Asking the right questions for mutagenicity prediction from BioMedical text.

Assessing the mutagenicity of chemicals is an essential task in the drug development process. Usually, databases and other structured sources for AMES mutagenicity exist, which have been carefully and laboriously curated from scientific publications. As knowledge accumulates over time, updating these databases is always an overhead and impractical. In this paper, we first propose the problem of predicting the mutagenicity of chemicals from textual information in scientific publications. More simply, given a chemical and evidence in the natural language form from publications where the mutagenicity of the chemical is described, the goal of the model/algorithm is to predict if it is potentially mutagenic or not. For this, we first construct a golden standard data set and then propose MutaPredBERT, a prediction model fine-tuned on BioLinkBERT based on a question-answering formulation of the problem. We leverage transfer learning and use the help of large transformer-based models to achieve a Macro F1 score of >0.88 even with relatively small data for fine-tuning. Our work establishes the utility of large language models for the construction of structured sources of knowledge bases directly from scientific publications.

DNA polymerase κ suppresses inflammation and inflammation-induced mutagenesis and carcinogenic potential in the colon of mice.

BACKGROUND: Chronic inflammation induces DNA damage and promotes cell proliferation, thereby increasing the risk of cancer. DNA polymerase κ (Pol κ), involved in translesion DNA synthesis, counteracts mutagenesis induced by inflammation in the colon of mice. In the present study, we examined whether Pol κ suppressed inflammation-induced colon tumorigenesis by treating inactivated Polk knock-in (Polk-/-) mice with dextran sulfate sodium (DSS), an inducer of colon inflammation. RESULTS: Male and female Polk-/- and Polk+/+ mice were administered 2% DSS in drinking water for six consecutive days, succeeded via a recovery period of 16 days, followed by 2% DSS for another two days. DSS treatment strongly induced colitis, and the severity of colitis was higher in Polk-/- mice than in Polk+/+ mice. The mice were sacrificed after 19 weeks from the initiation of the first DSS treatment and subjected to pathological examination and mutation analysis. DSS treatment induced colonic dysplasia, and the multiplicity of dysplasia was higher in Polk-/- mice than in Polk+/+mice. Some of the dysplasias in Polk-/- mice exhibited ß-catenin-stained nucleus and/or cytoplasm. Mutation frequencies in the gpt reporter gene were increased by DSS treatment in Polk-/- mice, and were higher than those in Polk+/+ mice. CONCLUSIONS: Pol κ suppresses inflammation and inflammation-induced dysplasia as well as inflammation-induced mutagenesis. The possible mechanisms by which Pol κ suppresses colitis- and colitis-induced dysplasia are discussed.

Optimizing machine-learning models for mutagenicity prediction through better feature selection.

Assessing a compound's mutagenicity using machine learning is an important activity in the drug discovery and development process. Traditional methods of mutagenicity detection, such as Ames test, are expensive and time and labor intensive. In this context, in silico methods that predict a compound mutagenicity with high accuracy are important. Recently, machine-learning (ML) models are increasingly being proposed to improve the accuracy of mutagenicity prediction. While these models are used in practice, there is further scope to improve the accuracy of these models. We hypothesize that choosing the right features to train the model can further lead to better accuracy. We systematically consider and evaluate a combination of novel structural and molecular features which have the maximal impact on the accuracy of models. We rigorously evaluate these features against multiple classification models (from classical ML models to deep neural network models). The performance of the models was assessed using 5- and 10-fold cross-validation and we show that our approach using the molecule structure, molecular properties, and structural alerts as feature sets successfully outperform the state-of-the-art methods for mutagenicity prediction for the Hansen et al. benchmark dataset with an area under the receiver operating characteristic curve of 0.93. More importantly, our framework shows how combining features could benefit model accuracy improvements.

o-Aminoazotoluene, 7,12-dimethylbenz[a]anthracene, and N-ethyl-N-nitrosourea, which are mutagenic but not carcinogenic in the colon, rapidly induce colonic tumors in mice with dextran sulfate sodium-induced colitis.

BACKGROUND: Several rodent models with chemically induced colon cancer have been developed. Among these models, dextran sulfate sodium (DSS), a colitis inducer, combined with azoxymethane as a colon mutagenic carcinogen, is commonly used. We previously reported that although benzo [a] pyrene (BP) is mutagenic but not carcinogenic in the colon, it rapidly develops colon tumors at a high incidence/multiplicity after treatment with DSS. In the present study, we examined whether other colon-mutagenic non-carcinogens (CMNCs) induced colon tumors after treatment with DSS. RESULTS: o-Aminoazotoluene, 7,12-dimethylbenz[a]anthracene, and N-ethyl-N-nitrosourea were selected as CMNCs. Male CD2F1 mice were orally administered CMNC for 5 consecutive days. After a 9-day dose-free period, mice were treated with 4% DSS in drinking water for 1 week. Three months after DSS treatment, colon samples were collected for histopathology and ß-catenin immunohistochemistry analyses. All CMNCs in combination with DSS induced colonic adenocarcinomas at a high incidence/multiplicity in the distal and middle parts of the colon, coinciding with the location of colitis. Unlike in normal cells where ß-catenin is exclusively located on the cell membrane, in adenocarcinoma cells, it was translocated to both the nucleus and cytoplasm or only to cytoplasm. The translocation of ß-catenin is closely associated with colon carcinogenesis in rodents and humans. No colonic tumors or dysplastic lesions were found after exposure to either CMNC or DSS alone. CONCLUSION: We provided further evidence clearly showing that CMNCs can rapidly induce colonic tumors in mice with DSS-induced colitis, even if they are not colonic carcinogens.

Bacterial mutagenicity test data: collection by the task force of the Japan pharmaceutical manufacturers association.

BACKGROUND: Ames test is used worldwide for detecting the bacterial mutagenicity of chemicals. In silico analyses of bacterial mutagenicity have recently gained acceptance by regulatory agencies; however, current in silico models for prediction remain to be improved. The Japan Pharmaceutical Manufacturers Association (JPMA) organized a task force in 2017 in which eight Japanese pharmaceutical companies had participated. The purpose of this task force was to disclose a piece of pharmaceutical companies' proprietary Ames test data. RESULTS: Ames test data for 99 chemicals of various chemical classes were collected for disclosure in this study. These chemicals are related to the manufacturing process of pharmaceutical drugs, including reagents, synthetic intermediates, and drug substances. The structure-activity (mutagenicity) relationships are discussed in relation to structural alerts for each chemical class. In addition, in silico analyses of these chemicals were conducted using a knowledge-based model of Derek Nexus (Derek) and a statistics-based model (GT1_BMUT module) of CASE Ultra. To calculate the effectiveness of these models, 89 chemicals for Derek and 54 chemicals for CASE Ultra were selected; major exclusions were the salt form of four chemicals that were tested both in the salt and free forms for both models, and 35 chemicals called "known" positives or negatives for CASE Ultra. For Derek, the sensitivity, specificity, and accuracy were 65% (15/23), 71% (47/66), and 70% (62/89), respectively. The sensitivity, specificity, and accuracy were 50% (6/12), 60% (25/42), and 57% (31/54) for CASE Ultra, respectively. The ratio of overall disagreement between the CASE Ultra "known" positives/negatives and the actual test results was 11% (4/35). In this study, 19 out of 28 mutagens (68%) were detected with TA100 and/or TA98, and 9 out of 28 mutagens (32%) were detected with either TA1535, TA1537, WP2uvrA, or their combination. CONCLUSION: The Ames test data presented here will help avoid duplicated Ames testing in some cases, support duplicate testing in other cases, improve in silico models, and enhance our understanding of the mechanisms of mutagenesis.

Revisiting the bacterial mutagenicity assays: Report by a workgroup of the International Workshops on Genotoxicity Testing (IWGT).

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to obtain consensus on unresolved issues associated with genotoxicity testing. At the 2017 IWGT meeting in Tokyo, four sub-groups addressed issues associated with the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471, which describes the use of bacterial reverse-mutation tests. The strains sub-group analyzed test data from >10,000 chemicals, tested additional chemicals, and concluded that some strains listed in TG471 are unnecessary because they detected fewer mutagens than other strains that the guideline describes as equivalent. Thus, they concluded that a smaller panel of strains would suffice to detect most mutagens. The laboratory proficiency sub-group recommended (a) establishing strain cell banks, (b) developing bacterial growth protocols that optimize assay sensitivity, and (c) testing "proficiency compounds" to gain assay experience and establish historical positive and control databases. The sub-group on criteria for assay evaluation recommended that laboratories (a) track positive and negative control data; (b) develop acceptability criteria for positive and negative controls; (c) optimize dose-spacing and the number of analyzable doses when there is evidence of toxicity; (d) use a combination of three criteria to evaluate results: a dose-related increase in revertants, a clear increase in revertants in at least one dose relative to the concurrent negative control, and at least one dose that produced an increase in revertants above control limits established by the laboratory from historical negative controls; and (e) establish experimental designs to resolve unclear results. The in silico sub-group summarized in silico utility as a tool in genotoxicity assessment but made no specific recommendations for TG471. Thus, the workgroup identified issues that could be addressed if TG471 is revised. The companion papers (a) provide evidence-based approaches, (b) recommend priorities, and (c) give examples of clearly defined terms to support revision of TG471.

Evaluation of acetone as a solvent for the Ames test.

BACKGROUND: Acetone is a common alternative solvent used in the Ames test when test chemicals are unstable or poorly soluble in water or dimethyl sulfoxide (DMSO). Yet, there is a very limited number of studies evaluating acetone as a solvent in the modified Ames test with preincubation (preincubation test). RESULTS: We evaluated the acetone as a solvent for the preincubation test. Fourteen mutagens dissolved in acetone was added each to the reaction mixture at 2 different volumes (25 or 50 µL) to examine mutagenicity using bacterial test strains recommended in the Organization for Economic Cooperation and Development (OECD) test guideline 471, and compared with DMSO (100 µL). Cytotoxicity of acetone was also examined in these bacterial strains. TA1537 was most sensitive to the cytotoxicity of acetone, the degree of which was moderate and similar to DMSO in TA1537 without S9 mix. In other strains, cytotoxicity was limited to a mild degree with or without S9 mix. Cytotoxicity of acetone did not affect detection of mutagenicity of any mutagens; many of them being comparable or less mutagenic than those with DMSO. CONCLUSIONS: These findings indicate that acetone is a viable candidate as a solvent for the preincubation test in the 5 bacterial strains.

Recommended criteria for the evaluation of bacterial mutagenicity data (Ames test).

A committee was constituted within the International Workshop on Genetic Toxicology Testing (IWGT) to evaluate the current criteria for a valid Ames test and to provide recommendations for interpretation of test results. Currently, determination of a positive vs. a negative result is made by applying various data evaluation procedures for comparing dosed plates with the concurrent solvent control plates. These evaluation procedures include a requirement for a specific fold increase (2- or 3-fold, specific to the bacterial strain), formal statistical procedures, or subjective (expert judgment) evaluation. After extensive discussion, the workgroup was not able to reach consensus recommendations in favor of any of these procedures. There was a consensus that combining additional evaluation criteria to the comparison between dosed plates and the concurrent solvent control plates improves test interpretation. The workgroup recommended using these additional criteria because the induction of mutations is a continuum of responses and there is no biological relevance to a strict dividing line between a positive (mutagenic) and not-positive (nonmutagenic) response. The most useful additional criteria identified were a concentration-response relationship and consideration of a possible increase above the concurrent control in the context of the laboratory's historical solvent control values for the particular tester strain. The workgroup also emphasized the need for additional testing to resolve weak or inconclusive responses, usually with altered experimental conditions chosen based on the initial results. Use of these multiple criteria allowed the workgroup to reach consensus on definitions of "clear positive" and "clear negative" responses which would not require a repeat test for clarification. The workgroup also reached consensus on recommendations to compare the responses of concurrent positive and negative controls to historical control distributions for assay acceptability, and the use of control charts to determine the validity of the individual test.

Demonstrating laboratory proficiency in bacterial mutagenicity assays for regulatory submission.

The bacterial reverse mutation test is a mainstay for evaluation of mutagenicity predicting the carcinogenic potential of a test substance and is recommended by regulatory agencies across the globe. The popularity of the test is due, in part, to the relatively low cost, rapid results and small amount of test material required compared to most other toxicological tests as well as the near universal acceptance of the toxicological significance of a clear positive or negative result. Most laboratories follow the Organization for Economic Cooperation and Development Test Guideline 471 (TG471) or national guidelines based on TG471. Regulatory agencies in most countries are obligated to consider results from tests which meet the recommendations laid out in TG471. Nonetheless, laboratories unfamiliar with the test sometimes have trouble generating reliable, reproducible results. TG471 is a test guideline, not a detailed test protocol. A group of experts from regulatory agencies and laboratories which use the assay has assembled here a set of recommendations which if followed, will allow an inexperienced laboratory to acquire proficiency in assay conduct. These include recommendations for how to create a cell bank for the 5 Salmonella typhimurium/Escherichia coli strains and develop a laboratory protocol to reliably culture each strain to ensure each culture has the characteristics which allow adequate sensitivity for detection of mutagens using the test as described in TG471. By testing compounds on the provided lists of positive and negative test substances, the laboratory will have surmounted many of the problems commonly encountered during routine testing of unknown chemicals and will have gained the experience necessary to prepare the detailed protocol needed for performing the test under Good Laboratory Procedures and the laboratory will have generated the historical positive and negative control databases which are needed for test reports which adhere to TG471.

DNA polymerase kappa counteracts inflammation-induced mutagenesis in multiple organs of mice.

In vitro studies indicate that DNA polymerase kappa (Polκ) is able to accurately and efficiently perform DNA synthesis using templates containing various types of DNA damage, including benzo[a]pyrene (BP)-induced N2 -deoxyguanosine adducts. In this study, we examined sensitivity of inactivated Polk knock-in (Polk-/- ) mice to BP carcinogenicity in the colon by administering an oral dose of BP plus dextran sulfate sodium (DSS), an inflammation causing promoter of carcinogenesis. Although colon cancer was successfully induced by BP plus DSS, there was no significant difference in tumor incidence or multiplicity between Polk-/- and Polk+/+ mice. Malignant lymphoma was induced in thymus by the treatment only in Polk-/- mice, but it lacked statistical significance. Mutant frequencies (MFs) in the gpt reporter gene were strongly enhanced in colon; almost to the same extent in both types of mice. Micronucleus formation in bone marrow at the high dose of BP and DNA adducts in colon and lung was not significantly different between two types of mice. Surprisingly, however, Polk-/- mice exhibited significantly higher MFs in colon and lung than did Polk+/+ mice when they were treated with DSS alone. The most prominent mutation induced by DSS treatment was G:C to C:G transversion, whose specific MF in proximal colon was 30 times higher in Polk-/- than in Polk+/+ mice. DSS alone did not enhance MF at all in Polk+/+ mice. The results indicate that Polκ does not suppress BP-induced mutagenesis and carcinogenesis in the colon, but counteracts inflammation-induced mutagenesis in multiple organs. Environ. Mol. Mutagen. 60:320-330, 2019. © 2019 Wiley Periodicals, Inc.

Negative and positive control ranges in the bacterial reverse mutation test: JEMS/BMS collaborative study.

A large-scale study was conducted by multiple laboratories affiliated with the Japanese Environmental Mutagen Society and the Bacterial Mutagenicity Study Group to investigate possible proficiency indicators for the bacterial reverse mutation test with a preincubation procedure. Approximately 30 laboratories generated negative and positive control count data and dose-response curves of the positive control articles for the bacterial reverse mutation test, with assays conducted annually from 2013 to 2016. Overall, the majority of the negative and positive control counts for Salmonella Typhimurium strains TA100, TA1535, TA98, and TA1537, and Escherichia coli strain WP2uvrA, with and without S9 mix, were within the range of the means ±2× standard deviation. The negative counts were normally distributed (strains TA100, TA98, and WP2uvrA) or followed Poisson distribution (strains TA1535 and TA1537), and the positive control counts for all strains were approximately normally distributed. In addition, the distribution of the negative and positive control counts was relatively constant over the 4 years. The number of revertant colonies increased in a dose-dependent linear or exponential fashion up to the recommended doses for the respective positive control articles in Japan. These data are valuable for determining the acceptance criteria and an estimation of the laboratory proficiency for the bacterial reverse mutation test.

The strains recommended for use in the bacterial reverse mutation test (OECD guideline 471) can be certified as non-genetically modified organisms.

The bacterial reverse mutation test, commonly called Ames test, is used worldwide. In Japan, the genetically modified organisms (GMOs) are regulated under the Cartagena Domestic Law, and organisms obtained by self-cloning and/or natural occurrence would be exempted from the law case by case. The strains of Salmonella typhimurium and Escherichia coli recommended for use in the bacterial reverse mutation test (OECD guideline 471), have been considered as non-GMOs because they can be constructed by self-cloning or naturally occurring bacterial strains, or do not disturb the biological diversity. The present article explains the reasons why these tester strains should be classified as non-GMOs.

Wif1 and Ifitm3 gene expression preferentially altered in the colon mucosa of benzo[a]pyrene pre-treated mice following exposure to dextran sulfate sodium.

Benzo[a]pyrene (BP) is highly mutagenic and yet does not lead to tumor development in the murine colon. We recently reported the generation of colonic tumors one week after treatment with BP followed by dextran sulfate sodium (DSS), a colitis-inducer. In this BP/DSS model, male CD2F1 mice were treated orally with BP at 125 mg/kg/day for 5 days, followed by 4% DSS in drinking water for one week. There has been no report so far on the molecular mechanisms involved in tumor development in this model. In the present study, we performed global gene expression analysis on the colonic mucosae obtained from BP-exposed mice one week after treatment with DSS and those treated with the vehicle, BP, or DSS alone. Global gene expression analysis revealed that there were 563 genes preferentially altered (≥2-fold vs vehicle group) in the colonic mucosae exposed to both BP and DSS. Furthermore, comparative gene expression analysis combined with Ingenuity Pathway Analysis™ identified 2 genes associated with Wnt/ß-catenin signaling pathway that were preferentially up-regulated (≥2-fold vs vehicle group) when BP and DSS were treated in combination in the distal part (site of predilection for tumor induction) of the colonic mucosae, especially in colonic tumors: WNT inhibitory factor 1 (Wif1; 14.6-fold increase) and interferon induced membrane protein 3 (Ifitm3; 5.7-fold increase). In colonic tumors, expression of Wif1 and Ifitm3 proteins were both confirmed by western blot analysis. These findings suggest that these genes are associated with rapid induction of colonic tumors in mice after exposure to BP/DSS, providing insights into the mechanisms of the BP/DSS short-term colon carcinogenesis.

Time course of the incidence/multiplicity and histopathological features of murine colonic dysplasia, adenoma and adenocarcinoma induced by benzo[a]pyrene and dextran sulfate sodium.

Benzo[a]pyrene (BP) is mutagenic but noncarcinogenic in the murine colon. Recently, we reported rapid induction of colonic tumors by treatment of CD2F1 mice with BP (125 mg/kg for 5 days) followed by a colitis inducer, dextran sulfate sodium (DSS) (4% in drinking water for 1 or 2 weeks). However, there are no reports on detailed time course and histopathological features of colonic proliferative lesions in this model. Here, we show the detailed time course of colonic dysplasia, adenoma and adenocarcinoma induced by treatment with BP, DSS, and a combination of the two (BP/DSS). In the colon of mice exposed to BP/DSS, 14.6 dysplastic foci per mouse were present one week after DSS treatment (week 4). The number of dysplastic foci decreased with time to 3.1 at week 9 and thereafter remained almost constant. At week 4, 1.5 adenocarcinomas were also observed, with a marked increase in numbers with time, reaching 29.3 at week 14. In contrast, the number of dysplastic foci induced by DSS alone showed a time course similar to that following BP/DSS treatment; however, only a few tumors appeared. Neither dysplastic foci nor neoplastic lesions were induced by BP only. In mice exposed to BP/DSS, ß-catenin was demonstrated immunohistochemically in the nucleus and/or cytoplasm of the tumor cells, and this translocation from the cell membrane was evident in subsets of dysplastic foci. In dysplastic foci induced by DSS alone, ß-catenin was absent in the nucleus/cytoplasm. These finding suggest that aberrant ß-catenin accumulation in dysplastic foci is associated with tumor progression in this BP/DSS model.

Rapid induction of colonic adenocarcinoma in mice exposed to benzo[a]pyrene and dextran sulfate sodium.

Previously, we reported that the mutation frequency was markedly increased in the colon after the oral treatment of mice with an environmental mutagen/carcinogen, benzo[a]pyrene (BP); however this was not followed by tumor development. The reasons for this are as yet unresolved. The purpose of the present study is to explore the mechanisms why a high frequency of mutations induced by BP in the colon is not associated with subsequent tumor development. We show in this study that oral administration of BP to CD2F(1) mice at 125 mg/kg/day for 5 days can lead to adenocarcinomas in the mouse colon both at Weeks 4 (5/8 mice) and 11 (100% of mice), but only in the presence of inflammation induced by 4% dextran sulfate sodium (DSS) in the drinking water for up to 2 weeks. These data indicate that, in this DSS model, BP induced mutagenic events lead to tumors in the mouse colon, a tissue which is not a BP target organ. DSS-induced inflammation in a tissue primed with mutagenic risk is a key to the induction of tumors in this model. This study provides a novel, rapid and useful colon carcinogenesis model (BP/DSS model).

Strategy for genotoxicity testing--metabolic considerations.

The report from the 2002 International Workshop on Genotoxicity Tests (IWGT) Strategy Expert Group emphasized metabolic considerations as an important area to address in developing a common strategy for genotoxicity testing. A working group convened at the 2005 4th IWGT to discuss this area further and propose practical strategy recommendations. To propose a strategy, the working group reviewed: (1) the current status and deficiencies, including examples of carcinogens "missed" in genotoxicity testing, established shortcomings of the standard in vitro induced S9 activation system and drug metabolite case examples; (2) the current status of possible remedies, including alternative S9 sources, other external metabolism systems or genetically engineered test systems; (3) any existing positions or guidance. The working group established consensus principles to guide strategy development. Thus, a human metabolite of interest should be represented in genotoxicity and carcinogenicity testing, including evaluation of alternative genotoxicity in vitro metabolic activation or test systems, and the selection of a carcinogenicity test species showing appropriate biotransformation. Appropriate action triggers need to be defined based on the extent of human exposure, considering any structural knowledge of the metabolite, and when genotoxicity is observed upon in vitro testing in the presence of metabolic activation. These triggers also need to be considered in defining the timing of human pharmaceutical ADME assessments. The working group proposed two strategies to consider; a more proactive approach, which emphasizes early metabolism predictions to drive appropriate hazard assessment; and a retroactive approach to manage safety risks of a unique or "major" metabolite once identified and quantitated from human clinical ADME studies. In both strategies, the assessment of the genotoxic potential of a metabolite could include the use of an alternative or optimized in vitro metabolic activation system, or direct testing of an isolated or synthesized metabolite. The working group also identified specific areas where more data or experiences need to be gained to reach consensus. These included defining a discrete exposure action trigger for safety assessment and when direct testing of a metabolite of interest is warranted versus the use of an alternative in vitro activation system, a universal recommendation for the timing of human ADME studies for drug candidates and the positioning of metabolite structural knowledge (through in silico systems, literature, expert analysis) in supporting metabolite safety qualification. Lastly, the working group outlined future considerations for refining the initially proposed strategies. These included the need for further evaluation of the current in vitro genotoxicity testing protocols that can potentially perturb or reduce the level of metabolic activity (potential alterations in metabolism associated with both the use of some solvents to solubilize test chemicals and testing to the guidance limit dose), and proposing broader evaluations of alternative metabolic activation sources or engineered test systems to further challenge the suitability of (or replace) the current induced liver S9 activation source.

Nitrogen-substitution effects on the mutagenicity and cytochrome P450 isoform-selectivity of chrysene analogs.

Nitrogen-containing analogs of chrysene, 1,10-diazachrysene (1,10-DAC) and 4,10-DAC, were tested for mutagenicity in Salmonella typhimurium TA100 in the presence of rat liver S9 and human liver microsomes to investigate the effect of nitrogen-substitution. Although these DACs could not be converted to the bay-region diol epoxide because of their nitrogen atoms in the bay-region epoxide or diol moiety, DACs were mutagenic in the Ames test with rat liver S9. Both DACs also showed mutagenicity in the Ames test using pooled human liver microsomes, although chrysene itself was not mutagenic in the presence of pooled human liver microsomes. The mutagenicity of DACs (50nmol/plate) in Ames tests using human liver microsome preparations from 10 individuals was compared with cytochrome P450 (CYP) activity in each microsome preparation to investigate the CYP isoform involved in the activation of DACs to the genotoxic forms. The numbers of induced revertants obtained by 1,10-DAC varied 6.2-folds (109-680) and those by 4,10-DAC 4.8-folds (155-751) among the 10 individuals. The number of induced revertants obtained by 1,10-DAC significantly correlated with the CYP1A2-selective catalytic activity (r=0.84, P<0.01) in each microsome preparation. On the other hand, the number of induced revertants obtained by 4,10-DAC significantly correlated with the combined activity of CYP2A6 and 1A2 (CYP2A6+0.51xCYP1A2; r=0.75, P<0.01). However, in Ames tests using microsomes from insect cells expressing various human CYP isoforms, the mutagenicity of 1,10-DAC was induced only by recombinant human CYP1A2, whereas both recombinant human CYP2A6 and 1A2 contributed to the mutagenicity of 4,10-DAC. These results suggest that 1,10-DAC shows the mutagenicity through involvement of CYP1A2 in human liver, and 4,10-DAC does so through both CYP2A6 and 1A2. In conclusion, our results suggested that the difference in the nitrogen-substituted position in the chrysene molecule might affect the mutagenic activity through influencing the ratio of participation of the metabolic activation enzyme isoforms of CYP.

Salmonella/human S9 mutagenicity test: a collaborative study with 58 compounds.

A large and extensive body of data on the use of human liver S9 fractions in the Salmonella mutagenicity test (Ames test) is presented; the data were obtained from a collaborative study by JEMS/BMS (Bacterial Mutagenicity Test Study Group) members and the Human and Animal Bridging Research Organization (HAB). In this study, the mutagenicity of 58 chemicals, many of which were judged to be human carcinogens by the IARC, was determined by the Ames test (the pre-incubation method at 37 degrees C for 20 min) in the presence of a selected human liver S9 fraction with a high drug-metabolic activity or a pooled human liver S9 fraction with a moderate drug-metabolic activity. For reference, mutagenicity was also examined in the presence of a phenobarbital/5,6-benzoflavone-pretreated rat liver S9 fraction, which is normally used in mutagenicity testing systems. The bacterial test strains consisted of Salmonella typhimurium TA100, TA98 or YG7108. The data indicated that the mutagenicity of chemicals in the rat and human liver S9 fractions varied considerably, depending on the chemicals in question. In addition, a large inter-individual diversity in the mutagenic response to mutagens, depending on the chemical structures of the mutagens, was also demonstrated using two selected human S9 fractions. Most of the mutagens tested in this study (75%; 36 out of 48 compounds that were judged to be mutagenic in at least one S9 fraction) were less mutagenic in the presence of the two human S9 fractions than in the presence of the rat S9 fraction. On the other hand, the other compounds (25%), including some aromatic amines and nitrosamines, showed a more potent mutagenicity in the presence of either one of the two human S9 fractions than in the presence of the rat S9 fraction. These data strongly suggest that the use of human liver S9 fraction in mutagenicity testing systems may be useful for a better understanding of the mutagenic effects of chemicals on humans.

Metabolic activation of 10-aza-substituted benzo[a]pyrene by cytochrome P450 1A2 in human liver microsomes.

We previously reported that 10-azabenzo[a]pyrene (10-azaBaP), a 10-aza-analog of BaP and an environmental carcinogen, showed greater mutagenicity than BaP in the Ames test using pooled human liver S9. To investigate the cytochrome P450 (CYP) isoform involved in the activation of 10-azaBaP to the genotoxic form, the mutagenicity of 10-azaBaP using nine individual donors' and pooled human liver microsome preparations was compared with each CYP activity. Induced revertants by 2.5 nmol per plate 10-azaBaP with 0.5 mg per plate human liver microsomal protein showed a large inter-individual variation (42-fold) among the nine donors. The number of induced revertants highly correlated with the CYP1A2-selective catalytic activity from each microsome preparation, and no correlation was observed with other CYP isoform-selective catalytic activities. Moreover, recombinant human CYP1A2 contributed to the mutagenicity of 10-azaBaP more markedly than recombinant human CYP1A1. These results suggest that CYP1A2 may be the principal enzyme responsible for the metabolic activation of 10-azaBaP in human liver microsomes. With regard to the proposal that BaP may be activated by human CYP1A1, our results suggest that the nitrogen-substitution at position-10 of BaP may cause the CYP enzyme-specificity in metabolic activation to change from CYP1A1 to CYP1A2.