In Vivo And In Vitro Comparison Essay

Abstract

Identification of nuclear receptor–mediated endocrine activities is important in a variety of fields, ranging from pharmacological and clinical screening, to food and feed safety, toxicological monitoring, and risk assessment. Traditionally animal studies such as the Hershberger and Allen-Doisy tests are used for the assessment of androgenic and estrogenic potencies, respectively. To allow fast analysis of the activities of new chemicals, food additives, and pharmaceutical compounds, high-throughput screening strategies have been developed. Here, a panel of mainly steroidal compounds, screened in different in vitro assays, was compared with two human U2-OS cell line–based CALUX® (Chemically Activated LUciferase eXpression) reporter gene assays for androgens (AR CALUX) and estrogens (ERα CALUX). Correlations found between the data of these two CALUX reporter gene assays and data obtained with other in vitro screening assays measuring receptor binding or reporter gene activation (CHO cell line–based) were good (correlation coefficients (r2) between 0.54 and 0.76; p < 0.0001). Good correlations were also found between the in vitro and in vivo data (correlation coefficient r2 = 0.46 for the AR CALUX vs. Hershberger assay and r2 = 0.87 for the ERα CALUX vs. Allen-Doisy assay). The variations in the results obtained with the reporter gene assays (CALUX vs. CHO cell line based) were relatively small, showing the robustness of these types of assays. Using hierarchical clustering, bioactivity relationships between compounds but also relationships between various bioassays were determined. The in vitro assays were found to be good predictors of in vivo androgenic or estrogenic activity of a range of compounds, allowing prescreen and/or possible reduction of animal studies.

androgen, estrogen, CALUX, bioassay, Hershberger, Allen-Doisy

Steroid hormones are essential for reproduction, stress management, salt and glucose balances, as well as several other physiological processes. Due to the relatively simple chemical structure and lipophilic nature of steroids, their regulatory pathways can easily be modified by pharmacological, environmental, and/or dietary agents. Because of this, steroids or steroid-mimicking compounds are applied in many fields, making identification of the endocrine activity of these compounds important. Analytical-chemical and immunological methods are commonly used to detect steroids in food and feed, clinical practice, environmental samples, or doping control. These methods have the drawback that they only quantify the compound of interest and are not able to determine biological activity of unknown compounds or their metabolites, this in contrast to biological assays.

Bioassays in rats, mice, or rabbits were developed a long time ago to determine the endocrine activity of compounds. Important examples are the assessment of vaginal smear types to define estrogenicity (Allen and Doisy, 1923) and of the prostate, seminal vesicle, and musculus levator ani (MLA) growth to determine androgenic and anabolic activities (Hershberger et al., 1953; van der Vies and de Visser, 1983). The contribution of animal studies, however, is hampered, particularly with respect to sensitivity, capacity, costs, the desire to limit animal use, and speed. To allow fast analysis of new chemicals, food additives, and pharmaceutical compounds, high throughput screening assays have been developed. These assays are based on the mechanism of action of compounds and are able to measure activation or inhibition of specific cellular pathways. These mechanism-based assays, in combination with rapid advance in automated screening technologies and bioinformatics, create new possibilities to limit animal studies. These in vitro detection systems are ideal for first-line screening, while positive hits can be tested more extensively using more specialized cell culture systems and animal models.

The mechanism of action of steroid hormones is well established, and opened opportunities for mechanism-based assays. Steroid hormones like estrogens and androgens are nuclear hormone receptor ligands that enter cells by diffusion where they bind to their cognate steroid receptors. Five major types of steroid receptors are known: those for estrogens, androgens, progestagens, glucocorticoids, and mineralocorticoids (Mangelsdorf et al., 1995; McKenna and O'Malley, 2002), now classified as members of the subfamily 3 within the nuclear receptor family (Nuclear Receptors Nomenclature Committee, 1999). Upon ligand binding these receptors become activated, and they will enter the nucleus and bind to recognition sequences in promoter regions of target genes, the hormone responsive elements. Depending on the presence of receptor-interacting proteins, so-called cofactors including coactivators as well as corepressors (Chang and McDonnell, 2005; McDonnell and Norris, 2002), the DNA-bound receptor will activate transcription of the target gene, leading to new protein synthesis and an altered cellular functioning. Besides the classical genomic-based action of steroid hormones involving nuclear hormone receptors, rapid nongenomic mechanisms of steroids might also occur via putative membrane-bound receptors, at least for estrogen and progesterone signaling (Luconi et al., 2004).

A whole range of so-called reporter gene assays have been developed by us and by others for compounds interacting with a range of steroid receptors, including the estrogen and androgen receptor (Balaguer et al., 1999; de Gooyer et al., 2003; Legler et al., 1999; Schoonen et al., 2000a,b; Sonneveld et al., 2005; Terouanne et al., 2000). In these reporter gene assays, DNA sequences containing specific hormone-responsive elements are linked to the gene of an easily measurable protein (the reporter gene; e.g., firefly luciferase). When stably introduced in a cell line expressing the cognate receptor, or by double transfection with a receptor of interest, a specific reporter cell line is generated allowing large scale screening of compounds. Similarly, simple receptor binding assays can be used to exert such screenings. However, the latter cannot distinguish between receptor interacting compounds that will lead to (partial) transcriptional activation or (partial) transcriptional inhibition of the receptor.

In this study we determined the suitability of two different reporter gene assays, either U2-OS or CHO cell line based, and two receptor binding assays using MCF-7 cells (Bergink et al., 1983) as a prescreen for, or limitation of animal studies (ECVAM working group on chemicals, 2002) in determining androgenic and estrogenic activities of compounds. The in vitro assays (AR/ER binding and AR/ERα reporter gene assays) were found to be good predictors of AR/ER in vivo agonist activity of a range of mainly steroidal compounds.

MATERIALS AND METHODS

Chemicals.

Androstenedione, diethylstilbestrol (DES), dexamethasone (DEX), 5α-dihydrotestosterone (DHT), 17α-estradiol, 17β-estradiol (E2), estriol, estrone, 17α-ethinyl-estradiol (EE), flutamide, genistein, levonorgestrel (LNG), 17α-methyl-testosterone (MT), mifepristone (RU486), norethynodrel (NE), progesterone, tamoxifen citrate, testosterone (T), and testosterone propionate (TP) were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Methyltrienolone (R1881) was obtained from Perkin Elmer (Perkin Elmer, Groningen, The Netherlands). Cyproterone acetate (CA), medroxyprogesterone acetate (MPA), and 19-nor-testosterone (nandrolone) were obtained from Steraloids Inc. (Newport, RI). All other used compounds were supplied by the Department of Medicinal Chemistry of N.V. Organon (Oss, The Netherlands). All chemicals were diluted in either ethanol or dimethylsulphoxide (DMSO, 99.9%, Acros, Geel, Belgium) and stored at −20°C. Neomycin (G418) was purchased from Life Technologies (Breda, The Netherlands).

Animals.

SPF-bred immature male and young female HSD/Cpb:ORGA rats were supplied by The Harlan Sprague-Dawley/Central Institute for the Breeding of Laboratory Animals of the Dutch Organization for Applied Scientific Research ((HSD-CPB), Zeist, The Netherlands). Rats were housed in light-, humidity- and temperature-controlled rooms (14 h light–10 h dark; 21–23°C), and given tap water and pelleted food (RMH-B, Hope farms, Linschoten, The Netherlands) ad libitum. Animal handling was in accordance with the Dutch law on Animal Experimentation and the European Directive for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (EU directive #86/606/CEE). The Committee for Experiments on Animals of N.V. Organon approved the experiments.

Cell culture.

Human MCF-7 cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium (DF, Gibco) supplemented with 5% fetal calf serum (FCS). U2-OS-based AR and ERα CALUX cells, stably expressing human AR and ERα and their corresponding luciferase reporter genes (e.g., multimerized responsive elements for the cognate receptor coupled to a minimal promoter element (the TATA box) and luciferase) (Legler et al., 1999; Sonneveld et al., 2005) were cultured in DF medium supplemented with 7.5% FCS and 200 μg/ml G418. Chinese Hamster Ovary CHO-AR and CHO-ERα cells stably expressing human AR and ERα and their corresponding luciferase reporter genes (e.g., the mouse mammary tumor virus promoter (MMTV) for AR and the rat oxytocin promoter (RO) for ERα coupled to the luciferase reporter gene), respectively, were cultured as described earlier (Schoonen et al., 2000a,b).

Reporter gene assays.

AR and ERα CALUX cells were plated in 96-well plates (8000 cells/well) with phenol red-free DF medium supplemented with 5% dextran-coated charcoal-stripped FCS (DCC-FCS; van der Burg et al., 1988) at a volume of 200 μl per well. Two days later, the medium was refreshed, and cells were incubated with the compounds to be tested (dissolved in ethanol or DMSO) in triplicate at a 1:1000 dilution. After 24 h the medium was removed, and cells were lysed in 30 μl Triton-lysis buffer and measured for luciferase activity using a luminometer (Lucy2; Anthos Labtec Instruments, Wals, Austria) for 0.1 min/well. For transactivation studies using CHO-derived reporter gene assays, stably transfected CHO-AR and CHO-ERα cells were used as described previously (Schoonen et al., 2000a,b). Data sets for the CHO reporter gene assays were collected between 1995 and 2003 at Organon.

Receptor binding assays.

For hAR and hER displacement analysis, MCF-7 cells were used. The cells were cultured, harvested, and cytosolic preparations were prepared as described previously (Schoonen et al., 1995). Prior to use, cytosol equivalent to 1 g of cells was diluted with buffer at a ratio of 1:10 for hER and 1:5 for hAR (w/v). Samples were counted in a Topcount microplate scintillation counter (Perkin Elmer). The specific 50% competition level of each compound was analyzed in the range of 0.121 up to 1000 nM with a two-fold dilution range. The relative binding affinities (RBAs) of the compounds were obtained by a three-point parallel line analysis (Finney, 1978) using three subsequent concentrations in the range of 25, 50, and 75% of competition for each individual compound in relation to the reference compound. The reference compound DHT, as well as E2, was measured in the range of 0.97, 1.95, and 3.90 nM (Schoonen et al., 1998, 2000a,b). Specific binding was determined by subtracting nonspecific from total binding. The mean RBA values of at least two different independent tests were calculated for each compound. The overall statistical deviation (SD) was within the 5% level. Data sets for the receptor binding assays were collected between 1983 and 2000 at Organon.

In vivo studies.

The assay for androgenic-anabolic activity of the compounds in immature male orchidectomized rats was performed according to the Hershberger test (Hershberger et al., 1953), with minor modifications (van der Vies and de Visser, 1983). Groups of six animals per compound dose were treated subcutaneously (sc) twice a day for seven consecutive days. At the end of the treatment period autopsy was performed, and the weights of the ventral prostate, seminal vesicles, and levator ani muscle were recorded. Testosterone was used as a reference for subcutaneous administration in a dose of 160 μg/kg as the minimal active dose (MAD). The MAD was determined as the dose at which the ventral prostate weight was 1.8 times higher than the placebo value. In vivo activities were calculated relative to testosterone. Data sets for the Hershberger assay were collected between 1970 and 2000 at Organon. The estrogenic activity of the compounds in ovariectomized rats was determined by scoring vaginal cornification (Allen-Doisy test) as described earlier (Allen and Doisy, 1923; van der Vies and de Visser, 1983). Female adult rats were ovariectomized and primed 3 weeks later with a single dose of 1 μg estradiol (sc). One week later the reference compound estradiol and the compounds to be tested were administered (sc) with three subsequent equal doses: one dose in the afternoon of the first day, and the next two doses in the morning and afternoon of the following day. Vaginal smears were taken at the end of the third day, twice the fourth day, and again on the morning of the last day (day 5). The smears were stained with Giemsa and evaluated (de Jongh and Laqueur, 1938). For estradiol a total dosing of 0.5 μg/kg was used to obtain the minimal active dose (MAD). Each total dose is divided equally over three administrations. Test compounds are administered in total doses of 0.05 μg/rat up to 1.0 mg/rat. The usual phases observed in the morning of the 4-day estrus cycle are di-estrus (score a), pro-estrus (score e) or estrus (score g). A rat is considered to give a positive score if at least one of the smears indicates a score of e, intermediate f or g. In total six rats per compound dose were treated, and a score of 1 or 2 positive animals out of six is called weakly active, while a score of 3 up to 6 out of six animals is called active. The total dose (sc) at which 50% of the animals showed one or more positive smears is given as the minimal active dose (MAD). In vivo activities were calculated relative to E2. Data sets for the Allen-Doisy assay were collected between 1970 and 2000 at Organon.

Statistical analysis.

Luciferase activity per well was measured as relative light units (RLUs). Fold induction was calculated by dividing the mean value of light units from exposed and nonexposed (solvent control) wells. For CALUX cells, luciferase induction as a percentage of maximal DHT (AR CALUX) or E2 (ERα CALUX) activity was calculated by setting the highest fold induction of DHT or E2 at 100%. Data are represented as mean values ± SEM from at least three independent experiments with each experimental point performed in triplicate. Dose-response curves were fitted using the sigmoidal fit y = (a0 + a1)/(1 + exp[−(x − a2)/a3]) in GraphPad Prism (version 4.00 for Windows, GraphPad Software, San Diego, CA), which determines the fitting coefficients by an iterative process minimizing the c2 merit function (least squares criterion). The EC50 values were calculated by determining the concentration by which 50% of maximum activity was reached using the sigmoidal fit equation. At least eight different concentrations covering the total S-curve were included for each compound. The relative transactivation activity (RTA) of each compound tested was calculated as the ratio of maximal luciferase reporter gene induction values of each compound and the maximal luciferase reporter gene induction value of reference compound of the specific assay. The transactivation activity of the reference compounds DHT or E2 was arbitrarily set at 100. The relative agonistic activities (RAA) for the CALUX reporter gene assays were calculated by dividing the EC50 concentration of the reference compound with the EC50 concentration of the compound of interest. Relative agonistic activity studies with CHO reporter gene assays were carried out with five concentrations of the standards DHT and E2 at 1.50 × 10−11, 3.00 × 10−11, 6.00 × 10−11, 1.21 × 1010, and 2.42 × 10−10 M and three subsequent concentrations of the compound of interest in the range of 1 pM up to 100 nM. The relative agonistic activities of the compounds were obtained by a 3-point parallel line analysis (Finney, 1978) using three up-following concentrations in the range of 25, 50, and 75% activation for each individual compound in relation to the reference compound (Schoonen et al., 1998, 2000a,b). The mean RAA values were calculated from at least two different independent tests. The overall SD was within the 5% level. In the in vivo Allen-Doisy and Hershberger tests, the mean scores per dose were calculated. The RAA values for in vivo compound testing were calculated by dividing the MAD of the standards testosterone (Hershberger assay) or E2 (Allen-Doisy assay) by the MAD of the compound of interest. Correlation coefficients (r2) and their correspondent p-values were calculated with GraphPad Prism (version 4.00 for Windows, GraphPad Software, San Diego, CA). Cutoff values were 0.0001 for Allen-Doisy comparison, and 0.001 for reporter gene, receptor binding, and Hershberger comparisons. Two-dimensional hierarchical clustering on the base 10 logarithm of the RAA data was performed using the correlation option within the clustergram function from the bioinformatics toolbox in Matlab (The Mathworks, the Netherlands).

RESULTS

Comparison of Different In Vitro Reporter Gene Assays for Determination of Androgenic and Estrogenic Activities

The results obtained in two different laboratories were compared by using a panel of mainly steroidal chemicals with in vitro reporter gene assays for androgen and estrogen receptors. The AR and ERα CALUX cell lines as well as the CHO cell lines are efficient tools to screen for agonistic and antagonistic effects of compounds toward the androgen receptor and estrogen receptor alpha, respectively (Schoonen et al., 2000a,b; Sonneveld et al., 2005). AR and ERα CALUX cells are human U2-OS cell line based with the same basal characteristics as other CALUX reporter gene assays (ERβ, PR, and GR CALUX), being robust, easy maintainable, stable and strongly responsive, and selective. The range in EC50 values measured with different ligands over time, including the positive controls DHT (AR CALUX) and E2 (ERα CALUX), is small, reflected by an inter-assay CV of 22% for the AR CALUX and 25% for the ERα CALUX reporter gene assay (Sonneveld et al., 2005).

Typical dose-response curves for several natural as well as synthetic androgens and estrogens using AR and ERα CALUX reporter gene assays, respectively, are shown in Figure 1. The AR CALUX cell line showed high sensitivity toward all androgens tested (Fig. 1A and Table 1), with the following range of potencies (EC50 values): dihydrotestosterone (DHT; 110 pM), testosterone (T; 657 pM), and its 19-nor derivatives nandrolone (19-nor-T; 301 pM), 19-nor-11-keto-T (2845 pM), and 11-methylene-19-nor-T (98 pM). The selectivity of the AR CALUX cells is high, since representative steroids for other hormone receptors (E2 and EE for ER, progesterone for PR, and dexamethasone for GR) showed no substantial agonistic response, with relative agonistic activities below 0.001, except for dexamethasone (0.003), which additionally has a relative transcriptional activity (RTA) of 8% compared to DHT (see Table 1; Sonneveld et al., 2005).

FIG. 1.

Dose response curves for different receptor activating compounds in the AR and ERα CALUX reporter gene assays. AR and ERα CALUX cells were plated in 96-well plates. AR CALUX cells (A) were treated with the androgenic compounds DHT (▪), testosterone (•), and its 19-nor derivatives 19-nor-11-keto-T (○), 11-methylene-19-nor-T (▾), and 19-nor-T (□), and ERα CALUX cells (B) were treated with the estrogenic compounds E2 (•), 11β-ethenyl-E2 (○), NET (▴), 19-nor-5α-NET (▪), and 3β-OH-5α-hydrogen-11β-ethenyl-NET (□) for 24 h using DF medium containing 5% DCC-FCS. Each point represents the mean of at least three independent experiments ± SEM.

FIG. 1.

Dose response curves for different receptor activating compounds in the AR and ERα CALUX reporter gene assays. AR and ERα CALUX cells were plated in 96-well plates. AR CALUX cells (A) were treated with the androgenic compounds DHT (▪), testosterone (•), and its 19-nor derivatives 19-nor-11-keto-T (○), 11-methylene-19-nor-T (▾), and 19-nor-T (□), and ERα CALUX cells (B) were treated with the estrogenic compounds E2 (•), 11β-ethenyl-E2 (○), NET (▴), 19-nor-5α-NET (▪), and 3β-OH-5α-hydrogen-11β-ethenyl-NET (□) for 24 h using DF medium containing 5% DCC-FCS. Each point represents the mean of at least three independent experiments ± SEM.

TABLE 1

AR CALUX LogEC50 Values, Relative Transcriptional Activity (RTA), and Relative Agonistic Activity (RAA)



Modification

AR CALUX

CHO-AR RAA 1 = DHT

AR binding RBA 1 = DHT

Hershberger RAA 1 = T
Compound
R3
R4–5
R6
R7
R11
R17
LogEC50(M)
RTA (%)
RAA 1 = DHT
Nandrolone derivatives
19-nor-T (Nandrolone) −9.5 92 0.486 0.530 0.474 0.080 
6α-methyl-NET −8.9 50 0.139 0.021 0.083 nd 
6α-methyl-19-nor-T −9.6 127 0.464 1.256 0.895 >0.064 
MENT = 7α-methyl-19-nor-T −10.1 121 1.983 2.630 1.405 1.280 
7α-methyl-NET −9.7 78 0.912 0.206 0.291 0.500 
7α-methyl-11β-methyl-NET −9.4 69 0.397 0.166 0.253 0.008 
7α-methyl-11-methylene-NET −9.1 74 0.218 0.064 0.133 0.008 
7α-methyl-11-ethylene-NET −8.5 69 0.054 0.016 0.097 0.250 
7α-methyl-17α-(2-propenyl)-19-nor-T −9.7 79 0.956 0.149 0.381 0.016 
11α-OH-19-nor-T −7.7 100 0.008 0.012 0.008 0.002 
11β-OH-19-nor-T −7.3 97 0.003 0.003 0.003 0.006 
11β-methyl-19-nor-T −9.6 120 0.734 1.200 0.620 <0.123 
11-methylene-19-nor-T −10.0 107 1.669 2.003 0.930 0.055 
11β-ethyl-NET −8.0 48 0.017 0.003 0.020 0.032 
11β-ethinyl-NET −8.5 64 0.051 0.020 0.046 0.051 
11β-ethenyl-NET −7.1 36 0.002 0.004 0.021 0.005 
11-keto-19-nor-T −8.6 102 0.059 0.065 0.109 0.007 
17α-methyl-19-nor-T −9.9 87 1.301 0.815 0.380 >0.032 
Norethisterone (NET) −8.2 69 0.026 0.011 0.034 0.064 
17α-(2-propenyl)-19-nor-T −8.4 51 0.047 0.003 0.111 0.008 
3-deoxy-11β-OH-19-nor-T −8.4 88 0.042 0.000 nd 0.032 
3-deoxy-11-keto-17α-ethyl-19-nor-T −8.0 44 0.017 0.000 nd 0.032 
5α-hydrogen-7α-methyl-NET −9.3 64 0.313 0.038 0.307 0.127 
5α-hydrogen-11β-methyl-NET −8.7 52 0.092 0.027 0.128 nd 
5α-hydrogen-11β-ethyl-NET −8.0 45 0.018 0.002 0.032 0.008 
5α-hydrogen-11β-ethinyl-17α- ethenyl-19-nor-T −8.7 53 0.092 0.040 0.025 nd 
5α-hydrogen-11β-ethinyl-NET −8.5 55 0.051 nd 0.070 0.016 
5α-hydrogen-19-nor-T −9.4 110 0.405 0.259 0.588 <0.008 
5α-hydrogen-NET −8.5 149 0.056 0.008 0.051 0.032 
5α-hydrogen-17α-(2-propenyl)-19-nor-T >−6.0 23 0.007 0.001 0.025 0.008 
Δ15-NET −8.3 57 0.036 0.001 0.025 0.043 
7α-methyl-androst-5(10)-ene-19-nor-T −9.4 102 0.466 0.809 nd 0.160 
Norethynodrel (NE) −8.2 48 0.027 0.006 0.007 0.008 
Testosterone derivatives
−9.2 94 0.146 0.168 0.171 1.000 
T propionate −9.1 81 0.200 nd nd >1.000 
Testosterone derivatives (continued)
11-methylene-17β-propionate-T −8.7 86 0.080 0.017 nd 1.000 
17α-methyl-T −9.1 108 0.197 0.195 0.206 >1.000 
T-17,17′-(2,2′-oxybisacetate) −9.3 81 0.319 0.014 nd 0.032 
7α-methyl-T −8.5 87 0.051 0.455 0.250 nd 
5α-hydrogen-T(DHT) −9.9 100 1.000 1.000 1.000 >0.250 
(14β,17α,20S)-20-OH-19-norpregna-4, 9-diene-3-one −9.7 90 0.975 0.530 0.400 0.127 
(11β,14β,17α,20S)-11-ethenyl-20-OH- 19-norpregna-4,9-diene-3-one −9.4 82 0.453 0.345 0.405 0.127 
Androst-4-ene-3,17-dione −8.4 82 0.057 nd 0.001 nd 
R1881 −9.9 69 1.063 0.976 0.595 nd 
Flutamide >−5.0 <5 0.000 <0.001 0.015 0.002 
17β-mercapto-androst-4-en-3-one >−6.0 41 0.000 <0.001 0.028 0.032 
11β-ethenyl-19-nor-androstenedione −8.3 71 0.036 0.026 nd 0.064 
5α-Androstane-3α,17β-diol −7.7 66 0.009 0.003 nd 0.500 
Progestagens
Progesterone >−5.0 36 0.000 <0.001 0.019 0.064 
Norgestimate −5.9 25 0.000 <0.002 0.027 nd 
MPA −8.2 75 

In Vivo vs In Vitro

Experiments are the methods that are used in scientific studies to aid in comparing two competing explanations of certain phenomena such as those that are found in certain scientific areas like biology wherein observations are made through testing and experiments.
In biology, the term “in situ” means that the examination and observation of a rare occurrence takes place where it occurs. Subjects are examined in position and are not moved to another tool or channel. An example is the observation of dolphins at sea. They are observed where they are found and are not moved to an aquarium or other container which is more convenient.
In cell science, in situ can mean something in between in vivo and in vitro. “In vivo” is a Latin word which means “within the living.” It is the experiment or observations done on the living tissue of the whole living organism in a controlled environment.
In vivo experiments are done in the organism’s natural environment or in the organism itself. It is done in a living organism and not in a dead or partial one. It is found to be more suited on experiments done on organisms that are alive.
One example is clinical testing or a clinical trial which can be a controlled testing of a new drug or device on human subjects. The subjects are given the drugs and are observed for a certain period of time. Another is animal testing which is an experiment which is done on animals usually rats, birds, frogs, and other animals.
It varies in duration from short-term and up to a lifetime exposure. In vivo experiments tend to be more expensive to do and are subject to several restrictions because it deals with live animals.
“In vitro,” on the other hand, is a Latin word that means “within the glass.” It is the experiment or observations done on the tissue outside of the living organism in a controlled environment, usually using Petri dishes and test tubes.
Most experiments in cellular biology are done through in vitro studies and are not conducted in the organism’s natural environment or inside a living organism. This results in the limited success of the experiments in simulating the actual conditions inside an organism and makes its outcome less precise. Compared to in vivo experiments, it is less expensive and provides quicker results.

Summary:

1.In vivo is an experiment or testing that is done inside the living organism or in its natural environment while in vitro is an experiment that is done outside of the living organism, usually in a test tube or Petri dish.
2.In vivo testing is more expensive and time consuming than in vitro testing which provides quicker results.
3.While most biological experiments are done in vitro, it is less precise than experiments done in vivo because it does not simulate the actual conditions inside the organism.
4.In vivo experiments and testing have many restrictions because it deals with live animals while in vitro does not.


Cite
Emelda M. "Difference Between In Vivo and In Vitro." DifferenceBetween.net. April 27, 2011 < http://www.differencebetween.net/science/difference-between-in-vivo-and-in-vitro/ >.

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