Reduced habituation of auditory evoked potentials indicate cortical hyper-excitability in Fragile X Syndrome.

Sensory hypersensitivities are common, clinically distressing features of Fragile X Syndrome (FXS). Preclinical evidence suggests this abnormality may result from synaptic hyper-excitability in sensory systems. This model predicts reduced sensory habituation to repeated stimulus presentation. Fourteen adolescents and adults with FXS and 15 age-matched controls participated in a modified auditory gating task using trains of 4 identical tones during dense array electroencephalography (EEG). Event-related potential and single trial time-frequency analyses revealed decreased habituation of the N1 event-related potential response in FXS, and increased gamma power coupled with decreases in gamma phase-locking during the early-stimulus registration period. EEG abnormalities in FXS were associated with parent reports of heightened sensory sensitivities and social communication deficits. Reduced habituation and altered gamma power and phase-locking to auditory cues demonstrated here in FXS patients parallels preclinical findings with Fmr1 KO mice. Thus, the EEG abnormalities seen in FXS patients support the model of neocortical hyper-excitability in FXS, and may provide useful translational biomarkers for evaluating novel treatment strategies targeting its neural substrate.

Effect of dicarboxylic (C6 and C9) acids on a human squamous carcinoma cell line in culture.

In tissue culture, azelaic acid (C9) has been shown to have an anti-proliferative and cytotoxic effect on human and murine malignant melanocytes, with inhibition of mitochondrial oxido-reductase enzymes and DNA synthesis, and damage to mitochondria. Recent reports of effects on differentiation of normal keratocytes have led to the present study of its effects on a squamous carcinoma cell line. Cells were exposed to single doses of disodium salts of azelaic (C9(2)Na) and adipic (C6(2)Na) acids at concentrations of 10(-2)M and 5 x 10(-2)M for 48 hrs. Only C9(2)Na at 5 x 10(-2) M for 4 hrs., and longer, significantly affected proliferation, and the cells exhibited massive swelling of mitochondria with loss of cristae. The results further confirm the probable value of azelaic acid as a general anti-tumoral agent rather than a specifically melanocytotoxic one. They could justify clinical studies on the effect of topical azelaic acid therapy on squamous cell carcinoma in vivo.

Effect of dicarboxylic acids (C6 and C9) on human choroidal melanoma in cell culture.

In cell culture, azelaic acid (C9) has been shown to have an antiproliferative and cytotoxic effect on human and murine malignant cutaneous melanocytes. Normal melanocytes are unaffected, as are normal choroidal melanocytes. Here, effects on cell kinetics and ultrastructure of cells of a human choroidal melanoma line have been studied. Cells were exposed to single doses of disodium salts of azelaic (C(9)2Na) and adipic (C(6)2Na) acids at concentrations of 10(-2) M and 5 X 10(-2) M for 48 hr. C(9)2Na at 5 X 10(-2) M had a significant effect on proliferation at 24 and 48 hr and this was not reversible on removal of diacid. At 5 X 10(-2) M for 24 hr, C(6)2Na had no effect and at 5 X 10(-2) M for 48 hr had an effect which was marginally significant, but reversible. Swelling and disruption of mitochondria was seen in cells exposed to C(9)2Na at 5 X 10(-2) M for 1 hr and longer, but even at 10(-1) M, cells exposed to C(6)2Na were minimally affected. The results could encourage further investigations of the feasibility of azelaic acid therapy for uveal and ocular adnexal melanoma.

Transmission and scanning electron microscopy and freeze-fracture replication of normal human melanocytes and human melanoma cells in tissue culture.

Basic LM, TEM, SEM, and FFR appearances of a pure line of normal human melanocytes derived from foreskin, and a human melanoma line, in cell culture are described. Normal melanocyte cultures exhibit side by side, cells of widely different melanogenic activities--possible clones--and melanosomes of bizarre shape and internal structure are frequent. Aggregates of melanosomes, with or without associated amorphous material, and with no discernible limiting membrane are present within many cells, and occasional simple specialised contacts occur between apposed cells. On replicas of plasma membrane of normal melanocytes, particle densities and diameters on P and E fracture faces were within the ranges for cells in general, and equivalent data for the melanoma cells were not significantly different. Similarly, there was no difference in density of distribution or diameter of nuclear pores between the normal and the tumoural cells.

Scanning electron microscopy of human and murine melanoma cells exposed to medium chain-length (C6-C12) dicarboxylic acids in tissue culture.

Human and murine (Harding-Passey and Cloudman) melanoma cells were exposed to various concentrations (1 x 10(-3) M-1 x 10(-1) M) of adipic (C6), azelaic (C9), and dodecanedioic (C12) acids for 1-6 hours in tissue culture, and the effects on shape and surface topography were examined by scanning electron microscopy. Effects, i.e., rounding up, concentration of microvilli, blebbing, and prominence of retraction fibrils were time and dose dependent, and for the same concentrations and exposure times, C12 had a greater effect than C9, and both a significantly greater effect than C6. These differential reactions to the three diacids parallel previously reported effects on cell kinetics and viability. The changes could be due to a prime effect on the cell membrane, or they might reflect phases of the cell cycle directed by action of the diacids on the nucleus; this latter seems unlikely. An effect on the cytoskeleton is possibly involved.

Observations on cell kinetics and viability of a human melanoma cell line exposed to dicarboxylic acids in tissue culture.

Cultures of human melanoma cell line B0008 were exposed to the disodium salts of azelaic acid (C9 2Na), adipic acid (C6 2Na) and dodecanediaic acid (C12 2Na) at 10(-2) M and 5 x 10(-2) M for 24 hrs. None of the diacid salts had a significant effect on growth rate or viability of the cells, at 10(-2) M for 24 hrs nor had C6 2Na any effect at 5 x 10(-2) M. At 5 x 10(-2) M for 24 hrs, both C9 2Na, and C12 2Na had a significant effect in reducing both growth and viability. These effects were accompanied by morphological evidence of cell death, and swelling of mitochondria and accumulation of lipid droplets within cytoplasm of still viable cells.

Effect of L-carnitine on cultured murine melanoma cells exposed to azelaic acid.

The cytotoxic effect of azelaic acid on murine melanoma cells in culture is due, at least in part, to an antimitochondrial action. We investigated the possibility that the addition of carnitine to the medium may increase the transport of azelaic acid into the mitochondria and thereby increase its cytotoxic effect. Using mitochondrial cross-sectional area measured from electron micrographs as a criterion for mitochondrial damage, we found that the addition of L-carnitine to the culture medium had no effect either alone or with a low (10(-3) M) concentration of azelaic acid. At a high concentration (5 X 10(-2) M) azelaic acid caused swelling and disruption of the mitochondria to such an extent that this was not increased by carnitine. At 10(-2) M azelaic acid, however, some swelling of the mitochondria occurred which was significantly increased by the addition of carnitine. This indicates that carnitine-mediated transport of the diacid into the mitochondria had occurred. We conclude that carnitine may reduce the time or concentration needed for azelaic acid to have a toxic effect on the malignant melanocyte.

Ultrastructural observations on the effect of azelaic acid on normal human melanocytes and a human melanoma cell line in tissue culture.

Azelaic acid has been shown clinically to have a cytotoxic effect on the abnormally active and malignant human melanocyte, but it has no apparent effect upon normal melanocytes. This difference in reactivity between normal and abnormal cells in vivo is further examined here in vitro. The disodium salt of azelaic acid (C(9)2Na) was added to pure and mixed cultures of normal human melanocytes and to cultured human melanoma cells, at 10(-3) M, 10(-2) M, 5 X 10(-2) M, and 10(-1) M for 1 and 6 h. Control cultures and cultures exposed to the same concentrations of the disodium salt of adipic acid (C(6)2Na) were also examined. No damage to cells of any line was observed with diacids at 10(-3) M or 10(-2) M up to 6 h. At 5 X 10(-2) M some mitochondria of melanoma cells appeared swollen. With C(6)2Na at 10(-1) M for I and 6 h, minimal swelling of mitochondria was observed in some cells of all lines. Pure normal melanocytes and melanocytes of mixed cultures exhibited greater swelling of mitochondria with 10(-1) M C(9)2Na at 1 and 6 h, but the mitochondria of the malignant melanocytes were massively swollen with destruction of cristae. Plasma and nuclear membranes and membranes of rough endoplasmic reticulum were intact, but Golgi membranes exhibited vesiculation. These results provide further evidence that azelaic acid damages the human malignant melanocyte and that one of its targets is the mitochondrion. Damage to normal melanocytes, found here, may be due to the fact that, in culture, they are more active than in intact epidermis.

Effect of dicarboxylic acids on Harding-Passey and Cloudman S91 melanoma cells in tissue culture.

Clinically, dicarboxylic acids have a cytotoxic effect on the abnormally hyperactive and malignant epidermal melanocyte, and diacids from C8 to C13 have been shown to inhibit mitochondrial oxidoreductases. Here, their effect on the growth kinetics and ultrastructure of murine melanoma cells in culture is examined. Cultures of Harding-Passey and Cloudman S91 melanoma cells were exposed to single doses of the disodium salts of C12, C9, and C6 (which does not significantly inhibit mitochondrial enzymes) dicarboxylic acids at concentrations of 10(-3) M to 10(-1) M. With C12 and C9, viability and cell proliferation over 3 days were significantly affected by concentrations greater than 10(-2) M. With exposure to C6 at 10(-1) M and to medium to which NaCl was added to produce equal osmolarity, the effect was much less. Electron microscopy of cells exposed to C9 at 10(-1) M for 1 h and 6 h revealed massive swelling of mitochondria with destruction of cristae, but plasma and nuclear membranes and membranes of endoplasmic reticulum were intact. Similar damage was not seen with C6 at 10(-1) M nor with equiosomolar NaCl. The results confirm (1) the cytotoxicity of dicarboxylic acids for malignant melanocytes, and (2) that the mitochondrion is a prime target for their action.

Analytical, ultrastructural, autoradiographic and biochemical studies on [3H]dicarboxylic acid added to cultures of melanoma cells.

Lentigo maligna and malignant melanoma can be treated by dicarboxylic acids (C9 and C12), which are competitive inhibitors of tyrosinase. We therefore studied the intracellular location and possible sites of action of dodecanedioic acid (C12) in murine melanoma cells, using EM autoradiography and biochemical analysis of lipid extracts by HPLC. Significant levels of radioactivity were found in the mitochondria and in the nuclei but not in association with membranes of rough endoplasmic reticulum, Golgi-associated endoplasmic reticulum, or Golgi apparatus, and not in coated vesicles or melanosomes. Biochemical analysis revealed that the diacid underwent beta-oxidation, which occurs only in mitochondria. The results suggest that the toxicity of dicarboxylic acids in melanoma cells is not related to anti-tyrosinase activity but may be due to interference with oxidoreductase enzymes in the mitochondria and possibly to inhibition of DNA synthesis in the nucleus.

Ultrastructural and biochemical observations on the effect of 4-hydroxyanisole plus tyrosinase on normal human melanocytes and keratocytes in tissue culture.

Cultures of melanocytes and keratocytes were exposed to 15 micrograms ml-1 tyrosinase and 4-hydroxyanisole (4-OHA) 5 X 10(-4) M to 5 X 10(-2) M for 1 to 24 h. No damage was suffered by either cell below 5 X 10(-3) M 4-OHA for 6 h, but higher concentrations and longer exposures extensively damaged both cells. Exposure of cells washed free of culture medium to tyrosinase and 4-OHA 1 X 10(-3) M for 1 h resulted also in extensive damage. This indicates that an early-formed toxic product of the reaction between tyrosinase and 4-OHA is inactivated by constituents of the medium. This was confirmed by Liquid Chromatography and Scanning Spectrophotometry which showed that a toxic 4-OHA quinone immediately reacted with nucleophilic substances in the medium resulting in products which, on accumulation, are probably responsible for the later (6 h plus) damage to melanocytes and keratocytes. A possible effect of allegedly specific melanocytotoxic drugs on keratocytes should always be borne in mind with tissue culture experiments.

The time sequence of response of the prolactin cells of the freshwater teleost, Poecilia reticulata, to an altered environmental salinity.

The time sequence of the ultrastructural changes in the prolactin cells of the adult guppy, Poecilia reticulata, was studied in freshwater fish transferred to 1/3 seawater for 1, 4, 11 and 28 days. The morphological changes were slight and only detectable by quantitative (morphometric) procedures. The secretory granules showed a "biphasic' response to the altered environmental salinity, but the volume density of the Golgi apparatus progressively declined throughout the experiment. After an initial decrease in the volume density of the RER, that organelle regained its original prominence by 11 days in 1/3 seawater. The volume density of the nucleus was markedly higher in fish 28 days after transfer to 1/3 seawater than in any other group. Cell volume estimations showed that a shrinking of the cytoplasm rather than a swelling of the nucleus accounted for the high volume density figure. The changes in the volume density of the mitochondria closely paralleled those of the RER. Profiles of exocytosed granules were rarely found in any of the groups, but were least frequent in fish kept in 1/3 seawater for 28 days, when dense (lysosomal?) bodies were most abundant. The quantitative methods high-lighted some discrepancies in the rate and magnitude of the changes shown by some organelles and have led the authors to suggest that during adaptation of fish to dilute seawater, synthesis and release may not be coupled processes in the prolactin cell of P. reticulata.