mRNA transfection retrofits cell-based assays with xenobiotic metabolism

DeGroot, Danica E.; Swank, Adam; Thomas, Russell S.; Strynar, Mark J.; Lee, Mi-Young; Carmichael, Paul L.; Simmons, Steven O.

doi:10.1016/j.vascn.2018.03.002

Cited by 34 publications

(21 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recently, some genetically modified HepG2 cell lines expressing human drug metabolizing genes have been developed and used for assessing drug metabolism-associated toxicity (Chen et al, 2018;Wu et al, 2016;Xuan et al, 2016). Human embryonic kidney cells (HEK293T) have also been engineered to transiently express human CYPs via mRNA transfection (DeGroot et al, 2018). However, their application may be restrained in highthroughput genotoxicity screening.…”

mentioning

confidence: 99%

Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

Li¹,

Chen

Guo³

et al. 2020

Toxicological Sciences

View full text Add to dashboard Cite

Abstract Metabolism plays a key role in chemical genotoxicity; however, most mammalian cells used for in vitro genotoxicity testing lack effective metabolizing enzymes. We recently developed a battery of TK6-derived cell lines that individually overexpress 1 of 8 cytochrome P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, and 3A4) using a lentiviral expression system. The increased expression and metabolic function of each individual CYP in each established cell line were confirmed using real-time PCR, Western blotting, and mass spectrometry analysis; the parental TK6 cells and empty vector (EV) transduced cells had negligible CYP levels. Subsequently, we evaluated these cell lines using 2 prototypical polyaromatic hydrocarbon mutagens, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P), that require metabolic activation to exert their genotoxicity. DMBA-induced cytotoxicity, phosphorylation of histone H2A.X, and micronucleus formation were significantly increased in TK6 cells with CYP1A1, 1B1, 2B6, and 2C19 expression as compared with EV controls. B[a]P significantly increased cytotoxicity, DNA damage, and chromosomal damage in TK6 cells overexpressing CYP1A1 and 1B1 when compared with EV controls. B[a]P also induced micronucleus formation in TK6 cells expressing CYP1A2. These results suggest that our CYP-expressing TK6 cell system can be used to detect the genotoxicity of compounds requiring metabolic transformation.

show abstract

mentioning

confidence: 99%

Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

Li¹,

Chen

Guo³

et al. 2020

Toxicological Sciences

View full text Add to dashboard Cite

show abstract

“…The discrepancy between PoD and C max for diclofenac in terms of its risk category from a consumer safety perspective highlights the importance of incorporating metabolism within in vitro assays, which is an on-going challenge in NGRA and may be addressed either by using more organotypic models ( Ramaiahgari et al , 2017 , 2019 ) that are metabolically competent, artificially increasing the expression of specific CYP enzymes ( DeGroot et al , 2018 ), or directly testing predicted metabolites.…”

Section: Discussionmentioning

confidence: 99%

Identifying and Characterizing Stress Pathways of Concern for Consumer Safety in Next-Generation Risk Assessment

Hatherell

Baltazar

Reynolds

et al. 2020

Toxicological Sciences

View full text Add to dashboard Cite

Many substances for which consumer safety risk assessments need to be conducted are not associated with specific toxicity modes of action, but rather exhibit nonspecific toxicity leading to cell stress. In this work, a cellular stress panel is described, consisting of 36 biomarkers representing mitochondrial toxicity, cell stress, and cell health, measured predominantly using high content imaging. To evaluate the panel, data were generated for 13 substances at exposures consistent with typical use-case scenarios. These included some that have been shown to cause adverse effects in a proportion of exposed humans and have a toxicological mode-of-action associated with cellular stress (eg, doxorubicin, troglitazone, and diclofenac), and some that are not associated with adverse effects due to cellular stress at human-relevant exposures (eg, caffeine, niacinamide, and phenoxyethanol). For each substance, concentration response data were generated for each biomarker at 3 timepoints. A Bayesian model was then developed to quantify the evidence for a biological response, and if present, a credibility range for the estimated point of departure (PoD) was determined. PoDs were compared with the plasma Cmax associated with the typical substance exposures, and indicated a clear differentiation between “low” risk and “high” risk chemical exposure scenarios. Developing robust methods to characterize the in vitro bioactivity of xenobiotics is an important part of non-animal safety assessment. The results presented in this work show that the cellular stress panel can be used, together with other new approach methodologies, to identify chemical exposures that are protective of consumer health.

show abstract

“…As such, caution should be exercised when extrapolating in-vitro-to-in-vivo findings for this chemical to avoid underestimation of health hazards relating to its reproductive toxicity. Research efforts are currently underway to incorporate metabolic competence into ToxCast assays and facilitate qualitative and quantitate characterization of chemcial hazards (DeGroot et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

A case study on the application of an expert-driven read-across approach in support of quantitative risk assessment of p,p’-dichlorodiphenyldichloroethane

Lizarraga

Dean

Kaiser

et al. 2019

Regulatory Toxicology and Pharmacology

View full text Add to dashboard Cite

Deriving human health risk estimates for environmental chemicals has traditionally relied on in vivo toxicity databases to characterize potential adverse health effects and associated doseresponse relationships. In the absence of in vivo toxicity information, new approach methods (NAMs) such as read-across have the potential to fill the required data gaps. This case study applied an expert-driven read-across approach to identify and evaluate analogues to fill non-cancer oral toxicity data gaps for p,p'-dichlorodiphenyldichloroethane (p,p'-DDD), an organochlorine contaminant known to occur at contaminated sites in the U.S. The source analogue p,p'dichlorodiphenyltrichloroethane (DDT) and its no-observed-adverse-effect level of 0.05 mg/kgday were proposed for the derivation of screening-level health reference values for the target chemical, p,p'-DDD. Among the primary similarity contexts (structure, toxicokinetics, and toxicodynamics), toxicokinetic considerations were instrumental in separating p,p'-DDT as the best source analogue from other potential candidates (p,p'-DDE and methoxychlor). In vitro highthroughput screening (HTS) assays from ToxCast were used to evaluate similarity in bioactivity profiles and make inferences toward plausible mechanisms of toxicity to build confidence in the read-across approach. This work demonstrated the value of NAMs such as read-across and in vitro HTS in human health risk assessment of environmental contaminants with the potential to inform regulatory decision-making.

show abstract

mRNA transfection retrofits cell-based assays with xenobiotic metabolism

Cited by 34 publications

References 28 publications

Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

Identifying and Characterizing Stress Pathways of Concern for Consumer Safety in Next-Generation Risk Assessment

A case study on the application of an expert-driven read-across approach in support of quantitative risk assessment of p,p’-dichlorodiphenyldichloroethane

Contact Info

Product

Resources

About