Transformation assay in Bhas 42 cells: a model using initiated cells to study mechanisms of carcinogenesis and predict carcinogenic potential of chemicals.

Transformation assays using cultured cells have been applied to the study of carcinogenesis. Although various cell systems exist, few cell types such as BALB/c 3T3 subclones and Syrian hamster embryo cells have been used to study chemically induced two-stage carcinogenesis. Bhas 42 cells were established as a clone by the transfection with the v-Ha-ras gene into mouse BALB/c 3T3 A31-1-1 cells and their subsequent selection based on their sensitivity to 12-O-tetradecanoylphorbol-13-acetate. Using Bhas 42 cells, transformed foci were induced by the treatment with nongenotoxic carcinogens, most of which act as tumor promoters. Therefore, Bhas 42 cells were considered to be a model of initiated cells. Subsequently, not only nongenotoxic carcinogens but also genotoxic carcinogens, most of which act as tumor initiators, were found to induce transformed foci by the modification of the protocol. Furthermore, transformation of Bhas 42 cells was induced by the transfection with genes of oncogenic potential. We interpret this high sensitivity of Bhas 42 cells to various types of carcinogenic stimuli to be related to the multistage model of carcinogenesis, as the transfection of v-Ha-ras gene further advances the parental BALB/c 3T3 A31-1-1 cells toward higher transforming potential. Thus, we propose that Bhas 42 cells are a novel and sensitive cell line for the analysis of carcinogenesis and can be used for the detection of not only carcinogenic substances but also gene alterations related to oncogenesis. This review will address characteristics of Bhas 42 cells, the transformation assay protocol, validation studies, and the various chemicals tested in this assay.

Mining-caused changes to habitat structure affect amphibian and reptile population ecology more than metal pollution.

Emissions from smelting not only contaminate water and soil with metals, but also induce extensive forest dieback and changes in resource availability and microclimate. The relative effects of such co-occurring stressors are often unknown, but this information is imperative in developing targeted restoration strategies. We assessed the role and relative effects of structural alterations of terrestrial habitat and metal pollution caused by century-long smelting operations on amphibian and reptile communities by collecting environmental and time- and area-standardized multivariate abundance data along three spatially replicated impact gradients. Overall, species richness, diversity, and abundance declined progressively with increasing levels of metals (As, Cu, and Ni) and soil temperature (T(s)) and decreasing canopy cover, amount of coarse woody debris (CWD), and relative humidity (RH). The composite habitat variable (which included canopy cover, CWD, T(s), and RH) was more strongly associated with most response metrics than the composite metal variable (As, Cu, and Ni), and canopy cover alone explained 19-74% of the variance. Moreover, species that use terrestrial habitat for specific behaviors (e.g., hibernation, dispersal), especially forest-dependent species, were more severely affected than largely aquatic species. These results suggest that structural alterations of terrestrial habitat and concomitant changes in the resource availability and microclimate have stronger effects than metal pollution per se. Furthermore, much of the variation in response metrics was explained by the joint action of several environmental variables, implying synergistic effects (e.g., exacerbation of metal toxicity by elevated temperatures in sites with reduced canopy cover). We thus argue that the restoration of terrestrial habitat conditions is a key to successful recovery of herpetofauna communities in smelting-altered landscapes.

Needle tract implantation of hepatoblastoma after percutaneous needle biopsy: report of a case.

A 13-year-old boy was referred to us for investigation of a giant liver mass, approximately 16 cm in diameter. Sonographically guided percutaneous needle biopsy was performed and histological examination revealed a fetal-type hepatoblastoma. After four courses of chemotherapy, we performed a left hepatic trisegmentectomy. Follow-up computed tomography, 55 months after the surgery, showed a 1-cm tumor on the route of the preoperative needle biopsy. A second laparotomy revealed a peritonealised tumor, which was excised. The histology of this tumor was identical to that of the primary hepatoblastoma. To our knowledge, this is only the second report of needle tract implantation of hepatoblastoma after percutaneous needle biopsy.

Closure of the Entire Fistula With Highly Effective Chemocauterization Using the Distal Hood Endoscope: A Novel Procedure for the Treatment of Pyriform Sinus Fistula.

BACKGROUND: Pyriform sinus fistula (PSF) causes a recurrent abscess in the neck. Endoscopic chemocauterization with trichloroacetic acid (TCA) for PSF is a simple, reproducible, and reliable procedure for treating PSF; however, there is concern about complications caused by TCA overflowing into the larynx. To prevent these complications, we devised a highly effective chemocauterization using a distal hooded endoscope (HuDHE). Our aim is to determine the efficacy and safety of HuDHE in children with PSF. METHODS: The main features of HuDHE are as follows (1) an endoscope with a translucent silicon hood at the tip was made; (2) TCA was endoscopically injected into the PSF; and (3) the color change of the mucosa into PSF was endoscopically evaluated. Data on children receiving HuDHE for PSF in the past seven years were collected from medical records. RESULTS: Data were obtained for eight children receiving HuDHE. The success rate of treatment for PSF after the first TCA chemocauterization was 87.5% (7/8) and the cumulative success rate after the second treatment was 100% (8/8). None of the children had recurrent PSF or serious complications such as vocal cord paralysis after HuDHE. CONCLUSION: HuDHE appears to be a less invasive, safe, and effective treatment for PSF.

Cell transformation assays for prediction of carcinogenic potential: state of the science and future research needs.

Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU Cosmetics Directive, have fuelled renewed interest in CTAs. Research is currently ongoing to improve the objectivity of the assays, reveal the underlying molecular changes leading to transformation and explore the use of novel cell types. The UK NC3Rs held an international workshop in November 2010 to review the current state of the art in this field and provide directions for future research. This paper outlines the key points highlighted at this meeting.

Photo catalogue for the classification of foci in the BALB/c 3T3 cell transformation assay.

This catalogue is a display of focus photos representative of the BALB/c 3T3 cell transformation assay (CTA). It is intended as a visual aid for the identification and the scoring of foci in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results.

Recommended protocol for the BALB/c 3T3 cell transformation assay.

The present protocol has been developed for the BALB/c 3T3 cell transformation assay (CTA), following the prevalidation study coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and reported in this issue (Tanaka et al. [16]). Based upon the experience gained from this effort and as suggested by the Validation Management Team (VMT), some acceptance and assessment criteria have been refined compared to those used during the prevalidation study. The present protocol thus describes cell culture maintenance, the dose-range finding (DRF) experiment and the transformation assay, including cytotoxicity and morphological transformation evaluation. Use of this protocol and of the associated photo catalogue included in this issue (Sasaki et al. [17]) is recommended for the future conduct of the BALB/c 3T3 CTA.

Prevalidation study of the BALB/c 3T3 cell transformation assay for assessment of carcinogenic potential of chemicals.

The cell transformation assays (CTAs) have attracted attention within the field of alternative methods due to their potential to reduce the number of animal experiments in the field of carcinogenicity. The CTA using BALB/c 3T3 cells has proved to be able to respond to chemical carcinogens by inducing morphologically transformed foci. Although a considerable amount of data on the performance of the assay has been collected, a formal evaluation focusing particularly on reproducibility, and a standardised protocol were considered important. Therefore the European Centre for the Validation of Alternative Methods (ECVAM) decided to coordinate a prevalidation study of the BALB/c 3T3 CTA. Three different laboratories from Japan and Europe participated. In the study the following modules were assessed stepwise: test definition (Module 1) consisted of the standardisation of the protocol, the selection of the cell lineage, and the preparation of a photo catalogue on the transformed foci. The within-laboratory reproducibility (Module 2) and the transferability (Module 3) were assessed using non-coded and coded 3-methylcholanthrene. Then, five coded chemicals were tested for the assessment of between-laboratory reproducibility (Module 4). All three laboratories obtained positive results with benzo[a]pyrene, phenanthrene and o-toluidine HCl. 2-Acetylaminofluorene was positive in two laboratories and equivocal in one laboratory. Anthracene was negative in all three laboratories. The chemicals except phenanthrene, which is classified by IARC (http://monographs.iarc.fr) as group 3 "not classifiable as to its carcinogenicity to human", were correctly predicted as carcinogens. Further studies on phenanthrene will clarify this discrepancy. Thus, although only a few chemicals were tested, it can be seen that the predictive capacity of the BALB/c 3T3 CTA is satisfactory. On the basis of the outcome of this study, an improved protocol, incorporating some changes related to data interpretation, has been developed. It is recommended that this protocol be used in the future to provide more data that may confirm the robustness of this protocol and the performance of the assay itself. During the study it became clear that selecting the most appropriate concentrations for the transformation assay is crucial.

An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity.

The Bhas 42 cell transformation assay is a sensitive short-term system for predicting chemical carcinogenicity. Bhas 42 cells were established from BALB/c 3T3 cells by the transfection of v-Ha-ras gene and postulated to have acquired an initiated state in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay is capable of detecting both tumor-initiating and tumor-promoting activities of chemical carcinogens. The full assay protocol consists of two components, the initiation assay and the promotion assay, to detect the initiating activity and the promoting activity, respectively. An international study was carried out to validate this cell transformation assay in which six laboratories from three countries participated. Twelve coded chemicals were examined in total and each chemical was tested by three laboratories. In the initiation assay, concordant results were obtained by three laboratories for eight out of ten chemicals and in the promotion assay, concordant results were achieved for ten of twelve chemicals. The positive results were obtained in all three laboratories with the following chemicals: 2-acetylaminofluorene was positive in both initiation and promotion assays; dibenz[a,h]anthracene was positive in the initiation assay; sodium arsenite, lithocholic acid, cadmium chloride, mezerein and methapyrilene hydrochloride were positive in the promotion assay. o-Toluidin hydrochloride was positive in the both assays in two of the three laboratories. d-Mannitol, caffeine and l-ascorbic acid were negative in both assays in all the laboratories, and anthracene was negative in both assays in two of the three laboratories except one laboratory obtaining positive result in the promotion assay. Consequently, the Bhas 42 cell transformation assay correctly discriminated all six carcinogens and two tumor promoters from four non-carcinogens. Thus, the present study demonstrated that the Bhas 42 cell transformation assay is transferable and reproducible between laboratories and applicable to the prediction of chemical carcinogenicity. In addition, by comparison of the present results with intra-laboratory data previously published, within-laboratory reproducibility using the Bhas 42 cell transformation assay was also confirmed.

A Bhas 42 cell transformation assay on 98 chemicals: the characteristics and performance for the prediction of chemical carcinogenicity.

The Bhas 42 cell transformation assay is a short-term system using a clone of the BALB/c 3T3 cells transfected with an oncogenic murine ras gene (v-Ha-ras). The assay has previously been reported to be capable of detecting the tumor-initiating and tumor-promoting activities of chemical carcinogens according to the different protocols, an initiation assay and a promotion assay, respectively. We applied this short-term assay to 98 chemicals to characterize the assay and evaluate its performance for the detection of chemical carcinogenicity. When the assay results were compared with the existing genotoxicity data, the Bhas 42 cell transformation assay could detect a considerable number of Ames-negative and Ames-discordant carcinogens: and the promotion assay detected most of those Ames-negative and -discordant carcinogens. This fact suggested that the Bhas 42 cells behaved as initiated cells in the transformation assay. The performance indices were calculated from the assay results of 52 carcinogens and 37 non-carcinogens. The concordance was 78%, sensitivity 73%, specificity 84%, positive predictivity 86%, negative predictivity 69%, false negative 27% and false positive 16%. Of these values, the concordance, specificity, negative predictivity and false positive were superior and the other performance indices were equivalent to those of conventional genotoxicity tests. From overall results, we concluded that the accuracy of prediction of chemical carcinogenicity would be improved by introducing the Bhas 42 cell transformation assay into the battery of in vitro assays.

Rapid evolution in the wild: changes in body size, life-history traits, and behavior in hunted populations of the Japanese mamushi snake.

Rapid evolution caused by human exploitation of wildlife is not usually addressed in studies of the impacts of such exploitation despite its direct relevance to population persistence. Japanese mamushi (Gloydius blomhoffii), an endemic venomous snake of the Japanese archipelago, has been heavily hunted by humans, and many populations appear to be declining or are already extirpated. We compared local populations that have been hunted regularly with populations that have not been hunted. Mamushi in hunted populations were smaller, had fewer vertebrae, produced more and smaller offspring, had increased reproductive effort among smaller females, and in nature fled at greater distances from an approaching human and were less defensive than mamushi in unhunted populations, as predicted from life-history theory. Heritability estimates for body size, number of vertebrae, and antipredator behavior were statistically significant, and neonates from hunted sites showed the same distribution of altered characters (compared with those from unhunted sites) as adults. Thus, distribution of the divergent trait between hunted and unhunted sites appeared in part to be genetically based, which suggests rapid evolution to human predation pressures. Trait distributions in hunted populations probably deviate from naturally (as opposed to anthropogenically) selected optima and, therefore, may have long-term negative repercussions on population persistence. Because rapid evolution affects a suite of parameters that characterize exploited populations, accurate understanding of the impacts of exploitation and effective resource management and conservation can only be achieved if evolutionary consequences are considered explicitly.

Comparison of sensitivity to arsenic compounds between a Bhas 42 cell transformation assay and a BALB/c 3T3 cell transformation assay.

A short-term cell transformation assay has recently been developed, using Bhas 42 cells which were established from BALB/c 3T3 cells transfected by v-Ha-ras gene and postulated to be initiated in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay has been reported to be capable of detecting initiating and promoting activities of chemical carcinogens, according to the different protocols, initiation assay and promotion assay, respectively. The assay is superior to classical transformation assays in cost and labor performance. The present study was carried out to compare its sensitivity with that of a classical BALB/c 3T3 cell system. We performed the Bhas 42 cell transformation assay with inorganic arsenic compounds which are potent environmental carcinogens in human but not mutagens in bacteria or weak mutagens in mammalian cells in vitro. Sodium arsenite, disodium arsenate, and their metabolites, monomethylarsonic acid and dimethylarsinic acid (DMAA) were included in the study. Sodium arsenite was positive in the initiation assay and all compounds except for DMAA were positive in the promotion assay. These results were compared with reported data in a two-stage BALB/c 3T3 cell transformation assay. The sensitivity of Bhas 42 cell transformation assay was found to be similar to that of the conventional BALB/c 3T3 cell transformation assay for the detection of initiating activities of arsenic compounds. For the detection of promoting activities, its sensitivity was equivalent to that of the two-stage BALB/c 3T3 cell transformation assay where the target cells were initiated with sub-threshold dose of 3-methylcholanthrene, confirming that Bhas 42 cells behave as initiated cells in the transformation assay.

Technical modification of the Balb/c 3T3 cell transformation assay: the use of serum-reduced medium to optimise the practicability of the protocol.

The two-stage Balb/c 3T3 model of cell transformation can mimic the two-stage carcinogenicity bioassay, and has been recognised as a screening method for detecting potential tumour initiators and promoters. A technical modification to the original protocol (which involved the use of M10F medium, consisting of MEM plus 10% fetal bovine serum [FBS]) has been previously proposed, in order to increase its efficacy, namely: the introduction of enriched, serum-reduced medium (DF2F medium, comprising DMEM/F12 plus 2% FBS and other supplements). The aim of this study was to further modify the protocol, so as to attain higher practicability for the assay. The protocol was further optimised by: a) reducing the number of plates required, through the use of larger plates; b) reducing the cost of the assay by retaining the reduced serum concentration and by using 2microg/ml insulin, rather than the more-complex insulin-transferrin-ethanolamine-sodium selenite (ITES) supplement (i.e. DF2F2I medium); and c) extending the culture period from 24-25 days to 31-32 days, resulting in clearer foci (the number of medium changes did not increase, as less-frequent medium changes were performed during the extended culture period). Growth curve construction revealed that variations in the saturation densities of the parental Balb/c 3T3 cell line and its three transformed clones were highest when M10F medium was replaced with DF2F2I medium just before cells reached confluence. We applied this newly-optimised protocol to the assessment of: a) the tumour initiating activity of 3-methylcholanthrene (MCA), N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C, methylmethane sulphonate, CdCl(2) and phenacetin, combining a post-treatment of 100ng/ml 12-O-tetradecanoylphorbol-13-acetate at the promotion stage; and b) the tumour promoting activity of insulin, lithocholic acid, CdCl(2) and phenobarbital, with pre-treatment of 0.2microg/ml MCA at the initiation stage. In the present study, only phenobarbital was negative when tested by using the modified protocol.

Yersinia pseudotuberculosis infection intractable by antibiotics: A rare case report.

INTRODUCTION: Yersinia pseudotuberculosis infection is usually cured spontaneously or with administration of antibiotics. PRESENTATION OF CASE: The patient is a twelve-year-old boy with right lower quadrant pain who had enterocolitis one month previously. Contrast-enhanced abdominal computed tomography showed a distended and edematous ileum and an intra-abdominal abscess adjacent to the mesentery with a normal appendix. The patient's general condition did not improve with antibiotics, so an ileocecectomy was performed. DISCUSSION: Yersinia pseudotuberculosis infection requiring an operation is rare. In our case, antibiotics were not effective in treating the abscess therefore surgery was required. An early diagnosis using serological studies, ultrasound of the abdomen, and fecal culture, with appropriate administration of antibiotics, may have avoided the need for surgery. Considering YP infection as a differential diagnosis is therefore important when encountering patients with enterocolitis, especially with right lower quadrant pain. Early diagnosis may assist in avoiding unnecessary operations. CONCLUSION: Diagnosis of YP infection may be missed or delayed because it is rare and difficult to detect, and must be distinguished from appendicitis. Although most YP infections are self-limiting, some rare cases will require surgery, therefore early diagnosis is essential.

Detection of initiating as well as promoting activity of chemicals by a novel cell transformation assay using v-Ha-ras-transfected BALB/c 3T3 cells (Bhas 42 cells).

Cell transformation assay using BALB/c 3T3 cells, C3H10T1/2 cells and others, can simulate the two-stage carcinogenesis utilized for formation of transformed foci. A sensitive cell transformation assay for tumor initiators as well as promoters has been developed using a v-Ha-ras-transfected BALB/c 3T3 cell line, Bhas 42; these cells are regarded as initiated in the two-stage paradigm of carcinogenesis. To distinguish between initiation and promotion, the initiation assay involves a 2-day treatment of low-density cells, obtained one day after plating, with a test chemical, and the promotion assay involves treatment of near-confluent cells with a test chemical for a period of 12 days (Day 3-14). When Bhas 42 cells were treated with tumor initiators, N-methyl-N'-nitro-N-nitrosoguanidine and 3-methylcholanthrene, transformed foci were induced in the initiation assay but not in the promotion assay. In contrast, tumor promoters, 12-O-tetradecanoylphorbol-13-acetate, lithocholic acid and okadaic acid, gave negative responses in the initiation assay but positive responses in the promotion assay. The results were reproducible with various treatment protocols. Sixteen polycyclic aromatic hydrocarbons were examined using both assays. Benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene induced focus formation only in the initiation assay. Increase of focus formation was observed in the promotion assay with benzo[e]pyrene, benzo[ghi]perylene, 1-nitropyrene and pyrene. Benz[a]anthracene, benz[b]anthracene, chrysene and perylene showed positive responses in both initiation and promotion assays. Results of initiation and promotion assays of acenaphthylene, anthracene, coronene, 9,10-diphenylanthracene, naphthalene and phenanthrene were negative or equivocal. The present Bhas assays for the detection of either/both initiating and promoting activities of chemicals are sensitive and of high performance compared with other cell transformation assays.

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.

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.

Minimally Conjoined Omphalopagus Twins with a Body Stalk Anomaly.

Introduction This report will discuss a case of minimally conjoined omphalopagus twins (MCOTs) with a body stalk anomaly (BSA). Case Report We experienced monochorionic diamniotic (MD) twins born at 31 weeks. One infant was suspicious of BSA before birth, and another infant was normal. But normal infant had anal atresia with small intestine which was inserted behind the umbilicus. Twins had very short common umbilicus and infant with BSA had intestinal conjunction, two appendixes at the site of the colon, and a blind-ending colon. We diagnosed MCOTs. Discussion On the basis of the Spencer hypothesis, the etiology of MCOTs was that MD twins shared a yolk sac. However, this could not explain the presence of a BSA. It is necessary to consider the possible reasons for a singleton BSA. In addition, intestinal fusion occurred unequally in this case, although two appendixes were found in the same place, which might have occurred because of the balanced fusion.

In vitro cytocompatibility assessment of beta-tricalcium phosphate/carboxymethyl-chitin composite.

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.

An assay method for the prediction of tumor promoting potential of chemicals by the use of Bhas 42 cells.

It has become an important task to develop a simple in vitro method for the detection of non-genotoxic carcinogens, among which tumor promoters are included. Bhas 42 cells are v-Ha-ras-transfected BALB/c 3T3 cells and are regarded as initiated cells in the 2-stage transformation paradigm. We designed a method for detecting tumor promoters by the use of Bhas 42 cells at advanced passage generation. In this method, the cells are cultured in six-well plates for 17 days during which test chemicals are added in the medium for 11 days from days 3 to 14. The end-point of the assay is the induction of transformed foci. When the tumor promoter TPA was used, a significant number of transformed foci were induced concentration-dependently, whereas only a few foci were observed in control cultures. When various chemicals were examined by the method, a reasonable correlation was observed with the reported tumor-promoting ability in animal experiments. We propose that the Bhas 42 cell transformation method is practical and useful for the detection of tumor promoters.

Relevance of chemical structure and cytotoxicity to the induction of chromosome aberrations based on the testing results of 98 high production volume industrial chemicals.

Over a 6-year period (1991-1996), the chromosomal aberration testing of high production volume (HPV) industrial chemicals had been conducted using Chinese hamster lung (CHL/IU) cells according to OECD HPV testing program and the national program in Japan. A total of 98 chemicals were tested for the induction of chromosome aberration (CA), consisting of structural CA and polyploidy. Of the 98 chemicals, structural CA and/or polyploidy were induced by 39 chemicals (40%). Anilines and phenols tended to induce only structural CA. p-tert-Butylphenol had a peculiar feature in inducing not only structural CA but also polyploidy at considerably high frequency (93.2%) after continuous treatment for 48 h, posing an aneugenic potential. Not all, but six of 11 carboxylic acids or esters also showed the simultaneous induction of structural CA and polyploidy. The majority of organic phosphates, alcohols or ethers, alkyl benzenes and non-cyclic alkanes had no CA induction activity. For chemicals which were negative in the bacterial reverse mutation assay (Ames test), the proportion of the chemicals that induced CA at a severely cytotoxic dose (doses manifesting more than 50% cytotoxicity) was similar to that of the CA-negative chemicals manifesting severe cytotoxicity, suggesting that severely cytotoxic chemicals do not always induce CA.