Total synthesis of isoprostanes: discovery and quantitation in biological systems

Isoprostanes (iP's), a new class of natural products isomeric with prostaglandins, are formed as the result of free radical oxygenation of polyunsaturated fatty acids. We have identified these iP's and developed analytical methodology to measure them in biological fluids. The approach we took, which led to the discovery and measurement of iP's, is as follows: (1) based on some biochemical and chemical considerations, we proposed possible structures for these isoprostanes; (2) we performed the total syntheses of some of these iP's, in particular Groups III through VI, and used them as markers for their discovery in biological fluids and developed a GC/MS and an LC/MS methodologies based on iPF2alpha-III, iPF2alpha-VI, and 8,12-iso-iPF2alpha-VI; (3) with the help of these assays, we measured elevated levels of iP's in Alzheimer's disease and atherosclerosis.     

Biomarkers of oxidative stress study III. Effects of the nonsteroidal anti-inflammatory agents indomethacin and meclofenamic acid on measurements of oxidative products of lipids in CCl4 poisoning

Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2alpha-VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.     

Quantitation of isoprostane isomers in human urine from smokers and nonsmokers by LC-MS/MS

A simple, rapid liquid chromatography-tandem mass spectrometry method was developed to identify and quantitate in human urine the isoprostanes iPF(2 alpha)-III, 15-epi-iPF(2 alpha)-III, iPF(2 alpha)-VI, and 8,12-iso-iPF(2 alpha)-VI along with the prostaglandin PGF(2 alpha) and 2,3-dinor-iPF(2 alpha)-III, a metabolite of iPF(2 alpha)-III. Assay specificity, linearity, precision, and accuracy met the required criteria for most analytes. The urine sample storage stability and standard solution stability were also tested. The methodology was applied to analyze 24 h urine samples collected from smokers and nonsmokers on controlled diets. The results for iPF(2 alpha)-III obtained by our method were significantly correlated with results by an ELISA, although an approximately 2-fold high bias was observed for the ELISA data. For iPF(2 alpha)-III and its metabolite 2,3-dinor-iPF(2 alpha)-III, smokers had significantly higher concentrations than nonsmokers (513 +/- 275 vs. 294 +/- 104 pg/mg creatinine; 3,030 +/- 1,546 vs. 2,046 +/- 836 pg/mg creatinine, respectively). The concentration of iPF(2 alpha)-VI tended to be higher in smokers than in nonsmokers; however, the increase was not statistically significant in this sample set. Concentrations of the other three isoprostane isomers showed no trends toward differences between smokers and nonsmokers. Among smokers, the daily output of two type VI isoprostanes showed a weak correlation with the amount of tobacco smoke exposure, as determined by urinary excretion of total nicotine equivalents.     

Indirect enhancement of neutrophil activity and adhesion to cultured human umbilical vein endothelial cells by isoprostanes (iPF2alpha-III and iPE2-III).

Isoprostanes are metabolites of arachidonic acid found in blood under various conditions of oxidative stress. Because arachidonic acid derivatives are major mediators of inflammation, we investigated the potential inflammatory effects of iPF2alpha-III (previously 8-isoPGF2alpha) and iPE2-III (8-isoPGE2) on human polymorphonuclear granulocytes (PMN), as well as on human umbilical vein endothelial cells (HUVECs). The early activation marker CD11b on PMN and the adhesion molecules ICAM-1, E-selectin, and P-selectin on HUVECs were quantified by flow cytometry. Levels of the cytokines interleukin (IL)-6 and IL-8 were measured in the culture supernatant by enzyme-linked immunosorbent assay. Furthermore, adhesion of PMN to HUVECs was assessed. Neither isoprostane showed any direct stimulatory effects on PMN or HUVECs at concentrations of 0.1 or 1 microM: there was no acute elevation in expression of CD11b or P-selectin and no change of ICAM-1 or E-selectin after 4 or 24 h of incubation, respectively. The levels of interleukin IL-6 and IL-8 were also unaltered. However, PMN adhesion was significantly enhanced both after 4 and 24 h of incubation of HUVECs with iPF2alpha-III, and CD11b expression on PMN was elevated by contact of these cells with the supernatant of pre-exposed HUVECs. Neither of these actions were inhibited by an endothelin receptor antagonist (bosentan) or a combined thromboxane A2/isoprostane-receptor antagonist (SQ29548). Thus, although not having a direct pro-inflammatory potential, isoprostanes might indirectly accentuate PMN stimulation. This seems to occur via a receptor-independent mechanism, perhaps the production of an active metabolite of isoprostanes by endothelial cells.     

Effects of 2 years of caloric restriction on oxidative status assessed by urinary F2‐isoprostanes: The CALERIE 2 randomized clinical trial

Calorie restriction (CR) without malnutrition slows aging in animal models. Oxidative stress reduction was proposed to mediate CR effects. CR effect on urinary F2‐isoprostanes, validated oxidative stress markers, was assessed in CALERIE, a two‐year randomized controlled trial. Healthy volunteers (n = 218) were randomized to prescribed 25% CR (n = 143) or ad libitum control (AL, n = 75) stratifying the randomization schedule by site, sex, and BMI. F2‐isoprostanes were quantified using LC‐MS/MS in morning, fasted urine specimens at baseline, at 12 and 24 months. The primary measure of oxidative status was creatinine‐adjusted 2,3‐dinor‐iPF(2α)‐III concentration, additional measured included iPF(2α)‐III, iPF2a‐VI, and 8,12‐iso‐iPF2a‐VI. Intention‐to‐treat analyses assessed change in 2,3‐dinor‐iPF(2α)‐III using mixed models assessing treatment, time, and treatment‐by‐time interaction effects, adjusted for blocking variables and baseline F2‐isoprostane value. Exploratory analyses examined changes in iPF(2α)‐III, iPF(2α)‐VI, and 8,12‐iso‐iPF(2α)‐VI. A factor analysis used aggregate information on F2‐isoprostane values. In CR group, 2,3‐dinor‐iPF(2α)‐III concentrations were reduced from baseline by 17% and 13% at 12 and 24 months, respectively; these changes were significantly different from AL group (p < .01). CR reduced iPF(2α)‐III concentrations by 20% and 27% at 12 and 24 months, respectively (p < .05). The effects were weaker on the VI‐species. CR caused statistically significant reduction in isoprostane factor at both time points, and mean (se) changes were ?0.36 (0.06) and ?0.31 (0.06). No significant changes in isoprostane factor were at either time point in AL group (p < .01 between‐group difference). We conclude that two‐year CR intervention in healthy, nonobese men and women reduced whole body oxidative stress as assessed by urinary concentrations of F2‐isoprostanes.     

Eicosapentaenoic-acid-derived isoprostanes: synthesis and discovery of two major isoprostanes

The stereospecific synthesis of two all-syn-EPA-derived isoprostanes (iPs), 5-epi-8,12-iso-iPF(3alpha)-VI 17 and 8,12-iso-iPF(3alpha)-VI 18, has been accomplished. These two synthetic probes have been used to discover and identify their presence in human urine. The eventual quantitative measurement of these two iPs may be a valuable index of oxidative stress in people with eicosapentaenoic acid- (EPA) and docosahexaenoic acid- (DHA) enriched phospholipids.     

Oxidized derivatives of omega-3 fatty acids: identification of IPF3 alpha-VI in human urine

Isoprostanes (iPs) are prostaglandin-like molecules derived from autoxidation of polyunsaturated fatty acids (PUFAs). Urinary iP levels have been used as indices of in vivo lipid peroxidation. Thus far, it has only been possible to measure iPs derived from arachidonic acid in urine, because levels of iPs/neuroprostanes (nPs) derived from omega 3-PUFAs have been found to be below detection limits of available assays. Because of the interest in omega3-PUFA dietary supplementation, we developed specific methods to measure nPF4 alpha-VI and iPF3 alpha-VI [derived from 4,7,10,13,16,19-docosahexaenoic acid (DHA) and 5,8,11,14,17-eicosapentaenoic acid (EPA)] using a combination of chemical synthesis, gas chromatography/mass spectrometry (GC/MS), and liquid chromatography tandem mass spectrometry (LC/MS/MS). Although nPF4 alpha-VI was below the detection limit of the assay, we conclusively identified iPF3 alpha-VI in human urine by GC/MS and LC/MS/MS. The mean levels in 26 subjects were approximately 300 pg/mg creatinine. Our failure to detect nPF4 alpha-VI may have been due to its rapid metabolism by beta-oxidation to iPF3 alpha-VI, which we showed to occur in rat liver homogenates. In contrast, iPF3 alpha-VI is highly resistant to beta-oxidation in vitro. Thus iPF3 alpha-VI can be formed by two mechanisms: i) direct autoxidation of EPA, and ii) beta-oxidation of nPF4 alpha-VI, formed by autoxidation of DHA. This iP may therefore serve as an excellent marker for the combined in vivo peroxidation of EPA and DHA.     

iPF2alpha-III-17,18,19,20-d4: total synthesis and metabolism

The first total synthesis of 17,18,19,20-d4-iPF2alpha-III 32, a deuterated analog of iPF2alpha-III, is described. We have used this analog in some beta-oxidation studies with rat liver homogenates and have shown that 32 was metabolized to 17,18,19,20-tetradeutero-2,3-dinor-iPF2alpha-III 36 and 17,18,19,20-tetradeutero-2,3-dinor-5,6-dihydro-iPF2alpha-III 37.     

Uncoupling protein-2 induction in rat hepatocytes after acute carbon tetrachloride liver injury

This study is focused on the role of UCP-2 in hepatic oxidative metabolism following acute CCl(4) administration to rats. UCP-2 mRNA, almost undetectable in the liver of controls, was significantly increased 24 h after CCl(4) administration, peaked at 72 h and then tended to disappear. UCP-2 protein, undetectable in controls, increased 48-72 h after CCl(4) treatment. Experiments with isolated liver cells indicated that in control rats UCP-2 was expressed in non-parenchymal cells and not in hepatocytes, whereas in CCl(4)-treated rats UCP-2 expression was induced in hepatocytes and was not affected in non-parenchymal cells. Addition of CCl(4) to the culture medium of hepatocytes from control rats failed to induce UCP-2 expression. Liver mitochondria from CCl(4)-treated rats showed an increase of H(2)O(2) release at 12-24 h, followed by a rise of TBARS. Vitamin E protected liver from CCl(4) injury and reduced the expression of UCP-2. Treatment with GdCl(3) prior to CCl(4), in order to inhibit Kupffer cells, reduced TBARS and UCP-2 mRNA increase in hepatic mitochondria. Our data indicate that CCl(4) induces the expression of UCP-2 in hepatocytes with a redox-dependent mechanism involving Kupffer cells. A role of UCP-2 in moderating CCl(4)-induced oxidative stress during tissue regeneration after injury is suggested.     

Effects of vitamin C and aspirin in ischemic stroke-related lipid peroxidation: results of the AVASAS (Aspirin Versus Ascorbic acid plus Aspirin in Stroke) Study

A condition of oxidative stress is known to occur in ischemic stroke, the current therapeutic intervention of which is largely limited to thrombolysis. To assess the effect of vitamin C - in conjunction to aspirin - in ischemic stroke-related lipid peroxidation, we measured plasma levels of ascorbate, of 8,12-isoprostanes F2alpha-VI (8,12-iPF2alpha-VI) and activities and levels of a broad spectrum of antioxidant enzymes and micronutrients in stroke patients randomized to receive, from stroke onset and up to three months, either vitamin C (200 mg/day) plus aspirin (300 mg/day) or only aspirin (300 mg/day). By the end of the first week, patients treated with vitamin C plus aspirin had higher vitamin C levels (p = 0.02) and lower 8,12-iPF2alpha-VI levels (p = 0.01) than patients treated with aspirin alone. The significance was maintained for the increase of vitamin C after three months of therapy (p < 0.01). The clinical functional outcome for both groups of patients similarly ameliorated after three months of treatment. We conclude that vitamin C, at the dose of 200 mg/day and in conjunction with aspirin, significantly decreases ischemic stroke-related lipid peroxidation in humans. Further studies are warranted to clarify whether the use of vitamin C may add clinical long-term beneficial effects in patients with stroke.     

Age- and gender-related oxidative status determined in healthy subjects by means of OXY-SCORE, a potential new comprehensive index.

Oxidative stress has been related to various diseases, gender and ageing, and has been measured by various markers. The authors developed a procedure to compute a global oxidative stress index (OXY-SCORE), reflecting both oxidative and antioxidant markers in healthy subjects. Its performance was tested in relation to age and gender and in coronary artery disease (CAD) patients. Eighty-two healthy subjects and 20 CAD patients were enrolled. Plasma free and total malondialdehyde (F- and T-MDA), glutathione disulphide/reduced form ratio (GSSG/GSH) and urine isoprostanes (iPF2alpha-III) levels were combined as oxidative damage markers (damage score). GSH, alpha- and gamma-tocopherol (TH) levels, and individual antioxidant capacity were combined as antioxidant defence indexes (protection score). The OXY-SCORE was computed by subtracting the protection score from the damage score. Among single parameters, T-MDA and iPF2alpha-III significantly correlated with age; only GSH and both tocopherols correlated with male gender in healthy subjects. The OXY-SCORE was positively associated with age (p=0.004) and male gender (p=0.03). As expected, the OXY-SCORE was higher in CAD with a very significant p-value (<0.0001), after adjusting for age, gender and smoking. Combining different markers can potentially provide a powerful index in the evaluation of oxidative stress related to age, gender and CAD status.     

Local and systemic increase in lipid peroxidation after moderate experimental traumatic brain injury

Traumatic brain injury is a common event associated with neurological dysfunction. Oxidative damage, may contribute to some of these pathologic changes. We used a specific and sensitive marker of lipid peroxidation, the isoprostane 8,12-iso-iPF(2alpha) -VI, to investigate whether local and also systemic lipid peroxidation were induced following lateral fluid percussion (FP) brain injury in the rat. Animals were anesthetized and subjected to lateral FP brain injury of moderate severity, or to sham injury as controls. Urine was collected before anesthesia (baseline), 6 and 24 h after injury. Blood was collected at baseline, 1, 6 and 24 h after injury. Animals were killed 24 h after surgery and their brains removed for biochemical analysis. No significant difference was observed at baseline (preinjury) for urine and plasma 8,12-iso-iPF(2alpha) -VI levels between injured and sham-operated animals. By contrast, plasma and urinary levels increased significantly already at 1 and further increased 24 h following brain injury, when compared to sham-operated animals. Finally, compared with sham, injured animals had a significant increase in brain 8,12-iso-iPF(2alpha) -VI levels. These results demonstrate that moderate brain injury induces widespread brain lipid peroxidation, which is accompanied by a similar increase in urine and plasma. Peripheral measurement of 8,12-iso-iPF(2alpha) -VI levels after brain injury may be a reliable marker of brain oxidative damage.     

Determination of isoprostanes in urine samples from Alzheimer patients using porous graphitic carbon liquid chromatography-tandem mass spectrometry

F2-isoprostanes (F2-iPs) comprise four classes of isomers produced non-enzymatically by free radical attack on arachidonic acid, a component of the cell membrane. This paper describes a new method for the quantification of F2-isoprostanes in urine samples from thoroughly diagnosed Alzheimer's disease (AD) patients. The sample pretreatment consisted of liquid extraction of 900 microl urine with diethyl ether, its subsequent evaporation, and finally, reconstitution in 50 microl water. Of this, 20 microl was injected into a HPLC system with a 15 mm x 1 mm porous graphitic carbon column coupled to a triple quadrupole mass spectrometer running in negative electrospray ionization mode. The F2-isoprostanes were separated in 15 min using a linear solvent gradient comprising water, methanol, acetonitrile and ammonium hydroxide at a pH of 9.5. The average recovery obtained was approximately 75%. The limit of detection (3S/N) was calculated for iPF2alpha-III to be 0.7 pg injected on column, corresponding to 0.1 nM. The average level of iPF2alpha was 241 +/- 163 pg/mg creatinine in the urine samples from AD patients (average +/- standard deviation). The corresponding control values were 216 +/- 101 pg/mg creatinine, i.e. no statistically significant difference was noticed. No correlation pattern specific to Alzheimer's disease was revealed by principal component analysis of the isoprostane peaks obtained either. The results from this study support earlier findings that levels of peripheral isoprostanes are not increased in patients with Alzheimer's disease.     

Racial Differences in Urinary F2‐Isoprostane Levels and the Cross‐Sectional Association With bmi

Levels of four urinary F₂‐isoprostanes (F₂‐IsoPs) were examined in a large sample of the Insulin Resistance Atherosclerosis Study (IRAS) multiethnic cohort: 237 African Americans (AAs), 342 non‐Hispanic whites (NHWs), and 275 Hispanic whites (HWs). F₂‐IsoP isomers — iPF2a‐III, 2,3‐dinor‐iPF2a‐III, iPF2a‐VI, and 8,12‐iso‐iPF2a‐VI — were measured in 854 urine samples using liquid chromatography with tandem mass spectrometry detection. In AAs, levels of all four F₂‐IsoPs were lower compared with NHWs and HWs (P values <0.05). When stratified by BMI, this gap was not observed among participants with normal BMI but appeared among overweight participants and increased among obese participants. Examining the slopes of the associations between BMI and F₂‐IsoPs showed no association between these variables among AAs (P values >0.2), and positive associations among whites (P values <0.05). Taking into account that positive cross‐sectional associations between systemic F₂‐IsoP levels and BMI have been consistently demonstrated in many study populations, the lack of such an association among AAs reveals a new facet of racial/ethnic differences in obesity‐related risk profiles.     

Serum 8,12-iso-iPF2α-VI Isoprostane Marker of Oxidative Damage and Cognition Deficits in Children with Konzo

We sought to determine whether motor and cognitive deficits associated with cassava (food) cyanogenic poisoning were associated with high concentrations of F2-isoprostanes, well-established indicators of oxidative damage. Concentrations of serum F2-isoprostanes were quantified by LC-MS/MS and anchored to measures of motor proficiency and cognitive performance, which were respectively assessed through BOT-2 (Bruininks/Oseretsky Test, 2^nd Edition) and KABC-II (Kaufman Assessment Battery for Children, 2^nd edition) testing of 40 Congolese children (21 with konzo and 19 presumably healthy controls, overall mean age (SD): 9.3 (3.2) years). Exposure to cyanide was ascertained by concentrations of its main metabolite thiocyanate (SCN) in plasma and urine. Overall, SCN concentrations ranged from 91 to 325 and 172 to 1032 µmol/l in plasma and urine, respectively. Serum isoprostanes ranged from 0.1 to 0.8 (Isoprostane-III), 0.8 to 8.3 (total Isoprostane-III), 0.1 to 1.5 (Isoprostane-VI), 2.0 to 9.0 (total Isoprostane-VI), or 0.2 to 1.3 ng/ml (8,12-iso-iPF2α-VI isoprostane). Children with konzo poorly performed at the BOT-2 and KABC-II testing relative to presumably healthy children (p<0.01). Within regression models adjusting for age, gender, motor proficiency, and other biochemical variables, 8,12-iso-iPF2α-VI isoprostane was significantly associated with the overall cognitive performance (β = −32.36 (95% CI: −51.59 to −13.03; P<0.001). This model explained over 85% of variation of the KABC-II score in children with konzo, but was not significant in explaining the motor proficiency impairment. These findings suggest that cognitive deficits and, possibly, brain injury associated with cassava poisoning is mediated in part by oxidative damage in children with konzo. 8,12-iso-iPF2α-VI isoprostane appears to be a good marker of the neuropathogenic mechanisms of konzo and may be used to monitor the impact of interventional trials to prevent the neurotoxic effects of cassava cyanogenic poisoning.     

Biomarkers of Oxidative Stress Study II: Are oxidation products of lipids,proteins, and DNA markers of CCl4 poisoning?

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.     

Urinary F₂-isoprostanes, obesity, and weight gain in the IRAS cohort

Obesity has been associated with increased F(2)-isoprostane (F(2)-IsoP) levels cross-sectionally. However, the prospective association may be inverse, based on our earlier finding that elevated urinary F(2)-IsoP levels predict lower risk of diabetes. This earlier finding led us to hypothesize that urinary F(2)-IsoPs reflect the intensity of oxidative metabolism and as such predict lower risk of both diabetes and weight gain. We examined cross-sectional relationships with obesity and prospective relationships with weight gain using the data from 299 participants of the Insulin Resistance Atherosclerosis Study (IRAS), all of whom were free of diabetes at baseline. Four urinary F(2)-IsoPs were assayed in stored baseline urine samples using liquid chromatography with tandem mass spectrometry: iPF(2α)-III, 2,3-dinor-iPF(2α)-III, iPF(2α)-VI, and 8,12-iso-iPF(2α)-VI (F(2)-IsoP 1-4, respectively). Baseline F(2)-IsoPs were positively associated with baseline measures of obesity; the strongest associations were found with two F(2)-IsoPs: odds ratios (95% confidence intervals) for overall and abdominal obesity were 1.74 (1.26-2.40) and 1.63 (1.18-2.24) for F(2)-IsoP2 and 1.47 (1.12-1.94) and 1.64 (1.22-2.20) for F(2)-IsoP4. F(2)-IsoP2 showed the strongest and significant inverse association with weight gain during the 5-year follow-up period: increase in F(2)-IsoP2 equal to 1 s.d. was associated with 0.90 kg lower weight gain (P = 0.02) and the odds ratios for relative (≥5%) and absolute (≥5 kg) weight gain were 0.67 (0.47-0.96) and 0.57 (0.37-0.87), respectively. The other three F(2)-IsoPs were consistently inversely associated with weight gain, although not significantly, suggesting that different F(2)-IsoPs vary in their ability to detect the association with weight gain.     

肝细胞生长因子对四氯化碳损伤肝细胞的保护作用及相关基因表达

利用无血清原代培养大鼠肝细胞,观察重组人肝细胞生长因子（ｒｈＨＧＦ）对ＣＣｌ４染毒肝细胞的保护作用。结果表明：（１）ｒｈＨＧＦ（５ｎｇ／ｍｌ）预自理后可显著提高ＣＣｌ４（１５ｍｍｏｌ／Ｌ）染毒肝细胞存活率,降低细胞内丙氨酸氨基转移酶（ＡＬＴ）、Ｋ＾＋的漏出;（２）表皮生长因子（ＥＧＦ,５０ｎｇ／ｍｌ）和ｒｈＨＧＦ（５ｎｇ／ｍｌ）合用预处理肝细胞,ＣＣｌ４染毒后细胞内ＡＬＴ、Ｋ＾＋漏出较ｒｈＨＧＦ和     

Early hypomethylation of 2''-O-ribose moieties in hepatocyte cytoplasmic ribosomal RNA underlies the protein synthetic defect produced by CCl4

Carbon tetrachloride (CCl4) treatment of rats produces an early defect in methylation of hepatocyte ribosomal RNA, which occurs concurrently with a defect in the protein synthetic capacity of isolated ribosomes. The CCl4-induced methylation defect is specific for the 2'-O-ribose position, and a corresponding proportional increase in m7G base methylation occurs in vivo. Undermethylated ribosomal subunits (rRNA) from CCl4-treated preparations can be methylated in vitro to a much greater extent than those from control preparations, and in vitro methylation restores their functional capacity. In vitro methylation of treated ribosomal subunits (which restores functional capacity) occurs at 2'-O-ribose positions (largely G residues). In contrast, in vitro methylation of control ribosomal subunits (which does not affect functional activity) represents base methylation as m7G, sites which are apparently methylated in treated preparations in vivo. Methylation/demethylation of 2'-O-ribose sites in rRNA exposed on the surface of cytoplasmic ribosomal subunits may represent an important cellular mechanism for controlling protein synthesis in quiescent hepatocytes, and it appears that CCl4 disrupts protein synthesis by inhibiting this 2'-O-ribose methylation.