An Interlaboratory Validation Study of the Improved Transformation Assay Employing Balb/c 3T3 Cells: Results of a Collaborative Study on the Two-stage Cell Transformation Assay by the Non-genotoxic Carcinogen Study Group

Tsuchiya, Toshiyuki; Umeda, Makoto; Nishiyama, Hiroshi; Yoshimura, Isao; Ajimi, Shozo; Asakura, Masanori; Baba, Hiroshi; Dewa, Yasuaki; Ebe, Youji; Fushiwaki, Yuichi; Hamada, Shuichi; Hamamura, Tetsuo; Hayashi, Makoto; Iwase, Yumiko; Kajiwara, Yoshitsugu; Kasahara, Y.; Kawabata, Masayoshi; Kitada, Emiko; Kubo, Kin‐ya; Mashiko, Kaori; Miura, Daisuke; Mizuhashi, Fukutaro; Mizuno, Fumio; Nakajima, Masahiro; Nakamura, Yoshiyuki; Nobe, Naoko; Oishi, Hidetoshi; Ota, Erina; Sakai, Ayako; Sato, M.; Shimada, Sawako; Sugiyama, Toshie; Takahashi, Chitose; Takeda, Yuko; Tanaka, Noriho; Toyoizumi, Chikako; Tsutsui, Takeki; Wakuri, Shinobu; Yajima, Satoshi; Yajima, Nobuhiro

doi:10.1177/026119299902700409

Cited by 18 publications

(15 citation statements)

References 8 publications

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“…The cells were cultured for 11 days with a change of medium every 3–4 days, and incubated in normal medium up to 7 days. After cultivation, the cells were fixed with methanol and stained with Giemsa solution, and then foci of transformed cells were counted in 10 dishes according to the previous method 31, 36…”

Section: Methodsmentioning

confidence: 99%

In vitro cytocompatibility assessment of β‐tricalcium phosphate/carboxymethyl‐chitin composite

Muramatsu

Nakajima²,

Kikuchi³

et al. 2004

J Biomedical Materials Res

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A novel bioabsorbable bone substitute composed of a beta-tricalcium phosphate (beta-TCP) and a carboxymethyl-chitin (CM-chitin) sodium has been developed. Rabbit tibia defects (4 mm in diameter) were repaired after 4 weeks more effectively by the composite compared with a sham-operation group. To further investigate the biological safety of the components, genotoxicity and carcinogenicity of an extract prepared from the composite were determined using four different in vitro assays. The main extract component was identified as CM-chitin sodium [average molecular weight (Mw) approximately 230 kDa] as determined by Fourier transform infrared spectroscopy and gel permeation chromatography analysis. The concentrations of P and Ca possibly derived from beta-TCP were 17.7 and 37.1 microg/g, respectively, as determined by inductively coupled plasma mass spectroscopy. Both the metabolic activation and nonactivation (-S9) systems of the rat microsome S9 fraction were used to perform a genotoxicity evaluation using the Ames test and chromosome aberration assay on Chinese hamster lung fibroblast cells treated with the extract. In these assays, no genotoxicity was detected with doses < or =5 mg/mL (maximum concentration). The cell transformation assay using BALB/c 3T3 cells and the metabolic cooperation assay with V79 cells both showed negative results for any tumor-promoting activity caused by the extract (approximately 5 mg/mL). These results indicate that the bioabsorbable beta-TCP/CM-chitin composite is a highly biocompatible bone substitute.

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Section: Methodsmentioning

confidence: 99%

In vitro cytocompatibility assessment of β‐tricalcium phosphate/carboxymethyl‐chitin composite

Muramatsu

Nakajima²,

Kikuchi³

et al. 2004

J Biomedical Materials Res

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“…CtAs have been devised using rodent cell lines (e.g. Balb/c 3t3 cells; tsuchiya and Umeda, 1995Umeda, , tsuchiya et al, 1999, and immortalised fibroblast cell lines of rodent or human origin (e.g. C3H10t1/2 cells; Reznikoff et al, 1973;landolph, 1985).…”

Section: Greenscreen Genotoxicity Assaysmentioning

confidence: 99%

Reverse transcriptase activity of hepatitis B virus polymerase in eukaryotic cell extracts in vitro

Favre¹

2008

ALTEX

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In hepadnaviruses, reverse transcription is primed by the viral reverse transcriptase (RT) and requires the specific interaction between the RT and the viral RNA encapsidation signal termed ε. To study the activity of the RT in vitro, the current procedure uses in vitro translated duck hepatitis B virus polymerase, but not the hepatitis B virus polymerase itself, in the rabbit reticulo-cyte lysate expression system. Here, the hepatitis B virus (HBV) polymerase has been successfully expressed in a translational extract that was obtained from monolayer human hepatocyte cells HuH-7. The translated polypeptide retained the RNA-directed polymerase (reverse transcriptase) activity on the viral RNA template containing the ε signal. We suggest that the reverse transcription event of the viral RNA coding for the polymerase and containing an ε structure is concomitant to the translation of the viral polymerase to the messenger RNA. In contrast to the duck polymerase, only a fraction of the reverse transcribed complementary DNA (cDNA) was covalently bound to the HBV polymerase in this system. When the ε signal was missing on the mRNA, the translated full-length HBV polymerase could not reverse transcribe the viral RNA template. A truncated HBV polymerase that was lacking the YMDD catalytic active site for the initiation of reverse transcription was unable to reverse transcribe the viral mRNA template containing the ε signal. The reverse transcription activity could also be partially inhibited by employing nucleoside analogues, such as 2'-3'-dideoxy-3'-thiacytidine (3TC; lamivu-dine) in the expression system. The procedure described here provides a method for the in vitro screening of new anti-HBV compounds directed against wild-type and mutants of this crucial viral protein, the HBV polymer-ase, without the use of animals (ducks) or animal extracts (rab-bit reticulocyte lysate). Zusammenfassung: In vitro reverse transkriptase Aktivität der Hepatitis B Virus Polymerase in eukariotischen Zellextrakten In Hepadnaviren wird die reverse Transkription von der viralen reversen Transkriptase (RT) ausgeführt. Hier findet eine spezifi-sche Interaktion zwischen der RT und dem viralen Verpackungs-signal ε statt. Um die Aktivität der RT in vitro zu untersuchen, wird im aktuellen Protokoll in vitro translatierte Entenhepatitis B Virus Polymerase, aber nicht die Hepatitis B Virus Polyme-rase selbst, in einem Kaninchen Retikulozytenlysat Expressions-system eingesetzt. In dieser Arbeit wurde die Hepatitis B Virus (HBV) Polymerase erfolgreich in einem translationalen Extrakt aus einer einlagigen Kultur der humanen Hepatozytenzellen HuH-7 exprimiert. Das translatierte Polypeptid behielt die RNA-gerichtete Polymerase (reverse Transkriptase) Aktivität auf dem viralen RNA Template mit einem ε Signal. Wir schlagen vor, dass die reverse Transkrip-tion der viralen RNA, welche die Polymerase kodiert und eine ε Struktur besitzt, gleichzeitig mit der Translation der viralen Polymerase in die Boten-RNA (mRNA) abläuft. Im Gegensatz zur Entenpolymerase ...

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“…Immediately following the workshop, a collaborative study on the use of a new protocol for the Balb/c 3T3 assay appeared (81), and this assay is being used by Sabbioni and his colleagues at ECVAM for the extensive testing of metals and related substances (82). In addition to these activities, a review of transformation assays involving the use of rodent cells has recently appeared (83).…”

Section: Cell Transformation Assaysmentioning

confidence: 99%

The ECVAM Workshops: A Critical Assessment of their Impact on the Development, Validation and Acceptance of Alternative Methods

Combes

2002

Altern Lab Anim

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ECVAM initiated its workshop programme in 1994, to enable it to become well informed about the state of the art of non-animal test development and validation, and about the possible incorporation of alternatives into regulatory requirements for safety testing. Fifty-one such workshops had been held on specific topics, up to 2002. In these workshops, the current status of in vitro tests and their potential uses were reviewed and recommendations were made as to the best ways forward to progress and enhance the use of in vitro methods. Reports for 46 of these workshops have been published in ATLA. Most of the workshops focused on in vitro replacement methods, although an increasing number have dealt with reduction and refinement. The recommendations in the ECVAM workshops have been progressed further by: a) the formation of ECVAM task forces; b) the organisation of further workshops; c) the activities of scientific committees; d) the provision of earmarked research funding; and e) the conduct of validation studies. Examples of each of these activities are discussed. Some individual workshops are covered in more detail, and several recommendations that have so far not been acted on are also considered. The workshops and their reports have had a substantial effect on the development and implementation of alternative methods, and have been a major factor in contributing to the success of the first nine years of ECVAM's existence. It is strongly recommended that ECVAM continues to organise workshops and to publish their findings, and suggestions are made for topics for future workshops.

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An Interlaboratory Validation Study of the Improved Transformation Assay Employing Balb/c 3T3 Cells: Results of a Collaborative Study on the Two-stage Cell Transformation Assay by the Non-genotoxic Carcinogen Study Group

Cited by 18 publications

References 8 publications

In vitro cytocompatibility assessment of β‐tricalcium phosphate/carboxymethyl‐chitin composite

In vitro cytocompatibility assessment of β‐tricalcium phosphate/carboxymethyl‐chitin composite

Reverse transcriptase activity of hepatitis B virus polymerase in eukaryotic cell extracts in vitro

The ECVAM Workshops: A Critical Assessment of their Impact on the Development, Validation and Acceptance of Alternative Methods

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