The sensitivity of metabolomics versus classical regulatory toxicology from a NOAEL perspective.

The identification of the no observed adverse effect level (NOAEL) is the key regulatory outcome of toxicity studies. With the introduction of "omics" technologies into toxicological research, the question arises as to how sensitive these technologies are relative to classical regulatory toxicity parameters. BASF SE and metanomics developed the in vivo metabolome database MetaMap®Tox containing metabolome data for more than 500 reference compounds. For several years metabolome analysis has been routinely performed in regulatory toxicity studies (REACH mandated testing or new compound development), mostly in the context of 28 day studies in rats (OECD 407 guideline). For those chemicals for which a toxicological NOAEL level was obtained at either high or mid-dose level, we evaluated the associated metabolome to investigate the sensitivity of metabolomics versus classical toxicology with respect to the NOAEL. For the definition of a metabolomics NOAEL the ECETOC criteria (ECETOC, 2007) were used. In this context we evaluated 104 cases. Comparable sensitivity was noted in 75% of the cases, increased sensitivity of metabolomics in 8%, and decreased sensitivity in 18% of the cases. In conclusion, these data suggest that metabolomics profiling has a similar sensitivity to the classical toxicological study (e.g. OECD 407) design.

Mechanistic analysis of metabolomics patterns in rat plasma during administration of direct thyroid hormone synthesis inhibitors or compounds increasing thyroid hormone clearance.

For identification of toxicological modes of action (MoAs) a database (MetaMap(®)Tox) was established containing plasma metabolome consisting of approximately 300 endogenous metabolites. Each five male and female Wistar rats per groups were treated with >500 reference compounds over a period of 28 days. More than 120 specific toxicity patterns of common metabolite changes associated with unique MoAs were established. To establish patterns predictive effects on the thyroid, animals have been treated with reference compounds directly acting on the thyroid hormone formation (such as methimazole, ethylenethiourea) as well as liver enzyme inducers leading to an increased excretion of thyroid hormones and therewith to a secondary response of the thyroid (such as aroclor 1254 and boscalid). Here we present the plasma metabolite changes which form the patterns for direct and indirect effects on the thyroid. It is possible to identify metabolites which are commonly regulated irrespective of an indirect or direct effect on the thyroid as well as groups of metabolites separating both MoAs. By putting the metabolite regulations in the context of affected pathways helps to identify thyroid hormone inhibiting MoAs even when the hormone levels are not consistently changed. E.g., direct thyroid hormone synthesis inhibitors affect some enzymes in the urea cycle, increase the ω-oxidation of fatty acids and decrease glutamate and oxoproline levels, whereas indirect thyroid hormone inhibiting compounds interact with the lipid mediated and liver metabolism.

The effects of pentachlorophenol (PCP) at the toad neuromuscular junction.

1. Effects of PCP at the frog neuromuscular junction were studied in vitro in sciatic nerve sartorius muscle of the toad Pleurodema-thaul. 2. Within the concentration 0.003-0.1 mM, PCP caused a dose-time-dependent block of evoked transmitter release acompanied by an increase in the rate of spontaneous quantal release. 3. PCP induced an increase in miniature endplate potential (MEPP) frequency and it was not antagonized in a Ca2(+)-free medium, indicating that it does not depend upon Ca2+ influx from the external medium, but may act by releasing Ca2+ from intraterminal stores. 4. The present data, together with previous results concerning PCP at eighth sympathetic ganglia indicate that 3,4-diaminopyridine (3,4-DAP) counteracts the effects of PCP on synaptic transmission. This result suggests that PCP interfering Ca2+ influx occurs during depolarization of motor nerve terminals.

Substance P-like immunoreactivity in sympathetic ganglion from toad.

Substance P-like immunoreactivity cellular in toad sympathetic ganglia was studied in normal and capsaicin-treated ganglia. In the eighth sympathetic ganglion substance P-like immunoreactive are found in mast cells and SIF cells. The effect of substance P (0.001-0.003 mM) caused increase of compound action potential during tetanical stimulation (50 Hz by 40 sec.) and post-tetanic potentiation (0.1 Hz). Our results show that substance P facilitates synaptic transmission in the sympathetic ganglia from Caudiverbera caudiverbera.

The actions of phenol and pentachlorophenol (PCP) on axonal conduction, ganglionic synaptic transmission, and the effect of pH changes.

1. A comparison of phenol, pentachlorophenol (PCP) and procaine effects on axonal conduction were studied in vitro in the sciatic nerves of toad. PCP and procaine were respectively 6.3 and 3.15 times more potent than phenol in blocking axonal conduction. 2. Effects of PCP on synaptic transmission were studied in vitro in the eighth sympathetic ganglion of toad. 3. Axonal conduction block and synaptic transmission block by phenol was reversible, but not that by PCP. 4. When the PCP ionization was increased, a lesser per cent reached the site of action, reducing its capacity to block the axonal conduction and ganglionic transmission. 5. PCP plus, 3,4-Diaminopyridine (3,4-DAP) decreased synaptic transmission block from post-ganglionic compound action potential (CAP) responses to supramaximal preganglionic stimulation.

The effects of hemicholinium-3 (Hc-3) during tetanic (TP) and posttetanic potentiation (PTP) on sympathetic ganglion.

An electrophysiological analysis has been made of the synaptic plasticity in the eighth sympathetic ganglion of the toad. The mean amplitude of Compound Action Potential (CAP) recorded extracellularly was taken as a measure of the synaptic transmission. Repetitive stimulation of the preganglionic axonal fibers at 50 Hz for 40 seconds in the presence of Hc-3 led to facilitatory and depressant actions on ganglionic synaptic transmission. Hc-3 in the presence of elevated extracellular Ca2+ concentration (5.4 mM) increased Tetanic Potentiation (TP) and Posttetanic Potentiation (PTP) on eighth sympathetic ganglion. Hc-3 plus 3,4-diaminopyridine (3,4-DAP) increased synaptic transmission, TP, and PTP from postganglionic CAP responses to supramaximal preganglionic stimulation.

Increase in transmitter release from motor nerve terminals induced by some pyridine derivatives.

The effects of 4-nitropyridine (4-NP), 4-aminopyridine-N-oxide (4-AP-N-O), 4-hydroxypyridine (4-HP), 2,6-diaminopyridine (2,6-DAP), 2,4-dihydroxypyridine (2,4-DHP) and pyridine on isolated sciatic nerve-sartorius muscle preparations were studied by means of intracellular and extracellular recording techniques. In junctions treated with (+) tubocurarine, 4-NP, 4-AP-N-O, 4-HP and 2,6-DAP reversibly increased the amplitude and the latency of end-plate potentials (EPPs) and induced repetitive EPPs in response to single nerve impulses. As shown by extracellular focal recordings, the increase in latency of EPPs was due to a prolongation of the minimum synaptic delay, while the appearance of repetitive EPPs was the result of repetitive firing of motor nerve terminals. 4-NP, 4-AP-N-O, 4-HP and 2,6-DAP increased dose-dependently the quantal content of EPPs, while 2,4-DHP and pyridine were found to be inactive. Comparison of the apparent rank order of potency in increasing quantal transmitter release indicates that the relative activity of the different pyridine derivatives studied is unrelated to their pK values. Spontaneous quantal transmitter release in resting junctions was unaffected by 4-NP, 4-AP-N-O, 4-HP and 2,6-DAP when applied at concentrations that enhanced evoked transmitter release. 4-NP differed from the other pyridine derivatives by producing in high concentrations a time-dependent increase in miniature end-plate potential frequency and a depolarization of the muscle fibres. In addition, 4-AP-N-O, 4-HP and 2,6-DAP were found to have no effect on MEPP frequency accelerated by increasing the external K+ concentration. In conclusion the data presented strongly suggest that 4-NP, 4-HP, 4-AP-N-O and 2,6-DAP facilitate evoked transmitter release from motor nerve terminals by a presynaptic action that seems related to an increased calcium influx secondary to the blockade of potassium channels in the nerve terminal.

Increase in transmitter release from motor nerve terminals induced by some pyridine derivatives.

The effects of 4-nitropyridine (4-NP), 4-aminopyridine-N-oxide (4-AP-N-O), 4-hydroxypyridine (4-HP), 2,6-diaminopyridine (2,6-DAP), 2,4-dihydroxypyridine (2,4-DHP) and pyridine on isolated sciatic nerve-sartorius muscle preparations were studied by means of intracellular and extracellular recording techniques. In junctions treated with (+) tubocurarine, 4-NP, 4-AP-N-O, 4-HP and 2,6-DAP reversibly increased the amplitude and the latency of end-plate potentials (EPPs) and induced repetitive EPPs in response to single nerve impulses. As shown by extracellular focal recordings, the increase in latency of EPPs was due to a prolongation of the minimum synaptic delay, while the appearance of repetitive EPPs was the result of repetitive firing of motor nerve terminals. 4-NP, 4-AP-N-O, 4-HP and 2,6-DAP increased dose-dependently the quantal content of EPPs, while 2,4-DHP and pyridine were found to be inactive. Comparison of the apparent rank order of potency in increasing quantal transmitter release indicates that the relative activity of the different pyridine derivatives studied is unrelated to their pK values. Spontaneous quantal transmitter release in resting junctions was unaffected by 4-NP, 4-AP-N-O, 4-HP and 2,6-DAP when applied at concentrations that enhanced evoked transmitter release. 4-NP differed from the other pyridine derivatives by producing in high concentrations a time-dependent increase in miniature end-plate potential frequency and a depolarization of the muscle fibres. In addition, 4-AP-N-O, 4-HP and 2,6-DAP were found to have no effect on MEPP frequency accelerated by increasing the external K+ concentration. In conclusion the data presented strongly suggest that 4-NP, 4-HP, 4-AP-N-O and 2,6-DAP facilitate evoked transmitter release from motor nerve terminals by a presynaptic action that seems related to an increased calcium influx secondary to the blockade of potassium channels in the nerve terminal.

A study of the actions of bromide on frog sympathetic ganglion.

The effects of sodium bromide on the bullfrog sympathetic ganglion were studied by extracellular and intracellular recording techniques. Equimolar replacement of sodium chloride (112 mM) by sodium bromide in Ringer's solution caused hyperpolarization (means = 7.4 mV) of ganglion cells, and antidromically evoked spikes showed increased rates of rise as well as prolonged post-spike positivity. These effects, rapid in onset, returned to control values after approximately 30 min of continued exposure to bromide Ringer. Orthodromic synaptic transmission was not impaired by bromide (84-112 mM); instead it produced increased post-spike negativity and occasionally, stimulus-bound repetitive postganglionic responses (SBR) to each single preganglionic stimulus. This synaptic enhancement persisted as long as exposure to bromide continued. Since ethanol also causes responses, its interaction with bromide was tested and pronounced synergism was found in which 95% of ganglion cells displayed repetitive orthodromic responses to each single preganglionic stimulus. The present and previous studies suggest that bromide and ethanol act at the unmyelinated presynaptic nerve terminal to generate stimulus-bound repetitive responses. The transient nature of the hyperpolarization produced by bromide argues against a mechanistic role in its anticonvulsant action. The persistent synaptic excitatory effects of bromide, however, may have implications for the role of chloride channels in anticonvulsant mechanisms or in epileptogenesis.

Effect of phenol on conduction and synaptic transmission block.

The effect of phenol on nerve conduction and ganglionic synaptic transmission has been studied on sciatic nerve and on the VIIIth sympathetic ganglion of the frog. Ganglionic synaptic transmission block occurred at lower concentration than axonal conduction block. Both, conduction and synaptic transmission recovered in Ringer's solutions after 10 minutes. CaCl2 5.4 mM antagonized partially the axonal conduction block and EGTA in Ringer's solution without CaCl2 increased the effect of phenol on axonal conduction block on desheated sciatic nerve. Early in the experiments phenol had a weaker effect than procaine but at 10 minutes the effect of procaine became steady state and the effect of phenol continued to increase.

Stimulus-bound repetitive synaptic firing caused by ethanol in sympathetic ganglion.

The effects of ethanol, 0.1 to 1.0 M, on synaptic transmission, preganglionic axonal fibers and the ganglion cell were examined by extracellular and intracellular recording in isolated bullfrog sympathetic ganglia. Within the concentration range 0.2 to 0.8 M ethanol caused stimulus-bound repetitive postganglionic responses (SBR) to single preganglionic stimuli. The presynaptic origin of ethanol-induced SBR was confirmed by recordings of repetitive synaptic potential responses to single stimuli, and by absence of repetitive responses in myelinated preganglionic axons and in ganglion cells stimulated antidromically. Ethanol acted synergistically with Cs+ to produce SBR more intense than that caused by either agent alone. The postganglionic SBR caused by ethanol was suppressed by concentrations of d-tubocurarine, lidocaine, or tetraethylammonium that had little or no effect on synaptic transmission. Ethanol also blocked synaptic transmission, but this occurred secondary to the initial excitatory effects, and in the concentration range 0.4 to 1.0 M. The present data, together with previous studies of ethanol at neuromuscular junction, indicate that synaptic excitatory effects of ethanol are unrelated to hyperosmolarity or cholinesterase inhibition and represent a primary action of ethanol on prejunctional nerve endings.