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Relationship: 2399

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Bulky DNA adducts, increase leads to Increase, Mutations

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Bulky DNA adducts, increase

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Increase, Mutations

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Bulky DNA adducts leading to mutations	non-adjacent			Carole Yauk (send email)	Under development: Not open for comment. Do not cite	Under Development

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Link
human and other cells in culture	human and other cells in culture	NCBI
human	Homo sapiens	NCBI
rat	Rattus norvegicus	NCBI
mouse	Mus musculus	NCBI
yeast	Saccharomyces cerevisiae	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Unspecific

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
All life stages

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Bulky DNA adducts occur when aromatic compounds are metabolically activated and interact with DNA bases. Not all of these bulky adductsare stable, however some have been found to persist and cause mutations during repair or replication. The specific mutation that occurs variesby bulky DNA adduct and by chemical. Exposure to the benzo(a)pyrene (B(a)P) or its metabolite anti-benzo(a)pyrene diol epoxide (BPDE)leads to (+/-)-trans-anti-BPDE-N-2-dG adducts, these adducts are associated with G→T transversions (Chiapperino et al. 2002; Zhang et al.2000, 2002), the occurrence of these transversions has been observed both in smokers (Anna et al. 2009; Hainaut and Pfeifer 2001) and in non-smokers (DeMarini et al. 2001). Exposure to aristicholic acid (AA) leads to the persistent DNA adduct 7-(deoxyadenosin-N6-yl) aristolactamI (dA–AAI) adducts and leads to AT→TA transversions (Arlt et al., 2002). Exposure to aflatoxin B1 has been leads to 8,9-dihydro-8- (N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adducts, which can lead to the AFB1-formamidopyrimidine (FAPY) adduct and ultimately causeG→T transversions (Bailey et al. 1996; Smela et al. 2002).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

There is a large body of evidence that describes the relationship between bulky DNA adducts and mutations (Alexandrov et al. 2002; Chen etal. 2008; Veglia, Matullo, and Vineis 2003; Yagi et al. 2017). The bulky DNA adducts preferentially pair with an erroneous base, resulting in amutation, the mutation that results depends on the specific bulky DNA adduct that occurs.

Aristolochic acid and plants containing aristolochic acid have been found to be carcinogenic to humans due to the specific DNA adducts and theA:T to T:A transversions found in renal tissues of exposed populations (IARC 2011). Exposure to AA leads to the formation of the adduct dA-AAI. In experiments with modified bacteriophage T7 DNA polymerase and with human DNA polymerase α, dA-AAI has been found to pairequally well with adenine or tyrosine (Broschard et al. 1994; Broschard, Wiessler, and Schmeiser 1995). Pairing with tyrosine results in a non-mutagenic event, therefore mutations resulting from dA-AAI are AT→TA transversion (Arlt, Stiborova, and Schmeiser 2002; Kohara et al.2002). Aristolochic acid and plants containing aristolochic acid are considered carcinogenic to humans due to the specific DNA adducts and theA:T to T:A transversions found in renal tissues of exposed populations (IARC, 2011).

B(a)P is a known to be carcinogenic to humans due to extensive experimental evidence in many animal species along with mechanisticevidence to support the biological plausibility of bulky DNA adducts leading to mutations that cause cancer in humans (IARC 2014). Exposureto the B(a)P or its metabolite anti-benzo(a)pyrene diol epoxide (BPDE) leads to (+/-)-trans-anti-BPDE-N-2-dG adducts. Human DNApolymerase eta has been found to insert an A across from the (+/-)-trans-anti-BPDE-N-2-dG adducts, resulting in the above mentioned GvTtransversions (Chiapperino et al. 2002; Zhang et al. 2000, 2002). Polymerase eta has been found to be unlikely to extend past the lesion (Chiapperino et al. 2002)and instead polymerase kappa has been found to work as the second step in the bypass of this lesion (Zhang et al.2002). Another common mutation occurring from the bulky DNA lesion (+)-trans -anti-BPDE-N-2-dG is a G→A transversion. Through molecularmodelling, it has been suggested DNA polymerase may be more likely to insert a T if the bulk of the adduct is in the major groove and an A ifthe bulk of the adduct is in the minor groove (Kozack, Shukla, and Loechler 1999).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

1. Dose concordance

MutaMouse models were exposed to 11 concentrations of BaP (0, 0.10, 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.50, 25.00, and 50.00 mgBaP/kg body weight (BW)/day) for 28 days (Long et al. 2018)
- Bulky DNA adducts were measured using ³²P post-labeling. A significant increase in bulky DNA adducts was observed in the mostsensitive tissue at 0.20 mg BaP/kg body weight (BW)/day and in all tissues at 1.56 mg BaP/kg body weight (BW)/day.
- Mutations were measured using the LacZ mutation assays. A significant increase in mutations was observed in the most sensitive tissue at 1.56 mg BaP/kg body weight (BW)/day and in all tissues 25.00 mg BaP/kg body weight (BW)/day.
- These results indicate that the formation of bulky DNA adducts occurs at lower doses than the occurrence of mutations as measured by the lacZ assay. This study also calculated BMDs for both assays. The BMD for bulky DNA adducts in bone marrow was 0.0286 (0.196 - 0.0661), the BMD for lacZ mutations in bone marrow was 2.22 (1.81 - 2.75). These BMDs support that bulky DNA adducts occur at lower doses than mutations.
TK6 cells exposed to BPDE (Akerman et al., 2004)
- After exposure to 0 uM, 0.01 uM, 0.10 uM and 1.00 uM BPDE for 4 hours with 4 hour recovery and exposure for 4 hours with 24 hour recovery, bulky DNA adducts were measured by ³²P post-labeling. Samples exposed to 0.01 uM, 0.10 uM and 1.00 uM BPDE had significantly more adducts than control.
- After exposure for 4 hours and 3 day recovery (TK gene) or exposure for 4 hours and 7 day recovery (HPRT gene), mutation frequency in the TK and HPRT genes was measured by only 1.00 uM BPDE had significantly more mutations than control
- Therefore after a 4 hour exposure of TK6 cells to BPDE, the frequency of bulky DNA adducts increased at lower doses than the frequency of mutations in the TK and HPRT genes increased.
Yeast p53 cDNA samples were treated with 0, 2.5 uM, 5 uM, 10 uM, 20 uM (+/-)-Anti-BPDE for 3 hours (Park et al. 2008)
- Stable (+)-Anti-BPDE-N-dGuo Adducts were detected with LC-MS and quantified with HPLC. A linear dose response was observed, ranging from 150 adducts per 10⁵dGuo at 2.5 uM anti-BPDE to 940 adducts per 10⁵dGuo at 20 uM.
- Mutations were detected with a yeast reporter system. A linear correlation (R=0.8411) was found between the incidence of thespecific bulky DNA adduct (+)-Anti-BPDE-N-dGuo and the percentage of mutated yeast colonies.
- These results suggest that the specific bulky DNA adduct (+)-Anti-BPDE-N-dGuo is incorporated into a mutagenic lesion in a dose dependant manner.

Temporal concordance

TK6 cells exposed to BPDE (Akerman et al., 2004)
- After exposure to 0 uM, 0.01 uM, 0.10 uM and 1.00 uM BPDE for 4 hours with 4 hour recovery and exposure for 4 hours with 24 hour recovery, bulky DNA adducts were measured by ³²P post-labeling. Samples exposed to 0.01 uM, 0.10 uM and 1.00 uM BPDE had significantly more adducts than control.
- After exposure for 4 hours and 3 day recovery (TK gene) or exposure for 4 hours and 7 day recovery (HPRT gene), mutation frequency in the TK and HPRT genes was measured by only 1.00 uM BPDE had significantly more mutations than control
- Therefore, after a 4 hour exposure of TK6 cells to BPDE, the frequency of bulky DNA adducts increased at earlier timepoints (4 or 24 hours) than the timepoints where mutations in the TK and HPRT genes increased (3 or 7 days).

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

There is some quantitative understanding of the amount of bulky DNA adducts that leads to mutations.

Broschard et al. 1994 and Broschard, Wiessler, and Schmeiser 1995 found that T7 DNA polymerase and with human DNA polymerase α,respectively, paired dA-AAI equally well with A or T, suggesting that there is a 50% chance of a dA-AAI lesion will lead to a mutation.

Zhang et al. 2000 found that polymerase eta predominately incorporated A opposite (+)-trans -anti-BPDE-N²-dG bulky adducts, lessfrequently a T was incorporated and least frequently a G or C was incorporated. Suggesting that it is most likely the persistence of a (+)-trans -anti-BPDE-N² -dG bulky adduct will lead to a mutation of a G to T transversion.

There are also studies demonstrating the quantitative dose-response between bulky DNA adducts and mutations, see Empirical Evidence.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Bulky DNA adducts can occur in any cell type that is able to metabolically activate the stressor. Bulky adducts and resulting mutation frequencyhave been observed in various cell lines in vitro (TK6, HeLa, CHO) as well as various organisms in vivo (yeast, rat, human and mouse). This is unspecific to sex and to life stage.

References

List of the literature that was cited for this KER description. More help

Akerman, G. S. et al. 2004. “Gene Expression Profiles and Genetic Damage in Benzo(a)Pyrene Diol Epoxide-Exposed TK6 Cells.” Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis 549(1–2): 43–64.

Alexandrov, Kroum et al. 2002. “CYP1A1 and GSTM1 Genotypes Affect Benzo[a]Pyrene DNA Adducts in Smokers’ Lung: Comparisonwith Aromatic/Hydrophobic Adduct Formation.” Carcinogenesis 23(12): 1969–77.

Anna, Lívia et al. 2009. “Relationship between TP53 Tumour Suppressor Gene Mutations and Smoking-Related Bulky DNA Adducts in aLung Cancer Study Population from Hungary.” Mutagenesis 24(6): 475–80.

Arlt, Volker M., Marie Stiborova, and Heinz H. Schmeiser. 2002. “Aristolochic Acid as a Probable Human Cancer Hazard in HerbalRemedies: A Review.” Mutagenesis 17(4): 265–77.

Bailey, Elisabeth A. et al. 1996. “Mutational Properties of the Primary Aflatoxin B1-DNA Adduct.” Proceedings of the National Academy of Sciences of the United States of America 93(4): 1535–39.

Broschard, Thomas H., Manfred Wiessler, Claus Wilhelm Von Der Lieth, and Heinz H. Schmeiser. 1994. “Translesional Synthesis on DNATemplates Containing Site-Specifically Placed Deoxyadenosine and Deoxyguanosine Adducts Formed by the Plant CarcinogenAristolochic Acid.” Carcinogenesis 15(10): 2331–40.

Broschard, Thomas H., Manfred Wiessler, and Heinz H. Schmeiser. 1995. “Effect of Site-Specifically Located Aristolochic Acid DNAAdducts on in Vitro DNA Synthesis by Human DNA Polymerase α.” Cancer Letters 98(1): 47–56.

Chen, Yanan et al. 2008. “Electronic Detection of Lectins Using Carbohydrate Functionalized Nanostructures: Graphene versus CarbonNanotubes.” Nano 6(9): 2166–71.

Cho, Eunnara et al. 2020. “Oxidative DNA Damage Leading to Chromosomal Aberrations and Mutations.” AOP Wiki:https://aopwiki.org/aops/296.

Chiapperino, Dominic et al. 2002. “Preferential Misincorporation of Purine Nucleotides by Human DNA Polymerase η OppositeBenzo[a]Pyrene 7,8-Diol 9,10-Epoxide Deoxyguanosine Adducts.” Journal of Biological Chemistry 277(14): 11765–71.http://dx.doi.org/10.1074/jbc.M112139200.

DeMarini, David M. et al. 2001. “Lung Tumor KRAS and TP53 Mutations in Nonsmokers Reflect Exposure to PAH-Rich Coal Combustion Emissions.” Cancer Research 61(18): 6679–81.

Hainaut, Pierre, and Gerd P. Pfeifer. 2001. “Patterns of P53→T Transversions in Lung Cancers Reflect the Primary Mutagenic Signature ofDNA-Damage by Tobacco Smoke.” Carcinogenesis 22(3): 367–74.

IARC. 2011. “Plants Containing Aristolochic Acid.” IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 100 A: 367–83.

IARC. 2014. “Benzo(a)Pyrene.” In Chemical Agents and Related Occupations IARC Monographs on the Evaluation of Carcinogenic Risksto Humans Volume 100F, 423–28.

Kohara, Arihiro et al. 2002. “Mutagenicity of Aristolochic Acid in the Lambda/LacZ Transgenic Mouse (MutaMouse).” Mutation Research- Genetic Toxicology and Environmental Mutagenesis 515(1–2): 63–72.

Kozack, Richard E., Rajiv Shukla, and Edward L. Loechler. 1999. “A Hypothesis for What Conformation of the Major Adduct of(+)-Anti-B[a]PDE (N2-DG) Causes G→T versus G→A Mutations Based upon a Correlation between Mutagenesis and Molecular Modeling Results.” Carcinogenesis 20(1): 95–102.

Long, Alexandra S. et al. 2018. “Benchmark Dose Analyses of Multiple Genetic Toxicity Endpoints Permit Robust, Cross-Tissue Comparisons of MutaMouse Responses to Orally Delivered Benzo[a]Pyrene.” Archives of Toxicology 92(2): 967–82.https://doi.org/10.1007/s00204-017-2099-2.

Mei, Nan et al. 2006. “DNA Adduct Formation and Mutation Induction by Aristolochic Acid in Rat Kidney and Liver.” Mutation Research -Fundamental and Molecular Mechanisms of Mutagenesis 602(1–2): 83–91.

Park, Jong Heum et al. 2008. “Erratum: The Pattern of P53 Mutations Caused by PAH o-Quinones Is Driven by 8-Oxo-DGuo FormationWhile the Spectrum of Mutations Is Determined by Biological Selection for Dominance (Chemical Research in Toxicology (2008) 21:5(1039-1049)).” Chemical Research in Toxicology 21(9): 1907.

Smela, Maryann E. et al. 2002. “The Aflatoxin B1 Formamidopyrimidine Adduct Plays a Major Role in Causing the Types of Mutations Observed in Human Hepatocellular Carcinoma.” Proceedings of the National Academy of Sciences of the United States of America 99(10):6655–60.

Veglia, Fabrizio, Giuseppe Matullo, and Paolo Vineis. 2003. “Bulky DNA Adducts and Risk of Cancer: A Meta-Analysis.” Cancer Epidemiology Biomarkers and Prevention 12(2): 157–60.

Yagi, Takashi et al. 2017. “Error-Prone and Error-Free Translesion DNA Synthesis over Site-Specifically Created DNA Adducts of Aryl Hydrocarbons (3-Nitrobenzanthrone and 4-Aminobiphenyl).” Toxicological Research 33(4): 265–72.

Zhang, Yanbin et al. 2000. “Error-Prone Lesion Bypass by Human DNA Polymerase η.” Nucleic Acids Research 28(23): 4717–24.

Zhang, Yanbin et al. 2002. “Two-Step Error-Prone Bypass of the (+)- and (-)-Trans-Anti-BPDE-N2-DG Adducts by Human DNAPolymerases η and κ.” Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis 510(1–2): 23–35.