Post on 21-Dec-2015
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
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
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
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 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
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
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
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
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
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
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
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?
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.