• Identify risk factors and outcome
• Biomarkers
• Carcinogen-macromolecular adducts
• Normal DNA sequence variants
• Mutations in target genes
• Measure in urine, serum or tissue
• Immunoassays
• GC/MS, LC/MS
• Florescence spectometry
May 2nd, 2005 Megan McBee-BE.450
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• Annual new cases ~600,000
• ~600,000 annual deaths
– 80% burden in Asia and subsaharan Africa
– 300,000+ cases in People’s
Republic of China
• High risk areas early age of onset 20’s
• Low risk areas early age of onset 50’s
May 2nd, 2005 Megan McBee-BE.450
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• Main causes in high risk areas
– HBV infection
– Aflatoxins in diet
• Synergism leading to increased risk
HBV vaccination aflatoxin exposure
May 2nd, 2005 Megan McBee-BE.450
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• Taiwan 1 : BsAg+ males with HCC compared to control subjects
• OR = 2.8 detectable vs. nondetectable aflatoxin metabolites
• OR = 5.5 high vs. low urinary metabolite levels
• Shanghai 2 : relative risk for HCC with presence aflatoxin metabolites = 3.8
1.
2.
Wang LY et al. Int J Cancer. 1996 Sep 4;67(5):620-5.
Ross RK et al. Lancet. 1992 Apr 18;339(8799):943-6.
May 2nd, 2005 Megan McBee-BE.450
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O
O
O
H
• Produced by fungi
– 1960 outbreak of “Turkey
‘X’ disease” in UK
– Aspergillus flavus
• Common in corn, peanuts, fermented soy products
May 2nd, 2005
Figure by MIT OCW.
Megan McBee-BE.450
O
H
O
Aflatoxin B
1
OCH
3
Cytochrome
P450
O
O
O
H
O
O
H O
OCH
3
Active DNA-modifying agent
6

• Primary
– Vaccination
– Reduced contamination
• Secondary
– Pharmaceuticals
– Natural products
May 2nd, 2005 Megan McBee-BE.450
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HCC Prevention/Intervention
Hepatitis B Virus vaccination
HBsAg (serum)
Insertional Mutagenesis
Aflatoxin B
1 chlorophyllin
1762 T /1764
(serum)
A
Double Mutation
X-Gene Mutations antioxidants
Chronic Inflammation
Reactive Intermediates oltipraz, ITCs
Promutagenic DNA Lesions p53 Gene Mutations
(G:T-T:A transversions)
Aflatoxin-Mercapturic
Acid (urine)
Aflatoxin-N 7 -guanine
(urine)
249 ser Mutations
(serum) antiviral drugs green tea polyphenols,
COX-2 inhibitors, ITCs, vitamin K analogs, retinoids
Selective Clonal Expansion and p53 Allelic Deletion
May 2nd, 2005
Cell Proliferation Chronic Hepatitis and/or
Cirrhosis green tea polyphenols,
COX-2 inhibitors, ITCs, vitamin K analogs, retinoids oltipraz
Hepatocellular
Carcinoma
Figure by MIT OCW.
Megan McBee-BE.450
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• HBV vaccination
– Taiwan HCC cases in 6-14 year olds
• Born 1981-1986 = 0.70
• Born 1986-1990 = 0.57
• Born 1990-1994 = 0.36
• Reduction of aflatoxins in food
May 2nd, 2005
Chang MH et al. N Engl J Med. 1997 Jun 26;336(26):1855-9.
Megan McBee-BE.450
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Oltipraz
N
N
H
3
C
S
S
S
R N C S
May 2nd, 2005
Secondary Intervention
Chlorophyllin
H
3
C C
2
H
5 H
3
C
C
2
H
5
Isothiocyanates
H
2
C=HC
H
3
C
N
H
3
C
N
Cu
N
N
CH
3
COO
-
Na +
CH
2
COO Na +
CH
2
CH
2
COO
-
Na +
H
2
C=HC
H
3
C
N
H
3
C
N
Cu
N
N
CH
3
CH
2
COO Na +
CH
2
CH
2
COO
-
Na +
Trisodium Copper Chlorin e
6
HO
HO
7
6 5
8
A
OH
Polyphenols
R
OH
O
1 2
C
4 3
2 +
B
3 +
4 +
5 +
R +
HO
O
OH
R
OH
O HO
R +
Disodium Copper Chlorin e
4
O
OH O
R
OH
R
R +
OH
R +
OH
OH O
Megan McBee-BE.450
OH
Figures by MIT OCW.
10

Secondary Intervention: Oltipraz
• Oltipraz
– Induces phase 2 enzymes
– Inhibits phase 1 enzymes
• Higher doses (500mg+) not more effective at induction or inhibition than lower doses (125mg and
250mg)
May 2nd, 2005 Megan McBee-BE.450
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Source
: Kensler,T. W. “Chemoprevention by inducers of carcinogen detoxication enzymes.” Environmental Health Perspective 105,
Supplement 4 (1997): 965-970. Reproduced with permission from Environmental Health Perspectives.
May 2nd, 2005 Megan McBee-BE.450
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Secondary Intervention: Oltipraz
• Phase IIa intervention trial
– Feasibility of biomarker measurements
– Dose response
– Tolerance/effectiveness longer term exposure
– Chronic toxicity
Source
: Kensler, T. W., et al. "Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas."
Gastroenterology 127, no. 5, Supplement 1 (2004): S310-318.
May 2nd, 2005 Megan McBee-BE.450
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Secondary Intervention: Oltipraz
Location: Dazin Township, Qidong,
People’s Republic of China
– Randomized, placebo-controlled, double blind
– 240 adults without history of chronic disease
– Detectable serum aflatoxin-albumin adducts
– 3 intervention groups
1) Placebo
2) 125mg once daily
3) 500mg once weekly
Source
: Kensler, T. W., et al. "Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas."
Gastroenterology 127, no. 5, Supplement 1 (2004): S310-318.
May 2nd, 2005 Megan McBee-BE.450
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Secondary Intervention: Oltipraz
• 500mg weekly after 1 month
– 51% decrease median levels aflatoxin M
1 excretion
– No effect on aflatoxin-mercapturic acid
– Inhibits activation
• 250mg daily after 1 month
– 2.6-fold increase in median levels of aflatoxinmercapturic acid
– Modest effect on aflatoxin M
1
– Increase phase 2 conjugation levels
Source
: Kensler, T. W., et al. "Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas."
Gastroenterology 127, no. 5, Supplement 1 (2004): S310-318.
May 2nd, 2005 Megan McBee-BE.450
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Secondary Intervention: Oltipraz
• Ongoing follow-up phase IIb trial
– Sustained expression enhancement of aflatoxin detoxification enzymes
– 250mg versus 500mg once weekly for 1 year
– Measuring multiple biomarkers for mechanisms of action
May 2nd, 2005 Megan McBee-BE.450
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Secondary Intervention: Chlorophyllin
• Mixture of sodium-copper salts of chlorophyll
• OTC drug
– Wound healing accelerant
– Controls body, fecal and urinary odor
• In vitro and in vivo antimutagen in short-term genotoxicity assays
May 2nd, 2005 Megan McBee-BE.450
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Secondary Intervention: Chlorophyllin
• Complexes with aflatoxin B1
• Reduction in bioavailability
• Needs molar excesses to carcinogen for efficacy
• In vitro inhibitor of cytochrome P450 enzymes
• Antioxidant-reduction in lipid peroxidation
May 2nd, 2005 Megan McBee-BE.450
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Secondary Intervention: Chlorophyllin
• Chemoprevention study in Qidong
– 180 healthy adults
– 100mg chlorophyllin or placebo 3-times daily for 4 months
– Endpoint of modulated aflatoxinN 7guanine adducts in urine after 3 months
• Resulted in 55% decrease in median urinary adduct levels
Source
: Kensler, T. W., et al. "Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas."
Gastroenterology 127, no. 5, Supplement 1 (2004): S310-318.
May 2nd, 2005 Megan McBee-BE.450
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Secondary Intervention:
Isothiocynates
Source: Family Cruciferae (mustards),
Genus Brassica (cauliflower, Brussels sprouts, broccoli, cabbage)
– Lower cancer rates in individuals consuming high levels of yellow and green vegetables
• Isothiocyanates
• Particularly glucosinolate precursors
• Sulforaphane induces phase 2 enzymes in rats
(glucoraphanin is precursor)
May 2nd, 2005 Megan McBee-BE.450
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Secondary Intervention:
Polyphenols
Source: Green tea
– Inverse association of consumption versus risk and development of cancer
– Green tea-derived polyphenols
(ongoing study)
• Reduce aflatoxin M
2 excretion
• Increase aflatoxin-mercapturic acid excretion
• Reduced 8-oxo-deoxyguanosine
May 2nd, 2005 Megan McBee-BE.450
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• HBV vaccination
– Only benefits younger generations
– Vertical transmission not prevented
• Reduced food contamination
– Requires infrastructure for production, processing and distribution
– Monitoring mycotoxins $$
– Not feasible in developing countries
May 2nd, 2005 Megan McBee-BE.450
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• Not practical for populations at highest risk
– SE Asia, China, Africa
• First-generation (oltipraz) expensive
• 2nd and 3rd generation dithiolethiones
– Cheaper
– 10-fold increase in potency over oltipraz
– Ongoing safety evaluations
• Long-term costs potentially high, chronic treatment
May 2nd, 2005 Megan McBee-BE.450
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• Practical for populations at highest risk
– SE Asia, China, Africa
• Inexpensive, diet-based
• Long-term compliance better
• Immediate impact
May 2nd, 2005 Megan McBee-BE.450
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• Reduction of aflatoxin-N7guanine
– Reduced risk HCC in animals
– Increased latency period
• Decreased aflatoxin exposure in Bejing correlated with later onset of HCC
Image removed due to copyright reasons.
Source
: Kensler, T .
W .
, et al. “Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas.”
Gastroenterology Review 127, no. 5, Supplement 1 (2004): S310-318.
May 2nd, 2005 Megan McBee-BE.450
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