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Ecotoxicological bioassays have been widely utilized to evaluate the toxicity of substances to organisms. However, the main challenge for researchers is finding native species to assess the effects of pollutants on aquatic biota. The tropical Oligochaeta, Allonais inaequalis, can be used as a test organism in bioassays to understand the effects of toxicants on aquatic ecosystems and their impact on native aquatic biota. In this study, we tested four methodological designs to validate the use of our "Allonais inaequalis reproduction test" as an ecotoxicological bioassay. For each sample, the assay consisted of a bottle containing 10 mg of sterilized fine sand, 60 mL of dechlorinated tap water and 6 organisms, fed at the beginning of the test and again after 5 days. The assay was first established in a controlled environment and then used to evaluate a stressed environment containing one of the following three toxicants suggested by the OECD (2008) and Corbi et al. (2015): zinc chloride, copper sulfate, or potassium chloride. Our results showed that the best experimental design for reproduction analysis was a static, long-term bioassay, which lasted 10 days without aeration and allowed for the reproduction of multiple generations (10 ± 5 new organisms). The observed inhibition reproduction by toxicants (EC50 ranging between 0.2 mg L-1 and 1.36 g L-1) validated the methods used in this paper. The use of a reproduction endpoint is a new contribution to the ecotoxicological toolbox, examining responses from a native organism to predict the effects of pollutants in an aquatic environment.

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Xylose reductase enzyme (EC 1.1.1.21) produced by Candida guilliermondii in sugarcane bagasse was extracted by reversed micelles of N-benzyl-N-dodecyl-N-bis (2-hydroxyethyl) ammonium chloride cationic surfactant. An experimental design was employed to evaluate the influences of the following factors on the enzyme extraction: temperature, cosolvent, and surfactant concentration. A model was used to represent the enzyme recovery and fit of the experimental data. The extraction yielded a total recovery of 130%, and the purity increased 4.8-fold. This study demonstrates that liquid-liquid extraction by reversed micelles is a process able to recover and increase the enzymatic activity and purity of XR produced by C. guilliermondii.

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Tianeptine, an antidepressant drug enhancing 5-HT uptake, was given to pregnant rats in the last 15 days of gestation and different neurotransmitter receptors as well as 5-HT2 receptor-linked inositol phosphate formation were measured in the brains of the offspring. Prenatal exposure to tianeptine significantly decreased the density of 3H-imipramine binding sites in the cerebral cortex of the pups without affecting beta-adrenoceptors, serotonin 5-HT2 and 5-HT1B receptors or inositol phosphate formation after a 5-HT challenge. Striatal dopamine D2 receptors labelled with 3H-spiroperidol were not changed but an apparent increase in the affinity of dopamine was noticed in the pups prenatally exposed to the drug. The results show that the neurochemical profile of tianeptine markedly differs from that of most antidepressants.

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Substance P (SP) antiserum (500 micrograms protein) was administered to rats on the second day of life and the animals were sacrificed 3 months later. This treatment produced a loss in SP immunoreactive fibers in the dorsal horn of the spinal cord, in the substantia nigra and in the periaqueductal gray matter, when compared to control animals receiving a neonatal treatment of non-specific immunoglobulins. In the dorsal horn, the observed depletion was greater in the superficial layers, lamina I and lamina IIo. Immunoreactivity for Met-enkephalin was apparently unchanged by SP antiserum. Results of this study provide cytochemical evidence for a specific and lasting deleterious effect of SP antiserum on different SP-containing neuronal systems.

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Acute or chronic treatment of young or adult rats with chlorimipramine, tianeptine or iprindole, antidepressants with different effects on 5-HT uptake mechanisms, did not modify the density or the affinity of 5-HT1B receptors of the frontal cortex. No significant receptor change was found after prenatal exposure to these antidepressants. The lack of effect of the antidepressants was not related to the density of 5-HT1B receptors, which was significantly lower in younger animals.

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Substance P (SP) antiserum was administered to rats on the second day of life. Three months later, the content of SP was significantly decreased in the dorsal part of the spinal cord and in the periaqueductal gray matter of these animals, as compared to control rats receiving a neonatal treatment of non-specific immunoglobulins. Further, the levels of Met-enkephalin and 5-hydroxyindole acetic acid (5-HIAA) were concomitantly increased in the same regions. SP receptor binding sites and opioid receptors, which appear earlier in development, were not modified in the two regions studied. On the other hand, the antinociceptive response to intracerebroventricular (i.c.v.) injection of SP or of the synthetic enkephalin analog D-Ala2,D-Leu5-enkephalin, as well as the hypertensive response to i.c.v. SP were blocked. The results suggest that, after administration to newborn rats, the antiserum is able to penetrate into SP neurons, producing a long-lasting SP suppression and a subsensitivity to the pharmacological effects of the neuropeptide. The modifications in the content of Met-enkephalin and 5-HIAA are possibly compensatory changes which subserve the functionality of central cardiovascular and pain regulatory systems after the immunolesion.

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The implication that opioid peptides are involved in the action of the antidepressants imipramine and iprindole was investigated in mice by using the forced swimming test as an experimental model of depression. Both the drugs were found to shorten the immobility time in this test. This effect of imipramine and iprindole was reversed by the opiate antagonist naloxone. Moreover, when subeffective doses of either imipramine or iprindole were given together with an intracerebroventricular injection of an inhibitor of their degradation (thiorphan or bestatin), the immobility time was again decreased. Interestingly, the reduction of the time of immobility was found to be not related to the effect of the drugs on locomotor activity. These data might be taken as further evidence for the involvement of opioid peptides in the pharmacological action of antidepressant drugs.

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The tail flick assay was used to evaluate pain perception in mice treated acutely with either of the two classical antidepressant drugs (AD) imipramine or amitriptyline, or the atypical antidepressant iprindole. A sustained hypoalgesic effect, sensitive to the opiate antagonist naloxone, was detected for the AD used in this study. Administration of the aminopeptidase inhibitor bestatin or the enkephalinase blocker thiorphan made subeffective doses of AD hypoalgesic. This synergistic effect was reversed by naloxone. The antinociceptive action of the AD wore off in mice rendered tolerant to morphine by subcutaneous implantation of a pellet of the opiate. Subchronic treatment with p-chlorophenylalanine did not alter the effect of AD on pain perception, but in animals whose serotonin (5-HT) receptors were blocked by methysergide AD did not produce any change in pain threshold. It was concluded on the basis of these findings that short-chain opioids and 5-HT appear to have a role in the hypoalgesic effect of either classical or atypical AD.

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Substance P (SP), physalaemin, SP4-11, SP5-11 and the SP5-11 analog DiMe-C7 induce an antinociceptive effect in rats after intraventricular administration. Other tachykinins and the N-terminal fragments of SP are inactive. All antinociceptive peptides increase the Met-enkephalin efflux from slices of rat periaqueductal gray matter and their antinociceptive potency is correlated with their capacity to release Met-enkephalin. The results, discussed in the light of current theories on different tachykinin receptors, suggest that the SP-P receptor subtype may be involved in the control of noxious stimulation elicited by SP at supraspinal levels.

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Chronic (21 consecutive days) and not acute administration of typical (clomipramine, desipramine, amitriptyline) or atypical (iprindole, nomifensin) antidepressant drugs was found to provoke a selective increase in [Met5]enkephalin-like immunoreactivity ([Met5]ELI) in striatum and nucleus accumbens of rat brain. In parallel experiments, following chronic treatment with clomipramine, iprindole and nomifensin striatal [Leu5]enkephalin-like immunoreactivity ([Leu5]ELI) was also significantly enhanced. No variations in enzymatic activity of either enkephalinase or aminopeptidase were detected when assayed in several brain parts of animals chronically treated with antidepressants. Elevation of ELI in discrete regions of the brain might play a part in the mechanism of action of these centrally acting agents.

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