莪术醇对肝星状细胞-T6细胞基因表达的影响

[目的]研究莪术醇对肝星状细胞(HSC)-T6细胞基因表达的影响.[方法]从基因库中查询50种肝纤维化相关基因的mRNA序列,用寡核苷酸探针设计软件设计探针,在PE8909DNA合成仪上合成寡核苷酸,用OGR-04点样仪及醛基化玻片制备成基因芯片.以莪术醇不同浓度含药培养液培养HSC-T6细胞,根据细胞毒性实验,确定细胞存活率在50%以上的药物浓度作为实验所用浓度,每组设空白对照,分别按1、6、12、24 h 4个时间段收集细胞.按操作步骤提取细胞总RNA,经反转录荧光标记,杂交和洗涤,用Genepix 4000B扫描仪扫描芯片,ImaGene 4.2软件进行数据分析和归一化处理,使用看家基因和阳性对照对Cy3和Cy5扫描结果进行校正.[结果]莪术醇1.562 5 μg/ml作用HSC-T6细胞12 h,可使基因转化生长因子β1、细胞色素P450a表达下调2.3、2.1倍.[结论]从分子水平揭示了莪术有效成分莪术醇的抗肝纤维化机制.     

抑制细胞因子RhoA的表达对肝星状细胞株HSC-T6胶原分泌的影响

目的:观察并比较两种方法抑制RhoA的表达对细胞外基质成分,如Ⅰ型胶原、透明质酸以及层粘连蛋白合成的影响,为肝纤维化的基因治疗寻求新的途径.方法:设计并合成针对RhoA相同靶点的反义寡核苷酸和小干扰RNA.分别转染肝星状细胞株HSC-T6,逆转录PCR技术检测细胞中RhoA和Ⅰ型胶原mRNA的表达:Western blot检测细胞中RhoA蛋白质的表达;ELISA检测培养上清中透明质酸(HA)、层黏蛋白(LN)及PIIIP的含量.结果:转染RhoA反义寡核苷酸和小干扰RNA Ratl质粒后.HSC-T6中RhoA mRNA的表达分别由0.892±0.051、0.937±0.044降为0.113±0.024、0.212±0.042;蛋白质表达水平均明显下调;细胞内Ⅰ型胶原mRNA的表达分别由0.709±0.067、0.695±0.087降为0.436±0.037、0.201±0.044;细胞外基质成分如HA、LN及PIIIP的表达水平明显下降.两者相比,小干扰RNA具有更强的生物学效应.结论:靶向抑制细胞因子RhoA的表达可明显减少肝星状细胞株HSC-T6细胞外基质的合成,且小干扰RNA的效果明显优于针对同一靶点的反义寡核苷酸,显示RNAi技术应用于肝纤维化基因治疗的良好前景.     

抑制性消减杂交技术筛选γ-氨基丁酸调节的肝星状细胞系靶基因

目的:应用抑制性消减杂交技术(SSH)筛选γ-氨基丁酸(GABA)作用肝星状细胞系HSC-T6后的差异表达基因.方法:10 umol/L的GABA作用于HSC-T6细胞24 h,提取mRNA,用分光光度计进行定量分析.以GABA处理和未处理的T6细胞mRNA为模板逆转录合成双链cDNA(dscDNA),并分别标记为Tester和Drivet,酶切后与接头连接,经两次杂交,构建消减杂交文库,将消减文库的扩增产物进行转化、克隆分析后应用生物信息学技术将测得序列再进行同源性分析.结果:15种基因表达上调,其中包括与DNA合成、线粒体、肿瘤抑制以及凋亡相关的4类基因,结果显示GABA可能促进HSC-T6细胞增殖而抑制凋亡.结论:用SSH技术可以成功获得GABA刺激肝星状细胞基因上调的监测,证明GABA能够影响肝星状细胞的基因表达谱.     

肝爽颗粒对HSC-T6细胞ColⅠ、ColⅢ、TIMP1基因及蛋白表达的影响

目的观察肝爽颗粒对HSC-T6细胞Ⅰ型胶原(Collagen Ⅰ,ColⅠ)、Ⅲ型胶原(Collagen Ⅲ,ColⅢ)、基质金属蛋白酶组织抑制因子1(Tissue Inhibitor of Metalloproteinase 1,TIMP1)基因及蛋白表达的影响.方法用浓度0.025、0.05、0.1、0.15、0.2、0.35、0.5、1.0、1.5、2.0、4.0、8.0、16.0mg/ml肝爽颗粒作用于HSC-T6细胞48小时,采用MTT比色法观察其对HSC-T6细胞生长的影响;以0.05、0.1、0.2mg/ml肝爽颗粒作用于HSC-T6细胞48小时,采用逆转录PCR与ELISA方法分别测定其对HSC-T6细胞ColⅠ、ColⅢ、TIMP1基因及蛋白表达的影响.结果空白对照组ColⅠ、ColⅢ、TIMP1基因表达水平分别为0.91±0.11、1.54±0.09、1.83±0.13辉度值,蛋白表达水平分别为(187.63±4.11)、(7.59±1.04)、(23.85±2.13)ng/ml,以0.05、0.1、0.2mg/ml肝爽颗粒作用于HSC-T6细胞48小时,浓度0.05mg/ml肝爽颗粒仅能降低ColⅠ蛋白的表达;当浓度升为0.10mg/ml时不仅可显著降低ColⅠ蛋白的表达,而且对ColⅢ mRNA与蛋白的表达亦产生明显抑制作用;当浓度达到0.20mg/ml时作用更明显,可显著降低ColⅠ、ColⅢmRNA,ColⅠ、ColⅢ、TIMP1蛋白的表达.结论肝爽颗粒能明显抑制HSC-T6细胞ColⅠ、ColⅢ基因表达,抑制ColⅠ、ColⅢ、TIMP1蛋白表达,从而可抑制Ⅰ、Ⅲ型胶原的合成,减弱TIMP1对基质金属蛋白酶的抑制作用,这可能是其抗纤维化的作用机制之一.     

Genistein对大鼠肝星状细胞的增殖及TGF-β1、MMP-2和TIMP-2表达的影响

目的:观察4,5,7-三羟基异黄酮(Genistein)对大鼠肝星状细胞(HSC)增殖及TGF-β1、MMP-2及TIMP-2基因表达的影响,探讨其抗肝纤维化的可能机制.方法:将体外培养的大鼠HSC-T6分别给予不同浓度的Genistein作用24 h后,用噻唑兰比色法(MTT)检测测定活细胞数,采用半定量RT-PCR法检测细胞中TGF-β1、MMP-2及TIMP-2的mRNA水平.结果:Genistein作用于大鼠HSC-T6后,活细胞数目减少,12.5、25及50 mg/L 3个作用浓度组与对照组的4值比较有显著性差异(0.306±0.0067,0.256±0.0085,0.1316±0.0049VS 0.4468±0.0299,均P<0.05).不同浓度的Genistein干预HSC-T6,MMP-2 mRNA表达水平与对照组比较均明显增强(均P<0.05);TGF-β1和TIMP-2 mRNA表达水平与对照组比较均明显降低(均P<0.05).结论:Geni Steill可能通过增强大鼠HSCMMP-2 mRNA的表达,同时抑制TGF-β与TIMP-2 mRNA的表达发挥抗纤维化的作用.     

调控Notch信号对肝星状细胞活化的影响

目的 探讨肝星状细胞(HSC-T6)内是否存在功能激活的Notch信号分子以及调控Notch信号转导对HSC-T6活化的影响. 方法 用PCR方法检测Notch信号分子mRNA在HSC-T6细胞内的表达,TaqMan探针检测转化生长因子(TGF)β 1刺激HSC-T6后Notch信号分子的表达变化.细胞免疫荧光法检测Notch3和Jagged1在HSC-T6内的表达定位.用携带Notch3胞内域(ICD)的真核表达质粒pcDNA3.1-N3ICD和特异性干扰Notch3的siRNA(Notch3-siRNA)分别转染HSC-T6细胞,Western blot检测Notch3、α-平滑肌肌动蛋白(SMA)和Ⅰ型胶原的表达变化.对数据采用t检验和方差分析. 结果 HSC-T6细胞内存在功能激活的Notch信号转导.TGF β 1(2.0 ng/ml)刺激HSC-T6导致Jagged1、Notch3和HES-1的mRNA水平明显上调,分别提高了2.9、3.2、2.2倍,F值分别为2543.482,287.982和1719.851,P值均＜0.01.pcDNA3.1-N3ICD转染后促使HSC-T6合成α-SMA和Ⅰ型胶原蛋白增加,较对照组分别提高了5.8倍和4.5倍,t值分别为13.157和9.810,P值均＜0.01;相反,Notch3- siRNA明显抑制HSC-T6表达α-SMA和Ⅰ型胶原蛋白,较对照组分别下降了约90％和84％,t值分别为19.863和10.376,P值均＜0.01.而且,Notch3-siRNA能拮抗TGFβ1诱导HSC-T6表达α-SMA和Ⅰ型胶原的作用.结论 Notch信号通路可能通过调控肝星状细胞的活化参与肝纤维化的形成,选择性地干预Notch3信号转导可能成为抗肝纤维化的新途径.     

银杏叶提取物对大鼠肝星状细胞细胞因子和胶原合成及细胞增殖的影响

目的研究银杏叶提取物抗肝纤维化的可能机理。方法用不同浓度(0,1,10,100,500μg·ml-1)的银杏叶提取物处理HSC-T6细胞24h和48h,采用RT-PCR法检测各组细胞转化生长因子β1(TGF-β1)、结缔组织生长因子(CTGF)、Ⅰ型胶原、Ⅲ型胶原mRNA的表达;应用流式细胞仪检测细胞增殖周期的变化。结果银杏叶提取物在10,100,500μg·ml-1浓度能明显抑制TGF-β1、CTGF、Ⅰ型胶原、Ⅲ型胶原mRNA的表达(P<0.01或P<0.05),在一定范围内呈剂量和时间依赖性,影响HSC-T6的细胞周期,降低其增殖活性。结论银杏叶提取物可明显抑制HSC-T6细胞的增殖,抑制细胞因子及细胞外基质成分基因的表达,由此发挥抗肝纤维化的作用。     

羟基喜树碱对大鼠肝星状细胞增殖抑制的最佳浓度

目的:探讨羟基喜树碱(HCPT)对体外培养的大鼠肝星状细胞(HSC)增殖的影响, 并观察其对大鼠正常肝细胞的毒性作用, 确定HCPT对大鼠肝星状细胞增殖抑制的最佳浓度.方法:大鼠肝星状细胞株(HSC-T6)和大鼠正常肝细胞株(BRL-3A)分别在实验组(分别以含HCPT 浓度为0.008, 0.016, 0.031, 0.063, 0.125,0.25, 0.5, 1, 2, 4, 8, 16, 32 mg/L的培养液作用)及对照组(单纯培养)中体外培养24, 48, 72 h.应用四甲基偶氮唑盐法(MTT法)检测细胞增殖情况, 找出对HSC-T6细胞增殖抑制作用最大的HCPT最佳浓度;光镜下观察对照组和实验组HSC-T6细胞的形态学变化.结果:24, 48, 72 h实验组HSC-T6细胞的增殖率均低于对照组, 差异具有显著性( t =6.07-46.98, 10.98-63.97, 20.76-107.68, 均P<0.01);HCPT对HSC-T6细胞和BRL-3A细胞的增殖抑制率均随着药物作用浓度的升高及药物作用时间的延长而逐渐升高;当HCPT浓度>0.5 mg/L时, 对BRL-3A细胞的毒性作用显著性增高(均P<0.05);0.25, 0.5, 1 mg/L的HCPT作用HSC-T6细胞24 h, 光镜下均可见细胞数量减少、体积缩小、核浓缩等变化, 且随着作用浓度的增加变化更加明显.结论:HCPT在体外可以明显的抑制HSC-T6细胞的增殖, 作用强度呈剂量-时间依赖性;0.5mg/L为对HSC-T6细胞增殖抑制作用最大的HCPT最佳浓度.     

积雪草酸对HSC-T6细胞Smad7和PPARγ的mRNA表达的影响

目的检测积雪草酸对HSC-T6细胞Smad-7和PPARγ的mRNA表达的影响,探讨积雪草酸抗肝纤维化的分子生物学机制。方法用MTT实验检测积雪草酸对HSC-T6细胞的增殖抑制作用,计算抑制率,确定积雪草酸作用浓度;设置空白对照组与积雪草酸干预组,用半定量RT-RCR检测两组HSC-T6细胞Smad7和PPARγ的mRNA表达差异。结果积雪草酸抑制HSC-T6细胞的增殖,药物浓度为10、20、30、40、50、70、90μmol/L时,抑制率分别为7.47%、12.42%、13.97%、21.32%、47.31%、51.18%、60.36%。空白对照组和积雪草酸干预组的半定量RT-PCR的结果分别为：Smad7/GAPDH mRNA空白对照组为0.267±0.037,积雪草酸干预组为0.358±0.035;PPARγ/GAPDH mRNA空白对照组为0.157±0.054,积雪草酸干预组为0.570±0.066。两组结果间比较差异具有统计学意义,P〈0.05。结论积雪草酸可以上调HSC-T6细胞Smad7和PPARγ的mRNA的表达,从而抑制HSC-T6细胞合成和分泌胶原,改善肝纤维化的病理表现。     

羟基喜树碱对HSC-T6细胞增殖与凋亡的影响

目的 探讨羟基喜树碱(HCPT)对体外培养大鼠肝星状细胞(HSC)的增殖与凋亡的影响.方法 大鼠肝星状细胞(HSC-T6)和大鼠正常肝细胞(BRL-3A)分别在实验组(分别以含HCPT浓度为0.008、0.016、0.031、0.063、0.125,0.25、0.5、1、2、4、8、16、32mg/L的培养液培养)和对照组(单纯培养)体外培养24 h.用四甲基偶氮唑盐法检测细胞增殖情况,找出HCPT对HSC-T6细胞增殖抑制的最佳作用浓度;流式细胞仪检测细胞凋亡率;透射电子显微镜下观察细胞凋亡的形态学变化情况;琼脂糖凝胶电泳法检测DNA片段化.多个样本均数的比较采用单因素方差分析,两样本均数的比较采用t检验.结果 HCPT对HSC-T6细胞和BRL-3A细胞的增殖抑制率随着药物浓度的升高而逐渐升高;当HCPT浓度＞0.5mg/L时,对BRL-3A细胞的毒性作用显著增高(P值均<0.05);0.5 mg/L对HSC-T6细胞增殖抑制作用最大,为HCPT对HSC-T6细胞增殖的最佳抑制浓度.0.125、0.25、0.5 mg/L的HCPT作用HSC-T6细胞24h,流式细胞仪检测显示细胞凋亡率分别为13.46%±2.42%、26.25%±5.65%、47.05%±8.76%,与对照组(4.89%±1.80%)相比,差异有统计学意义(F=34.24,P<0.01).0.5mg/L的HCPT作用HSC-T6细胞24 h,透射电子显微镜下可见细胞体积缩小,核仁消失,染色质浓缩聚集成团块状,沿核膜排列等凋亡形态学改变;琼脂糖凝胶电泳可见明显的DNA梯度带形成.结论 HCPT在体外可以明显地抑制HSC-T6细胞的增殖、诱导HSC-T6细胞凋亡,作用强度呈剂量依赖性.     

Three layer functional model and energy exchange concept of aging process

Relying on a certain degree of abstraction, we can propose that no particular distinction exists between animate or living matter and inanimate matter. While focusing attention on some specifics, the dividing line between the two can be drawn. The most apparent distinction is in the level of structural and functional organization with the dissimilar streams of ‘energy flow’ between the observed entity and the surrounding environment. In essence, living matter is created from inanimate matter which is organized to contain internal intense energy processes and maintain lower intensity energy exchange processes with the environment. Taking internal and external energy processes into account, we contend in this paper that living matter can be referred to as matter of dissipative structure, with this structure assumed to be a common quality of all living creatures and living matter in general. Interruption of internal energy conversion processes and terminating the controlled energy exchange with the environment leads to degeneration of dissipative structure and reduction of the same to inanimate matter, (gas, liquid and/or solid inanimate substances), and ultimately what can be called ‘death.’ This concept of what we call dissipative nature can be extended from living organisms to social groups of animals, to mankind. An analogy based on the organization of matter provides a basis for a functional model of living entities. The models relies on the parallels among the three central structures of any cell (nucleus, cytoplasm and outer membrane) and the human body (central organs, body fluids along with the connective tissues, and external skin integument). This three-part structural organization may be observed almost universally in nature. It can be observed from the atomic structure to the planetary and intergalactic organizations. This similarity is corroborated by the membrane theory applied to living organisms. According to the energy nature of living matter and the proposed functional model, the decreased integrity of a human body's external envelope membrane is a first cause of the structural degradation and aging of the entire organism. The aging process than progresses externally to internally, as in single cell organisms, suggesting that much of the efforts towards the restoration and maintenance of the mechanisms responsible for structural development should be focused accordingly, on the membrane, i.e., the skin. Numerous reports indicate that all parts of the human body, like: bones, blood with blood vessels, muscles, skin, and so on, have some ability for restoration. Therefore, actual revival of not only aging tissue of the human body's membrane, but the entire human body enclosed within, with all internal organs, might be expected. We assess several aging theories within the context of our model and provide suggestions on how to activate the body's own anti-aging mechanisms and increase longevity. This paper presents some analogies and some distinctions that exist between the living dissipative structure matter and inanimate matter, discusses the aging process and proposes certain aging reversal solutions.     

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: Attempts to define mechanisms

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm²) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.     

Melatonin and photoperiodic time measurement: Seasonal breeding in the sheep

Abstract: Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status—the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.     

Serological survey of HIV and syphilis in pregnant women in Madagascar

Objectives Peripartal transmission of human immunodeficiency virus (HIV) and Treponema pallidum, the causative agent of syphilis, leads to severe consequences for newborns. Preventive measures require awareness of the maternal infection. Although HIV and syphilis testing in Madagascar could be theoretically carried out within the framework of the national pregnancy follow‐up scheme, the required test kits are rarely available at peripheral health centres. In this study, we screened blood samples of pregnant Madagascan women for HIV and syphilis seroprevalence to estimate the demand for systemic screening in pregnancy. Methods Retrospective anonymous serological analysis for HIV and syphilis was performed in plasma samples from 1232 pregnant women that were taken between May and July 2010 in Ambositra, Ifanadiana, Manakara, Mananjary, Moramanga and Tsiroanomandidy (Madagascar) during pregnancy follow‐up. Screening was based on Treponema pallidum haemagglutination tests for syphilis and rapid tests for HIV, with confirmation of positive screening results on line assays. Results Out of 1232 pregnant women, none were seropositive for HIV and 37 (3%) were seropositive for Treponema pallidum. Conclusions Our findings are in line with previous studies that describe considerable syphilis prevalence in the rural Madagascan population. The results suggest a need for screening to prevent peripartal Treponema pallidum transmission, while HIV is still rare. If they are known, Treponema pallidum infections can be easily, safely and inexpensively treated even in pregnancy to reduce the risk of transmission.     

Variabilité des concentrations plasmatiques de l’angiotensinogène et risque d’hypertension artérielle chez le rat transgénique

Aim

Genetic polymorphisms of the human angiotensinogen gene are frequent and may induce up to 30% increase of plasma angiotensinogen concentrations with a blood pressure increase of up to 5 mmHg. Their role for the pathogenesis of human arterial hypertension remains unclear. High plasma angiotensinogen levels could increase the sensitivity to other blood pressure stressors.

Methods

Male transgenic rats with a 9-fold increase of plasma angiotensinogen concentrations and male non-transgenic rats aged 10 weeks were treated or not with NG-Nitro-L-arginine-methyl ester for 3 weeks in their drinking water (n = 3/group). Systolic blood pressure and body weight were measured at baseline and at the end of the study when left ventricular weight and ventricular expression of angiotensin I-converting enzyme and procollagen Iα1 were determined (polymerase chain reaction).

Results

At baseline, transgenic rats had +18 mmHg higher bood pressure and –8% lower body weight compared to non-transgenic rats (P < 0.05) without significant changes for the vehicle groups throughout the study (P > 0.05). NG-Nitro-L-arginine-methyl ester increased blood pressure, left ventricular weight and left ventricular weight indexed for body weight by +41%, +17.6% and +18.6% (P < 0.05) in transgenic and +25%, +5.3% and +6.7% (P > 0.05) in non-transgenic rats compared to untreated animals, respectively. Cardiac gene expression showed no differences between groups (P > 0.05).

Conclusion

Increased plasma angiotensinogen levels may sensitize to additional blood pressure stressors. Our preliminary results point towards an independent role of angiotensinogen in the pathogenesis of human hypertension and associated end-organ damage.     

Morphological and immunocytochemical heterogeneity of cultured pinealocytes from one-week-and two-month-old rats: Planimetric and densitometric investigations

Abstract: In vitro preparations of rat pinealocytes are widely used for biochemical analyses of signal transduction processes. This paper deals with morphological and immunocytochemical features of such preparations. Special attention was paid to the problems of whether pinealocytes represent a heterogeneous cell population and how such heterogeneity may develop during ontogeny. The investigations were performed with cells which were obtained from the pineal organ of one-week-and two-month-old rats, attached to synthetic peptide-coated coverslips or tissue culture chamber slides, and maintained under in vitro conditions overnight. The attached cells were then fixed with paraformaldehyde. These preparations yielded monolayers of spherical cells of different sizes; most cells were isolated, but some of them were aggregated and formed small clusters. On the average, the cells from the one-week-old animals were smaller than the cells from the two-month-old animals. Immunocytochemical demonstration of S-antigen, a pinealocyte-specific marker, showed that the majority of the cells from two-month-old animals were intensely or moderately labelled. Pinealocytes from one-week-old animals were less S-antigen immunoreactive. Only very few cells (less than 1% displayed glial fibrillary acidic protein (GFAP)-immunoreactivity. Planimetric investigations of the cell size and semiquantitative densitometric investigations of the intensity of the S-antigen immunoreaction revealed that (i) pinealocytes kept in vitro form a heterogeneous cell population, and that (ii) this heterogeneity increases during postnatal development from one-week-old to two-month-old animals. Two groups of pinealocytes can be distinguished based on their developmental fate: pinealocytes of one group grow dramatically, but show only a moderate increase in S-antigen immunoreactivity, and pinealocytes of the other group retain their size, but display a distinct increment in S-antigen immunoreacti vitv.     

MUTATION FREQUENCY IN NURSES AND PHARMACISTS WORKING WITH CYTOTOXIC DRUGS

Individuals occupationally exposed to cytotoxic drugs may be at risk owing to the effects of these agents on DNA. As an index of DNA damage, in vivo mutations were measured in lymphocytes from 24 oncology nurses or pharmacists and 24 matched controls. Mutation frequency was significantly increased in exposed individuals and appeared to be related to duration of exposure. However, the overall magnitude of the increase was small and its biological significance remains to be determined.