DNA Conformational Changes Research Articles

Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III

Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3'-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln(41) and Leu(81) as DNA lesion sensors.

Optimisation of an electrochemical impedance spectroscopy aptasensor by exploiting quartz crystal microbalance with dissipation signals

The response of an Electrochemical Impedance Spectroscopy (EIS) sensor using DNA aptamers is affected by many factors, such as DNA density, charge and conformational changes upon DNA-target binding, and buffer conditions. We report here for the first time on the optimisation of an EIS aptamer-based sensor by using Quartz Crystal Microbalance with Dissipation mode (QCM-D). As a case study, we employed a DNA aptamer against Prostate-Specific Antigen (PSA). PSA detection was achieved by functionalising the gold sensor surface via thiol chemistry with different ratios of thiolated-DNA aptamer and 6-mercapto-1-hexanol (MCH) used as spacer molecules. PSA binding efficiency can be monitored by measuring QCM-D signals which not only provide information about the mass of PSA bound on the sensor surface, but also crucial information about the aptamer conformation and layer hydration.Data generated through QCM-D analysis provided the optimal conditions in terms of aptamer/MCH ratio to maximise the PSA binding. The ratio of 1:200 for DNA aptamer/spacer molecule was found to be optimal for ensuring maximum PSA binding. However, this study showed how a maximum analyte binding does not necessarily correspond to a maximum EIS response, which revealed to be enhanced if a ratio of 1:100 for DNA aptamer/spacer molecule was used. Moreover, by monitoring the QCM-D signal, for the first time, a value of the dissociation constant (Kd), equal to 37nM, was found for the PSA DNA aptamer towards its target. The combination of QCM-D with EIS techniques provides further insight into the effects of mass loading and charge effects that govern the response of an EIS aptasensor, serving as a valuable support for future EIS aptamer-based applications.

PLGA-Loaded Gold-Nanoparticles Precipitated with Quercetin Downregulate HDAC-Akt Activities Controlling Proliferation and Activate p53-ROS Crosstalk to Induce Apoptosis in Hepatocarcinoma Cells.

Controlled release of medications remains the most convenient way to deliver drugs. In this study, we precipitated gold nanoparticles with quercetin. We loaded gold-quercetin into poly(DL-lactide-co-glycolide) nanoparticles (NQ) and tested the biological activity of NQ on HepG2 hepatocarcinoma cells to acquire the sustained release property. We determined by circular dichroism spectroscopy that NQ effectively caused conformational changes in DNA and modulated different proteins related to epigenetic modifications and c ell cycle control. The mitochondrial membrane potential (MMP), reactive oxygen species (ROS), cell cycle, apoptosis, DNA damage, and caspase 3 activity were analyzed by flow cytometry, and the expression profiles of different anti- and pro-apoptotic as well as epigenetic signals were studied by immunoblotting. A cytotoxicity assay indicated that NQ preferentially killed cancer cells, compared to normal cells. NQ interacted with HepG2 cell DNA and reduced histone deacetylases to control cell proliferation and arrest the cell cycle at the sub-G stage. Activities of cell cycle-related proteins, such as p21WAF, cdk1, and pAkt, were modulated. NQ induced apoptosis in HepG2 cells by activating p53-ROS crosstalk and induces epigenetic modifications leading to inhibited proliferation and cell cycle arrest.

Ten-atom silver cluster signaling and tempering DNA hybridization.

Silver clusters with ∼10 atoms are molecules, and specific species develop within DNA strands. These molecular metals have sparsely organized electronic states with distinctive visible and near-infrared spectra that vary with cluster size, oxidation, and shape. These small molecules also act as DNA adducts and coordinate with their DNA hosts. We investigated these characteristics using a specific cluster-DNA conjugate with the goal of developing a sensitive and selective biosensor. The silver cluster has a single violet absorption band (λ(max) = 400 nm), and its single-stranded DNA host has two domains that stabilize this cluster and hybridize with target oligonucleotides. These target analytes transform the weakly emissive violet cluster to a new chromophore with blue-green absorption (λ(max) = 490 nm) and strong green emission (λ(max) = 550 nm). Our studies consider the synthesis, cluster size, and DNA structure of the precursor violet cluster-DNA complex. This species preferentially forms with relatively low amounts of Ag(+), high concentrations of the oxidizing agent O2, and DNA strands with ≳20 nucleotides. The resulting aqueous and gaseous forms of this chromophore have 10 silvers that coalesce into a single cluster. This molecule is not only a chromophore but also an adduct that coordinates multiple nucleobases. Large-scale DNA conformational changes are manifested in a 20% smaller hydrodynamic radius and disrupted nucleobase stacking. Multidentate coordination also stabilizes the single-stranded DNA and thereby inhibits hybridization with target complements. These observations suggest that the silver cluster-DNA conjugate acts like a molecular beacon but is distinguished because the cluster chromophore not only sensitively signals target analytes but also stringently discriminates against analogous competing analytes.

Dielectric relaxation of hydration water in the Dickerson–Drew duplex solution probed by THz spectroscopy

The contribution of water to DNA conformational change, DNA–protein interactions, and DNA–ligand recognition has been underlined. Here, we investigate the relaxation process of water around the Dickerson–Drew B-DNA duplex using terahertz spectroscopy. By controlling the DNA concentration, the relaxation time for water reorientation and the width of the DNA hydration layer are determined. We found that a layer of weakly bound hydration water, with a relaxation time of 11.6ps, extends 7.6Å from the tight-binding water layer. This result demonstrates that a shell of hydration water, with reorientation time on the order of tens of picoseconds, exists beyond the structurally integrated water usually observed around DNA.

Photoinduced conformational changes in DNA by poly(vinyl alcohol) carrying a malachite green moiety for protecting DNA against attack by nuclease.

Light is a highly advantageous means of specific cell targeting. Though targeted gene delivery is an important characteristic of an ideal delivery vehicle, there has been little effort to develop a photoresponsive vector. Among nonviral vectors, cationic substances interact effectively with negatively charged DNA. With this property in mind, we designed copolymers of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG) with different molecular weights. Though PVAMG has no affinity for DNA in the absence of light, it undergoes photoionization in the presence of light to afford cationic DNA binding sites. The DNA-PVAMG complex was investigated with respect to DNA conformational changes and its protective nature, which are important properties for nonviral vectors. PVAMG irradiation promoted DNA conformational transitions from coils to partial globules to compacted globules. The complex had a protective effect against DNase I after PVAMG irradiation, while DNA was degraded under dark conditions. The effect on DNA transition and the protective nature were sensitive to the molecular weight of PVAMG. The data regarding binding constants and binding mode provided insight into the structure of the DNA-PVAMG complex. To withstand DNase I attacks, complexation results in the compaction of DNA, which is further covered with PVAMG.

DNA conformational changes induced by cationic gemini surfactants: the key to switching DNA compact structures into elongated forms

The extent of DNA decompaction induced by m-s-m gemini surfactants depend on the surfactant's tail length and on spacer's length.

Liquid Crystal Biosensor Based on Cd<sup>2+</sup> Inducing the Bending of PS-Oligo for the Detection of Cadmium

In this study, a novel liquid crystal (LC) biosensor was developed for the highly sensitive and selective detection of Cd2+ based on Cd2+ inducing the bending of PS-oligo. This strategy makes use of the DNA conformational change to enhance the disruption of orientation of LC leading to an amplified optical signal. DNA containing-SH was bound on the glass slide of the LC cell modified with the DMOAP/APTES. The DMOAP can effectively induce the homeotropic alignment of LC. In the presence of Cd2+, Cd2+ can induce DNA to bend and become a 2 nm spherical structure, which can greatly disrupt the orientational arrangement of LC, resulting in the correspond changes of the optical image of LC cell birefringent under the polarizing microscope. When the Cd2+ concentration is low to 0.1 nM, the optical signal of LC biosensor has an obvious change. But in the absence of Cd2+, there is no orientational response of LC and the optical image under the polarizing microscope is still a uniform dark background. Thus, this LC sensing method has a sensitive and clear distinction between positive and negative results and offers a highly sensitive detection of Cd2+ with a low detection limit down to 0.1 nM.

AFM-based quantification of conformational changes in DNA caused by reactive oxygen species.

Radical induced modification of DNA plays an important role in many pathological pathways like cancer development, aging, etc. In this work, we quantify radical-induced DNA damage that causes transitions from double to single stranded DNA using atomic force microscopy (AFM). The plasmid pBR322 is attacked by free hydroxyl radicals that are produced by Fenton's reaction; the strength of the radical attack is controlled via the ratio of hydroxyl radical molecules to DNA base pairs. The extent of DNA modification is assessed by AFM tapping mode (TM) imaging of the plasmids (after adsorption onto PAH-functionalized mica) in air. As single stranded DNA chains (height ∼2 Å) are much smaller than intact DNA strands (∼5 Å), their fraction can be quantified based on the height distribution, which allows a simplified data analysis in comparison to similar AFM-based approaches. It is found that the amount of damaged DNA strands increases with increasing strength of radical attack, and decreases if ROS scavengers like sodium acetate are added. Competition curves are calculated for the interaction of hydroxyl radicals with DNA and sodium acetate, which finally allows calculation of relative rate constants for the respective reactions.

Label-free and real-time imaging of dehydration-induced DNA conformational changes in cellular nucleus using second harmonic microscopy.

Dehydration-induced DNA conformational changes have been probed for the first time with the use of second harmonic microscopy. Unlike conventional approaches, second harmonic microscopy provides a label-free and real-time approach to detect DNA conformational changes. Upon dehydration, cellular DNA undergoes a transition from B- to A-form, whereas cellular nuclei change from invisible to visible under second harmonic microscopy. These results showed that DNA is a second order nonlinear optical material. We further confirmed this by characterizing the nonlinear optical properties of extracted DNA from human cells. Our findings open a new path for SHG imaging. DNA can change its conformations under many circumstances. For example: normal cells turning into cancerous cells and drug molecules binding with DNA. Therefore, the detection of DNA conformational changes with second harmonic microscopy will be a useful tool in cancer therapy and new drug discovery.

The phosphate clamp: sequence selective nucleic acid binding profiles and conformational induction of endonuclease inhibition by cationic Triplatin complexes.

The substitution-inert polynuclear platinum(II) complex (PPC) series, [{trans-Pt(NH3)2(NH2(CH2)nNH3)}2-μ-(trans-Pt(NH3)2(NH2(CH2)nNH2)2}](NO3)8, where n = 5 (AH78P), 6 (AH78 TriplatinNC) and 7 (AH78H), are potent non-covalent DNA binding agents where nucleic acid recognition is achieved through use of the ‘phosphate clamp' where the square-planar tetra-am(m)ine Pt(II) coordination units all form bidentate N–O–N complexes through hydrogen bonding with phosphate oxygens. The modular nature of PPC–DNA interactions results in high affinity for calf thymus DNA (Kapp ∼5 × 107 M−1). The phosphate clamp–DNA interactions result in condensation of superhelical and B-DNA, displacement of intercalated ethidium bromide and facilitate cooperative binding of Hoechst 33258 at the minor groove. The effect of linker chain length on DNA conformational changes was examined and the pentane-bridged complex, AH78P, was optimal for condensing DNA with results in the nanomolar region. Analysis of binding affinity and conformational changes for sequence-specific oligonucleotides by ITC, dialysis, ICP-MS, CD and 2D-1H NMR experiments indicate that two limiting modes of phosphate clamp binding can be distinguished through their conformational changes and strongly suggest that DNA condensation is driven by minor-groove spanning. Triplatin-DNA binding prevents endonuclease activity by type II restriction enzymes BamHI, EcoRI and SalI, and inhibition was confirmed through the development of an on-chip microfluidic protocol.

Development of a sensor to study the DNA conformation using molecular logic gates

This communication reports our investigations on the Fluorescence Resonance Energy Transfer (FRET) between two laser dyes Acriflavine and Rhodamine B in absence and presence of DNA at different pH. It has been observed that energy transfer efficiency is largely affected by the presence of DNA as well as the pH of the system. It is well known that with increase in pH, DNA conformation changes from double stranded to single stranded (denaturation) and finally form random coil. Based on our experimental results two different types of molecular logic gates namely, XOR and OR logic have been demonstrated which can be used to have an idea about DNA conformation in solution.

Sensing conformational changes in DNA upon ligand binding using QCM-D. Polyamine condensation and Rad51 extension of DNA layers.

Biosensors, in which binding of ligands is detected through changes in the optical or electrochemical properties of a DNA layer confined to the sensor surface, are important tools for investigating DNA interactions. Here, we investigate if conformational changes induced in surface-attached DNA molecules upon ligand binding can be monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. DNA duplexes containing 59-184 base pairs were formed on QCM-D crystals by stepwise assembly of synthetic oligonucleotides of designed base sequences. The DNA films were exposed to the cationic polyamines spermidine and spermine, known to condense DNA molecules in bulk experiments, or to the recombination protein Rad51, known to extend the DNA helix. The binding and dissociation of the ligands to the DNA films were monitored in real time by measurements of the shifts in resonance frequency (Δf) and in dissipation (ΔD). The QCM-D data were analyzed using a Voigt-based model for the viscoelastic properties of polymer films in order to evaluate how the ligands affect thickness and shear viscosity of the DNA layer. Binding of spermine shrinks all DNA layers and increases their viscosity in a reversible fashion, and so does spermidine, but to a smaller extent, in agreement with its lower positive charge. SPR was used to measure the amount of bound polyamines, and when combined with QCM-D, the data indicate that the layer condensation leads to a small release of water from the highly hydrated DNA films. The binding of Rad51 increases the effective layer thickness of a 59 bp film, more than expected from the know 50% DNA helix extension. The combined results provide guidelines for a QCM-D biosensor based on ligand-induced structural changes in DNA films. The QCM-D approach provides high discrimination between ligands affecting the thickness and the structural properties of the DNA layer differently. The reversibility of the film deformation allows comparative studies of two or more analytes using the same DNA layer as demonstrated here by spermine and spermidine.

Adsorption of polyethylenimine and its interaction with a genomic DNA on a silicon oxynitride surface characterized by dual polarization interferometry

Dual polarization interferometry was used to investigate the adsorption of polyethylenimine (PEI) on a silicon oxynitride chip surface, as well as to study the subsequent interaction between PEI and a genomic DNA. A compact homogeneous PEI layer formed initially as a result of their strong electrostatic attraction to the negatively charged chip surface. With the adsorption going on, a short time adsorption process with constant speed was observed and the subsequent PEI molecules loosely attached on the chip surface because of the electrostatic repulsion and crowded surface conditions. A loose and thick DNA layer formed on the PEI surface due to its rigidity. Further PEI deposition induced DNA conformation changes and the swelling of the PEI–DNA complex layer, which resulted in a dramatic layer thickness increase. Under high PEI concentration, the layer mass and thickness increments were partially lost as DNA was stripped off by PEI molecules. This result was interpreted in terms of overcharged DNA–PEI complex formation and the strong electrostatic repulsion between the first PEI layer and the newly formed overcharged DNA–PEI complexes. The explanation was proved by the case of the assembly process in PBS. No stripping effect was found even at a high PEI concentration in PBS because of the salt screening effect and the low charge density of PEI.

Tethered Fluorophore Motion: Studying Large DNA Conformational Changes by Single-fluorophore Imaging

We have previously introduced tethered fluorophore motion (TFM), a single-molecule fluorescence technique that monitors the effective length of a biopolymer such as DNA. TFM uses the same principles as tethered particle motion (TPM) but employs a single fluorophore in place of the bead, allowing TFM to be combined with existing fluorescence techniques on a standard fluorescence microscope. TFM has been previously been used to reveal the mechanism of two site-specific recombinase systems, Cre-loxP and XerCD-dif. In this work, we characterize TFM, focusing on the theoretical basis and potential applications of the technique. Since TFM is limited in observation time and photon count by photobleaching, we present a description of the sources of noise in TFM. Comparing this with Monte Carlo simulations and experimental data, we show that length changes of 100 bp of double-stranded DNA are readily distinguishable using TFM, making it comparable with TPM. We also show that the commonly recommended pixel size for single-molecule fluorescence approximately optimizes signal to noise for TFM experiments, thus enabling facile combination of TFM with other fluorescence techniques, such as Förster resonance energy transfer (FRET). Finally, we apply TFM to determine the polymerization rate of the Klenow fragment of DNA polymerase I, and we demonstrate its combination with FRET to observe synapsis formation by Cre using excitation by a single laser. We hope that TFM will be a useful addition to the single-molecule toolkit, providing excellent insight into protein-nucleic acid interactions.

English

Petiveria alliacea L. is a native herbaceous species from the Amazon region traditionally used in folk medicine for its various pharmacological activities. This study aimed to perform a comparative assessment of genotoxic and antigenotoxic potential of field- and in vitro-grown plants. Specimens from different populations from Rio de Janeiro State were used as donor plants for in vitro culture on Murashige and Skoog (MS) medium. Aqueous extracts from field and in vitro plants were evaluated by analyzing their effect on the integrity of pUC 9.1 plasmid. DNA genotoxic potential was assessed after treatment of plasmid DNA with increasing concentrations of aqueous extracts (1.0, 5.0 to 10.0 mg/ml) from the populations studied and the in vitro plants originated from them. The electrophoretic analysis showed changes in the structural conformation of plasmid DNA, indicating the occurrence of single- and double-strand breaks caused by the extracts, demonstrating the genotoxic potential at higher concentrations. The antigenotoxic potential was assessed based on plasmid protection induced by the extracts after DNA treatment with stannous chloride. Thus, data also pointed to a antigenotoxic effect of extracts against oxidative damage. In addition, the intensity of the effect of extracts on plasmid DNA varied according to origin of plants. Key words: Antigenotoxicity, bioactivity, DNA topology, genotoxicity, micropropagation

A structural insight into major groove directed binding of nitrosourea derivative nimustine with DNA: a spectroscopic study.

Nitrosourea therapeutics occupies a definite place in cancer therapy but its exact mechanism of action has yet to be established. Nimustine, a chloroethyl nitrosourea derivative, is used to treat various types of malignancy including gliomas. The present work focuses on the understanding of nimustine interaction with DNA to delineate its mechanism at molecular level. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) has been used to determine the binding sites of nimustine on DNA. Circular dichroism (CD) spectroscopy has been used to confirm conformational variations in DNA molecule upon nimustine-DNA interaction. Thermodynamic parameters of nimustine-DNA reaction have been calculated by isothermal titration calorimetry. Results of the present study demonstrate that nimustine is not a simple alkylating agent rather it causes major grove-directed-alkylation. Spectroscopic data suggest binding of nimustine with nitrogenous bases guanine (C6 = O6) and thymine (C4 = O4) in DNA major groove. CD spectra of nimustine-DNA complexes point toward the perturbation of native B-conformation of DNA and its partial transition into C-form. Thermodynamically, nimustine-DNA interaction is an entropy driven endothermic reaction, which suggests hydrophobic interaction of nimustine in DNA-major groove pocket. Spectral results suggest base binding and local conformational changes in DNA upon nimustine interaction. Investigation of drug-DNA interaction is an essential part of rational drug designing that also provides information about the drug’s action at molecular level. Results, demonstrated here, may contribute in the development of new nitrosourea therapeutics with better efficacy and fewer side effects.

Probing protein - DNA interaction by single molecule and structural analysis

In proteins, conformational change impacting their function has been well investigated in the past decades, and was named `allosteric effect'. However, in DNA-protein interaction, the concept of DNA conformational change caused by DNA-protein binding will affect another nearby DNA-binding protein has not been well investigated and understood. Combined with structural biology and Single Molecule Assays, we can now probe and study allosteric propagation through DNA which exists as a fundamental property in DNA-protein interaction, and this allosteric effect through DNA can fine tune gene expression. Therefore, DNA conformational changes should be seriously considered and analyzed for DNA –protein interactions in general.

Identification of genes in HepG2 cells that respond to DNA methylation and histone deacetylation inhibitor treatment.

Previous studies have demonstrated that epigenetics has an important role in the regulation of gene expression in cancer. Epigenetics is the study of reversible, heritable changes in gene function, which occur independently from changes in the DNA sequence. DNA methylation and histone deacetylation are the two most important epigenetic modifications. DNA methylation was one of the first discovered epigenetic modifications and it may lead to changes in chromatin structure, DNA conformation and DNA stability, thereby controlling gene expression. Sample data on the HepG2 cell line from the Gene Expression Omnibus database under GSE5230 accession number were obtained and GEOquery and the limma package were then used to analyze the data and identify differentially expressed genes using Gene Otology. This was conducted in order to investigate the effect on gene expression of inhibiting DNA methylation and histone deacetylation, and to explore the potential role of epigenetics in the development and treatment of hepatic carcinoma. It was found that inhibition of DNA methylation and histone deacetylation affected not only substance metabolism, but also the immune activity in HepG2 cells. Furthermore, common target sites for transcription factors were identified in the differentially expressed genes. It may be concluded that the inhibition of DNA methylation and histone deacetylation contributes to the treatment of hepatic carcinoma and may provide a novel therapeutic strategy for the treatment of hepatic cancer.

Erratum to: Study on the binding interaction between perfluoroalkyl acids and DNA

Abstract Perfluoroalkyl acids (PFAAs) are carcinogens,and elucidating their DNA binding properties is crucial forunderstanding PFAA genotoxicity. We have investigated thebinding mode and affinity of five PFAAs to seven DNAmolecules using fluorescence displacement and moleculardockinganalysis. DNA conformational changes uponPFAAbinding were also examined by circular dichroism (CD).The data revealed that DNA intercalation was the dominantinteraction mode of the PFAAs; however, these moleculesalso bound to grooves. The dissociation constants for thePFAAs ranged between 0.11 and 1,217.14 μM, and between3.46 and 2,141.21 μM for DNA intercalation and groovebinding, respectively. PFAAs that contain longer carbonchains had stronger DNA intercalation affinities. Bindingto DNA was stronger for perfluoroalkyl sulfonates than forperfluorcarboxyl acids that contain the same number ofcarbons. This observation is postulated to arise from thepresenceofmorefluorineandoxygenatomsinperfluoroalkylsulfonates acting as hydrogen bond donors that facilitatestronger DNA intercalation. The binding of the PFAAs toDNA showed some CT-DNA sequence selectivity. Moleculardocking analysis confirmed the DNA binding mode andaffinities of the PFAAs. CD analysis revealed that thePFAAs weakened DNA base stacking and loosened DNAhelicity.Thepresentstudyhasimprovedourunderstandingofthe formation of PFAA–DNA adducts.Keywords Perfluoroalkylacids .DNAbinding .Intercalation .Minorgroovebinding .DNAconformationalchange .DNAsequenceselectivityIntroductionPerfluoroalkyl acids (PFAAs) have been produced and usedfor over 50 years. Because of their specific physical andchemical properties, chemical and thermal inertness, lowsurface energy, and special surface-active properties,PFAAs have applications in numerous industrial and con-sumer products, ranging from carpet protection to fire-fighting foams (Lehmler 2005; Martin et al. 2004). PFAAspersist ubiquitously in the environment, resisting hydrolysis,photolysis, and biodegradation because of the high energyof the carbon–fluorine bond (Hekster et al. 2003; Martin etal. 2004; Quinete et al. 2010). Although the toxicologicaleffects of these chemicals are still under investigation, manystudieshavereportedontheirdevelopmentalandreproductivetoxicity, neurotoxicity, immunotoxicity, and carcinogenicityinanimalsbothinvivoandinvitro(Heksteretal.2003;Lauetal. 2004; Slotkin et al. 2008). PFAAs can cause peroxisomalproliferation, mitochondrial dysfunction, disturbance of fattyacid metabolism, hepatocellular hypertrophy, changes in