An Inter-laboratory Collaborative Study by the Non-Genotoxic Carcinogen Study Group in Japan,on a Cell Transformation Assay for Tumour Promoters Using Bhas 42 cells

Abstract

The Bhas promotion assay is a cell culture transformation assay designed as a sensitive and economical method for detecting the tumour-promoting activities of chemicals. In order to validate the transferability and applicability of this assay, an inter-laboratory collaborative study was conducted with the participation of 14 laboratories. After confirmation that these laboratories could obtain positive results with two tumour promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and lithocholic acid (LCA), 12 coded chemicals were assayed. Each chemical was tested in four laboratories. For eight chemicals, all four laboratories obtained consistent results, and for two of the other four chemicals, only one of the four laboratories showed inconsistent results. Thus, the rate of consistency was high. During the study, several issues were raised, each of which were analysed step-by-step, leading to revision of the protocol of the original assay. Among these issues were the importance of careful maintenance of mother cultures and the adoption of test concentrations for toxic chemicals. In addition, it is suggested that three different types of chemicals show positive promoting activity in the assay. Those designated as T-type induced extreme growth enhancement, and included TPA, mezerein, PDD and insulin. LCA and okadaic acid belonged to the L-type category, in which transformed foci were induced at concentrations showing growth-inhibition. In contrast, M-type chemicals, progesterone, catechol and sodium saccharin, induced foci at concentrations with little or slight growth inhibition. The fact that different types of chemicals similarly induce transformed foci in the Bhas promotion assay may provide clues for elucidating mechanisms of tumour promotion.

Keywords

Bhas 42 cells cell transformation assay inter-laboratory collaborative study tumour promoter

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References

Sugimura

(1992). Multistep carcinogenesis: a 1992 perspective. Science, New York 259, 603–607.

OECD (1997). Guidelines for Testing of Chemicals. Section 4. Health Effects. Paris, France: Organization for Economic Co-operation and Development. Website http://www.oecd.org/document/55/0,2340,en_2649_34377_2349687_1_1_1_1,00.html

Kakunaga

(1973). A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/3T3. International Journal of Cancer 12, 463–473.

Reznikoff

C.A.

, Bertram

J.S.

, Brankow

D.W.

, & Heidelberger

(1973). Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. Cancer Research 33, 3239–3249.

Hallowell

, Tu

, Pallotta

, Sivak

, Lubet

R.A.

, Curren

R.D.

, Avery

M.D.

, Jones

, Sedita

B.A.

, & Huberman

(1986). An interlaboratory comparison of transformation in Syrian hamster embryo cells with model and coded chemicals. Environmental Mutagenesis 8, 77–98.

Tsuchiya

, & Umeda

(1995). Improvement in the efficiency of the in vitro transformation assay method using BALB/3T3 A31-1-1 cells. Carcinogenesis 16, 1887–1894.

Yotti

L.P.

, Chang

C.C.

, & Trosko

J.E.

(1979). Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter. Science, New York 206, 1089–1091.

Murray

A.W.

, & Fitzgerald

D.J.

(1979). Tumor promoters inhibit metabolic cooperation in co-cultures of epidermal and 3T3 cells. Biochemical and Biophysical Research Communications 91, 395–401.

Umeda

, Noda

, & Ono

(1980). Inhibition of metabolic cooperation in Chinese hamster cells by various chemicals including tumor promoters. Japanese Journal of Cancer Research (Gann) 71, 614–620.

10.

Rovera

, Santoli

, & Damsky

(1979). Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester. Proceedings of the National Academy of Sciences of the USA 76, 2779–2783.

11.

Ito

, Yanase

, Fujita

, Harayama

, Takashima

, & Imanaka

(1981). A short-term in vitro assay for promoter substances using human lymphoblastoid cells latently infected with Epstein-Barr virus. Cancer Letters 13, 29–37.

12.

Ohmori

, Miyazaki

, & Umeda

(1998). Detection of tumor promoters by early antigen expression of EB virus in Raji cells using a fluorescence microplate-reader. Cancer Letters 132, 51–59.

13.

Busser

M.T.

, & Lutz

W.K.

(1987). Stimulation of DNA synthesis in rat and mouse liver by various tumor promoters. Carcinogenesis 81, 1433–1437.

14.

Ohmori

, Sasaki

, Asada

, Tanaka

, & Umeda

(2004). An assay method for the prediction of tumor promoting potential of chemicals by the use of Bhas 42 cells. Mutation Research 557, 191–202.

15.

Nagasawa

, Mori

, & Nakajima

(1980). Long-term effects of progesterone or diethylstilbestrol with or without estrogen after maturity on mammary tumorigenesis in mice. European Journal of Cancer 16, 1583–1589.

16.

Huseby

R.A.

(1980). Demonstration of a direct carcinogenic effect of estradiol on Leydig cells of the mouse. Cancer Research 40, 1006–1013.

17.

Asada

, Sasaki

, Tanaka

, Takeda

, Hayashi

, & Umeda

(2005). Detection of initiating as well as promoting activity of chemicals by a novel cell transformation assay using v-Ha-rastransfected BALB/c3T3 cells (Bhas 42 cells). Mutation Research 588, 7–21.