二甲基亚砜对原代培养大鼠肝细胞醋氨酚损伤的影响

醋氨酚（ＡＡＰ）引起肝细胞损伤时，肝细胞还原型谷胱甘肽（６ＳＨ）含量下降，胞浆游离Ｃａ２＋浓度（［Ｃａ２＋］）升高二甲亚砜（ＤＭＳＯ）对ＡＡＰ肝细胞损伤有明显的保护作用。对轻度损伤能完全拮抗，ＤＭＳＯ对ＧＳＨ含量下降有明显的拮抗作用，当ＧＳＮ维持在一定水平时，ＡＡＰ不引起［Ｃａ２＋］升高。提示ＤＭＳＯ可能通过保护ＧＳＨ等巯基物质而发挥拮抗ＡＡＰ肝细胞损伤的作用     

Enhanced formation of ethane and n-pentane by rat hepatocytes in the presence of dimethyl sulfoxide

Incubation of rat hepatocytes with 14 mM dimethyl sulfoxide (DMSO) produced an increase in the formation of ethane, measured by capillary column gas chromatography, to 18.0 pmoles/hr/10(7) cells from 11.2 pmoles/hr/1-(7) cells from 5.6 pmoles/hr/10(7) cells in control hepatocytes. This was about one-third the stimulation of ethane and n-pentane formation produced by incubation of hepatocytes with 13 mM carbon tetrachloride. DMSO-stimulated ethane and n-pentane formation was inhibited up to 63% by 0.1 microM alpha-tocopherol and up to 89% by N2. Formation of dimethylsulfide from DMSO by hepatocytes was the same in air and N2. DMSO increased methane production by hepatocytes to 31.3 pmoles/hr/10(7) cells from 6.9 pmoles/hr/10(7) cells in control hepatocytes. Although DMSO apparently stimulated lipid peroxidation by hepatocytes, as measured by ethane and n-pentane formation, there was no increase in the formation of thiobarbituric acid reactive material. DMSO was not toxic to hepatocytes, measured by release of cytosolic lactate dehydrogenase, over a 2-hr incubation. Possible mechanisms for the increase in alkane formation by DMSO are discussed.     

The toxicity of dimethyl sulphoxide (DMSO) for the dog,pig, rat and rabbit

Dimethyl sulphoxide (DMSO) was tested for oral toxicity in rats and dogs, and dermal toxicity in rabbits and pigs. Oral administration was by gastric intubation as a 50% aqueous solution, 5 days/week at levels equivalent to 9.0, 3.0 or 1.0 ml undiluted DMSO/kg/day. For dermal application 50% and 90% aqueous solutions were used to give levels equivalent to 8.1, 4.5, 2.7 or 1.5 ml DMSO/kg/day, as one daily application for rabbits, and divided into two applications/day for pigs. Dogs were dosed for approximately 2 years and pigs for 1 year, although half the animals of both species were dosed for only 18 weeks. Rats were dosed for 18 months, but some were used for interim sacrifice after a year. Rabbits received applications to normal and abraded skin for 6 months.Minor changes in bodyweight and haematological values were observed, together with a physiological diuretic response to DMSO, but the target organ was the eye, principally the lenticular nucleus. Ocular effects in dogs started after 5–10 weeks dosing at 9 ml/kg and consisted of central (nuclear) lens changes with alteration of the refractive index (myopia); transitory equatorial opacities during the 5th month; central (nuclear) opalescence; and changes in the vitreous humour. Similar changes occurred more slowly at 3 ml/kg, the alterations to the vitreous being first observed after 9–10 months at this level. Progressive nuclear refractive changes occurred after dosing for considerably longer than 6 months at 1 ml/kg, but none of the animals in this group manifested the opalescence. Biochemical investigation of the lenses revealed reduction of soluble protein (mainly α-crystallin), glutathione and water levels, and an increase of insoluble protein. Evidence of recovery was limited mainly to a reduction in the number of dioptres needed to correct nuclear refractive change. Cessation of dosing led to regression of refractive nuclear changes but did not prevent the appearance of opalescence at 3 ml/kg and above.Dogs were the most severely affected of the 4 species, with nuclear effects at 1 ml/kg, extensive changes in the lens, and involvement of the vitreous. Pigs and rabbits were affected by dose levels of 2.7 ml/kg and 1.5 ml/kg respectively. Rats occasionally showed minimal changes at 9 ml/kg.The importance of the findings in dogs is discussed in relation to general toxicology protocols. It is emphasised that reversibility of signs, and adequate duration of administration, must both be considered when ascertaining whether changes occur at levels approximating to those of human intake.     

Clozapine-induced hepatotoxicity in rat hepatocytes by gel entrapment and monolayer culture

Clozapine is limitedly used due to its adverse effect including agranulocytosis and hepatotoxicity. However, the mechanism of clozapine toxicity is still not clear. The previous in vitro studies on microsomes proposed a possible mediation of cytochrome P450 (CYP) in producing reactive metabolites. In this paper, clozapine toxicity was, respectively, examined in two cultures of rat hepatocytes. Gel entrapment culture of hepatocytes with higher expression on CYP activities showed higher sensitivity to clozapine treatment than hepatocyte monolayer, indicating the possible involvement of CYP in hepatotoxicity of clozapine. Moreover, in each culture, CYP inhibitors were used to confirm the possible mediation of CYP enzymes. Pretreatment of hepatocytes with CYP 3A inhibitor (ketoconazole), CYP 2E1 inhibitor (diethyldithiocarbamate, DDC) and non-specific inhibitor (cimetidine) significantly reduced the toxicity of clozapine. But the pretreatment with CYP 1A2 inhibitor (fluvoxamine) had no such protective effect indicative of non-function of CYP 1A2 in clozapine toxicity. In addition, glycyrrhizic acid (GA), a scavenger of reactive oxygen species (ROS), also inhibited the adverse response to clozapine, suggesting the positive involvement of oxidant pressure. Thus, it could be concluded that clozapine-induced toxicity was mediated by CYP, particularly CYP 3A and CYP 2E1, and oxidant pressure.     

Carrier-mediated uptake of pravastatin by rat hepatocytes in primary culture.

The transport mechanism of pravastatin, a new cholesterol-lowering drug, was compared in vitro with rat hepatocyte primary culture and mouse skin fibroblasts (L-cells). The uptake of 14C-labeled pravastatin by cultured hepatocytes was temperature- and dose-dependent. The temperature-dependent uptake as a function of [14C]pravastatin concentration showed saturation kinetics with Km = 32.2 microM and a maximal uptake rate of 68 pmol/mg protein/min. The uptake of pravastatin was inhibited significantly by metabolic inhibitors such as rotenone, oligomycin A, antimycin A, 2,4-dinitrophenol and KCN. Unlabeled pravastatin as well as R-416 and R-195, structural analogues of pravastatin, effectively competed for the hepatic uptake of [14C]pravastatin at 37 degrees. These results indicate that pravastatin is taken up by the liver by an active transport. In contrast, the transport of pravastatin by L-cells was temperature-independent and non-saturable, suggesting that the uptake of pravastatin by L-cells is mediated by passive diffusion. The marked difference in the uptake mechanism of pravastatin between hepatocytes and L-cells may account for a unique feature of this drug in that the uptake and inhibition of cholesterol biosynthesis occur selectively in the liver.     

Elevation of blood sugar in rats after single doses of dimethyl sulphoxide,dimethyl formamide,and dimethyl acetamide

Elevation of blood sugar occurred in rats after oral and intraperitoneal administration of dimethylsulphoxide (DMSO), dimethylformamide (DMF) and dimethylacetamide (DMA). Minimum effective doses were 4 ml/kg i.p. and 8 ml/kg p.o.; 0.5 ml/kg i.p. and 2 ml/kg p.o., and 1 ml/kg i.p. and 1 ml/kg p.o., respectively.     

Effect of dimethyl sulphoxide on oxidative stress, activation of mitogen activated protein kinase and necrosis caused by thioacetamide in the rat liver

Thioacetamide (400 mg/kg body weight, i.p.) was administered to rats. After 12 h the activity of plasma glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) was significantly higher than that of the control group, and after 24 h plasma GOT and GPT activities strongly increased. These results indicated that the necrotic process was initiated at about 12 h and developed thereafter. By co-administration of dimethyl sulphoxide (DMSO, 18 and 1 h before, and 8 h after administration of thioacetamide: each time, 2.5 ml/kg body weight, p.o.), plasma GOT and GPT were significantly decreased and were even comparable to the control group, showing that DMSO totally prevented the necrotic action of thioacetamide. After 12 and 24 h of thioacetamide administration, the hepatic level of vitamin C, the most sensitive chemical indicator of oxidative stress, decreased significantly, indicating that oxidative stress was significantly enhanced 12 h after thioacetamide intoxication and thereafter. DMSO totally restored the liver vitamin C level, demonstrating that DMSO effectively ameliorated the oxidative stress caused by thioacetamide, resulting in the prevention of necrosis of the liver. Phosphorylated c-Jun NH(2)-terminal kinase (JNK) significantly increased transiently 12 h after treatment with thioacetamide. These results indicated that oxidative stress and the activation of JNK took place almost simultaneously. Phosphorylated extracellular signal-related kinase (ERK) 2 was significantly increased 6-12 h after thioacetamide injection. Phosphorylated p38 MAPK (mitogen activated protein kinase) was significantly decreased 24 h after administration of thioacetamide. DMSO treatment inhibited the change of these MAPKs by thioacetamide, corresponding with the prevention of the liver necrosis as well as the attenuation of oxidative stress.     

Transfection of adult primary rat hepatocytes in culture

The use of adult primary hepatocytes in culture is of importance for the understanding of hepatic processes at the cellular and molecular levels, and the possibility to employ transient transfection of reporter constructs is invaluable for mechanistic studies on hepatic gene regulation. Although frequently used, there is a lack of reports addressing optimization and characterization of transfection of primary rodent hepatocytes. Here, we have shown that the efficiency of biochemical transfection reagents varies significantly and that Lipofectamine2000 was a superior transfection reagent for adult primary rat hepatocytes when using luciferase reporter vectors. The efficiency increased when the cells were allowed ample time to adapt to the in vitro milieu. Cotransfection of a second reporter gene indicated a risk for promoter competition, and we found that relating reporter activity to total cellular protein content gave consistent and reliable results. Differentiation of the cells, achieved by including biomatrix from the Engelbreth-Holm-Swarm mouse sarcoma in the culture system, was to a larger extent required for hormonal/drug responses of transfected constructs than for responses of endogenous genes and assured responses of transfected constructs. Dexamethasone (Dex) is most often included in hepatocyte culture media, but we could not demonstrate a general beneficial effect of Dex on expression of luciferease reporter contructs. Using the established protocol, we have demonstrated responses of transfected constructs to growth hormone, glucocorticoid and LXR stimuli.     

Induction of cytochrome P-450 by dimethyl sulfoxide in primary cultures of adult rat hepatocytes.

Treatment of hepatocyte cultures with dimethyl sulfoxide (DMSO) induced P-450IIE1-specific aniline 4-hydroxylase activity and P-450IA1-specific ethoxyresorufin O-deethylase activity at a concentration of 0.1% (v/v). The P-450IIB-specific pentoxyresorufin O-deethylase activity was induced only at the 2% (v/v) level. Dot blot analysis of the total cellular RNA and cycloheximide treatment of the culture suggested that induction of ethoxyresorufin O-deethylase activity by DMSO may be due to the increase of de novo synthesis of the P-450IA1 protein, not to accumulation of mRNA in the hepatocyte culture.     

Isoniazid-induced hepatotoxicity in rat hepatocytes of gel entrapment culture

Gel entrapment culture of rat hepatocytes in hollow fibers were evaluated as a potential in vitro model for studies on isoniazid-induced hepatotoxicity. After exposure to isoniazid (0.11 mM and 1.1 mM) for 24-96 h, gel entrapped hepatocytes were more severely damaged than hepatocyte monolayers according to the assays on methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, and albumin secretion. Furthermore, CYP 2E1 activity detected by 4-nitrocatechol (4-NC) formation maintained at least 7 days in gel entrapped hepatocytes but decreased to an undetectable level within 2 days in hepatocyte monolayer. And the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), significantly reduced isoniazid-induced GSH depletion in gel entrapped hepatocytes. In addition, the protective effects of N-acetylcysteine (NAC), GSH, liquorice extract and glycyrrhizic acid (GA), a purified compound from liquorice extract, against isoniazid hepatotoxicity were clearly observed in gel entrapped hepatocytes at 72 h incubation. Overall, gel entrapped hepatocytes were more susceptible to isoniazid-induced hepatotoxicity than hepatocyte monolayers by a possible mechanism that higher CYP 2E1 activity in gel entrapped hepatocytes could enhance isoniazid toxicity. This indicates that gel entrapped hepatocytes in hollow fibers could be a more effective model than hepatocyte monolayer for hepatotoxicity research in vitro.     

Some effects of dimethyl sulphoxide (DMSO) on the frog neuromuscular junction

1. Dimethyl sulphoxide (DMSO) partially reversed neuromuscular blockade brought about by the action of (+)-tubocurarine or Mg(2+) on the frog sartorius nerve-muscle preparation.2. The amplitude and duration of the endplate potential (e.p.p.) were increased by DMSO at concentrations of 70 mM or greater.3. Miniature endplate potentials were raised in frequency, prolonged in duration and increased in amplitude by DMSO at concentrations of 141 mM or greater, but the increase in amplitude was generally less than in the case of the e.p.p.4. The resting muscle membrane potential was significantly depolarized by DMSO at 70 mM or greater concentrations, both at the endplate and remote from an endplate.5. The reversal of neurmuscular blockade by DMSO can be explained in terms of its previously reported ability to inhibit cholinesterase activity, together with the depolarizing action on muscle.     

Antidotal effects of dimethyl sulphoxide against paracetamol-, bromobenzene-, and thioacetamide-induced hepatotoxicity

In mice the hepatotoxic effects of paracetamol (0·5–1·0 g kg^?1, orally) as evidenced by increased serum enzyme activities of the aminotransferases and sorbitol dehydrogenase were dose-dependently inhibited by simultaneous treatment with dimethyl sulphoxide (DMSO 0·25–1·0 g kg^?1, i.p.). DMSO was also active against bromobenzene- and thioacetamide-induced hepatotoxicity, but failed to protect mice against carbon tetrachloride-induced liver damage. Hepatic glutathione depletion in mice amounting to 94% after paracetamol (0·5 g kg^?1, orally) and to 60% after bromobenzene (0·25 ml kg^?1, orally) was dose-dependently reduced by the simultaneous administration of DMSO (0·25–1·0 g kg^?1, i.p.). This indicates less conjugation of the toxic metabolites of paracetamol and bromobenzene to liver glutathione (G-SH) in the presence of DMSO.     

Cardiac inotropism of dimethyl sulphoxide: osmotic effects and interactions with calcium ion

DMSO is a cryoprotectant drug which produces concentration dependent inotropic and chronotropic changes in a variety of cardiac muscle preparations. Electrically driven and rabbit guinea pig atria were used to elucidate the mechanisms of these changes. DMSO concentrations of 0.14 to 1.41 M generally produced significant positive inotropic responses in guinea pig atria, and significant negative inotropic responses in rabbit atria. The form and magnitude of the response to given concentrations of DMSO depended upon the manner in which DMSO was administered and removed, suggesting that osmotic effects played an important role in producing the responses. Inotropic responses to DMSO appeared to be independent of extracellular Ca²⁺ concentration if rabbit atria were equilibrated in the altered Ca²⁺ environment prior to addition of DMSO. When atria were placed in Ca²⁺-depleted media, the presence of DMSO significantly augmented the rate of loss of contractile tension. Kinetic analysis of contractile tension decay suggested that DMSO acted on a pool of myocardial Ca²⁺ responsible for regulating contractile strength. The magnitude of this effect was independent of DMSO concentration, and so may not be a direct result of osmotic mechanisms. These actions on contractile strength will be considered in relation to other reported possible mechanisms of DMSO cardiac actions.     

Influence of the isolation method on the stability of differentiated phenotype in cultured rat hepatocytes

Primary cultures of adult rat hepatocytes were established using two different isolation procedures: a two-step collagenase perfusion and a method using ethylenediaminetetraacetate (EDTA) as the dissociating agent. Both techniques provided good yields of hepatocytes with comparable viability. The evolution of hepato-specific protein levels and several drug-metabolizing enzyme activities were followed for 8 days in cultured hepatocytes obtained by both methods. EDTA-isolated hepatocytes maintained a low gamma glutamyltransferase (GGT) activity, whereas collagenase-treated cells acquired a high GGT level. Transferrin secretion and tyrosine aminotransferase (TAT), alanine aminotransferase (ALT), and microsomal epoxide hydrolase (mEH) activities were stable in both EDTA- and collagenase-isolated hepatocytes, whereas albumin secretion, aspartate amino transferase (AST) activity, total cytochromes P-450 content, IA1 and IIB1 P-450 isoenzymes, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) levels, and bilirubin glucuronidation decreased faster in collagenase-treated cells. The most important difference observed was the maintainance of the mixed-function oxidase system in EDTA-isolated hepatocytes. These results emphasize the critical role of isolation technique in stabilization of differentiated hepatocytes in primary culture.     

Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity

This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 μM which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 μM. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to β-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.