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Dynamically monitoring cellular γ-H2AX reveals the potential of carcinogenicity evaluation for genotoxic compounds

  • Genotoxicity and Carcinogenicity
  • Published:
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Abstract

Amongst all toxicological endpoints, carcinogenicity might pose the greatest concern. Genetic damage has been considered an important underlying mechanism for the carcinogenicity of chemical substances. The demand for in vitro genotoxic tests as alternative approaches is growing rapidly with the implementation of new regulations for compounds. However, currently available in vitro genotoxicity tests are often limited by relatively high false positive rates. Moreover, few studies have explored carcinogenicity potential by in vitro genotoxicity testing due to the shortage of suitable toxicological biomarkers to link gene damage with cancer risk. γ-H2AX is a recently acknowledged attractive endpoint (biomarker) for evaluating DNA damage and can simultaneously reflect the DNA damage response and repair of cells. We previously reported an ultrasensitive and reliable method, namely stable-isotope dilution-liquid chromatography–tandem mass spectrometry (ID-LC–MS/MS), for detecting cellular γ-H2AX and evaluating genotoxic chemicals. More importantly, our method can dynamically monitor the specific processes of genotoxic compounds affecting DNA damage and repair reflected by the amount of γ-H2AX. To clarify the possibility of using this method to assess the potential carcinogenicity of genotoxic chemicals, we applied it to a set of 69 model compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), with already-characterized genotoxic potential. Compared to conventional in vitro genotoxicity assays, including the Ames test, the γ-H2AX assay by MS has high accuracy (94–96%) due to high sensitivity and specificity (88% and 100%, respectively). The dynamic profiles of model compounds after exposure in HepG2 cells were explored, and a mathematical approach was employed to simulate and quantitatively model the DNA repair kinetics of genotoxic carcinogens (GCs) based on γ-H2AX time–effect curves up to 8 h. Two crucial parameters, i.e., k (rate of γ-H2AX decay) and t50 (time required for γ-H2AX from maximum decrease to half) estimated by the least squares method, were achieved. An open web server to help researchers calculate these two key parameters and profile simulated curves of the tested compound is available online (http://ccb1.bmi.ac.cn:81/shiny-server/sample-apps/prediction1/). We detected a positive association between carcinogenic levels and k and t50 values of γ-H2AX in tested GCs, validating the potential of using this MS-based γ-H2AX in vitro assay to help preliminarily evaluate carcinogenicity and assess genotoxicity. This approach may be used alone or integrated into an existing battery of in vitro genetic toxicity tests.

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Abbreviations

2,4-DAT:

2,4-Diaminotoluene

2-AAF:

2-Acetylaminofluorene

4-NQO:

4-Nitroquinoline-N-oxide

5-Fu:

5-Fluorouracil

AFB1:

Aflatoxin B1

ANOVA:

Analysis of variance

Arac:

Cytosine arabinoside

AZT:

Azidothymidine

BaP:

Benzo[a]pyrene

CA:

Chromosome aberration

CdCl2 :

Cadmium chloride

Cispt:

Cisplatin

CPA:

Cyclophosphamide

DMBA:

Dimethylbenzanthracene

DMEM:

Dulbecco’s modified Eagle’s medium

DMNA:

Dimethylnitrosamine

DSB:

DNA double-strand break

ECVAM:

European Center for the Validation of Alternative Methods

ENU:

N-Ethyl-N-nitrosourea

ESI:

Electrospray ionization

ETOP:

Etoposide

FBS:

Foetal bovine serum

GCs:

Genotoxic carcinogens

HepG2:

Human hepatoblastoma cells

IARC:

International Agency for Research on Cancer

ICH:

International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use

ID-LC–MS/MS:

Isotope dilution-liquid chromatography–tandem mass spectrometry

IQ:

2-Amino-3-methylimidazo(4,5-f)quinolone

LC–MS/MS:

Liquid chromatography–tandem mass spectrometry

k :

Rate of γ-H2AX decay

LSD:

Least significant difference

MEC:

Minimum effective concentration

MECs:

Minimum effective concentrations

MLA:

Mouse lymphoma assay

MMC:

Mitomycin C

MMS:

Methyl methanesulfonate

MN:

Micronucleus

MOA:

Mode of action

MRM:

Multiple reaction monitoring

NDCs:

Non-DNA damaging chemicals

OECD:

Organization for Economic Co-operation and Development

p-Chl:

p-Chloroaniline

PHHs:

Primary human hepatocytes

PhIP:

2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine

RCC:

Relative cell counts

SD:

Standard deviation

t 50 :

Time required for γ-H2AX from maximum decrease to half

t max :

Time to peak

VICH:

International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products

γ-H2AX:

Phosphorylated histone H2AX

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Funding

This work was supported by the National Key Research and Development Program (2018YFC1602600) and the National Natural Science Foundation of China (No. 21974151).

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Authors and Affiliations

  1. State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27 Taiping Road, Haidian District, Beijing, 100850, China

    Minmin Qu, Hua Xu, Jia Chen, Yajiao Zhang, Bin Xu, Zhi Li, Lei Guo & Jianwei Xie

  2. Beijing Institute of Basic Medical Sciences, 27 Taiping Road, Haidian District, Beijing, 100850, China

    Wuju Li & Tao Liu

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  1. Minmin Qu
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  2. Hua Xu
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Correspondence to Hua Xu or Jianwei Xie.

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Qu, M., Xu, H., Li, W. et al. Dynamically monitoring cellular γ-H2AX reveals the potential of carcinogenicity evaluation for genotoxic compounds. Arch Toxicol 95, 3559–3573 (2021). https://doi.org/10.1007/s00204-021-03156-3

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