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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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Negative

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