Antioxidant enzymatic systems in pigment tissue of amphibia

Superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities in pigmented and unpigmented liver tissues of frog and albino rat, respectively, were studied. Our results show that pigmented tissue is lacking in manganese superoxide dismutase activity and that the main enzymatic activity utilized in the cytosol by pigmented cells to reduce the hydrogen peroxide to water is represented by catalase; on the contrary, for the same reaction, the cells of albino rat liver primarily utilize the glutathione peroxidase activity. Both a low glutathione peroxidase activity and a low glutathione reductase activity were found in pigmented tissue of frog liver when compared with unpigmented tissue of rat liver. In light of our results, we also report a hypothetical interrelationship between melanin and reduced glutathione: We believe that in pigmented cells the melanin could act as a reducing physiological agent replacing the glutathione in the reduction of hydrogen peroxide. This reducing action of melanin could cause a diminished need for GSH and therefore could provoke the low glutathione peroxidase and reductase activities in pigmented tissue.     

Constraints on prostaglandin biosynthesis in tissues

The formation of prostaglandins by prostaglandin H synthase can be limited by the availability of the fatty acid substrate or the hydroperoxide activator and also by a self-catalyzed inactivation associated with the oxygenation reaction. Each pmol of synthase appeared able to form only about 1300 pmol of prostaglandin from arachidonate before it was inactivated. This extent of synthesis was not diminished when substrate fatty acid was complexed with cytosolic proteins even though the velocity of the oxygenation reaction was greatly decreased by the lower availability of substrate acid. When the availability of hydroperoxide activator was decreased by added glutathione peroxidase, the extent of oxygenation per mol of synthase was decreased irrespective of the amount of cytosolic protein present. Approximately 65% of the total prostaglandin synthesis by homogenates was suppressed with a glutathione peroxidase to prostaglandin H synthase ratio of about 90. The remaining prostaglandin synthetic activity was more resistant, being completely suppressed only when the ratio of peroxidase to synthase exceeded 750. The overall ratio of glutathione peroxidase (peroxide-removing) capacity to prostaglandin synthetic (peroxide-forming) capacity in selected tissues ranged from over 1800 in rat liver to less than 30 in leukocytes. A comparison between the daily urinary output of prostaglandin metabolites and tissue prostaglandin synthetic capacity suggested that prostaglandin H synthase inactivation along with glutathione peroxidase suppression of the extent of prostaglandin synthase may be important in limiting prostaglandin biosynthesis within cells.     

Evidence for suitability of glutathione peroxidase as a protective enzyme: studies of oxidative damage, renaturation, and proteolysis

The stability of glutathione peroxidase was assessed in vitro via oxidative inactivation by peroxides and a peroxidizing fatty acid and by renaturation and proteolysis. The stability of glutathione peroxidase to methyl ethyl ketone peroxide, H2O2, linoleic acid hydroperoxide, and peroxidizing methyl linolenate was compared with the stability of several other enzymes. Sulfhydryl enzymes were the most labile to all four treatments. Some of the enzymes tested were very stable to methyl ethyl ketone peroxide but very labile to linoleic acid hydroperoxide treatment. Glutathione peroxidase in the absence of glutathione was relatively slowly inactivated by each treatment. Linoleic acid hydroperoxide damage to glutathione peroxidase was characterized by release of a nonstoichiometric amount of selenite from the protein. Glutathione peroxidase samples lost all of their activity when (i) acidified to pH 2, (ii) heated 5 min at 100 degrees C, and (iii) treated with 6 M guanidinium hydrochloride or 8.5 M urea and heated 5 min at 100 degrees C. When the pH 2 sample was neutralized or the guanidinium hydrochloride-treated sample was diluted 101-fold, about 80% of the original activity was recovered in 30 min. The samples treated with urea and heat recovered no activity when diluted 101-fold. No loss of glutathione peroxidase occurred during treatment for 24 h within trypsin or thermolysin. Based on these results, glutathione peroxidase appears to be a relatively stable enzyme, and thus is is well-suited to perform its role in peroxide detoxification and prevention of oxidative deterioration of cells.     

Hydroperoxide metabolism in cyanobacteria

The enzymes involved in antioxidative activity and the cellular content of the antioxidants glutathione and ascorbate in the cyanobacteria Nostoc muscorum 7119 and Synechococcus 6311 have been examined for their roles in hydroperoxide removal. High activities of ascorbate peroxidase and catalase were found in vegetative cells of both species and in the heterocysts of N. muscorum. The affinity of ascorbate peroxidase for H2O2 was 15- to 25-fold higher than that of catalase. Increased activity of ascorbate peroxidase was observed in N. muscorum when H2O2 production was enhanced by photorespiration. Catalase activity was decreased in dilute cultures whereas ascorbate peroxidase activity increased. Ascorbate peroxidase activity also increased when the CO2 concentration was reduced. Ascorbate peroxidase appears to be a key enzyme in a cascade of reactions regenerating antioxidants. Dehydroascorbate reductase was found to regenerate ascorbate, and glutathione reductase recycled glutathione. In vegetative cells glutathione was present in high amounts (2-4 mM) whereas the ascorbate content was almost 100-fold lower (20-100 microM). Glutathione peroxidase was not detected in either cyanobacterium. It is concluded from the high activity of ascorbate peroxidase activity and the levels of antioxidants found that this enzyme can effectively remove low concentrations of peroxides. Catalase may remove H2O2 produced under photooxidative conditions where the peroxide concentration is higher.     

Coupling of Dopamine Oxidation (Monoamine Oxidase Activity) to Glutathione Oxidation Via the Generation of Hydrogen Peroxide in Rat Brain Homogenates

Abstract: Homogenates of perfused rat brain generated oxidized glutathione from reduced glutathione during incubation with dopamine or serotonin. This activity was blocked by pargyline. a monoamine oxidase inhibitor, or by catalase, a scavenger of hydrogen peroxide. These results demonstrate formation of hydrogen peroxide by monoamine oxidase and the coupling of the peroxide to glutathione peroxidase activity. Oxidized glutathione was measured fluorometrically via the oxidation of NADPH by glutathione reductase. In the absence of added dopamine or serotonin, a much smaller amount of reduced glutathione was oxidized: this activity was blocked by catalase, but not by pargyline. Therefore, endogenous production of hydrogen peroxide, not linked to monoamine oxidase activity, was present. These results indicate that glutathione peroxidase (linked to hexose monophosphate shunt activity) can function to eliminate hydrogen peroxide generated by monoamine oxidase and other endogenous sources in aminergic neurons.     

Prostaglandin hydroperoxidase, an integral part of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes.

The highly purified prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes had two still unresolved enzyme activities; the oxygenative cyclization of 8,11,14-eicosatrienoic acid to produce prostaglandin G1 and the conversion of the 15-hydro-peroxide of prostaglandin G1 to a 15-hydroxyl group, producing prostaglandin H1. The latter enzymatic reaction required heme and was stimulated by a variety of compounds, including tryptophan, epinephrine, and guaiacol, but not by glutathione. A peroxidatic dehydrogenation was demonstrated with epinephrine or guaiacol in the presence of various hydroperoxides, including hydrogen peroxide and prostaglandin G1. Higher activity and affinity were observed with the 15-hydroperoxide of eicosapolyenoic acid, especially those with the prostaglandin structure. Both the dehydrogenation of epinephrine or guaiacol and the 15-hydroperoxide reduction of prostaglandin G1 were demonstrated in nearly stoichiometric quantities. With tryptophan, however, such a stoichiometric transformation was not observed. The peroxidase activity as followed with guaiacol and hydrogen peroxide and the tryptophan-stimulated conversion of prostaglandin G1 to H1 were not dissociable as examined by isoelectric focusing, heat treatment, pH profile, and heme specificity. The results suggest that the peroxidase with a broad substrate specificity is an integral part of prostaglandin endoperoxide synthetase which is responsible for the conversion of prostaglandin G1 to H1.     

Evidence for glutathione peroxidase activities in cultured plant cells

Extracts from cultured plant cells of spinach, maize and sycamore and from Lemna plants contain detectable glutathione peroxidase activity, using either hydrogen peroxide or t-butyl hydroperoxide as substrates. Using extracts from cultured maize cells, two peaks of glutathione peroxidase activity could be resolved by a combination of gel filtration and ion exchange chromatography. One peak was eluted along with glutathione transferase activity; the second was distinct from both glutathione transferase and ascorbic acid peroxidase, and was active with both hydrogen peroxide and organic hydroperoxides. It seems likely that at least two enzymes with glutathione peroxidase activity exist in higher plant cells.     

Mechanism of acetaminophen inhibition of cyclooxygenase isoforms

Acetaminophen has similar analgesic and antipyretic properties to nonsteroidal antiinflammatory drugs (NSAIDs), which act via inhibition of cyclooxygenase enzymes. However, unlike NSAIDs, acetaminophen is at best weakly antiinflammatory. The mechanism by which acetaminophen exerts its therapeutic action has yet to be fully determined, as under most circumstances, acetaminophen is a very weak cyclooxygenase inhibitor. The potency of acetaminophen against both purified ovine cyclooxygenase-1 (oCOX-1) and human cyclooxygenase-2 (hCOX-2) was increased approximately 30-fold by the presence of glutathione peroxidase and glutathione to give IC50 values of 33 microM and 980 microM, respectively. Acetaminophen was found to be a good reducing agent of both oCOX-1 and hCOX-2. The results are consistent with a mechanism of inhibition of acetaminophen in which it acts to reduce the active oxidized form of COX to the resting form. Inhibition would therefore be more effective under conditions of low peroxide concentration, consistent with the known tissue selectivity of acetaminophen.     

Similarities in the optical spectra of prostaglandin H synthase during its cyclooxygenase and peroxidase reactions

The spectral behavior of the enzyme prostaglandin H synthase was studied in the Soret region under conditions that permitted comparison of enzyme intermediates involved in peroxidase and cyclooxygenase activities. First, the peroxidase activity was examined. The enzyme's spectral behavior upon reacting with 5-phenyl-pent-4-enyl-1-hydroperoxide was different depending on the presence or absence of the reducing substrate, phenol. In the reaction of prostaglandin H synthase with the peroxide in the absence of phenol, formation of the enzyme intermediate compound I is observed followed by partial conversion to compound II and then by enzyme bleaching. In the reaction with both peroxide and phenol the absorbance decreases and a steady-state spectrum is observed which is a mixture of native enzyme and compound II. The steady state is followed by an increase in absorbance back to that of the native enzyme with no bleaching. The difference can be explained by the reactivity of phenol as a reducing substrate with the prostaglandin H synthase intermediate compounds. Cyclooxygenase activity with arachidonic acid could not be examined in the absence of diethyldithiocarbamate because extensive bleaching occurred. In the presence of diethyldithiocarbamate, enzyme spectral behavior similar to that seen in the reaction of the peroxide and phenol was observed. The similarity of the spectra strongly suggests that the enzyme intermediates involved in both the peroxidase and cyclooxygenase reactions are the same.     

Quantitative similarities in the several actions of cyanide on prostaglandin H synthase

The effects of a heme ligand, cyanide, on pure ovine prostaglandin H synthase have been examined in detail as one approach to elucidating the role of the heme cofactor in cyclooxygenase and peroxidase catalysis by the synthase. Cyanide bound to the synthase heme with an affinity (Kd) of 0.19 mM, and inhibited the peroxidase activity of the synthase, with a KI value of 0.23 mM. Cyanide increased the sensitivity of the cyclooxygenase to inhibition by the peroxide scavenger, glutathione peroxidase. This increased sensitivity to inhibition reflected an increase in the level of peroxide required to activate the cyclooxygenase, from 21 nM in absence of cyanide to over 300 nM when 2.5 mM cyanide was present. The increase in peroxide activator requirement with increasing cyanide concentration closely paralleled the formation of the holoenzyme-cyanide complex. These effects of low levels of cyanide suggest that the heme prosthetic group of the synthase participates in the efficient activation of the cyclooxygenase by peroxide. Cyanide blocked the stimulation of cyclooxygenase velocity by phenol, but not the phenol-induced increase in overall oxygen consumption. This blockade by cyanide was noncompetitive with respect to phenol and was characterized by a KI of 4 mM. The higher KI value for this effect suggests that cyanide can also interact at a site other than the heme prosthetic group. The role of the heme prosthetic group in promoting efficient activation of the cyclooxygenase by peroxide appears to be central to the ability of the synthase to amplify the ambient peroxide concentration rapidly.     

Differences in activities of antioxidant superoxide dismutase, glutathione peroxidase and prooxidant xanthine oxidoreductase/xanthine oxidase in the normal corneal epithelium of various mammals

Under normal conditions, antioxidants at the corneal surface are balanced with the production of reactive oxygen species without any toxic effects. Danger from oxidative stress appears when natural antioxidants are overwhelmed leading to antioxidant/prooxidant imbalance. The aim of the present study was to examine the activities of enzymes contributing to the antioxidant/prooxidant balance in normal corneal epithelium of various mammals. The enzyme activities of antioxidant superoxide dismutase and glutathione peroxidase, as well as prooxidant xanthine oxidoreductase/xanthine oxidase were examined using biochemical methods. Results show that superoxide dismutase activity is high in rabbits and guinea pigs, whereas in pigs the activity is low and in cows it is nearly absent. In contrast, glutathione peroxidase activity is high in cows, pigs and rabbits, whereas in guinea pigs the activity is low. As far as prooxidant enzymes are concerned, elevated xanthine oxidoreductase/xanthine oxidase activities were found in rabbits, lower activities in guinea pigs, very low activity in cows and no activity in pigs. In conclusion, the above results demonstrate inter-species variations in activities of enzymes participating in antioxidant/prooxidant balance in the corneal epithelium. It is suggested that the levels of antioxidant and prooxidant enzymes studied in the corneal epithelium might be associated with the diurnal or nocturnal activity of animals. UV rays decompose hydrogen peroxide to damaging hydroxyl radicals and perhaps for this reason large animals with diurnal activity (cow, pig) require more effective peroxide removal (high glutathione peroxidase activity) together with the suppression of peroxide production (low superoxide dismutase activity, low xanthine oxidoreductase activity).     

Enzymatic enhancement of the catalytic rate of sulfhydryl oxidase

The rate of oxidation of glutathione by solubilized sulfhydryl oxidase was significantly enhanced in the presence of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). This enhancement was proportional to the amount of active peroxidase in the assay, but could not be attributed solely to the oxidation of glutathione catalyzed by the peroxidase. A change in the Soret region of the horseradish peroxidase spectrum was observed when both glutathione and peroxidase were present. Moreover, addition of glutathione to a sulfhydryl oxidase/horseradish peroxidase mixture resulted in a rapid shift of the absorbance maximum from 403 nm to 417 nm. This shift indicates the oxidation of horseradish peroxidase. Spectra for three isozyme preparations of horseradish peroxidase, two acidic and one basic, all underwent this red-shift in the presence of sulfhydryl oxidase and glutathione. Cysteine and N-acetylcysteine could replace glutathione. Addition of catalase had no effect on the oxidation of peroxidase, indicating that the peroxide involved in the reaction was not derived from that released into the bulk solution by sulfhydryl oxidase-catalyzed thiol oxidation. Further evidence for a direct transfer of the hydrogen peroxide moiety was obtained by addition of glutaraldehyde to a sulfhydryl oxidase/horseradish peroxidase/N-acetylcysteine mixture. Size exclusion chromatography revealed the formation of a high-molecular-weight species with peroxidase activity, which was completely resolved from native horseradish peroxidase. Formation of this species was absolutely dependent on the presence of both the cysteine-containing substrate and sulfhydryl oxidase. The observed enhancement of sulfhydryl oxidase catalytic activity by the addition of horseradish peroxidase supports a bi uni ping-pong mechanism proposed previously for sulfhydryl oxidase.     

A comparative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P

Glutathione peroxidase catalyzes the reduction of hydrogen peroxide and organic hydroperoxide by glutathione and functions in the protection of cells against oxidative damage. Glutathione peroxidase exists in several forms that differ in their primary structure and localization. We have also shown that selenoprotein P exhibits a glutathione peroxidase-like activity (Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K. (1999) J. Biol. Chem. 274, 2866-2871). To understand the physiological significance of the diversity among these enzymes, a comparative study on the peroxide substrate specificity of three types of ubiquitous glutathione peroxidase (cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, and extracellular glutathione peroxidase) and of selenoprotein P purified from human origins was done. The specific activities and kinetic parameters against two hydroperoxides (hydrogen peroxide and phosphatidylcholine hydroperoxide) were determined. We next examined the thiol specificity and found that thioredoxin is the preferred electron donor for selenoprotein P. These four enzymes exhibit different peroxide and thiol specificities and collaborate to protect biological molecules from oxidative stress both inside and outside the cells.     

Selenium supplementation sensitizes renca cells to tert-butylhydroperoxide induced loss of viability

Mouse renal carcinoma (renca) cells growing exponentially in foetal bovine serum (1%) supplemented with selenium (1 microM, sodium selenite) were exposed to oxidative insult. It was found that glutathione peroxidase activity increased (44%), while the activities of catalase, glutathione disulfide reductase, and level of total glutathione did not change due to selenium supplementation. Selenium supplementation made renca cells susceptible to tert-butylhydroperoxide induced cell death, while it did not affect the viability when the cells were exposed to hydrogen peroxide. It suggested that the contribution of glutathione peroxidase in antioxidant defense mechanism of renca cells was possibly not crucial and the function of catalase might be important especially against hydrogen peroxide.     

The VI international conference on prostaglandins and related compounds

The effects of a heme ligand, cyanide, on pure ovine prostaglandin H synthase have been examined in detail as one approach to elucidating the role of the heme cofactor in cyclooxygenase and peroxidase catalysis by the synthase. Cyanide bound to the synthase heme with an affinity (Kd) of 0.19 mM, and inhibited the peroxidase activity of the synthase, with a K_I value of 0.23 mM. Cyanide increased the sensitivity of the cyclooxygenase to inhibition by the peroxide scavenger, glutathione peroxidase. This increased sensitivity to inhibition reflect and increase in the level of peroxide required to activate the cyclooxygenase, from 21 nM in absence of cyanide to over 300 nM when 2.5 mM cyanide was present. The increase in peroxide activator requirement with increasing cyanide concentration closely paralleled the formation of the holoenzyme-cyanide complex. These effects of low levels of cyanide suggest that the heme prosthetic group of the synthase participates in the efficient activation of the cyclooxygenase by peroxide. Cyanide blocked the stimulation of cyclooxygenase velocity by phenol, but not the phenol-induced increase in overall oxygen consumption. This blockade by cyanide was noncompetitive with respect to phenol and was characterized by a K_I of 4 mM. The higher K_I value for this effect suggests that cyanide can also interact at a site other than the heme prosthetic group. The role of the heme prosthetic group in promoting efficient activation of the cyclooxygenase by peroxide appears to be central to the ability of the synthase to amplify the ambient peroxide concentration rapidly.     

A Photochemical System for Generating Free Radicals: Superoxide, Phenoxyl, Ferryl and Methyl

The photosensitizer flavin mononucleotide (FMN), in conjunction with the reducing agents diethylenetria-minepentaacetic acid (DTPA), hydrazine and hydroxylamines derived from nitroxides, generates superoxide radicals in a strictly light-dependent reaction in aerobic solution. Addition of superoxide dismutase (SOD) converts this system to a hydrogen peroxide generator. In the presence of horseradish peroxidase the latter system becomes a phenoxyl radical generator with appropriate phenolic substrates. Under anaerobic conditions FMN, hydrogen peroxide and an iron chelate generate ferryl and when this system is combined with dimethylsulfoxide, methyl radicals are produced. All the radicals can be generated with little contamination from other radicals, in high yields and the reaction can be terminated immediately upon cessation of illumination. Useful applications of this photochemical system include ESR studies of transient free radical species.     

Metabolism of paracetamol to a glutathione conjugate catalyzed by prostaglandin synthetase

Microsomes isolated from sheep seminal vesicles (SSV) were found to catalyze the metabolic activation of paracetamol as evidenced by rapid formation of paracetamol glutathione conjugate when SSV microsomes were incubated with paracetamol in the presence of arachidonic acid and GSH. The activity was inhibited by indomethacin indicating the involvement of prostaglandin synthetase in the reaction. The initial activity was very rapid, and the affinity for paracetamol in the reaction was high, since formation of the glutathione conjugate was optimal already at 0.2 mM drug concentration.It is concluded that the activation of paracetamol is due to the peroxidase activity of prostaglandin synthetase in SSV microsomes, since linolenic acid hydroperoxide was also able to support the reaction.     

Prostaglandin H synthase: an example of enzymic symbiosis

Reaction conditions which promote the heme-dependent peroxidase activity of prostaglandin H synthase appear to stimulate the heme-dependent cyclooxygenase activity also present in the synthase, even though the cyclooxygenase requires hydroperoxide for activity. However, aspirin-treated synthase, which retains only peroxidase activity, inhibited the cyclooxygenase activity of untreated synthase in the manner observed with similar levels of glutathione peroxidase. Any stimulatory effect of the synthase peroxidase on the synthase cyclooxygenase is thus likely to involve an intramolecular mechanism. Participation of peroxidase intermediates (Compounds I and II) in the initiation of a cyclooxygenase free radical chain reaction may provide an intramolecular mechanism for stimulation of the synthase cyclooxygenase by the synthase peroxidase.     

Cytosolic glutathione peroxidase from liver of pacu (Piaractus mesopotamicus), a hypoxia-tolerant fish of the Pantanal

Pacu (Piaractus mesopotamicus Holmberg, 1887, Characiformes) dwells in waters of Pantanal, in which it has adapted for alternate concentrations of dissolved oxygen. Intracellular antioxidant protection should be vital for such an adaptation. Accordingly, we found that cytosol from liver of pacu has the highest antioxidant glutathione peroxidase activity so far reported for fish and murine species. To clarify whether this activity was due to a selenium independent glutathione S-transferase or to a glutathione peroxidase, we purified it and studied its kinetics. The substrates cumene hydroperoxide and hydrogen peroxide were promptly reduced by the enzyme, but peroxidized phosphatidylcholine had to undergo previous fatty acid removal with phospholipase A(2). Augmenting concentrations (from 2 to 6 mM) of reduced glutathione activated the pure enzyme. Curves of velocity versus different micromolar concentrations of hydrogen peroxide in the presence of 2, 4 or 8 mM reduced glutathione indicated that at least 2.5 mM reduced glutathione should be available in vivo for an efficient continuous destruction of micromolar concentrations of hydrogen peroxide by this peroxidase. Molecular exclusion HPLC and SDS-polyacrylamide gel electrophoresis indicated that the purified peroxidase is a homotetramer. Data from internal sequences showed selenocysteine in its primary structure and that the enzyme was a homologue of the type-1 glutathione peroxidase found in rat, bull, trout, flounder and zebra fish. Altogether, our data establish that in liver cells of pacu, a hypoxia-tolerant fish from South America, there are high levels of a cytosolic GPX-1 capable of quenching hydrogen peroxide and fatty acid peroxides, providing an effective antioxidant action.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.