Risk Mamagement for Human Carcinogen Benzoapyrene Byung Mu

Risk Mamagement for Human Carcinogen: Benzo[a]pyrene Byung Mu Lee Div. of Toxicology, College of Pharmacy Sung. Kyun. Kwan University

Best estimate proportions of various risk factors associated w/ cancer deaths (Lee & Park, Mut. Res. 2003) (Adapted after Doll & Peto, 1981) 35% 1. Diet 30% 2. Tobacco 10% 3. Infection 7% 4. Reproductive & sexual behavior 4% 5. Occupation 3% 6. Geophysical factors 3% 7. Alcohol 2% 8. Pollution 1% 9. Medicines & medical procedures 1% 40% 35% 30% 25% 20% 15% 10. Industrial products <1% 11. Food additives <1% 12. Unknown 5% 0%

Chemical and physiological property of benzo(a)pyrene Chemical Name Benzo(a)pyrene CAS Registry Number 50 -32 -8 Synonyms 3, 4 -Benz(a)pyrene, 3, 4 -Benzopyrene, 3, 4 -Benzpyrene, 6, 7 Benzopyrene, Benzo(d, e, f)chrysene, B(a)P, Benzopyrene (nonspecific name) Chemical Family Aromatic hydrocarbon / polycyclic aromatic hydrocarbon / PAH Molecular Formula C 20 H 12 Molecular Weight 252. 32 Relative Density 1. 315 (water = 1) Melting Point Boiling Point Coefficient of Oil/Water Distribution 179 -179. 3 ℃ 310 -312 ℃ at 10 mm. Hg Solubility in Water Practically insoluble (3. 8 x 10 -6 g/L) Solubility in Other Liquids Soluble in benzene, toluene, xylene, cyclohexan, acetone, ether, dimethyl sulfoxide, dimethylformamide Log P(oct) = 6. 06

Historical Background of PAHs and Ba. P - 1775, Pott, chimney sweeper’s scrotal cancer coal tar - 1915, Yamagiwa & Ichigawa, experimental skin tumor in rabbits - 1930, Kennaway & Hieger, dibenzanthracene in coal tar skin tumor Cook et al. , identification of benzo(a)pyrene & ben(a)anthracene in coal tar skin tumor 1933, - 1951, Miller, protein binding theory carcinogen-protein adduct - 1964, Brookes & Lawley, DNA binding theory carcinogen-DNA adduct - 1970’s, Weinstein et al. , BPDE-DNA adduct :

List of PAHs Acenaphthene Acenaphthyne Anthracene Benz[a]anthracene Benzo[a]pyrene Benzo[e]pyrene Benzo[b]fluoranthene Benzo[g, h, I]perylene Benzo[i]fluouranthene Benzo[k]fluoranthene Chrysene Dibenz[a, h]anthracene Fluoranthene Fluorene Indeno[1, 2, 3 -c, d]pyrene Phenanthrene Pyrene These 17 PAHs were chosen to be included in ATSDR profile. • more information • more harmful • greater chance • the highest concentrations

Structure of PAHs Acenaphthene Benzo[a]pyrene : bay region Acenaphthyne Benzo[e]pyrene Anthracene Benzo[a]anthracene Benzo[b]fluoranthene Benzo[g, h, I]perylene

Structure of PAHs Benzo[i]fluouranthene Fluoranthene : bay region Benzo[k]fluoranthene Chrysene Fluorene Indeno[1, 2, 3 -c, d]pyrene Dibenz[a, h]anthracene Phenanthrene Pyrene

CERCLA Priority List of PAHs 300 250 200 150 PAH (8) BENZO(A)ANTHRACENE (35) BENZO(A)PYRENE (9) BENZO(B)FLUORANTHENE (10) DIBENZO(A, H)ANTHRACENE (15) ACENAPHTHENE (165) BENZO(K)FLUORANTHENE (61) CHRYSENE (140) FLUORANTHENE (115) FLUORENE (270) INDENO(1, 2, 3 -CD)PYRENE (183) PHENANTHRENE (249) PYRENE (259) 100 50 400 35 20 15 10 5 1997 1999 2001 2003 Ranking years The Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), The Agency for Toxic Substances and Disease Registry (ATSDR), the Centers for Disease Control & Prevention (CDC) <275 Chemicals>

PAH compounds and their TEFsa a. Data PAH TEF Naphthalene Acenaphthylene Acenaphthene 0. 001 Fluorene Phenanthrene Anthracene Fluoranthene 0. 001 Pyrene Cyclophenta[c, d]pyrene Benzo[a]anthrencene Chrysene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[e]pyrene 0. 001 ＿b 0. 1 0. 01 0. 1 ＿b Benzo[a]pyrene Perylene Indeno[1, 2, 3 -c, d]pyrene Dibenzo[a, h]anthracene Benzo[b]chrycene Benzo[ghi]perylene Coronene 1 ＿b 0. 1 1 ＿b 0. 01 ＿b from Nisbet and Lagoy (1992). b No TEF(toxic equivalent factor) has been suggested (Li et al. , Environ Health Perspect, 111, 483 -487, 2003)

Fig. 6. Structure of bay-region diol-epoxides of several polycyclic aromatic hydrocarbons. (From Conney. )

Carcinogens formed in cooking foods Classification Structure & chemical names Furfural Furans Nitrosamines Polycyclic Aromatic Hydrocarbons (PAHs) Dibenzofuran 2, 3, 7, 8 -Tetrachlorodibenzofuran 2, 3, 7, 8 -Hexachlorodibenzofuran 2, 3, 7, 8 -Heptachlorodibenzofuran 1, 2, 3, 7, 8 -Pentachlorodibenzofuran 2, 3, 4, 7, 8 -Pentachlorodibenzofuran 1, 2, 3, 4, 6, 7, 8, 9 -Octachlorodibenzofuran Dimethylnitrosamine (DMN) Diethylnitrosamine (DEN) N-nitroso-morpholine (NMOR) N-nitrosopyrrolidine (NPYR) N-nitrosopiperidine (NPIP) Acenaphthene, Acenaphthylene, Anthracene, Benz[a]anthracene, Benzo[a]pyrene, Benzo[e]pyrene, Benzo[b]fluoranthene, Benzo[g, h, i]perylene, Benzo[j]fluoranthene, Benzo[k]fluoranthee, Chrysene, Dibenz[a, h]anthracene, Fluoranthene, Fluorene, Indeno[1, 2, 3 -c, d]pyrene, Phenanthrene, Pyrene (17)

Carcinogens formed in cooking foods Classification Heterocyclic amines (HCAs) Acrylamide Structure & chemical names 2 -amino-3 -methylimidazo[4, 5 -f]quinoline (IQ) 2 -amino-3, 4 -dimethylimidazo[4, 5 -f]quinoline (Me. IQ) 2 -amino-3, 8 -dimethylimidazo[4, 5 -f]quinoxaline (Me. IQx) 2 -amino-3, 4, 8 -trimethylimidazo[4, 5 -f]quinoxaline (Di. Me. Qx) 2 -amino-1 -methyl-6 -phenylimidazo[4, 5 -b]pyridine (Ph. IP) 3 -amino-1, 4 -dimethyl-5 H-pyrido[4, 3 -b]indole (Trp-P-1) 2 -amino-9 H-pyrido[2, 3 -b]indole (A alpha C) 3 -amino-1 -methyl-5 H-pyrido[4, 3 -b]indole (Trp-P-2)

Carcinogen category OSHA- Occupational Safty and Health Administration, U. S. Department of Labor Group ORC : OSHA Regulated Carcinogen Group S : OSHA Select Carcinogen IARC- International Agency for Research on Cancer Group 1 : Carcinogenic to humans Group 2 A : Probably carcinogenic to humans Group 2 B : Possibly carcinogenic to humans NTP- National Toxicology Program, U. S. Department of Health and Human Services Group 1 : Known to be human carcinogens (K) Group 2 : Reasonably anticipated to be human carcinogens (R) ACGIH- American Conference of Governmental Industrial Hygienists Group A 1 : Confirmed human carcinogen Group A 2 : Suspected human carcinogen Group A 3 : Confirmed animal carcinogen with unknown relevance to humans USEPA : A, B 1, B 2, C, D, E

Carcinogen category of Individual Chemicals Chemical Name PAHs OSHA IARC NTP ACGIH Benz[a]anthracene S 2 A 2 Benzo[a]pyrene S 1 2 A 2 Benzo[b]fluoranthene S 2 B 2 A 2 Benzo[j]fluoranthene S 2 B 2 Benzo[k]fluoranthene S 2 B 2 Dibenz[a, h]anthracene S 2 A 2 Indeno[1, 2, 3 -c, d]pyrene S 2 B 2 Dec. 13, 2006

Toxicity of Benzo(a)pyrene (Ba. P) § Cancer – Lung, stomach, skin, pancreas, colon, breast § Mutation – Ba. PDE-protein adduct (Miller, 1954) Ba. PDE-DNA adduct (Weinstein et al. 1976) Ba. PDE-lipid-adduct (Kwack & Lee, 2000) § Reproductive disorders: Birth defects Low birth weight High incidence of sterility Impaired reproductive capacity § Systemic toxicity – Aplastic anemia, pancytopenia, Immune suppression § Endocrine disruption – (EDCs)

Toxic Effects - General Toxicity - Genetic Toxicity - Reproductive Toxicity - Developmental Toxicity - Carcinogenicity

Table 1. Acute Toxicity of Ba. P Species (Strain) Route Results Mouse (CD-1) Oral LD 50 - F 344 Rat Oral F 344 Rat Ref. Adverse hematological effects Salamone, 1981 25200 mg/kg, 6 to 96 h Acute neurotoxicityinhibition in the activity of superoxide dismutase and glutathione peroxidase and an enhancement in cataloes and lipid peroxidation Saunders et al. , 2006 Oral 0200 mg/kg, 2 to 96 h changes in neuromuscular, autonomic, sensorimotor and physiological functions Saunders et al. , 2001 Wistar Rat Gastrointesti nal 150 mg/kg Hepatic and Renal effects Nousiainen et al. , 1984 C 57 BL/ 6 Mouse Skin 0. 001 mg/c m 2, 1 to 2 days Induction of melanocytes Iwata et al. , 1981 Guinea pig Skin Slight contact hypersensitivity Oid et al. , 1963 232 mg/kg

Table 2. Subchronic Toxicity of Ba. P Species (Strain) Route Results Ref. Mouse (C 57 BL /6) Oral (Feed) 120 mg/k Decreased survival, Bone marrow depression, Proliferation leading to aplastic anemia, hemorrhage or infection Robinson et al. , 1975 F 344 rat (6 week old female) Oral 2 mg/kg Adverse endocrine effect of decreased numbers of thymic glucocorticoid receptors) Csaba et al. , 1991 Hamste rs inhalatio n 9. 8 mg/ m 3, 16 weeks Lack of weight gain Thyssen et al. , 1980 Human inhalatio n 0. 0001 m g/m 3, 6 months to >6 years Reduced lung function, abnormal chest X-ray, cough, blood vomit, throat and chest irritation Gupta et al. , 1993 Chicken Pigeons Intramu scular injection 0. 1100 mg/ kg, 6 months Increases in aorta plaque volume Adverse cardiovascular effects Revis et al. , 1984 OEHHA, 1994 g/day, 6 months

Table 3. Genetic toxicity of Ba. P in vivo Species Test system End points Results Ref. Mouse Dominant lethals Gene mutation + Epstein 1968; Generoso et al. 1982; Russell 1977 Mouse Spot test Gene mutation + Davidson & Dawson 1976, 1977 Rat hepatocytes Unscheduled DNA synthesis DNA damage - Miralis et al. , 1982 Mullaart et al. , 1989 Mouse germ cells Unscheduled DNA synthesis DNA damage - Sega 1979 Mouse Lung cell DNA binding, adduct formation + Mukhtar et al. , 1986 Mouse Skin cell DNA binding, adduct formation + Morse et al. , 1985; Rice et al. , 1984; Weyand & Bevan, 1987 Mouse Bone marrow, Spleen cells DNA binding + Wielgosz et al. , 1991

Species Test system End point Results Ref. Rat Liver parenchymal cell DNA single strand breaks + Mullaart et al. , 1989 Rat Liver nonparenchymal cell DNA single strand breaks - Mullaart et al. , 1989 Rat Intestinal cells DNA single strand breaks + Mullaart et al. , 1989 Mouse Bone marrow Chromosome aberrations + Adler & lngersen, 1989 Adler et al. , 1989 Mouse (ICR) Spleen cells Chromosome aberrations + Choi et al. , 1996 Mouse Embryoes Chromosome aberrations + Adler et al. , 1989 Chinese hamster Bone marrow Chromosome aberrations + Roszinsky-Kocher et al. , 1979; Bayer, 1978 Mouse Heritable translocation Chromosome aberrations - Generoso et al. , 1982 Mouse Bone marrow, spleen cells Chromosome aberrations + Wielgosz et al. , 1991

Species Test system End points Results Reference Chinese hamster Bone marrow Sister chromatid exchange + Roszinsky-Kocher et al. , 1979; Bayer 1978 Mouse Bone marrow Micronuclei + Salamone et al. , 1981 Mouse Bone marrow Micronuclei - Bruce and Heddle, 1979 Mouse Fetal liver Micronuclei + Harper et al. , 1989 Mouse Bone marrow Micronuclei + Awogi and Sato, 1989 Mouse Keratinocytes Micronuclei - He and Baker, 1991 Mouse Bone marrow Micronuclei + Shimada et al. , 1990 Mouse Peripheral blood reticulocytes Micronuclei + Shimada et al. , 1992 Rat Peripheral blood reticulocytes Micronuclei + Shimada et al. , 1992 Bone marrow Micronuclei - Bayer 1978 Chinese hamster

Species Test system Mouse End points Results Ref. Sperm abnormalities + Shimada et al. , 1992; Salamone & Logan, 1988 Mouse Bone marrow Micronuclei + Balansky et al. , 1994 Mouse Bone marrow Micronuclei + Koratkar et al. , 1993 Mouse papilloma cell Gene mutation + Colapiero et al. , 1993; Di. Giovanni et al. , 1993 Swiss albino mice Bone marrow Micronuclei, Chromosome aberrations + Samarth et al. , 2006 Rat Lung and Liver (in vivo and in vitro), electron spin resonance (ESR/EPR) spectrometry assay DNA adducts + Briede et al. , 2004 Oyster Embryo-larvae, metaphase chromosome aberration Gene mutation + Cheung et al. , 2006 Zebrafish embryos, comet assay DNA damage + Kosmehl et al. , 2006 Flounder Fish Blood cell, Comet assay DNA breakage, DNA damage + Woo et al. , 2006

Perna viridis ( 녹색담치) Haemocytes, Comet assay & micronucleus assay DNA strand breakage, Micronucleus formation + Siu et al. , 2004 Rat Primary culture, GC/MS analysis DNA adduct formation + Topinka et al. , 1998 CD-1 Mice Skin and lung DNA adduct formation + Booth et al. , 1999 Human MRC 5 CV 1 cell, comet assay DNA damage, DNA migration + Speit et al. , 1996 Human Lung DNA damage + Weston et al. , 1993 Human Hep. G 2 cell (liver hepatoma cell), single cell gel electrophoresis (SCGE) assay Comet assay DNA damage, DNA migration + + Park et al. , 2006; Mersch et al. , 2006; Uhl et al. , 2000 + Valentin et al. , 2003 DNA damage Human Jurkat T-cells, Comet assay DNA damage, Enzyme down regulation + Oh et al. , 2004

Table 3. Reproductive toxicity of Ba. P Species (Strain) Exposure/Duration/ Frequency (Route) Results Ref. Mouse (white swiss female mice) 19 to 29 days, feed, 133. 3 mg/kg ·day Reduced percentage of pregnant females Rigdon & Neal, 1965 Pregnant F 344 rats 0. 1% Ba. P in diet Stillbirth Rigdon & Neal, 1964 Zebrafish 0 -3 ug/L, for 56 days, waterborne exposure Altered gene expression, decreased spawning success, decreased ovarian somatic index (OSI) and lower circulating levels of 17 beta-estradiol and vitellogenin Hoffmann & Oris, 2006 F 344 Rats 25 -10 ug/m 3, inhalation, GD 11 to 20 Plasma progesterone, estrogen, and prolactin concentration declined Archibong et al. , 2002 F 344 Rats 50 mg/kg/day, pregnant rats significantly increased Glutathione S-transferase (GST) activity Cervello et al. , 1992 Pigeons Intramuscular injections for 3 to 5 months Infertility with ovarian abnormalities Hough et al. , 1993

Species (Strain) Mouse (CD-1) Exposure/Duration/ Frequency (Route) 10 days, gestation days 7 to 16 (Gavage), 160 mg/kg/day Results Reduced percentage of pregnant females Ref. Mackenzie and Angevine, 1981

Table 4. Developmental toxicity of Ba. P Species (Strain) Exposure/Duration/ Frequency (Route) Results Ref. F 344 Rats 50 mg/kg/day, pregnant rats Fetal resorptions and fetal wastage, decreasing the fetal weight. Cervello et al. , 1992 Mouse (B 6 AKF 1) 8 days in gestation days 2 to 10, 120 mg/kg/day Fetal resorption Legravverend et al. , 1984 Mouse (CD-1) 10 days in gestation days 7 to 16 (Gavage), 40 mg/kg/day Reduced pup weight at 20 days Mackenzie & Angevine, 1981 Mouse (NMR 1) 7 to 16 days of pregnancy (Oral intubations), 10 mg/kg/day Reduced fertility, few ovarian follicies Kristensen et al. , 1995 SD Rats 100 ug/m 3, inhalation, GD 15 to 21 Fetus brain development abnormality, Fetus is in utero neurotoxicity Hood et al. , 2000 Pregnant mice, inhalation Altered development of T lymphocytes Rodriquez et al. , 1999 Mouse

Table 5. Carcinogenic Effects and Levels of Ba. P Species (Strain) Exposure/Durati on/Frequency (Route) Dose without observable cancer Mouse (white swiss) Oral (Feed), 1 to 7 days 13. 3 33. 3 Gastric neoplasma Neal & Rigdon, 1967 Mouse (white swiss) Oral (Feed), 30 to 197 days 1. 3 2. 6 Gastric tumors Neal & Rigdon, 1967 Mouse (white swiss) Oral (Feed), 23 to 238 days 33. 3 Papillomas, Squamous cell carcinomas Rigdon & Neal, 1967 Mouse (white swiss) Oral (Feed), 80 to 140 days Forestomach tumors Rigdon & Neal, 1967 Rat (SD) 2 years (feed and gavage in a 1. 5% caffeine solution) Forestomach, Esophagus, Larynx tumors at all doses Brune et al. , 1981 mg/kg·day Lowest dose w/ observable cancer mg/kg·day Ref.

Species (Strain) Hamster (Syrian golden) Exposure /Duration/ Frequency (Route) Dose without observ able cancer Lowest dose with observable cancer Ref. 109 weeks 7 days/week 3 to 4. 5 hours/day, Respiratory digestive 9. 5 mg/m 3 34. 6% increase in respiratory tract tumors, 26. 9% in upper digestive tract neoplasmas Thyssen et al. , 1981 Mouse (SENCAR) First then 23 rd week, twice/week, Skin 20. 2 mg (F) 6 papilomas per mouse Cavalieri et al. , 1988 Mouse (white swiss) 20 week, twice/week, once/day, skin 0. 025 mg (F) Tumors in 90% Cavalieri et al. , 1988 Mouse (NMR 1) 17 to 22 months, 2 days/week, once/day, skin 1. 7 ug(0. 016 mg/kg·day) (F) 45% skin tumors Habs et al. , 1980 Mouse (C 3 H/Hr. J) 99 weeks, 2 days/week, once/day, Skin 12. 5 ug (F) Malignant tumors in 47/50 Warshawsky & Barkley, 1987

Species (Strain) Exposure/Durat ion/Frequency (Route) Dose without observable cancer Lowest dose with observable cancer Ref. Thyssen et al. , 1981 CD-1 mouse skin exposure 200 nmol, Mouse (SENCAR) First then 23 rd week, twice/week, Skin 20. 2 mg (F) 6 papilomas per mouse Cavalieri et al. , 1988 Mouse (white swiss) 20 week, twice/week, once/day, skin 0. 025 mg (F) Tumors in 90% Cavalieri et al. , 1988 Mouse (NMR 1) 17 to 22 months, 2 days/week, once/day, skin 1. 7 ug(0. 016 mg/kg·day) (F) 45% skin tumors Habs et al. , 1990 Mouse(C 3 H/Hr. J) 99 weeks, 2 days/week, once/day, Skin 12. 5 ug (F) Malignant tumors in 47/50 Warshawsky & Barkley, 1987

Schematic representation of the multistage process of carcinogenesis Carcinogens Ultimate carcinogen DNA damage Mutation Transformation Proliferation Hyperplasia Papilloma Carcinoma

Benzo(a)pyrene P 450 s(CYP 1 A 1, CYP 1 B 1) Epoxides GSH(GST) Glutathione MRP 2 Phenols UDPGA conjugates EH Discharge UGTs Diols UDPGA Glucuronides P 448 Diol epoxides Sulphates GSTs Radical cation Fe++ Ca++ Quinones OH • UGTs Glucuronides Oxidative DNA damage Phenol epoxides Tetraols Protein oxidation Glutathione MRP 2 Discharge conjugates Lipid peroxidation DNA adducts AKRs NAD(P)+ NAD(P)H Catechols O • 2 H 2 O 2 Sulphates Repair DNA adducts Oxidative damage O-semiquinone anion radical O 2 O • 2 Oxidative damage BP-7, 8 -Dione Stable adducts(N 2 -d. Guo, N 6 -d. Ado) Depurinating adducts(N 7 -Gua, N 7 -Ade) Abbreviation: Ade, Adenine; AKRs, Aldo-keto reductases; d. Guo, 2’-deoxyguanosine; EH, Epoxide hydrolase; GSH, Glutathione; GSTs, Glutathione-S-transferases; Gua, Guanine; MRP, Multidrug resistance proteins; P 450 s, Cytochrome P 450 s; UDPGA, Uridine diphosphate-glucuronic acid; UGTs, UDP-glucuronosyltransferases. (Ramos, Annu Rev Pharmacol Toxicol. 39, 243 -265. 1999; Wolf, Proc Natl Acad Sci U S A. 98, 2941 -2943, 2001)

(+) BPDE - I (-) BPDE - I (+) BPDE - II (-) BPDE - II Fig. 1. Chemical structures of (±) BPDE-I and -II.

Epidemiological data of Ba. P Study Sample type Case. PBLs from control -69 female pilot study breast cancer OR 3. 36 CI Result Ref. 1. 15 -9. 80 Women with reduced DRC Shi et may be at an increased al. , risk of developing breast 2004 cancer patients -79 controls Prospectiv -214 incident -Risk of lung e cohort cases of lung cancer among 0. 82 -3. 62 study cancer smokers of <20 -669 controls cigarettes/day: 1. 72 1. 07 -7. 33 -Homozygous deletion of GSTM 1 : 2. 80 the I 462 V polymorphism London is not related to lung et al. , cancer overall, but it 2000 might play a role at lower levels of cigarette smoking among subjects with impaired carcinogen detoxification as assessed by the GSTM 1 -null genotype PBLs, Peripheral blood lymphocytes; DRC, DNA repair capacity

Ba. PDE-DNA Adducts in Human Tissues Excisability & persistence of benzo(a)pyrene DNA adducts in epithelioid human lung cells (Feldman G, et al. , Nature, 274, 796 -8, 1978) Detection of smoking-related covalent DNA adducts in human placenta (Everson RB, et al. , Science, 231, 54 -7, 1986) Correlation of DNA adduct levels in human lung and cigarette smoking (Phillips DH, et al. , Nature, 336, 790 -2, 1988) Preferential formation of benzo(a)pyrene adducts at lung cancer mutational hotspots in P 53 (Denissenko MF, et al. , Science, 274, 430 -2, 1996) Association of Ba. PDE-DNA adducts & oxidative DNA damage w/ human stomach cancer (Lee BM et al. , Cancer Lett. , 125, 61 -68, 1998)

Ba. P DNA adduct detection in humans Country Parameter Sample(n) Levels Ref. Italy Anti-B[a]PDE Blood, -DNA adduct urine levels Padua municipal 1. 51± 2. 68 workers(438) Occupationally exposed 4. 07± 3. 78 workers(95) Pavanello et al. , 2005 France Level of BPDE-N(2)d. G non-cancerous bronchus of lung cancer(22) Lung parenchyma(14) 0. 9± 1. 5 Rojas et al. , 2004 Smoker(13) Non-smoker(3) 84. 7± 38. 4 37. 6± 22. 2 Bronchial epithelial cells 75. 8± 35. 8 USA DNA adduct formation Breast tissues Cancer cases(76) 134. 6± 21. 2 Noncancer controls(60) 66. 9± 7. 5 Li et al. , 2002 UK 14 C B[a]P DNA adduct levels Human breast tissue Administered 5 ug of Lightfoot et al. , 2000 14 C B[a]P 6. 61 -208. 38

Country Parameter Italy PAH DNA adduct (107 nts*) Sample(n) Oral cells Tobacco smokers(33) Nonsmokers(64) Levels 330± 98 286± 83 Ref. Romano et al. , 1999 1 -10 cigarettes/d(16) 305± 105 11 -20 group(14) 347± 77 > 20 group(3) 386± 112 Italy BPDE DNA adducts (108 nts*) Autopsy samples from the lungs Non-smokers(15) Ex-smokers(5) Smokers(10) Germany (+)-anti. BPDE DNA Adduct (108 nts*) Human colon mucosa Colon mucosa(7) 0. 2 -1. 0 Human pancreas(11) Alexandrov et al. , 1996 Korea Stomach tissues (N=211) -Tumor tissues(211) -Normaltissues(113) -Smokers H. pylory + (47) H. pylory - (66) Lee et al. , 1998 Lee et al. , 2005 BPDE-DNA adduct (fmol/ ug DNA) 1. 76± 1. 69 4. 04± 2. 37 4. 46± 5. 76 4. 20± 0. 59 2. 80± 0. 53 4. 92± 0. 82 2. 63± 1. 54 2. 58± 1. 21 Lodovici et al. , 1998

Ba. P & Polycyclic Aromatic Hydrocarbons (PAHs) 1) Exposure sources ▷ The incomplete burning of coal, oil, gas, wood, garbage, tobacco, ▷ Medicine, Dyes, Plastics, Pesticides, Asphalt, Crude oil, Coal tar pitch, Soot, Smoke, Creosote, Roofing tar, Rubber tire wire ▷Foods: Grilled or charred meats; contaminated cereals, flour, bread, vegetables, fruits, steak, beverages, & processed or pickled foods ▷ Heating protein, Carbohydrate, etc. 2) Exposure routes ▷ Air, Water, Soil, Foods ▷ Breathing, Eating, Drinking, Skin contact (ATSDR, 1995)

Benzo(a)pyrene conc. in foods Food Concentration (㎍ /kg dry weight) Remarks Ref. Larsson and Sahlberg, 1981 Lettuce 6. 2 Grown at 12 m from a Swedish highway (㎍ /kg fresh weight) Wheat grain 0. 3 From Broadbalk (UK) Jones et al. , 1989 Soybean oil 28. 4 Refinement Kolarovic and Traitler, 1982 105. 7 Refinement Kolarovic and Traitler, 1982 Groundnut oil Mussel of fish Egg 1 0. 0 Egg 2 7. 5 Mytills galloprovincialis from Fort Brescou (France) Peudotolithus elongatus from Lagos (Nigeria) Samples studied by Lodovici et al. Samples studied by Hudain et al. (㎍ /kg wet weight) 54. 2 Grilled on a log fire 0. 7 Fresh fish 44. 0 Smoked fish 48. 0 Frankfurters Baumard et al. , 1998 Akpan et al. , 1994 Lodovici et al. , 1995 Hudain et al. , 1997 Larsson et al. , 1983

Estimates of general population exposure to benzo(a)pyrene and PAHs (㎍/day) Source Benzo(a)pyrene Select Carcinogenic PAHsa Total PAHs Air 0. 0095 -0. 0435 0. 038 0. 207 Water 0. 0011 0. 0042 0. 027 Food 0. 16 -1. 6 - 1. 6 -16 a. Total of benzo(a)pyrene, benzo(j)fluoranthene and indeno(1, 2, 3 -c, d)pyrene. (Santodomito et al. , 1981)(Lee & Choi, 1994)

Estimated dietary exposureto PAHs from the whole diet (ng/kg bw/day) Average dietary exposure 1979 2000 B(a)Pa Adult High level dietary exposure 1979 2000 B(a)Pa 2. 4 0. 5 -1. 6 4. 4 1. 0 -2. 6 Agee- 15 -18 years 2. 3 0. 5 -1. 4 4. 2 1. 0 -2. 5 Agee- 11 -14 years 2. 8 0. 6 -1. 8 4. 7 1. 0 -3. 0 Agee- 7 -10 years 4. 1 1. 9 -2. 6 6. 8 1. 7 -4. 0 Agee- 4 - 6 years 5. 0 1. 0 -3. 3 7. 8 1. 9 -5. 0 Age 3. 5 -4. 5 4. 9 1. 0 -3. 1 7. 8 1. 8 -4. 8 Age 2. 5 -3. 5 5. 1 1. 0 -3. 4 8. 8 2. 1 -5. 4 Age 1. 5 -2. 5 5. 3 1. 0 -3. 8 9. 6 2. 2 -6. 2 Schoolchildren Toddlers a. Range represents lower bound to upper bound estimates (Committee on toxicity, 2002)

Regulations of Benzo(a)pyrene 종류 국가 기준 (maximum Level) 수질 미국 Benzo(a)pyrene등 9종의 합 : 0. 2 ppb 네덜란드 (1997) Maximum permissible concentrations in water : 0. 05 ppb 유럽 (EEC) 1. 2 ppb USEPA (1993) Clean Water Act Water Quality Criteria: Based on fish/shellfish and water consumption = 0. 0038 ug/L based on fish/shellfish consumption only = 0. 018 μg/L (benzo[a]anthracene, benzo[a]pyrene 등) 음용수 Safe Drinking Water Act Maximum Contaminant Level (MCL) = 0. 2 ug/L (benzo[a]pyrene) Arizona (USA, HSDB, 1993) California (USA, DHS, 1994) New Hampshire (USA, 1993) 0. 003 ppb 0. 2 ppb 0. 003 ppb 미국 (FDA) Maximum permissible level in bottled water : 0. 2 ppb WHO 0. 7 ppb (1996), 0. 2 ppb(PAHs, 1971) 유럽 (EEC) 0. 2 ppb

<계속> 종류 음용수 국가 기준 (maximum Level) 캐나다 0. 01 ppb 네덜란드 0. 1 ppb § Foods for infants and young children: 1 ppb § Baby foods and processed cereal-based foods for 식품 유럽 (EU, 2005) § § 독일 훈연식품 유럽 (EU, 2005) infants & young children(1) : 1 ppb Infant formulae and follow-on formulae, including infant milk & follow-on milk(2) : 1 ppb Dietary foods for special medical purposes(3) intended specifically for infants : 1 ppb Bivalve molluscs : 10 ppb In food as a result of the use of smoke flavorings: 0. 03 ppb 1 ppb § Smoked meats and smoked meat products : 5 ppb § Muscle meat of smoked fish and smoked fishery products(4) : 5 ppb § Muscle meat of fish(5), other than smoked fish : 2 ppb § Crustaceans, cephalopods, other than smoked : 5 ppb (1) Baby foods and processed cereal-based foods for infants and young children as defined in Article 1 of Directive 96/5/EC. The maximum level refers to the product as sold. 2 ( ) Infant formulae and follow-on formulae as defined in Article 1 of Directive 91/321/EEC. The maximum level refers to (3) the product as sold. Dietary foods for special medical purposes as defined in Article 1 of Directive 1999/21/EC. The maximum level refers to the product as sold. Fish and fishery products as defined in the category (b), (c), and (f) of the list of Article 1 of Regulation (EC) N° 104/2000. 5 ( ) Fish as defined in the category (a) of the list of Article 1 of Regulation (EC) N° 104/2000 (4)

<계속> 종류 국가 기준 (maximum Level) Oils and fats intended for direct human consumption or EU (2005) Olive-residue oil use an as ingredient in foods(6): 2 ppb 스페인 5 ppb 중국 10 ppb 한국 (2006. 10. 11 식약청 입 안예고) 2 ppb 스웨덴 (2005) 2 ppb (However, the sum of the specified PAHs may not exceed 5 ppb oil) 국제올리브오일협회 (IOOC) 권고치 2 ppb 캐나다 3 ppb (6) Cocoa butter is excluded from this category whilst investigations into the presence of benzo(a)pyrene in cocoa butter are made. This derogation will be reviewed by 1 April 2007.

Effect of broiling on the formation of benzo(a)pyrene in foods (units: ㎍ /㎏ ) Samples Broiling time (min) a) 0 Beef nd c) 5 10 15 20 0. 007± 0. 006 b) 0. 127± 0. 026** 0. 254± 0. 086 0. 632± 0. 091* Rice 0. 002± 0. 001 0. 003± 0. 003 0. 012± 0. 003* 0. 225± 0. 035** 0. 302± 0. 047 Soybean 0. 005± 0. 001 0. 010± 0. 001** 0. 138± 0. 063* 0. 400± 0. 149** 0. 604± 0. 171* Pacific saury nd 0. 008± 0. 001 0. 013± 0. 007 0. 108± 0. 017** 0. 550± 0. 149* Pork chop nd 0. 016± 0. 002 0. 213± 0. 017** 0. 365± 0. 016** 0. 596± 0. 115* Pork belly nd 0. 026± 0. 003 0. 243± 0. 003** 0. 533± 0. 046** 0. 851± 0. 417* Sample were broiled on frypan, b) Values are means± SD of three experiment, c) nd=not detected. *Significantly different from increasing broiling time (*: p<0. 05, **: 0. 01, student’s t-test). (Lee & Choi, J. Fd. Hyg. Safty, 9, 133 -139, 1994) a)

Effect of boiling on the formation of benzo(a)pyrene in foods (units: ㎍ /㎏ ) Broiling time (min) Samples a) 0 5 10 15 20 Beef Nd b) 0. 007 0. 015± 0. 004 0. 022± 0. 008 0. 021± 0. 006 Rice 0. 002± 0. 001 c) 0. 003± 0. 002± 0. 008 0. 010± 0. 009** 0. 024± 0. 009 Soybean 0. 005± 0. 001 0. 008± 0. 011± 0. 003 0. 016± 0. 010± 0. 007 Pacific saury Nd 0. 021± 0. 009 0. 027± 0. 010 0. 037± 0. 011 0. 031± 0. 012 Pork chop Nd 0. 018± 0. 009 0. 021± 0. 003 0. 028± 0. 002 0. 037± 0. 011 Pork belly nd 0. 026± 0. 010 0. 032± 0. 011 0. 033± 0. 003 0. 035± 0. 007 Sample were boiled in water, b) nd=not detected. c) Values are means± SD of three experiment. **Significantly different from 10 min boiling time (p<0. 01). (Lee & Choi, J. Fd. Hyg. Safty, 9, 133 -139, 1994) a)

Estimation of benzo[a]pyrene levels in broiled foods and expected daily intake in Koreans Samples B[a]P levels (㎍ /㎏) Beef 0. 19 0. 254 a) Rice 5. 51 0. 225 1. 240 Soybean 0. 74 0. 400 0. 296 Pacific saury 0. 86 0. 108 0. 093 Pork c) 0. 54 0. 449 0. 288 a)Measured b) Estimated human daily intake of B[a]P (ng/㎏ /day) Consumption of agricultural product(g/㎏ /day) 0. 048 b) from the samples broiled for 15 minutes, Estimated for adults (60㎏ body weight), c) Pork belly and pork chop are included. (Lee & Choi, J. Fd. Hyg. Safty, 9, 133 -139, 1994)

Estimation of benzo[a]pyrene levels in boiled foods and expected daily intake in Koreans Samples B[a]P levels (㎍ /㎏) Beef 0. 19 0. 021 a) 0. 004 b) Rice 5. 51 0. 024 0. 132 Soybean 0. 74 0. 010 0. 007 Pacific saury 0. 86 0. 031 0. 027 Pork c) 0. 54 0. 036 0. 020 a)Measured b) Estimated human daily intake of B[a]P (ng/㎏ /day) Consumption of agricultural product(g/㎏ /day) from the samples broiled for 15 minutes, Estimated for adults (60㎏ body weight), c) Pork belly and pork chop are included. (Lee & Choi, J. Fd. Hyg. Safty, 9, 133 -139, 1994)

Human daily intake and cancer risk assessment in Koreans (㎍ /㎏ ) a) Cancer risk assessment 0. 88 X 10 -3 1. 32 X 10 – 8 Boiled foods c) 0. 011 1. 65 X 10 – 7 Broiled foods d) 0. 118 1. 77 X 10 -6 Samples Raw foods b) Human daily intake a) Estimated for adults (60㎏ body weight), b) Foods were not heated, c) Foods were boiled for 20 min, d) Foods were broiled for 15 min. (Lee & Choi, J. Fd. Hyg. Safty, 9, 133 -139, 1994)

Identification of Ba. PDE-lipid Adducts

TLC analysis of total lipid treated in vitro with B(a)P and (±)BPDE-I. Triglyceride C PD wi E-I th m C ixe d ix e m P a) B( )B wi th d ts ra of d pi Li (± er um -I PD E )B (± B( a) P (±)BPDE-I-lipid adducts spot Basic molecular structure of triglyceride. (R 1, R 2 and R 3 are fatty acid chains of palmitic acid, stearic acid or oleic acid) (Kawck and Lee, Carcinogenesis, 21, 629 -632, 2000)

Two-dimensional TLC analysis of TG reacted with [1, 3 -3 H]BPDE-I isomers [1, 3 -3 H]BPDE-I-TG adducts Dose-related formation of [1, 3 -3 H]BPDE-I-TG in triglyceride (50 g) treated with [1, 3 -3 H]BPDE-I TG (±)BPDE-I TG HPLC analysis of (±)BPDE-I-TG adduct in TG treated with (±)BPDE-I (Kawck and Lee, Carcinogenesis, 21, 629 -632, 2000) (±)BPDE-I-TG adduct

Effect of broiling on the formation of benzo(a)pyrene in foods (units: ㎍ /㎏ ) Samples Broiling time (min) a) 0 5 10 15 20 Beef nd c) 0. 007± 0. 006 b) 0. 127± 0. 026** 0. 254± 0. 086 0. 632± 0. 091* Rice 0. 002± 0. 001 0. 003± 0. 003 0. 012± 0. 003* 0. 225± 0. 035** 0. 302± 0. 047 Soybean 0. 005± 0. 001 0. 010± 0. 001** 0. 138± 0. 063* 0. 400± 0. 149** 0. 604± 0. 171* Pacific saury nd 0. 008± 0. 001 0. 013± 0. 007 0. 108± 0. 017** 0. 550± 0. 149* Pork chop nd 0. 016± 0. 002 0. 213± 0. 017** 0. 365± 0. 016** 0. 596± 0. 115* Pork belly nd 0. 026± 0. 003 0. 243± 0. 003** 0. 533± 0. 046** 0. 851± 0. 417* Sample were broiled on frying pan, b) Values are means± SD of three experiment, c) nd=not detected. *Significantly different from increasing broiling time (*: p<0. 05, **: 0. 01, student’s t-test). a) (Lee & Choi, J. Fd. Hyg. Safty, 9, 133 -139, 1994)

Effect of boiling on the formation of benzo(a)pyrene in foods Samples (units: ㎍ /㎏ ) Boiling time (min) a) 0 5 10 15 20 Beef Nd b) 0. 007 0. 015± 0. 004 0. 022± 0. 008 0. 021± 0. 006 Rice 0. 002± 0. 001 c) 0. 003± 0. 002± 0. 008 0. 010± 0. 009** 0. 024± 0. 009 Soybean 0. 005± 0. 001 0. 008± 0. 011± 0. 003 0. 016± 0. 010± 0. 007 Pacific saury Nd 0. 021± 0. 009 0. 027± 0. 010 0. 037± 0. 011 0. 031± 0. 012 Pork chop Nd 0. 018± 0. 009 0. 021± 0. 003 0. 028± 0. 002 0. 037± 0. 011 Pork belly nd 0. 026± 0. 010 0. 032± 0. 011 0. 033± 0. 003 0. 035± 0. 007 Sample were boiled in water, b) nd=not detected. c) Values are means± SD of three experiment. **Significantly different from 10 min boiling time (p<0. 01). a) (Lee & Choi, J. Fd. Hyg. Safty, 9, 133 -139, 1994)

Estimation of benzo[a]pyrene levels in broiled foods and expected daily intake in Koreans Samples B[a]P levels (㎍ /㎏) Beef 0. 19 0. 254 a) Rice 5. 51 0. 225 1. 240 Soybean 0. 74 0. 400 0. 296 Pacific saury 0. 86 0. 108 0. 093 Pork c) 0. 54 0. 449 0. 288 a)Measured b) Estimated human daily intake of B[a]P (ng/㎏ /day) Consumption of agricultural product(g/㎏ /day) 0. 048 b) from the samples broiled for 15 minutes, Estimated for adults (60㎏ body weight), c) Pork belly and pork chop are included. (Lee & Choi, J. Fd. Hyg. Safty, 9, 133 -139, 1994)

Estimation of benzo[a]pyrene levels in boiled foods and expected daily intake in Koreans Samples B[a]P levels (㎍ /㎏ ) Beef 0. 19 0. 021 a) Rice 5. 51 0. 024 0. 132 Soybean 0. 74 0. 010 0. 007 Pacific saury 0. 86 0. 031 0. 027 Pork c) 0. 54 0. 036 0. 020 a)Measured b) Estimated human daily intake of B[a]P (ng/㎏ /day) Consumption of agricultural product(g/㎏ /day) 0. 004 b) from the samples broiled for 15 minutes, Estimated for adults (60㎏ body weight), c) Pork belly and pork chop are included. (Lee & Choi, J. Fd. Hyg. Safty, 9, 133 -139, 1994)

Human daily intake and cancer risk assessment in Koreans (㎍ /㎏ ) a) Cancer risk assessment 0. 88 X 10 -3 1. 32 X 10 – 8 Boiled foods c) 0. 011 1. 65 X 10 – 7 Broiled foods d) 0. 118 1. 77 X 10 -6 Samples Raw foods b) Human daily intake a) Estimated for adults (60㎏ body weight), b) Foods were not heated, c) Foods were boiled for 20 min, d) Foods were broiled for 15 min. (Lee & Choi, J. Fd. Hyg. Safty, 9, 133 -139, 1994)

BPDE-I-DNA adduct levels in stomach tissues of patients Gastric adenocarcinoma (Lee et al. , Cancer lett. , 125, 61 -68, 1998

8 -Hydroxydeoxyguanosine contents in stomach tissues of patients with gastric adenocarcinoma and normal subjects (Lee et al. , Cancer lett. 125, 61 -68, 1998)

Effects of Aloe barbadensis Miller in rat hepatocytes The formation of [3 H]Ba. P metabolites-DNA adduct Time course inhibitory effect (Kim and Lee, Carcinogenesis, 18, 771 -776, 1997)

Effects of Aloe barbadensis Miller in rat hepatocytes The uptake of [3 H]Ba. P The cellular and subcellular distribution of [3 H]Ba. P metabolites (Kim and Lee, Carcinogenesis, 18, 771 -776, 1997)

Liver Kidney Forestomach Lung Effect of Aloe barbadensis Miller on BPDE-I-DNA adduct formation and persistence in male ICR mice orally treated with benzo[a]pyrene (0. 5 g/kg/day) and Aloe (2. 5 g/kg/day) (Kim and Lee, Carcinogenesis, 18, 771 -776, 1997)

Effects of Aloe barbadensis Miller on the cellular and subcellular distribution of [3 H]Ba. P metabolites in rat hepatocytes (Kim and Lee, Carcinogenesis, 18, 771 -776, 1997)

Table II. Comparative chemopreventive effects of plant polysaccharides in Ba. P-DNA adduct & cancer related biomarkers Biomarkers APS LPS GPS CPS Ba. P-DNA adduct formation ++ – ± ± 8 -OHd. G formation + ± ± + TK activity + ± ± ± ODC activity + – ± ± Superoxide anion formation + ± ± + ++Strong positive effect in a dose-dependent manner (P<0. 01). +Positive effect in a dose-dependent manner (P<0. 05). ±Moderate effect in a dose-dependent manner, but not significant. –No effect. (Kim, H. S. , Kacew, S. , and Lee, B. M. Carcinogenesis, 20, 1637 -1640, 1999)

Conclusion · Benzo(a)pyrene (Ba. P) is one of critical human carcinogens formed during cooking process & Koreans are expected to be exposed to it at relatively high levels. · Exposure to Ba. P needs to be reduced as low as possible by modifying cooking process. · Regal limits should be set for not only olive oil, but other food items which are expected to be a major sources of Ba. P exposure · Research on Ba. P formation & reduction during cooking process, & exposure assessment are required especially in Korean foods. · Potential health risk for Ba. P exposure could be reduced by chemoprevention.