Potential new method of mixture effects testing using metabolomics and Caenorhabditis elegans

The development of superior tools for molecular and computational biology in recent years has provided an opportunity for the creation of faster toxicological screens that are relevant for, but do not rely on, mammalian systems. In this study, NMR spectroscopy and GC-MS based metabolomics have been used in conjunction with multivariate statistics to examine the metabolic changes in the nematode Caenorhabditis elegans following exposure to different concentrations of the heavy metal nickel, the pesticide chlorpyrifos, and their mixture. Novel metabolic profiles were associated with both exposure and dose level. The biochemical responses were more closely matched when exposure was at the same effect level, even for different chemicals, than when exposure was for different levels of the same chemical (e.g., low versus high dose). Responses to the mixture reflected the contribution of the chemicals to the overall exposure. In common with the metabolic responses of several other species exposed to the same chemicals, we observed changes in branch chain amino acids and tricarboxylic acid cycle intermediates. These results form the basis for a rapid and economically viable toxicity test that defines the molecular effects of pollution/toxicant exposure in a manner that is relevant to higher vertebrates.     

Age-specific threats induce CRF expression in the paraventricular nucleus of the hypothalamus and hippocampus of young rats

Young animals respond to threatening stimuli in an age-specific way. Their endocrine and behavioral responses reflect the potential threat of the situation at a given age. The aim of the present study was to determine whether corticotropin-releasing factor (CRF) is involved in the endocrine and behavioral responses to threat and their developmental changes in young rats. Preweaning 14-day-old and postweaning 26-day-old rats were exposed to two age-specific threats, cat odor and an adult male rat. The acute behavioral response was determined during exposure. After exposure, the time courses of the corticosterone response and of CRF expression in the paraventricular nucleus of the hypothalamus (PVN) and in extrahypothalamic areas were assessed. Preweaning rats became immobile when exposed to cat odor or the male rat, whereas postweaning rats became immobile to cat odor only. Male exposure increased serum corticosterone levels in 14-day-old rats, but cat odor failed to increase levels at either age. Exposure induced elevation of CRF mRNA levels in the PVN that paralleled changes in corticosterone levels. CRF may thus play a role in endocrine regulation and its developmental changes during early life. Neither cat odor nor the adult male altered CRF mRNA levels in the bed nucleus of the stria terminalis (BNST) or the amygdala, but both stimuli increased levels in the hippocampus. Hippocampal CRF mRNA expression levels did not parallel cat odor or male-induced immobility, indicating that CRF is not involved in this response in young rats but may be involved in aspects of learning and memory.     

Embryonic exposure to cypermethrin induces apoptosis and immunotoxicity in zebrafish (Danio rerio)

Cypermethrin (CYP) is widely used for control of indoor and field pests. As a result, CYP is one of the most common contaminants in freshwater aquatic systems. In the present study, we investigated the effects of CYP exposure on the induction of apoptosis and immunotoxicity in zebrafish during the embryo developmental stage. The mRNA levels of some key genes including P53, Puma, Bax, Apaf1, Cas9 and Cas3 on the mitochondrial pathway of cell apoptosis were significantly up-regulated at the concentration of 3 and 10 μg/l CYP. Correspondingly, the activities of Cas3 and Cas9 increased significantly after exposure to 3 or 10 μg/l CYP. In addition, the mRNA levels of iNOS and the total content of NO were also up-regulated significantly after CYP exposure. Moreover, it was also observed that the mRNA levels of IFN, CXCL-Clc, CC-chem and C3, which are closely related to the innate immune system, were affected in newly hatched zebrafish when exposed to 3 and 10 μg/l CYP, exhibiting CYP's prominent impacts on the innate immune system of zebrafish. Taken together, our results suggest that CYP has the potential to induce cell apoptosis and cause innate immune system disruption in zebrafish during the embryo stage. The information presented in this study will help elucidate the mechanism of CYP-induced toxicity in fish.     

Changes in Nuclear Receptor and Vitellogenin Gene Expression in Response to Steroids and Heavy Metal in Caenorhabditis elegans

To gain basic understanding of the reproductive and developmental effects of endocrine disrupting chemicals in invertebrates, we have used C. elegans as an animal model. The completion of the C. elegans genome sequence brings to bear microarray analysis as a tool for these studies. We previously showed that the C. elegans genome was responsive to vertebrate steroid hormones, and changes in gene expression of traditional biomarkers used in environmental studies were detected; i.e., vitellogenin (vtg), cytochrome P450 (cyp450), glutathione-S-transferase (gst) and heat shock proteins (hsp). The data were interpreted to suggest that exogenous lipophilic compounds can be metabolized via cytochrome P450 proteins, and that the resulting metabolites can bind to members of the Nuclear Receptor (NR) class of proteins and regulate gene expression. In the present study, using DNA microarrays, we examined the pattern of gene expression after progesterone (10(-5), 10(-7) M), estradiol (10(-5) M), cholesterol (10(-9) M) and cadmium (0.1, 1 and 10 μM) exposure, with special attention to the members of NRs. Of approximately 284 NRs in C. elegans, expression of 25 NR genes (representing 9% of the total NRs in C. elegans) was altered after exposure to steroids. Of note, each steroid activated or inhibited different subsets of NR genes, and only estradiol regulated NR genes implicated in neurogenesis. These results suggest that NRs respond to a variety of exogenous steroids, which regulate important metabolic and developmental pathways. The response of the C. elegans genome to cholesterol and cadmium was analyzed in more detail. Cholesterol is a probable precursor to signaling molecules that may interact with NRs and we focused on expression of genes related to lipid metabolism (cyp450), transport and storage (i.e., vitellogenin). Worms exposed to cadmium respond principally by activating the expression of genes encoding stress-responsive proteins, such as mtl-2 and cdr-1, and no significant changes in expression of NRs or vtg genes were observed. The possible implications of these results with regard to the evolution of steroid receptors, endocrine disruption and the role of vitellogenin as a lipid transporter are discussed.     

Water deficit and aluminum tolerance are associated with a high antioxidative enzyme capacity in Indica rice seedlings

Plant growth and productivity are greatly affected due to changes in the environmental conditions. In the present investigation, the interactive effects of two important abiotic stresses, i.e., water deficit and Al toxicity, were examined in the seedlings of two rice (Oryza sativa L.) cvs. Malviya-36 (water deficit/Al sensitive) and Vandana (water deficit/Al tolerant). When 15 days grown seedlings were exposed to water deficit (created with 15 % polyethylene glycol 6000) or Al (1 mM AlCl₃) treatment or both the treatments together for 48 h, the lengths of root/shoot, relative water content, and chlorophyll greatly declined in the seedlings of the sensitive cultivar, whereas in the tolerant seedlings, either little or insignificant decline in these parameters was observed due to the treatments. Seedlings subjected to water deficit or Al treatment alone or in combination showed increased intensity of the isoenzyme activity bands of superoxide dismutase (SOD), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX) in in-gel activity staining studies. Water deficit caused decrease in intensity of catalase (CAT) activity bands; however, when seedlings were exposed to AlCl₃ alone or in combination with water deficit, the intensity of the CAT isoforms increased in both the rice cultivars. The level of expression of the activity bands of SOD, CAT, GPX, and APX was always higher in the seedlings of tolerant cv. Vandana compared to the sensitive cv. Malviya-36 under both controls as well as stress treatments. Higher intensity of isozymes representing higher activity levels of antioxidative enzymes in the rice seedlings and their further increase under water deficit, Al exposure, or in combination of both the stresses appears to serve as useful marker for specifying a combination of water deficit and Al tolerance in rice.     

Evolution of male tail development in rhabditid nematodes related to Caenorhabditis elegans

The evolutionary pathway that has led to male tails of diverse morphology among species of the nematode family Rhabditidae was reconstructed. This family includes the well-studied model species Caenorhabditis elegans. By relating the steps of male tail morphological evolution to the phenotypic changes brought about by developmental mutations induced experimentally in C. elegans, the goal is to identify genes responsible for morphological evolution. The varying morphological characters of the male tails of several rhabiditid species have been described previously (Fitch and Emmons, 1995, Dev. Biol. 170:564-582). The developmental events preceding differentiation of the adult structures have also been analyzed; in many cases the origins of varying adult morphological characters were traced to differences during ontogeny. In the present work, the evolutionary changes producing these differences were reconstructed in the context of the four possible phylogenies supported independently by sequences of 18S ribosomal RNA genes (rDNA). Two or more alternative states were defined for 36 developmental and adult morphological characters. These characters alone do not provide sufficient data to resolve most species relationships; however, when combined with the rDNA characters, they provide stronger support for one of the four rDNA phylogenies. Assuming a model of ordered transformations for multistate developmental characters generally results in greater resolution. Transformations between character states can be assigned unequivocally by parsimony to unambiguous branches for most of the characters. Correlations are thereby revealed for some of the developmental characters, indicating a probability of a shared developmental or genetic regulatory pathway. Four of the unequivocal character state changes on unambiguously supported branches closely resemble the phenotypic changes brought about by known mutations in C. elegans. These mutations define genes that are known to act in genetic regulatory hierarchies controlling pattern formation, differentiation, and morphogenesis. Although these studies are still at an early stage, these results strongly suggest that parallel studies of developmental mutants in C. elegans and of morphological and developmental evolution among related nematodes will help define genetic changes underlying the evolution of form.     

Aluminum resistance in two cultivars of Zea mays L.: Root exudation of organic acids and influence of phosphorus nutrition

Root exudation of organic acids as Al-chelating compounds and P nutrition have been suggested to play a major role in Al-resistance in higher plants. Effects of Al exposure on maize plant growth, and organic acid root content and root exudation under various levels of P nutrition were examined. Sikuani, a Colombian maize cultivar tolerant to acid soils with high Al saturation, and Corso, a Swiss cultivar, were grown in sterile hydroponic conditions for 21 days. Al-caused inhibition of root growth was lower in Sikuani than in Corso. Al effect on plant growth was decreased with increasing P content in roots. Al content in roots increased with increasing P content and was higher in Sikuani than in Corso. When exposed to Al, the contents in root apices as well as the root exudation of citric and malic acids in Corso and citric, malic and succinic acids in Sikuani increased, and were higher in Sikuani than in Corso. Increased PEP carboxylase (PEPC) activity in root apices after Al exposure partially explained the variations of organic acid content in the roots. These Al-induced changes in PEPC activity, organic acid content and exudation were reduced in plants supplied with higher P concentrations during the 21 days prior to treatment. Increased secretion of organic acids after exposure to Al appeared to be specific to Al and was not totally explained by increased root content in organic acids.     

Hydroprene prolongs developmental time and increases mortality in wandering-phase Indianmeal moth (Lepidoptera: Pyralidae) larvae

Wandering phase Indianmeal moth, Plodia interpunctella (Hübner), larvae were exposed to the label rate of hydroprene (1.9 x 10(-3) mg [AI] /cm2) sprayed on concreted petri dishes. Larvae were exposed for 1, 3, 6, 12, 18, 24, and 30 h and maintained at 16, 20, 24, 28, and 32 degrees C and 57% RH until adult emergence. Larval developmental time and mortality were significantly influenced by temperature and exposure intervals. Maximum developmental time (47.2 +/- 1.3 d) occurred at 16 degrees C, and the minimum developmental time (7.0 +/- 0.5 d) occurred at 32 degrees C. Larval mortality generally increased at all of the five tested temperatures as exposure period increased. The greatest mortality (82.0 +/- 0.1%) occurred when larvae were exposed for 30 h at 28 degrees C, and minimum mortality (0.0 +/- 0.5%) occurred at 16 degrees C when larvae were exposed for 1 h. The relationships between temperature, exposure period, and developmental time were described by polynomial models, based on lack-of-fit tests. Hydroprene has potential to be an effective alternative to conventional insecticides in surface treatments for Indianmeal moth management. Response-surface models derived from this study can be used in simulation models to estimate the potential consequences of hydroprene on Indianmeal moth population dynamics.     

Combined effect of HEDTA and selenium against aluminum induced oxidative stress in rat brain

Aluminium (Al) is a potent neurotoxin and has together with other metals been suggested to be associated with Alzheimer's disease causality. The current study was carried out to investigate the potential role of N(2-hydroxyethyl) ethylenediamine triacetic acid (HEDTA) and Se in combination against Al induced toxicity. Animals were exposed to Al at a dose of 27 mg/kg/d i.p. for 60 days. HEDTA and Se were administered at a dose of 20mg/kg/d i.p. and 0.5mg/kg/d orally, respectively for 7 consecutive days. Induction of oxidative stress was recorded in the brain after Al exposure. Significant decrease was found in the levels of reduced glutathione activities of the enzymes glutathione reductase, glutathione peroxidase, catalase, superoxide dismutase, acetyl cholinesterase, and increased levels were observed in LPO and glutathione-S-transferase activity in brain and serum. These parameters responded positively to therapy with HEDTA, but more pronounced beneficial effects were observed when HEDTA was administered in combination with Se. The combination was effective in reducing the concentration of Al and level of DNA damage.     

谷氧还蛋白rbGrx可延缓CL4176秀丽隐杆线虫中β淀粉样蛋白诱导的毒性

阿尔茨海默病（Alzheimer’s disease,AD）是一种与衰老相关的神经退行性疾病,其中β-淀粉样蛋白（β-amyloid,Aβ）诱导的细胞毒性被认为是其发病的主要原因。本文以Aβ转基因秀丽隐杆线虫CL4176为模型,研究了重组荞麦谷氧还蛋白（recombinant buckwheat glutaredoxin, rbGrx）对Aβ诱导的毒性和氧化应激的影响。结果显示,4 μmol/L rbGrx可以延长CL4176线虫平均寿命达20%左右,并增加衰老虫体运动能力约43.6%,延迟产卵高峰期1 d,同时可以有效延缓Aβ毒性诱导的瘫痪表型。进一步研究发现,在正常条件和Aβ诱导毒性时,rbGrx均能降低CL4176线虫体内活性氧（reactive oxygen species,ROS）水平,并上调SOD活性和GSH含量。另外,rbGrx下调Aβ mRNA水平44.1％,减少Aβ沉积量,并且明显上调热激因子1 hsf-1（2.01倍）和hsp-16.2（2.65倍）mRNA表达水平。这表明,rbGrx通过降低CL4176线虫体内的ROS水平和上调热激蛋白质的转录表达水平,降低CL4176秀丽隐杆线虫中Aβ诱导的毒性。结果提示,rbGrx可能具有预防AD的潜力。     

Tracheal insufflation of tumor necrosis factor protects rats against oxygen toxicity

Tracheal insufflation of tumor necrosis factor (TNF; 5 micrograms or 1.2 x 10(5) U) markedly enhanced the survival of adult rats exposed to 100% O2: 12 of 17 rats (71%) survived for greater than 11 days, whereas 30 of 30 control (Hanks' balanced salt solution) insufflated rats (100%) died within 3 days of O2 exposure. Insufflation of gamma-interferon (5 micrograms) or intraperitoneal injection of up to 40 micrograms TNF did not afford any protection. At 55 h after O2 exposure, TNF-insufflated rats showed less pulmonary edema, as determined by the extravascular lung water content-to-bloodless lung dry weigh ratio and less alveolar capillary leak as determined by the protein content in the bronchoalveolar lavage fluid, than control insufflated rats similarly exposed. This protection against O2 toxicity by TNF insufflation was associated with increased lung superoxide dismutase, catalase, and glutathione peroxidase activities. The enhancement of lung antioxidant enzyme activities was noted at 55 h of O2 exposure, when control animals began to die of O2 toxicity. This temporal relationship suggests that TNF-induced increase in antioxidant enzyme activities contributes, at least in part, to the observed protection.     

Assessing developmental hazard: the reliability of the A/D ratio.

The quantitative relationship between developmentally toxic exposure levels and adult toxic exposure levels has been used as an index of developmental hazard and has figured prominently in discussions of legal regulation of developmentally toxic agents. Perhaps the most frequently cited index of developmental hazard is the A/D ratio. This index, a ratio of marginally toxic adult and developmental dose levels (e.g., NOAELs or LOAELs), is attractive because it is easily calculated from published toxicity assays and because it has been argued that A/D is relatively constant across species for a given agent. We explored some quantitative aspects of the A/D ratio and of the concept of developmental hazard by simulating 661,500 mammalian developmental toxicity assays on 441 hypothetical compounds. In our simulations, A/D often varied substantially among replicate assays: the median ratio of the upper and lower limits of the distribution of A/D values that include about 95% of the observed A/D values is 16. In addition, A/D did a poor job of predicting the relative developmental and adult responses at dosages lower than those used to calculate the index: among simulated compounds with A/Ds of about 1.0, the developmental response at 1/100th of the NOAEL ranged from about 0.1% to 20,000% of the adult response. Finally, we measured the concordance between pairs of four different indices of developmental hazard, including A/D. Concordance was greatest when the indices were based on the same portion of the dose response, and was much weaker between indices that examined different portions of the dose response. Therefore, it seems likely that no single index can quantify "developmental hazard," as defined by relative adult and developmental susceptibility, and more effort needs to be expended in refining the concept if it is to be useful for hazard assessment.     

Rye oxidative stress under long term Al exposure

Aluminium (Al) toxicity decreases plant growth. Secale cereale L. is among the most Al-tolerant crop species. In order to study the response to Al-long term exposure, two rye genotypes with different Al sensitivity (‘D. Zlote’ and ‘Riodeva’) were exposed to 1.11 and 1.85 mM Al and the antioxidant responses were followed for 2 and 3 weeks in roots and leaves. Al toxicity signals, such as a severe decrease in root growth, occurred sooner in ‘Riodeva.’ The antioxidant response was dependent on the genotype, the organ, Al concentration and the exposure period. Al-exposed roots of ‘D. Zlote’ showed earlier enhancements of APX, SOD and G-POX activities than those of ‘Riodeva.’ ‘D. Zlote’ roots showed stimulation of the AsA–GSH cycle after the second week (when root growth inhibition was less severe), while later (when severe root growth inhibition was observed), oxidation of AsA and GSH pools was observed. In leaves of both genotypes, CAT, SOD and G-POX activities increased with Al exposure. In these leaves, the effect of AsA–GSH was time dependent, with maximum oxidation at the second week, followed by recovery. We confirmed that the oxidation state of AsA and GSH pools is involved in the detoxification of Al-induced oxidative stress. Moreover, our data demonstrate that the production of ROS does not correlate with the Al-induced root growth decrease. Finally, the differences observed over time indicate that long term exposure may provide additional information on rye sensitivity to Al, and contribute to a better understanding of this species’ mechanisms of Al tolerance.     

利用模式生物线虫评价精对苯二甲酸废水的毒性

应用模式生物秀丽隐杆线虫,通过生命周期、半数致死天数、生殖速度、产卵数、头部摆动频率和身体弯曲次数等指标对精对苯二甲酸(PTA)废水毒性进行了研究.结果表明,与对照组相比,660 mg·L^-1 PTA废水暴露下的线虫生命周期有一定的延长,产卵时间延迟,头部摆动频率降低,身体弯曲次数减少(P<0.05),且PTA废水对线虫生殖能力的影响极显著(P<0.01),暴露于废水中的线虫产卵数大约只有正常产卵数的1/4.最敏感效应指标——产卵数,有望成为该类废水毒性预警预报的潜在生物标志物.     

Modulation by ellagic acid of DCA-induced developmental toxicity in the zebrafish (Danio rerio)

The ability of ellagic acid (EA) to modulate dichloroacetic acid (DCA)-induced developmental toxicity and oxidative damage was examined in zebrafish embryos. Embryos were exposed to 20 mM EA administered concomitantly with 32 mM DCA at 4 hours postfertilization (hpf) and 20 h later. Embryos were observed through 144 hpf for developmental malformations, and production of superoxide anion (SA) and nitric oxide (NO) was determined in embryonic homogenates. DCA was shown to produce developmental abnormalities and significant levels of SA and NO in zebrafish embryos. EA exposure alleviated the developmental malformations observed in treated embryos and decreased the levels of SA and NO in those same embryos. Less than 10% of DCA + EA exposed embryos showed developmental malformations compared to 100% of embryos treated with DCA alone. Animals in this group that developed malformations were shown to have fewer defects than those treated with DCA only. Taken together, the results confirm the involvement of oxidative stress in the developmental toxicity of DCA in zebrafish embryos, and suggest possible protection against those effects with the use of antioxidants.     

重复缺氧与过氧化氢对脑与突触体乳酸生成的影响

目的：观察重复缺氧对小鼠在体脑乳酸含量以及Ｈ２Ｏ２对荷兰猪离体突触体乳酸含量的影响,并分析其作用机制。方法：采用酶氧化方法对突触体培养液中乳酸含量进行测定。结果：单次急性缺氧鼠脑内乳酸含量高,但在重复缺氧的作用下,鼠脑内乳酸的含量并未随缺氧时间的延长而增加。在突触体培养液中分别加入Ｈ２Ｏ２、ＦＣＣＰ、Ｒｏｔｅｎｏｎ,乳酸浓度比正常对照组显著性的增高。对突触体进行长时间Ｈ２Ｏ２处理,乳酸含量未见显著升高或降低。同时在突触体培养液中加入Ｈ２Ｏ２和ＦＣＣＰ,乳酸浓度相当于单独在突触体培养液分别加入Ｈ２Ｏ２与ＦＣＣＰ产生乳酸之和。结论：重复缺氧与Ｈ２Ｏ２处理可能激发脑与突触体内的抗自由基损伤防御系统;突触体从有氧呼吸到无氧酵解的转换主要不是由于Ｈ２Ｏ２对突触体膜的损伤。     

The African sharptooth catfish Clarias gariepinus can tolerate high levels of ammonia in its tissues and organs during four days of aerial exposure

The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air breather, and can survive on land during drought. The objective of this study was to elucidate how C. gariepinus defends against ammonia toxicity when exposed to terrestrial conditions. During 4 d of aerial exposure, there was no accumulation of urea in its tissues, and the rate of urea excretion remained low. Thus, exposure to terrestrial conditions for 4 d did not induce ureogenesis or ureotely in C. gariepinus. Volatilization of NH(3) was not involved in excreting ammonia during aerial exposure. In addition, there were no changes in levels of alanine in the muscle, liver, and plasma of C. gariepinus; nor were there any changes in the glutamine levels in these tissues. However, there were extraordinarily high levels of ammonia in the muscle (14 micromol g(-1)), liver (18 micromol g(-1)), and brain (11 micromol g(-1)) of fish exposed to terrestrial conditions for 4 d. This is the first report on a fish adopting high tolerance of ammonia in cells and tissues as the single major strategy to defend against ammonia toxicity during aerial exposure. At present, it is uncertain how C. gariepinus tolerates such high levels of ammonia, especially in its brain, but it can be concluded that, contrary to previous reports on two air-breathing catfishes (Clarias batrachus and Heteropneustes fossilis) from India, C. gariepinus does not detoxify ammonia to urea or free amino acids on land.     

Metallothioneins are required for formation of cross-adaptation response to neurobehavioral toxicity from lead and mercury exposure in nematodes

Metallothioneins (MTs) are small, cysteine-rich polypeptides, but the role of MTs in inducing the formation of adaptive response is still largely unknown. We investigated the roles of metallothionein genes (mtl-1 and mtl-2) in the formation of cross-adaptation response to neurobehavioral toxicity from metal exposure in Caenorhabditis elegans. Pre-treatment with mild heat-shock at L2-larva stage effectively prevented the formation of the neurobehavioral defects and the activation of severe stress response in metal exposed nematodes at concentrations of 50 and 100 μM, but pre-treatment with mild heat-shock did not prevent the formation of neurobehavioral defects in 200 μM of metal exposed nematodes. During the formation of cross-adaptation response, the induction of mtl-1 and mtl-2 promoter activity and subsequent GFP gene expression were sharply increased in 50 μM or 100 μM of metal exposed Pmtl-1::GFP and Pmtl-2::GFP transgenic adult animals after mild heat-shock treatment compared with those treated with mild heat-shock or metal exposure alone. Moreover, after pre-treatment with mild heat-shock, no noticeable increase of locomotion behaviors could be observed in metal exposed mtl-1 or mtl-2 mutant nematodes compared to those without mild heat-shock pre-treatment. The defects of adaptive response to neurobehavioral toxicity induced by metal exposure formed in mtl-1 and mtl-2 mutants could be completely rescued by the expression of mtl-1 and mtl-2 with the aid of their native promoters. Furthermore, over-expression of MTL-1 and MTL-2 at the L2-larval stage significantly suppressed the toxicity on locomotion behaviors from metal exposure at all examined concentrations. Therefore, the normal formation of cross-adaptation response to neurobehavioral toxicity induced by metal exposure may need the enough accumulation of MTs protein in animal tissues.