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Food and Chemical Toxicology 49 (2011) 1904–1917

Daftar isi tersedia di ScienceDirect

Food and Chemical Toxicology


journalhomepage: www. elsev yaitu r. com / locate / foodchemtox

Penggunaan proses perlakuan tembakau baru untuk mengurangi hasil racun


dalam asap rokok

a,⇑ b c a a b
Chuan Liu , Yves DeGrandpré , Andrew Porter , Alexander Griffiths , Kevin McAdam , Richard Voisine , Prancis
b British
Côté , Christopher Proctor

a American Tobacco, Grup Pusat Penelitian dan Pengembangan, Regents Park Road, Southampton SO15 8TL, Inggris Raya

b Imperial Tobacco Kanada Ltd, 3711 St.-Antoine Street West, Montréal, Québec, Kanada H4C
c
3P6 3515 Connaught, Montréal, Québec, Kanada H4B 1X4

abstrak
artikel info

Institut Kedokteran AS telah mendorong pengejaran dan pengembangan potensi


Riwayat artikel:
pengurangan paparan produk (PREP) - produk tembakau yang secara substansial
mengurangi paparan terhadap satu atau lebih toksisitas tembakau dan dapat
Diterima 1 November 2010 Diterima 18 Februari 2011 Tersedia daring 16 Maret diharapkan untuk mengurangi th risiko satu atau lebih penyakit tertentu atau efek
2011
kesehatan buruk lainnya. Salah satu pendekatan potensial adalah untuk mengurangi
tingkat beberapa prekursor toksiktor asap, seperti protein dan polifenol, dalam
tembakau. Kami menjelaskan proses pengobatan yang melibatkan ekstraksi tembakau
berair dan pengobatan dengan protease; filtrasi ekstrak untuk menghilangkan peptida,
Kata kunci:
asam amino dan polifenol, dan rekombinasi ekstrak dan tembakau yang diolah. Proses
ini mengurangi tingkat nitrogen protein (59%), polifenol (33-78%) dan nikotin (12%)
Asap beracun sementara gula meningkat 16%. Hasil asap utama ISO dari 43 toxicants diukur dari
rokok yang mengandung tembakau olahan; hasil rendah tar, nikotin, karbon
Tembakau monoksida (16-20%), akrilonitril, amonia, amina aromatik, piridin, kuinolena dan
hidrogen sianida (33-51%), nitrosamin tembakau spesifik (25-32%); fenolik (24-
Pengurangan protein Pengurangan 56%), benzena (16%), toluena (25%) dan kadmium (34%) diperoleh. Ada
peningkatan yang signifikan dari formaldehid (49%) dan iso-prene (17%).
polifenol
Pengurangan dalam hasil sidestream dari toxican asap nitrogen dan peningkatan hasil
sidestream dari beberapa karbonil, benzo (a) pyrene dan isoprene juga diamati.

2011 Elsevier Ltd. Buka


akses di bawah Lisensi CC
BY-NC-ND.

berpotensi mengurangi paparan (PREPS) sebagai cara yang mungkin untuk


mengurangi bahaya yang disebabkan oleh penggunaan tembakau. IoM
mendefinisikan PREP sebagai '' Produk yang (1) menghasilkan pengurangan
substansial dalam paparan terhadap satu atau lebih racun tembakau dan (2)
1. Pendahuluan dapat diharapkan untuk mengurangi risiko satu atau lebih penyakit tertentu
atau efek samping lainnya. efek kesehatan '' (Stratton et al., 2001).
Pada tahun 2001, Institut Kedokteran AS (IOM) mengeluarkan laporan,
Clearing the Smoke, yang mendorong pengembangan produk-produk yang Dari lebih dari 5000 konstituen yang diidentifikasi dari asap tembakau
(Rodgman dan Perfetti, 2008), sekitar 150 dianggap sebagai racun (Rodgman
dan Green, 2003; Fowles and Dybing, 2003). Belum diketahui yang mana
dari racun ini yang paling banyak penting dalam kaitannya dengan penyakit Pengurangan keseluruhan dalam mesin merokok mengukur hasil racun
yang disebabkan oleh merokok, meskipun beberapa peneliti telah berusaha dapat dicapai dengan melarutkan asap menggunakan ventilasi filter atau
mengembangkan model penilaian risiko untuk mencoba dan menetapkan menggunakan kertas rokok dengan permeabilitas tinggi, dan, dalam kasus
prioritas (Fowles and Dybing, 2003; Mere-dith et al., 2008). racun yang terkait dengan fase partikel asap, dengan meningkatkan efisiensi
penyaringan filter. Selama bertahun-tahun, pemerintah dan otoritas kesehatan
masyarakat di berbagai belahan dunia menganggap rendahnya tar ISO yang
menghasilkan rokok sebagai cara untuk mengurangi risiko kesehatan merokok
bagi mereka yang tidak berhenti merokok. Namun, modifikasi produk ini
⇑ Penulis yang sesuai. Tel .: +44 23 8079 3597; faks: +44 23 8058 8856. Alamat e- belakangan ini telah banyak dikritik oleh berbagai badan, termasuk
mail: chuan_liu@bat.com (C. Liu).
Institut(Kanker Nasional ASKanker Nasional AS). Institute, 2001). Kelompok
0278-6915 2011 Elsevier Ltd. Buka akses di bawah Lisensi CC BY-NC-ND. Studi tentang Peraturan Produk Tembakau (TobReg) dari Organisasi
Kesehatan Dunia (WHO, 2008; Burns et al., 2008) telah mengusulkan
doi:10.1016 / j.fct.2011.02.015 pendekatan pengaturan yang akan membatasi hasil dari kelompok tertentu
racun tertentu. Kelompok ini juga merekomendasikan bahwa hasil racun
harus dibatasi berdasarkan hasil mereka diukur dengan rezim mesin merokok
intens dan ditentukan per mg nikotin.

Pendekatan untuk secara selektif mengurangi racun asap spesifik yang


terkait dengan hasil tar dan nikotin yang diukur dengan mesin sangat
tergantung pada sifat fisiokimia dari racun individu. Parameter desain rokok
konvensional menawarkan ruang terbatas untuk
C. Liu et al. / Food and Chemical Toxicology 49 (2011) 1904–1917 1905
tersubstitusi pada
pengurangan relatif pada toxicants asap. Sebagai contoh, dengan
meningkatkan efisiensi filter dari filter selulosa asetat konvensional,
konstituen fase partikulat dikurangi dengan tar dan nikotin dan tidak ada
pengurangan selektif yang terjadi. Dan, karena filter selulosa asetat memiliki
sedikit atau tidak berpengaruh pada toksisitas yang mudah menguap,
peningkatan efisiensi penyaringan meningkatkan rasio hasil relatif terhadap
tar dan nikotin.

Meningkatkan ventilasi filter memiliki efek bervariasi pada toxicants.


Hasil absolut dari semua racun racun berkurang, tetapi, relatif terhadap tar
atau nikotin, hasil dari sebagian besar emisi fase partikulat tidak berubah.
Hasil dari beberapa racun yang mudah menguap, seperti amonia dan karbon
monoksida, berkurang relatif terhadap tar dan nikotin, sedangkan hasil relatif
dari beberapa racun semi-volatile seperti amina aromatik dan fenol meningkat
(Norman, 1999).).

Banyak komponen fase uap yang mudah menguap, seperti aldehida vol-
atile dan hidrogen sianida dapat direduksi secara selektif menggunakan bahan
adsorben dalam filter seperti arang aktif atau resin tertentu (Horsewell, 1975;
Branton et al., 2009). Namun, gas permanen, seperti karbon monoksida dan
nitrat oksida, tidak dapat menerima adsorpsi pada suhu kamar, dan racun
dalam fase partikulat tidak dapat dikurangi secara selektif dengan
penyaringan karena sebagian besar terikat ke dalam partikel aerosol.

Pendekatan yang paling menjanjikan untuk mencapai pengurangan


spesifik substansial dalam partikulat racun dari rokok yang disaring secara
konvensional adalah dengan memodifikasi tembakau. Pergantian varietas
tembakau yang berbeda ke dalam campuran dapat berdampak pada hasil dari
beberapa racun asap. Sebagai contoh ada hasil yang lebih tinggi dari nitrogen
yang mengandung racun asap dari tembakau burley daripada dari flue-cured
atau oriental, dan hasil yang lebih tinggi dari formaldehida dan katekol dari
tembakau tembakau yang dikeringkan (Baker, 1999). Namun, de-lipatan
dalam satu racun atau serangkaian racun sering diimbangi dengan
peningkatan toksisitas lainnya. Untuk menghindari hal ini, akan berguna
untuk mengidentifikasi dan menghilangkan prekursor untuk menghisap racun
dari daun tembakau.

Dengan pengecualian racun logam (kromium, nikel, arsenik, selenium,


kadmium, merkuri dan timbal) dan beberapa nitrosamine tembakau spesifik
(seperti NAT dan NAB) yang ditransfer langsung dari daun, mayoritas asap
toxi -cants dibentuk oleh pyrosynthesis dari komponen daun. Dengan
demikian, prekursor utama untuk karbonil volatil, benzo (a) pirena, karbon
monoksida, benzena dan toluena adalah karbohidrat struktural seperti pektin
dan selulosa serta gula (Baker, 1999).

Toksik beracun nitrogen terbentuk dari prekursor nitrogen di daun, dan


ada bukti yang cukup besar bahwa pembakaran pro-tein dan asam amino
berkontribusi terhadap pembentukan beberapa nitrogen yang mengandung
racun pada daftar Health Canada (ditunjukkan pada Tabel 4). Protein dan
asam amino telah diubah menjadi prekursor untuk hidrogen sianida (Johnson
dan Kang, 1971; Tso et al., 1982), piridin dan kuinolin (Higman et al., 1970;
Schmeltz et al., 1972), 2-aminonaphthalene dan 4-aminobi-phenyl (Torikaiu
et al., 2005). Protein tembakau juga sangat terkait dengan pembentukan amina
heterosiklik mutagenik dan mutagenisitas kondensat asap yang dihasilkan
dalam uji TA98 Ames (Mizusaki et al., 1977; Yoshida dan Matsumoto, 1980;
Clapp et al., 1999).

Polifenol dalam tembakau adalah prekursor utama untuk senyawa asap


fenolik. Asam klorogenik, polyphe-nol yang paling melimpah dalam
tembakau yang disembuhkan, adalah prekursor utama untuk fenol, katekol
dan katekol tersubtitusi (Zane dan Wender, 1963; Sakuma et al., 1982;
Schlotzhauer dkk., 1982; Sharma dkk., 2002; Wooten et al., 2006; Torikaiu et
al., 2005), sementara hydroquinone juga dilaporkan sebagai produk pirolisis
asam klorogenat (Wooten et al., 2006; Sakuma et al., 1982; Torikaiu et al.,
2005). Rutin dan asam caffeic juga menghasilkan katekol dan katekol
pirolisis (Wooten dkk., 2006; Schlotzhauer dkk., 1982) tetapi karena rekonstitusi menjadi bahan lembaran.
konsentrasinya yang rendah pada tembakau dan karena hasil pirolitiknya yang
lebih rendah, kontribusinya terhadap katekol dapat disembuhkan. asap Makalah ini menjelaskan proses perawatan dan pengaruhnya pada kimia
tembakau jauh lebih sedikit daripada asam klorogenik. Resorcinol dikenal bokelat, termasuk kuantifikasi enzim residual pada tembakau, hasil dari asap
sebagai produk utama dari pirolisis rutin (Zane dan Wender, 1963). rokok utama dan sidestream dari rokok yang dibuat dengan tembakau ini
ketika diasapi dalam kondisi merokok ISO, dan estimasi transfer potensial
Ada laporan proses untuk menghilangkan protein dari tembakau. Kung enzim yang digunakan untuk merokok.
dan Tso (1978) menggambarkan proses untuk ekstraksi protein berair dari
daun tembakau yang tidak diawetkan, terutama sebagai cara menggunakan
tembakau sebagai sumber protein tetapi juga sebagai sarana untuk
mengurangi potensi daun untuk menghasilkan beberapa racun racun. Proses 2. Eksperimental
ini membutuhkan daun untuk dihomogenisasi sebelum ekstraksi dan
2.1. Perawatan tembakau
disembuhkan dalam proses bubur (Tso et al., 1975) sebelum konversi ke
bahan lembaran dilarutkan. Pirolisis dari protein rendah ke-bacco sheet 2.1.1. Bahan
menunjukkan beberapa pengurangan dalam produksi tembakau spesifik
nitrosamine dan fenol dan peningkatan nitrosamine volatil dan benzo (a) Tembakau: Tembakau yang digunakan untuk pengobatan adalah campuran
pyrene (Woodlief et al., 1984). tembakau all-lamina yang disembuhkan, dipotong pada 35 potongan per inci
(sekitar 0,7 mm lebarnya).
Clapp dkk. (1999) dijelaskan sebuah proses untuk mengekstraksi protein
dari tembakau yang diawetkan, menggunakan ekstraksi air diikuti oleh Novozym 80001: Persiapan protease, saat ini diidentifikasi menurut
pencernaan protease. Tembakau yang diekstraksi, dalam bentuk bubur, sistem klasifikasi enzim sebagai EC 3.4.21.62, tetapi sebelumnya termasuk
bersama dengan pelarut air, dibuat menjadi lembaran yang dilarutkan dan dalam EC 3.4.21.14. Ini adalah protease serin bakteri dari keluarga subtilisin
digunakan untuk membuat rokok. Meskipun racun dalam asap tidak dengan spesifisitas luas untuk ikatan peptida (Outtrup dan Boyce, 1990). Ini
direporting, aktivitas tar dalam uji mutagenisitas Ames TA98 ditentukan dan diproduksi dengan bahan baku food grade dan di bawah kondisi kualitas food
pengurangan signifikan dalam aktivitas spesifik dibandingkan dengan kontrol grade, dan dipasok oleh Novozymes Inc. (Denmark); itu selanjutnya disebut
yang tidak dirawat dilaporkan. sebagai pro-menggoda, atau enzim terapan. Enzim murni adalah protein
globular yang terdiri dari 269 asam amino dalam rantai tunggal dengan berat
Dalam makalah ini efek protein dan penghilangan polifenol dari tembakau molekul 27,6 kD. Enzim diberikan dalam larutan berair pekat-ous dengan
pada hasil racun asap telah diteliti. Perlakuan to-bacco dilakukan pada konsentrasi protein 40 g / L. Larutan protease yang digunakan untuk
potongan, tembakau yang sudah dikeringkan, dan ekstraksi tembakau dengan mengolah tembakau terdiri dari 0,1 L larutan enzim pekat dan 0,05 L larutan
air yang diikuti dengan pengobatan dengan larutan enzim protease berair. kalsium klorida 1,05 M pada 65 L air terdeionisasi. Setelah pencampuran, pH
Setelah perlakuan ekstrak tembakau dengan adsorben dan konsentrasi, disesuaikan menjadi 10,2 dengan 8 M larutan natrium hidroksida tepat
solubles diaplikasikan kembali ke tembakau yang diekstraksi. Tembakau yang sebelum digunakan.
dirawat merapikan struktur tembakau asli dan dibuat menjadi cig-arettes
menggunakan peralatan pembuatan rokok konvensional, tanpa perlu
1906 C. Liu dkk. / Food and Chemical Toxicology 49 (2011) 1904–1917
perawatandigabungkan dan kelebihan air dikeluarkan dengan sentrifu-gation
Bentonit: tanah liat (Volclay Air Purified Bentonite, Amcol Minerals selama 20 menit pada 3600 rpm, menghasilkan 1640 kg tungau tembakau
Europe Ltd., Cheshire, UK) dipasok sebagai bubuk halus. Bubur 0,48 kg (78,1% b / b air, 3,6 kg berat kering). disimpan semalam pada 1 - 4 LC
bentonit dalam 10 L air disiapkan 24 jam sebelum digunakan. sebelum rekombinasi dengan ekstrak yang diolah. Untuk membantu memecah
gumpalan tembakau yang dikeringkan, penyedot debu digunakan untuk
Polyvinylpolypyrrolidone: (PVPP, 2-pirolidon, 1-etenil-homopolimer, mengosongkan centrifuge.
CAS 9003-39-8) adalah polimer silang polyvi-nylpyrrolidone dan tidak larut
dalam air. Ini adalah bubuk mengalir bebas putih (dijual oleh BASF sebagai
Divergen RS) dan ditetapkan sebagai kualitas food grade. Sebelum digunakan 2.2.2. Ekstrak
dan setelah setiap siklus adsorpsi, PVPP diregenerasikan dengan larutan
natrium hidroksida panas, dinetralkan dan dibilas dengan air yang diionisasi. berair Ekstrak berair dari enam 4,5 kg ekstraksi (total 450 L, berdasarkan
volume ekstrak) digabungkan dan didinginkan hingga 0–10 LC, dan 10 L dari
bubur bentonit ditambahkan. Campuran diaduk selama 30 menit pada 5–10
2.2. Proses pengolahan tembakau LC; kemudian bentonit dihapus menggunakan centrifuge terus menerus (Clara
80 VNPX 507, Alfa Laval). PVPP (15,75 kg) ditambahkan ke cairan
Sebuah diagram proses perawatan ditunjukkan pada Gambar. 1. Sekitar supernatan yang terus-menerus disirkulasikan kembali melalui filter press
4,5 kg lamina tembakau yang sudah dikeringkan (15,8% b / b air, 3,8 kg berat (Novox 400, Filtrox AG). Re-sirkulasi dilanjutkan selama 30 menit pada 0-10
kering) diekstraksi selama 15 menit dengan 75 L air de-ion yang di 55-60 LC LC. The fil-trate kemudian dialihkan ke tangki induk dan terkonsentrasi
dalam mesin cuci ( Maytag Atlantis, Maytag) dimodifikasi dengan mesh menjadi sekitar 10 L hingga 55 LC dalam evaporator vakum (CT2, FT
stainless steel untuk mempertahankan tembakau selama ekstraksi. Tembakau Industri Pty Ltd). Ekstrak pekat disimpan semalam pada 1 - 4 LC sebelum
yang diekstrak (selanjutnya disebut '' serat tembakau '') dan ekstrak berair rekombinasi dengan serat tembakau.
dipisahkan menggunakan putaran siklus mesin cuci. Baik serat tembakau
maupun ekstrak disimpan secara terpisah untuk perawatan selanjutnya.
2.2.3. Menggabungkan serat tembakau dan ekstrak berair

2.2.1. Serat Glycerol (0,144 kg) ditambahkan ke setengah dari eksktrak yang
terkonsentrasi (kira-kira 7,0 kg) yang kemudian direkombinasi dengan figuran
tembakau Serat tembakau disimpan dalam mesin cuci dan 65 L larutan tembakau dari tiga perawatan enzimatik (3,59 kg berat kering) di ekstrak ke
protease encer ditambahkan. Serat tembakau direndam dalam larutan selama serat rasio 1: 1 (w / w). Penyedot debu kembali digunakan untuk
30 menit pada 55-60 LC, dan kemudian dicampur perlahan selama 15 menit. mengosongkan tembakau / ekstrak yang digabung untuk membantu
Serat tembakau dipisahkan dari fase aque-ous yang mengandung enzim pengerukan. Hasil tembakau dikeringkan hingga 37,5 LC untuk kadar air
menggunakan mesin pengering spin mesin cuci dan bagian berair itu dibuang. oven 11,5–14,5% (b / b) untuk memberikan 8,4 kg tembakau yang diolah.
Untuk menghilangkan enzim sisa, tembakau kemudian dikenakan serangkaian
bilasan, sebagai berikut: bilasan pertama dengan 65 L air selama 5 menit pada 2.2.4. Hasil
15–25 LC. Bilas kedua adalah dengan 65 L larutan air garam yang
mengandung 1,3 lM kalsium klorida dan 166 mM natrium klorida selama 10 Pada kelembaban tembakau akhir sebesar 13,7% (b / b), dan dengan
menit pada 65-75 LC. Kalsium klorida menghasilkan untaian yang lebih kuat memperhitungkan hitung berat humektan (0,144 kg), berat kering tembakau
secara subyektif, mungkin dengan menstabilkan pektin di daun. Air dan yang kering dan bebas dari tiga batch adalah 7,11 kg. Membandingkan berat
larutan air garam diulang dua kali lagi, diikuti dengan bilasan air akhir. akhir tembakau dengan berat kering tiga batch tembakau awal (3,8 kg = 11,4
Setelah setiap bilas, larutan bilas dipisahkan dari serat tembakau kg), hasil proses adalah 62%.
menggunakan pengering spin dan dis-carded. Untuk mendenaturasi enzim
yang tersisa, serat tembakau dipindahkan ke autoklaf di mana dipanaskan
pada 110 LC selama 1 menit. Batch dari serat tembakau yang dihasilkan dari 2.3. Metode analisis campuran tembakau
tigaenzimatik
Semua campuran tembakau dianalisis untuk gliserol, nikotin, total dan
nitrogen protein, mengurangi dan total gula, nikotin,

Cut
Lamina
tembaka Air
u
Eks Serat
trak
Ekstraksi

Mesin Cuci

Protein Protein Enzim Prot


ein
Removal Treatment
Bentonit, Centrifuge Mesin cuci

Polifenol Polifenol Panas


Penonaktifan
penghapusan Autoclave

PVPP, Filter Press

Konsentrat
Air Ekstrak Ekstrak Rekombinasi

Konsentrasi ganda Cone Blender

Evaporator

Olahan Tembakau Pengeringan Air


Tray Drier
Gambar. 1. diagram Arus untuk proses pengobatan tembakau.
C. Liu dkk. / Makanan dan Kimia Toksikologi 49 (2011) 1904–1917 1907
(Sigma), 1,25% v / v Teleostean gelatin (Sigma)). Gel-atin ditambahkan ke
polifenol (asam caffeic, asam klorogenat, rutin dan scopoletin), logam dan buffer PBS / Brij untuk mencegah pengikatan yang tidak spesifik dengan
elemen metaloid, dan tambang nitrosa spesifik tembakau. Metode yang antibodi.
digunakan adalah sebagai berikut:

Mengurangi dan total gula dan nikotin ditentukan oleh Analisis Aliran 2.3.6. ELISA
Kontinyu menggunakan metode neocuproine / cyanogen chloride seperti yang
dijelaskan dalam standar ISO yang relevan (Standar ISO) 15152, 2003; ISO Pelapisan semalam dari pelat 96-well (NUNC Maxisorp white opa-que,
Standard 15154, 2003). NUNC) dilakukan pada 4 LC dengan 100 ll / well antibodi anti-protease
poliklonal kelinci (Novozymes RA15-12302) diencerkan
Nitrogen total ditentukan dengan mengubah nitrogen dalam sampel
menjadi amonium sulfat dengan 'Pencernaan Blok Kering' pada 375 LC
dengan asam sulfat pekat (1 ml / 0,05 g sampel) dan katalis tembaga (II).
Konsentrasi amonium dalam digest diukur dengan Continuous Flow Analysis
dengan reaksi Berth-olet yang dimodifikasi (Krom, 1980).

Nitrogen protein ditentukan dengan terlebih dahulu mengekstrak nitrogen


non-protein dari tembakau menggunakan larutan panas 0,5% v / v asam
asetat. Nitrogen protein yang tersisa ditentukan sebagai '' total nitrogen '' (lihat
di atas).

Nitrosamine Tembakau Spesifik (TSNAs) ditentukan dari 8% v / v


metanol dalam ekstrak diklorometana dari tembakau tanah menggunakan
kromatografi gas yang dilengkapi dengan Thermal Energy Analyzer (Health
Canada, 1999).

2.3.1. Polifenol (asam klorogenat, asam caffeic, rutin dan scopoletin)


Tembakau ditumbuk di pabrik dengan 1 mm mesh dan 0,25 g sampel
diekstraksi dengan 25 ml larutan 1: 1 (v / v) air: metanol mengandung
coumarin sebagai standar internal, dan disaring sebelum analisis. Jumlah
polyphe-nol individu dalam sampel tembakau ditentukan oleh kalibrasi
standar internal dan fase balik HPLC (kolom: Luna C18 (2) 5 lm250 4,6 mm,
fase gerak: metanol kelas HPLC dan 2% v / v

HPLC grade asam asetat dalam air suling) dengan deteksi UV.

2.3.2. Logam dan metaloid

Sampel tembakau digiling menggunakan Retsch ZM200 Centrifugal Mill


dengan 0,50 mm mesh dan aksesoris titanium. Sampel dicerna di bawah
tekanan dalam Sistem Persiapan Sampel Microwave menggunakan 5,5 N
asam nitrat berair. Standar internal 1 lg / ml indium, lutetium dan yttrium
ditambahkan ke digest dan di-luted ke volume. Digest tersebut kemudian
dianalisis secara kuantitatif oleh ICP-MS untuk arsenik, cadmium, kromium,
timbal, merkuri, nikel dan selenium.

2.3.3. Enzyme Linked Immuno Sorbent Assay (ELISA) untuk protease


Tingkat residu enzim yang digunakan pada serat tembakau

ditentukan menggunakan ELISA sandwich ganda yang dimodifikasi.

2.3.4. Sampel uji tembakau

Lima ratus miligram serat tembakau (pre-or post auto-claved, 12–15%


kelembaban) diekstraksi di bawah agitasi selama 60 menit pada 150 rpm
dengan 10 ml 0,01 M Phosphate-Buffered Saline (PBS) (pH 7,2 M) ), 0,023%
v / v Brij (Sigma). Satu mililiter ekstrak disentrifugasi dan supernatan
digunakan untuk mengukur enzim yang digunakan setelah menggunakan
pengenceran sampel yang sesuai.

2.3.5. Standar protease

Pengenceran enzim secara bersamaan (Protein bubuk yang dimurnikan,


10,66 mg / g, Novozymes PXN 09279) berkisar 4,9-0,02 ng / ml disiapkan
dalam buffer PBS / Brij / Gelatin (0,1 M PBS (pH 7,2), 0,023% v / v Brij
1: 500 v / v di 0,05 M karbonat-bikarbonat penyangga (pH 9,6) (Sigma). Pelat glu-taric-boric buffer (pH 6,5). Garam dan peptida yang lebih kecil dari 27 kD
dicuci empat kali dengan 300 ll / cucian pencuci (0,01 M PBS, 0,05% v / v telah dihapus oleh ultrafiltrasi menggunakan Centriprep YM-30 unit filter
Tween 20) dan setelah dilapisi dengan buffer Superblock (Pierce) selama 20 (Millipore) disentrifugasi pada 4 LC untuk menghasilkan larutan protease
menit pada suhu kamar. Sampel uji ke-bacco dan standar protease yang dimurnikan dengan konsentrasi 11,4 mg / ml. Aliquot dari larutan ini
ditambahkan ke piring dan diinkubasi selama 60 menit pada suhu kamar. (3510 ll, 40 mg protease) diolah dengan larutan yang terbuat dari 109 ll dari
14
Setelah dicuci, 100 ll / well dari antibodi sekunder (antibodi poliklonal larutan C-formaldehida (26,0 mCi / mmol, Perkin-Elmer Life Sciences),
kambing anti-protease (Novozymes GO15-14104) ditambahkan pada 1600 ll 0,4 M larutan natrium cyanoboro-hydride dan 2781 ll dari buffer
pengenceran 1: 6000 v / v dalam PBS / Brij / Gelatin. Pelat diinkubasi selama dimethyl glutaric-boric, untuk menghasilkan 8 ml batch. The radiolabelling
60 min pada suhu kamar dan dicuci sebelum menambahkan 100 ll / well dari dilakukan semalam di 4 LC. Setiap 8 ml batch diencerkan sampai 15 ml dan
14
antibodi konjugasi (kelinci anti-kambing IgG-HRP, Dako P449) pada 1: 4000 dimurnikan dengan ultrafiltrasi.akhir Konsentrasi larutan proteaseC
pengenceran v / v dalam PBS / Brij / Gelatin dan diinkubasi selama 60 menit. termetilasi adalah 11,0 mg / ml.
Setelah mencuci tiga kali dengan 300 ll/ buffer pencuci sumur dan satu kali
dengan 300 ll/ well 0,1 M sitrat - buffer fosfat (pH 5,0) (Sigma), 100 ll reagen
chemiluminescence (Supersignal ELISA femto kit dari Pierce ) ditambahkan
ke setiap sumur, dan intensitas luminesensi diukur dengan lumi-nometer 14
2.3.7.2. Persiapan C solusi pengobatan protease yang diolololabel. Sebuah
(LMax, Molecular Devices) setelah inkubasi dalam kegelapan selama 1 menit protease solusi komersial (Novozymes, Novozym 80001) diperkaya dengan
dan waktu integrasi 1 s. Sinyal luminesensi diubah menjadi konsentrasi 14 14.
20% dari C protease alkohol untuk Ob-tain Csolusi pengobatan protease
protease aktif dengan menggunakan Kurva kalibrasi logistik 4-parameter dari 14
radiolabelled Sebuah 5 ml dari C solusi alkohol protease (55 mg protease)
luminescense vs. konsentrasi. Residual active protease konsentrasi pada serat
dicampur dengan 10,6 ml protease komersial (220 mg protease).
tembakau dihitung dalam bagian per juta (ppm).
Radioaktivitas solusi ini ditentukan dengan menambahkan 16 ml cairan kilau
(ScintiSafe Econo 2, Linear Alkylbenzene Base, Fisher Scien-tific) untuk 50
ll dari 14C solusi radiolabelled protease dan radioaktivitas dihitung dengan
counter kilau ( Model Tri-carb 2200CA, Perkin – Elmer). Setelah
Untuk menentukan tingkat enzim residu absolut pada tembakau yang pengenceran yang sesuai, aktivitas larutan juga ditentukan menggunakan
digunakan dalam rokok, baik faktor pengenceran (2) karena penambahan ELISA.
solubles ke serat tembakau, dan efisiensi ekstraksi protease (33% untuk
tembakau pra-diautoklaf dan 53% untuk tembakau setelah autoklaf - lihat
bagian selanjutnya) harus diperhitungkan. 2.3.7.3. Persiapan sampel tembakau. Dua puluh gram tembakau diekstraksi
dengan 570 ml air de-ionisasi pada 55-60 LC selama 15 menit, dikeringkan
2.3.7. Efisiensi ekstraksi selama 3 menit dan dipindahkan ke kantong muslin untuk sisa proses
perawatan. Serat tembakau basah direndam selama 30 menit dalam 470 ml air
14
14
proteinase 2.3.7.1. Persiapan C protease yang dirujuk dengan radiolabelled. de-terionisasi yang mengandung 600-800 ll dari C solusi pengobatan
Larutan stok protease (15,0 mg / ml) yang dibuat dengan bubuk protease protease radiolabelled dan 188 ll NaOH. Serat tembakau gelisah dalam
murni (Novo-zymes, PXN 09279) diencerkan (1: 2) dalam 212 mM dimethyl larutan
1908 C. Liu et al. / Food and Chemical Toxicology 49 (2011) 1904–1917
Tabel 1

selama 15 menit, dan kantong muslin dikeringkan dan diperas untuk


Spesifikasi dan data pengukuran rokok untuk analisis asap utama.
memindahkan kembali kelebihan cairan. Serat tembakau dibilas pertama
dengan 470 ml air pada 10 LC selama 5 menit, kemudian dengan 470 ml
larutan garam (250 mM NaCl + 0,15 mMCaCl2 2H2O dalam air de-terionisasi)
di 55- 60 LC. Urutan ekstraksi ini diulang tiga kali, diikuti oleh bilasan air
akhir. Setelah setiap bilas, cairan berlebih digerakkan kembali dengan
meremas kantong muslin. Serat tembakau kemudian diautoklaf pada 110 LC
selama 1 menit. Untuk masing-masing dari tujuh kumpulan serat tembakau,
tiga 0,5 g sampel serat tembakau diambil setelah bilasan akhir dan setelah
autoklaf, dan dikeringkan semalam dalam lemari aliran lamina. Kelembaban
(IR-200, Denver Instrumen) dan total radioaktivitas ditentukan oleh counter
sintilasi.
Panjang rokok 83 mm
Filter panjang 27 mm
panjang Tipping 32 mm
panjang batang Tembakau 56 mm
Lingkar 24,6 mm
Ventilasi 20%
Kertas permeabilitas 25 Coresta
tidak diobati Diobati

Jenis filter selulosa asetat selulosaasetat


tekananrokok menjatuhkan 115 mm WG 101 mmWG
penurunan tekananFilter 85 mm WG 85 mm WG
Berat tembakau 868 mg 864 mg

2.3.7.4. Ekstraksi sampel tembakau. Setiap sampel diekstraksi dalam 10 ml


Tabel 2
0,01 M PBS (pH 7,2), 0,023% (v / v) Brij, 1% v / v phen-
ylmethylsulfonylfluoride (PMSF) protease inhibitor selama 1 jam. Ekstraksi
dilakukan dalam 50 ml tabung sentrifugal dalam pengocok putar selama 60 Spesifikasi dan data pengukuran rokok untuk analisis asap sidestream.
menit pada 150 rpm. Sekitar 1,5-2 ml ekstrak dihilangkan dan disentrifugasi
pada 12.000 rpm selama 5 menit. Radioaktivitas 1 ml ekstrak dalam 16 ml
Panjang rokok 84 mm
sintilasi cair dihitung. Le-vel enzim aktif dalam ekstrak ditentukan oleh Panjang filter 25 mm
ELISA. Serat tembakau yang diekstrak dikeringkan (seperti di atas) dan Panjang tipping 30 mm
kelembaban dan radioaktivitas ditentukan. Ekstraksi efisiensi ditentukan Panjang batang tembakau 59 mm
Lingkar 25 mm
sebagai: total aktivitas ekstrak / (aktivitas total tembakau) 100 (%). Ventilasi 0%
Permeabilitas kertas 56 Coresta Tidak
Diobati Ditangani

2.4. Pengukuran fisik pada tembakau Penurunan tekanan rokok 115 mm WG 107 mm WG
Tekanan saringan turun 75 mm WG 71 mm WG
Berat tembakau 762 mg 782 mg
Analisis ukuran partikel tembakau dilakukan pada tembakau yang
dikondisikan pada 60% kelembaban relatif selama 48 jam menggunakan
Rotary Sieve Shaker dengan saringan 2,00, 1,40, 1,00, 0,71 dan 0,50 mm.
Lima ulangan dianalisis untuk menentukan hasil racun. Ringkasan metode
analisis yang digunakan diberikan pada Tabel 3 dan batas kuantifikasi (LOQ)
2.5. Pembuatan rokok
diberikan pada Tabel 4.
Hasil oksida nitrat (NO dan NOx) ditentukan oleh Lab-stat International
Untuk analisis kimia asap utama (kecuali NO / NOx), tembakau yang
Inc. (Kitchener, Ontario, Kanada) pada ciga-rettes yang digunakan untuk
dirawat dan tidak diobati dicampur dengan 20% batang yang diberi uap (STS)
pengukuran hasil sidestream (bagian berikutnya dan Tabel 4); tujuh ulangan
sebelum membuat rokok. Rokok dibuat dengan kertas yang memiliki
diukur. Metode untuk NO / NOx tersedia di situs web(Health
permeabilitas 48 unit Coresta. (Unit Coresta adalah jumlah udara dalam
CanadaKesehatan Kanada, 2010a). LOQ untuk metode ini ditampilkan dalam
sentimeter kubik yang melewati satu sentimeter persegi kertas dalam satu
Tabel 4.
menit pada tekanan konstan 1,0 kilopascal). Filter rokok dibuat dengan
selulosa asetat dan dilubangi laser untuk mencapai tingkat ventilasi 20%. Data 2.7. Pengumpulan asap dan analisisAnalisis
fisik rokok ditunjukkan pada Tabel 1.
asap Sidestreamasap sidestream dilakukan oleh Labstat Interna-tional Inc.
Semua analisis asap sidestream, dan analisis arus utama NO / NOx, (Kitchener, Ontario, Kanada). Metode analisis yang digunakan diterbitkan
dilakukan pada rokok yang dibuat dengan 100% tembakau yang diperlakukan oleh Health Canada, dan tersedia di situs web(Health CanadaHealth Canada,
dan tidak diobati, yaitu tanpa ditambahkan batang. Rokok-rokok ini dibuat 2010b). Tujuh ulangan dianalisis untuk setiap racun asap. The LOQs untuk
dengan kumpulan tembakau yang berbeda dari rokok yang digunakan untuk metode tersebut di-cluded pada Tabel 4.
analisis asap aliran utama dan tidak terventilasi. Kertas rokok yang digunakan
memiliki permeabilitas 56 unit Coresta. Data fisik untuk rokok ini
ditunjukkan pada Tabel 2. 2.8. Pengujian statistik

Penyimpangan standar untuk rasio racun / tar dihitung menggunakan


2.6. Pengumpulan dan analisisanalisis pendekatan seri Taylor terhadap teorema Fieller untuk varians rasio dua
variabel (Fieller, 1954). P values to deter-mine significances of differences in
asap arus utama Semuaasap utama dilakukan di R & D Laboratories blend composition between untreated and treated blends, and between
British American Tobacco Group, Southampton, Inggris, kecuali NO dan toxicant yields from cigarettes made with these tobaccos were calculated
NOx. Rincian lengkap tentang metode pengumpulan asap dan analisis dapat using Stu-dent's t-test.
ditemukan di situs web British American Tobacco (BAT, 2010). Singkatnya,
rokok dikondisikan menurut ISO Standard 3402 (1999) dan dinilai dalam
kondisi ISO ma-chine-smoking volume 35 puff, durasi 2 puff dan interval 60 3. Hasil dan diskusi
puff (ISO Standard 3308, 2000). Total Particulate Matter (TPM) dan karbon
monoksida (CO) hasil dari rokok ditentukan menggunakan Cerulean 350 3.1. Physical effect of treatment on the tobacco
Mesin Merokok dilengkapi dengan COA 200 ATCOM Sistem. Nikotin dan air
dalam TPM ditentukan dengan kromatografi gas megabore. Lima ulangan Results of sieve analyses of the tobacco blends before and after treatment
dianalisis untuk TPM, bebas nikotin, partikulat kering (NFDPM atau tar), are shown in Fig. 2. Treatment resulted in significant
nikotin, air dan karbon monoksida (CO).
C. Liu et al. / Food and Chemical Toxicology 49 (2011) 1904–1917 1
9
0
9
Table 3

*
Methods for mainstream smoke toxicant analyses .
Determination of: Cigs No of replicates Type of smoking Collection method Analysis method
per
port (ports) machine
Ammonia 5 5 Linear 20-port 92 mm Cambridge filter pad Potentiometric using ammonia specific
ion electrode
Benzo(a)pyrene 2 5 Rotary 20-port 92 mm Cambridge filter pad GC/MS selective ion monitoring
0
Carbonyls 2 5 Linear 20-port 2 Drechsel type traps HPLC with UV detection
Hydrogen cyanide 5 5 Linear 20-port Trapped in aqueous sodium hydroxide Colorimetric
Phenols 5 5 Rotary 20-port 44 mm Cambridge filter pad GC/MS selective ion monitoring
Pyridine, quinoline and 5 5 Rotary 20-port 44 Mm Cambridge filter pad GC/MS selective ion monitoring
styrene
Vapour phase 5 5 Rotary 20-port 44 mm Cambridge filter to remove GC/MS selective ion monitoring
particulate; vapour phase collected in
Tedlar bag
Tobacco specific 2 5 Rotary 20-port 44 mm Cambridge filter pad LC/MS/MS
0
nitrosamines
Aromatic amines 2 5 Rotary 20-port 92 mm Cambridge filter pad GC/MS selective ion monitoring
0
Metals 1 5 Linear 8-port Drechsel type traps ICP/MS
0
NO/NOx 1 7 Single port Direct analysis of unfiltered smoke Dual channel chemiluminescence
analyser

* These methods are described in reference BAT (2010) except for NO/NOx which is described in Health Canada (2010a).
Quinoline lg/cig 0.02 0.300
Styrene lg/cig 0.04 14.8
Table 4
1,3-Butadiene lg/cig 7.10 26.4
Limits of quantification (LOQ) for ISO mainstream and sidestream smoke toxicants. Acrylonitrile lg/cig 6.80 23.5
Toxicant LOQ value Isoprene lg/cig 55.4 12.0
B enzene lg/cig 7.60 9.74

Mainstream Sidestream Toluene lg/cig 57.9 16.0


Chromium ng/cig 1.2 103
Ammonia lg/cig 1.74 214 Nickel ng/cig 2.0 118
1-Aminonaphthalene ng/cig 5.1 2.13 Arsenic ng/cig 1.0 37.7
2-Aminonaphthalene ng/cig 3.4 2.76 Selenium ng/cig 4.1 58.4
3-Aminobiphenyl ng/cig 1.1 0.34 Cadmium ng/cig 1.9 13.4
4-Aminobiphenyl ng/cig 0.6 0.34 Mercury ng/cig 0.1 6.00
Benzo(a)pyrene ng/cig 0.4 1.96 Lead ng/cig 12.0 115
Formaldehyde lg/cig 1.4 2.10
Acetaldehyde lg/cig 0.43 5.67
Acetone lg/cig 4.17 4.94 degradation of the largest tobacco particles ie >2.00 mm mesh size, but the
Acrolein lg/cig 0.76 4.15 proportion of intermediate particles was increased, while dust (<0.50 mm)
Propionaldehyde was reduced. The treated tobacco was sim-
lg/cig 1.45 5.83
Crotonaldehyde lg/cig 1.67 5.76
Methyl ethyl ketone lg/cig 2.29 6.41
Butyraldehyde lg/cig 2.06 4.73
NO lg/cig 2.26 186
NOx lg/cig 7.5 179
Hydrogen cyanide lg/cig 1.94 13.1
NNN ng/cig 5 2.00
NAT ng/cig 4 7.75
NAB ng/cig 3 2.50
NNK ng/cig 8 15.5
Phenol lg/cig 0.11 5.02
o-Cresol lg/cig 0.03 0.644
m-Cresol lg/cig 0.03 1.34 (m + p)
p-Cresol lg/cig 0.07
Catechol lg/cig 0.24 4.23
Resorcinol lg/cig 0.06 1.38
Hydroquinone lg/cig 0.19 4.74
Pyridine lg/cig 0.15 9.89
Fig. 2. Particle size distributions for the untreated and treated tobacco blends.
5
0 ilar in appearance to the untreated tobacco and could be handled and made
into cigarettes without significant degradation.
4
5
S 4 Untreat 3.2. Tobacco chemical composition
i 0 ed
Treate Results of the chemical analyses of the untreated and treated tobaccos are
3
5 d shown in Tables 5 and 6. Nicotine levels in the treated tobacco were 12%
o 3 lower than in the untreated tobacco, which was due in part to some nicotine
n 0 losses during the bentonite extraction step. There were higher sugar levels in
the treated tobacco which partly reflect the losses of other soluble components
%Reten
and partly the relatively greater losses of tobacco fibre compared with solu-
1
bles. Chloride levels were unchanged which demonstrates the effi-cacy of the
tion

2 tobacco rinsing process.


5
2
0 Protein nitrogen was reduced by 59% and total nitrogen by 31%. Rutin
1 ( 79%) and scopoletin ( 78%) were extracted more effi-ciently than
5
chlorogenic acid ( 33%), the most abundant polyphenol.
5
The tobacco specific nitrosamines were not significantly af-fected by the
treatment. For the metals there were significant (p < 0.01) reductions in
arsenic ( 24%), cadmium ( 24%), chro-mium ( 39%), lead ( 12%) and
0 selenium ( 20%). In tobacco leaves, cadmium occurs predominately as water
2. 1.40 1.00 0.71 0.50 < soluble peptide com-plexes within the cell vacuoles (Wagner and Yeargan,
00 0.50 1986). Whether the cadmium–peptide complexes are extracted during the
initial water wash or during subsequent enzyme treatment was not
determined. The reasons for the reductions of the other metals during the
Sieve Aperture Size (mm)
process are also unknown.
1910 C. Liu et al. / Food and Chemical Toxicology 49 (2011) 1904–1917
Table 5

Tobacco chemistry for the treated and untreated blends on a dry weight basis.

Untreated blend Treated blend % Difference treated vs. untreated P value

M S Mean SD
ea D
n

Moisture % 15. – 13.7 – 13 N/A


8
Glycerol % 0.2 – 1.88 – +795 N/A
w/ 1
w
Nicotine % 2.6 0 2 0 12 <0.05
w/ . . .
w 0 3 0
2 1
Reducing sugar % 10. 0 12.6 0 +16 N/A
w/ 9 . .
w 0 0
7
Total sugars % 11. 0 13.3 0 +14 <0.05
w/ 7 . .
w 0 1
7 4
Protein nitrogen % 1.0 0 0.45 0 59 <0.01
w/ 9 . .
w 0 0
6 1
Total nitrogen % 2.7 0 1.87 0 31 <0.01
w/ 0 . .
w 0 0
4 2
Chloride % 0.6 0 0.58 0 3 NS
w/ 0 . .
w 0 0
8 5
Caffeic acid mg/ 0.1 0 0.06 0 68 N/A
g 9 . .
0 0
1 0
Chlorogenic acid mg/ 21. 0 14.4 0 33 <0.01
g 4 . .
4 2
7 5
Rutin mg/ 5.3 0 1 0 79 <0.01
g . . .
1 1 0
6 1
Scopoletin mg/ 0.6 0 0.14 0 78 N/A
g 4 . .
0 0
3 0
N/A: not applicable since standard deviations were zero.
NS: not significant.
Table 6

Tobacco specific nitrosamines and metals in the untreated and treated blends.

LOQ Untreated Treated blend % Difference treated vs. untreated P value


blend
Mean SD Mean S
D
NAB lg/g 0.0009 0.002 0. 0.001 0 50 –
00 .
0
0
NAT lg/g 0.0018 0.10 0. 0.08 0 20 –
01 .
0
0
NNK lg/g 0.0018 0.05 0. 0.06 0 +20 –
00 .
0
0
NNN lg/g 0.0018 0.06 0. 0.06 0 0 –
00 .
0
0
Arsenic lg/g 0.01 0.21 0. 0.16 0 24 <0.01
01 .
0
1
Cadmium lg/g 0.03 1.65 0. 1.26 0 24 <0.01
05 .
0
2
Chromium lg/g 0.01 0.44 0. 0.27 0 39 <0.01
06 .
0
5
Lead lg/g 0.01 0.41 0. 0.36 0 12 <0.01
02 .
0
1
Mercury lg/g 0.05 <LOQ N/A <LOQ N N/A –
/
A
Nickel lg/g 0.01 0.40 0. 0.47 0 +18 NS
04 .
0
5
Selenium lg/g 0.02 0.05 0. 0.04 0 20 <0.01
00 .
0
1

NS: not significant.

calculated as <0.009%. With a cigarette having the same tobacco weight, we


3.3. Protease residues on tobacco can estimate that the protease transfer from cigarettes with different tar yields
would be reduced or increased in propor-tion. For example, the maximum
3.3.1. Potential exposure considerations transfer for a cigarette yielding 10 mg tar under ISO smoking conditions
would be: 10/ 15 0.009% = 0.006%. The transfer would also be affected by
The enzyme molecule employed in the tobacco treatment pro-cess has a the puffing regime. A cigarette yielding 15-mg tar under ISO smoking
long history of use as an ingredient in laundry detergent (Peters and cigarette would, when smoked under the Health Canada intense smoking
Mackenzie, 1997). The technical formulation of the en-zyme applied in regime (two 55 ml puffs taken every minute with filter ventilation holes
laundry detergents has the trade name Savinase . It has also been well- blocked), be expected to yield up to 35 mg tar (Health Canada, 2006) and the
documented that active enzymes have the po-tential to cause allergic reactions maximum transfer of protease would be 35/15 0.009% = 0.021%.
in sensitised individuals (NRC, 1971; Johnsen et al., 1997). To ensure that the
level of enzyme res-idues on the treated tobacco were lower than the levels
Thus, for a cigarette containing 700 mg of treated tobacco the amount of
required to cause allergic reactions during smoking, levels of the applied pro- 6
protease in the cigarette would be: 30 (ppm) 700 (mg) 10 = 21 lg. If the
tease in smoke must be estimated.
cigarette yields 10 mg tar under ISO smoking conditions, the maximum
During preliminary experiments to remove active protease from treated protease transfer rate would be 0.006% and the maximum level of protease in
tobacco fibre, it was found that approximately 60 ppm pro-tease was left on mainstream smoke would be: 21 lg/cigarette 0.006% = 1.26 ng/cigarette. For
the same cigarette smoked under the Health Canada Intense re-gime the
the tobacco fibre after 3 saline and 3 water rinses. This is equivalent to a level
maximum level of protease in mainstream smoke would be 21 lg/cigarette
of 30 ppm on the final tobacco once the solubles are added back. Further
0.021% = 4.4 ng/cigarette.
reductions in levels of active pro-tease with additional washings were small.
To determine if 30 ppm of active protease on tobacco would be a level that The following approach (RJ Reynolds Tobacco Co., 1988), for
could cause an allergic reaction in a sensitised individual, it was first determining whether this dose is acceptable, is based on the con-cept of an
necessary to determine the maximum amounts that could be transferred to ''Acceptable Daily Intake'' (ADI), which is calculated from the published
smoke. Threshold Limit Value (TLV) for protease. Provided the TLV based ADI
(TLV-ADI) is significantly greater than the po-tential dose of protease from
Upper limits for transfer of Savinase into smoke have been pub-lished the cigarette, then there is assurance that this level of protease on the cigarette
previously (Voisine et al., 2004). Using a method that could detect 17.9 ng/mg tobacco is acceptable.
smoke condensate, no Savinase was detected in the condensate from a
cellulose acetate filter cigarette yielding 15 mg tar under ISO smoking The calculation for the TLV-ADI starts with the assumption that a person
conditions and with the tobacco (699 mg) spiked with 6000 ppm Savinase. 3
will respire 10 m of air during an eight-hour work period.
The upper limit for Savinase transfer into mainstream smoke with this
cigarette was
C. Liu et al. / Food and Chemical Toxicology 49 (2011) 1904–1917 1911
3.3.2. Measured levels of residual protease
3
The TLV for active subtilisins is 40 ng/m (EH40/2005, 2007).
Hence: Every batch of tobacco was analysed for levels of residual pro-tease both
before and after autoclaving to ensure, firstly, that the rinsing steps had
sufficiently removed protease from the tobacco and, secondly, that any
Daily exposure to protease at TLV ¼ 10ðm3=dayÞ 40ðng=m3Þ
enzyme remaining on the tobacco was denatured (deactivated) by the steam
treatment.
¼ 400 ng=day The pre- and post-autoclave ELISA results for a series of produc-tion runs
are shown in Figs. 3 and 4 masing-masing. The data shown have been
The potential dose of protease to the smoker was estimated by assuming: corrected for the extraction efficiencies involved with sample preparation
(33% extraction efficiency for pre-autoclaved tobacco and 53% for tobacco
(1) 100% of the protease in the inhaled smoke is absorbed by the smoker after autoclaving). With one exception (62 ppm) the pre-autoclave results are
and (2) a smoker smokes 60 cigarettes per day. consistently below 60 ppm. The post-autoclave results show more variability.
The majority of results obtained for post-autoclave enzyme protein content
Then, for a cigarette yielding 10 mg tar under the ISO smoking regime:
are be-low 60 ppb before solubles addback. There is only one result above 60
ppb and that was thought to be due to cross contamination of the samples. The
batches were all combined, ensuring that the overall protease content would
Maximum total exposure ¼ 60ðcig=dayÞ 1:26ðng=cigÞ be less than 30 ppb.

¼ 75:6 ng=day
3.4. Mainstream smoke results
For the same cigarette smoked under the Health Canada intense regime:
The results for the mainstream smoke yields of TPM, tar, nico-tine and
CO, and puff numbers for the cigarettes with the untreated and treated
Maximum total exposure ¼ 60ðcig=dayÞ 4:4ðng=cigÞ tobaccos are shown in Table 7. Yields of TPM ( 15%), tar ( 16%), nicotine
( 17%) and CO ( 20%) were all lower for the cigarettes with the treated
¼ 264 ng=day tobacco. Contributing to the yield dif-ferences was the lower puff number of
the treated cigarette (7.3 puffs) compared with untreated cigarette (7.9 puffs).
These calculations show that even if all the enzyme was active, the Yields of the smoke toxicants are shown in Table 8, which also shows the
amount to which the smoker is exposed is well below levels that could cause
concern. In addition, the enzyme is deactivated by steam during the treatment % differences in smoke toxicant yields as a result of tobacco treat-ment and
process to a level corresponding to <30 ppb of active protease on the treated the significance levels. With the exception of formalde-
tobacco (after solubles addback). The heat deactivated enzyme that remains
on the tobac-co is expected to have significantly lower allergenicity than
active enzyme (Debanne and Dolovich, 1974).

70

60

Active Savinase (ppm)


50

40

30

20

10

0 10 20 30 40 50 60
70 80

Batch No.

Fig. 3. Levels of active protease (ppm) on tobacco fibre prior to autoclaving.


Active Savinase (ppb)
2
0

9
1
0

8
0

7
0

6
0

5
0
0

0
4
0
0 10 20 30 40 50 60
70 80
3
0
Batch No.

Fig. 4. Levels of active protease (ppb) on tobacco fibre after autoclaving.


1912 C. Liu et al. / Food and Chemical Toxicology 49 (2011) 1904–1917
Given the approach recommended by TobReg (WHO, 2008; Burns et al.,
Table 7 2008), the toxicant yields relative to tar and nicotine have been calculated as
well as the % differences in these ratios as a result of tobacco treatment, and
Mainstream yields of TPM, water, tar, nicotine and CO and puff number for the cigarettes the results are presented in Table 9. Since the toxicant/tar and
prepared from untreated and treated tobacco blends.
toxicant/nicotine ratios are very similar, the remainder of this section will
focus on the effect of to-bacco treatment on toxicant/tar (or, tar-adjusted)
ratios, which are illustrated in Fig. 5. In this figure differences in yield ratios
Untreated Treated % Difference
which are significant at p < 0.01 are shown in red.
TPM mg/cig 11.4 9.7 15
Water mg/cig 0.97 0.90 7
Tar mg/cig 9.5 8.0 16
Nicotine mg/cig 0.93 0.77 17 3.4.1. Nitrogen containing toxicants
CO mg/cig 11.3 9.0 20
Puff number 7.9 7.3 There were significant reductions (p < 0.01) in tar-adjusted yields of
ammonia ( 29%), 1-aminonaphthalene ( 35%), 2-amino-naphthalene ( 38%),
3-aminobiphenyl ( 35%), 4-aminobiphenyl ( 39%), hydrogen cyanide ( 42%),
hyde, acrolein, propionaldehyde, isoprene and chromium, the yields of all the
pyridine ( 23%) and acryloni-trile ( 21%). Quinoline was also reduced ( 23%)
toxicants from the treated tobacco cigarette were reduced compared with
at a significance level of p < 0.05. The reductions in yields of these nitrogen
those from the untreated tobacco ciga-rette. Yields of acrolein,
contain-ing smoke components are consistent with pyrolysis studies which
propionaldehyde and chromium were in-creased but not significantly while
have shown that amino acids and proteins are precursors for sev-eral of the
yields of formaldehyde (+49%, p < 0.01) and isoprene (+17%, p < 0.05) were
nitrogen containing toxicants in smoke. Hydrogen cya-nide was shown to be a
significantly increased. Of the toxicant yields that were reduced by the
major product of amino acid pyrolysis (Johnson and Kang, 1971). There was
treatment, the reductions were all significant at p < 0.01 except for that of
also a strong correlation
NNK, that was significant at p < 0.05, and those of benzo(a)pyrene, acet-
aldehyde, acetone, crotonaldehyde, methyl ethyl ketone and ar-senic that were
not significant.

Table 8

Yields of mainstream smoke toxicants from cigarettes made with the untreated and treated tobacco blends.

Untreated Treated (%) Difference P value


Mean S Mean S
D D
Tar mg/cig 9.5 0 8 0 15.8 <0.01
. .
3 2
9 8
Nicotine mg/cig 0.93 – 0.77 – 17.2 –
Carbon monoxide mg/cig 11.3 0 9 0 20.4 <0.01
. .
2 2
8 2
Ammonia lg/cig 5.85 0 3.51 0 40.0 <0.01
. .
2 2
7 7
1-Aminonaphthalene ng/cig 10.9 0 5.95 0 45.4 <0.01
. .
9 5
1
2-Aminonaphthalene ng/cig 7.19 0 3.73 0 48.1 <0.01
. .
5 2
9 7
3-Aminobiphenyl ng/cig 1.7 0 0.93 0 45.3 <0.01
. .
1 1
2 1
4-Aminobiphenyl ng/cig 1.35 0 0.69 0 48.9 <0.01
. .
0 0
7 4
Benzo(a)pyrene ng/cig 5.68 0 5.34 0 6.0 NS
. .
3 2
5
Formaldehyde lg/cig 31.2 4 46.3 2 48.6 <0.01
. .
0 7
Acetaldehyde lg/cig 535 3 514 2 3.9 NS
5 0
. .
5 7
Acetone lg/cig 276 1 257 1 6.9 NS
7 4
. .
8 3
Acrolein lg/cig 60.3 3 62.9 1 4.3 NS
. .
5 7
4 6
Propionaldehyde lg/cig 47 2 47.1 2 0.2 NS
. .
8 0
2 4
Crotonaldehyde lg/cig 17.1 1 15.8 0 7.4 NS
. .
3 6
1
Methyl ethyl ketone lg/cig 67.7 4 65.3 4 3.6 NS
. .
5 0
6 9
Butyraldehyde lg/cig 32.1 1 24.3 1 24.1 <0.01
. .
8 2
9
Hydrogen cyanide lg/cig 96.3 5 47.2 1 51.0 <0.01
. .
8 5
4 8
NNN ng/cig 8.34 0 6.2 0 25.7 <0.01
. .
5 2
1 9
NAT ng/cig 21 .4 1 16.0 0 25.2 <0.01
. .
7 8
6 1
NAB ng/cig 1.5 0 1.02 0 32.0 <0.01
. .
0 1
8
NNK ng/cig 8.27 1 6.06 0 26.7 <0.05
. .
2 4
3
Phenol lg/cig 16.2 0 7.78 0 51.9 <0.01
. .
4 5
7 4
o-Cresol lg/cig 3.82 0 2.85 0 25.4 <0.01
. .
1 1
8
m-Cresol lg/cig 3.38 0 2.59 0 23.4 <0.01
. .
0 1
7 6
p-Cresol lg/cig 7.31 0 3.96 0 45.8 <0.01
. .
1 2
4 5
Catechol lg/cig 76.2 1 57.6 3 24.4 <0.01
. .
6 7
3 6
Resorcinol lg/cig 1.04 0 0.46 0 55.8 <0.01
. .
0 0
4 2
Hydroquinone lg/cig 65.2 1 42.0 2 35.5 <0.01
. .
7 8
5
Pyridine lg/cig 5.25 0 3.34 0 36.4 <0.01
. .
2 2
2 1
Quinoline lg/cig 0.32 0 0.208 0 35.0 <0.01
. .
0 0
5 2
Styrene lg/cig 9.73 0 7.82 0 19.6 <0.01
. .
3 5
8
1,3-Butadiene lg/cig 27.8 1 27.4 1 1.7 NS
. .
9 8
2
Acrylonitrile lg/cig 7.73 0 5.17 0 33.1 <0.01
. .
3 7
6 2
Isoprene lg/cig 148 1 173 1 16.9 <0.05
3 0
. .
6 8
Benzene lg/cig 38.6 1 32.6 1 15.7 <0.01
. .
5 9
1 6
Toluene lg/cig 60.2 7 45.2 4 24.9 <0.01
.
5
4
Chromium ng/cig 2.02 0 2.37 0 17.3 NS
. .
3 7
5 4
Nickel ng/cig LOQ – LOQ – – –
Arsenic ng/cig 7.4 1 5.56 1 24.9 NS
. .
4 5
8
Selenium ng/cig LOQ – LOQ – – –
Cadmium ng/cig 32.2 3 21.1 1 34.3 <0.01
. .
2 3
7 8
Mercury ng/cig LOQ – LOQ – – –
Lead ng/cig LOQ – LOQ – – –

NS: not significant.


C. Liu et al. / Food and Chemical Toxicology 49 (2011) 1904–1917 1913

Table 9

Ratios of yields of mainstream smoke toxicants to yields of tar and nicotine from cigarettes made with the untreated and treated tobacco blends.

Toxicant/tar % Difference P value Toxicant/nicotin % Difference


e
Untreated Trea Untreated Trea
ted ted
Nicotine mg/cig 0.098 0.09 1.7 NS 3.8
6
Carbon monoxide mg/cig 1.189 1.12 5.4 NS 12.2 11.7
5
Ammonia lg/cig 0.616 0.43 28.8 <0.01 6.29 4.56 27.5
9
1-Aminonaphthalene ng/cig 1.147 0.74 35.2 <0.01 11.7 7.73 34.1
4
2-Aminonaphthalene ng/cig 0.757 0.46 38.4 <0.01 7.73 4.84 37.3
6
3-Aminobiphenyl ng/cig 0.179 0.11 35.0 <0.01 1.83 1.21 33.9
6
4-Aminobiphenyl ng/cig 0.142 0.08 39.3 <0.01 1.45 0.90 38.3
6
Benzo(a)pyrene ng/cig 0.598 0.66 11.6 <0.05 6.11 6.94 13.5
8
Formaldehyde lg/cig 3.28 5.79 76.5 <0.01 33.5 60.2 79.5
Acetaldehyde lg/cig 56.3 64.3 14.1 <0.01 575 668 16.0
Acetone lg/cig 29.1 32.1 10.6 NS 297 334 12.5
Acrolein lg/cig 6.35 7.86 23.9 <0.01 64.8 81.7 26.0
Propionaldehyde lg/cig 4.95 5.89 19.0 <0.01 50.5 61.2 21.0
Crotonaldehyde lg/cig 1.80 1.98 9.9 NS 18.4 20.6 11.8
Methyl ethyl ketone lg/cig 7.131 8.16 14.4 <0.05 72.8 84. 16.4
8
Butyraldehyde lg/cig 3.38 3.04 9.8 <0.05 34.5 31.6 8.3
Hydrogen cyanide lg/cig 10.1 5.90 41.8 <0.01 103 61.3 40.8
NNN ng/cig 0.878 0.77 11.7 <0.05 8.97 8.05 10.2
5
NAT ng/cig 2.25 2.00 11.2 <0.05 23.0 20.8 9.6
NAB ng/cig 0.158 0.12 19.3 <0.01 1.61 1.32 17.9
8
NNK ng/cig 0.871 0.75 13.0 NS 8.89 7.87 11.5
8
Phenol lg/cig 1.703 0.97 42.9 <0.01 17.4 10.1 41.9
3
o-Cresol lg/cig 0.402 0.35 11.4 <0.01 4.11 3.70 9.9
6
m-Cresol lg/cig 0.356 0.32 9.0 <0.05 3.63 3.36 7.5
4
p-Cresol lg/cig 0.769 0.49 35.7 <0.01 7.86 5.14 34.6
5
Catechol lg/cig 8.03 7.20 10.2 <0.05 82.0 74.8 8.7
Resorcinol lg/cig 0.109 0.05 47.5 <0.01 1.12 0.60 46.6
8
Hydroquinone lg/cig 6.86 5.26 23.4 <0.01 70.1 54.6 22.1
Pyridine lg/cig 0.553 0.41 24.5 <0.01 5.65 4.34 23.2
8
Quinoline lg/cig 0.034 0.02 22.8 <0.05 0.34 0.27 21.5
6
Styrene lg/cig 1.02 0.98 4.6 NS 10.5 10.2 2.9
1,3-Butadiene lg/cig 2.93 3.42 16.7 <0.01 29.9 35.5 18.7
Acrylonitrile lg/cig 0.814 0.64 20.6 <0.01 8.31 6.71 19.2
6
Isoprene lg/cig 15.6 21.6 38.8 <0.01 159 225 41.2
Benzene lg/cig 4.07 4.07 0.1 NS 41.6 42.3 1.8
Toluene lg/cig 6.34 5.65 10.9 NS 64.7 58.7 9.3
Chromium ng/cig 0.213 0.29 39.3 NS 2.17 3.08 41.7
6
Nickel ng/cig N/A N/A N/A – N/A N/A N/A
Arsenic ng/cig 0.779 0.69 10.8 NS 7.96 7.22 9.3
5
Selenium ng/cig N/A N/A N/A – N/A N/A N/A
Cadmium ng/cig 3.39 2.65 21.9 <0.01 34.6 27. 20.6
5
Mercury ng/cig N/A N/A N/A – N/A N/A N/A
Lead ng/cig N/A N/A N/A – N/A N/A N/A

NS: not significant; N/A: not applicable since yields were <LOQ.
TT
or
Dd
i u

100

80

Significant at p<0.01

60

40

20

-20

-40

-60
NC A 1 2 3 4 B F A A A P C M B H N N N N P o m p C R H P Q S 1 A I B T C N A S C M
i O m - - - - ( o c c c r r e u y N A A N h - - - a e y y u t , c s e o h i r e a e

Fig. 5. % Difference in mainstream toxicant/tar ratios due to tobacco treatment.


1914 C. Liu et al. / Food and Chemical Toxicology 49 (2011) 1904–1917
The large reduction in phenol yield supports the results of pyro-lysis
found between yields of hydrogen cyanide in cigarette smoke and protein studies that have shown that for flue-cured tobacco, water soluble components
levels in the tobacco (Tso et al., 1982). Pyridine, as well as methyl and ethyl in general (Wooten et al., 2006) and chloro-genic acid (Torikaiu et al., 2005)
substituted pyridines and quinoline were found to be major pyrolysis products in particular are major precursors. The treated tobacco also contains 1%
of casein and collagen pyrolysis at 800–860 LC in nitrogen (Higman et al., glycerol which could increase the filtration efficiency of the tobacco rod for
1970; Schmeltz et al., 1972). The yields of the aromatic amines, 2- phenol.
aminonaphthalene and 4-aminobiphenyl, from mixtures of protein (bovine
serum albumin) and tobacco pyrolysed at 800 LC in nitrogen were highly The major precursors for catechol and hydroquinone in flue-cured tobacco
correlated with the level of protein added (Torikaiu et al., 2005), leading the smoke are the polyphenols (particularly chlorogenic acid), lignin, and, to a
authors to conclude that protein was a major precursor for these aromatic lesser extent, the polyphenol/protein pig-ment complexes (Sakuma et al.,
amines. Although Torikaiu et al. (2005) did not analyse the pyrolysates for the 1982; Schlotzhauer et al., 1982; Schlotzhauer and Snook, 1992). From
related amines, 1-aminonaphtha-lene and 3-aminobiphenyl, their similar tobacco pyrolysis studies Schlotzhauer and Snook (1992) estimated that for
reductions in the smoke of the treated tobacco would indicate that these flue-cured to-bacco with levels of chlorogenic acid in the range of 1.6–2.3%
amines are also generated from protein pyrolysis. approximately 50% of catechol from flue-cured cigarettes was de-rived from
chlorogenic acid in the leaf. The reduction in yield of catechol found in the
present study (10% relative to tar) is some-what lower than the approximately
Although mainstream nitric oxide (NO/NOx) yields were not determined 16% that would be predicted from a 33% reduction in chlorogenic acid. The
for this set of cigarettes, the cigarettes used for com-paring sidestream smoke reason for this is un-clear. There are a number of possible explanations: (1)
yields (Table 2) were analysed for main-stream NO/NOx yields and the the yields reported by Schlotzhauer and Snook were uncorrected for ISO tar,
results are shown in Table 10. The treated tobacco cigarettes had significantly (2) inhibitors of catechol formation (eg nitric oxide) were re-moved and (3)
(p < 0.01) lower yields of NO ( 41%) and NOx ( 38%) than the cigarettes other solubles may also generate catechol.
made with un-treated tobacco. The corresponding reductions in yields relative
to tar were 36% for NO and 32% for NOx. These results suggest that, for
flue-cured tobacco with relatively low nitrate levels, pro-tein and/or amino
acids are significant precursors for mainstream nitric oxide. This contrasts 3.4.3. Carbonyls
with results published by Umemura et al., 1986. These authors found that
after addition of insoluble protein and amino acids to cigarettes containing There were significant (p < 0.01) increases in tar-adjusted yields of
100% shredded cellulose instead of tobacco there were significant increases in formaldehyde (77%), acrolein (24%) and propionaldehyde (19%).
ni-tric oxide in sidestream smoke (next section) but not in main-stream
Pyrolysis and combustion studies have shown that a significant proportion
smoke. In fact these authors found lower levels of nitric oxide in mainstream
of formaldehyde is generated from the natural sugars found in flue-cured
smoke after treating the cellulose with the additives as well as higher puff
tobacco (Baker, 2006a,b). Baker found that addition of sugars to cigarettes at
numbers, which together suggest a change in the combustion characteristics
levels of 2.1–8.2% (w/w tobacco) caused significant increases (10–55%) in
that may have affected the yields that were reported.
formaldehyde yields when the cigarettes were smoked under ISO conditions.
However the in-crease in total sugar content of the tobacco (Table 5) as a
result of the treatment corresponds to about 1.6% (w/w tobacco) which is
The mainstream yields of the TSNA relative to tar were all re-duced by insufficient to account for the 76.5% (relative to tar) increase in
the tobacco treatment: NNN ( 12%, p < 0.05), NAT ( 11%, p < 0.05), NAB formaldehyde. It is also known that formation of formaldehyde is strongly
( 19%, p < 0.01) and NNK ( 13%), although the reduction in NNK was not inhibited by amino acids in the blend and by ammonia (generated, for
significant. In sidestream (next sec-tion) where the magnitudes of the TSNA example, from protein combustion), which compet-itively react with sugars to
yields are much greater, the effect of the tobacco treatment is much clearer. form Amadori compounds (Baker, 2006a,b). This is also supported by
Since the to-bacco treatment does not appear to change the TSNA levels in pyrolysis studies which have shown that protein addition to tobacco inhibits
the tobacco, the TSNA yield reductions in the smoke are likely to be due to a formaldehyde for-mation (Tarora et al., 2003; Yoshida et al., 2003). Thus
reduction in pyrosynthesis from leaf alkaloids and nitrogen oxides lower con-centrations of amino acids and proteins in the treated tobacco could
(Brunnemann and Hoffmann, 1991). This may be directly related to the also contribute significantly to the observed increase in the yield of
reduced yields of nitric oxide in the treated tobacco cigarettes. formaldehyde.

Increases in sugar also increase yields of acrolein and propion-aldehyde


3.4.2. Phenolic toxicants (Baker, 2006a,b) but the effect is much smaller than for formaldehyde and
does not explain the 24% and 19% increases in yields of these two carbonyls.
The treatment process resulted in significant (p < 0.01) reduc-tions in tar-
adjusted smoke yields of phenol ( 43%), o-cresol ( 11%), p-cresol ( 36%),
resorcinol ( 48%) and hydroquinone ( 23%). There were modest reductions in 3.4.4. Other toxicants
yields of catechol ( 10%) and m-cresol ( 9%) at significance levels of p <
0.05. The tar-adjusted yield of cadmium was reduced by 22% (p < 0.01), which
would be expected from the 24% reduction of cad-mium in the treated
tobacco. However, chromium which had a sig-

Table 10

Yields of mainstream tar, nicotine and nitric oxides from the cigarettes used for sidestream toxicant analyses.

Toxicant yields % Difference P value Toxicant/tar ratios % Difference P value

Untreate Treate Untreated Trea


d d ted

Mean SD Mean S
D
Tar mg/cig 16.1 1.61 14.9 1 7.4 NS – – – –
.
5
5
Nicotine mg/cig 2.12 0.21 1.90 0 10.3 NS – – – –
.
2
0
NO lg/cig 65.2 6 38.4 6 41.1 <0.01 4.05 2.58 36.3 <0.01
. .
7 5
NOx lg/cig 69.6 7 43.3 7 37.8 <0.01 4.32 2.91 32.6 <0.01
. .
5 0
C. Liu et al. / Food and Chemical Toxicology 49 (2011) 1904–1917 1915
4. Sidestream smoke results
nificantly lower concentration in the treated tobacco showed a non-significant
increase in smoke concentration. For the other metals (nickel, selenium, The results of sidestream smoke analyses are shown in Table 11 and the
mercury and lead) all the measurements were below the limit of differences in yields of toxicants between cigarettes con-taining the untreated
quantification. and treated tobaccos are shown in Fig. 6. Dif-ferences that are significant at p
< 0.01 are shown in red. There were some differences in the toxicants
Isoprene yields were increased by 39% (p < 0.01) relative to tar following measured in mainstream and sidestream smoke: chromium, nickel, arsenic
tobacco treatment. One possible explanation for this observation is that nitric and selenium were measured in mainstream (British American Tobacco Group
oxide reacts with isoprene, and that the lower yields of nitric oxide after Research and Development laboratories) but not in sidestream smoke.
treatment allow greater isoprene yields. For example Cueto et al. (1989) used
Fourier transform in-fra-red spectroscopy to monitor concentrations of nitric
oxide, nitrogen dioxide and isoprene in cigarette smoke and in mixtures of Cigarettes made with untreated and treated tobacco have sim-ilar
isoprene, nitric oxide and air. They proposed that nitric oxide is first oxidised sidestream yields of tar and nicotine, while for the treated to-bacco cigarette
to nitrogen dioxide which then reacts with iso-prene to form free radicals, there is a significant increase (12%, p < 0.01) in CO.
with subsequent autocatalytic genera-tion of nitrogen dioxide. However, the
concentrations of nitric oxide and isoprene used by Cueto et al. (1989) were Fig. 6 shows that the sidestream yields of all the nitrogen con-taining
much higher than could be obtained in the present study, and the reaction rate toxicants were decreased significantly (p < 0.01) as a result of the treatment:
of nitric oxide with oxygen to form nitrogen dioxide was too slow to account ammonia ( 39%), aromatic amines ( 28% to 41%), HCN ( 62%), TSNAs
for the reductions in isoprene observed in the present study. ( 16% to 31%), pyridine ( 14%), quin-oline ( 19%) and acrylonitrile ( 39%).
These results are consistent with the reductions in yields of the nitrogen
containing toxicants found for mainstream smoke. The yield of nitric oxide
was also sig-nificantly reduced ( 37%) in sidestream smoke, as would be ex-
Relative to tar, yields of 1,3 butadiene (17%, p < 0.01) and ben-
zo(a)pyrene (12%, p < 0.05) were increased following treatment.

Table 11

Yields of sidestream smoke toxicants.

Sidestream yields (per cigarette) Treated vs. untreated (%) P values


Untreated Treated
Average St. Dev. Average St. Dev.
Puff number 10 0.3 9.9 0.2 2.0
Tar mg/cig 29.5 2 28.9 1.6 NS
Nicotine mg/cig 7.54 0.33 7.58 0.3 0.5 NS
2
Carbon monoxide mg/cig 55.8 3.2 62.5 2.2 12.0 <0.01
Ammonia lg/cig 5847 141 3551 196 39.3 <0.01
1-Aminonaphthalene ng/cig 177 24 126 12 28.8 <0.01
2-Aminonaphthalene ng/cig 144 16 104 6 27.8 <0.01
3-Aminobiphenyl ng/cig 30.5 2.7 18.1 1.6 40.7 <0.01
4-Aminobiphenyl ng/cig 20.5 2.2 12.2 0.8 40.5 <0.01
Benzo[a]pyrene ng/cig 80.8 4.7 128 9 58.4 <0.01
Formaldehyde lg/cig 423 25 627 36 48.2 <0.01
Acetaldehyde lg/cig 1592 79 1874 130 17.7 <0.01
Acetone lg/cig 857 33 1027 47 19.8 <0.01
Acrolein lg/cig 392 17 404 27 3.1 NS
Propionaldeyde lg/cig 131 6 164 8 25.2 <0.01
Crotonaldehyde lg/cig 115 7 118 4 2.6 NS
Methyl ethyl ketone lg/cig 195 9 306 17 56.9 <0.01
Butyraldehyde lg/cig 86.5 5.7 73.7 8.7 14.8 <0.01
NO lg/cig 1392 116 881 51 36.7 <0.01
NOx lg/cig 1458 130 923 59 36.7 <0.01
Hydrogen cyanide lg/cig 157 7 59.6 1.3 62.0 <0.01
NNN ng/cig 48.3 3.8 36 2.6 25.5 <0.01
NAT ng/cig 39. 6 2.5 27.6 2.3 30.3 <0.01
NAB ng/cig 6.9 1.05 4.75 0.7 31.2 <0.01
4
NNK ng/cig 128 12 108 7 15.6 <0.01
Phenol lg/cig 359 16 275 9 23.4 <0.01
o-Cresol lg/cig 33.5 1.8 40.7 1.3 21.5 <0.01
m + p-Cresol lg/cig 89.3 2.6 79.4 2.1 11.1 <0.01
Catechol lg/cig 169 9 164 8 3.0 NS
Resorcinol lg/cig NQ NQ NQ NQ N/A –
Hydroquinone lg/cig 203 5 134 6 34.0 <0.01
Pyridine lg/cig 264 12 228 18 13.6 <0.01
Quinoline lg/cig 10.8 0.7 8.75 0.6 19.0 <0.01
2
Styrene lg/cig 87.6 13.7 75.9 6.4 13.4 NS
1,3-Butadiene lg/cig 263 16 287 29 9.1 NS
Acrylonitrile lg/cig 85.4 8.7 51.8 2.9 39.3 <0.01
Isoprene lg/cig 1244 90 1610 183 29.4 <0.01
Benzene lg/cig 248 19 265 25 6.9 NS
Toluene lg/cig 477 47 425 33 10.9 <0.05
Cadmium ng/cig 477 22 448 26 6.1 <0.05
Mercury ng/cig 13.8 1.2 9.25 1.1 33.0 <0.01
Lead ng/cig BDL BDL BDL BDL N/A –

NS: not significant.


1916 C. Liu et al. / Food and Chemical Toxicology 49 (2011) 1904–1917
8
s t 0
Significant at
i o
6 p<0.01
0

4
0
Dd
i u
2
0

% 0

-
2
0

-
4
0
-
6
0
-
8 CatecholResorcinolH

NN C A a a 3 4 B F A A A P C M B N N H N N N N P o m
quinonePyridineQuino
Styrene1,3ButadieneA

0
onitrileIsoprene

F i O m m m - - e o c c c r r E u O O C N A A N h - +
a K
m
i
n
o
-
b
i
p
h
e
n
y
l
1 2-
-

Fig. 6. % Difference in sidestream yields of smoke toxicants due to tobacco treatment.


process and the tobacco can be used with conventional cigarette making
pected from the report of Umemura et al. (1986) which found that addition of equipment. In addition, the process reduces any enzyme residues on the
insoluble protein and amino acids to cellulose ciga-rettes resulted in treated tobacco to levels below those that might cause concern.
significant increases in nitric oxide in sidestream smoke.
The reductions in the yields of a number of mainstream ciga-rette smoke
There were significant increases in the sidestream yields of many of the toxicants relative to smoke condensate and nicotine as a result of the tobacco
volatile carbonyls: formaldehyde (48%), acetaldehyde (18%), acetone (20%), treatment process are encouraging. How-ever, tobacco treatment is also
propionaldehyde (25%) and methyl ethyl ke-tone (57%). Yields of acrolein associated with increased yields of the volatile aldehydes, particularly
and crotonaldehyde however were not significantly affected by the treatment formaldehyde, and isoprene. Selective filters containing activated carbon
and the yield of butyr-aldehyde was significantly reduced ( 15%). These and/or resin adsor-bents have been found to be effective at reducing the yields
results were also consistent with the effects of the treatment on the of
mainstream yields on the carbonyls, except for acrolein.

Tobacco treatment resulted in reductions in sidestream yields of phenol


( 23%), m- and p-cresol ( 11%) and hydroquinone ( 34%), while yields of o-
cresol were increased by 21.5%. Resor-cinol yields were below the limit of
quantification for both the un-treated and treated cigarettes, and differences in
yields of catechol were not significant.

The sidestream yield of isoprene was increased (29%), as found for


mainstream smoke. Benzo(a)pyrene yields were also signifi-cantly increased
(58%) in sidestream smoke, compared with a much smaller increase in
mainstream smoke yields. Pyrosynthesis of benzo(a)pyrene is known to be
inhibited by nitrogen oxides (Rathkamp and Hoffmann, 1970) and the
reduction in nitric oxide yields from the treated tobacco could be responsible
for the ob-served increases in sidestream benzo(a)pyrene yields.

5. Conclusions

This tobacco treatment process removes more than half of the protein
nitrogen, and more than 40% of the total polyphenols from flue-cured
tobacco, while 12% of the nicotine is lost and total sug-ars increase by 14%.
The physical integrity of the tobacco is largely preserved during the treatment
these volatile toxicants and may represent an approach to the reduction of Conflict of Interest
these species.
The authors declare that there are no conflicts of interest.
It is not possible to judge from the smoke chemistry whether cigarettes
Acknowledgements
that included tobacco treated in this manner would rea-sonably be expected to
reduce the risk of one or more specific dis-eases or other adverse health Dr. Minoo Bilimoria of Imperial Tobacco Canada Ltd., Montréal, Québec,
effects associated with smoking. The 43 toxicants which were measured for Canada was responsible for much of the enzyme related work at the initiation
this study represent a small fraction of the around 5000 chemical constituents of this project.
that have been identified in mainstream smoke (Rodgman and Perfetti, 2008).
Rodgman (2003) and Rodgman and Green (2003) reviewed itu published Further work was carried out at the Group R&D Centre, British American
smoke yields and toxicity information for the approxi-mately 150 smoke Tobacco, Regents Park Road, Southampton, United King-dom with a team
toxicants which have been identified in tobacco smoke and concluded that, including Tom Woodman, Harriet Kimpton, Kav Ghanouni, Mark Earney,
currently, there is no scientific consen-sus as to the relationship between Jason Symonds, Anthony White, Paul Sungenis, Richard Salmon, Ben
specific smoke toxicants and the health risks associated with smoking. Blincoe, John McMullan, Michele Richards and Helen Barnor.

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Glossary

TPM: Total particulate matter of cigarette smoke, defined as that part of the smoke that is
trapped by a Cambridge filter

DPM: Dry particulate matter, determined by subtracting the weight of water from

TPM

NFDPM: Nicotine-free dry particulate matter, determined by subtracting the weight of nicotine