View source for Chemistry:In vitro toxicology

{{short description|Scientific analysis}}
{{DISPLAYTITLE:''In vitro'' toxicology}}
'''''In vitro'' [[Chemistry:Toxicology testing|toxicity testing]]''' is the scientific [[Scientific method|analysis]] of the toxic effects of [[Chemistry:Chemical substance|chemical substance]]s on cultured [[Biology:Bacteria|bacteria]] or mammalian [[Biology:Cell|cells]].<ref>{{cite journal |vauthors=Gavanji S, Bakhtari A, Famurewa AC, Othman EM |title=Cytotoxic Activity of Herbal Medicines as Assessed in Vitro: A Review |journal=Chemistry & Biodiversity |volume=20 |pages=3–27 |date=January 2023 |issue=2 |pmid=36595710 |doi=10.1002/cbdv.202201098|s2cid=255473013 |doi-access=free }}</ref> ''[[In vitro]]'' (literally 'in glass') testing methods are employed primarily to identify potentially hazardous chemicals and/or to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic [[Chemistry:Medication|drugs]], agricultural chemicals and food additives.

''In vitro'' assays for xenobiotic toxicity are recently carefully considered by key government agencies (e.g., EPA; NIEHS/NTP; FDA), to better assess human risks. There are substantial activities in using in vitro systems to advance mechanistic understanding of toxicant activities, and the use of human cells and tissue to define human-specific toxic effects.<ref>{{Cite web | title = Tox21 | publisher = Source: U.S. Environmental Protection Agency | url = http://www.epa.gov/ncct/Tox21/ | access-date = 2011-10-29}}</ref>

==Improvement over animal testing==
Most toxicologists believe that ''in vitro'' toxicity testing methods can be more useful, more time and cost-effective than toxicology studies in living animals<ref>{{Cite journal|last1=Mahony|first1=Catherine|last2=Ashton|first2=Randolph S.|last3=Birk|first3=Barbara|last4=Boobis|first4=Alan R.|last5=Cull|first5=Tom|last6=Daston|first6=George P.|last7=Ewart|first7=Lorna|last8=Knudsen|first8=Thomas B.|last9=Manou|first9=Irene|last10=Maurer-Stroh|first10=Sebastian|last11=Margiotta-Casaluci|first11=Luigi|date=July 2020|title=New ideas for non-animal approaches to predict repeated-dose systemic toxicity: Report from an EPAA Blue Sky Workshop|journal=Regulatory Toxicology and Pharmacology|language=en|volume=114|pages=104668|doi=10.1016/j.yrtph.2020.104668|pmid=32335207|doi-access=free}}</ref> (which are termed ''in vivo'' or "in life" methods). However, the [[Biology:In vitro to in vivo extrapolation|extrapolation from ''in vitro'' to ''in vivo'']] requires some careful consideration and is an active research area.

Due to regulatory constraints and ethical considerations, the quest for alternatives to animal testing has gained a new momentum. In many cases the in vitro tests are better than animal tests because they can be used to develop safer products.<ref>{{Cite web | title = Vision and Roadmap for the 21st Century | publisher = Source: [[Chemistry:National Toxicology Program|National Toxicology Program]] | url = http://ntp.niehs.nih.gov/go/vision | access-date = 2011-10-29}}</ref>

The United States Environmental Protection Agency studied 1,065 chemical and drug substances in their ToxCast program (part of the [[Biology:CompTox Chemicals Dashboard|CompTox Chemicals Dashboard]]) using ''in silica'' modelling and a human pluripotent [[Biology:Stem cell|stem cell]]-based assay to predict ''[[Biology:In vivo|in vivo]]'' developmental intoxicants based on changes in cellular [[Biology:Metabolism|metabolism]] following chemical exposure. Major findings from the analysis of this ToxCast_STM dataset published in 2020 include: (1) 19% of 1065 chemicals yielded a prediction of [[Chemistry:Developmental toxicity|developmental toxicity]], (2) assay performance reached 79%–82% accuracy with high specificity (> 84%) but modest sensitivity (< 67%) when compared with ''in vivo'' animal models of human prenatal developmental toxicity, (3) sensitivity improved as more stringent weights of evidence requirements were applied to the animal studies, and (4) statistical analysis of the most potent chemical hits on specific biochemical targets in ToxCast revealed positive and negative associations with the STM response, providing insights into the mechanistic underpinnings of the targeted endpoint and its biological domain.<ref>{{cite journal|last1=Zurlinden |first1=TJ|last2=Saili |first2=KS|last3=Rush |first3=N|last4=Kothiya| first4=P|last5=Judson|first5=RS|last6=Houck|first6=KA |last7=Hunter |first7=ES|last8=Baker|first8=NC |last9=Palmer|first9=JA |last10=Thomas|first10=RS|last11=Knudson |first11=TB|title=Profiling the ToxCast Library With a Pluripotent Human (H9) Stem Cell Line-Based Biomarker Assay for Developmental Toxicity|journal=Toxicological Sciences|date=2020|volume=174|issue=2|pages=189–209|doi=10.1093/toxsci/kfaa014|pmid=32073639|pmc=8527599}}</ref>

[[Image:Microtiter plate.JPG|thumb|A 96-well microtiter plate being used for ELISA.]]

==Examples of cell viability and other cytotoxicity assays used for ''in-vitro'' toxicology==
Many methods of analysis exist for [[Biology:Assay|assay]]ing test substances for cytotoxicity and other cellular responses.

===Hemolysis assay===
The hemolysis assay examines the propensity of chemicals, drugs or [[Chemistry:Medication|medication]], or any blood-contacting medical device or material to lyse [[Biology:Red blood cell|red blood cell]]s (erythrocytes). The lysis is easily detected due to the release of [[Biology:Hemoglobin|hemoglobin]].<ref>{{cite journal | vauthors = Sæbø PI, Bjørås M, Franzyk H, Helgensen E, Booth J | title = Optimization of the Hemolysis Assay for the Assessment of Cytotoxicity| journal = Int. J. Mol. Sci. | volume = 24 | issue = 3  | date = February 2, 2023 | page = 2914| pmid = 36769243| doi = 10.3390/ijms24032914 | pmc = 9917735 | doi-access = free }}</ref>

===MTT and MTS===
[[Biology:MTT assay|MTT assay]] is used often in determining cell [[Biology:Viability assay|viability]] and has been validated for use by international organisations.  MTT assay involves two steps of introducing the assay to the chemicals and then a solubilisation step.

The colorimetric MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium) in vitro assay is an updated version of the validated MTT method, MTS assay has the advantage of being soluble. Hence, no solubilisation step is required.

===ATP===
[[Physics:Bioluminescence#ATP bioluminescence|ATP assay]] has the main advantage of providing results quickly (within 15 minutes) and only requires fewer sample cells.  The assay performs lysis on the cells and the following chemical reaction between the assay and ATP content of cells produces luminescence.  The amount of luminescence is then measured by a photometer and can be translated into number cells alive since
*ATP assay assumes alive cells still have ATP inside them, and
*Luminescence level recorded is proportional to the ATP content in the sample cells.

===Neutral red===
Another cell viability endpoint can be neutral red (NR) uptake.  Neutral red, a weak cationic dye penetrates cellular membranes by non-diffusion and accumulates intercellularly in lysosomes.  Viable cells take up the NR dye, damaged or dead cells do not.

===Cytokine quantification via ELISA===
ELISA kits can be used to examine up and down regulation of proinflammatory mediators such as cytokines (IL-1, TNF alpha, PGE2)....

Measurement of these types of cellular responses can be windows into the interaction of the test article on the test models (monolayer cell cultures, 3D tissue models, tissue explants).

== Types of ''in vitro'' studies ==
Broadly speaking, there are two different types of ''in vitro'' studies depending on the type system developed to perform the experiment. The two types of systems generally used are : a) Static well plate system and b) the multi-compartmental perfused systems.

=== Static well plate system ===
The static well plate or layer systems are the most traditional and simplest form of assays widely used for in vitro study. These assays are quite beneficial as they are quite simple and provide a very accessible testing environment for monitoring chemicals in the culture medium as well as in the cell. However the disadvantage of using these simple static well plate assays is that, they cannot represent the cellular interactions and physiologic fluid flow conditions taking place inside the body.

=== Multi-compartmental perfused systems ===
New testing platforms are now developed to solve problems related to cellular interactions. These new platforms are much more complex based on multi-compartmental perfused systems.<ref>
{{cite journal
|author1=Leclerc E. |author2=Sakai Y. |author3=Fujii T. |title=Perfusion culture of fetal human hepatocytes in microfluidic environments
|journal=Biochemical Engineering Journal
|date=2004
|volume=20
|issue=2–3
|pages=143–148
|doi=10.1016/j.bej.2003.09.010}}</ref> 
The main objective of these systems is  to reproduce in vivo mechanisms more reliably by providing cell culture environment close to the in vivo situation. Each compartment in the system represent a specific organ of the living organism and thus each compartment has a specific characteristics and criteria. Each compartment in these systems are connected by tubes and pumps through which the fluid flows thus mimicking the blood flow in the in vivo situation. The draw back behind the use of these perfused systems is that, the adverse effects ( influence of both the biological and non-biological components of the system on the fate of the chemical under study) are more compared to the static systems. In order to reduce the effect of non-biological components of the system, all the compartments are made of glass and the connecting tubes are made up of teflon. A number of kinetic models have been proposed to take care of these non-specific bindings taking place in these in vitro systems.<ref>
{{cite journal
|author1=Ouattara D.A. |author2=Choi S.-H. |author3=Sakai Y. |author4=Pery A.R.R. |author5=Brochot C. |title=Kinetic modelling of ''in vitro'' cell-based assays to characterize non-specific bindings and ADME processes in a static and a perfused fluidic system 
|journal=Toxicology Letters
|date=2011
|volume=205
|issue=3
|pages=310–319
|doi=10.1016/j.toxlet.2011.06.021|pmid=21723928 }}</ref>

To improve the biological difficulties arising from the use of different culture in vitro conditions, the traditional models used in flasks or micro-well plates has to be modified. With parallel development in micro-technologies and tissue engineering, these problems are solved using new pertinent tools called "micro-fluidic biochips".<ref>
{{cite journal
|author1=Baudoin R. |author2=Corlu A. |author3=Griscom L. |author4=Legallais C. |author5=Leclerc E. |title=Trends in the development of microfluidic cell biochips for in vitro hepatotoxicity 
|journal=Toxicology in Vitro
|date=2007
|volume=21
|issue=4
|pages=535–544
|doi=10.1016/j.tiv.2006.11.004|pmid=17188836 }}</ref>

== References ==
{{Reflist}}
{{Toxicology}}

{{DEFAULTSORT:In Vitro Toxicology}}
[[Category:Toxicology]]

{{Sourceattribution|In vitro toxicology}}