Note: Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines

Note for: Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines

(doi: 10.1002/em.20656)

US Tox21 program -- evaluate the detection of genotoxic cpds.

This study focused on cytotoxicity by using DT40 with deficient in DNA repair pathway.

Using 7-isogenic DNA repair-deficient pw cell line to identify the types of DNA damage.

Criteria to judge;

1.clastogenicity (ability to break the DNA) in mutant and WT

2.ability to induce gH2Ax positive foci by melphalan

3.72-hr viability through the liquid assay

4.using more DNA repair deficient cell lines to clearly identify the damage induced

5.involvement of ROS in induction of DNA damage

At the end, it proposed that DT40 and theirs DNA-repaired deficient lines are useful tool to detect 1.genotoxic cmp. 2.identify the nature of DNA damage of the challenges 3.analyzing mechanism of mutagenesis

Rational;

Because of the number of chemical using is gradually high and some information regarding on those toxic compounds are lacking.

They want to use HTS as a tool for preliminarily screening the potential toxicity which may be harm to the human and before going down to examine the mechanism in depth.

there are numbers of methods which can quantify the DNA damage;

1. frequency of chromosomal aberration

2. micronuclei or gene mutation

3. DNA migration through comet assay

4. level of gH2Ax positive

They mentioned that all these assay could not yet be applied to qHTS.

They also mentioned that DNA repair process could be applied with qHTS. In each DNA lesion (damage) requires different repair pathway.

1. DB break -- HR and NHEJ

2. Base damage -- NER and BER

3. DNA interstrand cross-link -- FA and TLS

For the checkpoint

- stop cell cycle before (G1-S) and (G2-M)

Three major advantages that they mentioned by using DT40;

1. Doubling time 8 hr (at 40c)

2. During Log-phase; 70% is in S-phase but 50% in mammalian

3. Lack of G1-S checkpoint which normally appeared in mammalian cells

4. Having the same genetic background when non-functioning mutant has been generated.

Therefore, they can carry the damage throughout the cycle and we can measure the DNA repair process in this cell line.

The test based on the idea that cell deficient in DNA repair will increase the "cytotoxic" effects of DNA damage.

DNA-repair deficient being used in this study;

1. ATM (DNA damage checkpoint)

2. FancC (DNA intertrand cross-link)

3. PolB - 2 clones (BER)

4. Rad54+Ku70 (DSB)

5. Rev3 (TLS)

6. Ubc13 (DSB+TLS)

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Format being used;

1536 well plate

1408 compounds

14 points to collect

from 0.59 nM - 92 uM

24 hrs treat

measure the intracellular ATP amount

Output interpretations;

1.higher in sensitivity of KO compared with the parental cell line

2.IC50 -- in the concentration that can generate 50% of cell death.

3.to consider significantly different -- IC50 must be 6-fold different

By above criteria (duplicate), verify more with the positive hit with concentration ranging from 11pM to 92uM and triplicate.

5 independent confirm is conducted;

- 3 times with 24 hr exposure

- 2 times with 48 hr exposure (to observe the phenotype with longer contact time)

Decrease in IC50 value will be used to determine the potential of direct-acting genotoxicants.

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Further characterize with subset of positive;

1.numbers of chromosomal aberration on both KO and parental -- after treat for 24 hr at Mete-phase

2.concentration being used is the same concentration at IC50 obtained from qHTS

Another further characterization;

1. evaluate the specificity or sensitivity when exposed until 72 hrs

2. using more DNA repair-deficient clones to observe the cytotoxicity

3. examine the relationship of ROS in induction DNA damage

It sounds to me like this paper is conducted to test whether all the DNA-repair deficient DT40 could be used as representative for the genotoxicity testing by using the compound from the US Tox21.

the toxicity testing was conducted at 40C.

The dose-response cure is divided into 4 classes and each can be subdivided according to the criteria published by the other groups.

Criteria to judge positive cell for gH2Ax -- >4 foci/cell

the last confirmation platform;

1. cells 10,000 c/1000 ul/well in 24-well plate -- treated with 1-5 mL (ul maybe typo) of each compound dissolved in DMSO?

The reason that the authors wanted to find more in detail what causes the cytotoxicity -- ROS could trigger the cell death rather than DSB, therefore, we must differentiate on this (my guess they can look for the CAs or foci formation).

Actinomycin D is used to treat childhood cancers.

Mode of action - ActinomycinD;

1.inhibit mRNA synthesis by interacting with RNA polymerase

2.toxicity is detected by micronucleus assay

3.yeast DEL assay

4.not by Ames reverse mutation assay

Gap -- how ActD induced DNA damage is unk.

Nbs1 and CtIP --  involve in elimination of chemical modifications between polypeptide (topo-II) - oligonucleotide (DSB).

From the DNA repair deficient cell line panel;

- ActD reduced viability of Rad54/Ku70 mutant cells

- ActD increased number of Rad54+Ku70

Then the authors want to dissect more which repair pathway fix the ActD-induced damage.

They used Ku70,BRCA2,RAD54 individual KO to explore more on viability at 72 hrs.

ActD toward

Ku70 -- increased sensitivity

Rad54 -- not exhibit sig increased sensitivity

BRCA2 -- mild sensitivity

the authors concluded ActD-induced DSB are repaired majorly by NHEJ and partially by HR.

Analyse more on CtIP and Nbs1 --

CtIP+Nbs -- higher sensitivity

From this they proposed the mechanism of ActD by those sensitivity assays;

- ActD generates DSB by chemical modification which requires to be removed before fixing with HR or NHEJ dependent.

 - Chemical modification not related to ROS (there is the experiment that pretreat the cell with NAC (N-acetyl-L-cysteine [NAC], antioxidant) before adding ActD -- show no sign of restoring the cell from cytotoxicity from ActD.

Crosslinking can be divided to three categories;

1. interstrand crosslink-ICL (most harmful -- fixed by FA and TLS)

2.intrastrand crosslink -- rely on TLS

3.protein DNA crosslink -- rely on TLS

Therefore, it could say that if the FA(-/-) cell is sensitive to unk-genotoxic compound, once could say that it generates ICL.

After testing with sensitivity, the authors mentioned that sensitivity is not enough indicator to tell repair capability, therefore, they perform more experiments -- by which they looked at CAs in each DNA repair-deficient KO, treat and untreat and compared with WT.

The author hypothesized that longer exposure should increase by longer exposure period, therefore, they performed the viability assay extending up to 72 hrs.;

Some genotoxic substances showed sig cytotoxicity toward on some KO at 24 hr but not 48 and 72.

Some genotoxic substances  showed sig cytotoxicity toward on some KO at 48 and 72 but not 24 hr.

When the authors comparing the results between cytotoxicity and CAs -- they conclude that sensitivity at 24 hrs could not precisely represent genotoxicity of one particular compound.

They also raised the issue that observing CAs in Metaphase cells at 24hrs may represent the false-negative result (must observe in the longer period).

They gave the example of treating the FancC with Melphalan which showed

1.no sensitivity at 24hr

2.sensitivity at 48-72hr

3.CAs -- not sig different from WT but Mitotic index is very different (@24 hr)

Therefore, the authors point out the discordance between CAs@24 hrs+sensitivity@24 (false negative) with mitotic index which 1/3 lower from the WT (cell cycle of FancC is arrest) -- they prove more on the affect of Melphalan by using gH2Ax foci formation which indicating the appearance of DSB (incubating for 1 hr - collect and IF) -- more foci were observed in FancC.

Rev3-KO is the good candidate to see the affect on TLS.

Groups of genotoxic compounds were tested further with additional KOs -- Fen1 and XPA

Lovastatin;

1.drug used for reduce lipid level

2.no report on genotoxicity

3.Showed sensitivity toward UBC13 @24-48hr - but not at 72 hr

4.no CAs change compare to WT

From these results they conclude that shor-term exposure time data with DNA repair-deficient KO may not reflect the genotoxicity (@72 no affect).

The main idea for this publication is to investigate the relevance of qHTS based on cytotoxicity toward DNA-repair deficient KO with CAs.

1.qHTS by using representative of each DNA repair deficient pw

2.determine the short period at 24 -- using 6 fold different from the WT (IC50 -- to avoid the inherent variation w/i assay) to identify as positive.

However, the author also mentioned that the compounds giving lower 6-fold efferents are actually genotoxic (false negative)

Limitation of qHTS;

1. inability to incorporate of xenobiotic metabolism (require the conversion to show the toxic, esp. from group 1 xenobiotic metabolic enzymes --> they mentioned S9 mixture which is the mixture of metabolic enzyme which biotransform those cmp to the "activated" state - rich of ROS) -- they omit this since they need the homogenous assays.

Average of reproducibility for IC50 --> 24hr w/i 1.7 , 48hr w/i 1.5 fold

The panel is suite to use for "rapid" identification of genotoxic cmps and elucidate the mechanism of action.

Still having the drawback on false negative/positive -- it can replace some conventional method but it also requires the improvement as mentioned above.

Spectrum of sensitivity to each cmp amoung the DNA-repair deficient mutant -- good indicator to identify the type of DNA damage.

Ex. ActD -- Ku70+Rad54 -- very sensitive --> ActD may induce the DSB --> explore more on CAs and discard the possibility of cytotoxicity from ROS.

The author mentioned on the lovastatin which showed sensitivity at 24-48 hr but not 72 hr., no CAs (clastogenicity) through out all the DNA repair deficient cell lines -- therefore, they conclude that longer exposure is more reliable but hard to do in the 1536 platform since the liquid will be evaporate.

Clinical application receiving from this study;

ActD -- chemically modify DNA -- from this study, we know that NHEJ is used to fix the lesion generated by ActD -- finding the inhibitor toward the cancer cells which their viability relies NHEJ --> then inhibitor that inhibit the NHEJ would kill the cancer cells!.

Therefore, if there is another additional comp. tested with those DNA-repair deficient panels together with the genotoxic cmp --> then we could identify the novel compound through this -- which can target DNA repair protien?.

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