Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes.
Summary (2 min read)
Introduction
- It estimates the diffusion coefficient by fitting a straight line to experimental values of the MSD [14].
- The optimized least-squares fit (OLSF [17]) improves on the MSD estimator by including the optimal number of points in the fit [15].
- The increased resolution that their method offers enables us to see a clear negative correlation between a protein’s residence time on the DNA molecule and its diffusion coefficient.
II. HOW TO ANALYZE A TIME SERIES OF POSITIONS OF
- A SINGLE PARTICLE THAT DIFFUSES IN A MEDIUM WHICH IS AT REST OR FLUCTUATES.
- This section gives a “road map” to the practical use of this article, since few readers will need all its sections to analyze a given problem.
- An experimental time series of laboratory coordinates of a particle diffusing on a taut, fluctuating substrate can be analyzed as follows.
C. Diffusion on taut, fluctuating DNA
- An experimental time series of laboratory coordinates of a particle diffusing on a taut polymer, such as DNA, reveals the DNA’s transversal motion at the location of the particle.
- If the DNA’s motion has not already been characterized by independent measurements, it must be characterized using information in the time series (xn,yn)Nn=0.
- The second term describes localization errors associated with the time-averaged position given by the first term.
- D21 ), D̂ = d21/(2 t), is equal to the MLE of D and is optimal; it is unbiased and its precision reaches the CramérRao bound.
1. Spectrum of displacements for finite N
- For quantitative statistical testing of whether a recorded trajectory describes free diffusion, the MSDs and the covariance function of the single-time-lapse displacements are both impractical due to their complicated distributions and high inherent correlations.
- The comparison is made quantitative with Pearson’s χ2 goodness-of-fit test , taking into account that two parameters, D and σ 2, were fitted (one parameter if σ 2 was determined independently).
- Essentially all information in a time series is used to estimate its diffusion coefficient D. This reduces the standard error on estimates of the diffusion coefficient by a factor of up to 1.8 in the limit of high SNR and the absence of motion blur (R = 0).
4. Relationship between the CVE and the MSDs
- The variance of the MLE is to first order in 1/N given by the Cramér-Rao bound I(θ)−1, where I(θ) is the Fisher information matrix [18].
- The authors tested the CVE on Monte Carlo generated time series.
- Thus, it transforms the N displacements xn into N independent, normally distributed “transformed” displacements | xk with mean zero and variances given by Eq. (10).
2. Simulation results
- As shown in Sec. III A 2, the MSD-based estimators are suboptimal.
- The CVE and the MLEs practically reach the Cramér-Rao bound, and the MLEs even surpass it for high SNR [Figs. 4(a) and 4(b)]; this is possible because the MLEs are biased [Figs. 4(a) and 4(b)], which means that the total error of the MLE can be, and is here, smaller than that of any unbiased estimator.
- This extra precision comes at a cost, however: a systematic error in the estimate, a bias .
- There, CVE is the best estimator of diffusion coefficients.
- Monte Carlo simulations confirm that a more precise estimate of D can be obtained by using a priori knowledge of σ 2 [Figs. 4(c) and 4(d)].
3. Conclusion
- In conclusion, the authors find that the CVE is to be preferred in practice since (i) it is unbiased and practically optimal in experimentally relevant situations.
- (ii) The CVE is given by a simple analytical expression; it is thus regression-free and is orders of magnitude faster than the MLEs, the OLSF, and the GLS estimator.
- The authors then derive the covariance of single-time-lapse displacements of a particle diffusing on the substrate (Sec. IV B).
- Thus, without knowing the specific form of these spatial eigenmodes, the authors know the character of the dynamics of a given physical point of the substrate: Here τx(s) = (2πfc,x)−1, where fc,x is the so-called corner frequency of the Lorentzian power spectrum of this motion.
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Cites background from "Relevance of the incubation period ..."
...Primary human hepatocytes are still considered to represent a gold standard for hepatic biotransformation studies (Godoy et al. 2018; Gu et al. 2018), whereas HepG2 cells have been reported to represent a useful tool to study the regulation of drug-metabolizing enzymes (Wilkening et al....
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Cites background from "Relevance of the incubation period ..."
...Test strategy/AOP Gu et al., 2018 Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes The publication addresses a central scientific topic of EU-ToxRisk: how long do in vitro experiments need to run to predict human-repeated-dose toxicity?...
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17 citations
Cites background from "Relevance of the incubation period ..."
...Gu et al. (2018) address a central scientific topic of EU-ToxRisk: How long do in vitro experiments need to run to predict human repeated-dose toxicity? They explored how the variation of the incubation period of a test compound can significantly influence the results of in vitro tests....
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"Relevance of the incubation period ..." refers background in this paper
...Currently, primary human hepatocytes represent the gold standard model for in vitro testing of drug metabolism and cytotoxicity (LeCluyse 2001)....
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Frequently Asked Questions (12)
Q2. What are the future works in "Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes" ?
Therefore, 2 days may represent an adequate choice for cytotoxicity tests with human hepatocytes in future studies, offering the practical advantage that less culture medium changes are required. However, these results should be interpreted with caution and further reproduction is required before possible biological explanations are discussed.
Q3. Why was the function approximated according to the least square method?
Due to the non-linearity of the 4pLL model, the function was approximated according to the least square method with the Gauss–Newton algorithm.
Q4. How many cells were plated in 96-well plates?
After cell counting using Trypan blue to determine viability, 50,000 cells in FCS-containing medium were plated into each well of 96-well plates and kept at 37 °C for at least 3 h.
Q5. How long did the cells remain in the medium?
For single exposure, the cells were incubated with compounds for 24 h or 48 h; for repeated exposure, the compound-containing medium was renewed every 48 h and the cells were incubated for a total of 7 days.
Q6. How long does it take to wash out a test compound?
washout experiments with an initial exposure period with test compounds followed by test compound-free incubation or repeated exposure and washout periods may be considered.
Q7. What is the advantage of this procedure?
The advantage of this fitting procedure is that the left asymptote is used as a control level for calculation of EC50 and EC20 values which are more robust than just using the values of the solvent controls.
Q8. When was the solvent concentration increased to 0.5%?
Only when the cytotoxic test compound concentrations were not reached with 0.1% DMSO, the solvent concentration was increased to 0.5%.
Q9. What are the three compounds with a relatively strong decrease in EC50 after 7 days ?
Compounds with a relatively strong decrease in EC50 after 7 days compared to 1 day are busulfan, famotidine, and isoniazid (Fig. 2b).
Q10. What was the effect of the solvent on cell viability?
The applied solvent concentrations of 0.1% or 0.5% DMSO did not cause any cytotoxicity compared to cells cultivated in medium without DMSO.
Q11. What is the common cause of liver injury?
Dimethyl sulfoxide DILI Drug-induced liver injury EtOH Ethanol FAM Famotidine Glc Glucose HYZ Hydroxyzine INAH Isoniazid KC Ketoconazole LAB Labetalol LEV Levofloxacin MEL Melatonin MePa Methylparaben NAC N-Acetylcysteine NIM Nimesulide NFT Nitrofurantoin PhB Phenylbutazone PMZ Promethazine PPL Propranolol RIF Rifampicin TSN Triclosan VPA Valproic acid Vit C Vitamin CDrug-induced liver injury (DILI) is one of the principal reasons for drug withdrawal from the market (Godoy et al. 2013; Hewitt et al. 2007).
Q12. How long does it take to establish in vitro tests?
large efforts are undertaken to establish in vitro tests with the long-term goal to predict human toxicity (Daneshian et al.