Carcinogen risk assessment. Can we harmonise?

Harmonisation of risk assessment (RA) is one of the priorities for sound chemical management set by Chapter 19, Agenda 21 of the United Nations Conference on the Environment and Development (UNCED) 1992 Earth Summit. The benefits of harmonisation are self evident and include transportability and consistency of RA outcomes, transparency and efficiency of process, and credible science. The outcomes of carcinogen RA are a description or classification of the carcinogenic hazard, the conditions under which cancers may be induced, and an estimate of a dose or exposure which poses a minimal, or otherwise defined, risk in exposed human populations. Weight-of-evidence based systems which classify carcinogenic hazards are part of, but do not substitute for, the risk assessment process. Carcinogen RA is based on assessment of appropriate toxicological and exposure data sets, which may have much in common. However, national policy frameworks can differ to the extent that RA outcomes may be quite different for the same chemical(s). Historically, differences in science policy have been greater for cancer RA compared to other toxic endpoints, with a tendency to differentiate cancer RA on the basis of presumed mechanism (i.e. genotoxic or non-genotoxic) and relevance to humans (some carcinogenic responses in animals may be considered not relevant for human RA). Significant strides towards harmonisation are being made, with reassessment of some national policies and participation in international harmonisation programmes, such as the ones being managed by the International Programme for Chemical Safety (IPCS). Alternative approaches to quantitative carcinogen RA are being considered which are more amenable to harmonisation, and one such approach being developed in Australia in connection with contaminated sites will be discussed.

Modulation of the transient potassium current in rat hippocampal neurones by cholecystokinin.

Cholecystokinin (CCK) affects neuronal excitability in a variety of in vivo and in vitro preparations, apparently by modulating a resting potassium conductance. The data presented here show that CCK (applied as CCK8-S) also affects the transient potassium current in hippocampal neurones, by changing the voltage dependence of the inactivation and activation of the current. The way in which the voltage dependence is changed can lead to either an enhancement of the current or an attenuation, depending upon the voltage protocol used. This effect of CCK does not desensitise over a time period of minutes, and may therefore be important in controlling neuronal excitability in the CNS.

A voltage-dependent persistent sodium current in mammalian hippocampal neurons.

Currents generated by depolarizing voltage pulses were recorded in neurons from the pyramidal cell layer of the CA1 region of rat or guinea pig hippocampus with single electrode voltage-clamp or tight-seal whole-cell voltage-clamp techniques. In neurons in situ in slices, and in dissociated neurons, subtraction of currents generated by identical depolarizing voltage pulses before and after exposure to tetrodotoxin revealed a small, persistent current after the transient current. These currents could also be recorded directly in dissociated neurons in which other ionic currents were effectively suppressed. It was concluded that the persistent current was carried by sodium ions because it was blocked by TTX, decreased in amplitude when extracellular sodium concentration was reduced, and was not blocked by cadmium. The amplitude of the persistent sodium current varied with clamp potential, being detectable at potentials as negative as -70 mV and reaching a maximum at approximately -40 mV. The maximum amplitude at -40 mV in 21 cells in slices was -0.34 +/- 0.05 nA (mean +/- 1 SEM) and -0.21 +/- 0.05 nA in 10 dissociated neurons. Persistent sodium conductance increased sigmoidally with a potential between -70 and -30 mV and could be fitted with the Boltzmann equation, g = gmax/(1 + exp[(V' - V)/k)]). The average gmax was 7.8 +/- 1.1 nS in the 21 neurons in slices and 4.4 +/- 1.6 nS in the 10 dissociated cells that had lost their processes indicating that the channels responsible are probably most densely aggregated on or close to the soma. The half-maximum conductance occurred close to -50 mV, both in neurons in slices and in dissociated neurons, and the slope factor (k) was 5-9 mV. The persistent sodium current was much more resistant to inactivation by depolarization than the transient current and could be recorded at greater than 50% of its normal amplitude when the transient current was completely inactivated. Because the persistent sodium current activates at potentials close to the resting membrane potential and is very resistant to inactivation, it probably plays an important role in the repetitive firing of action potentials caused by prolonged depolarizations such as those that occur during barrages of synaptic inputs into these cells.

Excitation and inhibition of the heart of the snail, Lymnaea, by non-FMRFamidergic motoneurons.

1. The present paper extends the model of neuronal control of the Lymnaea heart by the use of intracellular recording techniques to identify further types of cardioactive neurons in the CNS that, like the previously described E heart excitor (Ehe) cells, influence the myogenic heartbeat. 2. Four new types of neuron that act on the heart are described. These are excitatory Hhe and She cells (H and S heart excitors) and the inhibitory Khi cell (K heart inhibitor). The fourth class, tonus pericardium excitor (Tpe), modulates the heart by action on pericardial tissue. 3. Pharmacologic, electrophysiological, and anatomic evidence is presented that shows that these cells are motoneurons, innervating heart muscle fibers directly: blocking central chemical synapses failed to prevent the actions of the neurons on the heart; simultaneous intracellular recordings showed unitary EJPs in heart muscle after 1:1 and with constant delay from evoked neuronal action potentials; intracellular injection of the dye Lucifer yellow showed all cells had axonal branches entering the intestinal nerve (which innervates the heart). 4. The use of selective antagonists to 5-hydroxytryptamine (5-HT) (cinanserin), dopamine (ergonovine), and acetylcholine (alpha-bungarotoxin) provided evidence that the actions of She and Hhe cells are mediated by 5-HT, whereas those of the Khi cell are mediated by acetylcholine. 5. A cyclically active network of three interneuronal inputs acting on the heart motoneurons is described. 6. One of these, input 3, is responsible for periodic excitation of the heart via its effects on the Hhe cells.

Pharmacology of the myogenic heart of the pond snail Lymnaea stagnalis.

1. We have used pharmacologic, immunologic, and biochemical techniques to examine the role of neurochemicals in modulating the myogenic heart of the snail, Lymnaea. 2. 5-HT [high-pressure liquid chromatography (HPLC) and immunocytochemistry], dopamine (HPLC), FMRFamide-related peptides (radioimmunoassay and immunocytochemistry) and substance P-related peptides (immunocytochemistry) were shown to be localized within heart tissue. 3. The pharmacologic actions of these substances on the auricle from an isolated heart preparation were examined together with other putative modulators, acetylcholine (ACh), small cardioactive peptides A and B (SCPA and SCPB), [Arg]8vasotocin (AVT), and Lymnaea native FMRFamide-related peptides [Phe-Met-Arg-Phe-NH2 (FMRFamide), Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFLRFamide) and Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDPFLRFamide)]. 4. The response to each substance could be distinguished by different effect on beat rate, amplitude, and diastolic tonus, as well as by the duration of responses to standard 1-min applications. ACh was inhibitory at low concentrations (threshold less than 10(-10) M) but excitatory at high concentrations (10(-6) M). AVT was alone in producing no dose-dependent response. At high concentrations (10(-4) M), AVT caused a massive tonic contraction and cessation of auricle beat. All other substances examined were excitatory. 5. Antagonists to 5-HT (cinanserin), dopamine (ergonovine), and ACh (alpha-bungarotoxin) were identified. 6. ACh, 5-HT, dopamine, and FMRFamide-related peptides all acted on the auricle at low concentrations, and the rapid onset and short duration of their excitatory effects (ACh inhibitory at low concentrations) suggested that they may have roles as neurotransmitters. SCPA and SCPB were also potent (threshold less than 10(-10) M) but produced long-duration responses suggesting a modulatory or hormonal role.

Regulation of heartbeat in Lymnaea by motoneurons containing FMRFamide-like peptides.

1. Intracellular stimulation of single neurons in the Lymnaea CNS was carried out to identify heart motoneurons. 2. Two of the identified motoneurons, the E heart excitor (Ehe) cells, were shown to contain Phe-Met-Arg-Phe-NH2 (FMRFamide)-like peptides by immunocytochemical staining of dye-marked neurons and by radioimmunoassay (RIA) applied to extracts of single dissected cells. 3. Bursts of spikes in the Ehe cells increased heart rate, beat amplitude and muscle tonus. This response was mimicked by perfusion of exogenous FMRFamide at low concentration (10(-6) to 5 x 10(-8) M) through the interior of the intact heart. 4. Application of selective antagonists to 5-hydroxytryptamine (5-HT) and dopamine failed to block Ehe cardiac effects. 5. Detailed evidence that the Ehe cells were heart motoneurons was obtained. 1) Anatomic mapping using the dye Lucifer yellow showed Ehe cells had peripheral projections restricted mainly to the intestinal nerve, the only nerve known to innervate the heart. 2) Perfusion of the CNS with a saline containing Co2+ blocked central chemical synapses but did not affect activity of Ehe cells on the heart. 3) Simultaneous intracellular recordings from Ehe cells and auricle muscle fibers showed unitary excitatory junction potentials following with constant latency from spikes in Ehe cells. 6. The present study elucidates the role of FMRFamide in cardioregulation and provides the first evidence that it acts as an excitatory neurotransmitter on the snail heart.

Cholecystokinin modulates voltage dependent K+ currents in cultured rat hippocampal neurones.

Much interest has been focussed recently on neuroactive peptides originally found in the peripheral nervous system, but now, increasingly, being shown to be present in considerable amounts in the mammalian CNS. One of these peptides, cholecystokinin (CCK), is present in large amounts in higher brain areas. Immunoreactivity to CCK has been demonstrated in the mammalian hippocampus and dentate gyrus, localised in nerve terminals, and increasingly this peptide is being suggested as having a role as a transmitter in the CNS. Generally, CCK appears to produce depolarisations of neurones: e.g. mesenteric ganglion cells, and hippocampal neurones [5], although the mechanism by which it does so remains unclear, there being reports of either a decrease in input resistance, an increase, or both.