Orion Corporation

Historically, by relying heavily upon costly and laborious animal-based toxicity assays, the field of toxicology has often neglected examinations of the cellular and molecular mechanisms of toxicity for most compounds—information that, if available, would strengthen risk assessment analyses.

It is undeniably concerning for ecosystems and human health that chemical pollutants are becoming more prevalent in the environment. The field of toxicology has historically neglected investigations into the cellular and molecular mechanisms of toxicity for most compounds by heavily relying on expensive and time-consuming animal-based toxicity assays. If such information were available, it would strengthen risk assessment analyses.

Functional toxicology, which assesses genetic needs for chemical tolerance using cells or creatures with gene deletions or depleted proteins, can enhance the science of toxicity testing by supplying information on the chemical causes of toxicity.

Functional approaches can provide fresh perspectives on chemical toxicity, promising pathways and processes, and potential genes for human toxicant sensitivity or resistance. With the aid of genetic techniques, functional toxicology can be carried out in yeasts, other fungi and bacteria, and more sophisticated eukaryotes, including zebrafish, fruit flies, rodents, and human cell lines. The need to employ simpler systems, like yeasts, to guide further research in more complicated systems, such as human cell lines, is highlighted.

To assess chemical toxicity, the science of toxicology currently uses time-consuming, prohibitively expensive animal-based procedures, which often only investigate several endpoints. It is unrealistic to rely on these conventional methods to fill data gaps when there are tens of thousands of chemicals in use that do not have sufficient toxicity data. The National Research Council (NRC) envisioned that toxicology should commit to mechanistically-based high-throughput cellular in vitro tests after identifying the necessity for more creative testing methods. This will accelerate testing, cut expenses, and use fewer animals while also improving our understanding of chemical toxicity.

High-throughput in vitro techniques undoubtedly represent a step forward in the field of toxicity testing, but they are still restricted to assays with already established endpoints, such as investigations of the stress response pathways brought on by oxidative species, heat shock, DNA damage, hypoxia, and unfolded proteins. Another method involves conducting focused and untargeted investigations into the chemical causes of toxicity using omics technologies including gene expression profiling, proteomics, lipidomics, and metabolomics. However, these assays are correlative and do not directly relate genes and their needs in the cellular toxicant response since they associate toxicant exposure with changes in mRNA, protein, lipid, or metabolite levels.

Functional toxicology bases its analysis of the genetic needs for toxicity tolerance on the high-throughput usage of cells and creatures that carry gene deletions or decreased protein levels. Any assayable trait that is responsive to a toxin can be assessed, but viability or fitness is the most common outcome. Functional techniques can provide different information than that presented by the aforementioned correlative procedures. Functional analyses can provide novel insight into the chemical mechanisms of action, define more precise toxicological endpoints, and guide future mechanistic-based assays. Functional analyses have been carried out in budding and fission yeast, bacteria, nematodes, fruit flies, zebrafish, and human cell lines.

Functional toxicological investigations can easily use genome-wide RNA interference (RNAi) screens, which have grown in importance as a tool in the drug discovery process. RNA interference (RNAi) techniques take advantage of already-existing cellular machinery to sever a target gene's mRNA, blocking translation and thus knocking down the activity of the target gene. Functional toxicology may benefit from RNAi screenings in cell lines. However, these procedures are difficult to carry out because off-target effects and inadequate knockdown of the target genes might make execution and analysis difficult. RNAi loss-of-function screens are the mainstay of functional genomic applications in human cells.

RNAi has been used in the past to explore cellular toxicity in Drosophila cell lines; examples include the identification of tiny compounds that impede the cell cycle and the discovery of genes that promote the aggregation of mutant Huntingtin proteins. Barcoded short hairpin RNA (shRNA) libraries make it possible to find shRNAs that, when subjected to toxicant selection, cause a certain phenotype. By hybridising to microarrays, the relative abundance of barcodes in the control and treatment populations can be determined. This method is effective at finding shRNAs that improve fitness, however, it is time consuming and needs a lot of tweaking to also find shRNAs that impair viability. RNAi-based screens have nevertheless revealed human genes whose silencing provides resistance to a variety of medications.

The use of computational analysis to understand toxicant mechanisms of action is a fundamental component of high-throughput functional toxicology approaches. High-throughput methods in zebrafish or human cell lines would not be achievable without computer analysis. Numerous research has used over-enrichment or the Cytoscape programme to identify yeast genomic networks that have been impacted by a toxin. Others have created chemical-phenotype networks by fusing information from numerous different yeast chemical-genetic datasets, which has assisted in identifying potential effects evoked by different chemicals.

Functional toxicological screening techniques, or those that pinpoint the genetic underpinnings of chemical tolerance, are potent, impartial instruments that offer unique mechanistic insights into the discipline of toxicology. Toxicologists can create theories about the corresponding mechanisms and pathways of toxicity of compounds with high throughput screening for unknown or chemicals with unknown toxicity. Without a doubt, automation and deep parallel sequencing technologies will boost the throughput of functional approaches. Implementing screening systems and analysing the generated data would necessitate significant computational resources and expertise. Integrating functional assays from other organisms can reveal conserved toxicity mechanisms and point investigations in the most pertinent direction to human health. Functional toxicology is well-positioned to help toxicologists meet the demand for more thorough chemical toxicity testing.