Lucia de Fatima Sobral Sampaio

Benzo(e)pyrene is a cytotoxic chemical to the eyes, while neurohormone melatonin may exhibit protective effects on this cytotoxicity. In the current study, we have investigated the cytotoxic effects of benzo(e)pyrene on the chicken embryonic optic cups formation and whether melatonin supplementation protects chicken embryos against this xenobiotic toxicity. Fertilized chicken eggs were incubated for 48 h and then, they were divided into different groups. These groups included basal (without any treatment), control (distilled water), benzo(e)pyrene, melatonin and benzo(e)pyrene + melatonin groups, respectively. The 10 µl of distilled water or same volume of solution containing treatment compounds were injected into the air sac of the chicken egg. After an additional 18 h of incubation, the chicken embryos were excised and analyzed. The cytotoxicity was measured by a colorimetric whole chick embryo trypan blue assay. In embryos from basal, control and melatonin (0.01, 1 and 100 µM) groups, the frequency of the embryos with normal optic cups was 100% and had no increase in the embryonic cell death observed in post excision. In contrast, the frequency of normal optic cups in the benzo(e)pyrene (0.02 to 1200 µM) groups was significantly reduced (log IC50= -4.24 ± 0.02, R2= 0.98) with concentration-responsive manner. In addition, an increase in the embryonic cell death was also observed (log IC50 = -7.23 ± 0.28; R2 = 0.63). Melatonin treatment dose-responsively inhibited the benzo(e)pyrene-induced optic cups abnormality by 22.35 ± 4.06, 76.38 ± 3.30 and 100 % at the concentrations of 0.01, 1 and 100 µM, respectively. This same phenomenon was also observed in benzo(e)pyrene-induced embryonic cell death, i.e., melatonin suppressed the embryonic cell death by 16.67 ± 4.17, 54.17 ± 4.17 and 100 % with the abovementioned concentrations, respectively. Thus, melatonin supplementation injected into the chicken eggs protected against the benzo(e)pyrene embryotoxicity. Different pathways can be involved in melatonin’s protective effects.

Calmodulin is vital for chick embryos morphogenesis in the incubation time 48 h to 66 h when the rudimentary C-shaped heart attains an S-shaped pattern and the optic vesicles develops to the optic cups. Melatonin is in the extraembryonic yolk sac of the avian egg. Melatonin binds calmodulin. The aim of this study was to investigate the melatonin functions in the formation of the chick embryo optic cups and S-shaped heart, by pharmacology and immunoassays methods. Mel1a melatonin receptor immunofluorescence was distributed in the optic cups and rudimentary hearts. Embryonated chicken eggs at 48 h of incubation were separated into basal, control, and drug-treated groups. The treatment was applied or not in the egg air sac. Embryos were excised from the 66 h incubation eggs and analyzed. Embryos from the basal, control (distilled water), melatonin and 6-chloromelatonin (melatonin receptors agonist) groups had regular optic cups and an S-shaped heart, while embryos from the calmidazolium (calmodulin inhibitor) group did not. Embryos from the luzindole (melatonin receptors antagonist) and prazosin (Mel1c melatonin receptor antagonist) groups did not have regular optic cups. Embryos from the 4-P-PDOT (Mel1b melatonin receptor antagonist) group did not have an S-shaped heart. Melatonin, 6-chloromelatonin, and forskolin prevented the appearing of the abnormal chick embryos from the calmidazolium, luzindole, prazosin, and 4-P-PDOT groups. However, 6-chloromelatonin and forskolin (adenylate cyclase enhancer) prevented only partially the appearing of embryos with defective eye cups from the calmidazolium group. Results suggested that melatonin modulated the chick embryo morphogenesis via calmodulin and membrane receptors.

NRH:quinone reductase (QR2) is present in the retinas of embryonic and post‐hatched (PH) chicks. 5‐Methoxycarbonylamino‐N‐acetyltryptamine (5‐MCA‐NAT) is a QR2 ligand that increases cAMP levels in developing retinas, but it does not affect cAMP levels in CHO‐QR2 cells. The dopamine quinone reductase activity of QR2 retrieves dopamine, which increases cAMP levels in developing retinas. The objective of the present study was to investigate whether 5‐MCA‐NAT increases endogenous dopamine levels in retinas from chick embryos and post‐hatched chicks. Endogenous dopamine was measured by enzyme‐linked immunosorbent assay (ELISA). 5‐MCA‐NAT increased retinal endogenous dopamine levels at all developmental stages studied and in PH chicks (−log EC50 = 11.62 ± 0.34 M). This effect was inhibited by non‐selective antagonists of receptors and melatonin binding sites N‐acetyl‐2‐benzyltryptamine (luzindole, 5 μM), but it was not inhibited by the Mel1b melatonin receptor antagonist 4‐phenyl‐2‐propionamidotetralin (4‐P‐PDOT, 10 nM). The QR2 cosubstrate, N‐methyl‐dihydronicotinamide (NMH) (−log EC50 = 6.74 ± 0.26 M), increased endogenous dopamine levels in controls and in retinas stimulated with 5‐MCA‐NAT (3 nM). The QR2 inhibitor benzo[e]pyrene inhibited endogenous dopamine levels in both control (−log IC50 = 7.4 ± 0.28 M) and NMH‐stimulated (at 100 nM and 1 μM benzo[e]pyrene concentrations) retinas. Theoretical studies using Molegro Virtual Docking software corroborated these experimental results. We conclude that 5‐MCA‐NAT increases the level of endogenous dopamine via QR2. We suggest that this enzyme triggers double reduction of the dopamine quinone, recovering dopamine in retinal development.

Melatonin inclusion into in vitro oocyte maturation (IVM) protocols has been suggested because it possesses a powerful free radical scavenger capability that improves the quality of the oocyte used in in vitro embryo production (IVP). The aim of our study was to investigate the presence of melatonin membrane receptors (MT1and MT2) and MT3, which is the melatonin binding site of NQO2 enzyme, in both oocytes and hatched blastocysts to consider an additional subcellular mechanism responsible for the effects of melatonin on IVP.

Rosewood oil (RO) (Aniba rosaeodora Ducke) is rich in linalool, a monoterpene alcohol, which has well studied anxiolytic, sedative and anticonvulsant effects. The inhibition of the increases in cAMP protects against seizures in a diversity of models of epilepsy. In this paper, the principal aim was to investigate the effects of RO, (±)‐linalool and (‐)‐linalool) on adenylate cyclase. They were tested in chick retinas and forskolin was used to stimulate the enzyme target. The phosphodiesterase inhibitor, 4‐(3‐butoxy‐4‐methoxybenzyl)‐imidazolidin‐2‐one, and the non‐selective adenosine receptor antagonist 3‐isobutyl‐methyl‐xanthine (IBMX), were used to control the participation of phosphodiesterase and adenosine receptors in the resulting effects, respectively. The cAMP accumulation was measured by enzyme immune assay (EIA). Rosewood oil, (‐)‐linalool and (±)‐linalool inhibited exclusively the cAMP accumulation stimulated by forskolin, even when adenosine receptors were blocked with IBMX. The IC₅₀ values (in μ m concentration range) calculated from their concentration response–curves were not statistically different, however, the compounds presented a different relative efficacy. These results extend the range of subcellular mechanisms underlying the relaxant action of linalool on the central nervous system. Copyright © 2011 John Wiley & Sons, Ltd.

We have previously shown that melatonin influences the development of α8 nicotinic acetylcholine receptor (nAChR) by measurement of the acetylcholine‐induced increase in the extracellular acidification rate (ECAR) in chick retinal cell cultures. Cellular differentiation that takes place between DIV (days in vitro) 4 and DIV 5 yields cells expressing α8 nAChR and results in a significant increase in the ECAR acetylcholine‐induced. Blocking melatonin receptors with luzindole for 48 h suppresses the development of functional α8 nAChR. Here we investigated the time window for the effect of melatonin on retinal cell development in culture, and whether this effect was dependent on an increase in the expression of α8 nAChR. First, we confirmed that luzindole was inhibiting the effects of endogenous melatonin, since it increases 2‐[¹²⁵I] iodomelatonin (23 pM) binding sites density in a time‐dependent manner. Then we observed that acute (15, 60 min, or 12 h) luzindole treatment did not impair acetylcholine‐induced increase in the ECAR mediated by activation of α8 nAChR at DIV 5, while chronic treatment (from DIV 3 or DIV 4 till DIV 5, or DIV 3.5 till DIV 4.5) led to a time‐dependent reduction of the increase in the acetylcholine‐induced ECAR. The binding parameters for [¹²⁵I]‐α‐bungarotoxin (10 nM) sites in membrane were unaffected by melatonin suppression that started at DIV 3. Thus, melatonin surges in the time window that occurs at the final stages of chick retinal cell differentiation in culture is essential for development of the cells expressing α8 nAChR subtype in full functional form.

Luzindole is an unselective antagonist of the melatonin receptors and melatonin's other binding sites, although some exceptions have been observed in chick retinal neurodevelopment, where this unselective antagonist does not block melatonin's inhibitory effect on the adenylate cyclase enzyme, probably due to the presence of some other melatonin receptor(s) or binding site(s). The present study investigated the modulation of cyclic adenosine 3'-5'-monophosphate (cAMP) levels via MT3 melatonin-binding sites, located within the QR2 (dihydronicotinamide riboside: quinone oxidoreductase 2) enzyme, by observing the response to luzindole. Embryonic and post-hatch retinas, incubated with a selective agonist for the MT3 melatonin-binding site 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT, 10 or 100 nM), had an increase in cAMP accumulation relative to control retinas. Luzindole (5microM) inhibited the 5-MCA-NAT stimulatory effect at all ages tested. The agonist 5-MCA-NAT enhanced the melatonin inhibitory effect on cAMP levels stimulated by forskolin (5microM), but not the stimulatory forskolin effect. The results suggest that MT3 melatonin-binding sites are present in embryonic and post-hatch chick retinas and that luzindole more selectively blocks the 5-MCA-NAT effect on cAMP accumulation than it blocks the melatonin inhibitory effect via G protein-coupled receptors in chick retinal neurodevelopment.

During vertebrate neurodevelopment, neuritogenesis and synaptogenesis are modulated by intracellular cAMP rises. Melatonin, which is implicated in neuronal differentiation, mainly inhibits this pathway. Here, an investigation about the profile of this effect during the vertebrate neurodevelopment is reported. In the embryonic chick retinas at days 8, 12, 14, 16 and at 2 days post‐hatched (E8, E12, E14, E16 and PH), those control embryonic retinas incubated only with the phosphodiesterase inhibitor at days corresponding to commencement of neuronal differentiation (E8, E12) and PH, presented cAMP levels inhibited by melatonin. While the cAMP accumulation stimulated by forskolin was inhibited in the embryonic retinas at all testing days. Neither the unselective antagonist N‐acetyl‐2‐benziltryptamine (luzindole) nor the selective Mel_1b antagonist 4‐phenyl‐2‐propionamidotetralin (4‐P‐PDOT) blocked the melatonin concentration‐dependent inhibitory effect on cAMP accumulation in the retinas initiating differentiation (E7 − E9), suggesting a tight binding between melatonin and their receptors. However, 4‐P‐PDOT competitively reverted the melatonin effect on cAMP stimulated by forskolin during synaptogenesis stages. Together, the melatonin effect on cAMP levels in chick retina, which is mainly through melatonin receptors, is depending on the developmental period observed, probably taking part in the mechanisms surrounding the melatonin action on neuronal differentiation.

The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5), while the Bmax value for [125I]-alpha-bungarotoxin was reduced. Despite the presence of alpha8-like immunoreactivity at DIV4, functional responses mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 microM) was essentially without effect at DIV4 and DIV5, while dihydro-ss-erythroidine (10-100 microM) blocked the response to acetylcholine (3.0 nM-2.0 microM) only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the alpha-bungarotoxin-sensitive response at DIV5. Therefore, alpha-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an alpha-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by alpha-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express alpha-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.