Functional characterization of two single nucleotide polymorphisms of acyl-coenzyme A:cholesterol acyltransferase 2.

BACKGROUND: Acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) plays a critical role in the formation of cholesteryl esters from cholesterol and fatty acids, and is a potential target for treating hypercholesterolemia. We recently reported the significant effects of two human ACAT2 gene polymorphisms, 41A>G (Glu(14)Gly, rs9658625) and 734C>T (Thr(254)Ile, rs2272296), on plasma lipid levels and coronary artery disease susceptibility in a case-control association study. In the present study, we evaluated the possible biological influence of the two polymorphism using two approaches. METHODS: In the first approach, the functional impact of the two polymorphisms was predicted in-silico using available web-based software, and in the second approach, the varying functions of the two polymorphisms were characterized in in vitro experiments, using ACAT2-deficient AC-29 cells. RESULTS: Our results show that the enzymatic activity of mutant Glu(14)Gly is approximately two times higher than wildtype, and that this increase is primarily due to the increased expression and/or stability of the mutant ACAT2 protein. CONCLUSIONS: These results suggest that the genetic variation at Glu(14)Gly is functionally important and may contribute to ACAT2 protein expression and stability.

Variant screening of the serum amyloid A1 gene and functional study of the p.Gly90Asp variant for its role in atherosclerosis.

OBJECTIVES: Serum amyloid A1 (SAA1) is a major acute-phase protein that is increasingly used as a reliable predictor of coronary artery disease (CAD). In this study we aim to screen the SAAI promoter and exons for genetic variants and to determine their association with CAD. In addition, we also carried out functional study on a variant of p.Gly90Asp encoded by the SAA1 gene. METHODS: Variant screening of SAA1 was performed using high resolution melting (HRM) analysis. Genetic association of p.Gly90Asp with CAD was determined in 800 CAD patients and 773 Chinese control subjects. Functional study of p.Gly90Asp was carried out using THP-1-derived macrophages and HL-60 promyelocytic leukemia cells. RESULTS: A total of 6 SNPs were identified, of which 2 were found to be novel (c.-913G > A and c.92-5T > G). The rare allele of p.Gly90Asp has a lower frequency of 0.013 in the CAD patients although this is not statistically significant. Functional studies of p.Gly90Asp revealed that the variant has decreased upregulation of key cytokines such as IL-8, MCP-1 and TNF-α as well as SERPINB2. CONCLUSIONS: We found the variant p.Gly90Asp SAA1 protein eliciting significantly reduced inflammatory responses in macrophages through a reduction in the secretion of inflammatory cytokines. Despite strong functional effects, the minor allele frequency is too low in the population to attain statistical significance difference between cases and controls.

Effect of serum amyloid A1 treatment on global gene expression in THP-1-derived macrophages.

OBJECTIVE: To investigate the effect of serum amyloid A1 (SAA1) on global gene expression in macrophages derived from THP-1 monocytes. MATERIALS AND METHODS: Global genetic expression in THP-1-derived macrophages was determined using Illumina HT-12 microarray chips and the results were validated by real-time PCR. Cytokine levels in cellular supernatant were quantified by ELISA. RESULTS: In total, 55 genes were upregulated with fold difference greater than two when THP-1-derived macrophages were incubated with SAA1 for 8 h. SAA1 is a strong cytokine inducer with significant upregulation of chemokines CCL1, CCL3, and CCL4 and this was confirmed by both real-time PCR and ELISA quantification. SAA1 also promotes the upregulation of genes involved in phagocytosis, anti-apoptosis, and tissue remodeling. CONCLUSIONS: SAA1 appears to play an important role during the immune response and in chronic inflammatory diseases through the stimulation of genes involved in cytokine production, phagocytosis, and anti-apoptosis.

Bioactivation and hepatotoxicity of nitroaromatic drugs.

Certain drugs containing a nitroaromatic moiety (e.g., tolcapone, nimesulide, nilutamide, flutamide, nitrofurantoin) have been associated with organ-selective toxicity including rare cases of idiosyncratic liver injury. What they have in common is the potential for multistep nitroreductive bioactivation (6-electron transfer) that produces the potentially hazardous nitroanion radical, nitroso intermediate, and N-hydroxy derivative. These intermediates have been associated with increased oxidant stress and targeting of nucleophilic residues on proteins and nucleic acids. However, other mechanisms including the formation of oxidative metabolites and mitochondrial liability, as well as inherent toxicokinetic properties, also determine the drugs' overall potency. Therefore, structural modification not only of the nitro moiety but also of ring substituents can greatly reduce toxicity. Novel concepts have revealed that, besides the classical microsomal nitroreductases, cytosolic and mitochondrial enzymes including nitric oxide synthase can also bioactivate certain nitroarenes (nilutamide). Furthermore, animal models of silent mitochondrial dysfunction have demonstrated that a mitochondrial oxidant stress posed by certain nitroaromatic drugs (nimesulide) can produce significant mitochondrial injury if superimposed on a genetic mitochondrial abnormality. Finally, there may be mechanisms for all nitroaromatic drugs that do not involve bioactivation of the nitro group, e.g., AHR interactions with flutamide. Taken together, the focus of research on the hepatic toxicity of nitroarene-containing drugs has shifted over the past years from the identification of the reactive intermediates generated during the bioreductive pathway to the underlying biomechanisms of liver injury. Most likely one of the next paradigm shifts will include the identification of determinants of susceptibility to nitroaromatic drug-induced hepatotoxicity.

Nimesulide-induced hepatic mitochondrial injury in heterozygous Sod2(+/-) mice.

Nimesulide, a preferential COX-2 inhibitor, has been associated with rare idiosyncratic hepatotoxicity. The underlying mechanisms of liver injury are unknown, but experimental evidence has identified oxidative stress as a potential hazard and mitochondria as a target. The aim of this study was to explore whether genetic mitochondrial abnormalities, resulting in impaired mitochondrial function and mildly increased oxidative stress, might sensitize mice to the hepatic adverse effects of nimesulide. We used heterozygous superoxide dismutase 2 (Sod2(+/-)) mice as a model, as these mice develop clinically silent mitochondrial stress but otherwise appear normal. Nimesulide was administered for 4 weeks (10 mg/kg, ip, bid), at a dose equivalent to human therapeutic dosage. We found that the drug potentiated hepatic mitochondrial oxidative injury (decreased aconitase activity, increased protein carbonyls) in Sod2(+/-), but not wild-type, mice. Furthermore, the nimesulide-treated mutant mice exhibited increased hepatic cytosolic levels of cytochrome c and caspase-3 activity, as well as increased numbers of apoptotic hepatocytes. Finally, nimesulide in vitro caused a concentration-dependent net increase in superoxide anion in mitochondria from Sod2(+/-), but not Sod2(+/+) mice. In conclusion, repeated administration of nimesulide can superimpose an oxidant stress, potentiate mitochondrial damage, and activate proapoptotic factors in mice with genetically compromised mitochondrial function.

Mitochondrial permeability transition as a source of superoxide anion induced by the nitroaromatic drug nimesulide in vitro.

Nimesulide, a widely used nonsteroidal anti-inflammatory drug containing a nitroaromatic moiety, has been associated with rare but serious hepatic adverse effects. The mechanisms underlying this idiosyncratic hepatotoxicity are unknown; however, both mitochondrial injury and oxidative stress have been implicated in contributing to liver injury in susceptible patients. The aim of this study was, first, to explore whether membrane permeability transition (MPT) could contribute to nimesulide's mitochondrial toxicity and, second, whether metabolism-derived reactive oxygen species (ROS) were responsible for MPT. We found that isolated mouse liver mitochondria readily underwent Ca2+-dependent, cyclosporin A-sensitive MPT upon exposure to nimesulide (at >or=3 microM). Net increases in mitochondrial superoxide anion levels, determined with the fluorescent probe dihydroethidium, were induced by nimesulide only in the presence of Ca2+ and were cyclosporin A-sensitive, indicating that superoxide production was a consequence, rather than the cause, of MPT. In addition, nimesulide caused a rapid dissipation of the inner mitochondrial transmembrane potential (at >or=3 microM), followed by a concentration-dependent decrease in ATP biosynthesis. Because nimesulide, unlike the related nitroaromatic drug nilutamide, did not produce any detectable ROS during incubation with mouse hepatic microsomes, we conclude that mitochondrial uncoupling causes MPT and that ROS production is a secondary effect.