Testicular DNA synthesis inhibition—an in vivo system for the detection of mutagenic and carcinogenic chemicals

By binding covalently to DNA chemical mutagens and carcinogens inhibit replication, which can be measured as a decrease in thymidine incorporation into DNA. This DNA synthesis inhibition (DSI) has been determined in testicular cells of mice for a large number of compounds and has been found to correlate very well with their known mutagenic and carcinogenic properties.

Not only could this test give a qualitative answer about potential carcinogenicity or mutagenicity, but, with regard to its in vivo characteristics, could furthermore give an indication of the carcinogenic and mutagenic potency and thus be of use in the risk evaluation of chemical substances; the relationship between the DNA synthesis inhibition and the underlying alkylation of guanine ‐0⁶ by different methylating agents is demonstrated.

As toxic effects decrease thymidine incorporation, too, means are discussed for distinguishing between “true”;, mutagenic, and “false”;, cytotoxic, DNA synthesis inhibition.

In conclusion, the implications of including the DSI‐test in a battery of mutagenicity tests are outlined.     

Experimental investigation of false positive errors in auditory species occurrence surveys

False positive errors are a significant component of many ecological data sets, which in combination with false negative errors, can lead to severe biases in conclusions about ecological systems. We present results of a field experiment where observers recorded observations for known combinations of electronically broadcast calling anurans under conditions mimicking field surveys to determine species occurrence. Our objectives were to characterize false positive error probabilities for auditory methods based on a large number of observers, to determine if targeted instruction could be used to reduce false positive error rates, and to establish useful predictors of among-observer and among-species differences in error rates. We recruited 31 observers, ranging in abilities from novice to expert, who recorded detections for 12 species during 180 calling trials (66,960 total observations). All observers made multiple false positive errors, and on average 8.1% of recorded detections in the experiment were false positive errors. Additional instruction had only minor effects on error rates. After instruction, false positive error probabilities decreased by 16% for treatment individuals compared to controls with broad confidence interval overlap of 0 (95% CI:--46 to 30%). This coincided with an increase in false negative errors due to the treatment (26%;--3 to 61%). Differences among observers in false positive and in false negative error rates were best predicted by scores from an online test and a self-assessment of observer ability completed prior to the field experiment. In contrast, years of experience conducting call surveys was a weak predictor of error rates. False positive errors were also more common for species that were played more frequently but were not related to the dominant spectral frequency of the call. Our results corroborate other work that demonstrates false positives are a significant component of species occurrence data collected by auditory methods. Instructing observers to only report detections they are completely certain are correct is not sufficient to eliminate errors. As a result, analytical methods that account for false positive errors will be needed, and independent testing of observer ability is a useful predictor for among-observer variation in observation error rates.     

生物样品体外致突变高通量检测方法研究进展

评述了几种常用的体外致突变检测方法及其用于生物样品检测的可行性,有些方法经改进可用于高通量检测生物样品体外致突变性.经典Ames实验受生物样品中组氨酸的影响,易产生假阳性结果,尽管经过修正可以排除组氨酸的干扰,但操作繁琐,不适合高通量检测.基于SOS反应的检测体系避开了组氨酸的影响,且简单易行,适合高通量检测:以β-半乳糖苷酶基因(lacZ)作为报告基因的检测体系灵敏度高,且经过离心洗涤或后培养的方式可降低样品颜色的影响;以绿色荧光蛋白(GFP)基因作为报告基因的检测体系避开了颜色的干扰,但这类方法灵敏度普遍不高,可以寻找信号更强的荧光蛋白以替代GFP;荧光素酶(lux)基因集lacZ和GFP的优点于一身,但检测时需要额外添加辅助因子,限制了其应用.也对单细胞凝胶电泳、tk基因突变实验、染色体损伤检测等方法进行了分析,有些适合生物样品高通量检测,但由于缺少国际通用的标准,很难推广使用.     

Ames测试不确定性分析

Ames测试具有实验周期短、简便的优点,是目前普遍采用的检测化合物致突变性的初筛方法,但Ames测试仍有一定的不确定性.本文主要依据本实验室的研究结果分析了Ames测试中测试菌株、统计回复子数目的时间及2倍判定标准给测试结果带来的不确定性,对影响Ames测试结论存在假阳性及假阴性的因素做了进一步分析与讨论.图6表1参19     

A Bayesian hierarchical approach to quantifying stakeholder attitudes toward conservation in the presence of reporting error

Stakeholder support is vital for achieving conservation success, yet there are few reliable mechanisms to monitor stakeholder attitudes toward conservation. Approaches used to assess attitudes rarely account for bias arising from reporting error, which can lead to falsely reporting a positive attitude toward conservation (false-positive error) or not reporting a positive attitude when the respondent has a positive attitude toward conservation (false-negative error). Borrowing from developments in applied conservation science, we used a Bayesian hierarchical model to quantify stakeholder attitudes as the probability of having a positive attitude toward wildlife notionally (or in abstract terms) and at localized scales while accounting for reporting error. We compared estimates from our model, Likert scores, and naïve estimates (i.e., proportion of respondents reporting a positive attitude in at least 1 question that was only susceptible to false-negative error) with true stakeholder attitudes through simulations. We then applied the model in a survey of tea estate staff on their attitudes toward Asian elephants (Elephas maximus) in the Kaziranga–Karbi Anglong landscape of northeast India. In simulations, Bayesian model estimates of stakeholder attitudes toward wildlife were less biased than naïve estimates or Likert scores. After accounting for reporting errors, we estimated the probability of having a positive attitude toward elephants notionally as 0.85 in the Kaziranga landscape, whereas the proportion of respondents who had positive attitudes toward elephants at a localized scale was 0.50. In comparison, without accounting for reporting errors, naïve estimates of proportions of respondents with positive attitudes toward elephants were 0.69 and 0.23 notionally and at local scales, respectively. False (positive and negative) reporting probabilities were consistently not 0 (0.22–0.68). Regular and reliable assessment of stakeholder attitudes–combined with inference on drivers of positive attitudes–can help assess the success of initiatives aimed at facilitating human behavioral change and inform conservation decision making.     

Mutagenicity assessment of textile dyes from Sanganer (Rajasthan)

Sanganer town, district Jaipur (Rajasthan, India) is famous worldwide for its hand block dyeing and textile printing industries. These industries use a variety of chemicals and dyes during processing and finishing of raw materials. Most of the textile dyes used by these industries have not been evaluated for their impact on health and the environment. The workers in these industries are exposed to such dyes with no control over the length and frequency of exposure. Further, untreated and sometimes even treated effluents from these industries are released into surface waters of Amani Shah drainage or through the drainage systems, seep into the ground water and adjoining water bodies. Since many textile dyes are known carcinogens and mutagens, a complete evaluation of the safety of these dyes in the human environment must include an evaluation of their genotoxicity or mutagenicity. A total of 12 textile dyes from Sanganer were tested for their mutagenicity, by Ames Salmonella reversion assay using strain TA 100 of Salmonella typhimurium. Only 1 dye, Red 12 B showed absence of mutagenic activity. The remaining 11 dyes were all positively mutagenic.     

Gentoxische Substanzen in Wässern

The contamination of waste waters with genotoxic compounds has raised concern about the toxicological impact on man and ecosystems. Some epidemiological studies showed a correlation between contamination of water with genotoxic compounds and incidence of cancer in man, fish, and mussels. This review of published literature summarizes results of the mutagenic potential of waste water from various industries. The Ames assay is frequently used as test system for mutagenicity of organic and inorganic compounds. Some investigations tried to identify the sources of mutagenic contamination. A variety of organic (e.g. nitroaromatic compounds, polycyclic aromatic hydrocarbons, azo-dyes, chlorinated hydroxyfuranes) and inorganic (e.g. heavy metals) mutagenes were identified but it was not possible to attribute the whole mutagenic potential to analytically identified compounds. Due to the lack of data it is presently not possible to perform a risk assessment for humans.     

Mutagenicity of diesel engine exhaust in the Ames / Salmonella assay using a direct exposure method

The aim of this study was to investigate the potential mutagenic activity of diesel engine exhaust in the Ames/Salmonella assay using a direct aerosol exposure system. So, TA 98 and TA 100 strains, with or without added S9 mix, were exposed to diesel emissions after varying degrees of filtration. Variants of these two strains, deficient in nitroreductase (TA 98NR and TA 100NR) or over-expressing O-Acetyl Transferase (YG 1024 and YG 1029), were also exposed to total (unfiltered) diesel exhaust to highlight the putative mutagenicity of any nitro-PAHs present in these emissions. Mutagenic activity of the diesel exhaust was demonstrated on Salmonella typhimurium, strains TA 100 and variants TA 100 NR and YG1029. The use of a particle filter did not modify the genotoxicity of the diesel emissions, indicating a major contribution of the gas phase to the mutagenicity of these diesel emissions. The prominent role of the particulate-associated nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) claimed by some authors working on diesel exhaust organic extracts was not confirmed by our results with native diesel exhaust exposure. Our results show that the gas phase is potentially more mutagenic than the particles alone.     

Assessment of mutagenic potency of source water treated by ozone and adsorption processes

This research utilized the Ames test to determine the mutagenicity of water treated by advanced processes, including ozonation and granular activated carbon (GAC). Raw water samples for this research included those obtained from the Pan Hsin waterworks as well as samples containing humic acids. Treated samples were collected from the pilot‐scale advanced treatment plant. The Ames test was used to measure the mutagenicity of the water after each treatment process. For the Pan Hsin raw water samples treated with ozone or GAC, it was indicated that, regardless of whether samples were preozonated or not, they all showed a mutagenic potency less than 2 once the S9 enzyme was added. This level of mutagenicity is insignificant. The prepared humic acid samples, on the other hand, demonstrated a significant reduction in mutagenicity after the pre‐ozonation process, indicating that preozonation can lower the degree of mutagenicity. Furthermore, the mutagenicity of the prepared humic acid samples gradually decreased after the advanced treatment process. However, when chlorine was added later to these samples, the mutagenicity increased again. This research shows that the use of O3/GAC processes to treat water can successfully lower mutagenicity, indicating a great potential for applications in the treatment of drinking water.     

彗星试验检测区域水源有机污染致突变量(Study on Mutagenic Threshold of Organic Pollutants in Typical Water Area by Comet Assay)

采用彗星试验检测了区域水源有机污染对人血淋巴细胞的致突变性,探讨了水源风险的评价方法.结果表明:Cr(VI)对人外周血淋巴细胞有强的致突变作用,并具有明显的剂量-效应关系;Cr(VI)剂量的对数与人外周血淋巴细胞DNA损伤的程度呈线性相关.可以用Cr(VI)作为阳性参照物量化表征水中有机污染混合物对人血淋巴细胞的致突变性.现行《地表水环境质量标准》、《污水综合排放标准》中Cr(VI)的限值可以用于评价水体中有机污染对人体健康的风险.     

Judicious Use of Multiple Hypothesis Tests

Abstract:  When analyzing a table of statistical results, one must first decide whether adjustment of significance levels is appropriate. If the main goal is hypothesis generation or initial screening for potential conservation problems, then it may be appropriate to use the standard comparisonwise significance level to avoid Type II errors (not detecting real differences or trends). If the main goal is rigorous testing of a hypothesis, however, then an adjustment for multiple tests is needed. To control the familywise Type I error rate (the probability of rejecting at least one true null hypothesis), sequential modifications of the standard Bonferroni method, such as Holm's method, will provide more statistical power than the standard Bonferroni method. Additional power may be achieved through procedures that control the false discovery rate (FDR) (the expected proportion of false positives among tests found to be significant). Holm's sequential Bonferroni method and two FDR-controlling procedures were applied to the results of multiple-regression analyses of the relationship between habitat variables and the abundance of 25 species of forest birds in Japan, and the FDR-controlling procedures provided considerably greater statistical power.     

Generalized site occupancy models allowing for false positive and false negative errors

Site occupancy models have been developed that allow for imperfect species detection or "false negative" observations. Such models have become widely adopted in surveys of many taxa. The most fundamental assumption underlying these models is that "false positive" errors are not possible. That is, one cannot detect a species where it does not occur. However, such errors are possible in many sampling situations for a number of reasons, and even low false positive error rates can induce extreme bias in estimates of site occupancy when they are not accounted for. In this paper, we develop a model for site occupancy that allows for both false negative and false positive error rates. This model can be represented as a two-component finite mixture model and can be easily fitted using freely available software. We provide an analysis of avian survey data using the proposed model and present results of a brief simulation study evaluating the performance of the maximum-likelihood estimator and the naive estimator in the presence of false positive errors.     

Confronting Uncertainty and Missing Values in Environmental Value Transfer as Applied to Species Conservation

Abstract: The nonuse (or passive) value of nature is important but time‐consuming and costly to quantify with direct surveys. In the absence of estimates of these values, there will likely be less investment in conservation actions that generate substantial nonuse benefits, such as conservation of native species. To help overcome decisions about the allocation of conservation dollars that reflect the lack of estimates of nonuse values, these values can be estimated indirectly by environmental value transfer (EVT). EVT uses existing data or information from a study site such that the estimated monetary value of an environmental good is transferred to another location or policy site. A major challenge in the use of EVT is the uncertainty about the sign and size of the error (i.e., the percentage by which transferred value exceeds the actual value) that results from transferring direct estimates of nonuse values from a study to a policy site, the site where the value is transferred. An EVT is most useful if the decision‐making framework does not require highly accurate information and when the conservation decision is constrained by time and financial resources. To account for uncertainty in the decision‐making process, a decision heuristic that guides the decision process and illustrates the possible decision branches, can be followed. To account for the uncertainty associated with the transfer of values from one site to another, we developed a risk and simulation approach that uses Monte Carlo simulations to evaluate the net benefits of conservation investments and takes into account different possible distributions of transfer error. This method does not reduce transfer error, but it provides a way to account for the effect of transfer error in conservation decision making. Our risk and simulation approach and decision‐based framework on when to use EVT offer better‐informed decision making in conservation.     

Determining Decision Thresholds and Evaluating Indicators when Conservation Status is Measured as a Continuum

Categorization of the status of populations, species, and ecosystems underpins most conservation activities. Status is often based on how a system's current indicator value (e.g., change in abundance) relates to some threshold of conservation concern. Receiver operating characteristic (ROC) curves can be used to quantify the statistical reliability of indicators of conservation status and evaluate trade‐offs between correct (true positive) and incorrect (false positive) classifications across a range of decision thresholds. However, ROC curves assume a discrete, binary relationship between an indicator and the conservation status it is meant to track, which is a simplification of the more realistic continuum of conservation status, and may limit the applicability of ROC curves in conservation science. We describe a modified ROC curve that treats conservation status as a continuum rather than a discrete state. We explored the influence of this continuum and typical sources of variation in abundance that can lead to classification errors (i.e., random variation and measurement error) on the true and false positive rates corresponding to varying decision thresholds and the reliability of change in abundance as an indicator of conservation status, respectively. We applied our modified ROC approach to an indicator of endangerment in Pacific salmon (Oncorhynchus nerka) (i.e., percent decline in geometric mean abundance) and an indicator of marine ecosystem structure and function (i.e., detritivore biomass). Failure to treat conservation status as a continuum when choosing thresholds for indicators resulted in the misidentification of trade‐offs between true and false positive rates and the overestimation of an indicator's reliability. We argue for treating conservation status as a continuum when ROC curves are used to evaluate decision thresholds in indicators for the assessment of conservation status. Determinación de Umbrales de Decisiones y Evaluación delos Indicadores cuando se Mide el Estado de de Conservación como un Continuo     

致突变的致癌剂和非致癌剂在引发DNA股间交联上的显著差别

借碱洗脱法证明：具有致突变能力的致癌剂1，2－二溴乙烷和肼在经过代谢活化后，均剂理相关地引起L1210细胞中DNA的股间交联。相反，动物致癌试验表现阴性的突变剂溴乙烷，以及通过各种致癌动物试验均显示阴性的传统常用致突变剂羟胺，在代谢活化以后的同样条件下均不能引起L1210细胞中DNA的股间交联。这证明了双区理论的致癌观点，即致癌剂必须是双官能烷化剂，其同时引起互补碱对的交联将引起癌变。相反，股内单一碱基的变异则可能引起致突变作用。并用APCI／SIM（大气压化学电离／选择离子质谱）证明：1，2－二溴乙烷与DNA碱基对的模型反应，与致突实验一致主要引起G－C对的交联和发生G－C→A－T突变。     

Synthesis of 2,5‐diaryl‐2,4‐pentadienenitriles and their spectral,fluorescence and mutagenic properties

The condensation of substituted cinnamaldehydes and arylacetonitriles using sodium ethoxide in ethanol at room temperature afforded 2,5‐diaryl‐2,4‐pentadienenitriles in good yield. The structure of the reaction products was established on the basis of their infrared, nuclear magnetic resonance and elemental analysis data. Two representative compounds were studied for their mutagenic activity. One of them showed a weak mutagenicity while the second showed a high mutagenic activity in TA97a Salmonella strain. Both were negative in TA100. Some of these pentadienenitriles showed fluorescence in solutions.     

A unified approach for including non-extractable residues (NER) of chemicals and pesticides in the assessment of persistence

All chemicals form non-extractable residues (NER) to various extents in environmental media like soil, sediment, plants and animals. NER can be quantified in environmental fate studies using isotope-labeled (such as ¹⁴C or ¹³C) tracer compounds. Previous NER definitions have led to a mismatch of legislation and state of knowledge in research: the residues are assumed to be either irreversibly bound degradation products or at least parts of these residues can be released. In the latter assumption, soils and sediments are a long-term source of slowly released residues. We here present a conceptual experimental and modeling approach to characterize non-extractable residues and provide guidance how they should be considered in the persistence assessment of chemicals and pesticides. Three types of NER can be experimentally discriminated: sequestered and entrapped residues (type I), containing either the parent substance or xenobiotic transformation products or both and having the potential to be released, which has indeed been observed. Type II NER are residues that are covalently bound to organic matter in soils or sediments or to biological tissue in organisms and that are considered being strongly bound with very low remobilization rates like that of humic matter degradation rates. Type III NER comprises biogenic NER (bioNER) after degradation of the xenobiotic chemical and anabolic formation of natural biomolecules like amino acids and phospholipids, and other biomass compounds. We developed the microbial turnover to biomass (MTB) model to predict the formation of bioNER based on the structural properties of chemicals. Further, we proposed an extraction sequence to obtain a matrix containing only NER. Finally, we summarized experimental methods to distinguish the three NER types. Type I NER and type II NER should be considered as potentially remobilizable residues in persistence assessment but the probability of type II release is much lower than that of type I NER, i.e., type II NER in soil are “operationally spoken” irreversibly bound and can be released only in minute amounts and at very slow rates, if at all. The potential of remobilization can be evaluated by chemical, physical and biological methods. BioNER are of no environmental concern and, therefore, can be assessed as such in persistence assessment. The general concept presented is to consider the total amount of NER minus potential bioNER as the amount of xenoNER, type I?+?II. If a clear differentiation of type I and type II is possible, for the calculation of half-life type I NER are considered as not degraded parent substance or transformation product(s). On the contrary, type II NER may generally be considered as (at least temporarily) removed. Providing proof for type II NER is the most critical issue in NER assessment and requires additional research. If no characterization and additional information on NER are available, it is recommended to assess the total amount as potentially remobilizable. We propose our unified approach of NER characterization and evaluation to be implemented into the persistence and environmental hazard assessment strategies for REACH chemicals and biocides, human and veterinary pharmaceuticals, and pesticides, irrespective of the different regulatory frameworks.     

广西速生桉施肥对林区内水体富营养物质的影响

施肥是种植速生桉(Eucalyptus)主要的营林技术措施之一。广西近几年速生桉发展面积较大,施肥量逐年增加,由于肥料通过其气态、淋洗和径流等途径流失到水体,增加了水体中富营养物质的含量。为了防止或减缓速生桉施肥加快对水体富营养化的目的,探索其有效的解决方法,课题组进行了速生桉的施肥数量、肥料颗粒形态、肥料养分含量、肥料养分配比等因子的田间试验,研究不同的施肥处理对林区内水体富营养物质含量的影响。试验结果表明:肥料养分含量越高、施肥量越大、颗粒化程度越低的处理区域水体总氮、总磷和总钾含量越高;桉树林区内水体的总氮、总磷和总钾含量中各有约40%、70%和50%来自桉树施肥,不合理的施肥可能会导致林区内水库或河流等水体富营养化;桉树施肥技术中施肥量对水体营养物质影响最大,其次分别为肥料养分和肥料颗粒形态。建议在一定范围内,速生桉施肥可提高肥料总养分含量、适当减少其施肥量,可防止或减缓桉树林区内水体富营养化。     

复方阿苯达唑/伊维菌素及其单药的体内外致突变性研究

农业生产上常用的阿苯达唑和伊维菌素均有一定的致突变性。为了解伊维菌素和阿苯达唑按1:24形成固定剂量复方的致突变性,以及复方和单药致突变性的差异,采用Ames试验、小鼠骨髓微核试验和小鼠精子畸变试验进行复方阿苯达唑/伊维菌素及其单药的致突变性研究。结果表明,复方阿苯达唑/伊维菌素能提高小鼠骨髓嗜多染红细胞的微核率和精子畸形率,但在单复方的比较中发现,单药的微核率和精子畸形率均高于复方,表明复方毒性有所降低。同时,无论是复方还是单药均不能提高组氨酸缺陷型沙门氏菌的突变率。以上表明,虽然复方阿苯达唑/伊维菌素致突变毒性低于单药,但仍然是一种能引起小鼠骨髓微核率和精子畸形率升高的致突变阳性药物,因此需警惕该药物的食品残留问题及对生态环境的危害。     

‘A powerful alternative to Williams’ test with application to toxicological dose-response relationships of normally distributed data

The comparison of increasing doses of a treatment to a negative control is frequently part of toxicological studies. For normally distributed data Williams (1971, 1972) introduced a maximum likelihood test under total order restriction. But until now there seems to have been no solution for the arbitrary unbalanced case. According to the idea proposed by Robertson et al. (1988) we will apply in this article the basic concept of Williams on the class of multiple contrast tests for the general unbalanced parametric set-up. Simulation results for size and power and two examples for estimating the minimal toxic dose (MTD) are given.