous in vivo determination of intravascular and local myocardial extracellular K+ activity, a capability not previously available.