Understanding genetic toxicity through data mining: the process of building knowledge by integrating multiple genetic toxicity databases.

ABSTRACT Genetic toxicity data from various sources were integrated into a rigorously designed database using the ToxML schema. The public database sources include the U.S. Food and Drug Administration (FDA) submission data from approved new drug applications, food contact notifications, generally recognized as safe food ingredients, and chemicals from the NTP and CCRIS databases. The data from public sources were then combined with data from private industry according to ToxML criteria. The resulting "integrated" database, enriched in pharmaceuticals, was used for data mining analysis. Structural features describing the database were used to differentiate the chemical spaces of drugs/candidates, food ingredients, and industrial chemicals. In general, structures for drugs/candidates and food ingredients are associated with lower frequencies of mutagenicity and clastogenicity, whereas industrial chemicals as a group contain a much higher proportion of positives. Structural features were selected to analyze endpoint outcomes of the genetic toxicity studies. Although most of the well-known genotoxic carcinogenic alerts were identified, some discrepancies from the classic Ashby-Tennant alerts were observed. Using these influential features as the independent variables, the results of four types of genotoxicity studies were correlated. High Pearson correlations were found between the results of Salmonella mutagenicity and mouse lymphoma assay testing as well as those from in vitro chromosome aberration studies. This paper demonstrates the usefulness of representing a chemical by its structural features and the use of these features to profile a battery of tests rather than relying on a single toxicity test of a given chemical. This paper presents data mining/profiling methods applied in a weight-of-evidence approach to assess potential for genetic toxicity, and to guide the development of intelligent testing strategies.

Does irritation potency contribute to the skin sensitization potency of contact allergens?

Chemicals that possess the capacity to cause skin sensitization have long been recognized to be reactive (electrophilic) or at least the precursor of an electrophile. The chemical species (hapten) covalently bound to skin protein then forms the antigen to which the immune system responds, with sufficient exposure ultimately leading to skin sensitization. However, for this process to occur, many have also considered that in addition to haptenation of skin protein, secondary stimuli (danger signals) are also necessary. Such signals might reasonably be expected to derive from keratinocytes and/or Langerhans cells perturbed by the chemical sensitizer. Whether this disturbance comes from the haptenation process itself or from other properties of the chemical is unknown. We hypothesized that chemicals that were stronger sensitizers might appear so, in part, as a consequence not only of greater (pro)electrophilic reactivity, but also if they were more able to produce inflammatory (danger) signals. To assess this, the sensitizing potency of 55 chemicals in the local lymph node assay was compared with their ability to produce pro-inflammatory signal release, measured as a function of their relative skin irritancy in guinea pigs. A limited trend was demonstrated, consistent with the hypothesis, but indicating that either skin irritation is a poor measure of danger signals, or that such signals are perhaps no more than a necessary requirement for the acquisition of skin sensitization rather than a key determinant of the relative potency of a skin sensitizing chemical. In addition, it is possible that irritancy alone does not represent a complete surrogate marker for the ability of a chemical to produce danger signals relevant to the induction of skin sensitization.

Sodium-dependent potassium channels in leech P neurons.

In leech P neurons the inhibition of the Na(+)-K(+) pump by ouabain or omission of bath K(+) leaves the membrane potential unaffected for a prolonged period or even induces a marked membrane hyperpolarization, although the concentration gradients for K(+) and Na(+) are attenuated substantially. As shown previously, this stabilization of the membrane potential is caused by an increase in the K(+) conductance of the plasma membrane, which compensates for the reduction of the K(+) gradient. The data presented here strongly suggest that the increased K(+) conductance is due to Na(+)-activated K(+) (K(Na)) channels. Specifically, an increase in the cytosolic Na(+) concentration ([Na(+)](i)) was paralleled by a membrane hyperpolarization, a decrease in the input resistance (R(in)) of the cells, and by the occurrence of an outwardly directed membrane current. The relationship between R(in) and [Na(+)](i) followed a simple model in which the R(in) decrease was attributed to K(+) channels that are activated by the binding of three Na(+) ions, with half-maximal activation at [Na(+)](i) between 45 and 70 mM. At maximum channel activation, R(in) was reduced by more than 90%, suggesting a significant contribution of the K(Na) channels to the physiological functioning of the cells, although evidence for such a contribution is still lacking. Injection experiments showed that the K(Na) channels in leech P neurons are also activated by Li(+).

Modulation of Ca2+ influx in leech Retzius neurons II. Effect of extracellular Ca2+.

We investigated the cytosolic free calcium concentration ([Ca2+]i) of leech Retzius neurons in situ while varying the extracellular Ca2+ concentration via the bathing solution ([Ca2+]B). Changing [Ca2+]B had only an effect on [Ca2+]i if the cells were depolarized by raising the extracellular K+ concentration. Surprisingly, raising [Ca2+]B from 2 to 10 mm caused a decrease in [Ca2+]i, and an increase was evoked by reducing [Ca2+]B to 0.1 mm. These changes were not due to shifts in membrane potential. At low [Ca2+]B moderate membrane depolarizations were sufficient to evoke a [Ca2+]i increase, while progressively larger depolarizations were necessary at higher [Ca2+]B. The changes in the relationship between [Ca2+]i and membrane potential upon varying [Ca2+]B could be reversed by changing extracellular pH. We conclude that [Ca2+]B affects [Ca2+]i by modulating Ca2+ influx through voltage-dependent Ca2+ channels via the electrochemical Ca2+ gradient and the surface potential at the extracellular side of the plasma membrane. These two parameters are affected in a counteracting way: Raising the extracellular Ca2+ concentration enhances the electrochemical Ca2+ gradient and hence Ca2+ influx, but it attenuates Ca2+ channel activity by shifting the extracellular surface potential to the positive direction, and vice versa.

Modulation of Ca2+ influx in leech Retzius neurons. I. Effect of extracellular pH.

We investigated the cytosolic free Ca2+ concentration ([Ca2+]i) of leech Retzius neurons in situ while varying the extracellular and intracellular pH as well as the extracellular ionic strength. Changing these parameters had no significant effect on [Ca2+]i when the membrane potential of the cells was close to its resting value. However, when the cells were depolarized by raising the extracellular K+ concentration or by applying the glutamatergic agonist kainate, extracellular pH and ionic strength markedly affected [Ca2+]i, whereas intracellular pH changes appeared to have virtually no effect. An extracellular acidification decreased [Ca2+]i, while alkalinization or reduction of the ionic strength increased it. Correspondingly, [Ca2+]i also increased when the kainate-induced extracellular acidification was reduced by raising the pH-buffering capacity. At low extracellular pH, the membrane potential to which the cells must be depolarized to evoke a detectable [Ca2+]i increase was shifted to more positive values, and it moved to more negative values at high pH. We conclude that in leech Retzius neurons extracellular pH, but not intracellular pH, affects [Ca2+]i by modulating Ca2+ influx through voltage-dependent Ca2+ channels. The results suggest that this modulation is mediated primarily by shifts in the surface potential at the extracellular side of the plasma membrane.

Chiral induction effects in ruthenium(II) amino alcohol catalysed asymmetric transfer hydrogenation of ketones: an experimental and theoretical approach

The enantioselective outcome of transfer hydrogenation reactions that are catalysed by ruthenium(II) amino alcohol complexes was studied by means of a systematically varied series of ligands. It was found that both the substituent at the 1-position in the 2-amino-1-alcohol ligand and the substituent at the amine functionality influence the enantioselectivity of the reaction to a large extent: enantioselectivities (ee values) of up to 95% were obtained for the reduction of acetophenone. The catalytic cycle of ruthenium(II) amino alcohol catalysed transfer hydrogenation was examined at the density functional theory level. The formation of a hydrogen bond between the carbonyl functionality of the substrate and the amine proton of the ligand, as well as the formation of an intramolecular H...H bond and a planar H-Ru-N-H moiety are crucially important for the reaction mechanism. The enantioselective outcome of the reaction can be illustrated with the aid of molecular modelling by the visualisation of the steric interactions between the ketone and the ligand backbone in the ruthenium(II) catalysts.

Effects of ATP and derivatives on neuropile glial cells of the leech central nervous system.

We investigated the effects of ATP (adenosine 5'-triphosphate) and derivatives on leech neuropile glial cells, focusing on exposed glial cells. ATP dose-dependently depolarized or hyperpolarized neuropile glial cells in situ as well as exposed neuropile glial cells. These potential shifts varied among cells and repetitive ATP application did not change their amplitude, duration or direction. In exposed neuropile glial cells, ATP most frequently induced a Na(+)-dependent depolarization and decreased the input resistance. The agonist potency ATP > ADP (adenosine 5'-diphosphate) > AMP (adenosine 5'-monophosphate) > adenosine indicates that P2 purinoceptors mediate this depolarization. The P2Y agonist 2-methylthio-ATP mimicked the ATP-induced depolarization, whereas the P2Y antagonist PPADS (pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid) reduced it. P2X agonists were without effect. Because the P1 antagonist 8-SPT (8-(p-sulphophenyl)-theophylline) also depressed ATP-induced depolarizations and some ATP-insensitive glial cells responded to adenosine, we suggest coexpression of metabotropic P2Y and P1 purinoceptors. The ATP-induced depolarization requires activation of Na(+) channels or nonselective cation channels, whereas the ATP-induced hyperpolarization indicates activation of K(+) channels. ATP also increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), that is independent of Ca(2+) influx but reflects intracellular Ca(2+) release possibly triggered by IP(3) formation. ADP and AMP also increased [Ca(2+)](i), but were less efficient than ATP; adenosine and 2-methylthio-ATP did not affect [Ca(2+)](i). In view of the mobilization of intracellular Ca(2+), ATP is clearly different from other leech neurotransmitters, because it enables intracellular Ca(2+) signaling without causing prominent changes in glial membrane potential. Thus disturbance of the extracellular microenvironment and the demand for metabolic energy are minimized.

Ionic mechanism of 4-aminopyridine action on leech neuropile glial cells.

Extracellular 4-aminopyridine (4-AP), tetraethylammonium chloride (TEA) and quinine depolarized the neuropile glial cell membrane and decreased its input resistance. As 4-AP induced the most pronounced effects, we focused on the action of 4-AP and clarified the ionic mechanisms involved. 4-AP did not only block glial K+ channels, but also induced Na+ and Ca2+ influx via other than voltage-gated channels. The reversal potential of the 4-AP-induced current was -5 mV. Application of 5 mM Ni2+ or 0.1 mM d-tubocurarine reduced the 4-AP-induced depolarization and the associated decrease in input resistance. We therefore suggest that 4-AP mediates neuronal acetylcholine release, apparently by a presynaptic mechanism. Activation of glial nicotinic acetylcholine receptors contributes to the depolarization, the decrease in input resistance, and the 4-AP-induced inward current. Furthermore, the 4-AP-induced depolarization activates additional voltage-sensitive K+ and Cl- channels and 4-AP-induced Ca2+ influx could activate Ca2+-sensitive K+ and Cl- channels. Together these effects compensate and even exceed the 4-AP-mediated reduction in K+ conductance. Therefore, the 4-AP-induced depolarization was paralleled by a decreasing input resistance.

Voltage-dependent Ca2+ influx into identified leech neurones.

We determined the relationships between the intracellular free Ca2+ concentration ([Ca2+]i) and the membrane potential (Em) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca2+]i was monitored by using iontophoretically injected fura-2. The membrane depolarization evoked by raising the extracellular K+ concentration ([K+]o) up to 89 mM caused a persistent increase in [Ca2+]i, which was abolished in Ca(2+)-free solution indicating that it was due to Ca2+ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca2+]i increase ranged between -40 and -25 mV. The different threshold potentials as well as differences in the relationships between [Ca2+]i and EM were partly due to the cell-specific generation of action potentials. In Na(+)-free solution, the action potentials were suppressed and the [Ca2+]i/Em relationships were similar. The K(+)-induced [Ca2+]i increase was inhibited by the polyvalent cations Co2+, Ni2+, Mn2+, Cd2+, and La3+, as well as by the cyclic alcohol menthol. Neither the polyvalent cations nor menthol had a significant effect on the K(+)-induced membrane depolarization. Our results suggest that different leech neurones possess voltage-dependent Ca2+ channels with similar properties.

Distribution and functional properties of glutamate receptors in the leech central nervous system.

1. The effect of kainate and other glutamatergic agonists on the membrane potential (Em), the intracellular Na+ activity (aNai), and the intracellular free Ca2+ concentration ([Ca2+]i) of identified leech neurons and neuropile glial cells was measured with conventional and ion-sensitive microelectrodes, as well as with the use of the iontophoretically injected fluorescent indicators sodium-binding benzofuran isophthalate and Fura-2. 2. In Retzius neurons, AE, L, 8, and 101 motoneurons, and in the unclassified 50 neurons (Leydig cells) and AP neurons, as well as in neuropile glial cells, bath application of 100 microM kainate evoked a marked membrane depolarization and an increase in aNai and [Ca2+]i. The kainate-induced aNai increase persisted in solutions with high Mg2+ concentration in which synaptic transmission is blocked. 3. A membrane depolarization as well as an increase in aNai and [Ca2+]i was also evoked by L-glutamate, quisqualate, and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). The agonist-induced [Ca2+]i increase was inhibited by 6,7-dinitroquinoxaline-2,3-dione (DNQX). 4. In Ca(2+)-free solution, the kainate-induced [Ca2+]i increase was abolished in the neurons and in neuropile glial cells, whereas membrane depolarization and aNai increase were unchanged. In Na(+)-free solution, kainate had no effect on Em, aNai, or [Ca2+]i in the neurons. 5. In the mechanosensory T, P, and N neurons, kainate induced considerably smaller membrane depolarizations than in the other neurons or in neuropile glial cells, and it had no significant effect on aNai or [Ca2+]i. 6. It is concluded that in leech segmental ganglia the majority of the neurons and the neuropile glial cells, but probably not the mechanosensory neurons, possess glutamate receptors of the AMPA-kainate type. In the neurons, the [Ca2+]i increase caused by glutamatergic agonists is due to Ca2+ influx through voltage-dependent Ca2+ channels that are activated by the agonist-induced membrane depolarization.