62nd Tobacco Science Research Conference

Chemical Composition and Biological Activity of Base Fractions Derived

from Cigarette Smoke Condensate (CSC)1 Lijia YANG, 2 Michael F. Borgerding, 2 William M. Coleman

III, 2 Betsy R. Bombick, 2 Jeremy B. Mabe, 2 Kathy P. Putnam, 1 Larry T. Taylor

1 Virginia Tech, 2 R.J. Reynolds Tobacco Co.

September 23rd, 2008Nashville, Tennessee

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Outline1. Research objectives2. Previous studies3. Fractionation schemes4. Biological activity (Ames & NRU) of CSC

fractions5. Chemical composition of CSC fractions6. Summary

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Research Objectives - Mutagenicity1. Identify constituents of mainstream

cigarette smoke that are (a) mutagenic (b) anti-mutagenic, or (c) not active in the Ames Salmonella assay

2. Determine the relative contribution of the active constituents to the aggregate mutagenicity observed for the complex mixture (i.e. cigarette smoke)

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Research Objectives - Cytotoxicity3. Identify constituents of mainstream

cigarette smoke that (a) are cytotoxic, (b) mitigate cytotoxicity, or (c) are not active in the neutral red uptake assay

4. Determine the relative contribution of the active constituents to the aggregate cytotoxicity observed for the complex mixture (i.e. cigarette smoke)

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Lee C.K., Munoz J.A., Fulp C., Chang K.M., Rogers J.C., Borgerding M.F., Doolittle D.J.

Mutation Research 322, 21-32, 1994

◼ CSC exerts a significant inhibitory effect on mutagens requiring bio-activation

◼ CSC fractionated into neutral, weakly acidic, acidic, and basic fractions

◼ Fractions were effective as anti-mutagens against Glu-P-1, a representative heterocyclic amine

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Lee C.K., Munoz J.A., Fulp C., Chang K.M., Rogers J.C., Borgerding M.F., Doolittle D.J.

Mutation Research 322, 21-32, 1994

Fraction Mass (%) Ames* (%)

CSC 100.0 100.0

N1 23.8 4.2

N2 3.5 5.6

B1 2.1 2.7

B2 1.3 11.1

A1 1.0 0.0

A2 2.2 0.3

InP 6.8 15.8

Recovery 40.7 39.8

CSC 1R4F

Hexane Soluble Hexane Insoluble

Hexane

Neutrals (N1, N2)

Base (B1)

Base (B2)

Acids (A1, A2)

Insoluble (InP)

* TA98 + S9

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Muto H., Honjo K., Yoshida S., Higashi N. et al., CORESTACongress, Paris, 2006

◼ Methanol, diethyl ether, water and cyclohexanewere used as solvents for extraction and separation

◼ Freeze drying and water extraction resulted in loss of Ames activity

2R4FCSC

MethanolSolubles

MethanolInsolubles

Diethyl EtherInsolubles (DEI)

Diethyl EtherSolubles (DES)

1. Dried

2. Diethyl Ether (DE)

MeOH

62% Ames Activity 21% Ames Activity

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CSC Collection◼ AMESA Type 1300C smoke generator

◼ 2R4F Kentucky reference cigarettes

◼ Standard Federal Trade Commission (FTC) smoking conditions

◼ Cigarette smoke was delivered to a CSC cold-trapping system with a temperature range from -10C to -70C

◼ Condensate was kept frozen at -20C, and then was extracted from the trap by rinsing with high-purity acetone

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Initial Diethyl Ether (DE) Fractionation Scheme – This Study

CSC

DE PhaseInsoluble Residue

HCl Aqueous Phase

B-DE Aq.Phase

1. NaOH (2N), adjust pH to 112. Diethyl Ether (DE) 3. Dichloromethane (CH2Cl2)4. Ethyl Acetate (EtOAc)

1. Diethyl Ether (DE)2. HCl (0.5N)

1.NaHCO3 (5%)

2.NaOH (0.5N)

NaHCO3 Phase NaOH Phase DE Phase

1.HCl (2N) to pH 2

2.Diethyl Ether (DE)

1.HCl (2N) to pH 2

2.Diethyl Ether (DE)

Strong Acids (SA) Weakly Acids (WA)

Hexane 2% EtOAc/Hexane(E/H) 5% E/H Methanol

Florisil

10%E/H 25%E/H 50%E/HN1 N2 N3 N4 N5 N6 N7

B-CH2Cl2

200 mL solvent for each fraction

B-EtOAc

Neutral Fraction (NF)

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Precaution1. Butylated hydroxytoluene (BHT) is a preservative

in diethyl ether (6-10 ppm)

2. Observed in all our fractions

3. Interferes with efforts (GC-MS) to identify components in individual fractions

4. Reported to be anti-mutagenic or non–mutagenic

• Y. Yoshida, Mutation Research, 242, 209-217, 1990

5. Are Ames results influenced by presence of BHT in the fractions?

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Final Methyl t-Butyl Ether (MTBE) Fractionation Scheme – This Study

CSC

MTBE PhaseInsoluble Residue

HCl Aqueous Phase

B-MTBE Aq.Phase

1. NaOH (2N), adjust pH to 112. Methyl t-Butyl Ether (MTBE) 3. Dichloromethane (CH2Cl2)4. Ethyl Acetate (EtOAc)

1. Methyl t-Butyl Ether (MTBE)2. HCl (0.5N)

1.NaHCO3 (5%)

2.NaOH (0.5N)

NaHCO3 Phase NaOH Phase MTBE Phase

1.HCl (2N) to pH 2

2.Methyl t-Butyl Ether (MTBE)1.HCl (2N) to pH 2

2.Methyl t-Butyl Ether (MTBE)

Strong Acids (SA) Weakly Acids (WA)

Hexane 2% EtOAc/Hexane(E/H) 5% E/H Methanol

Florisil

10%E/H 25%E/H 50%E/HN1 N2 N3 N4 N5 N6 N7

B-CH2Cl2

200 mL solvent for each fraction

B-EtOAc

Neutral Fraction (NF)

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Biological Assays• Ames Mutagenicity Assay

◼ TA98 +S9◼ Preincubation ± S9 activation (5%)◼ Methodology in accordance with OECD Guideline 471

• Neutral Red Cytotoxicity Assay

◼ 24 hr. exposures

◼ Chinese hamster ovary (CHO) cells

◼ Methodology recommended by ICCVAM (Interagency

Coordinating Committee on the Validation of Alternative

Methods)

◼ Results expressed as EC50: effective concentration of test sample that damages and/or kills 50% of the cells

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Ames Assay Results: MTBE Fractions

0

400

800

1200

1600

Am

es R

eve

rta

nts

/m

g

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Comparison of Ames Activity with DE versus MTBE FractionationDE (with BHT) MTBE (without BHT)

Fraction

Ames TA98

with S9 (Rev/mg)

Weight%

Weighted Revertants

(WR)Fraction

Ames TA98 with

S9 (Rev/mg)

Weight%

Weighted Revertants

(WR)

CSC* 634 100.0 634 CSC* 634 100.0 634Base-

DE 1,772 6.4 113 Base -MTBE 1,681 6.2 104

Base-CH2Cl2

1,831 3.0 55 Base -CH2Cl2

1,213 2.1 25

Base-EtOAc NA 0.6 NA Base -

EtOAc 649 0.3 2

Neutral 517 27.3 141 Neutral 536 31.3 168WA 404 16.2 65 WA 102 3.5 4SA 74 2.3 2 SA 79 1.6 1

* CSC was assayed “as is” without either DE or MTBE extraction.

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Recovery of Ames Activity vs. Recovery of Mass

(MTBE Fractions)

WR: Weighted Revertants

303 634

WA)SANFBBWR(B CSC EtOAcClCHMTBE 22

==

+++++

48%100634

303(Ames)Recovery ==

55%(mass)Recovery =

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Recovery of Ames Activity vs. Recovery of Mass

(DE Fractions)

WR: Weighted Revertants

376 634

WA)SANFBWR(B CSC22ClCHDE

==

++++

59%100634

376(Ames)Recovery ==

66%(mass)Recovery =

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Potential Sources of Lost Ames Activity

• Insoluble precipitate • Discarded aqueous phases• Volatile compounds lost during nitrogen

purge• Compounds irreversibly sorbed on the

Florisil stationary phase

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NRU Assay Results: MTBE Fractions

0

40

80

120

160

NR

U E

C5

0(µ

g/m

L)

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EC50 From Neutral Red Uptake Cytotoxicity Assay versus CSC Fraction

FractionsDE (With BHT)

(µg/mL)

MTBE (Without BHT)

(µg/mL)

CSC* 65.0

Base-DE/MTBE 37.1 69.7

NF 57.5 41.6

SA 68.6 84.0

WA 74.4 53.4

* CSC was assayed “as is” without either DE or MTBE extraction.

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Chemical Identification of Base Fraction Components

GC-MS Conditions

◼ Columns: DB-Waxetr (polar), DB-1701 (mid-polar), DB-1 (non-polar) , 30m x 0.25mm x 0.25m (J&W Scientific)

◼ Temperature programs: 1. 50C (1 min) 5C /min to 150C, 3C /min to 255C (10 min); 2. 37C (2 min) 2.5C /min to 255C (10 min)

◼ Helium flow: 1 mL/min◼ Injector temperature: 250C .◼ GC/MS interface temperature: 260C◼ Electron Impact mode ◼ MS scan range: 29-450 amu

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Components Identified In Base-MTBE Fraction versus Reported Ames Activity

1 Name in bold font indicates the compound was identified by both mass spectral and retention time match with authentic standard2 CCRIS: http://toxnet.nlm.nih.gov/ , 3 NA: Not available in CCRIS

Name1 Reported Ames 2

Nicotine -2,3'-Bipyridine + Myosmine NA 3

1H-Indole-2-acetonitrile, 1-methyl- NANorharmane -Harmane +?Nornicotine NATriacetin -Piperidine, 1-methyl-2-(3-pyridyl)- NA4-Hydroxy-benzeneethanol NA4,4'-Bipyridyl + Nicotyrine NA1,2-Benzenediol + Pyridine, 3-phenyl- NA4-Hydroxy-4-methyl-2-pentanone -4-Methyl-pyrrolo(1,2-A)Pyrazine NA

Name1 Reported Ames 2

Pentanamide, 4-methyl- NAAnabasine NA1H-Benzimidazole, 2-methyl- NA2-Ehyl-6-methyl-3-hydroxy pyridine NAQuinoline + Quinoline, 8-methyl- + Pyridine, 3-methyl- -Pyridine -1-Acetyl-beta-carboline NAPyridine, 3-ethenyl- -Pyridine, 2-methyl- -Quinoline, 4-methyl- + Pyridine, 3-butyl- NAFurfural + Corylone NA2-Furanmethanol -

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Components Identified In Base-MTBE Fraction versus Reported Ames Activity (cont.)

1 Name in bold font indicates the compound was identified by both mass spectral and retention time match with authentic standard

2 CCRIS: http://toxnet.nlm.nih.gov/3 NA: Not available in CCRIS

Name1 Reported Ames 2

4-Methylthio-2-butanone NA3

2,3-Dimethylcyclopent-2-en-1-one NAIsoquinoline -2-Propanone, oxime -2-Furancarboxaldehyde, 5-methyl- -Thiazole, 4,5-dimethyl- NAQuinoline, 2,6-dimethyl- NAPyridine, 3-ethyl- NAPyridine, 2,4-dimethyl- NAOxazole, trimethyl- NAMethional -2-Pentenal, 4,4-dimethyl- NAPyridine, 2-ethyl- NAThiocyanic acid, methyl ester NAAetonitrile, (dimethylamino)- NA

Name1 Reported Ames 2

Cyclopentanone NA3-Penten-2-one NA2-Methyl-2-cyclopenten-1-one NAQuinoline, 5-methyl- NAQuinoline, 5,6,7,8-tetrahydro- NAQuinoline, 1,2,3,4-tetrahydro- NAPyridine, 2,6-dimethyl- NAPyridine, 2,3-dimethyl- NA3-Penten-2-one, 4-methyl- NA2-Acetyl-pyridine -Pyridine, 4-methyl- -Pyrazine, 2,6-dimethyl +3-Acetylpyridine NAPyrazine, 2-methyl- -3-Methyl-2-cyclopenten-1-one NA

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Summary – Activity Found by Fraction

◼ MTBE Solvent: Base fractions exhibited the highest Ames mutagenic activity (rev/mg); Neutral fractions exhibited the greatest cytotoxicity

◼ DE Solvent: Base fractions exhibited the highest Ames mutagenic activity (rev/mg); Base fractions exhibited the greatest cytotoxicity

◼ The recovery of Ames activity for CSC fractions was about 50-60% of whole CSC

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Future Research◼ Determine biological activity of identified

compounds in base fractions◼ Spike identified compounds into base

fractions and CSC to assess potential antimutagenic activity and cytotoxicity mitigation potential

◼ Ascertain chemical composition and biological activity of neutral sub-fractions

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Acknowledgement

◼ This research was supported by a gift from R. J. Reynolds Tobacco Company

◼ Dr. Lijia Yang was selected as an RJRT-Harold McNair Fellow in 2007

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