Carcinogenicity of mutagens: predictive capability of the Salmonella mutagenesis assay for rodent carcinogenicity

A total of 224 chemicals that have been tested in long-term studies for carcinogenicity in rats and mice by the National Cancer Institute and the National Toxicology Program were tested for mutagenicity in Salmonella typhimurium. Correlations between mutagenicity and carcinogenicity were examined. The influences of chemical structure, rodent species and organ responses, and bacterial strain responses on the carcinogenesis/mutagenesis correlations were also examined. Not all carcinogens induced tumors in both rats and mice. A clear mutagenic or equivocal mutagenic response in Salmonella was predictive for 77% of the carcinogens or equivocal carcinogens, although only 54% of the 149 carcinogens or equivocal carcinogens were mutagens, and 58% of the nonmutagens were carcinogens or equivocal carcinogens. The proportion of mutagens and equivocal mutagens that were not carcinogenic or equivocal was 23%. There was no apparent way to distinguish the mutagenic carcinogens from the mutagenic noncarcinogens by the responses of the specific Salmonella strains. The proportions of different chemical classes in the data base strongly affected the correlations; 40% of the chlorinated carcinogens were mutagens, whereas 75% of the amines and 100% of the nitro-containing carcinogens were mutagens. Because 29% of the chemicals (30% of the carcinogens) were chlorinated, the poor correlation of this class was reflected in the overall correlation. It is concluded that the use of the Salmonella mutagenicity assay is warranted for the identification of carcinogens, but not for noncarcinogens. The proportion of carcinogens detected as mutagens is dependent on the specific classes of chemicals tested and on the rodent species used to define the carcinogens.     

Genotoxicity of a Variety of Hydrazine Derivatives in the Hepatocyte Primary Culture/DNA Repair Test Using Rat and Mouse Hepatocytes

The genotoxicity of a variety of hydrazine derivatives was examined in the DNA-repair test on rat or mouse hepatocytes. Out of 32 hydrazine derivatives, 6 chemicals, i.e., N -acetyl-4-(hydroxymethyl)phenylhydrazine, 1,2-dimethylhydrazine - 2HCl, 1-hydrazinophthalazine - HCl, methylhydrazine-sulfate, p, p '-oxybisbenzene disulfonylhydrazide and phenylhydrazine-HCl, elicited positive DNA repair responses in the test on rat hepatocytes. In the test on mouse hepatocytes, 4 more hydrazine derivatives, i.e., 1,1-dimethylhydrazine, hydrazine hydrate, hydrazine sulfate and 2-methyl-4-chlorophenoxyacetic acid hydrazide-HCl also generated positive responses, in addition to the 6 positive compounds in the rat assay. These results suggest that mouse hepatocytes are more susceptible to the genotoxicity of hydrazine derivatives, and that the species differences in genotoxicity appear to he in agreement with the in vivo carcinogenicity of these agents.     

Genotoxicity of a variety of hydrazine derivatives in the hepatocyte primary culture/DNA repair test using rat and mouse hepatocytes

The genotoxicity of a variety of hydrazine derivatives was examined in the DNA-repair test on rat or mouse hepatocytes. Out of 32 hydrazine derivatives, 6 chemicals, i.e., N'-acetyl-4-(hydroxymethyl)phenylhydrazine, 1,2-dimethylhydrazine.2HCl, 1-hydrazinophthalazine.HCl, methylhydrazine.sulfate, p,p'-oxybisbenzene disulfonylhydrazide and phenylhydrazine.HCl, elicited positive DNA repair responses in the test on rat hepatocytes. In the test on mouse hepatocytes, 4 more hydrazine derivatives, i.e., 1,1-dimethylhydrazine, hydrazine hydrate, hydrazine sulfate and 2-methyl-4-chlorophenoxyacetic acid hydrazide.HCl also generated positive responses, in addition to the 6 positive compounds in the rat assay. These results suggest that mouse hepatocytes are more susceptible to the genotoxicity of hydrazine derivatives, and that the species differences in genotoxicity appear to be in agreement with the in vivo carcinogenicity of these agents.     

A SEQUENTIAL TESTING PROGRAM FOR PREDICTING AND IDENTIFICATING CARCINOGENS AND ITS APPLICATION

In this paper our studies about the sequential testing program for predicting and identificating carcinogens, sequential discriminant method and cost- effectiveness analysis are summarized. The analysis of our database of carcinogeniclty and genotoxicity of chemicals demonstrates the uncertainty . of short- term tests ( STTs ) to predict carcinogens and the results of most routine STTs are statistically dependent. We recommend the sequential testing program combining STTs and carclnogenicity assay, the optimal STT batteries, the rules of the sequential discrimination and the preferal choices of STTs tor specific chemical class. For illustrative pmposes the carclnogenicity prediction of several sample chamicals is presented. The results of cost-effectiveness analysis suggest that this program has vast social-economic effectiveness.     

Interlaboratory comparison of IDH mutation detection

Isocitrate dehydrogenase (IDH) mutational testing is becoming increasingly important. For this, robust and reliable assays are needed. We tested the variation of results between six laboratories of testing for IDH mutations. Each laboratory received five unstained slides from 31 formalin-fixed paraffin-embedded (FFPE) glioma samples, and followed its own standard IDH diagnostic routine. All laboratories used immunohistochemistry (IHC) with an antibody against the most frequent IDH1 mutation (R132H) as a first step. Three laboratories then sequenced only IHC negative cases while the others sequenced all cases. Based on the overall analysis, 13 samples from 11 tumors had an R132H mutation and one tumor showed an R132G mutation. Results of IHC for IDH1 R132H mutations in all six laboratories were completely in agreement, and identified all R132H mutations. Upon sequencing the results of two laboratories deviated from those of the others. After a review of the entire diagnostic process, on repeat (blinded) testing one laboratory was completely in agreement with the overall result. A change in technique did only partially improve the results in the other laboratory. IHC for the IDH1 R132H mutation is very reliable and consistent across laboratories. IDH sequencing procedures yielded inconsistent results in 2 out of 6 laboratories. Quality assurance is pivotal before IDH testing is made part of clinical management of patients.     

Response of the ke test to NCI/NTP-screened chemicals. III. Complementary value of ke in screening for carcinogens.

The value of using a physico-chemical carcinogen-screening test, the ke test, in conjunction with the Salmonella typhimurium/microsome assay (the Ames test) and/or structural alerts of reactivity (the S/A test), is analyzed on the basis of the response of the three tests to 171 chemicals of known rodent carcinogenicity. The Ames test is widely used to screen chemicals for potential carcinogenicity; however, its relatively low sensitivity (proportion of true positives among carcinogens tested) has prompted a search for complementary tests that increase sensitivity without an unacceptable decrease in specificity (proportion of true negatives among non-carcinogens tested). The S/A test is a structural analysis based on recognition of chemicals groups likely to react with DNA. The S/A test does not complement the Ames test well, because of the high similarity of responses (dependence) between these two tests. The ke test measures the affinity of a test chemical for electrons, and has a sensitivity and specificity comparable to the Ames test. The ke test is shown in this work to complement both the Ames test and the S/A test. Addition of the ke test to either the Ames test or the S/A test results in a substantial decrease in false negatives and an approximately equal increase in false positives, which is a trade-off that would be desirable in all but the least risk averse situations. The S/A and ke battery has a sensitivity of > 0.9, and could be applied to untested chemicals without any biological testing. In view of these observations, it is proposed that the ke test be considered in developing future strategies to optimize the screening of potential carcinogens in the most cost-effective manner.     

Evaluation of the Toxicity Forecasting Capability of EPA's ToxCast Phase I Data: Can ToxCast In Vitro Assays Predict Carcinogenicity?

Long-term rodent bioassays have played a central role in protecting human health from carcinogens; for ethical and practical reasons their use is decreasing whereas genotoxicity testing has taken a pivotal role. However, this strategy—as presently implemented—is not sensitive enough to detect all genotoxic carcinogens, and cannot detect nongenotoxic carcinogens. Among the alternative approaches under study there is the ToxCast/Tox21 project. Following a previous study from our laboratory, here we present a new, more extensive analysis of ToxCast Phase I results, indicating that at the present state-of-art this approach is not able to predict the carcinogenicity of chemicals. Possible reasons for this mediocre performance are discussed, and opinions on ways to tune up the project in the next phases are presented.     

Aryl hydrocarbon receptor signaling plays a significant role in mediating benzo[a]pyrene- and cigarette smoke condensate-induced cytogenetic damage in vivo

This laboratory has previously reported data suggesting that aryl hydrocarbon receptor (AhR) signaling may have a net potentiating effect on the DNA damaging potential of cigarette smoke. The experiments described in this report extend these studies by testing whether the potent AhR antagonist 3'-methoxy-4'-nitroflavone (3'M4'NF) can modify the in vivo genetic toxicity of benzo[a]pyrene (B[a]P) and the complex mixture of chemicals in cigarette smoke condensate (CSC). Initial experiments were designed to determine 3'M4'NF doses which can antagonize AhR in vivo but which have little effect on constitutive cytochrome P4501A (CYP1A) activity. These experiments took three forms: (i) zoxazolamine paralysis tests, a functional assay of cytochrome P450 CYP1A activity in 3'M4'NF-treated C57Bl/6J mice; (ii) co-treatment of AHR: null allele mice with 150 mg/kg B[a]P plus a range of 3'M4'NF concentrations in order to evaluate the potential of the flavone to interact with non-AhR targets which may affect B[a]P toxicity; (iii) an evaluation of the in vivo AhR antagonist activity of 3'M4'NF using transgenic mice which carry a dioxin-responsive element-regulated lacZ reporter. Once an appropriate dose range was determined, C57Bl/6J mice were challenged with B[a]P or CSC with and without 3'M4'NF co-treatment. Chromosome damage was measured by scoring the frequency of micronuclei in peripheral blood reticulocytes. Data presented herein suggest that 3'M4'NF can protect mice from B[a]P-induced bone marrow cytotoxicity and genotoxicity. Furthermore, CSC-associated genotoxicity was abolished by the flavonoid. These data add support to our hypothesis that AhR signaling has a net potentiating effect on the genetic toxicity and, presumably, carcinogenicity of cigarette smoke.     

Chemicals classified by IARC: their potency in tests for carcinogenicity in rodents and their genotoxicity and acute toxicity.

Chemicals classified by the IARC to its groups 1, 2A, 2B and 3 were examined in an attempt to identify characteristics of their behaviour in experimental studies of carcinogenicity, genotoxicity and acute mammalian toxicity that correlate with those categories. Only those agents for which information on carcinogenic potency was available were studied. In both mice and rats, more chemicals were potent carcinogens if they had been categorized in Group 1 (human carcinogens) than if they had been put into one of the other categories. Not surprisingly, there was a weak association between carcinogenic potency and acute toxicity. Mice were especially sensitive to tumour induction by halides; the lower sensitivity of rats to any carcinogenic effect of halides could be due in part to their higher systemic toxicity in this species: a reduced differential of toxic and carcinogenic doses decreases the dose window in which carcinogenic effects may be demonstrated. It was notable that the human carcinogens were active in those genotoxicity tests with higher specificity for identifying rodent carcinogens. Predictive assays for carcinogenicity that were considered to be highly specific were tests for cytogenetic effects in vivo, unscheduled DNA synthesis in hepatocytes, mutation in any of the five commonly used strains of Salmonella typhimurium and mutation at the hprt locus in mammalian cells. None of the relationships was strong enough to form the basis of a simple categorization, but they could serve to alert investigators to chemicals of special toxicological interest and importance.     

化学品致癌性评分方案的比较研究

致癌性分类和分级是致癌危害性评定的主要步骤,对管理毒理学具有重要的意义。本文利用含47种化学品的致癌性数据库,比较了6种致癌性分类和评分方案。结果发现IARC分类和机理分类与致癌强度无显著的相关。TD50和多因素致癌性评分法具有某些局限性。对于遗传毒致癌物和非致癌物,多因素致癌性评分与遗传毒性联合评分有较好的相关(r=0.7872)。致癌性的综合评分方案应定量评价对人和动物的致癌性,还应考虑致癌作用机理、毒物动力学及生物学标志等研究资料。     

Effects of repeated applications of two semi-permanent hair dyes to the skin of A and DBAf mice

Two proprietary semi-permanent hair dyes were tested for carcinogenicity in A and DBAf mice by repeated topical applications in aqueous acetone. Mice of both strains developed lymphoid tumours but experimental differences were marked only in DBAf mice. A number of tumours of the ovary and uterus, and some skin papillomas near the penis, occured in dye-treated but not in control DBAf mice. As many hair-dye constituents are known mutagens, adequate carcinogenicity testing of these substances, and epidemiological study of exposed human populations, are needed for evaluating possible health hazard from hair dyeing.     

A peer comparison program for the quality assurance of human papillomavirus DNA detection using the Digene Hybrid Capture II/SurePath method shows excellent analytic interlaboratory correlation

BACKGROUND: Interlaboratory peer comparison programs are quality-assurance activities mandated by the Clinical Laboratory Improvement Amendments of 1988. No commercial program is available currently that was designed for cytology laboratories performing only human papillomavirus (HPV) DNA testing. In this report, the authors provide the results from a self-developed program between 2 cytology laboratories. METHODS: Between 4 and 11 SurePath liquid-based cervical cytology samples were selected at each of the 2 participating laboratories each quarter and exchanged without accompanying patient information. Samples were selected to test both positive and negative high-risk HPV DNA results in roughly equivalent numbers. Samples were run with the Hybrid Capture II method using each laboratory's standard procedure. The result obtained was compared with the originating laboratory's result. Correlation was compared on an ongoing basis as a method to assess analytic performance. RESULTS: Over a 3-year period, 12 exchanges took place, constituting 113 total specimens. Overall, there were 9 exchanges of 76 specimens that had 100% correlation, and 3 exchanges in which 4 of 37 specimens had discordant results. Overall, this represented a 97% correlation (109 of 113 specimens) of results between laboratories. All 4 discordant cases were reported as negative by the original laboratory and positive by the exchange laboratory (2 in each direction). CONCLUSIONS: The interlaboratory peer comparison result of 97% concordance demonstrated excellent analytic agreement between the HPV DNA-detection procedures of each laboratory. All discordant cases were "negative to positive" and were distributed equally by originating laboratory. The procedure was easily set up and provided assurance to each laboratory of ongoing performance for the detection of the HPV DNA analyte.     

Use of E mu-PIM-1 transgenic mice short-term in vivo carcinogenicity testing: lymphoma induction by benzo[a]pyrene, but not by TPA

E mu-pim-1 transgenic mice are predisposed to develop lymphomas. Due to their low spontaneous tumour incidence and their increased sensitivity towards the lymphomagen ethylnitrosourea these mice may present an interesting model for short-term carcinogenicity testing. Here, we report on the further exploration of this transgenic mouse model with two additional carcinogens known to have, among others, the lymphohaematopoietic system as target, i.e. benzo[a]pyrene (B[a]P) and 12-O-tetradecanoylphorbol-13-acetate (TPA). B[a]P, given three times a week (by gavage) for 13 weeks at 4.3, 13 or 39 mg/kg body weight, resulted in a dose-related increase in lymphomas up to a 90% incidence in E(mu)-pim-1 mice during the observation period of 40 weeks. B[a]P also induced tumours of the forestomach within this observation period, though at a lower incidence and apparently equally effective in wildtype and transgenic mice. TPA, on the other hand, was unable to induce lymphomas (or tumours in any other organ) in either transgenic or wildtype animals within the observation period of 44 weeks, when applied dermally at the maximum tolerated dose of 3 microg/mouse, twice a week for 35 weeks. Molecular analysis showed that B[a]P-induced lymphomas in transgenic mice were of T-cell origin, 80% of which had elevated levels of c-myc expression. None of the lymphomas had increased N-myc expression and mutation analysis of the ras-gene family revealed a K-ras mutation in only one out of eight tumours investigated. Also, none of the lymphomas showed aberrant expression of p53 as determined by immunohistochemistry. It is concluded that the E mu-pim-1 mouse model will not be very suitable for short-term carcinogenicity testing in general: only genotoxic chemicals that have the lymphohaematopoietic system as target for carcinogenesis in wild- type mice, appear to be efficiently identified.      

Assessing HER2 amplification in breast cancer: findings from the Australian In Situ Hybridization Program

In August 2006, the Australian government approved subsidized trastuzumab therapy for human epidermal growth factor receptor 2 (HER2)-positive early breast cancer, and it was mandated that HER2 testing should be performed using in situ hybridization (ISH) rather than immunohistochemistry (IHC). Here we review results of the first regulated, nationwide program to provide HER2 ISH testing for all newly diagnosed breast cancer patients, with a particular emphasis on cases where IHC and ISH results were discordant. Data from all laboratories participating in the program were collated. Cases with an equivocal ISH test result [by chromogenic ISH (CISH) or silver ISH (SISH)] were tested centrally by fluorescence ISH. Most laboratories also performed HER2 IHC, and 200 cases with discordant IHC and ISH results were selected for further analysis in a central laboratory. A total of 26 laboratories were involved and 53,402 tests were reported. Over a 4-year period the HER2 positivity rate decreased for primary cancers from 23.8 to 14.6 %, but remained relatively constant for samples from metastases. Average ISH reporting times were <5 days for all yearly reporting periods. Test-repeat rates decreased for CISH (8.9-3.6 %) and SISH (13.7-8.4 %). Only 12 of 196 cases remained discordant after retesting in a central laboratory. These findings demonstrate the successful implementation of a regulated, national program that continues to collect data on HER2 status. The results also highlight the differences in IHC interpretation between local laboratories and a central, more experienced, laboratory. This model could be used to establish future biomarker-testing programs in other countries.     

基于八腔室玻片的染色体畸变初筛方法的建立与验证

目的：为简化传统体外染色体畸变试验工序并减少受试物用量,建立基于八腔室玻片的染色体畸变初筛方法。方法：首先利用环磷酰胺（CP）和丝裂霉素C（MMC）作为阳性模式药物,分别建立CP作用6 h（＋S_9）和MMC作用24 h（-S_9）的八腔室玻片的染色体畸变试验方法。再用于检测5个已知遗传毒性物质苯并芘[B（a）P]、2-氨基蒽（2-AA）、甲基磺酸甲酯（MMS）、乙基磺酸甲酯（EMS）、依托泊苷（Eto）和2个非遗传毒性药物氨苄青霉素钠（AS）和氯化钠（SC）在±S_9两个条件下的染色体畸变效应,验证该法的灵敏度和特异性。结果：CP和MMC分别在±S_9活化系统中染色体结构畸变率呈浓度依赖性增加,与溶剂对照组相比,差异均具有统计学意义（P〈0.05或P〈0.01）。在验证试验中,B（a）P和2-AA在＋S_9系统中呈染色体畸变阳性,与溶剂对照组相比,差异均具有统计学意义（P〈0.05或P〈0.01）;MMS、EMS和Eto的染色体畸变率呈浓度依赖性增加（±S_9结果均为阳性）,与溶剂对照组相比,差异均具有统计学意义（P〈0.05或P〈0.01）;而2个已知非遗传毒性药物（AS和SC）在±S_9系统中均呈阴性结果。结论：通过上述物质的验证表明,八腔室玻片染色体畸变试验是一种简单、快速、有效的遗传毒性初筛方法。     

The impact of toxicity on carcinogenicity studies: implications for risk assessment

This paper explores the inter-relationship between toxicity,genotoxicity, and carcinogenicity in laboratory rodents. Toour knowledge this is the first attempt to integrate these factorsand evaluate their implications for the process of risk assessment.The evaluation is based on information obtained from 2-yearlaboratory-animal studies involving 99 chemicals. The data suggestthat only seven of the 53 positive carcinogenicity studies exhibitedthe types of target organ toxicity that could have been thecause of all observed carcinogenic effects. Furthermore, noapparent difference in mutagenicity as measured by the AmesSalmonella assay was observed between ‘high dose only’carcinogens and the entire set of carcinogens. These findingssuggest that the number of chemical carcinogens that we canidentify solely through rodent studies as being potential tumorinducers through some indirect mechanism is small. Generallyspeaking, the identification of histopathological effects isnot sufficient in itself for justifying mechanistic assumptions,and supplemental biological information will be necessary toreach definitive conclusions.     

A medium-term rat liver bioassay for rapid in vivo detection of carcinogenic potential of chemicals

A reliable medium-term bioassay system for rapid detection of carcinogenic potential of chemicals in the human environment has been developed. The 8-week-protocol consists of 2 stages; male F344 rats are given a single intraperitoneal injection of diethylnitrosamine (200 mg/kg) for initiation of liver carcinogenesis, followed by a 6-week test chemical treatment starting 2 weeks thereafter. Test chemicals are usually given in the diet or the drinking water and in the 2nd week of test chemical treatment, all rats are subjected to two-thirds partial hepatectomy in order to induce regenerative cell replication. The end-point marker is the glutathione S-transferase placental form (GST-P)-positive hepatic focus, the numbers and sizes of which are analyzed using an image-analyzer and expressed as values per unit liver section (1 cm²). When the yield of GST-P-positive foci is significantly enhanced (P<0.05) over the control value, a chemical is judged to possess carcinogenic or promotion potential for the liver. Among 313 chemicals already tested in this system in our laboratory, 30/31 (97%) mutagenic hepatocarcinogens and 29/33 (88%) non-mutagenic hepatocarcinogens gave positive results. Ten out of 43 (23%) agents known to be carcinogenic in organs other than the liver were also positive. It is particularly important that only one of 48 non-carcinogens gave a very weak positive result, so that the system has a very low false-positivity rate. It is now well documented that the assay system is highly effective for detecting hepatocarcinogens, bridging the gap between traditional long-term carcinogenicity tests and short-term screening assays. At the Fourth International Conference on Harmonization, our medium-term liver bioassay based on an initiation and promotion protocol was recommended in the guidelines as an acceptable alternative to the long-term rodent carcinogenicity test. (Cancer Sci 2003; 94: 3–8)     

Genetic effects of benzene and radiation in ICR and X/Gf mice

X/Gf mice (a tumor-resistant strain) were compared with ICR mice (moderately tumor-sensitive) for their sensitivity to chromosomal damage caused by benzene, cyclophosphamide (CP), benzo(a)pyrene (BP) and radiation. There was no difference between strains in the level of micronucleus formation caused by BP, CP or radiation. Although X/Gf mice metabolized somewhat less of the dose of benzene per weight than ICR mice, and had somewhat higher levels of genetic damage, it is not known whether X/Gf mice would be measurably more resistant to benzene carcinogenicity. Short-term genotoxicity tests are used as indicators of initiation, therefore, equal sensitivity to a set of standard clastogens suggests that tumor resistance in X/Gf mice is a function of later stages of carcinogenesis.