Applications of methods in toxicological research exam preparation

2024 exam

1. What do you always need to wear in the lab? (mark all correct)

   select one or more alternatives (1)

   • Goggles

   • Gloves

   • Shoes with closed toe

   • Lab coat

Answered by ChatGPT and reviewed by myself, corrected by Aileen, Jerry and Anna.

2. What do you need to consider before you start to work with a CMR at KI? (mark all correct)

   select one or more alternatives (1)

   • The possibility of substituting the CMR to a less hazardous substance must be investigated.
   • You need a permit
   • Work with CMR substances are only allowed in albs with a BSC 2 cabinet
   • A risk assessment must be done before starting the work

Answered by ChatGPT and reviewed by myself.

3. A transcriptomic experiment (bronchial epithelial cells exposed for 24 hours to silver nanoparticles) resulted in 451 differentially expressed genes. The top 5 enriched pathways are included in Table 1 below.

   Table 1. Top 5 Enriched canonical pathways

   Pathway name	Activation score(z-score)	p-value
   Acute phase response signaling	2.4	0.0034
   IL-6 signaling	3.2	0.0044
   Lung fibrosis	3.1	0.0053
   Apoptosis	-2.5	0.0065
   Granulocyte Adhesion and Diapedesis	3.0	0.0078

   What is the most significantly enriched pathway? Motivate your answer. (1)

   Acute phase response signaling is the most significantly enriched pathway.
   
   In pathway enrichment analysis, p-value indicates how statistically significant the enrichment is — lower p-value = higher significance. Acute phase response signaling is the most significantly enriched pathway due to having the lowest p-value.

   Based on the activation score, is this pathway activated or inhibited? Motivate your answer. (1)

   The Acute phase response signaling pathway is activated.
   The activation z-score indicates the direction of the predicted regulation:
   
   Positive z-score (> 0): → Predicted activation
   Negative z-score (< 0): → Predicted inhibition
   
   In this case, the activation score is +2.4, which is clearly positive — so the pathway is predicted to be activated.

Answered by ChatGPT and reviewed by myself.

4. The differentially expressed genes (treatment versus control) corresponding to the acute phase response signaling pathway are included in Table 2.

   Gene name	Log2foldchange	p-value	Adjusted p-value
   CCL2	2.3	0.00046	0.0021
   CXCL2	3.4	0.00032	0.0020
   CCL3L3	4.2	0.00025	0.0032
   CSF2	2.6	0.00024	0.0013
   IL1A	2.1	0.00018	0.0142
   CCL20	3.2	0.00015	0.0142
   IL1B	1.2	0.00021	0.0321
   IL6	1.3	0.00453	0.0023
   IL17A	0.76	0.00321	0.0012
   TGFB1	0.56	0.00142	0.0011
   CXCL9	0.89	0.00132	0.0011
   TNFSF12	0.75	0.00103	0.0121
   COL2A1	0.23	0.00102	0.0111
   COL2A2	0.25	0.00101	0.0167

   A. What does a positive log2foldchange represent? (1)

   A positive log2foldchange means that the gene is upregulated in the treatment group compared to the control group.

   B. The table includes both p-value and adjusted p-value. Why is that? (1)

   Because multiple genes are tested, the adjusted p-value controls for false positives due to multiple comparisons.

5. Based on the two tables shown in the previous questions, suggest one mechanism of toxicity for silver nanoparticles and one assay to validate the gene expression finding. Motivate your answer. (2)

   Based on the pathway and gene expression data, a suggested mechanism of toxicity for silver nanoparticles is inflammation, mediated through the acute phase response and IL-6 signaling pathways. Several pro-inflammatory genes, including IL1A, IL1B, IL6, CCL2, and CXCL2, are significantly upregulated, indicating activation of immune-related pathways in bronchial epithelial cells. To validate the gene expression findings at the protein level, an appropriate follow-up assay would be an ELISA or multiplex cytokine assay. These methods can quantitatively measure secreted inflammatory proteins (e.g., IL-6, IL-1β) in the culture medium, confirming that the mRNA changes also lead to functional protein-level responses.

Answered by ChatGPT and reviewed by myself.

6. You want to seed cells and expose them to nanoparticles for an experiment.

   You want to seed 50,000 cells per well in a 24-well plate. You have a cell suspension of 1 million cells per mL and want to seed 10 wells (0.5 mL per well). Which of the following options are correct? (mark all correct, 1p)

   select one or more alternatives:

   • You can take 1mL of the cell suspension and add 9 mL medium, then pipette 0.5 mL to each well
   • You need 5 million cells in total for the 10 wells
   • The total volume you need to prepare is 10 mL
   • 0.5 mL of the stock suspension is enough for the 10 wells but it it good to prepare for one extra well

Answered by ChatGPT and reviewed by myself.

7. The next day you want to expose your cells. Your particles stock is 1 mg/mL. You want three wells (0.5 mL each) with 50 µg/mL. How do you best proceed with the dilution of the stock and the exposure? (1) (select the best answer)

   Select one alternative:

   • You mix well and take 100 µL from the stock and add to a tube with 1900 µL, mix and then you pipette 0.5 mL of this to each of the three wells
   • You add 25 µL of the particle suspension to each well, then you add 475 µL fresh media
   • You mix the stock and take 25 µL to a tube with 750 µL, mix and add to one well. You then repeat for the remaining two wells

Answered by ChatGPT and reviewed by myself.

8. Name and briefly motivate two important strengths of using 3D (including organ-on-chip) compared to 2D cell models in toxicological research. (2)

   1. Physiological relevance:
   3D and organ-on-chip models replicate tissue architecture and cell–cell/matrix interactions more realistically than 2D cultures, resulting in more accurate toxicity responses.
   
   2. Modeling complex and long-term effects:
   These systems can simulate dynamic conditions like fluid flow and metabolism, allowing for better investigation of chronic toxicity, barrier function, and inter-organ interactions.

Answered by ChatGPT and reviewed by myself.

9. What steps are exclusively performed in the traditional Western Blot (WB) and not shared in In-Cell Western (ICW) version? Select one alternative. (1)

   Select one alternative.

   • Electrophoresis and transfer
   • Primary and secondary antibodies incubation
   • Fluorescence detection and blocking
   • Fixation and SDS-PAGE gel preparation

Answered by ChatGPT and reviewed by myself.

10. Which of the following statements are correct regarding flow cytometry? (mark all correct) (1)

    Select one or more alternative:

    • Side scatter is a measure of cell size
    • A shift in side-scatter can indicate particle uptake
    • Flow cytometry can be used to assess micronuclei formation
    • Dark particles may interfere with fluorescence
    • Flow cytometry analysis can be used with adherent cells without trypsinization

Answered by ChatGPT and reviewed by myself.

11. You are performing a toxicity testing of “drug A” and obtained the results below:

    

    In relation to Figure 1A, what do the three peaks represent? (1.5)

    Left peak (red): G₁ phase – cells with a diploid (2N) DNA content
    
    Middle peak (green): S phase – cells actively replicating DNA (between 2N and 4N DNA content)
    
    Right peak (blue): G₂/M phase – cells with replicated DNA (4N), either in G₂ or undergoing mitosis

    In relation to Figure 1B, what is yH2AX considered to be a measurement of? (1)

    γH2AX is a marker of DNA double-strand breaks (DSBs).

    How do you interpret the results shown in Figure 1? Describe the connections between results obtained by flow cytometry and In-Cell western. Also, describe an additional experiment that could have beed down to confirm their connection. (3)

    Figure 1A shows that Drug A causes a clear shift in cell cycle distribution, with a marked increase in the G₂/M population and a decrease in S phase, suggesting G₂/M arrest. Figure 1B demonstrates a dose-dependent increase in γH2AX, indicating elevated DNA double-strand breaks. Together, these results suggest that Drug A induces DNA damage, leading to cell cycle arrest at the G₂/M checkpoint. To confirm this link, a comet assay could be performed to directly assess DNA strand breaks.

Answered by ChatGPT and reviewed by myself.

12. You are interested in the toxicity of nanoparticles and read an article about cell toxicity Co3O4 nanoparticles.

    

    A. What is your conclusion regarding the results in A and B? Can you say something about the mechanism of cytotoxicity for Cobalt NPs? (1)

    Figure 1A shows that Co₃O₄ nanoparticles do not significantly increase LDH release, indicating that they do not cause substantial membrane damage or necrosis. However, Figure 1B shows a significant reduction in MTT activity at all concentrations, suggesting impaired mitochondrial function. Together, these results suggest that the cytotoxicity of Co₃O₄ NPs is likely due to mitochondrial dysfunction rather than direct membrane damage.

    B. Suggest a likely reason for the lack of clear dose-response observed in the MTT assay (1)

    The lack of a clear dose-response in the MTT assay could be due to interference by the nanoparticles, such as light scattering or interaction with the dye, which may affect the assay's readout. Additionally, mitochondrial function may already be maximally suppressed at lower doses, leading to an early saturation effect and a plateau in the MTT response across increasing concentrations.

    C. Explain the basic principle for the method used in D (why do your get more DNA in the tail) (1)

    The method used in Figure 1D is the Comet assay, which detects DNA strand breaks. During electrophoresis, fragmented DNA migrates out of the nucleus, forming a 'comet tail'. The more DNA damage a cell has, the more DNA moves into the tail region. Thus, an increase in DNA in the tail reflects increased genotoxicity caused by the nanoparticles.

    D. The doses are expressed as µg/mL. Why could it be relevant to also express the dose as µg/cm^2^? (1)

    Expressing the dose in µg/cm² is relevant because it accounts for the surface area to which adherent cells are exposed, providing a more accurate measure of the actual particle dose per cell layer. In contrast, µg/mL reflects the concentration in the medium but does not consider how particles settle and interact with cells on the surface.

    E. Suggest an experiment that would elucidate the causal link between C and D. (1)

    To investigate whether the ROS increase observed in Figure 1C causes the DNA damage seen in Figure 1D, one could perform a co-exposure experiment using an antioxidant such as N-acetylcysteine (NAC). If co-treatment reduces DNA damage in the Comet assay, it would suggest that ROS generation is a key upstream event leading to genotoxicity.

Answered by ChatGPT and reviewed by myself.

13. Zebrafish

    

    a) State six advantages using zebrafish embryos as experimental model in biomedical and toxicological research. (3)

    1. Transparent embryos – allow direct observation of internal development and organ structures.
    
    2. Rapid development – major organs form within 48–72 hours post-fertilization.
    
    3. Genetic similarity to humans – ~70% of human genes had zebrafish orthologues.
    
    4. Small size and high fecundity – easy to culture and enables high-throughput screening.
    
    5. Ethically favorable – embryos are not protected by animal welfare laws in early stages.
    
    6. Whole-organism context – enables assessment of systemic toxicity, not just cellular.

    b) Answer the following question in relation to the above figure. State four morphological effects seen in the exposed embryo, and name a xenobiotic compound that caused this type of effects. (2.5)

    Compared to the control, the exposed zebrafish embryo shows:
    
    Pericardial edema – visible swelling around the heart region.
    
    Small body size – shorted body length compared to control group.
    
    Curved body axis – abnormal spine curvature.
    
    Undeveloped swim bladder – failure of the swim bladder to inflate or develop properly.
    
    Example xenobiotic compound:
    Bisphenol A (BPA) or methylmercury are known to cause similar developmental defects in zebrafish embryos.

Answered by ChatGPT and reviewed by myself and corrected by Xiyuan.

14. NAMs are increasingly used in research and for regulatory purposes.

    a) List three aspects of NAMs that speak against and three that speaks for regulatory acceptance. (3)

    Three aspects that speak for regulatory acceptance:
    
    1. Animal-free testing – NAMs reduce or replace animal use, aligning with ethical and legal demands (e.g. 3Rs).
    
    2. Human relevance – Many NAMs use human cells or in silico models, potentially improving relevance to human health.
    
    3. Efficiency – They often allow faster, cheaper, and high-throughput screening of chemicals.
    
    Three aspects that speak against regulatory acceptance:
    
    1. Lack of historical data – Most regulatory frameworks rely on animal data with decades of precedent.
    
    2. Validation challenges – NAMs often lack standardized validation protocols and reproducibility across labs.
    
    3. Limited scope – Some NAMs cannot yet model complex systemic effects (e.g. chronic toxicity, metabolism, immunotoxicity).

    b) Mention and briefly describe one central approach that has been developed to increase the regulatory acceptance of NAMs. (2)

    Approach: Integrated Approaches to Testing and Assessment (IATA)
    
    IATA is a flexible, science-based framework that combines data from various sources — including NAMs, existing data, in silico models, and expert judgment — to guide regulatory decisions. It helps bridge gaps between traditional and new methods by organizing evidence in a structured, weight-of-evidence approach, increasing confidence in NAM-based assessments.

Answered by ChatGPT and reviewed by myself.

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