Impairment of mitochondrial bioenergetics and permeability transition induction caused by major long-chain fatty acids accumulating in VLCAD deficiency in skeletal muscle as potential pathomechanisms of myopathy.

cis-5-Tetradecenoic (cis-5) and myristic (Myr) acids predominantly accumulate in patients affected by very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. They commonly manifest myopathy with muscular pain and rhabdomyolysis, whose underlying mechanisms are poorly known. Thus, in the present study we investigated the effects of cis-5 and Myr on mitochondrial bioenergetics and Ca2+ homeostasis in rat skeletal muscle. cis-5 and Myr decreased ADP-stimulated (state 3) and CCCP-stimulated (uncoupled) respiration, especially when mitochondria were supported by NADH-linked as compared to FADH2-linked substrates. In contrast, these fatty acids increased resting respiration (state 4). Similar effects were observed in skeletal muscle fibers therefore validating the data obtained with isolated mitochondria. Furthermore, cis-5 and Myr markedly decreased mitochondrial membrane potential and Ca2+ retention capacity that were avoided by cyclosporin A plus ADP and ruthenium red, indicating that cis-5 and Myr induce mitochondrial permeability transition (MPT). Finally, docosanoic acid did not disturb mitochondrial homeostasis, indicating selective effects for Myr and cis-5. Taken together, our findings indicate that major long-chain fatty acids accumulating in VLCAD deficiency behave as metabolic inhibitors, uncouplers of oxidative phosphorylation and MPT inducers. It is presumed that these pathomechanisms contribute to the muscular symptoms and rhabdomyolysis observed in patients affected by VLCAD deficiency.

Melanogenesis of methyl myristate loaded niosomes in B16F10 melanoma cells.

The objective of this study was to compare the charge effect of methyl myristate loaded in neutral (Brij 72/cholesterol at 7:3), cationic (Brij 72/cholesterol/dimethyl dioctadecyl ammonium bromide at 7:3:0.65) and anionic niosomes (Brij 72/cholesterol/dicetyl phosphate at 7:3:0.65) for physicochemical characteristics, cytotoxicity in fibroblasts and B16F10 melanoma cells as well as melanogenesis induction activity. The maximum loading and percentage entrapment of methyl myristate were 4.5, 90.68 +/- 7.95 in neutral; 11.0, 92.54 +/- 6.32 in cationic and 0.1% w/w, 74.43 +/- 1.86% in anionic niosomes, respectively. All methyl myristate loaded niosomes were in unilamellar structure under transmission electron microscope and in nanosize at initial and after 3-month storage. The percentages of methyl myristate remaining in all niosomes kept at 4 +/- 2, 30 +/- 2 and 45 +/- 2 degrees C for 3 months were about 82, 74 and 72%, respectively, while the dry free methyl myristate indicated 97.82 +/- 1.74, 96.56 +/- 2.91 and 91.39 +/- 4.32%, respectively. Blank neutral, blank cationic and methyl myristate loaded neutral and cationic niosomes exhibited moderate cytotoxicity in fibroblasts and B16F10 melanoma cells at 56.64 +/- 3.19, 59.72 +/- 1.51; 73.81 +/- 2.86, 82.51 +/- 0.20; 47.34 +/- 2.13, 52.67 +/-2.78 and 73.20 +/- 3.49, 84.34 +/- 2.75% cell viability, respectively. Blank anionic and methyl myristate loaded anionic niosomes indicated no cytotoxicity in both cells. Cytotoxic ratio of cell viability in normal and cancer cells of all niosomes indicated no toxic effect to normal cells. Methyl myristate loaded cationic niosomes demonstrated the highest melanin induction with tyrosinase activity of 1.42 and 1.70 folds of the control and 1.14 and 1.59 folds higher than theophylline, respectively. This study has suggested the potential of methyl myristate loaded cationic niosomes for canities treatment.

Long-chain 3-hydroxy fatty acids accumulating in LCHAD and MTP deficiencies induce oxidative stress in rat brain.

Accumulation of long-chain 3-hydroxy fatty acids is the biochemical hallmark of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies. These disorders are clinically characterized by neurological symptoms, such as convulsions and lethargy, as well as by cardiomyopathy and muscle weakness. In the present work we investigated the in vitro effect of 3-hydroxydodecanoic (3HDA), 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, which accumulate in these disorders, on important oxidative stress parameters in cerebral cortex of young rats in the hope to clarify the mechanisms leading to the brain damage found in patients affected by these disorders. It was first verified that these compounds significantly induced lipid peroxidation, as determined by increased thiobarbituric acid-reactive substances levels. In addition, carbonyl formation was significantly increased and sulfhydryl content decreased by 3HTA and 3HPA, which indicates that these fatty acids elicit protein oxidative damage. 3HTA and 3HPA also diminished the reduced glutathione (GSH) levels, without affecting nitrate and nitrite production. Finally, we observed that the addition of the antioxidants and free radical scavengers trolox and deferoxamine (DFO) was able to partially prevent lipid oxidative damage, whereas DFO fully prevented the reduction on GSH levels induced by 3HTA. Our present data showing that 3HDA, 3HTA and 3HPA elicit oxidative stress in rat brain indicate that oxidative damage may represent an important pathomechanism involved in the neurologic symptoms manifested by patients affected by LCHAD and MTP deficiencies.

Identification of light-independent inhibition of human immunodeficiency virus-1 infection through bioguided fractionation of Hypericum perforatum.

BACKGROUND: Light-dependent activities against enveloped viruses in St. John's Wort (Hypericum perforatum) extracts have been extensively studied. In contrast, light-independent antiviral activity from this species has not been investigated. RESULTS: Here, we identify the light-independent inhibition of human immunodeficiency virus-1 (HIV-1) by highly purified fractions of chloroform extracts of H. perforatum. Both cytotoxicity and antiviral activity were evident in initial chloroform extracts, but bioassay-guided fractionation produced fractions that inhibited HIV-1 with little to no cytotoxicity. Separation of these two biological activities has not been reported for constituents responsible for the light-dependent antiviral activities. Antiviral activity was associated with more polar subfractions. GC/MS analysis of the two most active subfractions identified 3-hydroxy lauric acid as predominant in one fraction and 3-hydroxy myristic acid as predominant in the other. Synthetic 3-hydroxy lauric acid inhibited HIV infectivity without cytotoxicity, suggesting that this modified fatty acid is likely responsible for observed antiviral activity present in that fraction. As production of 3-hydroxy fatty acids by plants remains controversial, H. perforatum seedlings were grown sterilely and evaluated for presence of 3-hydroxy fatty acids by GC/MS. Small quantities of some 3-hydroxy fatty acids were detected in sterile plants, whereas different 3-hydroxy fatty acids were detected in our chloroform extracts or field-grown material. CONCLUSION: Through bioguided fractionation, we have identified that 3-hydroxy lauric acid found in field grown Hypericum perforatum has anti-HIV activity. This novel anti-HIV activity can be potentially developed into inexpensive therapies, expanding the current arsenal of anti-retroviral agents.

[Destabilization of the cytosolic calcium level and cardiomyocyte death in the presence of long-chain fatty acid derivatives].

It has been shown using the fluorescent microscopy technique that long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, exert the most toxic effect on rat ventricular cardiomyoctes. The addition of 20-50 microM acylcarnitines increases calcium concentration in cytoplasm ([Ca2+]i) and causes cell death after the 4-8 min lag-period. This effect is independent on extracellular calcium and L-type calcium channel inhibitors. Free acids (myristic and palmitic acids) at a concentration of 300-500 microM have a little effect on [Ca2+]i within 30 min. We suggest that the toxic effect is due to the activation of sarcoplasmic reticulum calcium channels by acylcarnitines and resulting acyl-CoA. Mitochondria play a role of calcium-buffer system in these conditions. The calcium capacity of this buffer determines the lag-period. Phosphate increases the calcium capacity of mitochondrial and the lag-period. In the presence of rotenone and oligomycin the elevation of [Ca2+]i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondrial leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine.

Toxicity studies of tetradecanoic acid, 2-sulfo-, 1-methylester, sodium salt (C14-MES).

We evaluated the toxicity of tetradecanoic acid methyl ester sodium salt (C14-MES), a major component of fabric detergents, following the test guidelines of the Organization for Economic Cooperation and Development. The rat acute oral LD(50) was 1,000 mg/kg in males and 500 mg/kg in females. Applying the combined repeated dose and reproductive/developmental toxicity screening test (ReproTox), we exposed groups of Crj:CD (SD) IGS rats to C14-MES in the diet at concentrations of 0, 0.3, 0.6, or 1.2%. We observed decreases in fibrinogen levels and longer prothrombin time at the 1.2% treated level in females and decreases in serum triglyceride levels in both sexes at the 0.6% and 1.2% treatment levels, but the effects were not clinically significant. The no-observed-effect-level (NOEL) for repeated dose toxicity was 0.3% (175 mg/kg body weight/day for males, 249 for females). The NOEL for reproduction/developmental toxicity was 1.2% (740 mg/kg for males, 1039 for females). C14-MES was negative in the reverse gene mutation assay and the chromosomal aberration test and did not induce skin sensitization in the guinea pig maximization test. These data confirm that C14-MES is of low hazard potential.

Application of oligo-(14-amino-3,6,9,12-tetraoxatetradecanoic acid) lipid conjugates as steric barrier molecules in liposomal formulations.

Lipid conjugates of oligo-(14-amino-3,6,9,12-tetraoxatetradecanoic acid) (ATTAn) were synthesized as monodisperse analogues of poly(ethylene glycol) (PEG) derivatives used in liposomal drug delivery systems. The new lipids were shown to be at least equivalent to MePEGA-2000-DSPE in assays designed to evaluate the effectiveness of polymers as steric barrier molecules in liposomes. Liposomes containing 1-5% of ATTA8-DSPE (octamer) showed comparable long circulation behavior relative to PEG-2000-DSPE analogues. Surprisingly, the shorter ATTA4-DSPE (tetramer) appeared to be quite effective in reducing clearance. Liver enzyme levels and systemic single dose tolerability of ATTA8-DSPE liposomes were comparable to controls, suggesting that the new materials are nontoxic. Prolonged exposure of ATTA8-DSPE liposomes to splenocytes in vitro showed no evidence of mitogenicity relative to controls or MePEGA-2000-DSPE liposomes. ATTA8-DSPE was as effective as MePEGC-2000-DSPE in preventing complement activation by cationic liposome systems. Repeat dosage in vivo regimes in ICR mice using DSPC/cholesterol liposomes, with and without 5% ATTA8-DSPE and MePEGC-2000-DSPE, showed no evidence of enhanced clearance on successive doses. Splenocytes recovered after repeat doses showed no significant evidence of mitogenicity on restimulation with liposomes. Cellular differentiation and activation marker levels in splenocytes recovered after the fourth in vivo administration were at normal levels. These results suggest that ATTAn oligomers do not induce an immune response in isolation. It was demonstrated that ATTA8-DSPE could be used to replace PEG-lipids in the formulation of doxorubicin, plasmid DNA and oligonucleotides using a variety of formulation techniques. The study demonstrates that ATTAn oligomers can be safely and effectively used in place of poly(ethylene glycol) as well-defined biomaterials in liposomal applications where reproducible behavior is critical.

The ability of the rat to metabolize myristoyl-methionine: an acylamino acid with potentially useful antibacterial properties.

Two experiments with Sprague Dawley rats tested their ability to hydrolyse myristoyl-methionine (M-M) into myristic acid and L-methionine (M). In the first experiment, lasting for 3 days. male rats were orally administered [9,10-3H]myristoyl-L-[35S]methionine. The recovery of radioactivity was approximately 90% for both isotopes; 19% of the administered 3H was recovered in the urine and 16% in the faeces, while the recovered 35S activity was 13 and 12%, respectively. The balance of the radioactivity was found among the tissues, organs and blood. In the second experiment, male and female rats received soybean-based diets which were supplemented with either 0.305% M-M or 0.2% M (both diets contained equal amounts of M) for periods up to 4 weeks. The growth rate of the rats receiving the 0.305% M-M diets was slightly slower than that for the rats on the 0.2% M diet, but the difference was not statistically significant (P > 0.05). The M-M rats had a transitory decrease in feed consumption, suggesting that palatability may have contributed to the growth difference and that a somewhat greater amount of M-M was necessary for the rat to attain the same growth rate as that produced by 0.2% M. When the amount of dietary M-M was increased to 3.05% M-M, a greater reduction in feed consumption and body weight gain was observed. This latter diet was an initial attempt to study the potential toxicity of M-M. None of the haematological, clinical chemistry or organ weight data suggested that M-M was overtly toxic per se, but longer-term feeding studies are needed to evaluate the potential toxicity of M-M more fully.

Toxicity of myristic acid analogs toward African trypanosomes.

New drugs are needed for treatment of diseases caused by African trypanosomes. One possible target for chemotherapy is the biosynthesis of the glycosyl phosphatidyl-inositol (GPI) of this parasite's variant surface glycoprotein (VSG). Unlike mammalian GPIs, the diacylglycerol moiety of the VSG anchor contains only myristate (tetradecanoate), added in unique remodeling reactions. We previously found that 11-oxatetradecanoic acid [i.e., 10-(propoxy)decanoic acid] is selectively toxic to trypanosomes. We have now assayed 244 different fatty acid analogs, most with chain lengths comparable to that of myristate, for trypanocidal effects. In these assays we surveyed the effects on toxicity of systematic alterations in the analogs' steric, conformational, and hydrophobic properties. We also used three 3H-labeled oxatetradecanoic acids to explore the mechanism of analog action. Their incorporation into VSG correlated roughly with toxicity, although they also were incorporated into phospholipids and other proteins. Myristate analogs are useful for studying the mechanism of GPI myristolyation, and they are candidates for antitrypanosomal chemotherapy.

Trypanocidal activity of a myristic acid analog in axenic cultures of Trypanosoma evansi.

The growth of bloodstream forms of Trypanosoma evansi in axenic culture was inhibited by incubation with 11-oxatetradecanoic acid (O-11), an analog of myristic acid. Parasites isolated from Asia, Africa and South America were affected to a similar extent in measurements using three different assay systems concerned with different aspects of trypanosome growth and metabolism. The concentration of O-11 that inhibited trypanosome growth by 50% (LD50) was 3.7 +/- 0.2 microM as measured by direct counting of survivors using a haemocytometer, 5.1 +/- 2.0 microM in a colorimetric test based on the formation of a formazan product, and 8.8 +/- 3.7 microM by estimation of pyruvate. The activity of the drug was enhanced by the addition of fatty-acid-free bovine serum albumin as a carrier protein to the culture medium at an optimal concentration of 5 mg/ml. Increasing amounts of the donor horse serum used for routine maintenance of these cultures, which is normally the only source of myristic acid for these trypanosomes, also affected the toxicity of the drug, in this case increasing the LD50.

Safety and short-term toxicity of a novel cationic lipid formulation for human gene therapy.

Among the potential nonviral vectors for human gene therapy are DNA-liposome complexes. In a recent clinical study, this delivery system has been utilized. In this report, a novel cationic lipid, dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), has been substituted into the DNA-liposome complex with dioleoyl phosphatidylethanolamine (DOPE), which both improves transfection efficiencies and allows increased doses of DNA to be delivered in vivo. The safety and toxicity of this DNA-liposome complex has been evaluated in two species, mice and pigs. The efficacy of DMRIE/DOPE in inducing an antitumor response in mice after transfer of a foreign MHC has been confirmed. No abnormalities were detected after administration of up to 1,000-fold higher concentrations of DNA and lipid than could be tolerated in vivo previously. Examination of serum biochemical enzymes, pathologic examination of tissue, and analysis of cardiac function in mice and pigs revealed no toxicities related to this treatment. This improved cationic lipid formulation is well-tolerated in vivo and could therefore allow higher dose administration and potentially greater efficiency of gene transfer for gene therapy.

Inhibition of varicella-zoster virus replication by an inhibitor of protein myristoylation.

Inhibitors of myristoylation and analogues of myristic acid inhibit the replication of some retroviruses including human immunodeficiency virus, but no studies with other virus families have been reported. We have shown that replication of varicella-zoster virus (VZV) in tissue is inhibited by DL-2-hydroxymyristic acid at concentrations similar to those required for inhibition with acyclovir. Protein synthesis is not inhibited, but protein myristoylation is non-specifically reduced. Despite this lack of specificity, DL-2-hydroxymyristic acid inhibits VZV replication without apparent cytotoxicity. This is in agreement with our earlier suggestion that non-specific inhibitors of myristoylation could have antiviral effects without toxicity to cells due to the stability of cellular myristoylproteins. This supports suggestions that myristoylation inhibitors have potential as antiviral drugs against the many viruses that produce myristoylproteins.

The effects of myristyl gamma-picolinium chloride on the rabbit retina: morphologic observations.

PURPOSE: This study was designed to localize the site of action of myristyl gamma-picolinium chloride (MGP) in the rabbit retina and to evaluate the extent of the structural damage induced by the drug. METHODS: The structural damage was assessed at the light microscopic level in eyes treated with various concentrations of MGP at different time intervals after intravitreal injection of the drug. Glial fibrillary acidic protein (GFAP) immunoreactivity was tested in the same eyes and served as an index of retinal damage. RESULTS: The rabbit retinas, examined about 1 mo after MGP injection, exhibited loss of photoreceptors and thinning of the retina in the regions close to the site of injection; remote retinal areas appeared morphologically intact or only slightly affected. Immunocytochemical analysis demonstrated the presence of GFAP in Müller (glial) cells throughout the entire retina. When the effects of MGP were examined at short time intervals (24 and 72 hr) after injection, severe morphologic damage in areas adjacent to the site of drug injection developed in parallel with the electroretinographic findings. However, GFAP could not be demonstrated. CONCLUSIONS: MGP, the preservative used in Depo-Medrol (Upjohn, Kalamazoo, MI), is highly toxic to the rabbit retina.

The effects of Depo-Medrol preservative on the rabbit visual system.

Periocular injections of corticosteroids play an important role in the management of various ophthalmologic diseases. The Depo-Medrol vehicle, injected into the vitreous, was shown to be toxic to the lens and to the retina when applied at double strength. The authors examined the effects of Depo-Medrol and one of the components of its vehicle, myristyl-gamma-picolinium chloride (MGP), on the functional integrity of the rabbit visual system. Visual function was assessed objectively from the electroretinogram (ERG) and the visual evoked potential (VEP). The experimental drugs were injected into the vitreous humor of one eye while saline was injected into the fellow eye for control. Depo-Medrol did not produce any measurable effects on the ERG or the VEP. When MGP solutions were injected in concentrations at least twice as large as that in the Depo-Medrol, significant reductions in the light- and dark-adapted ERG responses were seen. The effects of the drug on the ERG responses was seen as early as 3 days postinjection and developed to its maximal level within 1-2 weeks. No ERG recovery was seen over a period of more than 2 months. The VEP, elicited by applying light stimuli to the experimental eye, was characterized by low amplitude and delayed implicit time compared with the response obtained from the control eye.

Toxicity of free fatty acids for cultured rat heart muscle and endothelioid cells. I. Saturated long-chain fatty acids.

Capric (C10:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0), stearic (C18:0) and arachidic (C20:0) acids were compared for their toxic effects upon cultured rat heart muscle and endothelioid cells. The free fatty acids (FFA) were found to albumin (6:1) and tested at 5 x 10(-5)M. Reduction of cell viability (51Cr release) and in situ mitochondrial and lysosomal labilization were used as indices of injury. Reduction in viability of both cell types was produced by palmitic, stearic or arachidic acids, but only after exposures of from 12 to 36 h. These FFA also produced needle-like cytoplasmic inclusions. Mitochondria and lysosomes were labilized after shorter exposures. Capric, lauric and myristic acids, were relatively non-toxic, and protected endothelioid cell lysosomes from labilization.

Endotoxic activity of complexes of myristic acid and proteins.

Complexes of myristic acid and bovine serum albumin, myristic acid and concanavalin A, beta-hydroxymyristic acid and concanavalin A, or dimethyl myristamide and concanavalin A are lethal for male BALB/c mice treated with mithramycin. Prior treatment of mice with myristic acid-protein complexes renders the animals resistant to a dose of bacterial endotoxin that is lethal for untreated animals. Prior treatment of mice with bacterial endotoxin renders them resistant to a combination of mithramycin and a complex of myristic acid and bovine serum albumin or dimethyl myristamide and concanvalin A that is lethal for untreated animals. These data indicate that a fatty acid is an important functional component of the endotoxin toxophore.