Assessing the Carcinogenic Potential of Low Dose Exposures to Chemical Mixtures in the Environment: The Challenge Ahead

Abstract

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.
© The Author 2015. Published by Oxford University Press.

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Volume 36 Supplement 1 June 2015
www.carcin.oxfordjournals.org
Assessing the Carcinogenic Potential of Low-Dose Exposures
to Chemical Mixtures in the Environment: The Challenge Ahead
Volume 36 Supplement 1 June 2015
Carcinogenesis Integrative Cancer Research

CARCINOGENESIS SUPPLEMENT
Assessing the Carcinogenicity of Low-Dose
Exposures to Chemical Mixtures in the
Environment: The Challenge Ahead
Editor-in-Chief
Curtis C. Harris
We gratefully acknowledge the support of the National Institute of Health (NIH)-National Institute of
Environmental Health Sciences (NIEHS) conference grant travel support [R13ES023276]

EDITORIAL
Cause and Prevention of Human Cancer
Curtis C Harris ..............................................................................S1
REVIEWS
Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth
suppression
RitaNahta, FahdAl-Mulla, RabeahAl-Temaimi, AmedeoAmedei, RafaelaAndrade-Vieira, SarahBay,
DustinG.Brown, GloriaM.Calaf, RobertC.Castellino, KarineA.Cohen-Solal, AnnamariaColacci,
NicholaCruickshanks, PaulDent, RiccardoDiFiore, StefanoForte, GaryS.Goldberg, RoslidaA.Hamid,
HariniKrishnan, DaleW.Laird, AhmedLasfar, PaolaA.Marignani, LorenzoMemeo, ChiaraMondello,
ChristianC.Naus, RichardPonce-Cusi, JayadevRaju, DebasishRoy, RabindraRoy, ElizabethP.Ryan,
HosniK.Salem, A.IvanaScovassi, NeetuSingh, MonicaVaccari, RenzaVento, JanVondráček, MarkWade,
JordanWoodrick, and WilliamH.Bisson...........................................................S2
Disruptive chemicals, senescence and immortality
AmancioCarnero, CarmenBlanco-Aparicio, HiroshiKondoh, MatildeE.Lleonart, JuanFernando
Martinez-Leal, ChiaraMondello, A.IvanaScovassi, WilliamH.Bisson, AmedeoAmedei, RabindraRoy,
JordanWoodrick, AnnamariaColacci, MonicaVaccari, JayadevRaju, FahdAl-Mulla, RabeahAl-Temaimi,
HosniK.Salem, LorenzoMemeo, StefanoForte, NeetuSingh, RoslidaA.Hamid, ElizabethP.Ryan,
DustinG.Brown, JohnPierceWiseSr, SandraS.Wise, and HemadYasaei ...............................S19
The potential for chemical mixtures from the environment to enable the cancer hallmark of
sustained proliferative signalling
WilhelmEngström, PhilippaDarbre, StaffanEriksson, LindaGulliver, ToveHultman, MichalisV.Karamouzis,
JamesE.Klaunig, RekhaMehta, KimMoorwood, ThomasSanderson, HidekoSone, PankajVadgama,
GerardWagemaker, AndrewWard, NeetuSingh, FahdAl-Mulla, RabeahAl-Temaimi, AmedeoAmedei,
AnnaMariaColacci, MonicaVaccari, ChiaraMondello, A.IvanaScovassi, JayadevRaju, RoslidaA.Hamid,
LorenzoMemeo, StefanoForte, RabindraRoy, JordanWoodrick, HosniK.Salem, ElizabethRyan,
DustinG.Brown, and WilliamH.Bisson .........................................................S38
Causes of genome instability: the effect of low dose chemical exposures in modern society
SabineA.S.Langie, GudrunKoppen, DanielDesaulniers, FahdAl-Mulla, RabeahAl-Temaimi, AmedeoAmedei,
AmayaAzqueta, WilliamH.Bisson, DustinBrown, GunnarBrunborg, AmeliaK.Charles, TaoChen,
AnnamariaColacci, FirouzDarroudi, StefanoForte, LaetitiaGonzalez, RoslidaA.Hamid, LisbethE.Knudsen,
LucLeyns, AdelaLopezdeCerainSalsamendi, LorenzoMemeo, ChiaraMondello, CarmelMothersill,
Ann-KarinOlsen, SoaPavanello, JayadevRaju, EmilioRojas, RabindraRoy, ElizabethRyan, Patricia
Ostrosky-Wegman, HosniK.Salem, IvanaScovassi, NeetuSingh, MonicaVaccari, FrederikJ.VanSchooten,
MaharaValverde, JordanWoodrick, LuopingZhang, NikvanLarebeke, MichelineKirsch-Volders,
and AndrewR.Collins........................................................................S61
Disruptive environmental chemicals and cellular mechanisms that confer resistance to celldeath
KannanBadriNarayanan, ManafAli, BarryJ.Barclay, QiangCheng, LeandroD’Abronzo, Rita
Dornetshuber-Fleiss, ParamitaM.Ghosh, MichaelJ.GonzalezGuzman, Tae-JinLee, PoSingLeung, LinLi,
SuidjitLuanpitpong, EdwardRatovitski, YonRojanasakul, MariaFiammettaRomano, SimonaRomano,
RanjeetKumarSinha, ClementYedjou, FahdAl-Mulla, RabeahAl-Temaimi, AmedeoAmedei, DustinG.
Brown, ElizabethP.Ryan, AnnamariaColacci, RoslidaA.Hamid, ChiaraMondello, JayadevRaju, HosniK.Salem,
JordanWoodrick, IvanaScovassi, NeetuSingh, MonicaVaccari, RabindraRoy, StefanoForte, LorenzoMemeo,
SeoYunKim, WilliamH.Bisson, LeroyLowe, and HyunHoPark.......................................S89
JOURNAL OF CARCINOGENESIS
VOLUME 36, SUPPLEMENT 1
MAY 2015
Contents

Chemical compounds from anthropogenic environment and immune evasion
mechanisms: potential interactions
JuliaKravchenko, EmanuelaCorsini, MarcA.Williams, WilliamDecker, MasoudH.Manjili, TakemiOtsuki,
NeetuSingh, FahaAl-Mulla, RabeahAl-Temaimi, AmedeoAmedei, AnnaMariaColacci, MonicaVaccari,
ChiaraMondello, A.IvanaScovassi, JayadevRaju, RoslidaA.Hamid, LorenzoMemeo, StefanoForte,
RabindraRoy, JordanWoodrick, HosniK.Salem, ElizabethP.Ryan, DustinG.Brown, WilliamH.Bisson,
LeroyLowe, and H.KimLyerly ................................................................S111
The impact of low-dose carcinogens and environmental disruptors on tissue invasion and metastasis
JosiahOchieng, GladysN.Nangami, OlugbemigaOgunkua, IsabelleR.Miousse, IgorKoturbash, ValerieOdero-
Marah, LisaMcCawley, PratimaNangia-Makker, NuzhatAhmed, YunusLuqmani, ZhenbangChen,
SilvanaPapagerakis, GregoryT.Wolf, ChenfangDong, BinhuaP.Zhou, DustinG.Brown, AnnamariaColacci,
RoslidaA.Hamid, ChiaraMondello, JayadevRaju, ElizabethP.Ryan, JordanWoodrick, IvanaScovassi,
NeetuSingh, MonicaVaccari, RabindraRoy, StefanoForte, LorenzoMemeo, HosniK.Salem, AmedeoAmedei,
RabeahAl-Temaimi, FahdAl-Mulla, WilliamH.Bisson, and SakinaE.Eltom............................S128
The effect of environmental chemicals on the tumor microenvironment
StephanieC.Casey, MonicaVaccari, FahdAl-Mulla, RabeahAl-Temaimi, AmedeoAmedei, MaryHelen
Barcellos-Hoff, DustinG.Brown, MarionChapellier, JosephChristopher, ColleenCurran, StefanoForte,
RoslidaA.Hamid, PetrHeneberg, DanielC.Koch, P.K.Krishnakumar, EzioLaconi, VeroniqueMaguer-Satta,
FabioMarongiu, LorenzoMemeo, ChiaraMondello, JayadevRaju, JesseRoman, RabindraRoy, ElizabethP.Ryan,
SandraRyeom, HosniK.Salem, A.IvanaScovassi, NeetuSingh, LauraSoucek, LouisVermeulen, JonathanR.
Whiteld, JordanWoodrick, AnnamariaColacci, WilliamH.Bisson, and DeanW.Felsher...................S160
Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the
environment: focus on the cancer hallmark of tumor angiogenesis
ZhiweiHu, SamiraA.Brooks, ValérianDormoy, Chia-WenHsu, Hsue-YinHsu, Liang-TzungLin,
ThierryMassfelder, W.KimrynRathmell, MenghangXia, FahdAl-Mulla, RabeahAl-Temaimi,
AmedeoAmedei, DustinG.Brown, KalanR.Prudhomme, AnnamariaColacci, RoslidaA.Hamid,
ChiaraMondello, JayadevRaju, ElizabethP.Ryan, JordanWoodrick, A.IvanaScovassi, NeetuSingh,
MonicaVaccari, RabindraRoy, StefanoForte, LorenzoMemeo, HosniK.Salem, LeroyLowe, LasseJensen,
WilliamH.Bisson, and NicoleKleinstreuer.......................................................S184
Metabolic reprogramming and dysregulated metabolism: cause, consequence and/or enabler of
environmental carcinogenesis?
R.BrooksRobey, JudithWeisz, NancyKuemmerle, AnnaC.Salzberg, ArthurBerg, DustinBrown, LauraKubik,
RobertaPalorini, FahdAl-Mulla, RabeahAl-Temaimi, AnnamariaColacci, ChiaraMondello, JayadevRaju,
JordanWoodrick, IvanaScovassi, NeetuSingh, MonicaVaccari, RabindraRoy, StefanoForte, LorenzoMemeo,
HosniK.Salem, AmedeoAmedei, RoslidaA.Hamid, GraemeP.Williams, LeroyLowe, JoelMeyer,
FrancisL.Martin, WilliamH.Bisson, FerdinandoChiaradonna, and ElizabethP.Ryan ....................S203
Environmental immune disruptors, inammation and cancerrisk
PatriciaA.Thompson, MahinKhatami, CarolynJ.Baglole, JunSun, ShelleyHarris, Eun-YiMoon, FahdAl-Mulla,
RabeahAl-Temaimi, DustinBrown, AnnamariaColacci, ChiaraMondello, JayadevRaju, ElizabethRyan,
JordanWoodrick, IvanaScovassi, NeetuSingh, MonicaVaccari, RabindraRoy, StefanoForte, LorenzoMemeo,
HosniK.Salem, AmedeoAmedei, RoslidaA.Hamid, LeroyLowe, and WilliamH.Bisson ....................S232
Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment:
the challengeahead
WilliamH.GoodsonIII, LeroyLowe, DavidO.Carpenter, MichaelGilbertson, AbdulManafAli,
AdelaLopezdeCerainSalsamendi, AhmedLasfar, AmancioCarnero, AmayaAzqueta, AmedeoAmedei, AmeliaK.
Charles, AndrewR.Collins, AndrewWard, AnnaC.Salzberg, AnnamariaColacci, Ann-KarinOlsen, ArthurBerg,
BarryJ.Barclay, BinhuaP.Zhou, CarmenBlanco-Aparicio, CarolynBaglole, ChenfangDong, ChiaraMondello,
Chia-WenHsu, ChristianC.Naus, ClementYedjou, ColleenS.Curran, DaleW.Laird, DanielC.Koch, DanielleJ.
Carlin, DeanW.Felsher, DebasishRoy, DustinBrown, EdwardRatovitski, ElizabethP.Ryan, EmanuelaCorsini,
EmilioRojas, Eun-YiMoon, EzioLaconi, FabioMarongiu, FahdAl-Mulla, FerdinandoChiaradonna, FirouzDarroudi,
FrancisL.Martin, FrederikJ.VanSchooten, GaryS.Goldberg, GerardWagemaker, GladysNangami, GloriaM.
Calaf, GraemeWilliams, GregoryT.Wolf, GudrunKoppen, GunnarBrunborg, H.KimLyerly, HariniKrishnan,
HasiahAbHamid, HemadYasaei, HidekoSone, HiroshiKondoh, HosniK.Salem, Hsue-YinHsu, HyunHoPark,
IgorKoturbash, IsabelleR.Miousse, IvanaScovassi, JamesEKlaunig, JanVondráček, JayadevRaju, JesseRoman,

JohnPierceWiseSr., JonathanR.Whiteld, JordanWoodrick, JosephChristopher, JosiahOchieng,
JuanFernandoMartinez-Leal, JudithWeisz, JuliaKravchenko, JunSun, KalanR.Prudhomme,
KannanBadriNarayanan, KarineA.Cohen-Solal, KimMoorwood, LaetitiaGonzalez, LauraSoucek,
LeJian, LeandroS.D’Abronzo, Liang-TzungLin, LinLi, LindaGulliver, LisaJ.McCawley,
LorenzoMemeo, LouisVermeulen, LucLeyns, LuopingZhang, MaharaValverde, MahinKhatami,
MariaFiammettaRomano, MarionChapellier, MarcA.Williams, MarkWade, MasoudH.Manjili,
MatildeLleonart, MenghangXia, MichaelJGonzalez, MichalisV.Karamouzis, MichelineKirsch-
Volders, MonicaVaccari, NancyB.Kuemmerle, NeetuSingh, NicholaCruickshanks, NicoleKleinstreuer,
NikvanLarebeke, NuzhatAhmed, OlugbemigaOgunkua, P.K.Krishnakumar, PankajVadgama,
PaolaA.Marignani, ParamitaM.Ghosh, PatriciaOstrosky-Wegman, PatriciaThompson, PaulDent,
PetrHeneberg, PhilippaDarbre, PoSingLeung, PratimaNangia-Makker, Qiang(Shawn)Cheng,
R.BrooksRobey, RabeahAl-Temaimi, RabindraRoy, RafaelaAndrade-Vieira, RanjeetK.Sinha,
RekhaMehta, RenzaVento, RiccardoDiFiore, RichardPonce-Cusi, RitaDornetshuber-Fleiss,
RitaNahta, RobertC.Castellino, RobertaPalorini, RoslidaAbdHamid, SabineA.S.Langie,
SakinaEltom, SamiraA.Brooks, SandraRyeom, SandraS.Wise, SarahN.Bay, ShelleyHarris,
SilvanaPapagerakis, SimonaRomano, SoaPavanello, StaffanEriksson, StefanoForte,
StephanieC.Casey, SudjitLuanpitpong, Tae-JinLee, TakemiOtsuki, TaoChen, ThierryMassfelder,
ThomasSanderson, TizianaGuarnieri, ToveHultman, ValérianDormoy, ValerieOdero-Marah,
VenkataSabbisetti, VeroniqueMaguer-Satta, W.KimrynRathmell, WilhelmEngström, WilliamK.
Decker, WilliamH.Bisson, YonRojanasakul, YunusLuqmani, ZhenbangChen, and ZhiweiHu ..............S254

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Theo Colborn (March 28, 1927 - December 14, 2014)
We dedicate this special issue on the challenges associated with assessing the carcinogenic potential of low dose exposures to
chemical mixtures in the environment, to the memory of Dr Theodora (Theo) Colborn. Theo was a pioneer in the science of the
effects of low dose exposures to environmental chemicals and for the past twenty-ve years, was instrumental in the develop-
ment and integration of the eld of endocrine disruption. Theo introduced us to one another about four-years-ago which led to the
founding of Getting to Know Cancer, and ultimately the launch of the Halifax Project (which has been a tremendously productive
collaboration for the integration of cancer biology and environmental toxicology). So we want to thank her for her legacy of work in
this area, her inuence on our research, and her encouragement.
Theo was well known internationally for her tireless commitment to the protection of public health, but not everyone knew that
she was also a tremendously generous and insightful scientist who assembled researchers from a variety of specialties and allowed
them to discover for themselves what she had understood about the inuences of low-dose exposures to certain environmental
chemicals on embryonic and fetal development. Indeed, she nurtured cross-disciplinary collaboration and it was that collegiality
and spirit of sharing that produced seminal insights that opened up the entire eld of endocrine disruption. So we have attempted
to use a similar approach to help us understand the importance of ongoing low dose exposures to mixtures of chemicals in the
environment and their relevance for cancer and carcinogenesis. In other words, this is truly an extension of her work, and we want
to pay tribute and offer thanks for her wisdom, her generosity and her legacy.
Leroy Lowe and Michael Gilbertson,
Cofounders, Getting To Know Cancer
June, 2015

© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
Carcinogenesis, 2015, Vol. 36, Supplement 1, S1
doi: 10.1093/carcin/bgv047
Editorial
S1
Copyedited by: <CE_Initials>
Editorial
Cause and Prevention of Human Cancer
The relative contribution of the environment, genetic suscepti-
bility and DNA replication errors to cancer causation has been
a longstanding area of investigation in the elds of molecular
epidemiology of cancer and carcinogenesis. A recent report by
Tomasetti etal. (1) attributing DNA replication errors within stem
cells and ‘bad luck’ as a major cause of a select group of cancers
has stirred debate within community of cancer researchers espe-
cially those in cancer prevention (2–7). Tomasetti et al. (8) have
also written a balanced response to many of these concerns.
Carcinogenesis is joining this debate by publishing in this
issue a series of reviews on the carcinogenic potential of expo-
sure to low doses and mixtures of chemicals. The reviews utilize
a framework of the Hallmarks of Cancer (9) and are the prod-
uct of the Halifax Project Task Force initiated by Leroy Lowe
and Michael Gilbertson. They engaged international teams with
input of nearly 200 cancer biologists and toxicologist to review
the literature in each of the 11 Hallmarks of Cancer. The reviews
are multiauthored, condensed by a peer review and extensively
referenced. The primary recommendation is a research and
regulatory strategy using the Hallmarks of Cancer framework to
identify priority mixtures of chemicals, i.e. ‘….those with sub-
stantial carcinogenic relevance’, for future investigations ‘…. to
inform risk assessment practices worldwide’ (10).
Carcinogenesis will also publish a review of cancer prevention
this summer, which will be written by Christopher Wild, Director
of the International Agency on Research of Cancer.
References
1. Tomasetti, C. etal. (2015) Cancer etiology. Variation in cancer
risk among tissues can be explained by the number of stem
cell divisions. Science, 347, 78–81.
2. Crossan, G.P. etal. (2015) Do mutational dynamics in stem cells
explain the origin of common cancers? Cell Stem Cell, 16, 111–112.
3. O’Callaghan, M. (2015) Cancer risk: accuracy of literature. Sci-
ence, 347, 729.
4. Ashford, N.A. et al. (2015) Cancer risk: role of environment.
Science, 347, 727.
5. Song, M. etal. (2015) Cancer risk: many factors contribute. Sci-
ence, 347, 728–729.
6. Potter, J.D. etal. (2015) Cancer risk: tumors excluded. Science,
347, 727.
7. Wild, C. etal. (2015) Cancer risk: role of chance overstated. Sci-
ence, 347, 728.
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response. Science, 347, 729–731.
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10. Goodson, W.H. III, etal. (2015) Assessing the carcinogenesis
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environment: the challenge ahead. Carcinogenesis.
Curtis CHarris,
Editor-in-Chief

Received: August 7, 2014; Revised: January 23, 2015; Accepted: January 31, 2015
© The Author 2015. Published by Oxford University Press.
Carcinogenesis, 2015, Vol. 36, Supplement 1, S254–S296
doi:10.1093/carcin/bgv039
Review
S254
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
(http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any
medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Assessing the carcinogenic potential of low-dose
exposures to chemical mixtures in the environment: the
challengeahead
William H.Goodson III*, LeroyLowe1,2, David O.Carpenter3,
MichaelGilbertson4, AbdulManaf Ali5,
AdelaLopez de Cerain Salsamendi6, AhmedLasfar7,
AmancioCarnero8, AmayaAzqueta6, AmedeoAmedei9,
Amelia K.Charles10, Andrew R.Collins11, AndrewWard12,
Anna C.Salzberg13, AnnamariaColacci14, Ann-KarinOlsen15,
ArthurBerg13, Barry J.Barclay16, Binhua P.Zhou17,
CarmenBlanco-Aparicio18, Carolyn J.Baglole19, ChenfangDong17,
ChiaraMondello20, Chia-WenHsu21, Christian C.Naus22,
ClementYedjou23, Colleen S.Curran24, Dale W.Laird25, Daniel C.Koch26,
Danielle J.Carlin27, Dean W.Felsher28, DebasishRoy29,
Dustin G.Brown30, EdwardRatovitski31, Elizabeth P.Ryan30,
EmanuelaCorsini32, EmilioRojas33, Eun-YiMoon34, EzioLaconi35,
FabioMarongiu35, FahdAl-Mulla36,
FerdinandoChiaradonna37,38, FirouzDarroudi39, Francis L.Martin2,
Frederik J.Van Schooten40, Gary S.Goldberg41,
GerardWagemaker42, GladysNangami43, Gloria M.Calaf44,45,
GraemeWilliams46, Gregory T.Wolf47, GudrunKoppen48,
GunnarBrunborg15, H.Kim Lyerly49, HariniKrishnan41,
HasiahAb Hamid50, HemadYasaei51, HidekoSone52,
HiroshiKondoh53, Hosni K.Salem54, Hsue-YinHsu55,
Hyun HoPark56, IgorKoturbash57, Isabelle R.Miousse57, A.IvanaScovassi20,
James E.Klaunig58, JanVondráček59, JayadevRaju60, JesseRoman61,62,
John PierceWise Sr.63, Jonathan R.Whiteld64,
JordanWoodrick65, Joseph A.Christopher66, JosiahOchieng43,
Juan FernandoMartinez-Leal67, JudithWeisz68, JuliaKravchenko49,
JunSun69, Kalan R.Prudhomme70, Kannan BadriNarayanan56,
Karine A.Cohen-Solal71, KimMoorwood12, LaetitiaGonzalez72,
LauraSoucek64,73, LeJian74,75, Leandro S.D’Abronzo76, Liang-TzungLin77,

W.H.Goodson et al. | S255
Carcinogenesis, 2015, Vol. 36, Supplement 1, S254–S296
doi:10.1093/carcin/bgv039
Review
LinLi78, LindaGulliver79, Lisa J.McCawley80, LorenzoMemeo81,
LouisVermeulen82, LucLeyns72, LuopingZhang83,
MaharaValverde33, MahinKhatami84,
Maria FiammettaRomano85, MarionChapellier86, Marc A.Williams87,
MarkWade88, Masoud H.Manjili89, MatildeLleonart90, MenghangXia21,
Michael J.Gonzalez91, Michalis V.Karamouzis92,
MichelineKirsch-Volders72, MonicaVaccari14, Nancy B.Kuemmerle93,94,
NeetuSingh95, NicholaCruickshanks96, NicoleKleinstreuer97,
Nikvan Larebeke98, NuzhatAhmed99, OlugbemigaOgunkua43,
P.K.Krishnakumar100, PankajVadgama101, Paola A.Marignani102,
Paramita M.Ghosh76, PatriciaOstrosky-Wegman33, PatriciaThompson103,
PaulDent96, PetrHeneberg104, PhilippaDarbre105, PoSing Leung78,
PratimaNangia-Makker106, Qiang (Shawn)Cheng107, R.BrooksRobey93,94,
RabeahAl-Temaimi108, RabindraRoy65, RafaelaAndrade-Vieira102,
Ranjeet K.Sinha109, RekhaMehta60, RenzaVento110,111, RiccardoDi Fiore110,
RichardPonce-Cusi45, RitaDornetshuber-Fleiss112,113, RitaNahta114,
Robert C.Castellino115,116, RobertaPalorini37,38, RoslidaAbd Hamid50,
Sabine A.S.Langie48, SakinaEltom43, Samira A.Brooks117, SandraRyeom118,
Sandra S.Wise63, Sarah N.Bay119, Shelley A.Harris120,121,
SilvanaPapagerakis47, SimonaRomano85, SoaPavanello122,
StaffanEriksson123, StefanoForte81, Stephanie C.Casey26,
SudjitLuanpitpong124, Tae-JinLee125, TakemiOtsuki126, TaoChen127,
ThierryMassfelder128, ThomasSanderson129, TizianaGuarnieri130,131,132,
Tove  Hultman133, ValérianDormoy128,134, ValerieOdero-Marah135,
VenkataSabbisetti136, VeroniqueMaguer-Satta87,
W.KimrynRathmell117, WilhelmEngström137, William K.Decker138,
William H.Bisson70, YonRojanasakul139, YunusLuqmani140,
ZhenbangChen43 and ZhiweiHu141
California Pacic Medical Center Research Institute, 2100 Webster Street #401, San Francisco, CA 94115, USA, 1Getting to
Know Cancer, Room 229A, 36 Arthur Street, Truro, Nova Scotia B2N 1X5, Canada, 2Lancaster Environment Centre, Lancaster
University, Bailrigg, Lancaster LA1 4AP, UK, 3Institute for Health and the Environment, University at Albany, 5 University
Pl., Rensselaer, NY 12144, USA, 4Getting to Know Cancer, Guelph N1G 1E4, Canada, 5School of Biotechnology, Faculty of
Agriculture Biotechnology and Food Sciences, Sultan Zainal Abidin University, Tembila Campus, 22200 Besut, Terengganu,
Malaysia, 6Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Navarra, Pamplona 31008,
Spain, 7Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, State University of New
Jersey, Piscataway, NJ 08854, USA, 8Instituto de Biomedicina de Sevilla, Consejo Superior de Investigaciones Cienticas.
Hospital Universitario Virgen del Rocio, Univ. de Sevilla., Avda Manuel Siurot sn. 41013 Sevilla, Spain, 9Department of
Experimental and Clinical Medicine, University of Firenze, Florence 50134, Italy, 10School of Biological Sciences, University
of Reading, Hopkins Building, Reading, Berkshire RG6 6UB, UK, 11Department of Nutrition, University of Oslo, Oslo, Norway,
12Department of Biochemistry and Biology, University of Bath, Claverton Down, Bath BA2 7AY, UK, 13Department of Public
Health Sciences, College of Medicine, Pennsylvania State University, Hershey, PA 17033, USA, 14Center for Environmental
Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency, 40126 Bologna, Italy,
15Department of Chemicals and Radiation, Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo
N-0403, Norway, 16Planet Biotechnologies Inc., St Albert, Alberta T8N 5K4, Canada, 17Department of Molecular and Cellular
Biochemistry, University of Kentucky, Lexington, KY 40508, USA, 18Spanish National Cancer Research Centre, CNIO, Melchor
Fernandez Almagro, 3, 28029 Madrid, Spain, 19Department of Medicine, McGill University, Montreal, Quebec H4A 3J1,
Canada, 20Istituto di Genetica Molecolare, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy, 21Division of Preclinical Innovation,
National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Bethesda,
MD 20892–3375, USA, 22Department of Cellular and Physiological Sciences, Life Sciences Institute, Faculty of Medicine,
The University of British Columbia, Vancouver, British Columbia V5Z 1M9, Canada, 23Department of Biology, Jackson State
University, Jackson, MS 39217, USA, 24Department of Molecular and Environmental Toxicology, University of Wisconsin-
Madison, Madison, WI 53706, USA, 25Department of Anatomy and Cell Biology, University of Western Ontario, London,

S256 | Carcinogenesis, 2015, Vol. 36, Supplement 1
Ontario N6A 3K7, Canada, 26Stanford University Department of Medicine, Division of Oncology, Stanford, CA 94305, USA,
27Superfund Research Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27560,
USA, 28Department of Medicine, Oncology and Pathology, Stanford University, Stanford, CA 94305, USA, 29Department of
Natural Science, The City University of New York at Hostos Campus, Bronx, NY 10451, USA, 30Department of Environmental
and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523–1680, USA, 31Department of Head and
Neck Surgery/Head and Neck Cancer Research, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA,
32Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 20133 Milan, Italy,
33Department of Genomic Medicine and Environmental Toxicology, Institute for Biomedical Research, National Autonomous
University of Mexico, Mexico City 04510, México, 34Department of Bioscience and Biotechnology, Sejong University, Seoul
143–747, Korea, 35Department of Biomedical Sciences, University of Cagliari, 09124 Cagliari, Italy, 36Department of Pathology,
Kuwait University, Safat 13110, Kuwait, 37Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126
Milan, Italy, 38SYSBIO Centre of Systems Biology, Department of Biotechnology and Biosciences, University of Milano-Bicocca,
20126 Milan, Italy, 39Human Safety and Environmental Research, Department of Health Sciences, College of North Atlantic,
Doha 24449, State of Qatar, 40Department of Toxicology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht
University, Maastricht 6200, The Netherlands, 41Department of Molecular Biology, School of Osteopathic Medicine, Rowan
University, Stratford, NJ 08084, USA, 42Hacettepe University, Center for Stem Cell Research and Development, Ankara 06640,
Turkey, 43Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208, USA, 44Center for
Radiological Research, Columbia University Medical Center, New York, NY 10032, USA, 45Instituto de Alta Investigacion,
Universidad de Tarapaca, Arica, Chile, 46School of Biological Sciences, University of Reading, Reading, RG6 6UB, UK,
47Department of Otolaryngology - Head and Neck Surgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA,
48Environmental Risk and Health Unit, Flemish Institute for Technological Research, 2400 Mol, Belgium, 49Department of
Surgery, Pathology, Immunology, Duke University Medical Center, Durham, NC 27710, USA, 50Department of Biomedical
Sciences, Faculty of Medicine and Health Sciences, 43400 Universiti Putra Malaysia, Serdang, Selangor, Malaysia,
51Department of Life Sciences, College of Health and Life Sciences and the Health and Environment Theme, Institute of
Environment, Health and Societies, Brunel University Kingston Lane, Uxbridge, Middlesex UB8 3PH, UK, 52National Institute
for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibraki 3058506, Japan, 53Department of Geriatric Medicine, Kyoto
University Hospital 54 Kawaharacho, Shogoin, Sakyo-ku Kyoto, 606–8507, Japan, 54Department of Urology, Kasr Al-Ainy School
of Medicine, Cairo University, El Manial, Cairo 11559, Egypt, 55Department of Life Sciences, Tzu-Chi University, Hualien 970,
Taiwan, 56School of Biotechnology, Yeungnam University, Gyeongbuk 712-749, South Korea, 57Department of Environmental
and Occupational Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA, 58Department of
Environmental Health, Indiana University, School of Public Health, Bloomington, IN 47405, USA, 59Department of Cytokinetics,
Institute of Biophysics Academy of Sciences of the Czech Republic, Brno, CZ-61265, Czech Republic, 60Regulatory Toxicology
Research Division, Bureau of Chemical Safety, Food Directorate, Health Canada, Ottawa, Ontario K1A 0K9, Canada,
61Department of Medicine, University of Louisville, Louisville, KY 40202, USA, 62Robley Rex VA Medical Center, Louisville, KY
40202, USA, 63Department of Applied Medical Sciences, University of Southern Maine, 96 Falmouth St., Portland, ME 04104,
USA, 64Mouse Models of Cancer Therapies Group, Vall d’Hebron Institute of Oncology (VHIO), 08035 Barcelona, Spain,
65Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC 20057, USA, 66Cancer
Research UK. Cambridge Institute, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK, 67Department of Cell
Biology, Pharmamar-SAU, Avda. De los Reyes, 1.28770-Colmenar Viejo, Madrid, Spain, 68Departments of Obstetrics and
Gynecology and Pathology, Pennsylvania State University College of Medicine, Hershey PA 17033, USA, 69Department of
Biochemistry, Rush University, Chicago, IL 60612, USA, 70Environmental and Molecular Toxicology, Environmental Health
Science Center, Oregon State University, Corvallis, OR 97331, USA, 71Department of Medicine/Medical Oncology, Rutgers
Cancer Institute of New Jersey, New Brunswick, NJ 08903, USA, 72Laboratory for Cell Genetics, Vrije Universiteit Brussel, 1050
Brussels, Belgium, 73Catalan Institution for Research and Advanced Studies (ICREA), Barcelona 08010, Spain, 74School of Public
Health, Curtin University, Bentley, WA 6102, Australia, 75Public Health and Clinical Services Division, Department of Health,
Government of Western Australia, WA 6004, Australia, 76Department of Urology, University of California Davis, Sacramento,
CA 95817, USA, 77Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical
University, Taipei 11031, Taiwan, 78School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong
Kong SAR, The People’s Republic of China, 79Faculty of Medicine, University of Otago, Dunedin 9054, New Zealand,
80Department of Biomedical Engineering and Cancer Biology, Vanderbilt University, Nashville, TN 37235, USA, 81Department of
Experimental Oncology, Mediterranean Institute of Oncology, Via Penninazzo 7, Viagrande (CT) 95029, Italy, 82Center for
Experimental Molecular Medicine, Academic Medical Center, Meibergdreef 9, Amsterdam 1105 AZ, The Netherlands,
83Division of Environmental Health Sciences, School of Public Health, University of California, Berkeley, CA 94720-7360, USA,
84Inammation and Cancer Research, National Cancer Institute (NCI) (Retired), National Institutes of Health, Bethesda, MD
20892, USA, 85Department of Molecular Medicine and Medical Biotechnology, Federico II University of Naples, 80131 Naples,
Italy, 86Centre De Recherche En Cancerologie, De Lyon, Lyon, U1052-UMR5286, France, 87United States Army Institute of Public
Health, Toxicology Portfolio-Health Effects Research Program, Aberdeen Proving Ground, Edgewood, MD 21010-5403, USA,
88Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, Via Adamello 16, 20139 Milano, Italy,
89Department of Microbiology and Immunology, Virginia Commonwealth University, Massey Cancer Center, Richmond, VA
23298, USA, 90Institut De Recerca Hospital Vall D’Hebron, Passeig Vall d’Hebron, 119–129, 08035 Barcelona, Spain, 91University
of Puerto Rico, Medical Sciences Campus, School of Public Health, Nutrition Program, San Juan 00921, Puerto Rico,
92Department of Biological Chemistry, Medical School, University of Athens, Institute of Molecular Medicine and Biomedical
Research, 10676 Athens, Greece, 93White River Junction Veterans Affairs Medical Center, White River Junction, VT 05009, USA,

W.H.Goodson et al. | S257
94Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA, 95Advanced Molecular Science Research Centre (Centre
for Advanced Research), King George’s Medical University, Lucknow, Uttar Pradesh 226 003, India, 96Departments of
Neurosurgery and Biochemistry and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA,
97Integrated Laboratory Systems Inc., in support of the National Toxicology Program Interagency Center for the Evaluation of
Alternative Toxicological Methods, RTP, NC 27709, USA, 98Analytische, Milieu en Geochemie, Vrije Universiteit Brussel, Brussel
B1050, Belgium, 99Department of Obstetrics and Gynecology, University of Melbourne, Victoria 3052, Australia, 100Center for
Environment and Water, Research Institute, King Fahd University of Petroleum and Minerals, Dhahran 3126, Saudi Arabia,
101School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, London, E1 4NS, UK,
102Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada,
103Department of Pathology, Stony Brook School of Medicine, Stony Brook University, The State University of New York, Stony
Brook, NY 11794-8691, USA, 104Charles University in Prague, Third Faculty of Medicine, CZ-100 00 Prague 10, Czech Republic,
105School of Biological Sciences, The University of Reading, Whiteknights, Reading RG6 6UB, England, 106Department of
Pathology, Wayne State University, Detroit, MI 48201, USA, 107Computer Science Department, Southern Illinois University,
Carbondale, IL 62901, USA, 108Human Genetics Unit, Department of Pathology, Faculty of Medicine, Kuwait University, Jabriya
13110, Kuwait, 109Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037,
USA, 110Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies, Polyclinic Plexus, University of
Palermo, Palermo 90127, Italy, 111Sbarro Institute for Cancer Research and Molecular Medicine, Temple University,
Philadelphia, PA 19122, USA, 112Department of Pharmacology and Toxicology, University of Vienna, Vienna A-1090, Austria,
113Institute of Cancer Research, Department of Medicine, Medical University of Vienna, Wien 1090, Austria, 114Departments of
Pharmacology and Hematology and Medical Oncology, Emory University School of Medicine and Winship Cancer Institute,
Atlanta, GA 30322, USA, 115Division of Hematology and Oncology, Department of Pediatrics, Children’s Healthcare of Atlanta,
GA 30322, USA, 116Department of Pediatrics, Emory University School of Medicine, Emory University, Atlanta, GA 30322, USA,
117Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC 27599, USA, 118Department of
Cancer Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA, 119Program in
Genetics and Molecular Biology, Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, GA
30322, USA, 120Population Health and Prevention, Research, Prevention and Cancer Control, Cancer Care Ontario, Toronto,
Ontario, M5G 2L7, Canada, 121Departments of Epidemiology and Occupational and Environmental Health, Dalla Lana School
of Public Health, University of Toronto, Toronto, Ontario, M5T 3M7, Canada, 122Department of Cardiac, Thoracic and Vascular
Sciences, Unit of Occupational Medicine, University of Padova, Padova 35128, Italy, 123Department of Anatomy, Physiology and
Biochemistry, The Swedish University of Agricultural Sciences, PO Box 7011, VHC, Almas Allé 4, SE-756 51, Uppsala, Sweden,
124Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700,
Thailand, 125Department of Anatomy, College of Medicine, Yeungnam University, Daegu 705–717, South Korea,126Department
of Hygiene, Kawasaki Medical School, Matsushima Kurashiki, Okayama 701-0192, Japan, 127Division of Genetic and Molecular
Toxicology, National Center for Toxicological Research, United States Food and Drug Administration, Jefferson, AR 72079, USA,
128INSERM U1113, team 3‘Cell Signalling and Communication in Kidney and Prostate Cancer’, University of Strasbourg,
Faculté de Médecine, 67085 Strasbourg, France, 129INRS-Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, QC H7V
1B7, Canada, 130Department of Biology, Geology and Environmental Sciences, Alma Mater Studiorum Università di Bologna,
Via Francesco Selmi, 3, 40126 Bologna, Italy, 131Center for Applied Biomedical Research, S.Orsola-Malpighi University Hospital,
Via Massarenti, 9, 40126 Bologna, Italy, 132National Institute of Biostructures and Biosystems, Viale Medaglie d’ Oro, 305, 00136
Roma, Italy, 133Department of Biosciences and Veterinary Public Health, Faculty of Veterinary Medicine, Swedish University of
Agricultural Sciences, PO Box 7028, 75007 Uppsala, Sweden, 134Department of Cell and Developmental Biology, University of
California, Irvine, CA 92697, USA, 135Department of Biology/Center for Cancer Research and Therapeutic Development, Clark
Atlanta University, Atlanta, GA 30314, USA, 136Harvard Medical School/Brigham and Women’s Hospital, Boston, MA 02115,
USA, 137Department of Biosciences and Veterinary Public Health, Faculty of Veterinary Medicine, Swedish University of
Agricultural Sciences, PO Box 7028, 75007 Uppsala, Sweden, 138Baylor College of Medicine, Houston, TX 77030, USA,
139Department of Pharmaceutical Sciences, West Virginia University, Morgantown, WV, 26506, USA 140Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, PO Box 24923, Safat 13110, Kuwait and 141Department of
Surgery, The Ohio State University College of Medicine, The James Comprehensive Cancer Center, Columbus, OH 43210, USA
*To whom correspondence should be addressed. William H.Goodson III, California Pacic Medical Center Research Institute, 2100 Webster Street #401,
San Francisco, CA 94115, USA. Tel:+41 59 233925; Fax: +41 57 761977; Email: whg3md@att.net
Correspondence may also be addressed to Leroy Lowe. Tel:+90 28 935362; Fax: +90 28 935610; Email: leroy.lowe@gettingtoknowcancer.org
Part of the special issue on ‘Assessing the Carcinogenic Potential of Low-Dose Exposures to Chemical Mixtures in the Environment: The Challenge Ahead’
Abstract
Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the
World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers
attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to
mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed
11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical

S258 | Carcinogenesis, 2015, Vol. 36, Supplement 1
disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark
effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/
mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas
59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis
suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety
of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic
research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued
before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization
International Programme on Chemical Safety ‘Mode of Action’ framework should be revisited as it has inherent weaknesses
that are not fully aligned with our current understanding of cancer biology.
Introduction
Cancer is a burden on humanity and among the leading causes
of morbidity and mortality worldwide, with ~14 million new
cases and 8.2 million cancer-related deaths in 2012 (1). In gen-
eral, both genetic and environmental factors play a role in an
individual’s cancer susceptibility (2,3), so there has been a long-
standing emphasis on avoidable ‘lifestyle’ factors (i.e. those
that can be modied to reduce the incidence of the disease) and
a parallel focus on exogenous chemical exposures (e.g. agricul-
tural, occupational and so on) (4). But advances in our under-
standing of the complexity of cancer biology have resulted in
serious critiques of current risk assessment practices related
to exogenous exposures (5) along with calls for an expanded
focus on research that will allow us to evaluate the (potentially
carcinogenic) effects of in-utero exposures and low-level expo-
sures to combinations of chemicals that occur throughout our
lifetime (6,7).
The 2008–09 President’s Cancer Panel Annual Report in the
USA (8) opined that the ‘true burden of environmentally induced
cancer has been grossly underestimated’ (7), whereas Parkin
etal. (9) estimated in a British study that the fraction of cancer
that can now be attributed to both lifestyle and environmen-
tal factors is only 43% (i.e. the underlying cause of 57% of all
cancers is still unexplained). However, an expanded focus on
research that will allow us to evaluate the (potentially carcino-
genic) contribution of low-level exposures to combinations of
chemicals that occur in utero and throughout our lifetime is not
a trivial undertaking.
First of all, the number of chemicals to which we are exposed
is substantial, and many have not been adequately tested.
Christiani (6) cited increased and persistently high incidence
rates of various cancers and called on the National Institutes of
Health to expand their investigation of environmental causes
of cancer noting that ‘Massive gaps exist in toxicologic data,
even in the case of widely used synthetic chemicals. Only about
50% of chemicals classied by the Environmental Protection
Agency (EPA) as “high production volume” have undergone
even minimal testing for carcinogenicity’. But even though the
incidence of cancer attributable to environmental exposures
has not been denitively established (3,6), it remains an impor-
tant focus of our prevention efforts [with credible estimates
from the World Health Organization [WHO] and the IARC sug-
gesting that the fraction of cancers attributable to toxic envi-
ronmental exposures is between 7% and 19%] (10,11).
The possibility that unanticipated low-dose effects (LDE) are
also a factor in environmental carcinogenesis further compli-
cates matters. Vandenberg etal. (12) recently reviewed the accu-
mulating evidence that points to LDE that occur at levels that are
well below those used for traditional toxicological studies. This
review identied several hundred examples of non-monotonic
dose-response relationships (i.e. examples where the relation-
ship between dose and effect is complex and the slope of the
curve changes sign—from positive to negative or vice versa—
somewhere within the range of doses examined). Drawing on
the known actions of natural hormones and selected environ-
mental chemicals examined in cell cultures, animals and epi-
demiology, the authors emphasized that when non-monotonic
dose-response curves occur, the effects of low doses cannot be
predicted by the effects observed at high doses. However, endo-
crine disruption research to this point has been aimed primarily
at chemicals that disrupt developmental processes through a rel-
atively small subset of hormones (e.g. estrogen, androgen, thyroid
and so on), and thus, many commonly encountered chemicals
have not been tested at all for these effects (at environmentally
relevant dose levels) and, to date, mechanisms that relate to car-
cinogenesis have typically not been the focus of these studies.
Generally for chemical risk assessments, toxicity stud-
ies are conducted with individual chemicals in animal mod-
els based on regulatory test guidelines [e.g. Organization for
Economic Co-operation and Development (OECD) test guide-
lines (13)] with a key objective of providing a dose-response
assessment that estimates a point of departure [traditionally
the no-observed-adverse-effect level or the lowest-observed-
adverse-effect level (LOAEL)], which is then used to extrapo-
late the quantity of substance above which adverse effects can
be expected in humans. The no-observed-adverse-effect level,
combined with uncertainty factors (which acknowledge gaps
in the available data), is then used to establish safety criteria
Abbreviations
AhR aryl hydrocarbon receptor
BPA bisphenol A
EMT epithelial-mesenchymal transition
EPA environmental protection agency
HTS high-throughput screening
IARC International Agency for Research on Cancer
IL interleukin
LDE low-dose effects
LOAEL lowest-observed-adverse-effect level
LOEL lowest observed effect level
miRNA microRNAs
4-NP nonylphenol
MXC methoxychlor
NF-κB nuclear factor-κB
PBDE polybrominated diphenyl ethers
PPAR peroxisome proliferator-activated receptor
ROS reactive oxygen species

W.H.Goodson et al. | S259
for human exposure. However, in order to be able to detect
adverse effects utilizing classical toxicological endpoints, dose
selection has historically involved the use of high dose levels
and appropriate dose level spacing to obtain the LOAEL or no-
observed-adverse-effect level thresholds. Techniques such as
linear extrapolation or benchmark dose modeling (14) are then
employed to predict safety margins for low-dose exposures.
This approach to risk assessment depends on the use of appro-
priate and sensitive endpoints, and on valid assumptions for
extrapolation estimates (e.g. dose-response linearity) and cal-
culations, and on the existence of thresholds of effects (15–17).
So when the potential for non-linear dose-response relation-
ships is combined with the possibility of synergism between
and amongst low doses of mixtures of individual chemicals in
the environment, it appears plausible that chemicals that are
not individually carcinogenic may be capable of producing car-
cinogenic synergies that would be missed using current risk
assessment practices.
The complex nature of the biology of cancer adds another
layer of complexity for risk assessment. In a landmark paper
in 1979, Ames (18) noted that damage to DNA appeared to
be a major cause of most cancers and suggested that natu-
ral chemicals in the human diet and the tens of thousands
of man-made chemicals that had been introduced into the
environment in the preceding decades be tested for their abil-
ity to damage DNA. In doing so, he sketched out the difculty
of dealing with complex chemical mixtures and he proposed
the use of rapid mutagenicity assays to identify environ-
mental mutagens and carcinogens. The strategy was sound
at the time, but it led to a scientic and regulatory emphasis
on ‘mutagens as carcinogens’, whereas the issue of complex
environmental mixtures, or carcinogens that are not muta-
gens, was never vigorously pursued. Instead, what followed
was an international quest to nd individual chemicals and
a few well-dened mixtures (e.g. diesel exhaust) that could be
shown to be ‘complete’ carcinogens (i.e. substances that could
cause cancer on theirown).
However, advances in cancer biology have revealed the
limitations of this approach. Armitage and Doll rst laid out
a multistage theory of carcinogenesis in 1954 (19), and by
1990, initiation and promotion were well established as dis-
crete steps in the evolution towards malignancy, along with
the inuence of ‘free radicals’, proto-oncogenes, oncogenes,
epigenetic mechanisms and other synergistic or antagonistic
factors (20). In 2000, Hanahan etal. (21) gave structure to this
rapidly growing eld of research with the proposal that ‘the
vast catalog of cancer cell genotypes [could be organized into]
a manifestation of six essential alterations in cell physiology
that collectively dictate malignant growth’. They called these
alterations the Hallmarks of Cancer, dened as ‘… acquired
capabilities’ common to most cancers that ‘… incipient cancer
cells … [must acquire to] enable them to become tumorigenic
and ultimately malignant.’ The hallmarks delineated at the
time were as follows:
• Self-sufciency in growth signals (later renamed proliferative
signaling)—cancer cells grow at a seemingly unlimited rate.
• Insensitivity to antigrowth signals (evading growth suppres-
sors)—cancer cells are not subject to antigrowth signals or
withdrawal of normal growth signals.
• Evading apoptosis (resisting cell death)—cancer cells avoid the
usual process whereby abnormal or redundant cells trigger
internal self-destroying (as opposed to cell death) mecha-
nisms.
• Limitless replicative potential (enabling replicative immortal-
ity)—cancer cells do not senesce (or age) and die after a lim-
ited number of cell divisions.
• Sustained angiogenesis (inducing angiogenesis)—cancer cells
elicit new blood vessels to sustain growth.
• Tissue invasion and metastasis (activating invasion and
metastasis)—in situ or non-invasive cancers, e.g. ductal carci-
noma in situ in the breast or carcinoma in situ in colon polyps,
grow into pre-existing spaces but invasive tumors must cre-
ate a space to expand into normal tissue.
From this perspective risk assessments based on limited ‘mode
of action’ information, assumptions of linear dose-response
relationships and a focus on individual chemicals (as complete
carcinogens) appeared to be inadequate to estimate human can-
cer risks. So in 2005, a scientist at the United States EPA called
for a shift in risk assessment practices that would move the eld
towards the development of biomarkers directly related to the
pathways found within the Hallmarks of Cancer framework (22).
The Hallmarks of Cancer framework was subsequently revis-
ited by Hanahan et al. (21) and expanded to encompass addi-
tional areas suggested by subsequent cancer research (23). This
expansion included the following:
• Two enabling characteristics:
• Genome instability and mutation, which allows changes in one
cell to pass to daughter cells through mutation or epigenetic
changes in the parent cell DNA.
• Tumor-promoting inammation, which helps cancer cells grow
via the same growth signals normal cells provide to each
other during wound healing and embryonic growth; inam-
mation further contributes to the survival of malignant cells,
angiogenesis, metastasis and the subversion of adaptive
immunity (24).
• Two ‘emerging’ hallmarks:
• Avoiding immune destruction whereby tumor cells avoid
immune surveillance that would otherwise mark them for
destruction.
• Dysregulated metabolism, one of the most recognizable fea-
tures of cancer; its exclusion from the original list of hall-
marks (21) probably represented a signicant oversight, as it
constitutes one of the earliest described hallmarks of cancer
(25,26). It is needed to support the increased anabolic and cat-
abolic demands of rapid proliferation and is likely an enabler
of cancer development and its other associated hallmarks.
Unfortunately, risk assessment practices that are currently used
to assess the carcinogenic potential of chemicals have changed
very little (despite the vast literature that now underpins the
main tenants of the Hallmarks of Cancer framework). For exam-
ple, a chemical that disrupts DNA repair capacity might prove
to be non-carcinogenic at any level of exposure (when tested on
its own), but that very same chemical may have the potential to
be an important contributor to carcinogenesis (e.g. in the pres-
ence of mutagens that cause DNA damage). Similarly, a chemical
that has immuno-suppressive qualities may not be carcinogenic
on its own, but if it acts to suppress the immune response, it
may contribute to carcinogenesis (by dismantling an important
layer of defense) in the presence of other disruptive chemicals.
Considering the multistep nature of cancer and the acquired
capabilities implied by each of these hallmarks, it is therefore
a very small step to envision how a series of complementary
exposures acting in concert might prove to be far more carci-
nogenic than predictions related to any single exposure might
suggest (see Figure 1). Interacting contributors need not act

S260 | Carcinogenesis, 2015, Vol. 36, Supplement 1
simultaneously or continuously, they might act sequentially or
discontinuously. So a sustained focus on the carcinogenicity of
individual chemicals may miss the sorts of synergies that might
reasonably be anticipated to occur when combinations of disrup-
tive chemicals (i.e. those that can act in concert on the key mech-
anisms/pathways related to these hallmarks) are encountered.
To address the biological complexity issue associated with
chronic diseases, the EPA and other agencies have begun to pursue
risk assessment models that incorporate biological information.
This is the basis of the Adverse Outcome Pathway concept, a con-
struct that is gaining momentum because it ties existing knowl-
edge of disease pathology (i.e. concerning the linkage between
a direct molecular initiating event and an adverse outcome at a
biological level of organization) to risk assessment (27,28). This
line of thinking inspired a recent initiative by the EPA, where
the agency tested a proposal for characterizing the carcinogenic
potential of chemicals in humans, using in-vitro high-through-
put screening (HTS) assays. The selected HTS assays speci-
cally matched key targets and pathways within the Hallmarks
of Cancer framework. The authors tested 292 chemicals in 672
assays and were successfully able to correlate the most disrup-
tive chemicals (i.e. those that were most active across the vari-
ous hallmarks) with known levels of carcinogenicity. Chemicals
were classied as ‘possible’/‘probable’/‘likely’ carcinogens or des-
ignated as ‘not likely’ or with ‘evidence of non-carcinogenicity’
and then compared with in-vivo rodent carcinogenicity data in
the Toxicity Reference Database to evaluate their predictions. The
model proved to be a good predictive tool, but it was developed
only as a means to help the EPA prioritize many untested indi-
vidual chemicals for their carcinogenic potential (i.e. in order to
establish priorities for individual chemical testing (29)).
What is still needed, is an approach employing the Hallmarks
of Cancer framework that can be used to identify priority
mixtures (i.e. those with substantive carcinogenic potential).
Without a way to anticipate the carcinogenicity of complex
mixtures, an important gap in capability exists and it creates
a signicant weakness in current risk assessment practices.
Countries around the globe have made a signicant investment
in the regulatory infrastructure and risk assessment practices
that protect us from unwanted exposures to harmful chemicals
and carcinogens, so we wanted to review the biology of cancer
to map out the challenges associated with the development of
an approach that would help us assess the carcinogenic poten-
tial of low-dose exposures to chemical mixtures in the envi-
ronment. Such an approach was seen as a reasonable step to
provide impetus for progress in this area of research and ulti-
mately to inform risk assessment practices worldwide.
Materials and methods
In 2012, the non-prot organization ‘Getting to Know Cancer’ instigated
an initiative called ‘The Halifax Project’ to develop such an approach using
the ‘Hallmarks of Cancer’ framework as a starting point. The aim of the
project was to produce a series of overarching reviews of the cancer hall-
marks that would collectively assess biologically disruptive chemicals (i.e.
chemicals that are known to have the ability to act in an adverse manner
on important cancer-related mechanisms, but not deemed to be carcino-
genic to humans) that might be acting in concert with other seemingly
innocuous chemicals and contributing to various aspects of carcinogen-
esis (i.e. at levels of exposure that have been deemed to be safe via the
traditional risk assessment process). The reviews were to be written by 12
writingteams.
The writing teams were recruited by Getting to Know Cancer circu-
lating an email in July 2012 to a large number of cancer researchers, ask-
ing about their interest in the project. Respondents were asked to submit
personal details through a dedicated webpage that provided additional
project information. Atotal of 703 scientists responded to the email,
and from that group, 11 team leaders were selected to lead reviews of
each hallmark (10 Hallmarks plus an 11th team to consider the tumor
microenvironment as a whole) and one leader for the cross-validation
Figure1. Disruptive potential of environmental exposures to mixtures of chemicals. Note that some of the acquired hallmark phenotypes are known to be involved in
many stages of disease development, but the precise sequencing of the acquisition of these hallmarks and the degree of involvement that each has in carcinogenesis
are factors that have not yet been fully elucidated/dened. This depiction is therefore only intended to illustrate the ways in which exogenous actions might contribute
to the enablement of these phenotypes.

W.H.Goodson et al. | S261
team (see below). Writing group leaders were asked to form individual
teams drawn from the pool of researchers who expressed interest in
the project and from their own circles of collaborators. Leaders were
encouraged to engage junior researchers as well. Team leaders received
project participation guidelines and ongoing communication from the
project leaders, L.Lowe and M.Gilbertson. Each team included: a lead
author with a published expertise in the hallmark area; domain experts
who assisted in the production of the descriptive review of the biology;
environmental health specialists (e.g. specialists in toxicology, endo-
crine disruption, or other related disciplines) and support researchers.
Each writing team was charged to describe the hallmark, its systemic
and cellular dysfunctions and its relationships to other hallmarks. Apri-
ority list of relevant (i.e. prototypical) target sites for disruption was to
be developed by the team and a list of corresponding chemicals in the
environment that have been shown to have the potential to act on those
targets was requested, along with a discussion of related issues and future
research needed (in the context of project goals).
Selection of target sites for disruption
A ‘target’ was broadly dened as a procarcinogenic disruption at the sys-
tem level (e.g. the hypothalamic–pituitary–gonadal axis), organ level, tis-
sue level or cellular level. It was assumed from the outset that a project
intended to develop an approach for the assessment of the carcinogenic
potential of low-dose exposures to chemical mixtures in the environment
would encounter a practical upper limit to the number of potential targets
that any given team could realistically review. Therefore, each team was
asked to identify up to 10 relevant targets for their domain (bearing in
mind that each target would also serve as a starting point for the identi-
cation of a disruptive environmental chemical that had already shown
a demonstrated ability to act on that target). In theory, it was understood
that this could lead to as many as 110 targets for the entire project, and
as the teams were also asked to select one disruptive chemical for each
target, a maximum of 110 chemicals.
In this phase, teams were asked to focus on specic gene changes
common to many cancers as identied by The Cancer Genome Project
(30) in order to estimate how the function of specic genes might be
altered, not by specic gene mutations, but rather either by direct
action or by epigenetic changes that might lead to the same functional
ends. Most of these pathways and processes are found within both
the hallmarks of cancer and the genomic frameworks, so teams were
asked to evaluate both models and consider non-mutagenic/epigenetic
pathways of interference as well (given that epigenetic changes such as
DNA methylation and histone acetylation are relevant for cancer and
often inducible by chemicals and may be transmitted to daughter cells).
Selection of disruptive chemicals
Teams were then asked to identify ‘prototypical’ chemicals in the envi-
ronment that had demonstrated an ability to act on the selected targets.
During workshops in Halifax, the teams settled on the following criteria
to guide their choices:
• Chemicals should be ubiquitous in the environment because we
wanted the broadest possible relevance for the general popula-
tion.
• Chemicals should selectively disrupt individual targets such as
specic receptors, specic pathways or specic mechanisms. Hypo-
thetically speaking, a chemical could affect more than one pathway,
receptor and so on; indeed, we expected that most chemicals would
likely exert a multitude of actions. However, we used the term ‘selec-
tively disruptive’ to encourage teams to avoid choosing mutagens
that are randomly destructive in their action (i.e. unpredictable and
capable of producing varying types of damage across a wide range
of pathways).
• Chemicals should not be ‘lifestyle’ related, such as those encountered
from tobacco, poor diet choices (e.g. red meats, French fries, lack of
fruit and vegetables and so on), alcohol consumption, obesity, infec-
tions (e.g. human papillomavirus) and so on.
• Chemicals should not be known as ‘carcinogenic to humans’ (i.e. not
IARC Group1, carcinogens).
The choice to focus on environmental pollutants in this project was
intentionally restrictive. Countries around the globe have made sig-
nicant investments in regulatory infrastructure and risk assessment
practices to protect us from unwanted exposures to harmful chemicals
and carcinogens. Therefore, we focused on chemicals that are com-
monly encountered in the environment. Primarily, we wanted to gen-
erate insights that would be valuable for cancer researchers who are
specically interested in environmental chemical exposures to chemical
mixtures and/or those who are focused on risk assessment practices in
general.
Dose-response characterizations andLDE
Given that much of the evidence in the toxicological literature that docu-
ments the disruptive actions of various chemicals has been produced
under a wide range of differing experimental circumstances, we wanted
to assess the quality and relevance of data that were gathered for expo-
sures discussed in this review. Specically, for each chemical selected
and each mechanism identied, teams were additionally tasked to iden-
tify any dose-response characterization results and/or relevant low-dose
research evidence that might exist. The term ‘low dose’ was dened using
the European Food Safety Authority denition (i.e. responses that occur at
doses well below the traditional lowest dose of 1 mg/kg that are used in
toxicology tests) and the denition for ‘LDE’ was based on the EPA deni-
tion (31)—as follows:
Any biological changes occurring
(a) in the range of typical human exposuresor
(b) at doses lower than those typically used in standard testing proto-
cols, i.e. doses below those tested in traditional toxicology assess-
ments (32),or
(c) at a dose below the lowest dose for a specic chemical that has
been measured in the past, i.e. any dose below the lowest observed
effect level (LOEL) or LOAEL (33)
(d) occurring at a dose administered to an animal that produces blood
concentrations of that chemical in the range of what has been
measured in the general human population (i.e. not exposed oc-
cupationally, and often referred to as an environmentally relevant
dose because it creates an internal dose relevant to concentrations
of the chemical measured in humans) (34,35).
Each team was then asked to categorize each chemical by using one of
ve possible categories (to determine the relevance and relative strength
of the underlying evidence for each of the chemicals being considered).
The categories were as follows: (i) LDE (i.e. levels that are deemed relevant
given the background levels of exposure that exist in the environment);
(ii) linear dose-response with LDE; (iii) non-linear dose-response with LDE;
(iv) threshold (i.e. this action on this mechanism/pathway does not occur
at low-dose levels) and (v) unknown. Additional details of the descriptions
for each of these categories are shown in Table1.
Cross-hallmark relationships
In recognition of the network of signaling pathways involved and the
degree of overlap/interconnection between the acquired capabilities
described in each hallmark area, the project included a cross-validation
step to create a more complete mapping of the actions that might be
anticipated as the result of an action on the target sites identied or by
the disruptive effects of the chemicals selected. Given the diversity of the
targets involved in the 11 hallmark areas, it was anticipated that inhibit-
ing or stimulating a target relevant to one hallmark may have an impact
on other targets that are relevant, especially if both are linked via signal-
ing pathways.
Accordingly, the cross-validation team conducted additional back-
ground literature review of submitted targets and chemicals from each
writing team, searching for evidence to identify cross-hallmark activity.
Each potential target-hallmark or approach-hallmark interaction was
assessed to determine whether the inhibition or activation of each tar-
get and the corresponding biological activity of each chemical might
reasonably be expected to have either a procarcinogenic or anticarcinogenic
effect on key pathways/processes in the various hallmark areas.

S262 | Carcinogenesis, 2015, Vol. 36, Supplement 1
Table1. Dose-response characterization
Review team Chemical name Disruptive action on key mechanism/pathway Low-dose effect (LDE, LLDE, NLDE, threshold, unknown)
Angiogenesis Diniconazole Vascular cell adhesion molecule and cytokine signaling Threshold (H-PC) (36)
Ziram Vascular cell adhesion molecule and cytokine signaling Threshold (H-PC) (36,37)
Chlorothalonil Thrombomodulin, vascular proliferation and cytokine signaling Unknown (H-PC) (36), NLDE (A-in vivo) (38)
Biphenyl Angiogenic cytokine signaling Unknown (H-PC) (36)
Tributyltin chloride Vascular cell proliferation and adhesion molecule signaling Unknown (H-PC) (36)
Methylene bis(thiocyanate) Plasminogen activating system and cytokine signaling Unknown (H-PC) (36)
HPTE Vascular cell adhesion molecule and cytokine signaling Unknown (H-PC) (36), threshold (A-Ia) (39), LDE (A-Ia) (40)
PFOS Angiogenic cytokine signaling Threshold (H-PC) (36), LDE (H-CL) (41)
Bisphenol AF Matrix metalloproteinase expression and estrogen receptor signal-
ing
Unknown (H-PC) (36)
C.I.solvent yellow 14 AhR and hypoxic signaling Unknown (H-PC) (36)
Dysregulated metabolism Cypermethrin AR and ER expression, reduction of ATP and mitochondrial en-
zymes, mitochondrial membrane potential
LLDE (A-I) (42), NLDE (A-I) (42), NLDE (H-CL) (36,43,44)
Acrolein p53 activation, DNA repair inhibition, PERK phosphorylation, mito-
chondrial dysfunction, cell survival
LLDE (A-I, A-CL, H-PC, H-CL) (45–50), NLDE (49), threshold (46)
Rotenone Cell cycle, DNA damage response, proliferation, differentiation,
mitochondria
LLDE (H-CL) (51–53), NLDE (H-CL) (51,53), unknown (H-CL,H-
PC) (36)
Copper p53 activation, p21 up-regulation, cell viability LLDE (H-CL) (54–56)
Nickel Neutrophil apoptosis, E-cadherin regulation, matrix metallopepti-
dase (MMP) production
LLDE (H-CL) (57), NLDE (H-CL) (58), Threshold (H-CL) (58)
Cadmium p53-dependent apoptosis, cell proliferation LLDE (H-CL) (59), threshold (H-CL) (60)
Diazinon AChE activity, neuronal cytotoxicity Unknown (A-PC) (61), LLDE (H-CL) (62), threshold (H-CL) (36)
Iron KRAS mutations LLDE (A-I) (63)
Malathion Lymphocyte Mutations, Cytotoxicity Unknown (H-PC, H-E) (36,64)
Tissue invasion and
metastasis
BPA MMP-2 and MMP-9 expression, increased migration, invasion, EMT,
oxidative stress, ER signaling
LDE (H-CL) (65,66), threshold (H-CL, H-PC) (36)
Hexacholorobenzene Activation of c-Src, HER1, STAT5b and ERK1/2 signaling LLDE (H-CL, A-I) (67)
Sulfur dioxide MMP-9 expression Unknown (A-PC) (68)
Phthalates MMP-2 and MMP-9 expression LDE (H-CL) (66),Unknown (H-CL, H-PC) (36)
Iron ROI generation, NF-κB activation, uPA expression Unknown (H-CL) (69)
Biorhythms/melatonin GSK3β activation, EMT regulation Unknown (H-CL, H-E) (70,71)
Resistance to cell death BPA Inhibition of GJIC, activation of mTOR pathway, down-regulation of
p53, p21 and BAX, binding to ER-α, weakly binds to TH receptor
and AR, activation of ERK1/2 and p38
NLDE(H-CL, A-CL) (72–74)Threshold (H-CL, H-PC) (36)
Dibutyl phthalate Activation of PPAR-α, inhibition of GJIC, expression of cyclin D and
cdk-4, activation of AhR/HDAC6/c-Myc pathway
NLDE (H-CL) (75), unknown (H-CL, H-PC) (36)
Chlorothalonil Up-regulation of ErbB-2 tyrosine kinase and MAP kinase, aromatase
inhibitor
Threshold-based (i.e. non-linear) (A-I) (76), unknown (H-PC)
(36), threshold (H-CL) (36)
Lindane Induction of MAPK/ERK pathways Threshold-based (i.e. non-linear) (A-I) (77), threshold (H-CL)
(36)
Dichlorvos Expression of p16, Bcl-2 and c-myc LLDE (A-I) (78), threshold (H-CL) (36)
MXC Binding to ER-α receptor, up-regulation of cyclin D1, down-regula-
tion of p21
LLDE (H-CL, A-CL) (75,79), unknown (H-PC) (36), threshold
(H-CL) (36)
Oxyuorfen Expression of Cyp2b10 and Cyp4a10 transcripts (markers of PPAR-α
activation)
Threshold (A-I) (80), unknown (H-CL, H-PC) (36)
DEHP Activation of PPAR-α, inhibition of GJIC Threshold-based (i.e. non-linear) (A-I) (81)
Linuron Hypersecretion of LH, inhibition of GJIC Unknown (H-CL) (82)

W.H.Goodson et al. | S263
Review team Chemical name Disruptive action on key mechanism/pathway Low-dose effect (LDE, LLDE, NLDE, threshold, unknown)
Replicative immortality Nickel-derived compounds, (e.g.
nickel chloride)
Epigenetic silencing of p16 LLDE (H-CL, A-PC) (83)
Diethylstilbestrol Allelic loss and point mutation in ETRG-1 gene LLDE (A-I) (84)
Reserpine Epigenetic modications Unknown (A-PC) (85), threshold (H-CL) (36)
Phenobarbital Reduces expression of the CDKN1A product p21, CAR activation LLDE (A-I) (86,87)
Acetaminophen Cellular energy loss, mitochondrial damage, telomerase activation LDE (H-CL, A-I, A-CL) (88–92)
Cotinine Telomerase activation LLDE (H-PC) (93)
Nitric oxide p53 inactivation LLDE (H-PC, H-CL, A-CL, A-I) (94)
Na-selenite p53 promoter methylation LLDE (A-CL, A-I) (95,96)
Lead p53 inactivation LLDE (H-PC, H-CL, A-CL, A-I) (94)
Sustained proliferative
signaling
BPA Estrogen receptor activation, cell cycle/senescence LLDE (A-I, H-CL, H-E) (12,97), NLDE (A-I) (98,99), threshold (H-
CL) (36)
Cyprodinil Increased proliferation signaling, AhR activation Unknown (H-PC, H-CL) (36,100,101), threshold (H-CL) (36)
Imazalil AR signaling NLDE (A-I) (102,103), threshold (H-CL, H-PC) (36)
Maneb Nitric oxide signaling Unknown (A-CL, H-CL, H-PC) (36,104,105)
Methoxyclor ER signaling Threshold (H-CL) (36), LDE (A-I) (106,107), NLDE (A-I) (108)
PFOS Nuclear hormone receptors Threshold (H-CL) (36), LLDE (A-I) (109,110)
Phthalates CAR, ER signaling Unknown (H-CL) (36), LDE (A-I) (111–113)
Phosalone Increased proliferation, PXR signaling Unknown (H-PC, H-CL) (36,114,115)
PBDEs ER signaling LDE (A-I) (116,117)
Prochloraz ER signaling LDE (A-I) (118,119)
Trenbolone acetate Insulin-like growth hormone-1 and AR signaling Unknown, LDE (A-I, H-CL, H-E) (120,121)
Tumor-promoting inamma-
tion
BPA Immune cell proliferation, proinammatory cytokine induction Threshold (H-PC) (36), LDE (A-I, H-CL, H-E) (122–126)
Phthalates Immunomodulation of macrophages, lymphocytes, eosinophils and
neutrophils
Unknown (H-PC, H-CL, H-E) (36,127)
PBDEs Induction of pro-inammatory cytokines (IL-6, IL8 and CRP), inhibi-
tion of anti-inammatory cytokines (IL-10)
Threshold (H-PC, H-CL) (128–131)
Atrazine Immunomodulation of T cell and B cells, proinammatory cy-
tokines
Unknown (H-PC, A-I) (36,132,133)
Vinclozolin Proinammatory cytokine induction, NF-κB activation Unknown (H-PC, A-I) (36,134–136)
4-NP Proinammatory cytokine induction, NF-κB activation, iNOS induc-
tion
Unknown (A-CL, H-CL, H-PC) (36,137,138)
Immune system evasion Pyridaben Chemokine signaling, TGF-β, FAK, HIF-1a, IL-1a pathways Unknown (H-CL, H-PC, A-CL) (36,139,140), threshold (A-I) (141)
Triclosan Chemokine signaling, TGF-β, FAK, IL-1a pathways Threshold (H-CL, H-PC, A-I) (36,142–144), LDE (A-I, H-CL)
(145,146)
Pyraclostrobin Chemokine signaling, TGF-β, IL-1a pathways Unknown (H-CL, H-PC) (36)
Fluoxastrobin Chemokine signaling, EGR, HIF-1a, IL-1a pathways Unknown (H-CL, H-PC) (36)
BPA Chemokine signaling, TGF-β pathway Threshold (H-PC) (36), LDE (A-I) (12), NLDE (H-CL) (147), NLDE
(A-CL) (148–151), NLDE (A-I) (152–155)
Maneb PI3K/Akt signaling, chemokine signaling, TGF-β, FAK, IGF-1, IL-6,
IL-1a pathways
Unknown (H-CL, H-PC) (36,139,156–158), LDE (A-I) (159),
threshold (A-I) (139,160), threshold (A-CL, A-I) (161)
Table1. Continued

S264 | Carcinogenesis, 2015, Vol. 36, Supplement 1
Review team Chemical name Disruptive action on key mechanism/pathway Low-dose effect (LDE, LLDE, NLDE, threshold, unknown)
Evasion of antigrowth
signaling
DDT Induces MDM2, cyclin D1, E2F1 expression, disrupts gap junctions NLDE (A-I, H-CL, A-CL) (162–164)
Chlorpyrifos Increases proliferation LDE (H-CL, H-PC) (165,166)
Folpet Disrupts G1–S checkpoint kinases, down-regulates p53, promotes
proliferation
LDE(A-C) (167)
Atrazine Induces estrogen production and proliferation LDE(H-CL, A-I) (168–170)
BPA Reduced p53, reduced connexin 43 expression, increased prolifera-
tion
NLDE (H-CL, A-I) (171–174)
Tumor microenvironment Nickel ROS and cellular stress NLDE (A-I) (175)
BPA IL-6 expression, improper DC maturation and polarization, ROS
production
LLDE (A-I) (176), NLDE (A-I) (176)
Butyltins (such as tributyltin) NK cell inhibition LDE (A-I) (177)
MeHg Chronic oxidative stress LDE (H-PC, H-CL) (178,179)
Paraquat Chronic ROS production, cellular stress Unknown (A-I) (180)
Genome instability Lead Dysfunctional DNA repair, defect in telomere maintenance Unknown (A-CL) (181–183), threshold (H-CL, H-E) (184,185)
Acrylamide Inactivation of DNA repair proteins/enzymes Unknown (A-CL, A-I, H-CL) (186,187)
Quinones Affect free cysteine residues in catalytic center of DNA methyl-
transferases (DNMT)
Unknown (A-CL) (188)
Nickel Affect enzymes that modulate post-translational histone modica-
tion
LDE (H-E) (189,190), LDE (A-CL, H-CL) (191)
BPA Epigenetic changes via interactions with miRNA Threshold (H-PC) (192)
Alloy particles (tungsten/nickel/
cobalt)
Disruption of DNA damage/redox signaling involving Nrf, NF-κB,
Egr, and so on
LDE (A-I) (193)
Titanium dioxide NPs Decreased NADH levels and impaired mitochondrial membrane
potential and mitochondrial respiration, ROS generation
Unknown (A-PC) (194)
Benomyl Spindle defects leading to formation of micronuclei Threshold (H-CL) (195), Threshold (A-CL) (196)
Carbon nanotubes Spindle defects leading to formation of micronuclei LLDE (A-CL) (197,198), unknown (A-I) (198)
Each chemical in the table was categorized by using one of ve possible categories (to determine the relevance and relative strength of the underlying evidence for each of the chemicals being considered)—as follows: (1) LDE
(low-dose effect)—the ability of this chemical to exert this particular effect is not well characterized at a range of dose levels, but the evidence suggests that this chemical can exert this effect at low-dose levels (i.e. levels that
are deemed relevant given the background levels of exposure that exist in the environment and as further dened below). (2) LLDE (linear dose-response with low-dose effects)—the ability of this chemical to exert this particular
effect is well characterized at a range of dose levels and the evidence suggests that a linear dose-response relationship exists with effects at low-dose levels being evident (i.e. levels that are lower than the LOEL/LOAEL or thresh-
old and deemed relevant given the background levels of exposure that exist in the environment). Note: a linear dose-response model implies no threshold. Effects at low doses are the same as at higher doses even if at a lesser
extent. The effect is directly proportional to the dose. (3) NLDE (non-linear dose-response with low-dose effects)—the ability of this chemical to exert this particular effect is well characterized at a range of dose levels and the
evidence suggests that a non-linear dose-response relationship exists with exaggerated effects at low-dose levels being evident (i.e. levels that are lower than the LOEL/LOAEL or threshold and deemed relevant given the back-
ground levels of exposure that exist in the environment). Note: a non-linear dose-response with low-dose effect implies that the effect does not vary according to the dose of the agent. The effect at low doses may be the same
as at the higher doses or different. The non-linear dose-response may have or not have a threshold. It is represented by a sigmoid curve. The non-linear dose-response at low doses may be a non-monotonic dose-response. (4)
Threshold—the ability of this chemical to exert this particular effect is well characterized at a range of dose levels, and a threshold has been established for this chemical that suggests that this action on this mechanism/path-
way does not occur at low-dose levels (i.e. levels that are lower than the threshold and deemed relevant given the background levels of exposure that exist in the environment). (5) Unknown—although the ability of this chemi-
cal to exert this particular effect has been shown at higher dose levels, this effect is not well characterized at a range of dose levels, so a LOEL /LOAEL or a threshold has not been determined for this chemical and there is no
evidence showing that this chemical exerts this action at low-dose levels (i.e. levels that are lower than the LOEL/LOAEL or threshold and deemed relevant given the background levels of exposure that exist in the environment).
A-I, in-vivo animal models; A-CL, animal cell lines; A-PC, animal primary cells; H-PC, human primary cells; H-CL, human cell lines; H-E, human epidemiological studies. With respect to the human primary cell (H-PC) data from
ToxCast (36): unknown signies that the compound was tested across a range of doses and showed statistically signicant activity against the specied targets at the lowest test concentrations (~0.01µM); therefore, a threshold
could not be established. Threshold in this data set signies that there was no activity against the targets at one or more of the lowest concentrations tested.
aExtrapolated from in-vivo data on the parent compound, MXC.
Table1. Continued

W.H.Goodson et al. | S265
The cross-validation team also sought out controversial interactions
(i.e. mixed indications of hallmark-like effects) and instances where no
known relationship existed. It was our belief that target sites or chemicals
that demonstrated a substantial number of ‘anticarcinogenic’ effects in
other hallmark areas would be less suitable to serve as instigating con-
stituents in the design of carcinogenic mixtures (where procarcinogenic
synergy was being sought).
It is important to note that the cross-validation team was not
given any restrictions for literature selection for this effort, and con-
tributing authors were restricted neither to results from low-dose
testing, nor to that of cancer-related research. This approach was
taken because it was realized at the outset that this sort of breadth
and homogeneity (of low-dose evidence) does not yet exist in the lit-
erature. As a result, the types and sources of data gathered in this
effort varied considerably, resulting in an admixture of reviews and
original studies. Moreover, many studies that were cited in this effort
only considered a chemical’s ability to instigate or promote an action
that mimics a hallmark phenotype in a manner directionally consist-
ent with changes that have been associated with cancer. So, although
we have referred to these actions as procarcinogenic and anticarci-
nogenic, as these changes are frequently neither fixed nor specific
for cancer, the specificity of these changes and implications for car-
cinogenesis cannot and should not be immediately inferred from this
data set. Short-term toxicity and toxic responses—particularly in data
from in-vitro HTS platforms—must be distinguished from truly ‘carci-
nogenic’ long-term changes. In other words, the tabularized results
from this particular aspect of the project were only compiled to serve
as a starting point for future research. Where cross-hallmark effects
were reported (at any dose level and in any tissue type), we wanted
samples of that evidence to share with researchers who might be try-
ing to anticipate the types of effects that might be encountered in
future research on mixtures of chemicals (in a wide range of possible
research contexts).
Results
The results are presented roughly sequenced in a manner that
captures the acquired capabilities found in many/most cancers.
The section begins with two enabling characteristics found in
most cancers Genetic instability and Tumor-promoting inam-
mation, followed by Sustained proliferative signaling and
Insensitivity to antigrowth signals, the two related hallmarks
that ensure that proliferation is unabated in immortalized cells.
These sections are followed by Resistance to cell death and
Replicative immortality, two critical layers of defense that are
believed to be bypassed in all cancers and then by dysregulated
metabolism. Sections on Angiogenesis and Tissue invasion and
metastasis follow and speak to the progression of the disease,
and nally, the Tumor microenvironment and Avoiding immune
destruction sections offer summaries related to the very last
lines of defense that are defeated in most cancers. Additionally,
dose-response characterizations and evidence of LDE are then
presented for all of these areas and the results from the cross-
validation activity are summarized and reviewed.
Genetic instability
The phenotypic variations underlying cancer result from interac-
tions among many different environmental and genetic factors,
occurring over long time periods (199). One of the most important
effects of these interactions is genome instability—loosely dened
as an increased likelihood of the occurrence of potentially muta-
genic and carcinogenic changes in the genome. The term is used
to describe both the presence of markers of genetic change (such
as DNA damage and aneuploidy) and intrinsic factors that per-
mit or induce such change (such as specic gene polymorphisms,
defective DNA repair or changes in epigenetic regulation).
DNA damage—which can be caused by exposure to external
chemicals or radiation, or by endogenous agents such as reactive
oxygen or faulty replication—is an event that can initiate the
multistep process of carcinogenesis (200). Protection is afforded
at different levels; removal of damaging agents before they
reach the DNA, by antioxidant defenses and the phase I/phase
II xenobiotic metabolizing enzymes; a second line of defense,
DNA repair, operating on the damage that occurs despite the
primary protection; and as a last resort, apoptosis (programmed
cell death), disposing of heavily damagedcells.
A clear sign of genome instability is aneuploidy—a deviation
from the normal number of chromosomes (201). Aneuploidy is
a very common feature of human cancers. Another hallmark of
cancer is loss of the normal mechanism of telomere shortening,
which allows abnormal cells to escape senescence, by avoid-
ing the body’s ‘editing’ processes that normally eliminate aging
cells with their accumulated genome aberrations (202,203).
The genes of most signicance for cancer are the (proto)-
oncogenes which, if defective, or abnormally expressed, lead
to uncontrolled cell proliferation; tumor suppressor genes, the
normal products of which tend to switch off replication to allow
repair, and promote cell death if damage is excessive; and genes
such as those involved in DNA repair that can—if faulty—lead
to a ‘mutator phenotype’. Mutated proto-oncogenes and tumor
suppressor genes are found in most if not all cancers and
play key roles in cancer etiology (204–207). Rare mutations in
DNA repair genes greatly increase the risk of cancer (208,209).
However, the evidence for links between common variants of
repair genes and cancer is generally inconclusive (210).
The term ‘epigenetics’ refers to covalent modications of the
DNA (methylation of cytosine in ‘CpG islands’ within regula-
tory regions of genes) or of the histones. These modications
can control gene expression and the pattern of modications
is altered in many cancers (211,212). For instance, hypometh-
ylation of proto-oncogenes can lead to overexpression, which
is undesirable. MicroRNAs (miRNAs) are responsible for specic
down-regulation of gene expression at a post-transcriptional
level, by preventing translation from messenger RNAs. miRNAs
participate in DNA damage responses and some miRNAs are
deregulated in many cancers (213–215).
Mutations in germ and stem cells are potentially more seri-
ous than those in other cells as they are passed to the cells’
progeny within the developing embryo or regenerating tissue
(216,217). There is a presumed survival benet when stem cells
tend to show a particularly stringent maintenance of genome
integrity through cell cycle regulation and enhanced responses
to DNA damage (218).
The selected ‘chemical disruptors’ that induce genome
instability include chemicals that not only directly damage DNA
or cause mutations, but act indirectly, via pathways such as DNA
damage signaling, DNA repair, epigenetic regulation or mito-
chondrial function. They include the following:
Metals such as lead, nickel, cobalt and mercury (common
water pollutants) are known to disrupt DNA repair (181,219),
whereas nickel also affects epigenetic histone modication
(189,191) and lead causes defective telomere maintenance
(184,220). Alloy particles, containing tungsten, nickel and cobalt,
can be inhaled and disrupt redox signaling (193,221). Titanium
dioxide nanoparticles are also common in many consumer prod-
ucts and foods and have been reported to disrupt mitochondrial
function and increase oxidative stress, as well as inhibit DNA
repair and disrupt mitosis (194,222,223).
Acrylamide occurs in many fried and baked food products,
and (apart from the well-known DNA adduct formation) can

S266 | Carcinogenesis, 2015, Vol. 36, Supplement 1
inactivate many critical proteins by binding sulfhydryl groups
(186).
Bisphenol A (BPA) is a plasticizer used for manufactur-
ing polycarbonate plastics and epoxy resins, and it can leach
from plastics into food and water. It is implicated in disruption
of DNA methylation, histone acetylation and disturbance of
miRNA binding (192,224,225), redox signaling (226) and induc-
tion of micronuclei through spindle defects in mitosis (227).
The fungicide benomyl is metabolized to carbendazim;
both are classied as possible human carcinogens at present.
The route of exposure is most likely ingestion via residues in
crops. Benomyl disrupts the microtubules involved in the func-
tion of the spindle apparatus during cell division, leading to
production of micronuclei (Frame,S.R. etal., unpublished report,
Schneider,P.W. etal., unpublished report, (228)).
Halobenzoquinones are disinfection by-products in chlo-
rinated drinking water (229). Quinones are electrophilic com-
pounds, known to react with proteins and DNA to form adducts.
These electrophylic chemicals can interact with functional thiol
groups via Michaelis–Menton type addition, causing modica-
tion of enzymes involved in methylation and demethylation
(188). This mechanism might be shared by other xenobiotics
that increase reactive oxygen species(ROS).
Human exposure to nano-sized materials used in cosmetics,
biomedical compounds, textiles, food, plastics and paints has
increased not only in a conscious way but also passively by the
leakage of nanomaterials from different objects. Nanoparticles
can induce genome instability via mitochondrial-related apop-
tosis (230), decreased DNA repair (222,230,231), hypoacetyla-
tion of histones (232), disruption of DNA methylation (231),
up-regulation of miRNA (233), reducing telomerase activity
(220) and—more specically for carbon nanotubes—interact-
ing with components of the mitotic spindle during cell division
or interacting with proteins directly or indirectly involved in
chromosome segregation (197,234). Nano-sized materials can
also produce inammation and alteration of the antioxidant
defenses that can lead to genome instability.
Tumor-promoting inammation
One of the earliest hypothesized causes of tumors subsequently
supported experimentally was the irritation hypothesis pro-
posed by Virchow. Although it was recognized initially that injury
alone was insufcient for carcinogenesis, it was also recognized
that ‘irritation may have an accessory or predisposing inuence
in tumor formation, and that it may be enough nally to upset
the balance of a group of cells which for some other reason were
already hovering on the brink of abnormal growth’ (235). Indeed,
it is now recognized that inammatory responses, similar to
those associated with wound healing or infection, support the
development of invasive carcinomas by altering the microen-
vironment in favor of proliferation, cell survival, angiogenesis
and tumor cell dissemination while also disrupting antitumor
immune surveillance mechanisms. In other words, inamma-
tion plays a critical role in tumorigenesis (23,24).
Inammation is an immediate and necessary host defense
mechanism in response to infection or tissue injury by noxious
stimuli. In tumor-associated inammation, both the epithelium
and the immune cells express receptors that signal the activa-
tion and production of a wide array of biologically active proteins
most analogous to an unhealed wound. The sustained or uncon-
trolled release of potent and reactive molecules such as prosta-
glandins, cytokines, ROS and chemokines from both the tumor
cell and the microenvironment constituents lead to progressive
genomic instability, alterations in the integrity and function of
the microenvironment including alterations in the vasculature
(e.g. vascular hyperpermeability, neovascularization and angio-
genesis), as well as alterations in local immune dynamics. The
cellular and molecular mechanisms include a diverse array of
immune- and tumor-cell-derived effector molecules such as the
proinammatory reactive oxygen and nitrogen species, a num-
ber or cytokines, chemokines as well as cyclooxygenase-2 and
its product, prostaglandin E2.
In general, there is a paucity of experimentation, and when
present, inconsistent ndings for the role of environmental
chemicals as proinammatory molecules and more so for a pro-
inammatory action as a co-factors in carcinogenesis. However,
some recent studies provide a credible mechanistic basis, par-
ticularly early life exposures that might act by disrupting the
immune cell balance toward inammation, and that manifest in
adulthood. One example is BPA, one of the most abundant and
best studied environmental endocrine disruptors, and its con-
troversial role as an immune disruptor. Specically, studies in
male rats found that early life BPA exposure leads to the devel-
opment of prostate intraepithelial neoplasia (a prostate cancer
precursor lesion) through a pathological process that includes
BPA-dependent epigenetic reprogramming of genes involved in
the development of lateral prostate inammation in adulthood
(236,237).
This work in prostate is complemented by a much more
extensive study of BPA effects on immune cell components,
particularly the T-cell compartment, demonstrating that BPA
acts as an immune disruptor by promoting ‘immune’ cell pro-
liferation though the exact nature of the effect on specic cells
of the immune system is poorly delineated. Most interesting is
the work by Yan etal. (122), who reported ndings suggesting
that the timing of BPA exposure during development (prenatally,
early life or adult) alters the effect of BPA on regulatory T cells.
BPA actions also map over to the effects on the immune sys-
tem including the promiscuity of BPA for a number of nuclear
receptors relevant to immune cells such as the estrogen recep-
tor and the aryl hydrocarbon receptor (AhR). As well, bulky BPA
analogs may act as antagonists of members of the peroxisome
proliferator-activated receptor (PPAR) family, an important fam-
ily of nuclear receptors with potent anti-inammatory function
(238,239). Effects on the PPAR nuclear receptors may also explain
inammation-associated phenotypes observed with exposures
to certain phthalates and nonylphenol (4-NP).
A second example is the reported immunotoxic effects of
atrazine (6-chloro-N-ethyl-N-(1-methylethyl)-1,3,5-triazine-2,4-
diamine) (240), a chemical that is the most commonly detected
triazine herbicide in USA soil and water. Atrazine is banned
by the European Union and drinking water exposures are sup-
posed to be limited in the USA to <3µg/l (although exposures
exceed this limit regularly), but the use of this chemical is high
and increasing in Asia and other countries. Thus, atrazine is an
important pesticide to which humans are exposed. Atrazine
exhibits weak mutagenicity and low oncogenic properties, but
research by a number of authors is emerging that suggests that
immune system disruption might be a concern (132,240,241).
Although the majority of work on atrazine has been focused
on its endocrine disrupting properties, there is also evidence
to support immunotoxicity including effects on T-lymphocytes
composition with oral dosing (242,243), modulation of nitric
oxide production (244) and potential generation of ROS (245,246).
The local production of reactive nitrogen species and ROS
by mast cells and macrophages are among the better stud-
ied immune modulatory molecules for which recent evidence

W.H.Goodson et al. | S267
supports important roles both in the tumor microenvironment
and in the tumor progression (247–249). Notably, these reactive
species trigger oxidative/nitrosative modications, which can
initiate redox signaling that tightly modulates the inammatory
response in a manner that is highly relevant for carcinogenesis
(250,251).
We also looked at polybrominated diphenyl ethers (PBDEs)
and their effects on inammatory cytokines. Peltier etal. (128)
recently found that placental explants treated with a mix-
ture of the cogeners BDE-47, -99 and -100 and then exposed
to Escherichia coli were ‘reprogrammed’ toward a proinam-
matory response (increased IL-1β and tumor necrosis factor
α) and away from the expected anti-inammatory response
(decreased IL-10) compared with untreated placenta. Although
these studies are preliminary, chronic PBDE exposure may
lower the threshold for bacteria to stimulate a proinammatory
response, which has potential relevance given the established
link between bacteria and certain cancers (e.g. Helicobacter pylori
and gastric cancer), where tumor development is dependent on
inammation.
Vinclozolin was also of particular interest as an environmen-
tal chemical because transient early life exposures in utero have
been linked to both adult-onset disease and transgenerational
disease that involves inammation (134,135). For example, tran-
sient vinclozolin exposure in utero has been shown to promote
inammation in the prostate (prostatitis) of postpubertal rats
coupled with a down-regulation of the androgen receptor and
increase in nuclear factor-κB (NF-κB). The late or delayed effect
of exposure is hypothesized to reect a mechanism whereby
vinclozolin exposure during a critical development window
imprints an irreversible alteration in DNA methyltransferase
activity, leading to reprogramming of the androgen receptor (AR)
gene(s), which manifest as inammation in early adult life with
adverse effects on spermatid number.
Similarly, 4-NP has been shown to increase progenitor white
adipose levels, body weight and overall body size in rodents
exposed prenatally. Like vinclozolin, 4-NP effects on adipogen-
esis in the perinatal period confer transgenerational inheritance
of the obesogenic effects observable in F2 offspring, consistent
with genome reprogramming through an epigenetic process
(252) and others have reported immune and inammation-
related effects (137,138) making it relevant to carcinogenesis a
deserving further investigation.
Sustained proliferative signaling
Sustained proliferative potential is an essential component of
cancerous growth. Progressive conversion of normal cells into
cancer cells requires a series of genetic alterations, where each
alteration confers one or more types of growth advantage. One
such alteration that affords the transformed cell a distinct
growth advantage over its normal counterparts is the acquired
capacity of the cancer cell to proliferate in a sustained manner,
so as to crowd out and outnumber the normal cell population
(23). One of the fundamental differences between a normal and
a transformed cell is that normal cells halt proliferation when
subjected to growth inhibitory signals or in the absence of
growth stimulatory signals (253). But tumor cells act to sustain
proliferative signaling in several different ways. They can acti-
vate specic genes to produce relevant growth factors, which in
turn bind to signaling receptors giving rise to an autocrine loop
(254). Growth factors produced by tumor cells can also stimulate
the proliferation of stromal cells that in turn produce growth
factors to sustain tumor cell proliferation (255). Sustained pro-
liferation can additionally be maintained at the receptor level
by truncation of signaling receptor proteins whereby the ligand-
activated switch is missing (256). Alternatively, the number of
high-afnity receptor proteins may be increased to levels that
will sustain proliferative signaling in otherwise normal growth
factor levels. Finally, sustained proliferative signaling may well
be the result of perpetual activation of the intracellular sign-
aling chain independent of growth factors or receptors (e.g.
mutated ras (257) or truncated src (258) are intermediaries of a
normal proliferation signaling chain responsible for sustained
proliferation).
We hypothesized that disruptive environmental chemicals
acting in a procarcinogenic manner by inducing what is referred
to as ‘sustained cell proliferation’ likely exerted their action by
interfering with some basic control mechanisms (23,253). For
instance, they could achieve this by positively regulating tar-
gets within and outside the cell known to promote cell prolif-
eration or negatively regulating targets within and outside the
cell known to halt cell proliferation. In this way, such chemicals
could confer proliferative advantage to a distinct cell population
and contribute to that population’s capability to successfully
breach innate anticancer defense mechanisms and to become
progressively autonomous.
Specically, we identied a total of 15 ubiquitous chemical
disruptors capable of producing sustained cell proliferation. The
majority of these chemicals interacted with multiple targets,
and we have tabled this information in our review. In summary,
we identied several commonly used insecticides and fungi-
cides capable of causing sustained proliferation. These included
cyprodinil, etoxazole, imazalil, lactofen, maneb, methoxychlor
(MXC), phosalone, prochloraz and pyridaben, all of which tar-
geted estrogen receptor α and frequently other steroid hormone
receptors such as androgen receptor (102,259–275). Most of
these chemicals also targeted growth factors and their recep-
tors (264,267) and induced cytokines and cytokine receptors
(identied by ToxCast high throughput assay). Top disrupting
chemical fungicides and insecticides were MXC and cyprodinil,
which each interacted with a total of six individual targets that
further included the AhR (100), B-lymphocyte markers (ToxCast
2009 high-throughput assay, both chemicals), AP-1 proteins/
transcription/translation regulators, downstream signaling
molecules and cell cycle regulators (276,277). Other strong per-
formers for sustained proliferation were BPA (activated all tar-
gets activated by the insecticides and fungicides above except
growth factors and their receptors, B lymphocyte markers and
PPAR, but included cell cycle regulators alongside AP-1 proteins/
transcription/translation regulators and downstream signaling)
(272,276,278,279) (also identied in ToxCast high-throughput
assay, 2009), polyuorinated octinoid sulfate and polybromi-
nated diphenylethers (ame retardants) that either activated
AhR (280,281) or up to ve other targets that included steroid
receptors, growth factors, cytokines and cell cycle regulators
(109) (ToxCast high-throughput assay 2009). Three other con-
tenders were phthalates (plasticizers that acted via three tar-
gets that included AhR, steroid hormone receptors and PPAR)
(282–285), trenbolone acetate (a synthetic anabolic steroid
that unsurprisingly acted through steroid hormone receptors)
(120,286–290) and nally, edible oil adulterants (food contami-
nants produced during food processing that acted via down-
stream signaling) (291,292).
We have shown estrogen and androgen receptors to be
important targets in relation to sustained proliferative signaling
(293), and note that environmental estrogens and androgens are
frequently recognized as prototypical disruptor(s) of this hall-
mark. Although this is a small sample, there are a great number

S268 | Carcinogenesis, 2015, Vol. 36, Supplement 1
of chemicals in the environment, both naturally occurring and
man-made, are estrogenic, interact with estrogen receptor and
produce estrogen metabolites (just as naturally derived ovarian
estrogen does during metabolic breakdown). Catechol estrogens
(hydroxyl derivatives of estrogens), which are formed during
estradiol metabolism, are also potentially important mediators
of endogenous estradiol levels, and therefore of sustained pro-
liferative signaling and oncogenesis (294).
Insensitivity to antigrowth signals
Cell cycle arrest is important for maintaining genomic integrity
and for preventing genetic errors from being propagated. The
normal cell cycle contains multiple checkpoints to safeguard
against DNA-damaging agents. Specic proteins at these check-
points are activated in response to harmful stimuli, ensuring
that cellular proliferation, growth and/or division of cells with
damaged DNA are blocked.
There are multiple key mediators of growth inhibition that
may become compromised during carcinogenesis. Some, such
as p53, RB1, and checkpoint kinases cause cells to arrest at the
G1–S phase transition when they are activated by DNA damage.
Mutations in the p53 gene occur in ~50% of all cancers, although
certain tumor types, such as lung and colon, show a higher than
average incidence (295). Similarly, pRb hyperphosphorylation
(296), direct mutations (297), loss of heterozygosity (298) and dis-
ruption of the INK4–pRb pathway (INK4–CDK4/6–pRb–E2Fs) (299)
are common events in the development of most types of can-
cer. Cancer cells may also evade the growth inhibitory signals
of transforming growth factor-β (TGF-β) (300) and modulate the
action of downstream effectors as well as crosstalk with other
pathways.
Cells also receive growth inhibitory signals through intercel-
lular communication via gap junctions. Gap junctions disperse
and dilute growth-inhibiting signals, thereby suppressing cell
proliferation. In contrast, loss of gap junctions increases intra-
cellular signaling, leading to enhanced proliferation and tumor
formation. The molecular components of gap junctions are the
connexin proteins (301). Connexins are recognized as tumor sup-
pressors and have been documented to reduce tumor cell growth.
Numerous environmental stimuli have been reported to directly
affect gap junction intercellular communication. Adherens junc-
tion machinery mediates contact inhibition of growth, and loss
of contact inhibition is a mediator of tumor cell growth.
Chemicals that may contribute to insensitivity to antigrowth
signals through multiple targets of this hallmark are BPA, a
common constituent of everyday plastics, and pesticides such
as DDT, folpet and atrazine. BPA promotes proliferation by dis-
rupting the growth inhibitory signals of p53 and gap junction
communication (171,302). DDT has also been shown to enhance
proliferation by increasing the expression of Ccnd1 (cyclin D1)/
E2f, inducing phosphorylation of pRb, increasing the expression
of p53-degrading protein Mdm2 (a negative regulator of p53)
(162) and disrupting gap-junctional intercellular communica-
tion (163,164). Folpet down-regulates the functions of p53 and
ATM/ATR checkpoint kinases (167) and promotes proliferation,
whereas atrazine shows genotoxic effects at subacute dose on
Wistar rats. Genotoxicity was also associated with increased
transcription of connexin accompanied with increased oxida-
tive stress (303).
Resistance to celldeath
Cell death is an actively controlled and genetically regulated
program of cell suicide that is essential for maintaining tis-
sue homeostasis and for eliminating cells in the body that are
irreparably damaged. Cell death programs include: apoptosis,
necrosis, autophagy senescence and mitotic catastrophe (21).
Defects in these pathways are associated with initiation and
progression of tumorigenesis. Normally, cells accumulate from
an imbalance of cell proliferation and cell death, permissive cell
survival amidst antigrowth signals such as hypoxia and con-
tact inhibition, resistance to the killing mechanisms of immune
cell attack and anoikis resistance (304). Increased resistance to
apoptotic cell death involves inhibition of both intrinsic and
extrinsic apoptotic pathways.
The link between malignancy and apoptosis is exemplied
by the ability of oncogenes, such as MYC and RAS, and tumor
suppressor genes, such as TP53 and RB, to engage both apop-
tosis and the aberrant alterations of apoptosis regulatory pro-
teins such as BCL-2 and c-FLIP in various solid tumors (305). This
variety of signals driving tumor evolution provides the selective
pressure to alter apoptotic programs during tumor development.
Some chemical carcinogens and sources of radiation cause DNA
damage and increase genetic and/or epigenetic alterations of
oncogenes and tumor suppressor genes leading to loss of cel-
lular homeostasis (306). Other signals include growth/survival
factor depletion, hypoxia, oxidative stress, DNA damage, cell
cycle checkpoint defects, telomere malfunction and oncogenic
mutations, and exposure to chemotherapeutic agents and heavy
metals (307,308).
Cancer cells resist apoptotic cell death by up-regulation of
antiapoptotic molecules and the down-regulation, inactivation
or alteration of pro-apoptotic molecules. Activation of p53 usu-
ally induces expression of pro-apoptotic proteins (Noxa and
PUMA) and facilitates apoptotic cell death (309). Antiapoptotic
Bcl-2 family proteins suppress pro-apoptotic Bax/Bak [which
would otherwise inhibit mitochondrial outer membrane perme-
abilization]. Mitochondrial outer membrane permeabilization
releases cytochrome c and triggers apoptosis through an intrin-
sic pathway (310). Thus, regulation of apoptosis can be achieved
by inhibiting the antiapoptotic Bcl-2 family proteins and Bcl-XL
proteins as this restores a cell’s ability to undergo apoptosis. In
the process, mitochondrial outer membrane permeabilization,
mitochondrial proteins (Smac/DIABLO and Omi/HtrA2), which
inhibit the X-linked inhibitor of the apoptosis protein, are leaked
to trigger caspase activity in apoptosis (311,312).
Normal cellular metabolism is important for the sur-
vival of cells, whereas dysregulated metabolism in cells (see
Dysregulated metabolism) can induce either apoptosis or resist-
ance to apoptotic stimuli (313). In the liver, nearly every enzyme
in glycolysis, in the tricarboxylic acid cycle, in the urea cycle, in
gluconeogenesis and in fatty acid and glycogen metabolism is
found to be acetylated, and this N-α-acetylation confers sensi-
tivity to apoptotic stimuli (314). The antiapoptotic protein, Bcl-xL
reduces the efux of acetyl-CoA from the mitochondria to the
cytosol in the form of citrate and decreases N-α-acetylation of
apoptotic proteins, which enables cells less sensitive toward
apoptotic stimuli to mediate cell proliferation, growth and sur-
vival. Thus, N-α-acetylation might be a major factor in overcom-
ing apoptotic resistance in cancer cells (315,316).
Death receptor ligands such as TRAIL—which is bound to
DR4/DR5—induce receptor oligomerization and recruitment of
FADD and caspase-8 to form death-inducing signaling com-
plex, which leads to subsequent cell death via apoptosis. Thus,
expression of death receptors and their decoy receptors (Dcr1/2)
mediates apoptosis in tumor cells (317). When normal cells lose
contact with their extracellular matrix or neighboring cells, they
undergo an apoptotic cell death pathway known as ‘anoikis’
(304). During the metastatic process, cancerous cells acquire

W.H.Goodson et al. | S269
anoikis resistance and dissociate from primary sites, travel
through the vascular system and proliferate in distant target
organs.
A blockage of gap junction intracellular communication
(GJIC) between normal and preneoplastic cells also creates an
intra-tissue microenvironment in which tumor-initiated prene-
oplastic cells are isolated from growth controlling factors of nor-
mal surrounding cells resulting in clonal expansion (318). Gap
junction channels and Cxs control cell apoptosis by facilitating
the inux and ux of apoptotic signals between adjacent cells
and hemi-channels between the intracellular and extracellular
environments, and Cx proteins in conjunction with their intra-
cytoplasmic localization, may act as signaling effectors that are
able to activate the canonical mitochondrial apoptotic pathway
(319).
Several anthropogenic chemicals can affect resistance to cell
death. For example, BPA has been shown to strikingly impair
TP53 activity and its downstream targets, cell cycle regulators,
p21WAF1 and RB, or pro-apoptotic BAX, thereby enhancing the
threshold for apoptosis (172).
Chlorothalonil, a broad-spectrum fungicide that is used on
vegetables, fruit trees and agricultural crops, is considered to be
non-genotoxic but classied as ‘likely’ to be a human carcino-
gen by all routes of exposure (29). In a eukaryotic system, chlo-
rothalonil reacted with proteins and decreased cell viability by
formation of substituted chlorothalonil-reduced glutathione
derivatives and inhibition of specic nicotinamide adenine dinu-
cleotide thiol-dependent glycolytic and respiratory enzymes (320).
Caspases (cysteine-dependent proteases) and transglutaminase
are some of the thiol-dependent enzymes involved in apoptosis,
so inhibition of these thiol-dependent enzymes in tumor-initiated
cells may disrupt apoptotic cell death and aid in tumor survival.
Dibutyl phthalate and diethylhexyl phthalate (DEHP) are
diesters of phthalic acid and commonly referred to as phtha-
lates. In general, mimic the function or activity of the endoge-
nous estrogen 17β-estradiol (E2) and bind to estrogen receptors.
Interestingly, phthalates can mimic estrogen in the inhibition
of TAM-induced apoptosis in human breast cancer cell lines by
increasing intracellular Bcl-2/Bax ratio in breast cancer (321).
Lindane, an organochlorine pesticide, bioaccumulates in
wildlife and humans. Exposure to lindane induces tumor for-
mation in the mouse 42GPA9 Sertoli cell line by disrupting the
autophagic pathway and sustained activation of the mitogen-
activated protein kinase (MAPK)/extracellular signal-regulated
kinase (ERK) pathway (322).
MXC (1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane) is a
DDT derivative that was developed after the ban of DDT and
it exhibits antiandrogenic and estrogenic activity. MXC stimu-
lates proliferation and human breast cancer cell growth by the
up-regulation of genes that involve cell cycle (cyclin D1), and the
down-regulation of genes p21 and Bax affecting G1/S transition
and apoptosis, respectively, through ERα signaling (323).
Replicative immortality
Cellular senescence is a state of irreversible arrest of cellular
proliferation characterized by changes in transcription, chro-
matin conformation, cytoplasmic and nuclear morphology,
DNA damage signaling and a strong increase in the secretion of
proinammatory cytokines (324) Senescence is the rst line of
defense against potentially transformed cells (325). Progression
to malignancy correlates with a bypass of cellular senescence.
Thus, senescence inhibits the activation of the tumorigenic
process (325). Senescence has been observed in vitro and in vivo
in response to various stimuli, including telomere shortening
(replicative senescence), oncogenic stress, oxidative stress and
chemotherapeutic agents (326).
Cellular senescence exhibits several layers of redundant
regulatory pathways. These pathways converge to arrest the cell
cycle through the inhibition of CDKs. The best-known effector
pathways are the p16INK4a/pRB, the p19ARF/p53/p21CIP1 and
the PI3K/mammalian target of rapamycin (mTOR)/FOXO path-
ways (327–330), which show a high degree of interconnection.
Additionally, the pRb and the mTOR pathways are two routes
that have been proposed to be responsible for permanent arrest
of the cell cycle (331). More pathways and genes are being dis-
covered, increasing the complexity of our knowledge of this
physiological process (329). Most, if not all of these genes have
been related to human tumorigenesis.
Despite the relevance of senescence as a gatekeeper in the
process of tumorigenesis, there is not a large body of infor-
mation exploring the effect of chemicals on this safeguard.
Little research has been undertaken on chemicals that alter
gene expression regulating senescence and few genes have
been identied (e.g. telomerase, p53, pRb, INK4a) (83,332,333).
Traditional protocols for the assessment of the carcinogenic
risk rely on the detection of tumors induced by agents that
alter many different pathways at the same time (includ-
ing senescence). These agents are mainly unspecic muta-
gens or epigenetic modiers. The effect of some compounds
is being explored including nickel-derived compounds (e.g.
nickel chloride), diethylstilbestrol, reserpine or phenobarbital
(83,334–337).
There may be environmental chemicals that are not muta-
gens or epigenetic modiers, but that target specic proteins on
the senescence pathways and may affect the initiation of tumo-
rigenesis by other compounds allowing senescence bypass. The
contribution of these compounds to the carcinogenesis process
is largely unknown. A few compounds bypass senescence in
this specic manner—acetaminophen, cotinine, nitric oxide,
Na-selenite and lead. Other chemicals known to alter senes-
cence only are mostly unknown (86,88–91,338–341).
Senescence has strong fail-safe mechanisms, and experi-
mental attempts to bypass senescence are usually recognized
as unwanted signals and trigger a senescence response anyway.
However, these conclusions are based on the interpretations of
experimental designs in which acute molecular or cellular alter-
ations are produced. There are few experiments regarding the
effects of chronic, low-dose alterations and even fewer studies
that consider the different cellular and molecular contexts that
can arise over the course of a lifetime.
Dysregulated metabolism
The highly glycolytic cancer phenotype described by Warburg
etal. (25) in the early 20th century determined much of the initial
direction in cancer research (26). Other characteristic metabolic
abnormalities have also been described (25,26,342,343) and have
recently garnered increased attention (344–348). These changes
are neither xed nor specic for cancer (349–351), but the uni-
versality of metabolic dysregulation suggests major roles in can-
cer genesis, maintenance and progression. Precise denitions of
what constitutes cancer metabolism, and when such changes
rst occur during the course of cancer development, are lacking.
From a teleological perspective, alterations in both intermediary
metabolism and its control are not surprising insofar as highly
proliferative cancer cells exhibit increased energy demands
and expanded requirements for macromolecular precursors to

S270 | Carcinogenesis, 2015, Vol. 36, Supplement 1
support nucleic acid and protein biosynthesis, as well as mem-
brane biogenesis, for increased biomass. Metabolic reprogram-
ming ostensibly equips cancer cells to cope with these demands,
as well as accompanying cellular stresses. Although much of
the attention on cancer metabolism has focused on enhanced
glucose utilization via glycolytic and pentose phosphate path-
ways, cancer cells are also capable of the oxidative utilization of
carbohydrates, lipids and peptides, and the metabolism of these
individual substrate classes remain intimately intertwined as in
normal cells (26,345,352).
Major control of glycolysis is traditionally ascribed to glu-
cose transport, hexokinase, phosphofructokinase and pyru-
vate kinase (352). Glyceraldehyde-3-phosphate dehydrogenase
also normally couples glycolytic ux to mitochondrial metabo-
lism in the presence of oxygen and to lactate generation in its
absence, but this relationship is fundamentally altered in can-
cer (26,345,353,354). Given the central importance of the pen-
tose phosphate pathway to anabolic metabolism and redox
homeostasis, glucose-6-phosphate dehydrogenase and its redox
coupling partners represent similarly attractive carcinogenic
targets (355). In addition, the enzymes of the tricarboxylic acid
cycle, such as fumarate hydratase, succinate dehydrogenase
and isocitrate dehydrogenase, play crucial roles in oxidative
energy metabolism and the interconversion of metabolic inter-
mediates, making them appealing candidates for study as well
(356,357).
The central importance of the mitochondrial electron trans-
port chain to oxidative energy metabolism and its established
role in toxic responses and dysregulated mitochondrial func-
tion in cancer makes its assembly and function attractive topics
for study (358–360). Despite well-established roles for lipid and
amino acid metabolism in cancer development and progression,
they have historically received less attention than carbohydrate
metabolism (26). Lipogenic, lipolytic and lipophagic pheno-
types are now widely recognized (344,361–363), so targets such
as acetyl-CoA carboxylase, fatty acid synthase, cellular lipases
and lipid transporters represent additional attractive targets
for study. Amino acid metabolism—particularly glutamine and
serine metabolism—also has well-established roles in cancer
(364–366), providing additional potential targets for study that
include 3-phosphoglycerate dehydrogenase (346,365,367,368)
and cellular transaminase coupling mechanisms. Study of
both lipid and protein metabolism must accommodate the fact
that cancer cells exhibit substrate preferences, including well-
described endogenous lipid- and protein-sparing effects of
exogenous glucose availability in cancercells.
The metabolic capacity of both normal cells and cancer cells
generally exceed their catabolic and anabolic requirements
(364,369,370), and only a fraction of the available potential energy is
ultimately required for cell survival (371,372). Moreover, very small
changes in metabolic ux can have profound phenotypic conse-
quences, and metabolic control analysis has suggested that the
importance of increased cancer-associated glycolytic and glutami-
nolytic uxes may lie not in their magnitudes, but in the mainte-
nance and control of smaller branched pathway uxes (364). For
these reasons, rigorous functional validation is needed for all can-
cer-associated changes in gene expression or metabolite accumu-
lation. Well-described moonlighting functions for many metabolic
enzymes (373–375), including the novel antiapoptotic roles of mito-
chondrial hexokinases (376), cannot be simply extrapolated from
our knowledge of classical roles in cellular metabolism.
These enzymes and their pathways constitute broad cat-
egories of potential targets for disruption that could serve to
enable the observed metabolic phenotypes of cancer cells (377).
Although metabolic control is broadly distributed over all indi-
vidual steps for a given pathway (352,378), the most obvious
targets for conceptual and experimental scrutiny involve major
rate-controlling elements of pathways capable of supporting the
anabolic and catabolic needs of rapidly proliferating cancercells.
Numerous studies have demonstrated cancer-associated
changes in metabolism or related gene expression (26). We
looked at acrolein, copper, cypermethrin, diazinon, hexythi-
azox, iron, malathion and rotenone as chemicals that had been
reported to show relevant disruptive potential (51,379–383);
however, the toxicological data that are available for many
suspected or known environmental disruptors, generally lacks
mechanistic information regarding their potential roles as
determinants of the observed metabolic hallmarks of cancer.
Even prior metabolic screening platforms, including tetrazolium
reduction assays, have limited specicity and can be profoundly
inuenced by experimental screening conditions. Unfortunately,
standardized chemical screening has typically not been con-
ducted under controlled or limiting substrate conditions that
would directly inform our understanding of the functional rel-
evance of observed changes. None have established unambigu-
ous causal relationships between specic chemical exposures
and the parallel or sequential development of dysregulated
metabolism of cancer in the same model, and most observed
changes in gene expression with potential relevance to cancer
metabolism have not been accompanied by validating func-
tional studies.
Angiogenesis
Angiogenesis, the process of formation of new blood vessels
from existing blood vessels, is a critical process for normal organ
function, tissue growth and regeneration (e.g. wound healing,
female menstruation, ovulation and pregnancy) as well as for
pathological conditions (e.g. cancer and numerous non-cancer-
ous diseases, such as age-related macular degeneration, dia-
betic retinopathy, rheumatoid arthritis, endometriosis, diabetes
and psoriasis) (384,385).
Tumor angiogenesis is an early critical event for tumor
development: Atumor cannot grow beyond 1 mm3 (by estimate)
without angiogenesis (386). Tumor growth, invasion and metas-
tasis depend on blood vessels and neovascular development
to provide nutrients, oxygen and removal of metabolic waste
as tumors grow in primary sites, invade adjacent tissues and
metastasize to distant organs (387,388). Inhibition or eradication
of tumor angiogenesis by antiangiogenic inhibitors (389,390) or
by antineovascular agents (such as vascular-disrupting agents
(391–393) and fVII/IgG Fc (394), the latter also called ICON (395–
397)) can treat pathological angiogenesis-dependent diseases,
including cancer and many non-cancerous diseases.
Under physiological conditions, angiogenesis is well bal-
anced and controlled by endogenous proangiogenic factors
and antiangiogenic factors. Factors produced by cancer cells
can shift the balance to favor tumor angiogenesis. Such factors
include vascular endothelial growth factor (VEGF) and tissue
factor (TF). VEGF, one of the most potent proangiogenic factors
produced by cancer stem cells and cancer cells, binds to vascular
endothelial cells via its receptor VEGFR, initiating VEGF/VEGFR
intracellular signal transduction pathways and activating many
gene transcriptions and translations toward angiogenesis. TF
is a transmembrane receptor (398) not expressed on quiescent
endothelial cells (399,400). Upon stimulation of VEGF, TF is selec-
tively expressed by angiogenic endothelial cells, the inner layer
of the tumor neovasculature. Thus, TF is a specic biomarker
for tumor angiogenesis (408–410). Both of the membrane-bound

W.H.Goodson et al. | S271
receptors VEGFR and TF can mediate separate intracellular sign-
aling pathways that contribute to tumor angiogenesis.
Environmental exposures can promote tumor development,
but the role of chemicals in tumor angiogenesis, particularly the
role of low-dose non-carcinogens, is largely unknown. Some food-
use pesticides that are non-genotoxic act as tumor promoters,
and other chemicals affect various hallmarks such as apoptosis,
proliferative signaling, evading growth suppression, enabling
replicative immortality, metastasis, avoiding im