Molecular biology of liver tumors following chemical exposure robert c sills dvm phd diplomate acvp
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Molecular Biology of Liver Tumors Following Chemical Exposure Robert C. Sills, DVM, PhD Diplomate, ACVP. Overview. Molecular Pathology Review Molecular Pathology Case Studies Oxazepam (Mice) Hepatocellular neoplasms Hepatoblastomas Riddelliine (Rats and Mice) Liver hemangiosarcomas

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Molecular biology of liver tumors following chemical exposure robert c sills dvm phd diplomate acvp l.jpg

Molecular Biology of Liver Tumors Following Chemical ExposureRobert C. Sills, DVM, PhDDiplomate, ACVP


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Overview

  • Molecular Pathology Review

  • Molecular Pathology Case Studies

    • Oxazepam (Mice)

      • Hepatocellular neoplasms

      • Hepatoblastomas

    • Riddelliine (Rats and Mice)

      • Liver hemangiosarcomas

  • Research Team


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Neoplastic Liver LesionsB6C3F1 Mouse

Hepatocellular Adenoma

Hepatocellular Carcinoma

Hepatoblastoma

Hemangiosarcoma



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Major Genes Involved in Carcinogenesis

Proto-oncogenes

Tumor suppressor genes

  • 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


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Case Study in Mice

OxazepamNon-genotoxic


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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


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Incidences of Neoplasams and Nonneoplastic Lesions of the Liverof Female B6C3F1 Mice in the 2-Year Feed Study of Oxazepam

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

2-Year Study

Liver (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


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Approach for Evaluating the Liver Mechanism of Carcinogenesis B6C3F1 Mouse

Oxazepam

Tissue

Cellular

Molecular

?

Liver

Centrilobular

Hypertrophy

Cytochrome p450

Hepatocellular

Neoplasms

Hepatoblastomas

Bucher et al, 1994

Fund. Appl. Tox, 23, 280,1994

Griffen et al, 1995

Tox. Let. 76, 251,1995


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OXAZEPAM Liver

METABOLITES

OXAZEPAM

CANCER GENES

Cytochrome P450Oxidative Damage


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Isoprostane (oxidative damage marker) in Livers of LiverB6C3F1 Mice Following 6-months Oxazepam Exposure

70

60

(n=3)

50

40

(n=3)

30

20

10

0

Control

Oxazepam

Tomer, Devereaux, NIEHS, 2000


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Cytochrome P450 LiverOxidative Damage

OXAZEPAM

METABOLITES

OXAZEPAM

H-ras proto-oncogene

Hepatocellular Tumors Hepatoblastomas


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Ras Genes and Cancer Liver

  • 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


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Ras Signal Transduction Pathway Liver

Growth factor

Growth factor receptor

GTP

GDP

Inactivation by

Hydrolysis of GTP

Activation of

MAP kinase pathway

BLOCKED IN

MUTANT RAS

Activation of transcription

Cell cycle progression

Robbins and Cotran

Pathologic Basis of Disease, 2005



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H-ras Mutations in Hepatocellular Adenomas Liver

and Carcinomas from Oxazepam Treated Mice

Treatment

Tumor Incidence

Tumors with Mutations

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%)

Historical Control - H-ras mutations, 80/126 (63%)

Hepatoblastomas - No H-ras Mutations


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Revisit Hypothesis: LiverAssessment of Mutations in Cancer Genes b-catenin Gene

Oxazepam

Exposure

Liver

Tumors

Induction of

Cytochrome

p450

b–Catenin

Mutations

Oxygen

Radicals

DNA

Damage

Genetic

Alterations in

Cancer Genes


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Assessment of Mutations in Cancer Genes from LiverOxazepam 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


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b Liver-catenin ProteinCancer

APC

Cancer Cell

Normal Cell

Adapted, Science 281: 1439, 1998


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Assessment of Mutations in Cancer Genes from LiverOxazepam 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


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Strategy for Evaluating DNA from LiverTumors for Mutations



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SSCA Analysis Liverb-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


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G Liver

G

T

T

A

A

C

C

Codon

41

C

C

A

C

C

A/G

Normal

Mutation

Direct Sequencingb-catenin Mutations


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Mutation Frequency of Liverb-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


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Examples of Liverb-Catenin Mutations in B6C3F1Mouse Hepatocelluar Neoplasms

Tumor

Group Codon Mutation Bases

Control 32 GAT to GCT A to C

33 TCT to TTT C to T

Oxazepam 32 GAT to GGT A to G

32 GAT to GTT A to T

32 GAT to CAT G to C

32 GAT to AAT G to A

33 TCT to TAT C to A

33 TCT to TTT C to T

34 GGA to AGA G to A

34 GGA to GTA G to T

34 GGA to GAA G to A

41 ACC to ATC C to T

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


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Assessment of Mutations Liver

  • 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


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Oxazepam LiverHepatoblastomasb-Catenin Protein Expression


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Immunohistochemical Detection Liverb-catenin ProteinB6C3F1 Mouse

Cancer cells

Hepatoblastoma

Science, 281, 1439, 1998

Anna, Sills, Devereux et al. Cancer Res., 60, 2864, 2000


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b Liver-catenin Mutations in Hepatoblastomas

From B6C3F1 Mice Treated with Oxazepam

Tumor Group

Codon mutation (amino acid)

Frequency

Codon 32 GAT to GGT (Asp to Gly)

+ Del. Codons 5-8

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

Oxazepam

1

2

3

4

5

6

7

8

8/8 (100%)

Anna, C.H., Sills, R.C., Devereux et al.,

Cancer Res., 60, 2864-2868, 2000


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Western Blot Analysis of Proteins Associated Liverwith b-Catenin Mutations and Cancer

Methylene Chloride

Oxazepam

1

2

3

4

5

6

7

8

9

10

b-Catenin

N

+

+

+

+

+

+

+

Mutation

Cyclin D1

4

1

0

0

0

1

15

18

30

43

C-Myc

Actin

N

A

C

C

C

C

C

A

A

C

Anna, CH., Ida, M., Sills, R.C., Devereux, T.R.,

Tox. Appl. Pharmacology, 190: 135-145, 2003


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Summary Liver

Oxazepam

Tissue

Cellular

Molecular

Hepatocellular

Neoplasms

Hepatoblastomas

Liver

Endogenous

Source of

Superoxide

Anion Radicals

Centrilobular

Hypertrophy

Cytochrome p450

DNA Damage

b-catenin gene

  • b-catenin protein

  • Cyclin D1


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Case Study in Rats and Mice LiverHemangiosarcomas

RiddelliineGenotoxic


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Riddelliine Liver

  • 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


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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


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Metabolism of Riddelliine to Activated Metabolite Hemangiosarcomas in F344 Rats and B6C3F1Mice


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Metabolism of Riddelliine to Activated Metabolite Hemangiosarcomas in F344 Rats and B6C3F1Mice



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Relationships Between Administered Dose, Adduct Riddelliine for 3 and 6 MonthsLevels, and Hemangiosarcoma Incidence of Rats


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Tumor Incidence in Rats and Mice Riddelliine for 3 and 6 Months

Rats

Mice

Cho, M.W., Chan, P. et al.,

Cancer Letters, 193: 119-125, 2003


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Molecular Level Riddelliine for 3 and 6 MonthsAssessment of Mutations in Cancer Genes Hypothesis

Riddelliine

Exposure

Liver

Hemangiosarcomas

Induction of

Cytochrome

p450

P53 Mutations

Ras Mutations

Dehydroretronecine

(DHR)

Metabolite

DNA

DHR-Derived

Adducts

– Cancer Genes


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Rationale for Evaluating Cancer Genes Riddelliine for 3 and 6 Monthsin 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


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Mutation Analysis of K-ras Oncogene Riddelliine for 3 and 6 MonthsHemangiosarcomas B6C3F1 Mice

K-ras

Codon 12

Mutation

Frequency

0/13a

GGT (Normal Sequence)

Spontaneous

Hemangiosarcomas

7/12

GGT GTT (Mutation)

Riddelliine

Hemangiosarcomas

aVarious Hemangiosarcomas in control B6C3F1 mice in NTP studies


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Direct Sequencing Riddelliine for 3 and 6 MonthsK-ras Mutations

G

G

T

T

A

A

C

C

K-ras

C

G

G

Codon

13

T

G/T

G

T

G

G

Codon

12

Normal

Mutation


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S

Cell

Cycle

p53

G2

G1

M

Apoptosis

p53 Gene

Nature Reviews, Genetics 2001


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P53 Protein Expression arrest, promotes apoptosis and loss of function causes genomic instabilityRiddelliineHemangiosarcomas


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Relevance of Mechanistic Data to Humans arrest, promotes apoptosis and loss of function causes genomic instability

  • Do human liver microsomes metabolize riddelliine?

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


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Metabolism of Riddelliine to Activated Metabolite arrest, promotes apoptosis and loss of function causes genomic instability


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Human Liver Microsomal Metabolism arrest, promotes apoptosis and loss of function causes genomic instability

0.7

DHR

0.6

N-oxide

0.5

0.4

0.3

0.2

0.1

0

MR

FR

M1

M2

M3

M4

F1

F2

F3

F4

Rat

Human

Human


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32 arrest, promotes apoptosis and loss of function causes genomic instabilityP-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.


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Summary arrest, promotes apoptosis and loss of function causes genomic instability

  • 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.


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Research Team arrest, promotes apoptosis and loss of function causes genomic instability


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