Automated quantification of DNA demethylation effects in cells via 3D mapping of nuclear signatures and population homogeneity assessment.

Today's advanced microscopic imaging applies to the preclinical stages of drug discovery that employ high-throughput and high-content three-dimensional (3D) analysis of cells to more efficiently screen candidate compounds. Drug efficacy can be assessed by measuring response homogeneity to treatment within a cell population. In this study, topologically quantified nuclear patterns of methylated cytosine and global nuclear DNA are utilized as signatures of cellular response to the treatment of cultured cells with the demethylating anti-cancer agents: 5-azacytidine (5-AZA) and octreotide (OCT). Mouse pituitary folliculostellate TtT-GF cells treated with 5-AZA and OCT for 48 hours, and untreated populations, were studied by immunofluorescence with a specific antibody against 5-methylcytosine (MeC), and 4,6-diamidino-2-phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei (n = 163). Cell images were processed utilizing an automated 3D analysis software that we developed by combining seeded watershed segmentation to extract nuclear shells with measurements of Kullback-Leibler's (K-L) divergence to analyze cell population homogeneity in the relative nuclear distribution patterns of MeC versus DAPI stained sites. Each cell was assigned to one of the four classes: similar, likely similar, unlikely similar, and dissimilar. Evaluation of the different cell groups revealed a significantly higher number of cells with similar or likely similar MeC/DAPI patterns among untreated cells (approximately 100%), 5-AZA-treated cells (90%), and a lower degree of same type of cells (64%) in the OCT-treated population. The latter group contained (28%) of unlikely similar or dissimilar (7%) cells. Our approach was successful in the assessment of cellular behavior relevant to the biological impact of the applied drugs, i.e., the reorganization of MeC/DAPI distribution by demethylation. In a comparison with other metrics, K-L divergence has proven to be a more valuable and robust tool for categorization of individual cells within a population, with potential applications in epigenetic drug screening.

Recombinant rat and mouse growth hormones: risk assessment of carcinogenic potential in 2-year bioassays in rats and mice.

Recombinant rat growth hormone (rrGH) and recombinant mouse growth hormone (rmGH) were developed to evaluate the potential carcinogenicity of each biologically active growth hormone (GH) as assessed in the respective species. Biological activities of rrGH and rmGH were demonstrated by showing an increase in body weight gain and serum levels of insulin-like growth factor-1 (IGF-1) in hypophysectomized rats receiving daily sc injections for 6 days. With the exception of pharmacologically mediated weight gain, rrGH and rmGH had no adverse effects in 5-week oral toxicity studies and no production of anti-recombinant GH antibodies. The high doses selected for the carcinogenicity studies provided systemic exposures of GH up to approximately 10-fold over basal levels. In the 105-week mouse carcinogenicity study, daily sc injections of rmGH at 0.1, 0.2, or 0.5 mg/kg/day were well tolerated and had no effects on survival or incidence of tumors. In the 106-week rat carcinogenicity study, daily sc injections of rrGH at 0.2, 0.4, or 0.8 mg/kg/day had a favorable effect on longevity in female rats administered 0.4 or 0.8 mg/kg/day, an increased weight gain in females and males, and no increase in the incidence of tumors. The absence of carcinogenic effects of recombinant GH administered daily for 2 years to rodents was consistent with publications of clinical experience, indicating a lack of convincing evidence for an increased risk of cancer in children receiving human recombinant GH replacement therapy.

Development of an enzyme-linked immunosorbent assay for the measurement of plasma growth hormone (GH) levels in channel catfish (Ictalurus punctatus): assessment of environmental salinity and GH secretogogues on plasma GH levels.

We report the development of a sensitive, and specific, competitive, antigen-capture enzyme-linked immunosorbent assay for the measurement of channel catfish (Ictalurus punctatus) growth hormone (cfGH). The detection limit of the assay (90% binding) was 2.0ng/ml and the ED(50) value (standard curve range 150-0.59 ng/ml) was 67.3 ng/ml. Recovery of cfGH-spiked plasma samples was determined to be 102%. Dose-response inhibition curves using serially diluted pituitary homogenates and plasma samples consistently showed parallelism with the standard curves using purified cfGH. The GH antibody (rabbit anti-catfish GH) specificity was demonstrated in competitive binding curves employing heterologous hormones and purified channel catfish prolactin (cfPRL). These studies show that there was no significant (0.006%) binding of cfPRL (competitive inhibition of cfGH binding), or heterologous hormones, within the working range of the assay. To physiologically validate the assay, catfish were injected (100 microg/g body weight, 3 injections every 5 days) with either bovine GHRH(1-29)-amide or the synthetic hexapeptide GHRP-2 (KP-102: D-Ala-D-beta-Nal-Ala-Trp-D-Phe-Lys-NH(2)) suspended in corn oil. Following the last injection, half of the animals were sampled for plasma and the remaining transferred from fresh water (FW) to 12 ppt seawater (BW: brackish water). Twenty-four hours after transfer to BW, animals were again sampled for plasma. Plasma GH levels were significantly (p<0.001) elevated in all the BW groups (control, KP-102, and bGHRH), compared with the FW (fresh water) groups. In addition, plasma GH levels were significantly (p<0.001) elevated by treatment with either of the GH secretogogues, KP-102 or bGHRH. Our findings demonstrate that two regulatory mechanisms of GH elevation, one which is seen in euryhaline teleosts (salinity-induced GH levels) and another, which has been recently described in teleosts (GHRP-induced GH levels), are present in the stenohaline channel catfish.

Immunohistochemical and radioimmunological assessment of thyrotrophs in the pituitary of aging rats.

Thyrotrophs were studied by quantitative immunohistochemistry in the pituitary gland of young (4 months), old (20 months) and very old (29 months) male rats. An attempt was also made to correlate morphometric parameters with serum levels of thyrotropin (TSH), thyroxine (T4) and triiodothyronine (T3). Cells were immunostained by the peroxidase-antiperoxidase method and hormones were measured in serum by specific radioimmunoassays. There was a marked age-related reduction in TSH cell number, volume density and surface density but a significant increase in TSH cell area and perimeter. Basal serum levels of TSH increased, T4 decreased and T3 remained unchanged with age. There was a highly significant (p < 0.001) negative correlation between serum TSH and T4, but no significant correlation was found between TSH and morphometric parameters. The present results suggest that aged rats possess a reduced but functionally preserved thyrotrophic cell population. The coexistence of high circulating levels of TSH with reduced serum T4 suggests that aging brings about a progressive desensitization of the thyroid to TSH.

Assessment of bioactivity of WHO/NIH research standard for inhibin (code 86/690) using two separate pituitary cell-culture systems.

The Research Standard for inhibin, porcine (No. 86/690) distributed by the National Institute for Biological Standards and Control, U.K. for the bioassay of inhibin was tested for its bioactivity in vitro in two rat pituitary cell culture systems. The system A was obtained from pituitaries of 12 day old immature rats while system B was obtained from pituitaries of mature male rats. The inhibin preparation failed to inhibit basal secretion of FSH in the system A. Instead it stimulated the release of both LH and FSH. 1 ng LHRH induced release of LH was inhibited by 32, 80 and 43% by 0.1, 1 and 10 IU inhibin, respectively. 10 ng LHRH induced release of FSH was inhibited in a none-dose related manner and the maximum inhibition was by 10 IU inhibin (25%). The same dose of 10 IU inhibin stimulated LHRH-induced release of LH by 35%. In system B, 1, 10, 50, 100 and 200 IU inhibin suppressed basal secretion of FSH by 0, 44, 72, 70 and 48%, respectively, while LH was suppressed by 13, 24, 46, 22 and 21% respectively. The pattern of inhibition of 10 ng LHRH induced release of LH and FSH by inhibin was similar to its effect on basal secretion. A specific dose-related inhibition of FSH was not observed. Our data question the utility of the inhibin standard (86/690).

[Evaluation of a technic for the isolation of mammotropic cells using dopamine as a ligand]. / Valoración de una técnica de aislamiento de células mamotropas utilizando dopamina como ligando.

Pituitary cell suspension from female Wistar rats were allowed to enter the 6MB sepharose gel with added dopamine, which acts ligand attaching the mammotrophs to the gel grains. After eluting the column with 3 different dopamine buffer concentration, 3 different cellular fractions were obtained. Fraction F1 contains 5% of mammotroph cells, fraction F2 2-4% and fraction F3 90%, as determined by immunocytochemistry. The absolute amount of mammotroph cells contained in F3 fraction increases by gradually warming up the column from room temperature to 37 degrees C. The eluted F3 fraction mammotroph cells from female lactating rats is observed to be twice the amount eluted in experiments performed with virgin rats. No ultrastructural differences between fraction F1 and fraction F3 mammotroph cells have been found.