Long-term protective effect of PACAP in a fetal alcohol syndrome (FAS) model.

Prenatal ethanol exposure provokes teratogenic effects, due to oxidative stress and massive neuronal apoptosis in the developing brain that result in lifelong behavioral abnormalities. PACAP exerts anti-oxidative and neuroprotective activities on neuronal cells, and prevents ethanol neurotoxicity. The present study focused on the ability of PACAP to protect the brain of 30-day-old mice (P30) from prenatal alcohol exposure induced oxidative damage and toxicity. Pregnant mice were divided randomly into 4 groups, i.e. control group, ethanol group (1.5 g/kg ip daily injection), PACAP group (5 µg intrauterine daily injection) and an ethanol plus PACAP group. Offspring prenatally exposed to ethanol had decreased body weight and reduced cell survival. Moreover, production of ROS was sharply enhanced in the brain of prenatal ethanol-exposed animals, associated with an elevation in the activity of the antioxidant enzymes, and an increase of oxidative damages as shown by the accumulation of the lipid oxidation marker malondialdehyde and of protein carbonyl compounds. Intrauterine administration of PACAP during the gestational period restored the endogenous antioxidant system, prevented ROS overproduction and promoted the survival of dissociated cells from animals prenatally exposed to ethanol. Behavioral tests revealed that P30 animals exposed to ethanol during the prenatal period exhibited reduced motor activity, altered exploratory interest and increased anxiety. However, PACAP treatment significantly attenuated these behavioral impairments. This study demonstrates that PACAP exerts a potent neuroprotective effect against alcohol toxicity during brain development, and indicates that PACAP and/or PACAP analogs might be a useful tool for treatment of alcohol intoxication during pregnancy.

The pathological effects of Heminecrolysin, a dermonecrotic toxin from Hemiscorpius lepturus scorpion venom are mediated through its lysophospholipase D activity.

We have previously identified Heminecrolysin, a sphingomyelinase D (SMaseD), as the major protein responsible for the main pathological effects observed following Hemiscorpius (H.) lepturus scorpion envenomation. We aimed herein to further investigate the kinetics and molecular mechanisms triggered by Heminecrolysin to initiate hematological disorders and inflammatory reaction. We show that Heminecrolysin highly hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) and choline, with a Vmax = 1481 ± 51 µmol/min/mg and a Km = 97 ± 16.78 µM, at a much lesser extend sphingomyelin but not phosphatidylcholine substrates. Its lysophospholipase D (lysoPLD) catalytic efficiency, up to three orders of magnitude higher, comparatively to spider's SMaseDs (newly referred as phospholipases D; PLDs), could explain its strong hemolytic capacity. Chelating agents such as EDTA, EGTA, and 1, 10-phenantroline blocked Heminecrolysin-induced LPC hydrolysis at 98, 48, and 70% respectively. Hemolysis blockade occurs only when the toxin is added to erythrocytes in the presence of serum, source of LPC and complement, indicating that the production of LPA and the presence of complement are mandatory for hemolysis. Moreover, we show that Heminecrolysin efficiently binds to erythrocyte's membrane and provokes phosphatidylserine (PS) translocation without cleavage of glycophorin A, suggesting that, unlike spider's PLDs, complement was activated only via the classical pathway. Interestingly, Heminecrolysin was found to induce PS exposure on human nucleated Jurkat T cells, to stimulate secretion of the pro-inflammatory (TNF-α, IL-6), and anti-inflammatory (IL-10) cytokines by human monocytes, and to provoke a disseminated intravascular coagulation on chick embryo chorioallantoic membrane model system. Taken together, our results indicate that Heminecrolysin evokes the major characteristic clinical features of H. lepturus envenomation by using mainly its lysoPLD, rather than its SMaseD's, activity.