Alternative Toxicity Assays: Replacing Rodent Models

Posted on Jan 11, 2025 in Biotechnology

Describe 5 alternative toxicity assays that complement or might be able to replace rodent assays

Several alternatives to the traditional rodent bioassay have been proposed, of which the most promising are non-animal assays such as quantitative structure-activity relationship expert systems, in vitro assays, the use of cDNA microarrays to detect genetic expression changes, human clinical trials, & epidemiological research.

1. QSAR expert systems, particularly for initial screening, should be further developed & expanded from their traditional reliance on chemical analogues to include info on the structural properties of cellular receptors facilitating toxicity, as this becomes available. Toxicity testing data should be used retrospectively to enlarge QSAR databases.
2. Cell & tissue assays, particularly those using human cell lines, the SHE cell transformation assay, others sensitive to nongenotoxic carcinogens, & the Saccharomyces GreenScreen assay, should be further developed & implemented. The availability of human cells & tissues for toxicity testing should be increased.
3. Research into improving cDNA microarray data reproducibility & interpretation should continue.
4. Predictive biomarkers of toxicity should be identified through genomic, proteomic & clinical research, thereby allowing speedier generation of results, well prior to more invasive endpoints, & facilitating increased understanding of carcinogenesis mechanisms.
5. Increased human epidemiological research is also required, in order to identify more known human carcinogens & presumed non-carcinogens, thereby increasing the data set available for validation studies & QSAR predictive systems. Cancer Centers should be financially supported to establish tumor registries focused on identifying new human carcinogens, & post-marketing surveillance should be required for all pharmaceuticals, with mandatory reporting of adverse side effects.

Explain the difficulties in implementing the alternative assays

• Fear of lack of acceptance of alternatives by regulatory agencies is discouraging the use of alternative assays.
• Cumbersome validation process required of alternative assays, made more difficult by attempts to match outcomes to the variable & inconsistent results of animal bioassays.

List the design criteria of the new assays

• Develop a more robust scientific basis for assessing health effects of environmental agents (mechanistic data).
• Provide broad coverage of chemicals, chemical mixtures, outcomes, & life stages.
• Reduce the cost & time of testing.
• Base decisions on human rather than rodent biology & focus on more relevant dose levels.

Explain the new strategy of toxicity testing

Toxicity pathway: A cellular response pathway that, when sufficiently perturbed, is expected to result in an adverse health effect.

Toxicity Pathways: Endogenous hormones; DNA damage; PXR, CAR, PPAR & AhR receptors; Hypo-osmolarity; Nrf2 oxidative stress; Heat-shock proteins; P38 MAPK

Describe the antioxidant response pathway with some key proteins

• Normally, Nrf2 is bound to the cytoplasmic protein Keap1 – an Ub E3 ligase.
• When challenged with oxidant stressors, Nrf2 is released, going to the nucleus & guides expression of antioxidant stress genes.
• Both, sub & superphysiological effects have been noted to be detrimental.
• Nrf2 nuclear translocation requires the activation of several translocation pathways, including mitogen-activated protein kinases (MAPKs). ( MAPK cascades integrate cell signalling pathways that govern cell kinetics)
• Controlled adaptive stress responses govern activation & perturbation of signalling pathways!

Describe the components of the alternative experiments with their advantages

• Chemical Characterization
  1. Compile Data on physical & chemical properties, use characteristics, environmental concentrations, possible metabolites & breakdown products, & possible toxic properties.
  2. Predict properties & characteristics, where possible & appropriate, by using computational tools.
  3. Answer key questions concerning compound´s stability, potential for human exposure & bioaccumulation, & toxicity of chemical & possible metabolites.
• Toxicity Testing: Toxicity pathways + Targeted Testing
• Toxicity Pathways
  1. Evaluation of perturbations in toxicity pathways rather than apical end points.
  2. Emphasis on high-throughput approaches using cells or cell lines, preferably of human origin.
  3. Use of medium-throughput assays of more integrated cellular responses.
• Targeted Testing
  1. Testing  conducted to evaluate metabolites, assess target tissues, & develop understanding of affected processes at genomics level.
  2. Limited types & duration of in vivo studies, focusing on up to 14-day exposures.
  3. More extensive testing for representative compounds in novel chemical classes.
• Dose-Response & Extrapolation Modelling
  1. Empirical dose-response models will be developed on the basis of data from in vitro, mechanistically based assays.
  2. Physiologically based pharmacokinetic (PBPK) models will equate tissue-media concentrations from toxicity tests with tissue doses expected in humans.
  3. Dose-response models for toxicity pathways will reliably predict concentrations expected to cause measurable precursor-effect responses.
  4. PBPK & toxicity-pathway models will identify biomarkers of susceptibility for sensitive subpopulations.
• Implementation of Strategy:
  1. Comprehensive suite of in vitro tests, preferably based on human cells, cell lines, or components.
  2. Computational models of signal transduction in toxicity pathways to support application of in vitro test results in risk assessments.
  3. Physiologically based pharmacokinetic (PBPK) models to assist in vitro to in vivo extrapolations.
  4. Validation of toxicity pathway tests & test strategies.

Explain the conclusions of the future in vitro assessments

• Paradigm shift away from apical endpoints in test animals to perturbation of toxicity pathways in human cells.
• Providing much broader coverage of the universe of environmental agents that warrant our attention.
• Has consequences for toxicity testing & in the search for alternatives to animal testing.
• Already, at this point in time, this vision is an applied sciences problem, rather than a research-driven process.

Describe the cytotox and apoptosis assays (LDH, MTT, Caspase)

LDH Cytotoxicity Assay

• Tests levels of lactate dehydrogenase, an enzyme which is present in all cells in the medium
• Facilitates conversion of lactate into pyruvate, creating NADH in the process.
• LDH is usually impermeable to the cell membrane.
• When the cell membrane is damaged, LDH is released into surrounding medium.
• NADH is used to convert a tetrazolium salt (INT) into a formazan product (purple colour).
• Protocol:

1. Samples of cell-free medium are taken at 0, 12, 48, 72 & 96 hours after exposure & frozen.
2. Samples are thawed & placed into 96-well plate.
3. LDH dye solution is added & incubated for 30 min develop colored crystals.
4. Analyzed with a microplate reader at 490 nm.

MTT Cell Proliferation Assay

• Tests for metabolic activity of viable cells
• Yellow tetrazolium salt MTT is cleaved into purple formazan by mitochondrial dehydrogenases found in active cells
• Similar idea than the LDH Cytotoxicity Assay
• Protocol:

1. 10 uL of 5 mg/ml MTT solution is added to test & control wells
2. Incubation for 4 hours -> black crystals form on bottom of flask
3. Isopropanol & HCl solution (or DMSO) is added to dissolve crystals & negate neutralize medium colour
4. Colour development is analysed by spectrophotometer at 570 nm

Caspase 3/ CPP32 Colorimetric Assay

• Caspase 3 is a known mediator of apoptosis (programmed cell death)
• Member of the asparate-specific cysteinyl proteases
• Can also cleave artificial substrates consisting of an appropriate sequence of four amino acids
• Resulting compounds can be analysed fluorometrically or colorimetrically

Compare whole embryo cultures with embryonic stem cells as tools for cytotoxicity assays

Whole Embryo Culture:

Embryonic Stem Cell Test (EST)

• Embryonic stem cells are a permanent cell line established from the ICM (inner cell mass) of embryos
• The basis of the EST is to determine 3 endpoints
  1. IC50 for inhibition of ESC (embryonic stem cell) differentiation to beating heart cells
  2. IC50 for induction of cell death in ESC
  3. IC50 for induction of cell death in 3T3 cells
• Cytotoxicity Assay: Grow D3 mouse embryonic stem cells & 3T3 embryonic fibroblasts in presence of test chemical to determine IC50 for each cell line. Endpoint: cell viability after 10 days.
• Differentation Assay: Allow D3 cells to differentiate for 10 days in presence of chemical to determine ID50. Endpoint: microscopic inspection of beating cardiomyocyte development.

Explain the case of ‘TGN1412’

TGN1412 (also known as CD28-SuperMAB) is the working name of an immunomodulatory drug which was withdrawn from development after inducing severe inflammatory reactions in the first-in-man study in London in March 2006. The developing company, TeGenero Immuno Therapeutics, entered into insolvency proceedings later that year.

Originally intended for the treatment of B cell chronic lymphocytic leukemia (B-CLL) and rheumatoid arthritis, TGN1412 is ahumanised monoclonal antibody that not only binds to, but is a strong agonist for, the CD28 receptor of the immune system’s T cells. CD28 is the co-receptor for the T cell receptor; it binds to receptors on the interacting partner in the reaction through one of its ligands (B7 family).

In its first human clinical trials, it caused catastrophic systemic organ failure in the subjects, despite being administered at a supposed sub-clinical dose of 0.1 mg per kg; some 500 times lower than the dose found safe in animals. Six volunteers were hospitalized on 13 March 2006, at least four of these suffering from multiple organ dysfunction. Tentative opinions from an as-yet uncompleted inquiry suggest that the problems resulted from “unforeseen biological action in humans”, rather than breach of trial protocols, and the case therefore has had important ramifications for future trials of potentially powerful clinical agents.

Scientists in early 2007 put forth the theory that the drug acted in a different fashion in humans as compared with the laboratory animals in which the drug was first tried. The severe reactions in humans could have only occurred, they believe, in animals with memory T lymphocytes. Animals raised in a sterile lab would presumably have no ‘memory’ of previous illnesses, thus would not exhibit the severe reactions that occurred in the human subjects. However this is a misunderstanding of the research: the research says that non-human animals studied have fewer memory T cells than humans, and that stimulation through the CD28 receptor alone in memory T cells causes them to infiltrate organs and also activates them.

The drug, which was designated as an orphan medical product by the European Medicines Agency in March 2005, was developed by TeGenero Immuno Therapeutics, tested by Parexel and manufactured by Boehringer-Ingelheim. TeGenero announced the first elucidation of the molecular structure of CD28 almost exactly one year prior to commencement of the TGN1412 phase I clinical trial.