Duckweed Lemna minor as a tool for testing toxicity and genotoxicity of surface waters

In this investigation growth parameters and certain endpoints (pigment content, peroxidase activity, lipid peroxidation and alkaline comet assay) were used to detect the toxic and genotoxic effects of surface water samples on duckweed plants. The surface waters of different origin and pollutant burdens were collected monthly over a 3-month monitoring period at three sampling sites along the river Sava and its confluents (Croatia). Physicochemical characterization of the water samples included measurements of conductivity, chemical and biological oxygen demand, levels of total suspended solids, nitrate, nitrite, ammonium, Kjeldahl nitrogen and orthophosphate. Surface water samples collected from three stations caused reduction of duckweed growth rates, chlorophylls and carotenoid contents and peroxidase activity. In contrast, damage to membrane lipids (estimated by malondialdehyde content) and especially to DNA (estimated by tail extent moment) markedly increased in duckweed exposed to industrial wastewater samples. The results from the study indicate the ability of selected biomarkers to predict the phyto- and genotoxic effects of complex water mixtures on living organisms as well as the relevance of duckweed as a sensitive indicator of water quality.     

Assessment of genotoxic potency of sulfate-rich surface waters on medicinal leech and human leukocytes using different versions of the Comet assay

The aim of the present study was to investigate how exposure to sulfate-rich surface waters affects the level of primary DNA damage in hemocytes of leech Hirudo medicinalis. Samples of surface water were collected at two sites near a gypsum factory (Knin, Croatia) and two reference sites. In the laboratory, samples were subjected to detailed chemical analysis and used in toxicity testing. For that purpose, previously acclimatized individuals of H. medicinalis were sub-chronically exposed (for 28 days) to tested water samples. Levels of primary DNA damage were evaluated using the alkaline Comet assay in hemocytes collected on days 7, 14, 21 and 28 of exposure and compared with their baseline values. Genotoxic potency of the water sample with the highest sulfate concentration was further evaluated using the alkaline, neutral and hOGG1-modified Comet assay on human peripheral blood leukocytes exposed ex vivo for 30 min. The purpose was to explore which mechanisms are responsible for DNA damage. Chemical analysis revealed that sulfate concentrations in two water samples collected in Mali Kukar Lake (1630 mg/L SO₄) and Kosov?ica River (823.3 mg/L SO₄) exceeded the WHO and US EPA defined limits for sulfate in drinking water. Increased levels of metals were found only in the water sample collected in Mali Kukar Lake. However, of the 65 elements analyzed, only nickel and titanium exceed the value legally accepted in Croatia for drinking water.The levels of DNA damage, estimated by the alkaline Comet assay in hemocytes of medicinal leech, increased with the duration of exposure to two sulfate-rich water samples. Since hemocytes responded sensitively to treatment, they could be used for biomonitoring purposes. As observed on treated human peripheral blood leukocytes, all versions of the Comet assay were effective in detecting DNA damage, which was measured in samples with sulfate concentrations equal to or higher than the legally accepted levels for drinking water. Based on the obtained results, it can be assumed that genotoxicity was a consequence both of direct (single- and double-strand DNA breaks) and indirect effects (oxidative damage) caused by the combined effects of all contaminants present in the tested water samples. Our results indicate the need for in situ monitoring and purification of gypsum mine water prior to its release in the natural environment.     

Polycyclic aromatic hydrocarbons as inducers of cytochrome P4501A enzyme activity in the rainbow trout liver cell line, RTL-W1, and in primary cultures of rainbow trout hepatocytes

In order to investigate cell-specific differences in the response of in vitro models to environmental toxicants, we compared the capacity of nine polycyclic aromatic hydrocarbons (PAHs) to induce cytochrome P4501A (CYPIA) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes and a rainbow trout liver cell line, RTL-W1. Induction of CYPIA was estimated from the catalytic activity of 7-ethoxyresorufin-O-deethylase (EROD) and compared by median effective concentration (EC50) values, induction spans, and benzo[a]pyrene induction equivalency factors for inducing PAHs. The influence of culture conditions was investigated with respect to the presence or absence of serum and varying exposure times. Both in vitro systems lead to an identical classification of the PAHs in noninducing (anthracene, fluoranthene, phenanthrene, and pyrene) and inducing compounds with a similar ranking of inducing PAHs. Mean EC50 values in RTL-W1 cells were, respectively, 343 and 266 nM for benzo[a]anthracene, 57 and 92 nM for BaP, 134 and 283 nM for benzo[b]fluoranthene, 455 and 270 nM for chrysene, and 98 and 116 nM for 3-methylcholanthrene. Compared to primary hepatocytes, the RTL-W1 cell line was more sensitive in its EROD response to the presence or absence of serum and to the increase in exposure time, which led to higher EC50 values.     

Assessment of tryptophol genotoxicity in four cell lines in vitro: a pilot study with alkaline comet assay

Tryptophol is an aromatic alcohol and secondary metabolite of the opportunistic fungus Candida albicans. Although its toxicity profile at cell level has been poorly investigated, recent data point to cytotoxic, cytostatic, and genotoxic effects in lymphocytes and the induction of apoptosis in leukaemic blood monocytes. In this pilot study we evaluated the genotoxicity of tryptophol in vitro on four permanent cell lines of animal and human origin: ovary cells, alveolar epithelium, liver cells, and blood monocytes using the alkaline comet assay. We selected cells that might be principal targets of tryptophol and other low-molecular geno(toxins) secreted by Candida albicans during host invasion. Our results suggest that tryptophol applied in vitro at 2 mmol L(-1) for 24 h damages DNA in HepG2, A549 and THP-1 cells, obviously due to bioactivation and/or decomposition of the parent compound, which results in the formation of more genotoxic compound(s) and production of reactive species that additionally damage DNA. On the other hand, notably lower levels of primary DNA damage were recorded in CHO cells, which lack metabolic activity. Future studies with tryptophol should look further into mechanisms involved in its toxic action and should focus on other cell types prone to infection with Candida spp. such as vaginal epithelial cells or keratinocytes of human origin.     

A novel contact assay for testing genotoxicity of chemicals and whole sediments in zebrafish embryos

Broad consensus exists that whole-sediment exposure protocols represent the most realistic scenario to simulate in situ exposure conditions. So far, however, several endpoints including genotoxicity in vertebrate-based systems could be tested only after transfer of particle-bound substances into the aqueous phase. The present study was carried out to develop a protocol for generating a suspension of single cells from sediment-exposed zebrafish embryos that is suitable for detecting particle-bound genotoxicity in the alkaline single cell gel electrophoresis (comet assay). In this solid-phase genotoxicity assay, a whole-body cell suspension derived from zebrafish embryos exposed to native (whole) sediments is assayed in the comet assay. Several chemical and mechanical isolation procedures were compared to optimize cell yield and minimize DNA damage by the method itself. If compared to collagenase isolation, mechanical cell dissociation gave less DNA damage; trypsinization resulted in similarly low DNA damage but significantly lower cell yield. In order to test the optimized protocol, effects of well-known genotoxicants (4-nitroquinoline-N-oxide, nitrofurantoin, hydrogen peroxide, benzo[a]pyrene) and of two sediments from the upper Rhine River (Germany) on zebrafish embryos were investigated. Results documented clear-cut genotoxicity for all four substances and for one of the two whole-sediment samples. An ultraviolet (UV) light exposure of whole embryos and primary cultures from embryos elucidated only minor effects for the whole embryos compared to the primary cells. Consequently, UV irradiation cannot be suggested as a positive control in intact zebrafish embryos. In conclusion, the newly developed sediment contact assay can be recommended for the detection of both single substances but also the bioavailable fraction of the total hazard potential of sediments.     

鱼外周血彗星试验及其在多溴联苯醚污染水体中的运用

目的通过鱼外周血彗星试验方法的摸索,为地面水污染遗传毒性效应检测提供生物学试验手段。方法红鲫鱼环磷酰胺腹腔注射染毒,设1个对照组(磷酸盐缓冲液,PBS)和5个剂量组(环磷酰胺剂量分别为:12.50、25.00、50.00、100.00、200.00 mg/kg),分别在0、2、24、72 h尾静脉采血,彗星试验检测DNA损伤效应。分别于丰水期和枯水期,在轻污染区A、中污染区B、重污染区C采样,分别获取3尾该地鲫鱼,并以3尾实验室内驯养4周的鲫鱼作为阴性对照,进行鱼肉多溴联苯醚(PBDEs)定量分析及鱼外周血彗星试验。结果在给药后2 h时间点,即能在各剂量组引起DNA损伤效应,最大的DNA损伤效应(15.42±1.30)%出现在50.00 mg/kg剂量组、24 h时间点。重污染区C的鱼外周血细胞DNA损伤程度在丰水期和枯水期皆为最高,且显著高于对照组,差异有统计学意义(P0.01)。污染区B在丰水期与对照组相比,差异有统计学意义(P0.05)。鱼肉中PBDEs含量与预判断的污染程度相关。结论鱼外周血彗星试验是较为方便、直观、敏感的DNA损伤试验,可用于对多种污染物污染的水体进行生物学评价。     

Tail moment versus tail length--application of an in vitro version of the comet assay in biomonitoring for genotoxicity in native surface waters using primary hepatocytes and gill cells from zebrafish (Danio rerio)

In order to investigate the suitability of an in vitro version of the comet assay with primary hepatocytes and gill cells from zebrafish (Danio rerio), cells were isolated by immersion in trypsin/EDTA solution after whole-body perfusion with phosphate-buffered saline. Within the scope of an 18-month biomonitoring study, primary cells were used to identify the genotoxic potential of native water samples from different sites along the major German rivers, Rhine and Elbe, and to evaluate the sensitivity and practicability of the chosen assay. Depending on the endpoint measured, considerable differences were detected with respect to the number of genotoxic surface water samples: Whereas no differences could be recorded for tail moment and relative DNA contents of head and tail, the number of positively tested native surface water samples significantly increased with tail length as endpoint.     

A sensitive zonagenetic assay for rapid in vitro assessment of estrogenic potency of xenobiotics and mycotoxins.

Mounting evidence confirms that hepatic biosynthetic processes are essential for female sexual maturation in fish, which is directly controlled by estrogens. These oogenetic events (zonagenesis and vitellogenesis) are induced in both sexes by estrogens. In this paper, we report the induction of zona radiata (zr) proteins and vitellogenin in primary hepatocytes from Atlantic salmon (Salmo salar L.) exposed to xenoestrogens and mycotoxins. Cells were treated with doses of 1, 5, and 10 microM 4-nonylphenol (4-NP), o, p'-DDT, lindane ([gamma]-HCH), and bisphenol A (BPA), which all induced zr proteins and vitellogenin in an approximate dose-dependent manner. Hepatocytes were also treated with combinations of xenoestrogens at 1 or 2 microM, resulting in elevated levels of both zr proteins and vitellogenin, compared to single treatment. The estrogenic activity of the mycotoxin zearalenone (ZEA) and its metabolites [alpha]-ZEA) and ss-zearalenol (ss-ZEA)], with regard to zonagenesis and vitellogenesis, was assessed in this assay system. Mycotoxins were used at concentrations of 10, 100, or 1,000 nM. All induced zr proteins and vitellogenin, with [alpha]-ZEA being the strongest inducer. When cells were treated with xenoestrogens or mycotoxins in combination with an estrogen receptor inhibitor (ICI 182,780), the induction of both zr proteins and vitellogenin was inhibited in all cases. Thus, the reported estrogen effects are bonafide estrogen responses. Zona radiata proteins were more responsive than vitellogenin to both xenoestrogens and mycotoxins. The versatility and sensitivity of the hepatocyte assay demonstrates that biosynthesis of zr proteins provides a new supplementary method for estimating xenoestrogenicity and mycotoxin action.     

用Ames试验和小鼠原代细胞彗星试验检测A市生活饮用水遗传毒性

目的　用Ames试验和小鼠原代肝细胞彗星试验检测A市生活饮用水遗传毒性并比较两种方法的敏感性。方法　采用Ames试验和彗星试验分别对A市南郊水厂的水源水、出厂水、自来水的有机浓集物的诱变性进行检测。结果　出厂水和自来水的Ames试验于试样浓度为 3 0L/皿时才表现为阳性结果 ,而肝细胞的彗星试验在水样量为0 1L时就出现明显的阳性结果 (与阴性对照比P <0 0 1) ,后者的浓度比前者低 3 0倍。水源水的Ames试验于试样浓度为 6 0L/皿时仍表现为阴性结果 ,而原代肝细胞的彗星试验在水样量为 0 5L时就出现明确的阳性结果。结论　Ames试验只能检测出氯化消毒后饮用水的致突变阳性 ,而彗星试验能同时检测出水源水和氯化消毒后饮用水的致突变性 ,后者的敏感性远远高于前者。     

Monitoring of endocrine disruptors in surface waters by the yeast recombinant assay

Endocrine disruptors exert physiological effects at very low concentrations. Surface waters present often a mixture of high concentrations of low-potency disruptors and low amounts of very powerful ones, making their chemical analysis complicated and expensive. We developed a recombinant yeast assay (RYA) for estrogenic compounds using 96-well microtiter plates. This assay is based on three yeast strains, transformed with self-propagating plasmids. One strain contains an expression plasmid for the human estrogen hormone receptor and an appropriate reporter; it detects estrogenic and antiestrogenic activities. The two other yeast strains, one expressing the human progesterone receptor and a second based on the yeast activator Gal4p, served to analyze the nature of antiestrogenic activities. We applied this technique to water samples from two tributaries on the Llobregat river (NE Spain) as well as from four sewage treatment plants discharging on them. Our results indicate that the efficiency of sewage treatment plants for eliminating estrogenic compounds varied notably, being in at least one case completely inefficient. We also observed a prevalence of an inhibitory activity all through the two rivers; this inhibition was hormone specific. These results were consistent to previously obtained chemical analyses of the same samples, although chemical and in vivo analyses showed rather different levels of sensitivity for some compounds. Our findings demonstrate the utility of the yeast recombinant assay for analyzing complex natural samples; at the same time, they stress the necessity of a panel of different yeast systems to adequately describe endocrine-disruptor activities.     

他克莫司在体外对HepG2细胞增殖及细胞周期的影响

目的研究他克莫司在体外对肝癌细胞HepG2的细胞增殖、细胞周期及细胞周期蛋白A(cyclin A)表达的生物学影响。方法选择肝癌细胞HepG2进行体外培养,经含有不同浓度的他克莫司(低、中和高浓度组剂量分别为50μg/L、100和500μg/L、1 000和3 000μg/L,对照组0μg/L)的培养基干预后,采用四甲基偶氮唑盐微量酶反应比色法(MTT)及流式细胞技术,分别检测细胞增殖、细胞周期及cyclin A水平。结果①中浓度(500μg/L)和高浓度(1 000和3 000μg/L)的他克莫司对HepG2细胞有增殖抑制作用,且抑制作用随浓度的增加而增加,而低浓度的他克莫司则无抑制作用。②他克莫司对HepG2细胞周期的影响:中浓度(500μg/L)和高浓度(1 000和3 000μg/L)的他克莫司作用时,HepG2细胞停止在G0/G1期,从而对肿瘤细胞的生长有抑制作用,且抑制作用随浓度的增加而增加,而低浓度的他克莫司则无抑制作用。③他克莫司可以降低HepG2细胞中cyclin A的表达,且与浓度相关,他克莫司浓度越高,cyclin A表达越少。结论他克莫司在体外对HepG2增殖有抑制作用,其中cyclin A发挥一定的作用。     

Toxicity and genotoxicity of nano-SiO(2) on human epithelial intestinal HT-29 cell line

Silica mesoporous nanoparticles have been recently selected for a wide range of applications from electronics to medicine due to their intrinsic properties. Among medical applications, drug delivery using SiO(2) nanoparticles by oral route is under study. Major benefits are expected including higher specificity and sensitivity together with side effect reduction. Since literature shows that very complex and unexpected interactions could occur between nanomaterials and biological systems, one critical issue is to control the nanoparticle cytotoxicity/genotoxicity for normal tissues and specially stomach and intestine when oral route is considered. The aim of the work is to study the cytotoxicity and genotoxicity of SiO(2) nanoparticles on HT29 human intestine cell line, using conventional and innovative methodologies, for measuring cell viability and proliferation, global metabolism, genotoxicity, and nanoparticles uptake. Core-dye doped SiO(2) nanoparticles of 25 and 100 nm were specifically synthesized to track nanoparticles incorporation by confocal and video microscopy. Besides conventional approaches (sulforhodamine B, flow cytometry, and γ-H2Ax foci), we have performed a real-time monitoring of cell proliferation using an impedance-based system which ensure no interference between measures and nanoparticles physicochemical characteristics. Overall, our results showed that SiO(2)-25nm and SiO(2)-100nm induced a rather limited cytotoxic and genotoxic effects on HT-29 cells after a 24 h exposure. However, regarding cell viability and genotoxicity, inverse dose-dependant relationships were observed for SiO(2)-100nm nanoparticles. In conclusion, it seems that the higher the dose of SiO(2)-100nm, the lower the cytotoxic/genotoxic effects, data that well illustrate the complexity in identifying and understanding the hazards of nanoparticles for human health.     

Assessing the genotoxicity of imidacloprid and RH-5849 in human peripheral blood lymphocytes in vitro with comet assay and cytogenetic tests

A combined approach employing comet assay and micronucleus (MN) and sister chromatid exchanges (SCE) tests was utilized to assess the genotoxicity of two pesticides, imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitro-imidazolidin-2-ylideneamine] and RH-5849 [2'-benzoyl-1'-tert-butylbenzoylhydrazine], on human peripheral blood lymphocytes in vitro. No significant difference in the frequencies of MN and SCE from the negative groups (P>0.05) was observed at low dose levels (i.e., 0.05 mg/L for imidacloprid and 5mg/L for RH-5849). As the concentrations of imidacloprid and RH-5849 were increased to 0.1 and 25 mg/L, respectively, significant effects to the frequencies of MN and SCE (P<0.05) were achieved relative to those of the negative controls. MN and SCE frequencies increased similarly in a dose-related manner with both pesticides. With the comet assay, however, the distribution of DNA damage grades in all the pesticide-treated groups was significantly different from those in the control (P<0.01). DNA damage scores increased with the exposure levels of both pesticides, and linear dose-effect relationships were observed for both imidacloprid (r2=0.98) and RH-5849 (r2=0.92). The cytogenetic techniques and comet assay revealed potential adverse effects of both imidacloprid and RH-5849 in human peripheral blood lymphocytes in vitro. Combination of the comet assay and cytogenetic tests appears commendable to assess the potential risks of human exposure to the pesticides.     

Monoclonal antibody enzyme-linked immunosorbent assay to quantify vitellogenin for studies on environmental estrogens in the rainbow trout (Oncorhynchus mykiss)

Vitellogenin (VTG) induction has proved to be a valuable biomarker for assessing exposure to environmental estrogens in fish. The widespread use of VTG in this regard has lead to the need for standardized assays to quantify VTG, and monoclonal antibodies have the potential to help accomplish this. A VTG enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody prepared against Atlantic salmon (Salmo salar) VTG (MAb BN-5) and its ability to quantify VTG in the rainbow trout (Oncorhynchus mykiss) compared with a rainbow trout vitellogenin (rt-VTG) ELISA that employed homologous polyclonal antibodies (PAb). In routine protocols, the working range of the homologous rt-PAb VTG ELISA was between 9 ng/ml and 70 ng/ml (80- 20% relative maximum binding [B/Bo]) with a 50% B/Bo of 25+/-0.9 ng/ml and inter- and intraassay variations at 50% B/Bo of 7% (n = 7) and 8% (n = 15), respectively. The working range of the MAb BN-5 VTG ELISA was between 60 ng/ml and 850 ng/ml (80-20% B/Bo) with a 50% B/Bo of 227+/-22 ng/ml and inter- and intraassay variations at 50% B/Bo of 5% (n = 10) and 9% (n = 12), respectively. In the routine protocols, detection limits for measurement of plasma VTG in rainbow trout (at 80% B/Bo; and given the requirement to dilute plasma to a minimum of 1:10 for the assays) were 90 ng/ml for the polyclonal rt-VTG assay and approximately 600 ng/ml in the monoclonal antibody assay. In juvenile female rainbow trout exposed to a series of doses of estradiol-17beta (E2) and 4-tert nonylphenol (4-NP), there were no differences in the vitellogenic responses measured in the PAb and MAb BN-5 VTG ELISAs. The monoclonal MAb BN-5 VTG ELISA is likely to be of considerable value for studies on environmental estrogens in juvenile female rainbow trout in standardized tests.     

Short-term test for predicting the potential of xenobiotics to impair reproductive success in fish

Short-term screening tests with the zebra fish (Brachydanio rerio) have been developed for predicting the potential of xenobiotics to impair reproductive success in fish. The aim was to find simple and sensitive test parameters and to simulate exposure situations typical for anadromous fish species (salmonids), which generally cross heavily polluted coastal areas or estuaries before they reach uncontaminated upstream spawning areas. Therefore, particular attention was directed to tests designed to assess adverse effects induced during gametogenesis in adult fish. the test protocol involves exposure of adults prior to, but not during, spawning and the effects are measured in the offspring as alterations in hatching frequency and hatching rate of eggs, and survival and stress tolerance of embryos and larvae. Some representative examples of the application of these tests are given, and it is shown that impairment of reproductive success can be induced by exposure of parent fish prior to spawning at concentrations of xenobiotics at least five times lower than those yielding effects during direct exposure of embryos and larvae. It is suggested that, in hazard assessment programs, tests of the effect of xenobiotics on the offspring of preexposed adults be routinely incorporated.     

Distribution and elimination of naphthalene and 2-methylnaphthalene in rainbow trout during short- and long-term exposures

The accumulation and elimination of 14C in rainbow trout tissues following short- and long-term exposures to aqueous 14C-naphthalene or 14C-2-methylnaphthalene were studied. After exposure for eight hr to 0.005 mg/L or 0.023 mg/L of 14C-naphthalene most tissues of fingerling rainbow trout studied contained 14C at 20 to 100 times the water levels while fat and bile contained 14C at several hundred times water levels. The half-lives for elimination of 14C from all tissues except fat were less than 24 hr. Exposure of fingerling rainbow trout to 14C-naphthalene or 14C-2-methylnaphthalene for four weeks in a continuous-flow delivery system resulted in maximum tissue levels of these chemicals of from 40 to 300 times the water concentration. Maximum bile 14C levels were 13,000 and 23,500 times the water concentration for 14C-naphthalene and 14C-2-methylnaphthalene exposure, respectively. Elimination of 14C accumulated from 14C-naphthalene in this long-term exposure was much slower than after short-term exposures, while elimination of 14C accumulated from 14C-2-methylnaphthalene was biphasic. The presence of parent compounds and metabolites in acetone extracts of muscles was determined by TLC. The data suggest that the biphasic release of 14C from muscle of trout exposed to 14C-2-methylnaphthalene may be due to differential elimination of parent compound and metabolites.