Chrisovalantis Papaefthimiou

Comparison of two screening bioassays, based on the frog sciatic nerve and yeast cells, for the assessment of herbicide toxicity

Two different test systems, one based on the isolated sciatic nerve of an amphibian and the other on a microbial eukaryote, were used for the assessment of herbicide toxicity. More specifically, we determined the deleterious effects of increasing concentrations of herbicides of different chemical classes (phenoxyacetic acids, triazines, and acetamides), and of 2,4-dichlorophenol (2,4-DCP), a degradation product of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), on electrophysiological parameters and the vitality of the axons of the isolated sciatic nerve of the frog (Rana ridibunda) and on the growth curve of the yeast Saccharomyces cerevisiae based on microtiter plate susceptibility assays. The no-observed-effect-concentration (NOEC), defined as the maximum concentration of the tested compound that has no effect on these biological parameters, was estimated. In spite of the different methodological approaches and biological systems compared, the NOEC values were identical and correlated with the lipophilicity of the tested compounds. The relative toxicity established here, 2,4-DCP > alachlor, metolachlor >> metribuzin > 2,4-D, 2-methyl-4-chlorophenoxyacetic acid (MCPA), correlates with the toxicity indexes reported in the literature for freshwater organisms. Based on these results, we suggest that the relatively simple, rapid, and low-cost test systems examined here may be of interest as alternative or complementary tests for toxicological assessment of herbicides.

AN IN VITRO METHOD FOR RECORDING THE ELECTRICAL ACTIVITY OF THE ISOLATED HEART OF THE ADULT DROSOPHILA MELANOGASTER

SummaryA recording chamber for monitoring the electrophysiological properties of the isolated heart of adult Drosophila melanogaster has been developed. Spontaneously generated field potentials of constant amplitude can be recorded for 6–8 h (n=14); in very few cases, records were maintained stable for over 10 h (n=4), and in some cases below 6 h (n=5). The chamber consists of the tip of a micropipette, which allows for monitoring the field potential generated by the spontaneously contracting heart. The method can produce accurate information about the heart rate and the amplitude of the cardiac action potential. The preparation can be used for pharmacological studies on the heart of D. melanogaster since it responds, with an increase in the heart rate, to unusually low concentrations of octopamine, 1 nM, a compound with cardioacelerating properties for insect heart. The recording system can be easily modified for experiments on the heart of other insects. Finally, the isolated heart of D. melanogaster provides a simple method for identifying mutations that affect heart physiology.

Octopamine--a single modulator with double action on the heart of two insect species (Apis mellifera macedonica and Bactrocera oleae): Acceleration vs. inhibition.

The effects of octopamine, the main cardioacceleratory transmitter in insects, were investigated, in the isolated hearts of the honeybee, Apis mellifera macedonica, and the olive fruit fly, Bactrocera oleae. Octopamine induced a biphasic effect on the frequency and force of cardiac contractions acting as an agonist, with a strong acceleratory effect, at concentrations higher than 10(-12)M for the honeybee and higher than 50×10(-9)M for the olive fruit fly. The heart of the honeybee is far more sensitive than the heart of olive fruit fly. This unusual sensitivity is extended to the blockers of octopaminergic receptors, where phentolamine at 10(-5)M stopped the spontaneous contractions of the honeybee heart completely and permanently, while the same blocker at the same concentration caused only 50% inhibition in the heart of the olive fruit fly. Phentolamine and mianserin at low concentrations of 10(-7)M also blocked the heart octopaminergic receptors, but for a short period of time, of less than 15.0 min, while a partial recovery in heart contraction started in spite of the presence of the antagonist. The unusual response of the honeybee heart in the presence of phentolamine and/or mianserin suggests excitatory effects of octopamine via two different receptor subtypes. At lower concentrations, 10(-14)M, the agonist octopamine was converted to an antagonist, inducing a hyperpolarization in the membrane potential of the honeybee cardiac pacemaker cells and inhibiting the firing rate of the heart. The inhibitory effects of octopamine on certain parameters of the rhythmic bursts of the heart of the honeybee, were similar to those of mianserin and phentolamine, typical blockers of octopaminergic receptors. The heart of the olive fruit fly was 10(5) times less sensitive to octopamine, since a persistent inhibition of heart contractions occurred at 10(-9)M. In conclusion, the acceleration of the insect heart is achieved by increasing the levels of octopamine, while there is a passive but also an active decrease in heart activity due to the minimization of octopamine.

The bite of the honeybee: 2-heptanone secreted from honeybee mandibles during a bite acts as a local anaesthetic in insects and mammals.

Honeybees secrete 2-heptanone (2-H) from their mandibular glands when they bite. Researchers have identified several possible functions: 2-H could act as an alarm pheromone to recruit guards and soldiers, it could act as a chemical marker, or it could have some other function. The actual role of 2-H in honeybee behaviour remains unresolved. In this study, we show that 2-H acts as an anaesthetic in small arthropods, such as wax moth larva (WML) and Varroa mites, which are paralysed after a honeybee bite. We demonstrated that honeybee mandibles can penetrate the cuticle of WML, introducing less than one nanolitre of 2-H into the WML open circulatory system and causing instantaneous anaesthetization that lasts for a few minutes. The first indication that 2-H acts as a local anaesthetic was that its effect on larval response, inhibition and recovery is very similar to that of lidocaine. We compared the inhibitory effects of 2-H and lidocaine on voltage-gated sodium channels. Although both compounds blocked the hNav1.6 and hNav1.2 channels, lidocaine was slightly more effective, 2.82 times, on hNav.6. In contrast, when the two compounds were tested using an ex vivo preparation–the isolated rat sciatic nerve–the function of the two compounds was so similar that we were able to definitively classify 2-H as a local anaesthetic. Using the same method, we showed that 2-H has the fastest inhibitory effect of all alkyl-ketones tested, including the isomers 3- and 4-heptanone. This suggests that natural selection may have favoured 2-H over other, similar compounds because of the associated fitness advantages it confers. Our results reveal a previously unknown role of 2-H in honeybee defensive behaviour and due to its minor neurotoxicity show potential for developing a new local anaesthetic from a natural product, which could be used in human and veterinary medicine.

The action of 2,4-Dichlorophenoxyacetic acid on the isolated heart of insect and amphibia

The action of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the isolated heart of the frog (Rana ridibunda) and two insects, the honeybee (Apis mellifera macedonica) and the beetle (Tenebrio molitor), was investigated using basic electrophysiological methods. The results of this study showed that a concentration of 1 μM 2,4-D was required to reduce the force and the frequency of the isolated heart of the honeybee to about 70% of the initial contraction in less than 20 min. To cause the same effects on the atria of the frog, 45 μM 2,4-D was required and on the isolated heart of the beetle, over 1000 μM of 2,4-D. The presence of an extensive system of gap junctions found in the honeybee is most probably the cause of the unusual sensitivity of its heart to 2,4-D, compared with the heart of the beetle, where no gap junctions were identified.

Triazines facilitate neurotransmitter release of synaptic terminals located in hearts of frog (Rana ridibunda) and honeybee (Apis mellifera) and in the ventral nerve cord of a beetle (Tenebrio molitor)

Three triazine herbicides, atrazine, simazine and metribuzine, and some of their major metabolites (cyanuric acid and 6-azauracil) were investigated for their action on synaptic terminals using three different isolated tissue preparations from the atria of the frog, Rana ridibunda, the heart of the honeybee, Apis mellifera macedonica, and the ventral nerve cord of the beetle, Tenebrio molitor. The results indicate that triazines facilitate the release of neurotransmitters from nerve terminals, as already reported for the mammalian central nervous system. The no observed effect concentration, the maximum concentration of the herbicide diluted in the saline that has no effect on the physiological properties of the isolated tissue, was estimated for each individual preparation. According to their relative potency, the three triazines tested can be ranked as follows: atrazine (cyanuric acid), simazine>metribuzine (6-azauracil). The action of these compounds on the cholinergic (amphibians, insects), adrenergic (amphibian) and octopaminergic (insects) synaptic terminals is discussed.

In vitro assessment of the effects of cadmium and zinc on mammalian nerve fibres

Zinc and cadmium are environmental contaminants that have a wide range of effects on the nervous system, but zinc is also considered to be an important metal in the human body. In this study the effect of CdCl(2) and ZnCl(2), at concentrations of 50,150, 250 and 500 microM, on the nerve fibres of the sciatic nerve of the rat isolated in a three-chamber recording bath were studied. At the same concentrations, CdCl(2) and ZnCl(2) were found to have almost the same inhibitory effect on the compound action potential (CAP) of the nerve fibres. Their concentration-effect curves almost overlap and there was no significant difference in their EC(50) which for CdCl(2) is 250.1+/-18 microM (n=5) and for ZnCl(2) is 282.2+/-25 microM (n=5) correspondingly (P>0.05). The no-observed-effect concentration (NOEC) was estimated to be 50-100 microM for both metals. The identical inhibitory effect of both metals on the sciatic nerve fibres indicates a common mode of action which is related to their potential to generate reactive oxygen species (ROS).

Differences in the morphology of prothoracic and propodeal spiracles in three strains of Apis mellifera: possible relation to resistance against Acarapis woodi

SUMMARY The prothoracic and propodeal spiracles of three honey bee strains used commercially in Greece (from N Greece, N Italy and Slovenia) were examined with a light microscope in order to detect possible morphological differences between the strains. This is an attempt to explain the apparent resistance against Acarapis woodi shown by the Greek Macedonian (N Greece) strain but not by the others. The propodeal spiracles of honey bees from N Greece and N Italy were examined, in addition, using a scanning electron microscope. Certain dimensions, like the length and the area of aperture in the prothoracic spiracle, were found to be significantly larger in honey bees from N Italy and Slovenia compared with the honey bees from N Greece. The length of aperture was found to be 648 μm in the honey bees from N Greece, while it was 13% larger in the honey bees from N Italy and 10% larger in the honey bees from Slovenia; the area of the aperture was found to be 103000 μm2 in the honey bees from N Greece, while it was 26% larger in the honey bees from N Italy and 27% larger in the honey bees from Slovenia. The dimensions of the propodeal spiracle were found to be similar between the strains examined. The fact that the prothoracic spiracle of honey bees from N Greece was significantly smaller compared to other honey bee groups (from N Italy and Slovenia), is discussed as a possible mechanism for resistance developed by this strain against Acarapis woodi.

Inhibitory vs. protective effects of N-acetyl-l-cysteine (NAC) on the electromechanical properties of the spontaneously beating atria of the frog (Rana ridibunda): an ex vivo study.

The results of this study have shown that N-acetyl-l-cysteine (NAC), a compound used for protection of tissues or cell cultures against the deleterious effects of various environmental pollutants, has certain unusual effects on the contraction of the spontaneously beating atria of the frog isolated in saline (ex vivo): (1) NAC, 6.0 and 10.0mM, eliminated, in a concentration-dependent manner, the contractile properties of the atria (force and frequency) within minutes, without affecting its electrical properties; (2) the IC(50) of NAC for the force was 5.09+/-1.01 mM (n=6) [4.98-5.19 mM, 95% confidence interval (CI)], significantly lower than the IC(50) for the frequency, 6.15+/-1.01 mM, (6.02-6.29 mM, 95% CI), indicating that working atria cells are more sensitive to NAC than autorhythmic cells. The no-observed-effect concentration (NOEC) was 1-2mM; (3) the pattern of NAC-induced inhibition of electromechanical activity was similar to that of verapamil, an indication that NAC possibly affects L-type voltage-gated calcium channels; (4) NAC at 2mM protected against cadmium-induced inhibition of atria contraction. The IC(50) for cadmium was 17.9+/-1.1 microM (n=6) (16.9-19.0 microM, 95% CI), while in the presence of 2mM NAC, it became 123.3+/-1.0 microM (n=6) (114.8-132.4 microM, 95% CI). The same concentration of NAC failed to exert any protective effects against rotenone (5 microM)-induced inhibition of atria contraction. The protective effects of NAC are probably due to chelation of cadmium, rather than scavenging of oxidants.

The force of the spontaneously contracting zebrafish heart, in the assessment of cardiovascular toxicity: Application on adriamycin

The heart of the zebrafish has been used extensively to assess the cardiotoxic effect of compounds, using the frequency of heart contractions as the main index of cardiac response to drugs. In this study, the force and the frequency generated by the spontaneously contracting zebrafish heart, isolated in saline, were found to be 0.87 ± 0.05 mN and 1.54 ± 0.03 Hz (n=6) respectively within the first hour of recording. Both values of force and frequency remained constant for over 8h. The advantage of prolonged vitality in the assessment of cardiovascular toxicity was shown using the well-known anticancer drug adriamycin, which has severe cardiotoxic side effects. At 10.0 μM there was a 21.05 ± 4.42% (p=0.02, n=4) decrease in the force of contraction, while the frequency was not affected after 3h treatment (p>0.05). At 50.0 and 100.0 μM there was a 33.24 ± 3.0 and 46.6 ± 4.80% irreversible decrease in force (p<0.05, n=4), while a 18.02 ± 4.07% and 16.16 ± 4.07% reversible increase was observed in the frequency (p=0.02, n=4). These contradictory positive chronotropic and negative inotropic responses indicate the strong inhibitory effect of adriamycin on ventricular cardiomyocytes and its excitatory effects on auto-rhythmical pacemaker cells. If heart frequency was the only parameter used to assess the cardiotoxic effect of adriamycin, at the above range of concentrations, this compound would have been classified as non-cardiotoxic.