kanemaru y., otsu y., fukushima t. - jt science · hci kanemaru y., otsu y., fukushima t. japan...
TRANSCRIPT
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KANEMARU Y., OTSU Y., FUKUSHIMA T.
Japan Tobacco Inc., R&D group, Scientific Product Assessment Center
1. Introduction and Objective
2. Materials & Methods
3. Results
4. Discussion
Evaluation of cigarette smoke-induced initiation and promotion potency in Bhas 42 cell transformation assay
5. Conclusion
Reference
DMSO (SC) TPA* (PC)
TPM and GVP sample preparation
Acknowledgement
Chemical carcinogenesis is known to be a multistage process consisting of
initiation, promotion, and progression. There are many in vitro assays for
detecting initiation (genotoxic) potency, which are adopted by regulatory
authorities. In contrast, there are few validated in vitro assays for detecting
promotion and/or progression potencies despite a significant number of
carcinogens being considered to act via non-genotoxic mode of action.
Bhas 42 cell transformation assay (CTA) is a simple in vitro assay for
predicting carcinogenicity of test chemicals by measuring morphologically
transformed foci in v-Ha-ras-transfected BALB/c-3T3 cells. The assay
protocol consists of two components; initiation assay and promotion assay
for detecting activity at each step of the carcinogenesis process. Both
assay protocols have been validated internationally [1 and 2].
The objective of this study is to evaluate the initiation and promotion
potency of cigarette smoke total particulate matter (TPM) and gas vapor
phase (GVP) in the Bhas 42 CTA (Objective 1). We also investigated the
assays capability to discriminate the promotion potencies of TPM collected
from combustible and heated cigarettes (HC) (Objective 2).
The mainstream smoke of Kentucky 3R4F and a heated cigarette were
generated under ISO and Health Canada Intense (HCI) smoking regime. The
TPM was collected on glass fiber filters and extracted with DMSO. The GVP
which passed through the filter was collected by bubbling into ice-cold PBS.
Cell culture
Cell transformation assay
This work was conducted in the Food and Drug Safety Center (FDSC) where the Bhas 42 cell
have been established. We are grateful to the member of FDSC for their technical assistance.
Bhas 42 cells were obtained from Japanese Cancer Research Resources
Bank. The cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s
F12 supplemented with 5% fetal bovine serum in a 5% CO2 incubator at 37ºC.
CTA was performed according to OECD draft test guideline [3]. The
experimental schedule was shown in Fig.1. Six wells were used for each test
substance concentration. The cells were treated with test substances for 3
days at growth phase in the initiation assay, and for 10 days at stationary
phase in the promotion assay. After 21 day culture, the cells were fixed in
methanol and stained with 5% Giemsa solution. The number of transformed
foci in each well was counted and average value of six wells were recorded.
*TPA: 12-O-Tetradecanoyl- phorbol-13-acetate
The Bhas 42 CTA detected the promotion potency of cigarette TPM.
The results of product comparison in this assay is consistent with that of the in
vivo skin painting assay.
The Bhas 42 CTA is a useful tool to assess the promotion potency of tobacco
products, and could potentially be used to replace the in vivo skin-painting assays.
Fig.1. Schematic protocol and morphological characteristics of transformed
focus in the Bhas 42 CTA.
Transformed focus - > 100 cells - Spindle-shaped - Basophilic - Piling up - Criss-cross - Invasive
Normal cells - Monolayer - Contact inhibition
1. A. Sakai et al., Mutat Res. 725 (2011) 57–77
2. EURL ECVAM Recommendation on the Cell Transformation Assay based on the Bhas 42 cell line.
3. OECD draft test guideline of Bhas 42 cell transformation assay, which is to be adopted as a test guidance
4. Reporting Harmful and Potentially Harmful Constituents in Tobacco Products and Tobacco Smoke Under
Section 904(a)(3) of the Federal Food, Drug, and Cosmetic Act, draft guidance
5. H. Tsuji et al., Food Chem Toxicol. 72 (2014) 187-94
●: No of foci/well
○: Relative cell growth (%)
■: Positive controls
- MCA(1μg/mL) for Ini.
- TPA(50ng/mL) for Pro.
Mean ± S.D. are presented.
* represents significant
difference compared to SC
by Dunnett’s test (p < 0.05). Fig. 2. Initiation potency of 3R4F TPM and GVP
1. Initiation and promotion potency of cigarette smoke in the Bhas 42 CTA
2. Product comparison using the promotion assay
Carbon heat source
Charcoal filter
Acetate filter
Tobacco leaves Aluminum wrapper
Figure and table. Schematic representation and TNCO yeilds of HC
3R4F HC 3R4F HC
TPM 10.1 0.72 43.9 49.5
Water 0.89 0.13 16.1 28.6
Tar 8.50 0.57 25.9 20.6
Nicotine 0.71 0.01 1.95 0.37
CO 11.3 1.64 30.6 21.2
ISO HCI
(mg/cig.)
The chemical analysis was performed according to a modified Health Canada official method.
3R4F TPM (ISO)
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3R4F GVP (ISO)
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3R4F GVP (ISO)
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Tox.
3R4F TPM (ISO)
Positive *
*
* *
Fig. 3. Promotion potency of 3R4F TPM and GVP
Representative wells in the promotion assay of TPM
Fig. 4. Relative amounts of HC smoke constituents included in the
abbreviated list of Harmful and Potentially Harmful Constituents (HPHCs) [4]
Fig. 5. Comparison of the promotion potency of 3R4F and HC in the Bhas 42 CTA
DMSO (0.33%)
TPA (50ng/mL)
3R4F (2μg/mL)
3R4F (4μg/mL)
3R4F (8μg/mL)
3R4F (16μg/mL)
1-1. Initiation potency of cigarette smoke in the Bhas 42 CTA
No dose-related increase of transformed foci was observed in both TPM and
GVP fractions (Fig.2), while many of genotoxicity assays such as Ames and
micronucleus gave positive results to cigarette TPM or GVP. The reason why
this assay did not detect the initiation potency of cigarette smoke is uncertain,
but one possible explanation is that continuous stimuli by promotors at
stationary growth phase may be necessary for malignant transformation of the
cigarette smoke-initiated Bhas 42 cells.
1-2. Promotion potency of cigarette smoke in the Bhas 42 CTA
TPM but not GVP induced significant increase of transformed foci (Fig.3). This
results suggested that the impact of GVP on the promotion potency of
cigarette smoke might be negligible.
Fig. 6. Incidence of gross
skin mass in the in vivo
skin painting assay †
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TPM (HCI)
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3R4F HC
TPM (ISO)
* * *
* *
3R4F
HC
2. Product comparison using the promotion assay
The ranking of promotion potency was 3R4F > HC
(Fig. 5) in consistent with the reduced amounts of
toxicants of HC (Fig. 4). The Bhas 42 CTA can
discriminate the promotion potency of tobacco
products in different categories. The ranking of
promotion potency (3R4F > HC) in the Bhas 42 CTA
were comparable to that of the tumor incidence in
the in vivo skin painting assay reported from Tsuji
et.al [5]. (Fig. 6).
* *
*
† The tar sample collected by HCI regime was applied to SENCAR mice
for 30 wks. * p < 0.05 compared to negative control (DMBA + vehicle).
Normal cell Initiated cell
Initiation Promotion
Tumor cell DNA damage Proliferation
Bhas 42 CTA
1-1. Initiation assay
1-2. Promotion assay
3R4F
▼ : Less than limit of detection in HC.
▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲
Initiation assay
Promotion assay
Day 21 0 14 11 7 4 1
Treat at log growth phase
Treat at stationary phase
(0.4×104 cells/well)
(1.4×104 cells/well) Exposure
Medium change
Add chemical