Acute toxicological studies of the main organosulfur compound derived from Allium sp. intended to be used in active food packaging.

Some plant extracts have been proposed as potential alternative to the use of synthetic preservatives in the food industry. Among those, extracts from Allium species exhibit interesting antimicrobial and antioxidant properties for the food packaging industry. The present work aims to assess the usefulness and potential safety of the major organosulfur compound present in a commercial Allium sp. extract (PROALLIUM AP®), namely propyl thiosulfinate oxide (PTSO). For this purpose, its antimicrobial activity was studied in a wide range of microorganisms. Moreover, cytotoxicity and ultrastructural cellular damages caused by PTSO were studied in two human cell lines, Caco-2 and HepG2, being the colonic cells more sensitive to this compound. Finally, the protective role of PTSO against an induced oxidative situation was evaluated in the human intestinal Caco-2 cells. The results revealed damage at high concentration, although no significant adverse effects were recorded for the concentration to be used in food packaging. Moreover, the in vivo study also revealed the potential safety use at the established concentrations. In addition, the antimicrobial properties and the antioxidant role of PTSO were confirmed. Therefore, this compound could be considered as a good natural alternative to synthetic preservatives used in the food packaging industry.

Differential Changes in Expression of Stress- and Metabolic-Related Neuropeptides in the Rat Hypothalamus during Morphine Dependence and Withdrawal.

Chronic morphine treatment and naloxone precipitated morphine withdrawal activates stress-related brain circuit and results in significant changes in food intake, body weight gain and energy metabolism. The present study aimed to reveal hypothalamic mechanisms underlying these effects. Adult male rats were made dependent on morphine by subcutaneous implantation of constant release drug pellets. Pair feeding revealed significantly smaller weight loss of morphine treated rats compared to placebo implanted animals whose food consumption was limited to that eaten by morphine implanted pairs. These results suggest reduced energy expenditure of morphine-treated animals. Chronic morphine exposure or pair feeding did not significantly affect hypothalamic expression of selected stress- and metabolic related neuropeptides - corticotropin-releasing hormone (CRH), urocortin 2 (UCN2) and proopiomelanocortin (POMC) compared to placebo implanted and pair fed animals. Naloxone precipitated morphine withdrawal resulted in a dramatic weight loss starting as early as 15-30 min after naloxone injection and increased adrenocorticotrophic hormone, prolactin and corticosterone plasma levels in morphine dependent rats. Using real-time quantitative PCR to monitor the time course of relative expression of neuropeptide mRNAs in the hypothalamus we found elevated CRH and UCN2 mRNA and dramatically reduced POMC expression. Neuropeptide Y (NPY) and arginine vasopressin (AVP) mRNA levels were transiently increased during opiate withdrawal. These data highlight that morphine withdrawal differentially affects expression of stress- and metabolic-related neuropeptides in the rat hypothalamus, while relative mRNA levels of these neuropeptides remain unchanged either in rats chronically treated with morphine or in their pair-fed controls.

Changes in metabolic-related variables during chronic morphine treatment.

To reveal neuroendocrine/neurochemical changes that are responsible for the robust metabolic alterations seen during chronic morphine treatment we followed hormonal-, transcriptional- and behavioral changes during chronic morphine administration in adult male Wistar rats. Animals were implanted with increasing amount of slow release morphine tablets for 8 days. Morphine treated animals gain significantly less weight than placebo implanted controls. This weight loss is due to the dramatic decrease in the food intake and caloric efficiency in the first days of drug administration and to the lasting disregulated feeding pattern. Changes in feeding behavior included increase of diurnal and decrease of nocturnal feeding frequency in morphine treated rats. Significantly less leptin and insulin plasma levels were detected in morphine implanted animals than in placebo implanted controls, while adiponectin and ACTH concentration remain unchanged. Morphine treated rats display an increase of FosB/Delta FosB immunoreactivity at brain sites that have been implicated regulation of food intake and energy expenditure, including hypothalamic arcuate, paraventricular and ventromedial nuclei and in the lateral hypothalamic area as well as in the caudal brainstem. However, morphine-induced long-term metabolic alterations were not accompanied with any significant changes in the expression of anorexigenic neuropeptides POMC and CART in the hypothalamus and in the brainstem. The disregulated feeding pattern was not reflected in changes of orexin transcription, however, a compensatory upregulation was revealed in hypothalamic NPY expression.

Elevated glucocorticoid levels are responsible for induction of tyrosine hydroxylase mRNA expression, phosphorylation, and enzyme activity in the nucleus of the solitary tract during morphine withdrawal.

Chronic opiate exposure induces neurochemical adaptations in the noradrenergic system. Enhanced responsiveness of the hypothalamo-pituitary-adrenal axis after morphine withdrawal has been associated with hyperactivity of ascending noradrenergic input from the nucleus of the solitary tract (NTS-A(2)) cell group to the hypothalamic paraventricular nucleus (PVN). This study addressed the role of morphine withdrawal-induced corticosterone (CORT) release in regulation of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis in adrenalectomized (ADX) rats supplemented with low CORT pellet (ADX plus CORT). Present results show that in sham-ADX rats, noradrenergic neurons in the NTS-A(2) became activated during morphine withdrawal, as indicated by increased TH mRNA expression. However, this induction of TH expression is not detected in ADX plus CORT rats that are unable to mount CORT secretory response to morphine withdrawal. Total TH protein levels were elevated in the NTS-A(2) from sham-operated rats during morphine dependence and withdrawal, whereas we did not find any alteration in ADX plus CORT animals. Furthermore, high levels of TH phosphorylated (activated) at Ser31 (but not at Ser40) were found in the A(2) area from sham-morphine withdrawn rats. Consistent with these effects, we observed an increase in the enzyme activity of TH in the PVN. However, induction of morphine withdrawal to ADX plus CORT animals did not alter the phosphorylation (activation) of TH in NTS-A(2) and decreased TH activity in the PVN. These results suggest the existence of a positive reverberating circle in which elevated glucocorticoids during morphine abstinence play a permissive role in morphine withdrawal-induced activation of noradrenergic pathway innervating the PVN.

Regulation of serine (Ser)-31 and Ser40 tyrosine hydroxylase phosphorylation during morphine withdrawal in the hypothalamic paraventricular nucleus and nucleus tractus solitarius-A2 cell group: role of ERK1/2.

Our previous studies have shown that naloxone-induced morphine withdrawal increases the hypothalamic-pituitary-adrenocortical (HPA) axis activity, which is dependent on a hyperactivity of noradrenergic pathways [nucleus tractus solitarius (NTS) A(2)] innervating the hypothalamic paraventricular nucleus (PVN). Short-term regulation of catecholamine biosynthesis occurs through phosphorylation of tyrosine hydroxylase (TH), which enhances enzymatic activity. In the present study, the effect of morphine withdrawal on site-specific TH phosphorylation in the PVN and NTS-A(2) was determined by quantitative blot immunolabeling and immunohistochemistry using phosphorylation state-specific antibodies. We show that naloxone-induced morphine withdrawal phosphorylates TH at Serine (Ser)-31 but not Ser40 in PVN and NTS-A(2), which is associated with both an increase in total TH immunoreactivity in NTS-A(2) and an enhanced TH activity in the PVN. In addition, we demonstrated that TH neurons phosphorylated at Ser31 coexpress c-Fos in NTS-A(2). We then tested whether pharmacological inhibition of ERK activation by ERK kinase contributes to morphine withdrawal-induced phosphorylation of TH at Ser31. We show that the ability of morphine withdrawal to stimulate phosphorylation at this seryl residue is reduced by SL327, an inhibitor of ERK(1/2) activation. These results suggest that morphine withdrawal increases noradrenaline turnover in the PVN, at least in part, via ERK(1/2)-dependent phosphorylation of TH at Ser31.