A randomized controlled laboratory study on the long-term effects of methylphenidate on cardiovascular function and structure in rhesus monkeys.

BACKGROUND: Whether long-term methylphenidate (MPH) results in any changes in cardiovascular function or structure can only be properly addressed through a randomized trial using an animal model which permits elevated dosing over an extended period of time. METHODS: We studied 28 male rhesus monkeys (Macaca mulatta) approximately 7 years of age that had been randomly assigned to one of three MPH dosages: vehicle control (0 mg/kg, b.i.d., n = 9), low dose (2.5 mg/kg, b.i.d., n = 9), or high dose (12.5 mg/kg, b.i.d., n = 10). Dosage groups were compared on serum cardiovascular and inflammatory biomarkers, electrocardiograms (ECGs), echocardiograms, myocardial biopsies, and clinical pathology parameters following 5 years of uninterrupted dosing. RESULTS: With the exception of serum myoglobin, there were no statistical differences or apparent dose-response trends in clinical pathology, cardiac inflammatory biomarkers, ECGs, echocardiograms, or myocardial biopsies. The high-dose MPH group had a lower serum myoglobin concentration (979 ng/mL) than either the low-dose group (1882 ng/mL) or the control group (2182 ng/mL). The dose response was inversely proportional to dosage (P = .0006). CONCLUSIONS: Although the findings cannot be directly generalized to humans, chronic MPH exposure is unlikely to be associated with increased cardiovascular risk in healthy children.

Green tea epigallocatechin gallate binds to and inhibits respiratory complexes in swelling but not normal rat hepatic mitochondria.

Epigallocatechin gallate (EGCG), the major flavonoid in green tea, is consumed via tea products and dietary supplements, and has been tested in clinical trials. However, EGCG can cause hepatotoxicity in humans and animals by unknown mechanisms. Here EGCG effects on rat liver mitochondria were examined. EGCG showed negligible effects on oxidative phosphorylation at 7.5-100µM in normal mitochondria. However, respiratory chain complexes (RCCs) were profoundly inhibited by EGCG in mitochondria undergoing Ca(2+) overload-induced mitochondrial permeability transition (MPT). As RCCs are located in mitochondrial inner membranes (IM) and matrix, it was reasoned that EGCG could not readily pass through IM to affect RCCs in normal mitochondria but may do so when IM integrity is compromised. This speculation was substantiated in three ways. (1) Purified EGCG-bound proteins were barely detectable in normal mitochondria and contained no RCCs as determined by Western blotting, but swelling mitochondria contained about 1.5-fold more EGCG-bound proteins which included four RCC subunits together with cyclophilin D that locates in mitochondrial matrix. (2) Swelling mitochondria consumed more EGCG than normal ones. (3) The MPT blocker cyclosporine A diminished the above-mentioned difference. Among four subunits of RCC II, only SDHA and SDHB which locate in mitochondrial matrix, but not SDHC or SDHD which insert into the IM, were found to be EGCG targets. Interestingly, EGCG promoted Ca(2+) overload-induced MPT only when moderate MPT already commenced. This study identified hepatic RCCs as targets for EGCG in swelling but not normal mitochondria, suggesting EGCG may trigger hepatotoxicity by worsening pre-existing mitochondria abnormalities.

Inhibition of cytochrome P450s enhances (+)-usnic acid cytotoxicity in primary cultured rat hepatocytes.

(+)-Usnic acid (UA) is consumed as a dietary supplement to promote weight loss; however, dietary supplements containing UA have been associated with clinical cases of severe liver injury. UA has been shown to be hepatotoxic in rats and is extensively metabolized by hepatic cytochrome P450s (CYPs); therefore, we examined if UA metabolism results in the formation of cytotoxic metabolites or if metabolism is a detoxification process in primary rat hepatocytes. When CYP activity was suppressed by the non-isoenzyme-selective inhibitor SKF-525A (20 µM), or the CYP1A inhibitor alpha-naphthoflavone (10 µM), or the CYP3A inhibitor ketoconazole (25 µM), the cytotoxicity of UA at 3~6 µM after 3~20 h of exposure was significantly increased as measured by lactate dehydrogenase (LDH) leakage. At 2 h after UA exposure, an earlier time point prior to LDH release, these CYP inhibitors potentiated UA-induced inhibition of cellular respiration as determined by the Clark type oxygen electrode. Cellular adenosine triphosphate (ATP) depletion by UA was also exacerbated by these CYP inhibitors. The CYP2B/2C inhibitor, ticlopidine at 20 µM, showed no effects in parallel experiments. These data demonstrate that UA is bio-transformed to less toxic metabolites in rat primary hepatocytes, probably mainly by CYP1A and 3A, but not 2B/2C. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Metabolomics evaluation of the effects of green tea extract on acetaminophen-induced hepatotoxicity in mice.

Green tea has been purported to have beneficial health effects including protective effects against oxidative stress. Acetaminophen (APAP) is a widely used analgesic drug that can cause acute liver injury in overdose situations. These studies explored the effects of green tea extract (GTE) on APAP-induced hepatotoxicity in liver tissue extracts using ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. Mice were orally administered GTE, APAP or GTE and APAP under three scenarios. APAP alone caused a high degree of hepatocyte necrosis associated with increases in serum transaminases and alterations in multiple metabolic pathways. The time of GTE oral administration relative to APAP either protected against or potentiated the APAP-induced hepatotoxicity. Dose dependent decreases in histopathology scores and serum transaminases were noted when GTE was administered prior to APAP; whereas, the opposite occurred when GTE was administered after APAP. Similarly, metabolites altered by APAP alone were less changed when GTE was given prior to APAP. Significantly altered pathways included fatty acid metabolism, glycerophospholipid metabolism, glutathione metabolism, and energy pathways. These studies demonstrate the complex interaction between GTE and APAP and the need to employ novel analytical strategies to understand the effects of dietary supplements on pharmaceutical compounds.

Mouse liver protein sulfhydryl depletion after acetaminophen exposure.

Acetaminophen (APAP)-induced liver injury is the leading cause of acute liver failure in many countries. This study determined the extent of liver protein sulfhydryl depletion not only in whole liver homogenate but also in the zonal pattern of sulfhydryl depletion within the liver lobule. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase levels, liver necrosis, and glutathione depletion in a dose-dependent manner. Free protein sulfhydryls were measured in liver protein homogenates by labeling with maleimide linked to a near infrared fluorescent dye followed by SDS-polyacrylamide gel electrophoresis. Global protein sulfhydryl levels were decreased significantly (48.4%) starting at 1 hour after the APAP dose and maintained at this reduced level through 24 hours. To visualize the specific hepatocytes that had reduced protein sulfhydryl levels, frozen liver sections were labeled with maleimide linked to horseradish peroxidase. The centrilobular areas exhibited dramatic decreases in free protein sulfhydryls while the periportal regions were essentially spared. These protein sulfhydryl-depleted regions correlated with areas exhibiting histopathologic injury and APAP binding to protein. The majority of protein sulfhydryl depletion was due to reversible oxidation since the global- and lobule-specific effects were essentially reversed when the samples were reduced with tris(2-carboxyethy)phosphine before maleimide labeling. These temporal and zonal pattern changes in protein sulfhydryl oxidation shed new light on the importance that changes in protein redox status might play in the pathogenesis of APAP hepatotoxicity.

Mechanisms for epigallocatechin gallate induced inhibition of drug metabolizing enzymes in rat liver microsomes.

Epigallocatechin gallate (EGCG) inhibits drug metabolizing enzymes by unknown mechanisms. Here we examined if the inhibition is due to covalent-binding of EGCG to the enzymes or formation of protein aggregates. EGCG was incubated with rat liver microsomes at 1-100µM for 30min. The EGCG-binding proteins were affinity purified using m-aminophenylboronic acid agarose and probed with antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, cytochrome P450 (CYP) 1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A, catechol-O-methyltransferase (COMT) and microsomal glutathione transferase 1 (MGST1). All but actin and soluble COMT were positively detected at ≥1µM EGCG, indicating EGCG selectively bound to a subset of proteins including membrane-bound COMT. The binding correlated well with inhibition of CYP activities, except for CYP2E1 whose activity was unaffected despite evident binding. The antioxidant enzyme MGST1, but not cytosolic GSTs, was remarkably inhibited, providing novel evidence supporting the pro-oxidative effects of EGCG. When microsomes incubated with EGCG were probed on Western blots, all but the actin and CYP2E1 antibodies showed a significant reduction in binding at ≥1µM EGCG, suggesting that a fraction of the indicated proteins formed aggregates that likely contributed to the inhibitory effects of EGCG but were not recognizable by antibodies against the intact proteins. This raised the possibility that previous reports on EGCG regulating protein expression using GAPDH as a reference should be revisited for accuracy. Remarkable protein aggregate formation in EGCG-treated microsomes was also observed by analyzing Coomassie Blue-stained SDS-PAGE gels. EGCG effects were partially abolished in the presence of 1mM glutathione, suggesting they are particularly relevant to the in vivo conditions when glutathione is depleted by toxicant insults.

Green tea extract can potentiate acetaminophen-induced hepatotoxicity in mice.

Green tea extract (GTE) has been advocated as a hepatoprotective compound and a possible therapeutic agent for acetaminophen (APAP) overdose. This study was conducted to determine if GTE can provide protection against APAP-induced hepatotoxicity. Three different exposure scenarios were tested. The first involved administering APAP (150 mg/kg, orally) to mice followed 6h later by GTE (500 or 1000 mg/kg). The other two involved administering GTE prior to the APAP dose. GTE (500 or 1000 mg/kg, orally) was administered 3h prior to APAP (200 mg/kg, orally) or for three consecutive days (once-daily) followed by APAP (300 mg/kg) on the fourth day. Indices of hepatotoxicity were assessed 24h after the APAP dose. GTE potentiated APAP-induced hepatotoxicity when administered after the APAP dose. GTE caused significant glutathione depletion and this effect likely contributed to the observed potentiation. In contrast, GTE provided protection against APAP-induced hepatotoxicity when administered prior to the APAP dose. GTE dramatically decreased APAP covalent binding to protein indicating that less reactive metabolite was available to cause hepatocellular injury. These results highlight the potential for drug-dietary supplement interactions and the importance of testing multiple exposure scenarios to adequately model different types of potential interactions.

Changes in mouse liver protein glutathionylation after acetaminophen exposure.

The role of protein glutathionylation in acetaminophen (APAP)-induced liver injury was investigated in this study. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase and aspartate aminotransferase levels and liver necrosis in a dose-dependent manner. The ratio of GSH to GSSG was decreased in a dose-dependent manner, suggesting that APAP produced a more oxidizing environment within the liver. Despite the increased oxidation state, the level of global protein glutathionylation was decreased at 1 h and continued to decline through 24 h. Immunohistochemical localization of glutathionylated proteins showed a complex dynamic change in the lobule zonation of glutathionylated proteins. At 1 h after APAP exposure, the level of glutathionylation decreased in the single layer of hepatocytes around the central veins but increased mildly in the remaining centrilobular hepatocytes. This increase correlated with the immunohistochemical localization of APAP covalently bound to protein. Thereafter, the level of glutathionylation decreased dramatically over time in the centrilobular regions with major decreases observed at 6 and 24 h. Despite the overall decreased glutathionylation, a layer of cells lying between the undamaged periportal region and the damaged centrilobular hepatocytes exhibited high levels of glutathionylation at 3 and 6 h in all samples and in some 24-h samples that had milder injury. These temporal and zonal pattern changes in protein glutathionylation after APAP exposure indicate that protein glutathionylation may play a role in protein homeostasis during APAP-induced hepatocellular injury.

Kava extract, an herbal alternative for anxiety relief, potentiates acetaminophen-induced cytotoxicity in rat hepatic cells.

The widely used over-the-counter analgesic acetaminophen (APAP) is the leading cause of acute liver failure in the United States and due to this high incidence, a recent FDA Advisory Board recommended lowering the maximum dose of APAP. Kava herbal dietary supplements have been implicated in several human liver failure cases leading to the ban of kava-containing products in several Western countries. In the US, the FDA has issued warnings about the potential adverse effects of kava, but kava dietary supplements are still available to consumers. In this study, we tested the potential of kava extract to potentiate APAP-induced hepatocyte cytotoxicity. In rat primary hepatocytes, co-treatment with kava and APAP caused 100% loss of cell viability, while the treatment of kava or APAP alone caused ∼50% and ∼30% loss of cell viability, respectively. APAP-induced glutathione (GSH) depletion was also potentiated by kava. Co-exposure to kava decreased cellular ATP concentrations, increased the formation of reactive oxygen species, and caused mitochondrial damage as indicated by a decrease in mitochondrial membrane potential. In addition, similar findings were obtained from a cultured rat liver cell line, clone-9. These observations indicate that kava potentiates APAP-induced cytotoxicity by increasing the magnitude of GSH depletion, resulting in oxidative stress and mitochondrial dysfunction, ultimately leading to cell death. These results highlight the potential for drug-dietary supplement interactions even with widely used over-the-counter drugs.