The National Institute of Environmental Health SciencesThe National Institutes of HealthThe Department of Health and Human Services

NTPThe National Toxicology ProgramThe Department of Health and Human Services

Molecular Biology of Liver Tumors

Following Chemical Exposure

Robert C. Sills, DVM, PhDDiplomate, ACVP

Overview

Molecular Pathology Review Molecular Pathology Case Studies

Oxazepam (Mice) Hepatocellular neoplasms Hepatoblastomas

Riddelliine (Rats and Mice) Liver hemangiosarcomas

Research Team

Neoplastic Liver LesionsB6C3F1 Mouse

Hepatocellular Carcinoma

Hemangiosarcoma

Hepatocellular Adenoma

Hepatoblastoma

Molecular Pathology Review

Major Genes Involved in Carcinogenesis

Tumor suppressor genesProto-oncogenes

Involved in cellular growth and differentiation

Must be activated in cancer Point mutation Chromosomal translocation Gene amplification

Negative growth regulator

Must be inactivated or lost in cancer

Point mutation Loss of gene or chromosome Methylation

Case Study in Mice

OxazepamNon-genotoxic

Oxazepam

Central nervous system depressant

Prescribed widely for treatment of anxiety

Metabolite of benzodiazepines (valium)

Valium has been prescribed at an annual rate of greater than 25 million times in US

Incidences of Neoplasams and Nonneoplastic Lesions of the Liver

of Female B6C3F1 Mice in the 2-Year Feed Study of Oxazepam

Dose (ppm) 0 125 2,500 5,000

2-Year StudyLiver (Number of rats examined) 50 50 50 50

Centrilobular Hypertophy 0 2(1.5) 11**(2.5) 29**(2.9)

Hepatocellular Adenoma 25 35* 35* 36*

Hepatocellular Carcinoma 9 5 49** 44**

Hepatoblastomas 0 1 8** 8**

*P<0.05**P<0.01

Approach for Evaluating the Mechanism of Carcinogenesis

B6C3F1 Mouse

OxazepamOxazepam

TissueTissue CellularCellular MolecularMolecular

Liver Hepatocellular

Neoplasms

Hepatoblastomas

?

Bucher et al, 1994Fund. Appl. Tox, 23, 280,1994Griffen et al, 1995Tox. Let. 76, 251,1995

Centrilobular

Hypertrophy

Cytochrome p450

Cytochrome P450Oxidative Damage

OXAZEPAMOXAZEPAM

METABOLITES

CANCER GENES

Isoprostane (oxidative damage marker) in Livers of

B6C3F1 Mice Following 6-months Oxazepam Exposure

0

10

20

30

40

50

60

70

(n=3)

(n=3)

Control Oxazepam

Tomer, Devereaux, NIEHS, 2000

Cytochrome P450Oxidative Damage

OXAZEPAMOXAZEPAM

METABOLITES

H-ras proto-oncogene

Hepatocellular Tumors Hepatoblastomas

Ras Genes and Cancer

Commonly mutated in both animal and human tumors

Contributes to understanding of pathogenesis of cancer

Links between chemical exposure and signature mutations in cancer

Robust spontaneous data base for comparison with chemically induced tumors

Ras Signal Transduction Pathway

Robbins and CotranPathologic Basis of Disease, 2005

Activation of MAP kinase pathway

BLOCKED INMUTANT RAS

Inactivation byHydrolysis of GTP

GDP

GTP

Activation of transcription

Cell cycle progression

Growth factor

Growth factor receptor

H-ras Proto-Oncogene Evaluation

H-ras Mutations in Hepatocellular Adenomasand Carcinomas from Oxazepam Treated Mice

Treatment

Control, 0 p.p.m.Oxazepam 125 p.p.m.Oxazepam 2500 p.p.m.Oxazepam 5000 p.p.m.

11/20 (55%)13/37 (35%)2/25 (8%)0/21 (0%)

28/50 (56%)36/50 (72%)50/50 (100%)47/50 (94%)

Tumors with Mutations

Tumor Incidence

Historical Control - H-ras mutations, 80/126 (63%)Hepatoblastomas - No H-ras Mutations

Revisit Hypothesis:Assessment of Mutations in Cancer Genes

-catenin Gene

OxazepamExposure

Induction ofCytochrome

p450

OxygenRadicals

LiverTumors

–CateninMutations

GeneticAlterations inCancer Genes

DNADamage

Assessment of Mutations in Cancer Genes from Oxazepam Induced Liver Tumors

-Catenin gene First cancer gene where mutations identified

in both mouse and human hepatocellular neoplasms

Mutations of -catenin also a major factor in colon cancer and melanomas

Hot spot for mutations: Codons 32-45

De La Coste, et al., PNAS, 95: 8847-8851, 1998

-catenin ProteinCancer

Adapted, Science 281: 1439, 1998

APC

Normal Cell Cancer Cell

Assessment of Mutations in Cancer Genes from Oxazepam Induced Liver Tumors

-Catenin gene Determine the mutation frequency and pattern

of

-catenin mutations in spontaneous and oxazepam

induced hepatocellular neoplasms and

hepatoblastomas

Determine if the -catenin protein accumulated in oxazepam induced liver tumors

Strategy for Evaluating DNA from Tumors for Mutations

Single-Strand Conformational Analysis (SSCA)

SSCA Analysis-catenin Mutations

N

Devereux, T.R, Sills, R.C., Barrett, J.C et al., Oncogene, 18: 4726-4733, 1999

1 2 3 4 5 6 7 8 9 10

Direct Sequencing-catenin Mutations

A C G T A C G T

Normal Mutation

CCA

CCA/G

Codon41

Mutation Frequency of -Cateninin Hepatocellular Tumors of B6C3F1

Treatment Mutation Frequency

Control 2/22 (9%)Oxazepam 18/42 (41%)

a

ap<0.001 when comparing mutation frequency to controls

Devereaux, Sills, Barret et al., Oncogene 18, 4726, 1999

Examples of -Catenin Mutations in B6C3F1Mouse Hepatocelluar Neoplasms

Devereux, Sills, Barrett et al., Oncogene 18, 4726-33, 1999

TumorGroup Codon Mutation Bases

Control 32 GAT to GCT A to C33 TCT to TTT C to T

Oxazepam 32 GAT to GGT A to G32 GAT to GTT A to T32 GAT to CAT G to C32 GAT to AAT G to A33 TCT to TAT C to A33 TCT to TTT C to T34 GGA to AGA G to A34 GGA to GTA G to T34 GGA to GAA G to A41 ACC to ATC C to T

Assessment of Mutations

Increase in point mutations at guanine bases following oxazepam exposure is consistent with the theory that oxygen radicals contributed to their formation as these genetic lesions can arise from oxidative damage

Mutations can occur as the result of oxidative damage to guanine residues resulting in the production of 8-oxoguanine

Oxazepam Hepatoblastomas

-Catenin Protein Expression

Immunohistochemical Detection-catenin Protein

B6C3F1 Mouse

Anna, Sills, Devereux et al. Cancer Res., 60, 2864, 2000Science, 281, 1439, 1998

Cancer cells Hepatoblastoma

-catenin Mutations in Hepatoblastomas From B6C3F1 Mice Treated with Oxazepam

Tumor Group

Oxazepam

1

2345678

Codon 32 GAT to GGT (Asp to Gly)+ Del. Codons 5-8

Del. Codons 5-7Del. Codons 36-48Del. Codons 23-49Codon 34 GGA to GTA (Gly to Val)Del. Codons 5-13Del. Codons 16-36Del. Codons 21-43

8/8 (100%)

Frequency

Codon mutation (amino

acid)

Anna, C.H., Sills, R.C., Devereux et al.,Cancer Res., 60, 2864-2868, 2000

Western Blot Analysis of Proteins Associated

with -Catenin Mutations and Cancer

Cyclin D1

C-Myc

Actin

N

–

A C C C C C A A C

N – + + + + + + +21 3 4 5 6 7 8 9 10

Methylene Chloride

Oxazepam

Mutation-Catenin

4 1 0 0 0 1 15 18 30 43

Anna, CH., Ida, M., Sills, R.C., Devereux, T.R.,Tox. Appl. Pharmacology, 190: 135-145, 2003

Summary

OxazepamOxazepam

TissueTissue CellularCellular MolecularMolecular

Liver Centrilobular

Hypertrophy

Cytochrome p450

Endogenous

Source of

Superoxide

Anion Radicals

DNA Damage

-catenin gene -catenin protein Cyclin D1

Hepatocellular

Neoplasms

Hepatoblastomas

Case Study in Rats and MiceHemangiosarcomas

RiddelliineGenotoxic

Riddelliine

Belongs to a class of pyrrolizidine alkaloids

Isolated from plants of the genera Crotalaria, Amsinckia,

and Senecio

Plants may contaminate human food sources, and intact plants and their seeds may contaminate commercial grain

Strategy for Examining Molecular Mechanisms of Liver Hemangiosarcomas in F344 Rats and

B6C3F1Mice

Mechanistic Studies (NCTR) Identification of activated riddelliine metabolites 32P-postlabeling/HPLC method for identification of

riddelliine-derived DNA adducts Detection and quantification of adducts in livers of

F344/N rats orally gavaged with riddelliine for 3 or 6 months

Molecular Studies (NIEHS) Identification of K-ras/p53 mutations in hemangiosarcomas

in B6C3F1 mice

Relevance of Mechanistic Studies to Humans

Metabolism of Riddelliine to Activated Metabolite

Metabolism of Riddelliine to Activated Metabolite

Total HPLC DHR-derived DNA Adducts in Liver DNA of Rats Fed Riddelliine for 3

and 6 Months

Relationships Between Administered Dose, Adduct

Levels, and Hemangiosarcoma Incidence of Rats

Tumor Incidence in Rats and Mice

Cho, M.W., Chan, P. et al.,Cancer Letters, 193: 119-125, 2003

Rats Mice

Molecular LevelAssessment of Mutations in Cancer Genes

Hypothesis

RiddelliineExposure

Induction ofCytochrome

p450

Dehydroretronecine(DHR)

Metabolite

LiverHemangiosarcomas

P53 MutationsRas Mutations

DHR-DerivedAdducts

– Cancer GenesDNA

Rationale for Evaluating Cancer Genes in Hemangiosarcomas of Mice

Vinyl chloride induce similar tumors in humans and rats

Vinyl chloride genotoxic intermediates DNA etheno adducts N2-ethenoguanine: K-ras G A transitions

N6-etheno adduct: p53 A T transversions

Mutation Analysis of K-ras Oncogene Hemangiosarcomas

B6C3F1 Mice

SpontaneousHemangiosarcomas

RiddelliineHemangiosarcomas

7/12

0/13a

Mutation Frequency

K-ras Codon 12

GGT (Normal Sequence)

aVarious Hemangiosarcomas in control B6C3F1 mice in NTP studies

GGT GTT (Mutation)

Direct SequencingK-ras Mutations

A C G T A C G T

Normal Mutation

CGG T

G/TG

Codon13

TGG

Codon12

K-ras

p53 Gene

p53 G1

S

G2

M

CellCycle

Apoptosis

Nature Reviews, Genetics 2001

p53 Gene is a tumor suppressor gene which causes G1 and G2 arrest, promotes apoptosis and loss of function causes genomic instability

Mutated p53 gene increased half life of protein detected by immunohistochemistry

Most commonly mutated tumor suppressor gene in human cancer

P53 Protein ExpressionRiddelliine

Hemangiosarcomas

Relevance of Mechanistic Data to Humans

Do human liver microsomes metabolize riddelliine?

Do human liver microsomes with DNA and riddelliine form DHR-derived DNA adducts?

Metabolism of Riddelliine to Activated Metabolite

Human Liver Microsomal Metabolism

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

MR FR M1 M2 M3 M4 F1 F2 F3 F4

DHRN-oxide

Rat Human Human

32P-Postlabeling/HPLC Analysis of DHR-Derived Adducts Formed from Metabolism of Riddelliine by

Liver Microsomes

The liver microsomes were from female F344/N rats or male or female humans.

Summary

Riddelliine induces liver hemangiosarcomas through a genotoxic mechanism.

Riddelliine-derived DNA adducts are dose-dependent and persistent, and responsible for liver hemangiosarcoma induction.

The greater DNA adduct levels, K-ras and p53 mutations in endothelial cells correlate with riddelliine-induced liver hemangiosarcomas in rats and mice.

Research Team