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Molecular Biology of Liver Tumors
Following Chemical Exposure
Robert C. Sills, DVM, PhD
Diplomate, ACVP
The National Institute of Environmental Health Sciences
The National Institutes of Health
The Department of Health and Human Services
NTP
The National Toxicology Program
The Department of Health and Human Services
Overview


Molecular Pathology Review
Molecular Pathology Case Studies

Oxazepam (Mice)



Riddelliine (Rats and Mice)


Hepatocellular neoplasms
Hepatoblastomas
Liver hemangiosarcomas
Research Team
Neoplastic Liver Lesions
B6C3F1 Mouse
Hepatocellular Adenoma
Hepatocellular Carcinoma
Hepatoblastoma
Hemangiosarcoma
Molecular Pathology Review
Major Genes Involved in Carcinogenesis
Proto-oncogenes


Involved in cellular growth
and differentiation
Must be activated in
cancer
Tumor suppressor genes

Negative growth regulator

Must be inactivated or lost
in cancer

Point mutation

Point mutation

Chromosomal
translocation

Loss of gene or
chromosome

Gene amplification

Methylation
Case Study in Mice
Oxazepam
Non-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
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**
2-Year Study
Liver (Number of rats examined)
*P<0.05
**P<0.01
Approach for Evaluating the
Mechanism of Carcinogenesis
B6C3F1 Mouse
Oxazepam
Tissue
Liver
Cellular
Centrilobular
Molecular
?
Hepatocellular
Hypertrophy
Neoplasms
Cytochrome p450
Hepatoblastomas
Bucher et al, 1994
Fund. Appl. Tox, 23, 280,1994
Griffen et al, 1995
Tox. Let. 76, 251,1995
Cytochrome P450
Oxidative Damage
OXAZEPAM
OXAZEPAM
METABOLITES
CANCER GENES
Isoprostane (oxidative damage marker) in Livers of
B6C3F1 Mice Following 6-months Oxazepam Exposure
70
60
(n=3)
50
40
(n=3)
30
20
10
0
Control
Oxazepam
Tomer, Devereaux, NIEHS, 2000
Cytochrome P450
Oxidative Damage
OXAZEPAM
OXAZEPAM
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
Growth factor
Growth factor receptor
GTP
GDP
Inactivation by
Hydrolysis of GTP
BLOCKED IN
MUTANT RAS
Activation of
MAP kinase pathway
Activation of transcription
Cell cycle progression
Robbins and Cotran
Pathologic Basis of Disease, 2005
H-ras Proto-Oncogene Evaluation
H-ras Mutations in Hepatocellular Adenomas
and 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.
Tumors with Mutations
11/20 (55%)
13/37 (35%)
2/25 (8%)
0/21 (0%)
Historical Control - H-ras mutations, 80/126 (63%)
Hepatoblastomas - No H-ras Mutations
Tumor Incidence
28/50 (56%)
36/50 (72%)
50/50 (100%)
47/50 (94%)
Revisit Hypothesis:
Assessment of Mutations in Cancer Genes
b-catenin Gene
Oxazepam
Exposure
Liver
Tumors
Induction of
Cytochrome
p450
b–Catenin
Mutations
Oxygen
Radicals
Genetic
Alterations in
Cancer Genes
DNA
Damage
Assessment of Mutations in Cancer Genes from
Oxazepam Induced Liver Tumors

b-Catenin gene



First cancer gene where mutations identified in both
mouse and human hepatocellular neoplasms
Mutations of b-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
b-catenin Protein
Cancer
APC
Normal Cell
Cancer Cell
Adapted, Science 281: 1439, 1998
Assessment of Mutations in Cancer Genes from
Oxazepam Induced Liver Tumors

b-Catenin gene

Determine the mutation frequency and pattern of
b-catenin mutations in spontaneous and oxazepam
induced hepatocellular neoplasms and
hepatoblastomas

Determine if the b-catenin protein accumulated in
oxazepam induced liver tumors
Strategy for Evaluating DNA from
Tumors for Mutations
Single-Strand Conformational Analysis (SSCA)
SSCA Analysis
b-catenin Mutations
N
1
2
3
4
5
6
7
8
9
10
Devereux, T.R, Sills, R.C., Barrett, J.C et al.,
Oncogene, 18: 4726-4733, 1999
Direct Sequencing
b-catenin Mutations
A
C
G
Codon C
C
41
T
A
C
G
T
C
C
A/G
A
Normal
Mutation
Mutation Frequency of b-Catenin
in Hepatocellular Tumors of B6C3F1
Treatment
Control
Oxazepam
Mutation Frequency
2/22 (9%)
18/42 (41%)
a
a
p<0.001 when comparing mutation frequency to controls
Devereaux, Sills, Barret et al., Oncogene 18, 4726, 1999
Examples of b-Catenin Mutations in B6C3F1Mouse
Hepatocelluar Neoplasms
Tumor
Group
Codon
Mutation
Bases
Control
32
33
GAT to GCT
TCT to TTT
A to C
C to T
Oxazepam
32
32
32
32
33
33
34
34
34
41
GAT to GGT
GAT to GTT
GAT to CAT
GAT to AAT
TCT to TAT
TCT to TTT
GGA to AGA
GGA to GTA
GGA to GAA
ACC to ATC
A to G
A to T
G to C
G to A
C to A
C to T
G to A
G to T
G to A
C to T
Devereux, Sills, Barrett et al., Oncogene 18, 4726-33, 1999
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
b-Catenin Protein Expression
Immunohistochemical Detection
b-catenin Protein
B6C3F1 Mouse
Cancer cells
Science, 281, 1439, 1998
Hepatoblastoma
Anna, Sills, Devereux et al. Cancer Res., 60, 2864, 2000
b-catenin Mutations in Hepatoblastomas
From B6C3F1 Mice Treated with Oxazepam
Tumor
Group
Frequency
Oxazepam
8/8 (100%)
Codon mutation (amino acid)
1
Codon 32 GAT to GGT (Asp to Gly)
+ Del. Codons 5-8
2
3
4
5
6
7
8
Del. Codons 5-7
Del. Codons 36-48
Del. Codons 23-49
Codon 34 GGA to GTA (Gly to Val)
Del. Codons 5-13
Del. Codons 16-36
Del. Codons 21-43
Anna, C.H., Sills, R.C., Devereux et al.,
Cancer Res., 60, 2864-2868, 2000
Western Blot Analysis of Proteins Associated
with b-Catenin Mutations and Cancer
Methylene Chloride
b-Catenin
Mutation
Oxazepam
1
N
2
–
3
–
4
+
5
+
6
+
7
+
8
+
9
+
10
+
4
1
0
0
0
1
15
18
30
43
N
A
C
C
C
C
C
A
A
C
Cyclin D1
C-Myc
Actin
Anna, CH., Ida, M., Sills, R.C., Devereux, T.R.,
Tox. Appl. Pharmacology, 190: 135-145, 2003
Summary
Oxazepam
Tissue
Liver
Cellular
Molecular
Centrilobular
Endogenous
DNA Damage
Hepatocellular
Hypertrophy
Source of
b-catenin gene
Neoplasms
Cytochrome p450
Superoxide

b-catenin protein
Anion Radicals

Cyclin D1
Hepatoblastomas
Case Study in Rats and Mice
Hemangiosarcomas
Riddelliine
Genotoxic
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)




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 activated riddelliine metabolites
32P-postlabeling/HPLC method for identification of
riddelliine-derived DNA adducts
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
Rats
Mice
Cho, M.W., Chan, P. et al.,
Cancer Letters, 193: 119-125, 2003
Molecular Level
Assessment of Mutations in Cancer Genes
Hypothesis
Riddelliine
Exposure
Liver
Hemangiosarcomas
Induction of
Cytochrome
p450
P53 Mutations
Ras Mutations
Dehydroretronecine
(DHR)
Metabolite
DHR-Derived
Adducts
– Cancer Genes
DNA
Rationale for Evaluating Cancer Genes
in Hemangiosarcomas of Mice

Vinyl chloride induce similar tumors in humans and rats

Vinyl chloride
adducts
genotoxic intermediates

N2-ethenoguanine: K-ras G

N6-etheno adduct: p53 A
DNA etheno
A transitions
T transversions
Mutation Analysis of K-ras Oncogene
Hemangiosarcomas
B6C3F1 Mice
Mutation
Frequency
K-ras
Codon 12
Spontaneous
Hemangiosarcomas
0/13a
GGT (Normal Sequence)
Riddelliine
Hemangiosarcomas
7/12
aVarious
GGT
GTT (Mutation)
Hemangiosarcomas in control B6C3F1 mice in NTP studies
Direct Sequencing
K-ras Mutations
A C G T
A C G T
K-ras
Codon
13
C
G
G
Codon
12
T
G
G
T
G/T
G
Normal
Mutation
p53 Gene

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
immunohistochemistry

Most commonly mutated tumor suppressor gene in human cancer
detected by
S
p53
G1
Cell
Cycle
G2
M
Apoptosis
Nature Reviews, Genetics 2001
P53 Protein Expression
Riddelliine
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
0.7
DHR
N-oxide
0.6
0.5
0.4
0.3
0.2
0.1
0
MR FR M1 M2 M3 M4 F1 F2 F3 F4
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