应用16S rRNA基因技术研究仔猪结肠梭菌Ⅳ群变化

【目的】研究从7日龄到35日龄(断奶后两周)仔猪结肠中梭菌Ⅳ群(柔嫩梭菌群,Clostridium Leptumgroup)结构以及数量的变化,探究该菌群与结肠中丁酸浓度的关系。【方法】选取3窝新生仔猪,分别在7、14、21(断奶)、24和35日龄每窝随机屠宰1头,收集结肠内容物,利用气相色谱测定挥发性脂肪酸(Volatilefatty acid,VFA);提取结肠内容物总细菌DNA,利用基于16S rRNA基因的变性梯度凝胶电泳(Denaturinggradient gel electrophoresis,DGGE)和Real-time PCR技术定性、定量分析梭菌Ⅳ群。【结果】对仔猪结肠中梭菌Ⅳ群DGGE图谱进行相似性分析显示,7日龄仔猪样品归于一簇,数值达90%以上,而与其他日龄的样品间相似性较低,断奶前(21日龄)和断奶后3天菌群则没有显著变化。Real-time PCR定量分析显示,仔猪结肠总细菌拷贝数在断奶后3天显著降低,这一趋势与结肠中的总挥发性脂肪酸和丁酸浓度变化相似,而梭菌Ⅳ群拷贝数变化则不显著。克隆和测序分析表明,仔猪结肠梭菌Ⅳ群中的优势菌为Faecalibacteriumprausnitzii,Subdoligranulum variabile以及一些未培养细菌。【结论】7日龄至14日龄仔猪结肠中梭菌Ⅳ群发生改变,但断奶对其影响不大;该菌群和结肠丁酸浓度关系还需进一步研究。     

高脂肪日粮和产甲烷菌抑制剂对C57BL/6J小鼠盲肠微生物及脂肪代谢的影响

【目的】本文旨在研究产甲烷菌抑制剂溴氯甲烷(BCM)对采食高、低脂肪水平日粮小鼠的肠道微生物以及脂肪代谢的影响。【方法】选取32只21日龄的雌性C57BL/6J小鼠,随机分成4组:对照低脂日粮组、对照低脂日粮加BCM组、高脂日粮组及高脂日粮加BCM组,每组8个重复,BCM通过饮水添加。饲养周期为6周,试验期结束,采集小鼠血液样品,用于生化指标分析;采集小鼠盲肠内容物,用变性梯度凝胶电泳(DGGE)和real-time PCR技术分析其菌群;气相色谱法分析粪样中短链脂肪酸含量;real-time PCR技术分析肝脏中脂肪代谢相关基因表达。【结果】高脂日粮和BCM处理对小鼠体增重均无显著影响;DGGE图谱中低脂日粮组和高脂日粮组样品聚于不同簇,但对各组总细菌、产甲烷菌和硫还原菌数量无显著影响。高脂日粮组粪样中乙酸占总挥发酸比例显著降低(P0.05);BCM处理显著增加小鼠粪样丙酸比例(P0.05);高脂日粮处理显著影响高密度脂蛋白胆固醇、低密度脂蛋白胆固醇和总胆固醇含量(P0.05);高脂日粮处理促进了甘油三酯水解酶表达(P0.05)。【结论】高脂日粮对小鼠肠道菌群结构以及脂肪代谢有明显影响,而BCM处理影响较小。     

高脂肪日粮和产甲烷菌抑制剂对C57BL/6J小鼠盲肠微生物及脂肪代谢的影响

摘要:【目的】本文旨在研究产甲烷菌抑制剂溴氯甲烷(BCM)对采食高、低脂肪水平日粮小鼠的肠道微生物以及脂肪代谢的影响。【方法】选取32只21日龄的雌性C57BL/6J小,随机分成4组:对照低脂日粮组、对照低脂日粮加BCM组、高脂日粮组及高脂日粮加BCM组,每组8个重复,BCM通过饮水添加。饲养周期为6周,试验期结束,采集小鼠血液样品,用于生化指标分析;采集小鼠盲肠内容物,用变性梯度凝胶电泳(DGGE)和real-time PCR技术分析其菌群;气相色谱法分析粪样中短链脂肪酸含量;real-time PCR技术分析 肝脏中脂肪代谢相关基因表达。【结果】高脂日粮和BCM处理对小鼠体增重均无显著影响;DGGE图谱中低脂日粮组和高脂日粮组样品聚于不同簇,但对各组总细菌、产甲烷菌和硫还原菌数量无显著影响。高脂日粮组粪样中乙酸占总挥发酸比例显著降低(P＜0.05);BCM处理显著增加小鼠粪样丙酸比例(P＜0.05);高脂日粮处理显著影响高密度脂蛋白胆固醇、低密度脂蛋白胆固醇和总胆固醇含量(P＜0.05);高脂日粮处理促进了甘油三酯水解酶表达(P＜0.05)。【结论】高脂日粮对小鼠肠道菌群结构以及脂肪代谢有明显影响,而BCM处理影响较小。     

健康与腹泻仔猪粪样中挥发性脂肪酸和菌群区系的比较

[目的]本试验通过对比健康与腹泻仔猪粪样挥发性脂肪酸( Volatile fatty acid,VFA)以及菌群的差异,初步探讨腹泻对仔猪后肠环境的影响.[方法]采集腹泻和健康仔猪粪样,气相色谱测定挥发性脂肪酸浓度;提取粪样总细菌核酸,利用变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)和Real-time PCR技术定性、定量分析总细菌和梭菌Ⅳ菌群.[结果]与健康仔猪相比,腹泻仔猪粪样中乙酸含量有升高的趋势,支链脂肪酸(Branched chain fatty acid,BCFA)含量显著降低(p＜0.05),总VFA、丙酸和丁酸含量有降低的趋势,但差异均不显著;乙酸占总挥发酸比例显著升高(p＜0.05),而丙酸及BCFA占总挥发酸比例则显著降低(p＜0.05),丁酸占总挥发酸比例有降低趋势.PCR-DGGE指纹技术分析表明:腹泻发生后,仔猪粪样中总细菌和梭菌Ⅳ菌群在DGGE图谱上并没有特异性的条带消失或出现,但相似性分析显示,腹泻仔猪样品趋于归类于同一簇.Real-time PCR定量分析显示:仔猪腹泻后,粪样中的总细菌和梭菌Ⅳ菌群的数量显著下降(p＜0.05);而大肠杆菌和乳酸杆菌的数量变化不显著.[结论]与健康仔猪相比,腹泻仔猪粪样挥发性脂肪酸组成发生改变,并且微生物区系中的一些菌群也发生变化.     

我国8个典型水稻土中产甲烷古菌群落组成的空间分异特征

【目的】产甲烷古菌主导稻田甲烷生成,是稻田生态系统的模式微生物类群之一,具有重要的生态学意义。然而,水稻土产甲烷古菌群落组成的空间分异却鲜有报告。【方法】本研究沿20.55°N至47.43°N梯度,采集了我国不同纬度上8个典型水稻土,利用PCR-DGGE指纹图谱和系统发育树分析揭示不同地点水稻土中产甲烷古菌群落的组成;结合多个环境因子,利用生物信息学,典范对应分析(Canonical Correspondence Analysis,CCA)和维恩图(Venn diagram)明确产甲烷古菌的空间分异规律。【结果】研究发现p H值是驱动水稻土中产甲烷古菌群落组成分异的主要因子;此外,沿纬度梯度,8个地区的产甲烷古菌群落组成也呈现出规律性变化。【结论】本研究首次阐明了稻田中产甲烷古菌群落分布情况,并揭示其主要驱动因子。该认知不仅有助于我们更好地了解产甲烷古菌的生物地理学分布,还有助于从微生物学机制上阐明我国温度梯度带上有机质转化空间的差异。     

青藏高原三个盐碱湖的产甲烷菌群和产甲烷代谢途径分析

【目的】分析青藏高原不同类型盐碱湖中的优势产甲烷菌群和优势产甲烷代谢途径。【方法】以不同盐度和植被类型的公珠错、昆仲错和无植被的兹格塘错的沉积物为研究对象,通过高通量测序和q PCR定量古菌16S r RNA多样性分析优势古菌类群;模拟原位盐浓度及p H,比较不同产甲烷底物(甲醇、三甲胺、乙酸和H_2/CO_2)富集沉积物的产甲烷速率,分析其优势产甲烷菌代谢类型。通过添加产甲烷抑制剂(2-溴乙烷磺酸盐),检测沉积物中产甲烷底物积累,确定不同盐碱湖中主要的产甲烷途径。【结果】昆仲错的优势菌群包括甲基/乙酸型的甲烷八叠球菌科(Methanosarcinaceae,11%),乙酸型的甲烷鬃菌科(Methanosaetaceae,7.9%)和氢型甲烷菌甲烷杆菌目(Methanomicrobiales,7.4%);公珠错和兹格塘错的优势菌群为甲烷鬃菌科(Methanosaetaceae)分别占15%和15.3%,及甲烷杆菌属(Methanobacterium)和甲基型的甲烷叶菌属(Methanolobus)。公珠错和昆仲错分别以乙酸和甲醇产甲烷速率最高,而兹格塘错从不同底物产甲烷速率无差异。抑制甲烷产生后,公珠错主要积累乙酸,昆仲错主要积累甲醇;兹格塘错不仅甲烷排放低,也无产甲烷物质显著积累。【结论】昆仲错沉积物中的甲烷主要来自甲醇,公珠错中的甲烷主要来自乙酸,而兹格塘错产甲烷和底物积累不活跃。因而推测高原盐碱湖主要的产甲烷途径和菌群可能与周围植被类型的相关性更高,而与盐度的直接相关性较低。     

T-RFLP分析厌氧真菌传代频率对共存产甲烷菌菌群的影响

【目的】建立瘤胃产甲烷菌T-RFLP多样性分析方法,并研究厌氧真菌与产甲烷菌共培养液在不同时间传代对共存产甲烷菌菌群的影响。【方法】利用产甲烷菌mcrA基因特异性引物PCR扩增后,选择合适内切酶对扩增产物进行内切,分析内切后末端片段长度多态性,测定共培养液在不同传代频率时共存产甲烷菌多样性的变化。【结果】利用Msp I内切酶分析发现,末端片段长度约为470 bp的产甲烷菌为共培养液中的优势甲烷菌,共培养液传代至第15代时,片段长度约为130 bp和200 bp的产甲烷菌也成为共培养中的优势菌株;比较发现,Taq I能更好地内切共培养液中甲烷菌mcrA基因序列,瘤胃内容物及3 d传代共培养液中产甲烷菌主要为末端片段长度约为70、100、200、270、300、330和470 bp的菌株,共培养液在体外传代培养过程中,末端片段长度约为70、100、270和470 bp的产甲烷菌变化更为显著。Taq I比较分析不同传代频率(3、5和7 d)对共培养液中产甲烷菌菌群结构的影响表明,3 d传代的共培养液中产甲烷菌菌群与瘤胃内容物较为相似,而5 d和7 d传代的共培养液中产甲烷菌菌群间差异较小,但与瘤胃内容物差异较大,导致不同传代频率的共培养液中产甲烷菌菌群间显著差异的最主要菌株为末端片段长度约为100 bp的产甲烷菌,其次为末端片段长度约为70 bp和270 bp的产甲烷菌。【结论】利用建立的快速可行的瘤胃产甲烷菌T-RFLP方法分析表明,传代频率显著影响厌氧真菌与产甲烷菌共培养液中产甲烷菌的菌群结构,3 d传代共培养液内产甲烷菌菌群与瘤胃内容物更相似。     

模拟增温对青海湖鸟岛土壤产甲烷菌群落特征的影响

甲烷生成与产甲烷菌群落关系密切,探究温度升高对产甲烷菌群落的影响可以预测产甲烷菌群落特征对气候变暖的响应.以青海湖鸟岛为研究对象,通过高通量测序方法开展研究.结果表明,基于97％的序列相似度聚类,共得到697个操作分类单元.鸟岛土壤产甲烷菌群落以甲烷微菌目(Methanomicrobiales)、甲烷八叠球菌目(Met...     

典型煤层水微生物产甲烷潜力及其群落结构研究

内蒙古自治区二连盆地、海拉尔盆地是我国重要的煤层气产区,其中生物成因煤层气是煤层气的重要来源,但复杂物质转化产甲烷相关微生物群落结构及功能尚不清楚。【目的】研究煤层水中的微生物代谢挥发性脂肪酸产甲烷的生理特征及群落特征。【方法】以内蒙古自治区二连盆地和海拉尔盆地的四口煤层气井水作为接种物,分别添加乙酸钠、丙酸钠和丁酸钠厌氧培养;定期监测挥发性脂肪酸降解过程中甲烷和底物的变化趋势,应用高通量测序技术,分析原始煤层气井水及稳定期产甲烷菌液的微生物群落结构。【结果】除海拉尔盆地H303煤层气井微生物不能代谢丙酸外,其他样品均具备代谢乙酸、丙酸和丁酸产生甲烷的能力,其生理生态参数存在显著差异,产甲烷延滞期依次是乙酸丁酸丙酸;最大比产甲烷速率和底物转化效率依次是丙酸乙酸丁酸。富集培养后,古菌群落结构与煤层气井水的来源显著相关,二连盆地优势古菌为氢营养型产甲烷古菌Methanocalculus (相对丰度13.5%–63.4%)和复合营养型产甲烷古菌Methanosarcina (7.9%–51.3%),海拉尔盆地的优势古菌为氢营养型产甲烷古菌Methanobacterium(24.3%–57.4%)和复合营养型产甲烷古菌Methanosarcina(29.6%–66.5%);细菌群落则与底物类型显著相关,硫酸盐还原菌Desulfovibrio(12.0%–41.0%)、互营丙酸氧化菌Syntrophobacter(39.6%–75.5%)和互营丁酸菌Syntrophomonas(8.5%–21.9%)分别在乙酸钠、丙酸钠和丁酸钠处理组显著富集。【结论】煤层气井水微生物可降解挥发性脂肪酸(乙酸、丙酸和丁酸)并具有产甲烷潜力;乙酸可能被古菌直接代谢产甲烷,而丙酸和丁酸通过互营细菌和产甲烷古菌代谢产甲烷。Desulfovibrio、Syntrophobacter和Syntrophomonas分别在乙酸、丙酸和丁酸代谢过程中发挥了重要作用。这些结果为煤层气生物强化开采提供了一定的微生物资源基础。     

培养法和免培养法联合检测油藏环境烃降解菌和产甲烷菌群多样性

烃降解菌和产甲烷菌是油藏环境微生物生态系统中重要的功能菌群, 采用DGGE和FISH方法分析了不同油藏样品中两类菌群的多样性和产甲烷活性。DGGE结果表明, 不同水样的alkB基因多样性相差较大, 而且注水井条带明显多于采油井。FISH结果表明, 油藏水样中产甲烷菌含量明显高于烃降解菌, 且两者空间分布的位置较近; 说明油藏环境中烃降解菌和产甲烷菌结成一定的相互关系。富集培养表明, 胜利油田产出液接种物培养130 d后, 石油烃降解率达到50%以上, 产甲烷的最大速率达到1.57×10^?2 mmol/(L?d)。利用分子生物学方法分析油藏环境功能菌群的多样性, 可以为开展微生物采油技术的应用提供有用信息。     

The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans

Polycyclic aromatic hydrocarbons (PAHs) appear to be significant contributors to the genotoxicity and carcinogenicity of air pollution present in the urban environment for humans. Populations exposed to environmental air pollution show increased levels of PAH DNA adducts and it has been postulated that another contributing cause of carcinogenicity by environmental air pollution may be the production of reactive oxygen species following oxidative stress leading to oxidative DNA damage. The antioxidant status as well as the genetic profile of an individual should in theory govern the amount of protection afforded against the deleterious effects associated with exposure to environmental air pollution. In this study we investigated the formation of total PAH (bulky) and B[a]P DNA adducts following exposure of individuals to environmental air pollution in three metropolitan cities and the effect on endogenously derived oxidative DNA damage. Furthermore, the influence of antioxidant status (vitamin levels) and genetic susceptibility of individuals with regard to DNA damage was also investigated. There was no significant correlation for individuals between the levels of vitamin A, vitamin E, vitamin C and folate with M₁dG and 8-oxodG adducts as well as M₁dG adducts with total PAH (bulky) or B[a]P DNA adducts. The interesting finding from this study was the significant negative correlation between the level of 8-oxodG adducts and the level of total PAH (bulky) and B[a]P DNA adducts implying that the repair of oxidative DNA damage may be enhanced. This correlation was most significant for those individuals that were non smokers or those unexposed to environmental air pollution. Furthermore the significant inverse correlation between 8-oxodG and B[a]P DNA adducts was confined to individuals carrying the wild type genotype for both the GSTM1 and the GSTT1 gene (separately and interacting). This effect was not observed for individuals carrying the null variant.     

Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO(3)(-)-N mg of mixed-liquor volatile suspended solids (MLVSS)(-1) h(-1) to a steady-state value of 0.06 mg of NO(3)(-)-N mg of MLVSS(-1) h(-1) over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [(13)C]methanol to biomark the DNA of the denitrifiers. The extracted [(13)C]DNA and [(12)C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [(13)C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [(12)C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [(14)C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.     

DNA oxidative damage and strand breaks in young healthy individuals: a gender difference and the role of life style factors

The aim of this study was to analyze background levels of DNA damage in young (19-31 years) non-smoking individuals and to correlate damage to gender and life style. DNA single strand breaks (SSB) and alkali labile sites (ALS) were measured in 99 subjects living in Stockholm, Sweden. Further, oxidative DNA damage was analyzed using the DNA repair glycosylase FPG as well as HPLC-ECD for specific analysis of 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxodG). We found that males had higher (P < 0.001) levels of SSB + ALS than females, but no difference was seen for oxidative lesions. There was no correlation between FPG sites and 8-oxodG. For females, there was a positive correlation between FPG levels and body mass index and a negative correlation between SSB + ALS and fruit intake. We conclude that the background level of oxidative DNA damage, analyzed with improved methods, is low and that gender, fruit intake and BMI can affect DNA damage.     

Microsatellite polymorphisms in DNA repair genes XRCC1, XRCC3 and XRCC5 in patients with gynecological tumors: association with late clinical radiosensitivity and cancer incidence

This study investigates the association of microsatellite polymorphisms in XRCC1, XRCC3 and XRCC5 with the development of late radiation-induced radiotherapy reactions and examines the correlation between these microsatellites and cancer incidence. Sixty-two women with cervical or endometrial cancer treated with radiotherapy were included in the study. According to the CTCAEv3.0 scale, 22 patients showed late adverse radiotherapy reactions (grade 2 or more). PCR on lymphocyte DNA followed by automated fragment analysis was performed to examine the number of tandem repeat units at each locus. No significant association was found between the repeat length at any of the microsatellites in XRCC1, XRCC3 or XRCC5 and the incidence of late radiotherapy complications. Since higher odds ratios (ORs) were found for the rare XRCC1 [AC]11 and [AC]21 repeats (OR = 2.65, P = 0.325 and OR = 8.67, P = 0.093, respectively), the possible involvement of these small and large repeats in clinical radiosensitivity cannot be completely ruled out. When specific numbers of repeats were examined, no significant correlation was found between the microsatellite repeat length in XRCC1 and XRCC5 and cancer incidence. A weak correlation between XRCC3 [AC]16 homozygotes and cancer incidence was found (OR = 2.56, P = 0.055). A large-scale multicenter study of cancer patients with a high number of radiosensitive individuals is needed to clarify the value of rare polymorphic microsatellite repeats in XRCC1 and XRCC3 as a biomarker of clinical radiosensitivity or increased cancer risk.     

Inhibition of human topoisomerase II by anti-neoplastic benzazolo[3,2-a]quinolinium chlorides

Previously we reported [20] that there is no correlation between the cytotoxic activity of four new structural analogs of the antitumor DNA intercalator 3-nitrobenzothiazolo[3,2-a]quinolinium chloride (NBQ-2) and their interaction with DNA. In the present study, we present evidence suggesting that the molecular basis for the anti-proliferative activity of these drugs is the inhibition of topoisomerase II. The NBQ-2 derivatives inhibited the relaxation of supercoiled DNA plasmid pRYG mediated by purified human topoisomerase II. Inhibition of the decatenation of kinetoplast DNA mediated by partially purified topoisomerase II extracted from the human histiocytic lymphoma U937 (a cell line previously shown to be sensitive to the drugs) was also caused by these drugs. The potency of the benzazolo[3,2-a]quinolinium drugs against topoisomerase II in vitro was the following: 7-(1-propenyl)-3-nitrobenzimidazolo[3,2-a]quinolinium chloride (NBQ-59) > 4-chlorobenzothiazolo[3,2-a]quinolinium chloride (NBQ-76) > 7-ethyl-3-nitrobenzimidazolo[3,2-a]quinolinium chloride (NBQ-48) > 7-benzyl-3-nitrobenzimidazolol[3,2-a]quinolinium chloride (NBQ-38). This rank of potency for topoisomerase II inhibition correlated very well with the cytotoxicity elicited by these drugs. Furthermore, significant levels of topoisomerase II/DNA cleavage complex induced by these drugs in vivo were detected when U937 cells were treated with NBQ-59 and NBQ-76 whereas NBQ-38 and NBQ-38 and NBQ-48 produced negligible amounts of the cleavage complex. Our results strongly suggest that topoisomerase II is the major cellular target of this family of compounds.     

Activity of some bromomethylated aromatic hydrocarbons on the in vitro synthesis of DNA and RNA

The effect of a series of bromomethylated polycyclic hydrocarbons on in vitro DNA and RNA synthesis has been studied by measurement of the incorporation of [³H]-dTMP or [¹⁴C]-AMP into new chains. The inhibition of RNA synthesis was less than 12% for 9-bromomethylanthracene, 9-methyl-10-bromomethylanthracene and 12-bromomethylbenzo(a)acridine, and more than 37% for 7-bromomethylbenzo(a)anthracene, 7,12-dibromomethylbenzo(a)anthracene and 7-bromomethylbenzo(c)acridine. Analogous results were found for the inhibition of DNA synthesis, except for 7-bromomethylbenzo(c)acridine which had little effect. Apart from this exception a good correlation was found between the inhibitory action of the bromo derivatives and the carcinogenicity of the non-halogenated parent hydrocarbons.     

The association of bacterial mutagenicity of hydrocarbon-derived 'bay-region' dihydrodiols with the Iball indices for carcinogenicity and with the extents of DNA-binding on mouse skin of the parent hydrocarbons.

The mutagenic activities of benz[alpha]anthracene, 7-methylbenz[alpha]anthracene, 7,12-dimethylbenz[alpha]anthracene, 3-methylcholanthrene and benzo[alpha]pyrene, together with those of the trans-dihydrodiols derived from these hydrocarbons that would be expected to yield 'bay-region' vicinal diolepoxides on further metabolism have been examined in assays with S. typhimurium TA100 using post-mitochondrial supernatant fractions prepared from the livers of 3-methylcholanthrene-treated rats. Mutagenic activities obtained have been compared with: (a) the extents of reaction with DNA that occur in mouse skin following treatment with these hydrocarbons; (b) the carcinogenicities of the hydrocarbons expressed as Iball indices; (c) their activities as tumour-initiating agents on mouse skin. Close positive associations were found between the microsome-mediated mutagenicities of the dihydrodiols that could yield "bay-region" diol-epoxides and: (a) the extents of reaction with DNA in hydrocarbon-treated mouse skin; (b) the carcinogenic potencies of the parent hydrocarbons; although these correlations are not perfect, the mutagenic activities of the hydrocarbons themselves in microsome-mediated assays with S. typhimurium show no correlation with their extents of DNA binding on mouse skin and a poor correlation with their activities as initiating agents. These comparisons also indicated a statistically-significant positive correlation between carcinogenicity and the in vivo DNA binding on mouse skin treated with the hydrocarbons. Differences in the metabolic pathways by which polycyclic hydrocarbons are activated in vivo and in vitro are discussed in relation to the improved correlations found with the dihydrodiols.     

Comparing the genotoxic sensitivities of human peripheral blood lymphocytes and mucosa cells of the upper aerodigestive tract using the Comet assay

Carcinogenesis in the upper aerodigestive tract is influenced by multiple factors. Besides tobacco and alcohol consumption, specific pollutants such as phthalates, nitrosamines, and polycyclic aromatic carbohydrates may be important in tumor initiation. Genetic factors related to mutagen sensitivity and DNA repair capacity also play a role. The aim of this study was to investigate whether human peripheral blood lymphocytes and mucosal epithelium of the upper aerodigestive tract, the target for volatile and liquid xenobiotics, are equally sensitive to genotoxic agents.The Comet assay was used to detect for DNA damage induced by genotoxic agents in mucosal epithelial cells and peripheral blood lymphocytes of 60 volunteers. Mucosa was harvested from larynx, oropharynx, and inferior nasal turbinates. Xenobiotics investigated were dibutylphthalate (DBP), diisobutylphthalate (DiBP), N'-nitrosodiethylamine (NDELA), benzo[a]pyrene (B[a]P), and N'-methyl-N'-nitro-N-nitrosoguanidine (MNNG). DBP, DiBP, B[a]P, NDELA and MNNG induced a significant increase in DNA migration in both cell populations. Peripheral blood lymphocytes were more sensitive than mucosal cells to DBP and DiBP, but not to NDELA and B[a]P. The correlation, in terms of DNA migration, between lymphocytes and mucosal cells among volunteers was relatively poor. Based on the poor correlation in response between the two cell types, the sensitivity of peripheral blood lymphocytes to genotoxic agents appears to be a poor predictor of sensitivity in the target cells of the upper aerodigestive tract. Further attention should be focused on intra-individual mutagen sensitivities and inter-individual genetic differences as regards susceptibility to upper aerodigestive tract cancer.     

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part I: acellular assay

Acellular assay of calf thymus DNA ± rat liver microsomal S9 fraction coupled with ³²P-postlabelling was used to study the genotoxic potential of organic compounds bound onto PM10 particles collected in three European cities—Prague (CZ), Kosice (SK) and Sofia (BG) during summer and winter periods. B[a]P alone induced DNA adduct levels ranging from 4.8 to 768 adducts/10⁸ nucleotides in the concentration dependent manner. However, a mixture of 8 c-PAHs with equimolar doses of B[a]P induced 3.7–757 adducts/10⁸ nucleotides, thus suggesting the inhibition of DNA adduct forming activity by interaction among various PAHs. Comparison of DNA adduct levels induced by various EOMs indicates higher variability among seasons than among localities. DNA adduct levels for Prague collection site varied from 19 to 166 adducts/10⁸ nucleotides, for Kosice from 22 to 85 and for Sofia from 6 to 144 adducts/10⁸ nucleotides. Bioactivation with S9 microsomal fraction caused 2- to 7-fold increase in DNA adduct levels compared to −S9 samples, suggesting a crucial role of indirectly acting genotoxic EOM components, such as PAHs. We have demonstrated for the first time a significant positive correlation between B[a]P content in EOMs and total DNA adduct levels detected in the EOM treated samples (R = 0.83; p = 0.04). These results suggest that B[a]P content in EOM is an important factor for the total genotoxic potential of EOM and/or B[a]P is a good indicator of the presence of other genotoxic compounds causing DNA adducts. Even stronger correlation between the content of genotoxic compounds in EOMs and total DNA adduct levels detected (R = 0.94; p = 0.005) was found when eight c-PAHs were taken into the consideration. Our findings support a hypothesis that a relatively limited number of EOM components is responsible for a major part of its genotoxicity detectable as DNA adducts by ³²P-postlabelling.