Coupling Genome-wide Transcriptomics and Developmental Toxicity Profiles in Zebrafish to Characterize Polycyclic Aromatic Hydrocarbon (PAH) Hazard.

Polycyclic Aromatic Hydrocarbons (PAHs) are diverse environmental pollutants associated with adverse human health effects. Many studies focus on the carcinogenic effects of a limited number of PAHs and there is an increasing need to understand mechanisms of developmental toxicity of more varied yet environmentally relevant PAHs. A previous study characterized the developmental toxicity of 123 PAHs in zebrafish. Based on phenotypic responses ranging from complete inactivity to acute mortality, we classified these PAHs into eight bins, selected 16 representative PAHs, and exposed developing zebrafish to the concentration of each PAH that induced 80% phenotypic effect. We conducted RNA sequencing at 48 h post fertilization to identify gene expression changes as a result of PAH exposure. Using the Context Likelihood of Relatedness algorithm, we inferred a network that links the PAHs based on coordinated gene responses to PAH exposure. The 16 PAHs formed two broad clusters: Cluster A was transcriptionally more similar to the controls, while Cluster B consisted of PAHs that were generally more developmentally toxic, significantly elevated cyp1a transcript levels, and induced Ahr2-dependent Cyp1a protein expression in the skin confirmed by gene-silencing studies. We found that cyp1a transcript levels were associated with transcriptomic response, but not with PAH developmental toxicity. While all cluster B PAHs predominantly activated Ahr2, they also each enriched unique pathways like ion transport signaling, which likely points to differing molecular events between the PAHs downstream of Ahr2. Thus, using a systems biology approach, we have begun to evaluate, classify, and define mechanisms of PAH toxicity.

Systematic developmental neurotoxicity assessment of a representative PAH Superfund mixture using zebrafish.

Superfund sites often consist of complex mixtures of polycyclic aromatic hydrocarbons (PAHs). It is widely recognized that PAHs pose risks to human and environmental health, but the risks posed by exposure to PAH mixtures are unclear. We constructed an environmentally relevant PAH mixture with the top 10 most prevalent PAHs (SM10) from a Superfund site derived from environmental passive sampling data. Using the zebrafish model, we measured body burden at 48 hours post fertilization (hpf) and evaluated the developmental and neurotoxicity of SM10 and the 10 individual constituents at 24 hours post fertilization (hpf) and 5 days post fertilization (dpf). Zebrafish embryos were exposed from 6 to 120 hpf to (1) the SM10 mixture, (2) a variety of individual PAHs: pyrene, fluoranthene, retene, benzo[a]anthracene, chrysene, naphthalene, acenaphthene, phenanthrene, fluorene, and 2-methylnaphthalene. We demonstrated that SM10 and only 3 of the individual PAHs were developmentally toxic. Subsequently, we constructed and exposed developing zebrafish to two sub-mixtures: SM3 (comprised of 3 of the developmentally toxicity PAHs) and SM7 (7 non-developmentally toxic PAHs). We found that the SM3 toxicity profile was similar to SM10, and SM7 unexpectedly elicited developmental toxicity unlike that seen with its individual components. The results demonstrated that the overall developmental toxicity in the mixtures could be explained using the general concentration addition model. To determine if exposures activated the AHR pathway, spatial expression of CYP1A was evaluated in the 10 individual PAHs and the 3 mixtures at 5 dpf. Results showed activation of AHR in the liver and vasculature for the mixtures and some individual PAHs. Embryos exposed to SM10 during development and raised in chemical-free water into adulthood exhibited decreased learning and responses to startle stimulus indicating that developmental SM10 exposures affect neurobehavior. Collectively, these results exemplify the utility of zebrafish to investigate the developmental and neurotoxicity of complex mixtures.

Comparative developmental toxicity of a comprehensive suite of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that occur in complex mixtures. Several PAHs are known or suspected mutagens and/or carcinogens, but developmental toxicity data is lacking for PAHs, particularly their oxygenated and nitrated derivatives. Such data are necessary to understand and predict the toxicity of environmental mixtures. 123 PAHs were assessed for morphological and neurobehavioral effects for a range of concentrations between 0.1 and 50 µM, using a high throughput early-life stage zebrafish assay, including 33 parent, 22 nitrated, 17 oxygenated, 19 hydroxylated, 14 methylated, 16 heterocyclic, and 2 aminated PAHs. Additionally, each PAH was evaluated for AHR activation, by assessing CYP1A protein expression using whole animal immunohistochemistry (IHC). Responses to PAHs varied in a structurally dependent manner. High-molecular weight PAHs were significantly more developmentally toxic than the low-molecular weight PAHs, and CYP1A expression was detected in five distinct tissues, including vasculature, liver, skin, neuromasts and yolk.

Aerobic Bioremediation of PAH Contaminated Soil Results in Increased Genotoxicity and Developmental Toxicity.

The formation of more polar and toxic polycyclic aromatic hydrocarbon (PAH) transformation products is one of the concerns associated with the bioremediation of PAH-contaminated soils. Soil contaminated with coal tar (prebioremediation) from a former manufactured gas plant (MGP) site was treated in a laboratory scale bioreactor (postbioremediation) and extracted using pressurized liquid extraction. The soil extracts were fractionated, based on polarity, and analyzed for 88 PAHs (unsubstituted, oxygenated, nitrated, and heterocyclic PAHs). The PAH concentrations in the soil tested, postbioremediation, were lower than their regulatory maximum allowable concentrations (MACs), with the exception of the higher molecular weight PAHs (BaA, BkF, BbF, BaP, and IcdP), most of which did not undergo significant biodegradation. The soil extract fractions were tested for genotoxicity using the DT40 chicken lymphocyte bioassay and developmental toxicity using the embryonic zebrafish (Danio rerio) bioassay. A statistically significant increase in genotoxicity was measured in the unfractionated soil extract, as well as in four polar soil extract fractions, postbioremediation (p < 0.05). In addition, a statistically significant increase in developmental toxicity was measured in one polar soil extract fraction, postbioremediation (p < 0.05). A series of morphological abnormalities, including peculiar caudal fin malformations and hyperpigmentation in the tail, were measured in several soil extract fractions in embryonic zebrafish, both pre- and postbioremediation. The increased toxicity measured postbioremediation is not likely due to the 88 PAHs measured in this study (including quinones), because most were not present in the toxic polar fractions and/or because their concentrations did not increase postbioremediation. However, the increased toxicity measured postbioremediation is likely due to hydroxylated and carboxylated transformation products of the 3- and 4-ring PAHs (PHE, 1MPHE, 2MPHE, PRY, BaA, and FLA) that were most degraded.

Site-selective synthesis of in situ Ni-filled multi-walled carbon nanotubes using Ni(salen) as a catalyst source.

The synthesis of Ni-filled multi-walled carbon nanotubes was performed by atmospheric pressure chemical vapor deposition with propane on Si at 850 °C using a simple mixture of (N, N'-bis(salicylidene)-ethylenediiminato) nickel(II), commonly known as Ni(salen), and a conventional photoresist. Analysis of the carbon nanotubes using scanning electron microscopy together with high-resolution transmission electron microscopy show that the nanotubes have grown by a tip-growth mechanism and exhibit a multi-walled structure with partial Ni filling. The high quality of the Ni-filled nanotubes is evidenced by Raman spectroscopy. The magnetic properties of Ni-filled nanotubes were analyzed using a superconducting quantum interference device which revealed their ferromagnetic behavior with large coercivity. A scalable as well as site-selective growth of high quality Ni-filled carbon nanotubes is achieved by a simple photolithographic method.

Delayed Cerebral Infarction Following Trans-Sylvian Surgery for Craniopharyngioma Presenting as Status Epilepticus.

The postoperative period after craniopharyngioma surgery has a high likelihood of complications and its management can be challenging. We present the case of a 10-year-old boy who was operated for craniopharyngioma. In the postoperative period he developed lacunar infarct, endocrine disturbances, delayed vasospasm leading to cerebral infarction and status epilepticus. The likely cause of status epilepticus was likely delayed cerebral infarction compounded with dyselectrolytemia. We discuss the possible etiology of delayed cerebral infarction.

Aryl Hydrocarbon Receptor Mediates Larval Zebrafish Fin Duplication Following Exposure to Benzofluoranthenes.

The aryl hydrocarbon receptor (AHR) mediates developmental toxicity of several xenobiotic classes including polycyclic aromatic hydrocarbons. Using embryonic zebrafish, we previously identified 4 polycyclic aromatic hydrocarbons that caused a novel phenotype among AHR ligands-growth of a lateral, duplicate caudal fin fold. The window of sensitivity to the most potent inducer of this phenotype, benzo[k]fluoranthene (BkF), was prior to 36 h postfertilization (hpf), although the phenotype was not manifest until 60 hpf. AHR dependency via Ahr2 was demonstrated using morpholino knockdown. Hepatocyte ablation demonstrated that hepatic metabolism of BkF was not required for the phenotype, nor was it responsible for the window of sensitivity. RNA sequencing performed on caudal trunk tissue from BkF-exposed animals collected at 48, 60, 72, and 96 hpf showed upregulation of genes associated with AHR activation, appendage development, and tissue patterning. Genes encoding fibroblast growth factor and bone morphogenic protein ligands, along with retinaldehyde dehydrogenase, were prominently upregulated. Gene Ontology term analysis revealed that upregulated genes were enriched for mesoderm development and fin regeneration, whereas downregulated genes were enriched for Wnt signaling and neuronal development. MetaCore (Clarivate Analytics) systems analysis of orthologous human genes predicted that R-SMADs, AP-1, and LEF1 regulated the expression of an enriched number of gene targets across all time points. Our results demonstrate a novel aspect of AHR activity with implications for developmental processes conserved across vertebrate species.

Molecular orbital studies on nucleoside analogs. II. Conformation of 6-azapyrimidine nucleosides.

PCILO (Perturbative Configuration Interaction using Localised Orbitals) computations have been carried out for three 6-azapyrimidine nucleosides, 6-azauridine, 6-azacytidine and 6-azathymidine, for both C(2')-endo and C(3')-endo pucker of the sugar ring. The results indicate a syn (chiCN=180 degrees) conformation followed by chiCN=90 degrees and gg conformation for C(3')-endo 6-aza analogs as compareed to the anti (chiCN=0 degrees) and gg conformation preferred by the corresponding pyrimidine nucleosides. For C(2')-endo sugar geometry, 6-azauridine and 6-azacytidine prefer, respectively, chiCN=0 degrees (anti) and phi C(4')-C(5')=60 degrees C (gg) and chiCN-240 degrees (syn) and phi C(4')-C(5')=120 degrees. The corresponding nucleosides, uridine and cytidine, show a preference for syn (chiCN=240 degrees) and gg and anti(chiCN=0 degrees) and gg , respectively. The X-ray crystallographic conformations of 6-azauridine and 6-azacytidine have been attributed to intermolecular hydrogen bonding and crystal packing forces. The results of PMR, CD and ORD studies on 6-azauridine and 6-azacytidine in aqueous solutions are in agreement with the PCILO predictions.

Molecular orbital studies on the structure of nucleoside analogs. I. Conformation of 8-azapurine nucleosides.

PCILO (perturbative configuration interaction using localized orbitals) computations have been carried out for the conformational properties of 8-azapurine nucleosides. The results indicate an anti conformation for Xcn and a gg conformation for phiC(4')-C(5') for C(2')-endo 8-aza analogs compared to the syn and gg conformation for the corresponding purine nucleosides. For C(3')-endo sugar puckering, both molecules prefer the syn conformation due to intramolecular hydrogen bonding between O(5')-H of the sugar and N(3) of the base, the preference being more profound in 8-aza analogs. The crystallographic conformation 8-azaadenosine has been attributed to crystal forces. The available NMR data on 8-azapurine nucleosides are in agreement with the PCILO predictions.

Fractal studies on the protein secondary structure elements.

The fractal dimensions of the protein secondary structure elements based on the positional distributions were calculated. The seven secondary structure elements could be grouped into two distinct classes based on their fractal dimensions. A negative correlation was observed between the dimensional values of the secondary structures and their abundances. Markov model was applied, to check the persistence of transitions of the secondary structure elements, in the protein sequences. The results suggest the presence of long-range correlations in the protein sequences that may be useful in prediction algorithms.

Pair-preferences: a quantitative measure of regularities in protein sequences.

We present here the results obtained by applying several different methods to quantitatively measure regularities in protein sequences based on pair-preferences. We have studied the distribution of amino acid residues, singly as well as in pairs in a large data base and have attempted this task. We confirmed the existence of well-defined pair-preferences in proteins which were shown to be remarkably absent in simulated random sequences of similar amino acid distribution. The analysis of the sequences from the SWISS-PROT data base using simple statistical tests. Fourier analysis, fractal analysis and statistical thermodynamical tests were used to derive parameters to define a natural sequence. As a consequence of the existence of pair-preferences, parameters like fractal dimension (D), spectral exponent (beta), scaling parameter (H) and entropy (statistical) were found to be characteristic for natural sequences. For a reference state we chose a randomised state devoid of any pair-preference. The pair-preferences qualified well to be used as quantitative measures of regularities in protein sequences.

Muscle relaxant properties of chloramphenicol.

Experiments with the guinea pig ileum, guinea pig trachea, rat fundal strip, rat colon, rat vas deferens, and toad heart indicated that chloramphenicol inhibited smooth muscles, decreasing both the height and frequency of spontaneous contraction. Chloramphenicol-induced relaxation was not mediated through adrenergic, cholinergic, or histaminergic mechanisms. The degree of muscle relaxation was related to the concentration of chloramphenicol, and the relaxant effect could be reversed by removing chloramphenicol from site of action by washing. Its action appears to be direct on the muscle, possibly by interfering with the energy-generating mechanism.

Smooth muscle relaxant properties and vasomotor actions of 2-amino-1-p-nitrophenylpropane-1,3-diol.

Experiments with the guinea pig ileum, trachea, and vas deferens, the rat fundal strip, the rabbit jejunum and aortic strip, and the toad heart indicated that 2-amino-1-p-nitrophenylpropane-1,3-diol, the hydrolytic product of chloramphenicol, inhibited smooth muscles. Its action was direct and not through any mediators. After intravenous administration, the compound produced vasodepression followed by an overshooting rise of blood pressure. Vasodepression was not mediated by adrenergic, cholinergic, or histaminergic mechanisms. Hypertension was a sympathomimetic effect. Muscle relaxant and cardiovascular effects of the compound were similar to those of chloramphenicol, although it had no antibacterial effect.

Inhibition of rat liver mitochondrial monoamine oxidase by chloramphenicol and 2-amino-1-p-nitrophenylpropane-1,3-diol.

Inhibition of rat liver mitochondrial monoamine oxidase by chloramphenicol and its hydrolytic product, 2-amino-1-p-nitrophenylpropane-1,3-diol, was studied. The enzyme activity and its inhibition by these two compounds were optimum at pH 7.0 after preincubation for 60 min, the time needed for maximum enzyme--inhibitor complex formation. Enzyme activity could be restored after prolonged dialysis. Monamine oxidase inhibition by chloramphenicol and its hydrolytic product was noncompetitive and reversible. Deamination of various monamines was not to the same degree by these compounds. Of the different antimicrobials studied, only chloramphenicol and its hydrolytic produced has a strong inhibitory effect on monoamine oxidase.

The effect of chloramphenicol on mucosal transference of glucose in mice; the role of intestinal alkaline phosphatase in this process.

The effect of chloramphenicol (CAP) on the mucosal transference of glucose in mice and its relation with the activities of different small intestinal enzymes were studied. CAP produced an increase in the mucosal transference of glucose in jejunum and a decrease in ileum. However, CAP reduced the activity of Na(+)-K(+)-ATPase in both of these segments. Treatment with ouabain could not alter the effects of CAP. Under similar experimental conditions, the activity of alkaline phosphatase (AP) was reduced in jejunum but increased in ileum. In presence of theophylline, the CAP-induced increase in the transference in jejunum was further enhanced, whereas in acute experiments with ileum theophylline counteracted the reduced transference to the control level. In presence of Zn2+, CAP-induced changes in jejunum were reversed whereas in ileum the decrease was more pronounced. Like AP, CAP altered the activity of Ca(2+)-ATPase in both segments. It is proposed that in presence of CAP an inverse relationship exists between the activity of AP and the glucose transference in these segments. It is further revealed that such differential changes in the transference of glucose may be due to site specific alterations in the activity of AP.

Mechanism of inhibition of smooth muscle of guinea-pig taenia coli by chloramphenicol.

The effects of antibiotic chloramphenicol (CAP) on Ca(2+)-ATPase activity and muscle tension were examined in guinea-pig taenia coli. In general, when CAP was added to the resting tissue no inhibition was observed except when a tonus was present, caused by either ouabain, high K+ or acetylcholine. Ouabain and high K(+)-induced sustained contractions were concentration-dependently inhibited by CAP. The sustained contraction induced by high K+ was more strongly inhibited by CAP than ouabain (IC50 value: high K+ 0.29 mumol/ml; ouabain 0.34 mumol/ml). In Ca(2+)-free solution, inhibition of ouabain-induced sustained contracture by CAP was more pronounced. CAP increased the activity of Ca(2+)-ATPase in taenia coli in all experiments. In presence of cystine, CAP-induced inhibition and increase in Ca(2+)-ATPase activity could not be observed. CAP analogue thiamphenicol (TAP), devoid of p-NO2 group, showed insignificant response on smooth muscle inhibition and Ca(2+)-ATPase activity. These findings suggest that CAP inhibits smooth muscle contractility by decreasing cytosolic Ca2+ ([Ca2+]i) level through a cGMP mediated increase in Ca(2+)-ATPase activity and this action is possibly related with the p-NO2 group present in its molecule like other nitro-compounds.

Effects of different intensities of cold stress on certain physiological phenomena related to skeletal health in a hypogonadal rat model.

Intestinal transference pattern of calcium and associated changes in the activities of intestinal mucosal enzymes, rate of bone turnover and bone metabolism were evaluated in ovariectomized rats exposed to cold stress of various intensities i.e., mild (15 degrees C) or stronger (8 degrees C and 4 degrees C) for 5 min everyday for 7 consecutive days. Except mild cold stress-induced group (15 degrees C), rats of other two groups (8 degrees C and 4 degrees C) showed considerable decrease in the rate of in situ intestinal transference of calcium. Likewise, in these groups, the activities of intestinal mucosal enzymes, alkaline phosphatase (AP) and calcium ATPase (Ca2+-ATPase) were decreased significantly in all the segments of small intestine in a descending gradient. Also significant changes in bone turnover and bone resorption were confirmed in these animals by marked alterations in plasma AP activity, urinary calcium and phosphate excretion and urinary calcium to creatinine ratio (Ca: Cr). The skeletal changes were further ascertained by examining other physical and biochemical parameters of bone metabolism viz., body mass, bone density, ratio of mineral to matrix and mineral content of bone ash (calcium and phosphate) in the ovariectomized rat model. All these results suggest that stronger cold stress (8 degrees C and 4 degrees C) may be an important ecological factor in the development of earlier bone loss in hypogonadal rats.

Monoamine oxidase inhibition by the tremorogenic drug--LON 954.

N-Carbamoyl-2-(2,6-dichlorophenyl)acetamidine HCl (LON 954), a tremorogenic drug, inhibited MAO activity in various tissue preparations in a reversible, competitive manner showing some degree of selectivity towards type-B MAO.

Cold stress facilitates calcium mobilization from bone in an ovariectomized rat model of osteoporosis.

In an ovariectomized rat model of osteoporosis, the effects of cold stress on intestinal Ca2+ transference and rate of bone turnover were evaluated. In the ovariectomized rats, a significant reduction in intestinal transference of Ca2+ was associated with decreased activities of intestinal mucosal enzymes, alkaline phosphatase (AP), and calcium ATPase (Ca2+-ATPase) in all the different segments of small intestine in a descending gradient. The development of a high rate of bone turnover and osteoporosis in these animals was confirmed by significant alteration in plasma AP activity and calcium (Ca) level, urinary excretion of Ca and phosphate, and Ca : creatinine ratio. Cold stress in this model, apart from its unique influence in elevating plasma corticosterone and thyroid hormone level, enhanced all the above referred parameters studied in connection with intestinal transference of Ca2+, bone turnover rate, and osteoporosis. The results of this study emphasize that cold stress may have a positive influence on bone loss for an early development of hypogonadal osteoporosis in rats.

High-lipid diet intake is a possible predisposing factor in the development of hypogonadal osteoporosis.

The effects of a high-lipid diet on the intestinal transference of Ca2+ and the activities of intestinal enzymes were studied in ovariectomized rats. The plasma Ca2+ level and urinary loss of Ca2+ were also observed. Ovariectomy decreased both alkaline phosphatase (AP) and calcium ATPase (Ca(2+)-ATPase) activity, and also reduced the mucosal transference of Ca2+ in all parts of the small intestine. Although the plasma Ca2+ level did not change significantly, the urinary loss of Ca2+ was enhanced. A high-lipid diet supplement enhanced all these parameters in ovariectomized rats. It is therefore believed that a high-lipid diet may promote osteoporosis in ovariectomized rats as the result of Ca2+ transfer and enhanced urinary Ca2+ loss.