HEPATOTOXICITY ASSESSMENT OF THE AZO DYES DISPERSE ORANGE 1 (DO1), DISPERSE RED 1 (DR1,) AND DISPERSE RED 13 (DR13) IN HEPG2 CELLS

During the dyeing process in baths approximately 10 to 15% of the dyes used are lost and reach industrial effluents, thus polluting the environment. Studies showed that some classes of dyes, mainly azo dyes and their by-products, exert adverse effects on humans and local biota, since the wastewater treatment systems and water treatment plants were found to be ineffective in removing the color and reducing toxicity of some dyes. In the present study, the toxicity of the azo dyes disperse orange 1 (DO1), disperse red 1 (DR1), and disperse red 13 (DR13) was evaluated in HepG2 cells grown in monolayers or in three dimensional (3D) culture. Hepatotoxicity of the dyes was measured using 3-(4,5-dimethylthiazol-2yl)2,5-diphenyltetrazolium (MTT) and cell counting kit 8 (CCK-8) assays after 24, 48, and 72 h of incubation of cells with 3 different concentrations of the azo dyes. The dye DO1 only reduced the mitochondrial activity in HepG2 cells grown in a monolayer after 72 h incubation, while the dye DR1 showed this deleterious effect in both monolayer and 3D culture. In contrast, dye DR13 decreased the mitochondrial activity after 24, 48, and 72 h of exposure in both monolayer and 3D culture. With respect to dehydrogenase activity, only the dye DR13 diminished the activity of this enzyme after 72 h of exposure in both monolayer and 3D culture. Our results clearly demonstrated that exposure to the studied dyes induced cytotoxicity in HepG2 cells.

CYP2E1 genotype and isoniazid-induced hepatotoxicity in patients treated for latent tuberculosis

OBJECTIVE: To determine whether pharmacogenetic tests such as N-acetyltransferase 2 (NAT2) and cytochrome P450 2E1 (CYP2E1) genotyping are useful in identifying patients prone to antituberculosis drug-induced hepatotoxicity in a cosmopolite population. METHODS: In a prospective study we genotyped 89 patients treated with isoniazid (INH) for latent tuberculosis. INH-induced hepatitis (INH-H) or elevated liver enzymes including hepatitis (INH-ELE) was diagnosed based on the clinical diagnostic scale (CDS) designed for routine clinical practice. NAT2 genotypes were assessed by fluorescence resonance energy transfer probe after PCR analysis, and CYP2E1 genotypes were determined by PCR with restriction fragment length polymorphism analysis. RESULTS: Twenty-six patients (29%) had INH-ELE, while eight (9%) presented with INH-H leading to INH treatment interruption. We report no significant influence of NAT2 polymorphism, but we did find a significant association between the CYP2E1 *1A/*1A genotype and INH-ELE (OR: 3.4; 95% CI:1.1-12; p = 0.02) and a non significant trend for INH-H (OR: 5.9; 95% CI: 0.69-270; p = 0.13) compared with other CYP2E1 genotypes. This test for predicting INH-ELE had a positive predictive value (PPV) of 39% (95% CI: 26-54%) and a negative predictive value (NPV) of 84% (95% CI: 69-94%). CONCLUSION: The genotyping of CYP2E1 polymorphisms may be a useful predictive tool in the common setting of a highly heterogeneous population for predicting isoniazid-induced hepatic toxicity. Larger prospective randomized trials are needed to confirm these results.

Severe hepatotoxicity following ingestion of Herbalife nutritional supplements contaminated with Bacillus subtilis

BACKGROUND/AIMS: Nutritional supplements are widely used. Recently, liver injury after consumption of Herbalife preparations was reported but the underlying pathogenesis remained cryptic. METHODS: Two patients presented with cholestatic hepatitis and pruritus, and cirrhosis, respectively. Viral, alcoholic, metabolic, autoimmune, neoplastic, vascular liver diseases and synthetic drugs as the precipitating causes of liver injury were excluded. However, both patients reported long-term consumption of Herbalife products. All Herbalife products were tested for contamination with drugs, pesticides, heavy metals, and softeners, and examined for microbial contamination according to standard laboratory procedures. Bacteria isolated from the samples were identified as Bacillus subtilis by sequencing the 16S rRNA and gyrB genes. RESULTS: Causality between consumption of Herbalife products and disease according to CIOMS was scored "probable" in both cases. Histology showed cholestatic and lobular/portal hepatitis with cirrhosis in one patient, and biliary fibrosis with ductopenia in the other. No contamination with chemicals or heavy metals was detected, and immunological testing showed no drug hypersensitivity. However, samples of Herbalife products ingested by both patients showed growth of Bacillus subtilis of which culture supernatants showed dose- and time-dependent hepatotoxicity. CONCLUSIONS: Two novel incidents of severe hepatic injury following intake of Herbalife products contaminated with Bacillus subtilis emphasize its potential hepatotoxicity.

Serum metabolomics reveals irreversible inhibition of fatty acid beta-oxidation through the suppression of PPARalpha activation as a contributing mechanism of acetaminophen-induced hepatotoxicity

Metabolic bioactivation, glutathione depletion, and covalent binding are the early hallmark events after acetaminophen (APAP) overdose. However, the subsequent metabolic consequences contributing to APAP-induced hepatic necrosis and apoptosis have not been fully elucidated. In this study, serum metabolomes of control and APAP-treated wild-type and Cyp2e1-null mice were examined by liquid chromatography-mass spectrometry (LC-MS) and multivariate data analysis. A dose-response study showed that the accumulation of long-chain acylcarnitines in serum contributes to the separation of wild-type mice undergoing APAP-induced hepatotoxicity from other mouse groups in a multivariate model. This observation, in conjunction with the increase of triglycerides and free fatty acids in the serum of APAP-treated wild-type mice, suggested that APAP treatment can disrupt fatty acid beta-oxidation. A time-course study further indicated that both wild-type and Cyp2e1-null mice had their serum acylcarnitine levels markedly elevated within the early hours of APAP treatment. While remaining high in wild-type mice, serum acylcarnitine levels gradually returned to normal in Cyp2e1-null mice at the end of the 24 h treatment. Distinct from serum aminotransferase activity and hepatic glutathione levels, the pattern of serum acylcarnitine accumulation suggested that acylcarnitines can function as complementary biomarkers for monitoring the APAP-induced hepatotoxicity. An essential role for peroxisome proliferator-activated receptor alpha (PPARalpha) in the regulation of serum acylcarnitine levels was established by comparing the metabolomic responses of wild-type and Ppara-null mice to a fasting challenge. The upregulation of PPARalpha activity following APAP treatment was transient in wild-type mice but was much more prolonged in Cyp2e1-null mice. Overall, serum metabolomics of APAP-induced hepatotoxicity revealed that the CYP2E1-mediated metabolic activation and oxidative stress following APAP treatment can cause irreversible inhibition of fatty acid oxidation, potentially through suppression of PPARalpha-regulated pathways.

{\it In vivo\/} magnetic resonance studies of experimental liver disease: Carbon tetrachloride hepatotoxicity and alcohol-induced fatty liver in rat

Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) were used to non-invasively determine if cirrhosis induced by carbon tetrachloride (CCl$\sb4$) and phospholipase-D (PLD) could be distinguished from fatty infiltration in rat. MRS localization and water suppression methods were developed, implemented and evaluated in terms of their application to in vivo proton NMR studies of experimental liver disease. MRS studies were also performed to quantitate fatty infiltration resulting from carbon tetrachloride (CCl$\sb4$) or alcohol (ethanol) administration and the MRS results were confirmed using biochemical total lipid analysis and histology. $\rm T\sb1$ weighted MR images acquired weekly, 48 hours post administration, demonstrated only a slight increase in overall liver intensity with CCl$\sb4$ or alcohol administration, which is consistent with previously reported results. The MR images were able to detect nodules resulting from CCl$\sb4$+PLD induced cirrhosis as hypointense regions, also consistent with previous reports. Localized in vivo water and lipid proton $\rm T\sb1$ relaxation time measurements were performed and demonstrated no statistically significant trends for either agent. In vivo proton spectra were also acquired using stimulated echo techniques to quantitatively follow the changes in liver lipid content. The changes in liver lipid content observed using MRS were verified by total lipid analysis using the Folch technique and histology. The in vivo $\rm T\sb1$ and lipid quantification data str inconsistent with the previous hypothesis that the changes in $\rm T\sb1$ weighted images were the result of increased "free" water content and, therefore, increased water $\rm T\sb1$ relaxation times. These data indicate that the long term changes are more likely the result of changes in lipid content. The data are also shown to agree with the accepted hypothesis that the time course and mechanism of fatty infiltration are different for CCl$\sb4$ and alcohol. The hypothesis that the lipids resulting from either protocol are from the same lipid fraction(s), presumably triglycerides, is also supported. And lastly, on the basis of MR images and quantitative MRS lipid information, it was shown that cirrhosis could be distinguished from fatty infiltration. ^

Eculizumab hepatotoxicity in pediatric aHUS.

BACKGROUND Eculizumab is a humanized anti-C5 antibody approved for the treatment of atypical hemolytic uremic syndrome (aHUS). Its use is increasing in children following reports of its safety and efficacy. METHODS We reviewed biochemical and clinical data related to possible drug-induced liver injury in 11 children treated with eculizumab for aHUS in a single center. RESULTS Elevated aminotransferases were observed in 7 children aged 6 to 11 years following eculizumab treatment for aHUS. Internationally accepted liver enzyme thresholds for drug-induced liver injury were exceeded in 5 cases. In all cases, liver injury was classified as mixed hepatocellular and cholestatic. Infectious and other causes were excluded in each case. One patient with no pre-existing liver disease developed tender hepatomegaly and liver enzyme derangement exceeding 20 times the upper limit of normal following initiation of eculizumab. Recurrent liver injury following re-challenge with eculizumab necessitated its discontinuation and transition to plasma therapy. CONCLUSIONS Hepatotoxicity in association with eculizumab is a potentially important yet previously unreported adverse event. We recommend monitoring liver enzymes in all patients receiving eculizumab. Further research is required to clarify the impact of this adverse event, to characterize the mechanism of potential hepatotoxicity, and to identify which patients are most at risk.

Oral thearubigins do not protect against acetaminopheninduced hepatotoxicity in mice

Purpose: To investigate the potential protective effect of oral repeated doses of thearubigins against acetaminophen-induced hepatotoxicity in mice. Methods: Mice were randomly divided into six groups (n=8) and administered the following: Control group (saline), acetaminophen group (saline), N-acetylcysteine group (500 mg/kg/day), and thearubigins groups (60, 70, 100 mg/kg/day). The drugs were given orally by gavage for seven days. On day 7, 1 h after the last dose of treatment, the mice (except control group) were given a single dose of acetaminophen (n-acetyl-p-aminophenol, APAP) orally by gavage (350 mg/kg) and then sacrificed 4 h post-APAP intake. Blood was collected for biochemical measurements and their liver were subjected to biochemical and histopathological assessment. Results: The acetaminophen group showed significant increases (p < 0.001) in serum alanine aminotransferase level, hepatic cytochrome P2E1 level, and serum and hepatic malondialdehyde levels. Moreover it showed significant decrease (p < 0.001) in serum and hepatic glutathione levels. Morphologically, the liver sections showed cellular necrosis, vacuolization, and degeneration around the centrilobular veins. Pretreatment with N-acetylcysteine reversed all acetaminophen-induced changes (p < 0.001 for all biomarkers except for hepatic MDA (p = 0.014) while pretreatment with thearubigins failed to reverse any of them. Conclusion: Oral repeated doses of thearubigins failed to protect against acetaminophen-induced hepatotoxicity in mice and didn\'t affect hepatic cytochrome P2E1 level.

The Kava Anxiety Depression Spectrum Study (KADSS): A randomized, placebo-controlled, cross-over trial using an aqueous extract of Piper methysticum.

Rationale: Piper methysticum (Kava) has been withdrawn in European, British, and Canadian markets due to concerns over hepatotoxic reactions. The WHO recently recommended research into “aqueous” extracts of Kava. Objective: The objective of this study was to conduct the first documented human clinical trial assessing the anxiolytic and antidepressant efficacy of an aqueous extract of Kava. Design and participants: The Kava Anxiety Depression Spectrum Study was a 3-week placebo-controlled, double-blind crossover trial that recruited 60 adult participants with 1 month or more of elevated generalized anxiety. Five Kava tablets per day were prescribed containing 250 mg of kavalactones/day. Results: The aqueous extract of Kava reduced participants' Hamilton Anxiety Scale score in the first controlled phase by −9.9 (CI = 7.1, 12.7) vs. −0.8 (CI = −2.7, 4.3) for placebo and in the second controlled phase by −10.3 (CI = 5.8, 14.7) vs. +3.3 (CI = −6.8, 0.2). The pooled effect of Kava vs. placebo across phases was highly significant (p < 0.0001), with a substantial effect size (d = 2.24, η² [sub]p[sub] = 0.428). Pooled analyses also revealed highly significant relative reductions in Beck Anxiety Inventory and Montgomery–Asberg Depression Rating Scale scores. The aqueous extract was found to be safe, with no serious adverse effects and no clinical hepatotoxicity. Conclusions: The aqueous Kava preparation produced significant anxiolytic and antidepressant activity and raised no safety concerns at the dose and duration studied. Kava appears equally effective in cases where anxiety is accompanied by depression. This should encourage further study and consideration of globally reintroducing aqueous rootstock extracts of Kava for the management of anxiety.