MASOT 2019 Ambassador Award

Chemical Carcinogenesis: Where have we been, Where are we now

James E. Klaunig, Ph.D., ATS, IATP Professor, Environmental Health Professor, School of Public and Environmental AffairsIU School of Public Health Bloomington , IN 47405

Klauniglab.comjklauni@indiana.edu

MASOT 2019 Ambassador Award

Chemical Carcinogenesis: Where have I been, Where is my lab now

James E. Klaunig, Ph.D., ATS, IATP Professor, Environmental Health Professor, School of Public and Environmental AffairsIU School of Public Health Bloomington , IN 47405

Klauniglab.comjklauni@indiana.edu

Causes of Human Cancer

Klaunig, J.E. ,and Wang, Z., (2018). Chapter 8: Chemical Carcinogenesis. In Klaassen (Ed.), Casarett & Doull’s Toxicology: The Basic Science of Poisons (9th Edition) pp 705-813 . New York: McGraw-Hill

• Cristian Tomasetti, Bert Vogelstein, Science 02 Jan 2015:Vol. 347, Issue 6217, pp. 78-81

Cancer is Bad Luck

• Variation in cancer risk among tissues can be explained by the number of stem cell divisions

• Random errors occurring during DNA replication in normal stem cells

• This “bad luck” component explains a far greater number of cancers than do hereditary and environmental factors.

• Correlation with cell division rate and cancer

Benjamin Franklin Trump, MDChair of Pathology University of Maryland

Ames BN, Durston WE, Yamasaki E, Lee FD (August 1973). "Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection". Proceedings of the National Academy of Sciences of the United States of America. 70 (8): 2281–5

Multistage Aspect of Carcinogenesis1970s - Mutation

- “all carcinogens are mutagens, - not all mutagens are carcinogens”

- Role of Metabolism (activation) - Multistage cancer process

- (initiation, promotion , progression)

How do Chemicals Cause Cancer

J H Weisburger and G M Williams The distinct health risk analyses required for genotoxic carcinogens and promoting agents. Environ Health Perspect. 1983 Apr; 50: 233–245.

Classification of Chemical Carcinogens based on mechanism of action - 1980s Concept of Genotoxic and Non-Genotoxic Carcinogens

Hepatic Carcinogenesis in Rodents

Mechanisms of Nongenotoxic Carcinogens

• Increase cell proliferation• increased DNA synthesis • mitogenic• cytolethal and compensatory hyperplasia • decreased apoptosis

• Modulation of intercellular communication• hormonal disruption/modification• inhibition of gap junctional intercellular communication

• Modulation of gene expression• hypomethylation• transcription factor activation

• Induction of oxidative stress• direct radical formation and interaction• decrease in antioxidants • MFOS, futile cycling• inflammatory cell involvement

7

Cancer = Mutation and Cell Proliferation (cell growth)

• Klaunig Lab

• Our lab’s research for the past 40 years has been directed to:

• Understanding how do chemicals cause cancer?

• Specifically examine possible mechanisms for the action of Nongenotoxic carcinogens on tumor development and modulation

• Using the rodent liver as an in vivo and in vitro model.

• How do we use the rodent results for determination of potential human risk?

• Mechanisms involved in the early stages of Carcinogenesis (Role of cell growth)

• 1980 –• Modulation of cell to cell communication • Factors effecting preneoplastic lesion growth

• Selective clonal expansion • 1990 -

• Role of gene expression• Oxidative stress

• 2005 –• Receptor mediated processes (CAR, PPAR)• Mode of Action• RNA seq• Pathway Analysis

• 2016 –• Role of NASH in carcinogenesis • Computer Modeling of multistage Carcinogenesis

Rodent Liver Tumors

• Most common endpoint in cancer bioassays

• Very good model system to study carcinogenesis

• Liver is often the most sensitive target in 2-yr bioassays

• Mouse: • 45% of all chemicals

• Rat: • 37% of all chemicals

• Strain-dependent background liver tumor incidence.

• C3H/HeJ – 100%• B6C3F1 – 10-50%• C57Bl/6 – close to 0-2 %• Rat - 5 -10%

WHY STUDY LIVER?

Klaunig, J.E., Ruch, R.J., Weghorst, C.M. (1990). Comparative Effects of Phenobarbital, DDT, and Lindane on Mouse Hepatocyte Gap Junctional Intercellular Communication. Toxicology and Applied Pharmacology, 102 (3), 553-563.

De Feijter, A.W., Ray, J.S., Weghorst, C.M., Klaunig, J.E., Goodman, J.J., Chang, C.C., Ruch, R.J., Trosko, J.E. (1990). Infection of Rat Liver Epithelial Cells with V-Ha-ras: Correlation between Oncogene Expression, Gap Junctional Communication, and Tumorigenicity. Molecular Carcinogenesis, 3(2), 54-67.

Ruch, R.J., Klaunig, J.E., Kerckaert, G.A., LeBoeuf, R.A. (1990). Modification of Gap Junctional Intercellular Communication by Changes in Extracellular pH in Syrian Hamster Embryo Cells. Carcinogenesis, 11(6), 909-913.

Ruch, R.J., Fransson, R., Flodstrom, S., Warngard, L., Klaunig, J.E. (1990). Inhibition of Hepatocyte Gap Junctional Intercellular Communication by Endosulfan, Chlordane, and Heptachlor. Carcinogenesis, 11(7), 1097-1101.

Klaunig, J.E., Ruch, R.J., Hampton, J.A., Weghorst, C. M., Harnett, J.A. (1990). Gap Junctional Intercellular Communication and Murine Hepatic Carcinogenesis. Progress in Clinical and Biological Research, 331, 277-291.

Ruch, R.J., Klaunig, J.E. (1992). Enhancement of Rodent Hepatocyte Gap Junctional Intercellular Communication by Dexamethasone. In Vitro Toxicology, 5(2), 103-111.

Ruch, R.J., Madhukar, B.V., Trosko, J.E., Klaunig, J.E. (1993). Reversal of ras-Induced Inhibition of Gap Junctional Intercellular Communication, Transformation, and Tumorigenesis by Lovastatin. Molecular Carcinogenesis, 7(1), 50-59.

Kwiatkowski, A.P., Baker, T.K, Klaunig, J.E. (1994). Comparison of Glucocorticoid-Mediated Changes in the Expression and Function of Rat Hepatocyte Gap Junctional Proteins. Carcinogenesis, 15(8), 1753-1757

Trosko, J.E., Chang, C.C., Madhukar, B.V., Klaunig, J.E. (1990). Chemical, Oncogene, and Growth Factor Inhibition Gap Junctional Intercellular Communication: An Integrative Hypothesis of Carcinogenesis. Pathobiology, 58(5), 265-278.

Klaunig, J.E. (1991). Alterations in Intercellular Communication during the Stage of Promotion. Proceedings of the Society for Experimental Biology and Medicine, 198(2), 688-692.

Baker, T.K., Kwiatkowski, A.P., Madhukar, B.V., Klaunig, J.E. (1995). Inhibition of Gap Junctional Intercellular Communication by 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) in Rat Hepatocytes. Carcinogenesis, 16(10), 2321-2326

Baker, T.K., Bachowski, S., Stevenson, D.E., Walborg, E.F. Jr., Klaunig, J.E. (1995). Modulation of Gap Junctional Intercellular Communication in Rodent, Monkey and Human Hepatocyte by Nongenotoxic Compounds. Progress in Clinical and Biological Research, 391, 71-80.

Gap Junction mediated cell to cell communication

Jim Trosko

- Gap junction involved in cell growth and maintenance of cell homeostasis

- Tumor cells appear to have less or no GJIC

- Klaunig Lab examined the effects of nongenotoxic agents on cell to cell communication in liver cells

- First to use primary hepatocytes- Linked the inhibition of GJIC to the in

vivo tumor results - Showed a dose responsive inhibition of

GJIC - Threshold - Reversible- Organ / species specific - Compound specificity

• Role of Oxidative Stress/Damage in Carcinogenesis

Carcinogenesis is a Multistage Process

Peroxisomes • Cellular Organelles

• They are involved in metabolism of• fatty acids,

• Reduction of reactive oxygen species –specifically hydrogen peroxide

• Peroxisomal genes of b-oxidation• Acyl CoA oxidase (ACO)• Genes of lipid transport/metabolism

• LPL• apoA1

• Peroxisomes were identified• First seen by Swedish doctoral

student, Rhodin in 1954• Reported by the Belgian cytologist

Christian de Duve in 1967

Peroxisomes - Role in Pharmacology and Toxicology

• Peroxisomes• Show an increase in number (peroxisome

proliferation in response to several chemicallydiverse group of compounds

• Peroxisome Proliferators are frequently rodent liver carcinogens

History • 1975 Hypolipidemic

• J K Reddy Science Nov 21;190(4216):787-9. • 1980 Liver Cancer

• JK Reddy, et al Nature. 1980 283(5745):397-8• 1990 - Cloned PPAR

• Isseman and Green, Nature 347:645; 1990• 1995 - PPAR alpha Knock Out

Hypolipidemic ClofibratesGenfibrozilNafenopinWy-14,643

Fluro compoundsPFOAPFOSGenX

PlasticizersDEHP

SolventsTrichloroacetic acidDichloroacetic acidTrichloroethyleneTetrachloroethylene

Fatty acids Linoleic acidArachidonic acid

All are • Non genotoxic• Induce peroxisome proliferation • Dependent on PPAR alpha • Induce Tumors in the Liver of Rats and

Mice

Hepatic Carcinogens - PPAR alpha activators (peroxisome proliferators)

Klaunig, J.E, Babich, M.A., Baetcke, K.P., Cook, J.C., Corton, J.C., David, R.M., DeLuca, J.G., Lai, D.Y., McKee, R.H., Peters, J.M., Roberts, R.A., Fenner-Crisp, P.A. (2003). PPAR-alpha Agonist-Induced Rodent Tumors: Modes of Action and Human Relevance. Critical Reviews in Toxicology, 33(6), 655-780

PFOA C8

• Perfluorooctanoic acid (PFOA)• perfluorinated carboxylic acid• industrial surfactant • PFOA persists in the

environment• PFOA has been detected in

the blood of more than 98% of the general US population parts per billion (ppb) range

• Higher levels in chemical plant employees and surrounding subpopulations.

Biegel et al. (2001) Butenhoff et al. (2012)Lesion Control PFOA 300ppm Control PFOA

30ppmPFOA

300ppmLiver

Adenoma 2/80 (3%) 10/76* (13%) 0/49 (0%) 0/50 (0%) 0/50 (0%)

Carcinoma 0/80 (0%) 0/76 (0%) 3/49 (6%) 1/50(2%) 5/50 (10%)

Combined 2/80 (3%) 10/76* (13%) 3/49 (6%) 1/50(2%) 5/50 (10%)

PancreasHyperplasia 14/80

(18%)30/76*(39%) 3/46 (7%) 1/46 (2%) 10/47*(21%)

Acinar adenoma 0/80 (0%) 7/76* (9%) 0/49 (0%) 0/50 (0%) 0/50 (0%)

Acinar carcinoma 0/80 (0%) 1/76 (1%) 0/49 (0%) 0/50 (0%) 0/50 (0%)

Combined 0/80 (0%) 8/76* (11%) 0/49 (0%) 0/50 (0%) 0/50 (0%)

TestesLeydig cell adenoma 0/80 (0%) 8/76* (11%) 0/49 (0%) 2/50(4%) 7/50* (14%)

Two Year Chronic Bioassay

Figure 1. Histopathological evaluation of rat liver exposed to PFOA. Liver sections were stained with H&E. Whole tissue slides were digitalized by Aperio CS2 Digital Pathology Slide Scanners. Microvesicular steatosis was identified in rats treated by 5 and 15 mg/kg/d PFOA. Hepatocellular hypertrophy was found in all PFOA groups after 14 and 28 days. Focal necrosis was found in two out of five rats in the high dose group after 28 days of treatment. A: microvesicular steatosis. B: hepatocellular hypertrophy. C: focal necrosis.

Xilin Li, Zemin Wang, Qiangen Wu and James E. Klaunig Investigation of the Mode of Action (MOA) of Perfluorooctanoic acid (PFOA) Induced Liver and Pancreatic Tumors in Rats Submitted for publication 2019

Surrogate: 7,14, 28 day treatment : 0,1,5,15 mg/kgbw

PFOA

Liver

Cyp4a1 Acox1 Cyp1a1 Cyp1a2 Cyp2b1 Cyp3a10123

10203040506070

PFOA 7dPFOA 14dPFOA 28d

** *

**

*

*

*

* *

*

*

*ControlR

ecep

tor m

edia

ted

gene

exp

ress

ion

7,14, 28 day treatment : 0,1,5,15 mg/kgbw

Nuclear receptors

Ox StressAcetyl COA activity

Cell proliferation

BMDexpress

• Plot pathways- most sensitive to less sensitive – based on median gene in pathway• Example (PPARa activator) based on GO terms

7days 30 days 60 days 0

1

2

3

4

5

Hepa

tic f

ocal

lesi

on V

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control5 mg/kg/d15 mg/kg/d

PFOA Rat Liver Focal Lesions

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Effect of Wy-14,643 on the incidence of mice with hepatocellular neoplasms in wild-type

(1/1) and PPARa knockout (–/–) mice

Group Diet Adenomas Carcinomas

PPARa (+/+) Control 0/9 0/9

PPARa (+/+) Wy-14,643 6/6 3/6

PPARa (–/–) Control 0/9 0/9

PPARa (–/–) Wy-14,643 0/9 0/9

Peters et al ,Carcinogenesis 1997

BrDU labeling of hepatic cells. Wild-type (1/1) or PPARa (–/–) mice were treated for either 1 week (1A) or 5 weeks (1B) with or without dietary Wy-14,643,

Labelling Index

• Are PPAR alpha agonist (activators) Human Carcinogens?

Several Workshops were Held to Address This Question

Purpose Describe the state of the science on the rodent MOA of liver tumor induction and human relevance by PPARa activators

Corton J.C., Cunningham M.L., Hummer B.T., Lau C., Meek B., Peters J.M., Popp J.A., Rhomberg L., Seed J., Klaunig J.E. (2013). Mode of action framework analysis for receptor-mediated toxicity: The peroxisome proliferator-activated receptor alpha (PPARa) as a case study. Critical Reviews in Toxicology . Crit Rev Toxicol. 2014 Jan;44(1):1-49.

Corton JC, Peters JM, Klaunig JE. (2018) The PPARα-dependent rodent liver tumor response is not relevant to humans: addressing misconceptions. Arch Toxicol. 2018 Jan;92(1):83-119.

PPARalpha activation is a key event

In Rodents, PPARalpha regulates genes involved inPeroxisome proliferation

Cell proliferationLipid metabolismChronic exposure leads to promotion of preneoplastic foci

• Large number of studies showed that PPARalpha is required for the cancer effects of peroxisome proliferator chemicals

• Klaunig et al., 2003• Corton et al 2014• Felter et al 2018 • Corton 2018• PPARalpha activation is a key event• In Rodents, PPARalpha regulates genes

involved in• Peroxisome proliferation • Cell proliferation• Lipid metabolism

• Chronic exposure leads to promotion of preneoplastic foci

• Foci from spontaneous damage or indirectly initiated through increases in oxidative stress?

• Knock out • Abolishment of gene

expression changes• Increases in cell

proliferation• Hypolipidemic response• Oxidative stress

In Humans:

PPARalpha is also activated However, PPARalpha regulates genes involved inLipid metabolism (hypolipidemic effects)No Cell proliferation is seen No peroxisome proliferation is seen Therefore there is no increase in Foci or Tumors

Conclusions: PPARalpha Activators

1. In Rodents:• Liver tumor induction follows the

PPARα MOA• PPAR alpha activation • Cell growth (proliferation/apoptosis) • Selective clonal expansion of foci • Adenomas

2. In Humans:• The Key causal event perturbation of

cell growth (proliferation and/or apoptosis) does not occur

• Increased peroxisome proliferation does not occur

3. Conclusion:• The rodent MOA for PPARa

activator induced hepatic neoplasia does not occur in human liver

• Question • In individuals with Hepatic steatosis or Non alcoholic fatty liver disease

(NAFLD), will exposure to PPAR alpha chemicals modulate the liver function and pathology?

• Experimental approach: Using our NAFLD model in mice we treated mice already on high fat diet (16 wks) with PFOA for 2,8 and 16 wks.

Modulation of PPAR

Cell proliferation

• Results:• PFOA activated PPAR alpha

• Increased AcylCoaoxidase and Cyp4a10 activity

• PFOA decreased hepatic steatosis

• PFOA reduced the fibrosis induced by HFD

• PFOA with HFD showed enhanced Cell proliferation

Li X, Wang Z, Klaunig JE. 2019 The effects of perfluorooctanoate on high fat diet induced non-alcoholic fatty liver disease in mice. Toxicology. Mar 15;416:1-14

Key Events

Receptor Activation

Associative Events Associative Events

HypertrophyCYP induction

Decreased apoptosis

Altered epigenetic changes specific to

the receptor activation

Altered epigenetic changes specific to receptor activation

Cell proliferation

Clonal expansion of initiated cells

Liver adenomas/carcinomas

Associative Events orModulating Factor

Inhibition of gapJunction communication

34

Modes Of Action of Hepatic CarcinogensA. Genotoxic/DNA reactive (AFB-1)

B. Nongenotoxic/non-DNA reactive• Receptor Mediated (activation)

• CAR (Phenobarbital)• AhR (TCDD)• PPARα (DEHP)• Estrogen (DES/Estradiol)• Other

• Non Receptor Mediated• Cytotoxicity (CHCl3, CCl4)

• Oxidative stress (Fe, Cu overload)• Infection (HBV/HCV)• Immunosuppression• Fatty Liver• Other

Indiana University Bloomington

Yinbin Liu Rami Saadeh Shawn (Xilin) LiJohn FyolekLuma MeloRaj Sellamuthu*Zemin Wang*Shaoyu Zhou*Jianyong Zhang*Zuoqing Yuan*Ebenezer Olatunde Farombi*Maud De Meyer*Karen Timant*

Indiana University School of Medicine

Solomon Owumi Ann Kwiatowski *Gulam Kalimi *Lisa Kamendulis *David Ackley *JuLee Kaster *JJ Park *BaoGuo Ren *Guimeng Li *Barbara Gottschling*Zhenquan Jiao*Patrick Klein * Xinzhu Pu*Solomon Owumi*Binu Philip*Zemin Wang*

Klaunig Lab Colleagues

Medical College of Ohio (Pathology)

Randall J. RuchChristopher WeghorstXie Jian Lydia D. Schafer

Indiana University School of Medicine

Karen Steinmetz Stephen BachowskiKyle KolajaCraig ZwicklJiazhong Jiang Jason Isenberg Elizabeth JulianJiazhong ZhangKristin HornMinerva MercadoAditya Bansal

* Post doc/ visiting professor