Use of a high-throughput umu-microplate test system for rapid detection of genotoxicity produced by mutagenic carcinogens and airborne particulate matter

In the present study, we developed a rapid umu-microplate test system that uses the nitroreductase- and O-acetyltransferase-overproducing Salmonella typhimurium strain NM3009 and the O-acetyltransferase-overproducing S. typhimurium strain NM2009 to detect genotoxic activity in small volume samples. The assay was used to test the genotoxicity of several standard mutagens and environmental samples. Exponentially growing cultures of NM3009, NM2009, and the parental strain TA1535/pSK1002 were incubated in 96-well microplates with test chemicals both in the presence and in the absence of rat liver S9. The relative beta-galactosidase activities were then determined colorimetrically using either chlorophenol red-beta-D-galactopyranoside (CPRG) or O-nitrophenyl-beta-D-galactopyranoside (ONPG) as a measure of umuC gene induction activity. The sensitivities of NM3009 without S9 mix and NM2009 with S9 mix to nitroarenes and aromatic amines were up to 24- to 75-fold higher than those of the parent strain. Induction of umuC gene expression was detected more readily with CPRG than ONPG. The umu-microplate assay also detected genotoxicity in organic extracts of particulate matter from air samples collected in Osaka City, Japan. The pattern of the responses suggested that the genotoxic activity of the particulate extract was due primarily to nitrated polycyclic aromatic hydrocarbons. Our results indicate that the umu-microplate assay may be a useful way of carrying out rapid screens for genotoxicity in small-volume environmental samples.     

Effect of chemical composition on the induction of DNA damage by urban airborne particulate matter

Airborne particulate matter (PM) contains a large number of genotoxic substances capable of endangering human health. In the present study, we have investigated the ability of chemically characterized water-soluble and organic-soluble fractions of two particle sizes (PM2.5 and PM10) from different regions of Mexico City to induce DNA damage in a human lung epithelial cell line. We also evaluated associations between the physicochemical parameters of the PM and its genotoxicity. The airborne particulate samples were collected from four regions of the city; a HiVol air sampler was used to collect PM10 on glass fiber filters and a tapered element oscillating system coupled to an automatic cartridge collection unit was used to collect PM2.5 on teflon filters. PM mass was determined by gravimetric analysis of the filters. Filters containing PM2.5 and one section of each PM10 filter were agitated either with deionized water to extract water-soluble compound, or with dichloromethane to prepare organic-soluble compounds. The chemical composition of the extracts was determined by ion and gas chromatography and atomic adsorption spectroscopy. A549 human type II alveolar epithelial cells were exposed to different concentrations of the PM2.5 and PM10 extracts, and alkaline single cell gel electrophoresis or the Comet assay was performed to measure DNA damage and repair. These analyses indicated that soluble transition metals and the organic-soluble PM fractions are crucial factors in the DNA damage induced by PM. PM composition was more important than PM mass for producing genotoxicity. The results of this study showed that the constituents of the water-soluble PM extract are more likely to induce DNA damage than the organic compounds.     

空气细颗粒物暴露水平与健康成人大脑灰质体积和认知功能的关联研究

目的:应用磁共振成像(MRI)技术探究空气细颗粒物PM2.5暴露水平对健康成年人大脑灰质体积(GMV)和认知功能的影响。方法:选取487例健康成人,采集一般人口学信息及详细居住信息,并根据居住信息获取各受试近6个月和8岁～15岁期间的PM2.5暴露水平,对所有研究对象完成高分辨率结构MRI扫描,使用中文版精神分裂症认知功能成套测验-共识版(MCCB)进行认知测查,使用相关和回归分析探索空气PM2.5暴露水平与GMV和认知功能的关联。结果:近6个月PM2.5暴露水平与大脑右侧额下回三角部(brodmann分区[BA] 45)和右侧颞中回(BA 22)GMV存在负性关联(FWE校正,P<0.05,团块>10体素);与MCCB认知评估的信息处理速度-连线测试(r=-0.17,P<0.01)和推理和问题解决-迷宫测验评分(r=-0.22,P<0.01)负相关。8～15岁时的累积空气PM2.5暴露量与左侧梭状回(BA 18)和右侧舌回(BA 17)GMV负相关(FWE校正,P<0.05,团块>10体素),且与信息处理速度-连线测试、推理和问题解决-迷宫测验评分...     

Profiling the pattern of human TRB/IGH-CDR3 repertoire in liver transplantation patients via high-throughput sequencing analysis

Immune processes in liver transplantation remain poorly understood. Acute allograft rejection in liver transplantation is a kind of T cell–mediated inflammatory disease accompanied by inflammatory cell infiltration. However, the effect of acute allograft rejection on the immunological characteristics of TCRs in peripheral blood mononuclear cell is unknown. In this study, we characterized the pattern of the human T cell receptor beta chain (TRB) and immunoglobulin heavy chain (IGH) complementarity-determining region 3 (CDR3) repertoires via high-throughput sequencing in 11 acute allograft rejection (AG) cases, 23 patients with stable allograft liver function (ST) who had liver transplantation performed and 20 healthy controls (HC). The diversity of TRB-CDR3 was significantly reduced in the AG group compared with the ST group and healthy controls (HC). The CDR3 and N-addition length distribution were not significantly different between the AG and ST groups. However, N-addition length distribution was significantly changed compared to HC. It seemed that AG used more short N-additions and healthy people used more long N-additions in TRB-CDR3 repertoire. Our findings suggested that the TRB-CDR3 region of AG had distinctive V gene use compared with that of HC. The characteristics of ST seemed to be in between those of AG and HC although the difference is not significant. Cluster analysis showed that the TRB repertoire could not effectively distinguish AG from ST. This research might give to a better understanding of the immune process of liver transplantation.     

结节病肉芽肿构成细胞内空气细颗粒物的观察

目的探讨空气细颗粒物（PM2．5）与结节病之间的关系。方法收集下列病例的石蜡包埋组织标本：结节病49例,因肺外疾病死亡的成人尸检肺标本10例,胎儿尸检肺标本10例。标本行（1）HE染色,观察肉芽肿及尘细胞;（2）Warthin—Starry（W—S）银染色,显示细胞内的PM2．5;（3）免疫组化染色标记肉芽肿和尘细胞;（4）制作超薄切片,在透射电镜下观察证实细胞内的PM2．5。结果经W—S染色,在结节病肉芽肿细胞内观察到明显的PM2．5,并在电镜观察中得到进一步的证实,肉芽肿细胞和尘细胞CD68抗体标记阳性。结论结节病肉芽肿细胞内有PM2．5,为结节病空气颗粒物相关学说提供了形态学证据,提示结节病可能与PM2．5有关。     

新一代高通量测序技术在遗传相关性智力障碍诊断中的应用

智力低下的病因复杂且大多尚未明了,大部分智力低下属遗传性,具有高度特异性.根据遗传特点将智力低下原因分为:染色体数目和结构异常、代谢缺陷、单基因缺陷、非综合征智力低下.因此快速有效地去诊断智力障碍是遗传诊断学的一大挑战.传统的诊断方式如:Southern印迹、连锁分析、染色体核型分析技术、PCR、微阵列-比较基因技术均因各自的缺陷不能在临床上得到有效推广.1987年,美国ABI公司推出第一台自动化测序仪-ABi370的问世实现了基因诊断史上质的飞跃.但由于一代测序通量低、成本高,终将面临淘汰.在此基础上,以高通量、低成本为特点的二代测序全面发展,与传统技术相比二代测序适用于大规模测序,适用于病因不明的突变基因检测,对遗传性智力障碍可能致病基因进行并行测序,向着快速有效地诊断智力障碍迈进一大步.     

Differential mRNA expression profiling of oral squamous cell carcinoma by high-throughput RNA sequencing

Differentially expressed genes are thought to regulate the development and progression of oral squamous cell carcinomas (OSCC). The purpose of this study was to screen differentially expressed mRNAs in OSCC and matched paraneoplastic normal tissues, and to explore the intrinsic mechanism of OSCC development and progression. We obtained the differentially expressed mRNA expression profiles in 10 pairs of fresh-frozen OSCC tissue specimens and matched paraneoplastic normal tissue specimens by high-throughput RNA sequencing. By using Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, the functional significance of the differentially expressed genes were analyzed. We identified 1,120 significantly up-regulated mRNAs and 178 significantly down-regulated mRNAs in OSCC, compared to normal tissue. The differentially expressed mRNAs were involved in 20 biological processes and 68 signal pathways. Compared to adjacent normal tissue, the expression of MAGEA11 was up-regulated; TCHH was down-regulated. These findings were verified by real-time PCR. These differentially expressed mRNAs may function as oncogenes or tumor suppressors in the development and progression of OSCC. This study provides novel insights into OSCC. However, further work is needed to determine if these differentially expressed mRNAs have potential roles as diagnostic biomarkers and candidate therapeutic targets for OSCC.     

High-level ambient particulate matter before influenza attack with increased incidence of Aspergillus antigenemia in Southern Taiwan, 2016

We found significant correlation between the incidence of severe influenza and Aspergillus antigenemia among medical intensive care unit patients for 7-month observation (coefficient γ = 0.976, p < 0.001). High-level ambient pollution was noticed for 2 months before the epidemic, highlighting that influenza patients might coinfect with aspergillosis in the community.     

Unexpected inorganic elements in oral lesions: Results of X-ray energy spectroscopy (XES) on particulate matter in paraffin sections

X-ray energy spectroscopy (XES) of all particulate matter found by light microscopy in histological sections of 222 oral lesions revealed a much wider range of elements than expected from dental amalgam. Subsequent XES of a variety of endodontic materials, impression materials and toothpastes indicated possible sources for virtually all elements identified in the histopathological lesions. The range of materials identified is tabulated; histopathological lesions and probable source substances are correlated; light microscopic pointers to the elemental nature of particulate matter in sections are indicated—titanium and bismuth have characteristic appearances; possible implications of local implantation and large scale ingestion of such substances are discussed.     

Genotoxicity and physicochemical characteristics of traffic-related ambient particulate matter

Exposure to ambient particulate matter (PM) has been linked to several adverse health effects. Since vehicular traffic is a PM source of growing importance, we sampled total suspended particulate (TSP), PM(10), and PM(2.5) at six urban locations with pronounced differences in traffic intensity. The mutagenicity, DNA-adduct formation, and induction of oxidative DNA damage by the samples were studied as genotoxicological parameters, in relation to polycyclic aromatic hydrocarbon (PAH) levels, elemental composition, and radical-generating capacity (RGC) as chemical characteristics. We found pronounced differences in the genotoxicity and chemical characteristics of PM from the various locations, although we could not establish a correlation between traffic intensity and any of these characteristics for any of the PM size fractions. Therefore, the differences between locations may be due to local sources of PM, other than traffic. The concentration of total (carcinogenic) PAHs correlated positively with RGC, direct and S9-mediated mutagenicity, as well as the induction of DNA adducts and oxidative DNA damage. The interaction between total PAHs and transition metals correlated positively with DNA-adduct formation, particularly from the PM(2.5) fraction. RGC was not associated with one specific PM size fraction, but mutagenicity and DNA reactivity after metabolic activation were relatively high in PM(10) and PM(2.5), when compared with TSP. We conclude that the toxicological characteristics of urban PM samples show pronounced differences, even when PM concentrations at the sample sites are comparable. This implies that emission reduction strategies that take chemical and toxicological characteristics of PM into account may be useful for reducing the health risks associated with PM exposure.     

Mutagenicity of airborne particles from a nonindustrial town in Italy

The mutagenic activity of airborne particulate matter collected in Pisa, a small nonindustrial town located in Italy, has been monitored over 1 year using the Ames Salmonella Test. Airborne particulate was collected on fibreglass filters using a Hi-Vol sampler and extracted by sonication and Soxhlet acetone extraction in sequence. TA 98 and TA 100 salmonella strains gave positive results with the great majority of samples. The mutagenicity trend fits with a harmonic regression with a peak during December/January and inversely correlates with the temperature. No correlations were observed with other meteorological conditions such as wind, cloud, rainfall, atmospheric pressure, and humidity. The ratio between mutagenicity/microgram of particulate matter with S9 and that without S9 remains more or less constant regardless of seasonal fluctuations, suggesting that during cold months quantitative increases of mutagens onto particulate matter have probably occurred. The comparison of air mutagenicity in different sites suggests that motor vehicle exhaust fumes are the major source of air pollution. Finally, because of high-traffic volume, air mutagenicity at street level is comparable to that observed in several metropolitan areas all over the world.     

高通量测序技术分析肺结核患者PBMC基因表达谱差异

目的:应用Illumina高通量测序技术研究肺结核患者和健康人群PBMC基因表达谱差异,以寻找与肺结核发病相关的基因.方法:分离肺结核患者和健康人群PBMC,提取总RNA,并制备cDNA文库,通过接头序列固定在Flow cell上进行桥式PCR扩增,再用Illumina测序仪进行深度测序,结合生物信息方法分析两类人群基因表达谱的差异情况.结果:测序产生的原始数据经过滤、比对、注释后得到肺结核样本36 390类标签12 270个基因,正常对照样本35 009类标签12 244个基因.按照FDR≤0.001,l log2 Ratiol≥1筛选标准,筛选出3 097个差异基因,其中上调基因1 601个,下调基因1 469个.增大筛选标准至4倍,并对表达量进行控制,筛选出33个差异极显著基因,其中上调基因16个,下调基因17个.结论:差异基因大部分位于细胞内,起着连接功能(蛋白质连接),具有酶催化活性,在代谢中起着重要作用,并主要位于MAPK信号通路和趋化因子信号通路中.这些差异基因可以为今后肺结核领域筛选诊断标识和药物作用靶点做参考.     

Ultrafine but not fine particulate matter causes airway inflammation and allergic airway sensitization to co-administered antigen in mice

BACKGROUND: Airborne particulate matter (PM) is an important factor associated with the enhanced prevalence of respiratory allergy. The PM adjuvant activity on allergic sensitization is a possible mechanism of action involved, and the induction of airway inflammation is suggested to be of importance in PM-induced adjuvant activity. OBJECTIVE: Because differently sized PM have different toxic potentials, we studied the role of particle size in the induction of airway inflammation and allergic sensitization. This was done using fine (0.250 and 0.260 micro m) and ultrafine (0.029 and 0.014 micro m) titanium dioxide (TiO(2)) and carbon black particles (CBP) with known differences in airway toxicity. METHODS: Mice were intranasally exposed to ovalbumin (OVA) alone or in combination with one of the different particles. The induction of airway inflammation and the immune adjuvant activity were studied in the lungs and lung-draining peribronchial lymph nodes (PBLN) at day 8. OVA-specific antibodies were measured at day 21, and the development of allergic airway inflammation was studied after OVA challenges (day 28). RESULTS: When administered at the same total particle mass (200 micro g), exposure to ultrafine TiO(2) and CBP-induced airway inflammation, and had immune adjuvant activity. The latter was shown by increasing both the PBLN cell numbers and the production of OVA-specific T-helper type 2 (Th2) cytokines (IL-4, IL-5, IL-10 and IL-13). Whereas OVA-specific IgE and IgG1 levels in serum were only increased in animals exposed to the ultrafine TiO(2), allergic airway inflammation could be detected in both ultrafine TiO(2)-and CBP-treated groups after challenges with OVA. CONCLUSION: Our data show that only the ultrafine particles, with a small diameter and a large total surface area/mass, cause airway inflammation and have immune adjuvant activity in the current model supporting the hypothesis that particle toxicity is site-dependent and related to adjuvant activity.     

Contaminating viral sequences in high-throughput sequencing viromics: a linkage study of 700 sequencing libraries

ObjectivesSample preparation for high-throughput sequencing (HTS) includes treatment with various laboratory components, potentially carrying viral nucleic acids, the extent of which has not been thoroughly investigated. Our aim was to systematically examine a diverse repertoire of laboratory components used to prepare samples for HTS in order to identify contaminating viral sequences.MethodsA total of 322 samples of mainly human origin were analysed using eight protocols, applying a wide variety of laboratory components. Several samples (60% of human specimens) were processed using different protocols. In total, 712 sequencing libraries were investigated for viral sequence contamination.ResultsAmong sequences showing similarity to viruses, 493 were significantly associated with the use of laboratory components. Each of these viral sequences had sporadic appearance, only being identified in a subset of the samples treated with the linked laboratory component, and some were not identified in the non-template control samples. Remarkably, more than 65% of all viral sequences identified were within viral clusters linked to the use of laboratory components.ConclusionsWe show that high prevalence of contaminating viral sequences can be expected in HTS-based virome data and provide an extensive list of novel contaminating viral sequences that can be used for evaluation of viral findings in future virome and metagenome studies. Moreover, we show that detection can be problematic due to stochastic appearance and limited non-template controls. Although the exact origin of these viral sequences requires further research, our results support laboratory-component-linked viral sequence contamination of both biological and synthetic origin.     

Urban particulate matter stimulation of human dendritic cells enhances priming of naive CD8 T lymphocytes

Epidemiological studies have consistently shown associations between elevated concentrations of urban particulate matter (UPM) air pollution and exacerbations of asthma and chronic obstructive pulmonary disease, which are both associated with viral respiratory infections. The effects of UPM on dendritic cell (DC) ‐stimulated CD4 T lymphocytes have been investigated previously, but little work has focused on CD8 T‐lymphocyte responses despite their importance in anti‐viral immunity. To address this, we examined the effects of UPM on DC‐stimulated naive CD8 T‐cell responses. Expression of the maturation/activation markers CD83, CCR7, CD40 and MHC class I on human myeloid DCs (mDCs) was characterized by flow cytometry after stimulation with UPMin vitro in the presence/absence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF). The capacity of these mDCs to stimulate naive CD8 T‐lymphocyte responses in allogeneic co‐culture was then assessed by measuring T‐cell cytokine secretion using cytometric bead array, and proliferation and frequency of interferon‐γ (IFN‐γ)‐producing T lymphocytes by flow cytometry. Treatment of mDCs with UPM increased expression of CD83 and CCR7, but not MHC class I. In allogeneic co‐cultures, UPM treatment of mDCs enhanced CD8 T‐cell proliferation and the frequency of IFN‐γ⁺ cells. The secretion of tumour necrosis factor‐α, interleukin‐13, Granzyme A and Granzyme B were also increased. GM‐CSF alone, and in concert with UPM, enhanced many of these T‐cell functions. The PM‐induced increase in Granzyme A was confirmed in a human experimental diesel exposure study. These data demonstrate that UPM treatment of mDCs enhances priming of naive CD8 T lymphocytes and increases production of pro‐inflammatory cytokines. Such UPM‐induced stimulation of CD8 cells may potentiate T‐lymphocyte cytotoxic responses upon concurrent airway infection, increasing bystander damage to the airways.     

Prenatal exposure to particulate matter and ozone: Bulky DNA adducts,plasma isoprostanes,allele risk variants,and neonate susceptibility in the Mexico City Metropolitan Area

Mexico City's Metropolitan Area (MCMA) includes Mexico City and 60 municipalities of the neighbor states. Inhabitants are exposed to emissions from over five million vehicles and stationary sources of air pollutants such as particulate matter (PM) and ozone. MCMA PM contains elemental carbon and organic carbon (OC). OCs include polycyclic aromatic hydrocarbons (PAHs), many of which induce mutagenic and carcinogenic DNA adducts. Gestational exposure to air pollution has been associated with increased risk of intrauterine growth restriction, preterm birth or low birth weight risk, and PAH-DNA adducts. These effects also depend on the presence of risk alleles. We investigated the presence of bulky PAH-DNA adducts, plasma 8-iso-PGF_2α (8-iso-prostaglandin F_2α) and risk allele variants in neonates cord blood and their non-smoking mothers' leucocytes from families that were living in a highly polluted area during 2014–2015. The presence of adducts was significantly associated with both PM_2.5 and PM₁₀ levels, mainly during the last trimester of gestation in both neonates and mothers, while the last month of pregnancy was significant for the association between ozone levels and maternal plasma 8-iso-PGF_2α. Fetal CYP1B1*3 risk allele was associated with increased adduct levels in neonates while the presence of the maternal allele significantly reduced the levels of fetal adducts. Maternal NQO1*2 was associated with lower maternal levels of adducts. Our findings suggest the need to reduce actual PM limits in MCMA. We did not observe a clear association between PM and/or adduct levels and neonate weight, length, body mass index, Apgar or Capurro score. Environ. Mol. Mutagen. 60:428–442, 2019. © 2019 Wiley Periodicals, Inc.     

结节病肉芽肿细胞内空气细颗粒物元素组成及来源分析

目的 分析结节病肉芽肿细胞(吞噬细胞、类上皮细胞、多核巨细胞)内空气细颗粒物(PM2.5)的元素组成及来源,探讨结节病与PM2.5的相关性.方法 收集50例结节病病变组织、10例非结节病成人尸检肺组织、18例PM2.5支气管灌注染毒大鼠肺组织(有肉芽肿病灶)石蜡包埋标本,采集大气中PM2.5,分别行HE染色、沃森-斯塔理(Warthin-Starry,WS)银染色,应用共聚焦显微镜拉曼光谱和透射电镜-能谱对上述部分病例标本肉芽肿细胞和尘细胞内的PM2.5及大气PM2.5的成分进行分析,用X线荧光分析技术对所采集大气PM2.5的主要元素成分进行分析.结果 结节病病变组织、非结节病成人尸检肺组织、PM2.5支气管灌注染毒大鼠肺组织及大气中PM2.5共4组标本中PM2.5的拉曼光谱和元素组成非常相似,均以碳质成分为主要特征.结论 为结节病肉芽肿细胞中的PM2.5来源于大气PM2.5提供了进一步的依据,从而不能完全除外结节病患者可能是对PM2.5易感的个体的可能性.

Abstract：

Objective To explore the source of the fine particulate matter (PM2.5) in the sarcoidosis granulomatous cell and the relationship between the sarcoidosis and the PM2.5 in the atmosphere.Methods Paraffin-embedded tissues of 50 cases of human sarcoidosis biopsy samples, 10 cases of nonsarcoidosis autopsy lung samples, 18 cases of lung tissues (with granulomatous lesions) of rats exposed to PM2.5 by bronchial infusion, and the free PM2.5 sample in the atmosphere were collected. The characteristics of tissues above mentioned were observed under the light microscopy, which stained by HE staining and Warthin-Starry silver staining. The characteristics of the PM2.5 in the four groups were analyzed using confocal Raman microscopy. The component of the PM2.5 in the sarcoidosis granuloma was analyzed using transmission electron microscope-energy dispersive X-ray detector (TEM-EDX), and the component of the PM2.5 in the atmosphere was analyzed with X-ray fluorescence separately. Results The PM2.5 in the four groups have the similar Raman spectrum, they share the feature of carbonaceous composition, the element component of PM2.5 in the human sarcoidosis was the same as PM2.5 in the atmosphere. Conclusion The study provided the further evidence that the PM2.5 in the sarcoidosis lesion was from PM2.5 in the atmosphere,and it should be not excepted that sarcoidosis may be a sensitive individual reaction to the PM2.5 inhaled from the atmosphere.     

Genotoxicity of fine and coarse fraction ambient particulate matter in immortalised normal (TT1) and cancer‐derived (A549) alveolar epithelial cells

Human exposure to airborne particulate matter (PM) is associated with adverse cardiopulmonary health effects, including lung cancer. Ambient PM represents a heterogeneous mixture of chemical classes including transition metals, polycyclic aromatic hydrocarbons (PAHs) and their derivatives such as nitro‐PAHs, many of which are classified as putative carcinogens. As the primary site of human exposure to PM is the lungs, we investigated the response of two alveolar epithelial cell lines, the tumour‐derived A549 and newly described TT1 cells, to fine and coarse PM collected from background and roadside locations. We show that coarse PM elicits a genotoxic response in the TT1 cells, with the strongest signal associated with the background sample. This response could be recapitulated using the organic extract derived from this sample. No responses were observed in PM‐challenged A549 cells. Fine PM failed to elicit a genotoxic response in either cell line despite the higher PAH concentrations within this fraction. Consistent with the lack of a simplistic association between PM PAH content and the observed genotoxic response, TT1 cells treated with benzo[a]pyrene (BaP) demonstrated no increase in the selected markers. In contrast, a pattern of response was observed in TT1 cells challenged with 3‐nitrobenzanthrone (3‐NBA) similar to that with coarse PM. Together, these data illustrated the suitability of the TT1 cell line for assessing PM‐induced genotoxicity and challenge the contention that fine roadside PM poses the higher cancer risk. Furthermore, the response to 3‐NBA and not BaP suggests a major contribution of nitro‐PAHs to the overall toxicity of PM. Environ. Mol. Mutagen. 59:290–301, 2018. © 2018 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society