Ataluren treatment of patients with nonsense mutation dystrophinopathy.

INTRODUCTION: Dystrophinopathy is a rare, severe muscle disorder, and nonsense mutations are found in 13% of cases. Ataluren was developed to enable ribosomal readthrough of premature stop codons in nonsense mutation (nm) genetic disorders. METHODS: Randomized, double-blind, placebo-controlled study; males ≥ 5 years with nm-dystrophinopathy received study drug orally 3 times daily, ataluren 10, 10, 20 mg/kg (N=57); ataluren 20, 20, 40 mg/kg (N=60); or placebo (N=57) for 48 weeks. The primary endpoint was change in 6-Minute Walk Distance (6MWD) at Week 48. RESULTS: Ataluren was generally well tolerated. The primary endpoint favored ataluren 10, 10, 20 mg/kg versus placebo; the week 48 6MWD Δ=31.3 meters, post hoc P=0.056. Secondary endpoints (timed function tests) showed meaningful differences between ataluren 10, 10, 20 mg/kg, and placebo. CONCLUSIONS: As the first investigational new drug targeting the underlying cause of nm-dystrophinopathy, ataluren offers promise as a treatment for this orphan genetic disorder with high unmet medical need.

Interactive toxicity of usnic acid and lipopolysaccharides in human liver HepG2 cells.

Usnic acid (UA), a natural botanical product, is a constituent of some dietary supplements used for weight loss. It has been associated with clinical hepatotoxicity leading to liver failure in humans. The present study was undertaken to evaluate the interactive toxicity, if any, of UA with lipopolysaccarides (LPS), a potential contaminant of food, at low non-toxic concentrations. The human hepatoblastoma HepG2 cells were treated with the vehicle control and test agents, separately and in a binary mixture, for 24 h at 37°C in 5% CO2. After the treatment period, the cells were evaluated by the traditional biochemical endpoints of toxicity in combination with the toxicogenomic endpoints that included cytotoxicity, oxidative stress, mitochondrial injury and changes in pathway-focused gene expression profiles. Compared with the controls, low non-toxic concentrations of UA and LPS separately showed no effect on the cells as determined by the biochemical endpoints. However, the simultaneous mixed exposure of the cells to their binary mixture resulted in increased cytotoxicity, oxidative stress and mitochondrial injury. The pathway-focused gene expression analysis resulted in the altered expression of several genes out of 84 genes examined. Most altered gene expressions induced by the binary mixture of UA and LPS were different from those induced by the individual constituents. The genes affected by the mixture were not modulated by either UA or LPS. The results of the present study suggest that the interactions of low nontoxic concentrations of UA and LPS produce toxicity in HepG2 cells.

Effects of usnic acid exposure on human hepatoblastoma HepG2 cells in culture.

Usnic acid, a natural botanical product, is a constituent of some dietary supplements used for weight loss. It has been associated with clinical hepatotoxicity leading to liver failure in humans. The present study was undertaken for metabolism and toxicity evaluations of usnic acid in human hepatoblastoma HepG2 cells in culture. The cells were treated with the vehicle control and usnic acid at concentrations of 0-100 µm for 24 h at 37 °C in 5% CO2 . Following the treatment period, the cells were evaluated by biochemical and toxicogenomic endpoints of toxicity that included cytochrome P450 activity, cytotoxicity, oxidative stress, mitochondrial dysfunction and changes in pathway focused gene expression profiles. Usnic acid exposure resulted in increased P450 activity, cytotoxicity, oxidative stress and mitochondrial dysfunction in HepG2 cells. The pathway-focused gene expression analysis resulted in significantly altered expression of six genes out of a total of 84 genes examined. Of the six altered genes, three genes were up-regulated and three genes down-regulated. A marked up-regulation of one gene CCL21 associated with inflammation, one gene CCNC associated with proliferation and carcinogenesis and one gene UGT1A4 associated with metabolism as well as DNA damage and repair were observed in the usnic acid-treated cells compared with the vehicle control. Also a marked down-regulation of one gene CSF2 associated with inflammation and two genes (CYP7A1 and CYP2E1) associated with oxidative metabolic stress were observed in the usnic acid-treated cells compared with the control. The biomarkers used in this study demonstrate the toxicity of usnic acid in human hepatoblastoma HepG2 cells, suggesting an oxidative mechanism of action.

Comparative hepatotoxicity of deoxynivalenol in rat, mouse and human liver cells in culture.

The present study was undertaken to assess, in vitro, the hepatotoxic potential of the food-borne mycotoxin, deoxynivalenol (DON), using rat (Clone9 and MH1C1), mouse (NBL CL2) and human (WRL68 and HepG2) liver cells in culture. The cells were treated with DON for 24 h at 37 degrees C in 5% CO(2) at concentrations of 0-25 microg ml(-1). Following the treatment period, the cells were assayed for biochemical markers of hepatotoxicity that included three independent cytotoxicity assays, oxidative stress and mitochondrial dysfunction. Concentration-dependent cytotoxicity of DON was observed in each of the five different liver cells derived from three different species (rat, mouse and human) over the entire concentration range studied, beginning at 0.1 microg ml(-1). At these concentrations DON did not induce a biologically significant increase in oxidative stress in these liver cells, and showed a significant decrease in the mitochondrial function only in the rat liver MH1C1 cells compared with the control. The results of this in vitro study suggest that DON is a potential hepatotoxin for the rat, mouse and human liver cells in the concentration range tested in this study. The liver cells used in this study showed distinct endpoint-sensitivity to DON related to the species.

Free fatty acids profile of the fetal brain and the plasma, liver, brain and kidneys of pregnant rats treated with sodium arsenite at mid-organogenesis.

Free fatty acids (FFAs) are known to be markers of cellular membrane degradation through lipid peroxidation and are substrates for the production of reactive oxygen species (ROS). Oxidative stress, due to overproduction of ROS, may facilitate cellular insult by various toxicants. The ability of the rat conceptus to respond to toxic stress may be critical for normal development. In this study, the effects of the environmental toxicant sodium arsenite (NaAsO2) on FFAs were investigated after administering a single oral dose, in water and in a lipid medium, to pregnant rats on gestational day (GD) 10, a time point at mid-organogenesis. NaAsO2 was administered in deionized water (AsH2O) or in half and half dairy cream (AsHH) at a dose of 41 mg sodium arsenite (NaAsO2)/kg body weight. Control animals were treated with either dairy cream (HH) or deionized water (H2O). The animals were sacrificed on GD 20. The fetal brain and the maternal liver, brain, plasma and kidneys were harvested. The FFAs were extracted and analyzed by gas chromatography. In the liver, there was an increase of myristic acid (1200%), myristoleic acid (174%), palmitic acid (47%), elaidic acid (456%), oleic acid (165%) and docosahexaenoic acid (224%) in the AsH2O group as compared to the AsHH group. Oleic acid and arachidonic acid were increased by 192% and 900%, respectively, in the AsH2O group as compared to the H2O group, and myristic acid was decreased by 90% in the AsHH group as compared to the HH group. In the maternal brain, myristoleic acid was decreased by 91% in the AsH2O group as compared to the H2O group, and DHA increased by 148% in the AsHH group as compared to the HH group. In the fetal brain, myristic and stearic acids were decreased by 87% and 89%, respectively, in the AsH2O group as compared to the AsHH group. Myristic, stearic and arachidonic acids were increased by 411%, 265%, and 144%, respectively, in the AsHH group as compared to the HH group. There was no effect on the fatty acids concentrations in the kidney or plasma as compared to controls. This study shows that NaAsO2 produced a differential effect on the fatty acid profiles in rats. Further investigation is needed to elucidate the role of fatty acids in differential signaling and regulation by either the palmitoylation or myristoylation process of cellular functions in these target organs.

Validation of an in vitro model for assessment of androstenedione hepatotoxicity using the rat liver cell line clone-9.

Androstenedione, a naturally occurring steroid hormone, has been used to enhance athletic performance. Little is known, however, about its hepatotoxicity. Clone-9 cells, a non-transformed epithelial cell line that was originally isolated from normal liver of a 4-week old Sprague-Dawley rat, were used as an in vitro model to assess the hepatotoxic potential of androstenedione. The cultures were treated with androstenedione for 24 h at 37 degrees C in 5% CO(2) at concentrations of 0-100 microg ml(-1). After the treatment period, the cells and the culture supernatants were assayed for markers of cytotoxicity which included: release of liver enzymes, cell viability, cellular double-stranded DNA content, oxidative stress, steatosis, cellular ATP content, caspase-3 activity, the mitochondrial permeability transition and induction of cytochrome P450 activity. Significant concentration-dependent differences from control were observed in some endpoints at medium concentrations of 10 microg ml(-1) and above. These in vitro findings were compared with comparable endpoints obtained from an in vivo study of androstenedione toxicity in female Sprague-Dawley rats. Of the eight endpoints that could be compared between the two studies, only three (lipid accumulation, ATP depletion and P450 activity) appeared to be concordant. This suggests that, under the experimental conditions used, the clone-9 cells were not a good model for androstenedione hepatotoxicity.

Prooxidant activity and toxicity of nordihydroguaiaretic acid in clone-9 rat hepatocyte cultures.

Nordihydroguaiaretic acid (NDGA) is a polyphenol. It is present at high concentrations in the leaves of the evergreen desert shrub, Larrea tridentate (Creosote bush), which has a long history of medicinal use traditionally by the native Americans and Mexicans. It is generally believed that the antioxidant properties of NDGA are responsible for the medicinal value of this desert shrub. The clone-9 rat hepatocyte cultures were used as an in vitro model to assess the hepatotoxic potential of NDGA and to determine whether it exhibits any prooxidant activity. The hepatocyte cultures were treated with NDGA for 2 h at 37 degrees C at concentrations of 0-100 microM. After the treatment period the cells, the culture supernatants and cell lysates were assayed for evaluation of prooxidant activity and toxicity of NDGA. Oxidative stress level and oxidative cell injury as measured by the peroxidation of membrane lipids and DNA double-strand breaks were used to index prooxidant activity. Cytotoxicity as measured by the leakage of the liver enzyme lactate dehydrogenase (LDH) into the culture medium, mitochondrial function and extent of cell proliferation were used as the endpoints of toxicity. Significant concentration-dependent differences were observed in these biomarkers over the concentration range examined demonstrating the prooxidant activity and toxicity of NDGA in clone-9 rat hepatocyte cultures.

Hepatotoxicity of androstenedione in pregnant rats.

Androstenedione, a naturally occurring steroid hormone, is a dietary supplement used to enhance athletic performance. Little is known, however, about the safety of its use by young adults including women of child bearing age. To test the possible hepatotoxic effects of androstenedione use, this study was undertaken using a rat model. Pregnant rats (six rats/dose) were exposed to androstenedione in corn oil by gastric intubation at 0, 5, 30 or 60 mg/kg body weight/day beginning 2 weeks before mating and continuing through gestation day 19. On gestation day 20, blood and livers were collected from the pregnant rats for analysis of hepatotoxicity endpoints: serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), glutathione (GSH) and glutathione S-transferase (GST), total microsomal P450, nuclear DNA damage and lipid peroxidation. Under these experimental conditions, no significant differences were observed in any of these biomarkers over the concentration range examined.