Gene Expression over Time during Cell Transformation Due to Non-Genotoxic Carcinogen Treatment of Bhas 42 Cells.

The Bhas 42 cell transformation assay (Bhas 42 CTA) is the first Organization for Economic Cooperation and Development (OECD)-certificated method used as a specific tool for the detection of the cell-transformation potential of tumor-promoting compounds, including non-genotoxic carcinogens (NGTxCs), as separate from genotoxic carcinogens. This assay offers the great advantage of enabling the phenotypic detection of oncotransformation. A key benefit of using the Bhas 42 CTA in the study of the cell-transformation mechanisms of tumor-promoting compounds, including non-genotoxic carcinogens, is that the cell-transformation potential of the chemical can be detected directly without treatment with a tumor-initiating compound since Bhas 42 cell line was established by transfecting the v-Ha-ras gene into a mouse fibroblast cloned cell line. Here, we analyzed the gene expression over time, using DNA microarrays, in Bhas 42 cells treated with the tumor-promoting compound 12-O-tetradecanoylphorbol-13-acetate (TPA), and NGTxC, with a total of three repeat experiments. This is the first paper to report on gene expression over time during the process of cell transformation with only a tumor-promoting compound. Pathways that were activated or inactivated during the process of cell transformation in the Bhas 42 cells treated with TPA were related not only directly to RAS but also to various pathways in the hallmarks of cancer.

Chemical carcinogen safety testing: OECD expert group international consensus on the development of an integrated approach for the testing and assessment of chemical non-genotoxic carcinogens.

While regulatory requirements for carcinogenicity testing of chemicals vary according to product sector and regulatory jurisdiction, the standard approach starts with a battery of genotoxicity tests (which include mutagenicity assays). If any of the in vivo genotoxicity tests are positive, a lifetime rodent cancer bioassay may be requested, but under most chemical regulations (except plant protection, biocides, pharmaceuticals), this is rare. The decision to conduct further testing based on genotoxicity test outcomes creates a regulatory gap for the identification of non-genotoxic carcinogens (NGTxC). With the objective of addressing this gap, in 2016, the Organization of Economic Cooperation and Development (OECD) established an expert group to develop an integrated approach to the testing and assessment (IATA) of NGTxC. Through that work, a definition of NGTxC in a regulatory context was agreed. Using the adverse outcome pathway (AOP) concept, various cancer models were developed, and overarching mechanisms and modes of action were identified. After further refining and structuring with respect to the common hallmarks of cancer and knowing that NGTxC act through a large variety of specific mechanisms, with cell proliferation commonly being a unifying element, it became evident that a panel of tests covering multiple biological traits will be needed to populate the IATA. Consequently, in addition to literature and database investigation, the OECD opened a call for relevant assays in 2018 to receive suggestions. Here, we report on the definition of NGTxC, on the development of the overarching NGTxC IATA, and on the development of ranking parameters to evaluate the assays. Ultimately the intent is to select the best scoring assays for integration in an NGTxC IATA to better identify carcinogens and reduce public health hazards.

Identification and detection of genetically modified papaya resistant to papaya ringspot virus strains in Thailand.

Many lines of genetically modified (GM) papaya (Carica papaya Linnaeus) have been developed worldwide to resist infection from various strains of papaya ringspot virus (PRSV). We found an unidentified and unauthorized GM papaya in imported processed papaya food. Transgenic vector construct that provides resistance to the PRSV strains isolated in Thailand was detected. An original and specific real-time polymerase chain reaction method was generated to qualitatively detect the PRSV-Thailand-resistant GM papaya.

Interlaboratory validation study of an event-specific real-time polymerase chain reaction detection method for genetically modified 55-1 papaya.

Genetically modified (GM) papaya line 55-1 (55-1) is resistant to papaya ringspot virus infection, and is commercially available in several countries. A specific detection method for 55-1 is required for mandatory labeling regulations. An event-specific real-time PCR method was developed by our laboratory. To validate the method, interlaboratory validation of event-specific qualitative real-time PCR analysis for 55-1 was performed in collaboration with 12 laboratories. DNA extraction and real-time PCR reaction methods were evaluated using 12 blind samples: six non-GM papayas and six GM papayas in each laboratory. Genomic DNA was highly purified from all papayas using an ion-exchange column, and the resulting DNA sample was analyzed using real-time PCR. Papaya endogenous reference gene chymopapain (CHY) and the event-specific 55-1 targeted sequence were detected in GM papayas whereas CHYalone was detected in non-GM papayas in all laboratories. The cycle threshold values of CHYand the 55-1 targeted sequence showed high repeatability (RSD, 0.6-0.8%) and reproducibility (RSDR 2.2-3.6%). This study demonstrates that the 55-1 real-time PCR detection method is a useful and reliable method to monitor 55-1 papaya in foods.

Identification and detection method for genetically modified papaya resistant to papaya ringspot virus YK strain.

Unauthorized genetically modified (GM) papaya (Carica papaya LINNAEUS) was detected in a commercially processed product, which included papaya as a major ingredient, in Japan. We identified the transgenic vector construct generated based on resistance to infection with the papaya ringspot virus (PRSV) YK strain. A specific detection method to qualitatively monitor papaya products for contamination with the GM papaya was developed using the real-time polymerase chain reaction.

Deep neural network for the determination of transformed foci in Bhas 42 cell transformation assay.

Bhas 42 cell transformation assay (CTA) has been used to estimate the carcinogenic potential of chemicals by exposing Bhas 42 cells to carcinogenic stimuli to form colonies, referred to as transformed foci, on the confluent monolayer. Transformed foci are classified and quantified by trained experts using morphological criteria. Although the assay has been certified by international validation studies and issued as a guidance document by OECD, this classification process is laborious, time consuming, and subjective. We propose using deep neural network to classify foci more rapidly and objectively. To obtain datasets, Bhas 42 CTA was conducted with a potent tumor promotor, 12-O-tetradecanoylphorbol-13-acetate, and focus images were classified by experts (1405 images in total). The labeled focus images were augmented with random image processing and used to train a convolutional neural network (CNN). The trained CNN exhibited an area under the curve score of 0.95 on a test dataset significantly outperforming conventional classifiers by beginners of focus judgment. The generalization performance of unknown chemicals was assessed by applying CNN to other tumor promotors exhibiting an area under the curve score of 0.87. The CNN-based approach could support the assay for carcinogenicity as a fundamental tool in focus scoring.

Development of a novel method for specific detection of genetically modified Atlantic salmon, AquAdvantage, using real-time polymerase chain reaction.

Genetically modified (GM) Atlantic salmon, AquAdvantage (AquAd), was the first GM animal approved officially for human consumption. Many countries monitor the use of this product under their GM regulations, but a pragmatic system for AquAd-specific detection is needed. Here, we developed a real-time polymerase chain reaction method with high sensitivity for detection of AquAd in foods. This method showed high specificity for the AquAd transgene and the detection limit was 12.5-25 targeted DNA copies per test reaction. An inter-laboratory study using the method developed demonstrated reproducibility at >0.1% (w/w) AquAd content.

Data representing applicability of developed growth hormone 1 (GH1) gene detection method for detecting Atlantic salmon (Salmo salar) at high specificity to processed salmon commodities.

This article is referred to the research article entitled "Development of a novel method for specific detection of genetically modified Atlantic salmon, AquAdvantage, using real-time polymerase chain reaction" by Soga et al. (2020). Applicability of the developed growth hormone 1 (GH1) and 18S ribosomal DNA (18S rDNA) detection methods using real-time polymerase chain reaction (PCR) for detecting Atlantic salmon (Salmo salar) to processed food commodities was examined. DNAs extracted and purified from 24 commodities labelled to include salmon as an ingredient were used as template. Yield and purity of DNAs obtained and Cq values from real-time PCR analyses were provided.

[Method for DNA extraction and purification from corn-processed food using an ion-exchange resin type kit].

The yield of genomic DNA extracted from corn-processed foods, such as corn flake and one of the corn snack what is called "Jumbo corn", using an ion-exchange resin type kit (Gtip) has been reported to be very low, and it is thought to be difficult to detect the intrinsic corn gene "Zein" in the foods. Therefore, we developed a new method using Gtip, which we called the "KNG-Gtip method," by modification of the Ministry of Health, Labour and Welfare (MHLW) method using Gtip (MHLW-Gtip method). We compared the KNG-Gtip method, MHLW-Gtip method, the Gtip method for detection of allergen (ALG-Gtip method), and the Gtip method according to the Ministry of Agriculture, Forestry and Fisheries (MAFF) (JAS-Gtip method) in terms of the yield and quality of genomic DNA and the detection probabilities of the PCR-amplified Zein gene. The concentrations of DNA and the detection probabilities of the PCR-amplified Zein gene of genomic DNA extracted from 4 g corn flake and 4 g Jumbo corn by the KNG-Gtip method were larger than those by using the conventional methods. In addition, the PCR-amplified Zein gene from 4 g of corn starch could be detected by the KNG-Gtip method. We propose that the KNG-Gtip method, in which requires sample weight of four grams, is practical and useful to extract genomic DNA from corn flake and Jumbo corn.

DNA extraction method using a silica-base resin type kit for the detection of genetically modified papaya.

Genetically modified (GM) papaya has not yet been approved for importation into, or cultivation in the European Union (EU) and Japan. A DNA extraction method using the Qiagen DNeasy Plant Mini Kit (PM method) and a method using a buffer containing cetyltrimethyl ammonium bromide (CTAB method) have been adopted as the official Japanese methods for detecting GM foods. However, the amounts of DNA extracted from papaya by these methods are very low. Therefore, we investigated an extraction method to obtain a high yield of DNA from raw or freeze-dried fresh papaya using the Promega Wizard DNA Clean-Up Resin System (WCR). The incubation for the extraction was carried out at 58 degrees C without proteinase K for 15 min. The extract was applied to a mini-column, then the column was washed with 80% isopropyl alcohol, and genomic DNA adsorbed on the column was eluted with TE buffer. The WCR method gave a higher yield of genomic DNA, and was simpler and faster than the PM method or CTAB method. In addition, it could be used to extract genomic DNA from fresh papaya at various stages of ripeness. Based on these results, we propose that the present method using WCR is the most practical and useful way to extract genomic DNA for the purpose of detecting GM papaya.

Indicated detection of two unapproved transgenic rice lines contaminating vermicelli products.

We analyzed the DNA fragments extracted from four rice vermicelli products. The Bacillus thuringiensis (Bt) rice line, which has a construct similar to the GM Shanyou 63 line, was detected in some vermicelli products by identification of the junction region sequence between rice Act1 promoter and the Cry1Ac gene, and that between Cry1Ac and nos. In addition, we also detected a different Bt rice line by means of the junction region sequence between the maize ubiquitin promoter and cry1Ab gene and that between the cauliflower mosaic virus 35S promoter and the hygromycin phosphotransferase in some vermicelli products. Accordingly, we for the first time have detected the two transgenic Bt rice lines contaminating rice vermicelli samples. Furthermore, we developed a duplex real-time polymerase chain reaction (PCR) method for the simultaneous detection of both Bt rice lines.

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.

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.

Characteristics of the transformation frequency at the tumor promotion stage of airborne particulate and gaseous matter at ten sites in Japan.

We used a high-volume air sampler in the summer of 2007 and the winter of 2008 at ten Japanese sites (Sapporo, Sendai, Maebashi, Tsukuba, Shinjuku, Sagamihara, Shizuoka, Touhaku, Kitakyushu, and Kagoshima) to collect total suspended particulate (TSP) and gaseous matter for evaluation. We evaluated the transformation frequency at the tumor promotion stage of these samples in a cell transformation assay using Bhas 42 cells, which were established from BALB/c 3T3 cells transfected with the v-Ha-ras oncogene. All samples collected from the gaseous matter were negative for transformed foci. There were several patterns of transformation frequency at the tumor promotion stage by area for the TSP samples. At Sapporo, the transformation frequency at the tumor promotion stage was remarkably higher in winter than in summer as well as in winter at the other sites. At six urban cities from Sendai to Shizuoka, the levels of transformed frequencies per µg of suspended particulates in winter were almost the same, and were higher than those of the remaining three sites. At three sites, Touhaku, Kitakyushu and Kagoshima, the transformation results in winter were judged as negative. The characteristics of the transformed frequencies of the compounds adsorbed on particulate matter at the sampling sites were significant in winter. We also studied the correlation between the transformation frequency at the tumor promotion stage of the TSP samples and the results of quantitative analysis of 16 polyaromatic hydrocarbons (PAHs) at the ten sites. We found that the transformation frequency at the tumor promotion stage of airborne samples could not be predicted based on the quantitative results of the PAHs in those samples. These data suggest that direct risk assessment of air samples with a bioassay is more valuable than quantitative analysis of compounds such as PAHs for predicting carcinogenicity.

Application of a qualitative and quantitative real-time polymerase chain reaction method for detecting genetically modified papaya line 55-1 in papaya products.

Genetically modified (GM) papaya (Carica papaya L.) line 55-1 (55-1), which is resistant to papaya ringspot virus infection, has been marketed internationally. Many countries have mandatory labeling regulations for GM foods, and there is a need for specific methods for detecting 55-1. Here, an event- and construct-specific real-time polymerase chain reaction (PCR) method was developed for detecting 55-1 in papaya products. Quantitative detection was possible for fresh papaya fruit up to dilutions of 0.001% and 0.01% (weight per weight [w/w]) for homozygous SunUp and heterozygous Rainbow cultivars, respectively, in non-GM papaya. The limit of detection and quantification was as low as 250 copies of the haploid genome according to a standard reference plasmid. The method was applicable to qualitative detection of 55-1 in eight types of processed products (canned papaya, pickled papaya, dried fruit, papaya-leaf tea, jam, puree, juice, and frozen dessert) containing papaya as a main ingredient.

Evaluation of real-time PCR detection methods for detecting rice products contaminated by rice genetically modified with a CpTI-KDEL-T-nos transgenic construct.

Genetically modified (GM) rice (Oryza sativa) lines, such as insecticidal Kefeng and Kemingdao, have been developed and found unauthorised in processed rice products in many countries. Therefore, qualitative detection methods for the GM rice are required for the GM food regulation. A transgenic construct for expressing cowpea (Vigna unguiculata) trypsin inhibitor (CpTI) was detected in some imported processed rice products contaminated with Kemingdao. The 3' terminal sequence of the identified transgenic construct for expression of CpTI included an endoplasmic reticulum retention signal coding sequence (KDEL) and nopaline synthase terminator (T-nos). The sequence was identical to that in a report on Kefeng. A novel construct-specific real-time polymerase chain reaction (PCR) detection method for detecting the junction region sequence between the CpTI-KDEL and T-nos was developed. The imported processed rice products were evaluated for the contamination of the GM rice using the developed construct-specific real-time PCR methods, and detection frequency was compared with five event-specific detection methods. The construct-specific detection methods detected the GM rice at higher frequency than the event-specific detection methods. Therefore, we propose that the construct-specific detection method is a beneficial tool for screening the contamination of GM rice lines, such as Kefeng, in processed rice products for the GM food regulation.