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Tuesday, January 23, 2007

Screening Methods for Androgens Part 1b

*The whole animal (in vivo),
*A combination of the whole animal and isolated organ(s) (ex vivo),
*Isolated and cultured whole or sections/minced organs, i.e., testis (in vitro),
*Isolated and cultured cells from the testis (in vitro), and
*Cell lines (in vitro).

Screening Methods for Androgens Part 1a

Whole Animal Methods (In Vivo)
Intact animals used to measure androgenic activity (Allen and Doisy, 1924; Hershberger et al., 1953).
In vivo methods
- Take advantage of animal’s innate hormone-directed tissue development and maintenance responses and
- Use these responses to identify whether administered substances alter the normal response.
Animal’s inherent capacity to produce these hormones must be removed.
# Castration.
# Immature or juvenile animals used

(M. Chaturvedia, P.C. Mali, A.S. Ansarib; Pharmacology: 2003; 68:38-48)
For androgenicity evaluation of test subs. rats were divided into four groups:
Group I- animals were castrated 30 days before the experiment to serve as controls
Group II, III and IV subjected to castration 30 days before the experiments, followed by administration of test subs., testosterone propionate (0.01 mg/rat/alternate day s.c.), and test subs. with testosterone propionate, respectively, for 30 days.
Studied-- cauda epididymis sperm motility and density,
Number of pups,
Fertility,
The weights of testes, epididymis, seminal vesicle, and prostate
Histoarchitecture of the testes

Endocrine challenge test (ECT). (Anderson et al., 1992).
Bioassay - to evaluate gonadal response to a substance by measuring the steroid hormone production and release following administration of test subs.
The intact mature animal is challenged with GnRH or an LH- or FSH-like substance that stimulates a hormonal response.
Serial blood samples collected and measured to evaluate whether the substance being tested causes increased testosterone
Techniques used include tail vein sampling, tail vein treatments and/or jugular catheterization (Fail et al., 1995; Fail and Anderson, 2002).
A number of different endpoints have been used.
* Plasma testosterone, before (basal) and after stimulation. Testosterone concentrations measured by RIA (Fail et al., 1995; 1996).
*Epididymis weight, caudal sperm count, testicular sperm heads, and sperm motility
(Fail et al., 1995; 1996).

Screening for Androgens- part 3

Screening for AR activity using Gene expression assays

*Assay involves transient transfection of a tester cell line with a plasmid base receptor and the reporter, followed by chemical exposure and measurement of the modulation of gene expression.
· KB2 assay (Vickie S. Wilson; 2002: Toxicological Sciences 66, 69-81)
· Describe the effects of androgens in a stably transformed cell line that they developed (MDA-KB2), which expresses the human AR (hAR) and an AR-responsive promoter linked to a luciferase reporter gene (MMTV-luc).
· Main advantage of KB2 assay -- it employs a genetically modified cell line, which eliminates the effort and inherent variability associated with repeated transient transfections.
· The breast cancer cell line, MDA-MB-453, was stably transformed with the MMTV.luciferase.neo reporter gene construct.
· Since both GR and AR are present in the MDA-MB-453 cells, and both receptors can act through the MMTV promoter, compounds that act through either AR or GR activate the MMTV luciferase reporter.
· AR agonists such as dihydrotestosterone (DHT), and GR agonists such as dexamethasone (DEX), corticosterone, and aldosterone induce luciferase expression at appropriate concentrations.



· DHT consistently produced 3–9-fold induction at concentrations from 0.1 to 10 nM.
· To distinguish AR- from GR-mediated ligands, chemicals were assayed concurrently with the antiandrogen, hydroxyflutamide (OHF), which blocks AR- but not GR-mediated responses.
· These cells are relatively easy to culture and maintain.
· Responsiveness was monitored over time and was stable for more than 80 passages.
· Some advantages -- relatively rapid (2 days), eliminates the need for transfection, can be conducted in a 96-well plate format, produces consistent reproducible results

* Construction and verification of reporter plasmid containing MMTV. neo.luc-- The neomycin gene was removed from pcDneo and inserted into pMMTV.luc

* Transformation, isolation, and screening of stable clones.
MDA-MB-453 cells (ATCC No. HTB 131) used for transformation.
Cells were seeded at 2 x 105 cells per 60-mm culture dish in maintenance medium, then transformed using Fugene 6 (Roche) per manufacturer's protocol, with 5 µg pMMTV.luc.neo per dish.
To screen clones for responsiveness to an androgen agonist, cells grown from individual colonies were plated at 1 x 104 cells per well in 96-well luminometer plates, allowed to attach. When cells were attached, the medium was replaced with freshly prepared dosing medium containing ethanol only (negative control), an agonist (dihydrotestosterone, DHT; 1 nM), or the agonist plus a known competitor (hydroxyflutamide, OHF; 1 M).
Cells were dosed with 100 µl of medium/well and incubated for 20–24 h, --harvested with
25 µl/well of lysis buffer and assayed for luciferase reporter activity.
Relative light units per well determined using a 96-well MLX Luminometer.
Clones with a minimum 2-fold response to 1 nM DHT were retained in culture.
The final clone (MDA-kb2) was chosen based on appropriate ligand responsiveness and genetic stability over time.

*Maintenance of cultures and transcriptional activation assays.
-MDA-kb2 cells stably transformed with the pMMTV.neo.luc reporter gene construct were maintained in L-15 media supplemented with, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C, without CO2.
-For experiments, cells were plated at 1 x 104 cells per well in 100 µl of medium in 96-well luminometer plates.
-When cells were attached (4–6 h), medium was removed and replaced with dosing medium. Stock solutions for each chemical were prepared at 1000x in ethanol.
- Dosing medium was prepared at the time of treatment by aliquoting 1 µl of stock solution into 1 ml of maintenance medium
-Vehicle control wells contained 100 µl medium/ well with1 µl of ethanol/ml of medium. Each plate also contained either 0.1 or 1.0 nM DHT, or DHT/plus 1 µM OHF as agonist control.
-Cells were incubated overnight at 37°C without CO2.
Luciferase activity was assayed --using an MLX microtiter plate luminometer and quantified as relative light units (RLU).
-Relative light units were converted to fold induction above the vehicle control value for each replicate for statistical analysis.
-Stability of Androgenic Response and Baseline Activity Androgen-induced luciferase activity measured over approximately 9 months of continuous culture to assess the stability and responsiveness of the MDA-kb2 clone.

-cells were assayed over more than 80 passages for their response to 1 nM DHT.

-At early passages, luciferase induction relative to solvent controls was about 10 fold.

-In continuous culture, luciferase induction dropped over time, but responsiveness stabilized at 30 to 40 passages.

-In later passages, a minimum of 5–6-fold induction in response to 1 nM DHT was consistently maintained over the remaining test period for more than 80 passages, demonstrating stable expression of the luciferase gene.
P. C. Hartig,1, Toxicological Sciences 66, 82-90 (2002)
*Attempt to deliver the genes via replication-defective adenovirus transduction
*describe the responses of several androgens in two AR-responsive in vitro assays. These assays used transduced MDA-453 and CV-1 cells, cell lines.
* Ad/mLuc7 virus used, which contains the luciferase gene regulated by the glucocorticoid-inducible hormone response element found in the mouse mammary tumor virus (MTV) LTR
*Transduction assays were performed in 96-well plates.
· Twenty-four h prior to transduction, 5 x 104 cells were plated per well.
· Medium was removed and replaced with 20 µl of control medium or medium with diluted virus.
· MDA cells (which contain endogenous AR) were transduced with Ad/mLuc7 reporter virus at a multiplicity of infection (MOI) of 50 (i.e., 50 virions per cell).
· CV-1 cells (which lack native AR) were transduced with Ad/mLuc7 at a MOI of 50 and Ad5hAR at a MOI of one.
· Dishes were rocked every 15 min for 1 h, incubated 3 additional h,
· 200 µl medium/medium + test chemicals added to each well
· followed by a 48-h incubation.
· Plates were either frozen at –80°C, or assayed immediately for luciferase activity.
· Luciferase activity was quantitated in an MLX microtiter plate luminometer, data expressed in relative light units (RLU).
· For androgen agonists, which stimulate luciferase expression, treatments compared to the media/ethanol control group
· Relative light units were converted to fold induction above the media value for each replicate


(L. G. Parks; Toxicological Sciences 62, 257-267 (2001)
COS whole cell human androgen receptor (hAR) binding assay

* In 3 blocks, COS cells (SV-40 transformed monkey kidney line ATCC # CRL-1650) were transiently transfected with hAR expression vector pCMVhAR
* COS cells were plated at 200,000 cells/well in 12 plates and transfected with 1 µg of pCMVhAR
* After a 3-h transfection period, cells were washed and incubated overnight
*Twenty-four hours later, medium replaced with 200 µl of serum-free/phenol red-free DMEM with R1881 plus 200 microliters of medium containing tast substance (400-µl incubation volume, with a concentration factor of 40x).
* Cells were incubated for 2 h with 5 nM [3H] R1881 at 37°C under an atmosphere of 5% CO2.
* Cells were washed in phosphate-buffered saline and lysed in 200 µl ZAP
(0.13 M ethylhexadecyldimethylammonium bromide with 3% glacial acetic acid).
* The lysate was added to 5 ml OPTI-fluor scintillation cocktail and radioactivity was counted using a Beckman LS 5000 TD counter
· 1 µM hydroxyflutamide was added to half of the samples to see if this potent antiandrogen would block the test substance-induced luciferase activity.
· Cells also were exposed to 1 nM DHT as a positive control.

CV-1 AR and GR 40-dependent transcriptional activation assays
*Experiments, each with several replicates, were conducted to determine induced AR- or GR-dependent gene expression in CV-1 cells (monkey kidney line, ATCC # CCL-70).
* 200,000 CV-1 cells were plated in a 60-mm dish and then transiently cotransfected with 1 µg pCMVhAR and 5 µg MMTV-luciferase reporter using 5 µl Fugene reagent in 95 µl serum-free medium as per the manufacturer's protocol.
· Twenty-four h after transfection, medium was aspirated and replaced with 2 ml of medium containing test substance.
· Cells then incubated at 37°C under 5% carbon dioxide.
· After 5 h of exposure, medium was removed, and cells were washed once with phosphate-buffered saline and harvested with 500 µl lysis buffer.
· Relative light units of 0.05-ml aliquots of lysate determined using a Monolight 2010 luminometer.

COS cell immunocytochemistry
Experiment conducted to visualize, by immunofluorescence, the ligand-induced nuclear translocation of hAR in COS cells.

· Two chamber slides were seeded with 100,000 cells/chamber in 2 ml DMEM supplemented with 10% FBS
· Cells transfected with 0.5 µg pCMVhAR
· Following transfection, 2 ml medium containing test substance, added to slides,
· incubated for 24 h at 37°C under 5% CO2.
· The next day, medium removed, cells washed once with DPBS (Dulbecco's phosphate buffered saline), allowed to dry for 45 min at room temperature, fixed for 10 min with 95% ethanol (-20°C), blocked with 5% BSA, and incubated overnight with primary AR antibody (1:1000) at 4°C.
· The following day, cells were washed once with DPBS and incubated with fluorescently labeled secondary antibody for 30 min at room temperature.
· To visualize the nuclei, cells were counter-stained with DAPI, a DNA stain, mounted with fluoromount
· slides examined using Microscope at 200 x magnification.
· The localization of the AR was classified as either perinuclear or nuclear in a blinded fashion from 10 randomly selected fields from a slide for each site.
· In another experiment, cells were exposed to 1 nM DHT, as a positive control.

Novel yeast bioassay system for detection of androgenic compounds.
Lee HJ, Lee YS, Kwon HB, Lee K.;Toxicol In Vitro. :2003 Apr;17(2):237-44.

Report the development of a rapid, simple, effective yeast detection system for androgenic compounds, based on the yeast two-hybrid protein interaction.
* A yeast strain, ARhLBD-ASC1, was established by co-transformation of yeast cells harboring a lacZ reporter plasmid with two vectors expressing each of LexA fused hinge-ligand binding domain (hLBD) of androgen receptor (AR) and B42 fused ASC-1 that interacts with AR-hLBD in an androgen-dependent manner.
* In this strain, androgens, but not other hormones, strongly stimulated the beta-galactosidase activity in a dose-dependent manner.

Screening for Androgens- part 2

Combination of Whole Animal and Isolated Organs Method (ex vivo)

* First involves treating immature or adult animals according to selected dosing regimen.
* Route, duration of exposure are not constrained.
* In addition to the acute exposures, exposures for ex vivo studies can actually begin as early as gestation and continue for as long as the life expectancy of animal being tested.
* During the in-life exposure period, blood samples can be collected at specified times for serum hormone analysis.
* After the exposure period is completed, animal is killed, at which time final necropsy
specimens collected, and the testis isolated.
* The gonads are then processed according to the type of in vitro preparation that was selected to assess the test substance’s effect.

Isolated Organ Methods (in vitro)

*The organs where steroidogenesis occurs removed from animal & kept viable, thereby providing an isolated organ method for assessing substances as stimulants of the
steroidogenic pathway.
* Organs, once isolated, can be used whole or further processed into sections or minced organ preparations
* These preparations allow effect of given test substance to be measured without influence of other organs or systems/other physiological factors.
* The integrity and interrelationship of the cells and tissues within the organ remain intact.
*These preparations retain the cellular and biochemical pathways that involve the receptor and second messenger.

# way in which the organ is processed.
*Whole organ-- after the organ is removed and placed in media, no further processing occurs
*Sectioned organ --For the testes, an organ section refers to an organ that has been cut such that each section constitutes 1/8 to ½ the size of the whole organ, i.e., 50 to 250 mg (rat testis). *Minced organ -- organ that has been cut into very small sizes, i.e., 1 to 50 mg (rat testis).

The perfused testis

# Provides a method to simulate biosynthesis and secretion of testicular steroid hormones
Developed for the rabbit by Vandemark and Ewing (1963), modified for the rat by Chubb and Ewing (1979).
# Apparatus is sterilized and then assembled.
# The system must be air-free to prevent bubbles from entering the perfusate and blocking flow.
# Testis is removed, spermatic artery cannulated, organ flushed of blood with perfusate solution.
# The testis is placed in the organ chamber and perfusion initiated.
# Test substance given after a preliminary perfusion for approximately 1 hour, the temperature (370 C), rate of flow (20 mL/hr), and pH (7.4) are monitored and maintained.
# The perfusion can be maintained for 6 to 10 hours.
# Samples are collected from the perfusate for analysis.

Simple Whole Organ Incubation technique

* Involves removal of the testis and incubation of the organ in medium for testing.
* For the whole testis (Deb et al., 1980), animal anesthetized or euthanized & testes removed.
* The testes are incubated in Kreb-Ringer-bicarbonate solution (pH 7.6) at 370 C & atmosphere of 95 percent O2 and 5 percent CO2
* Testes are incubated with or without the substance being tested, as well as with or without stimulant, e.g., LH.

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