Effect of penicillin G on the biliary excretion of cholephilic compounds in rats

Aim

Penicillin G is reported to increase bile flow by increasing biliary glutathione excretion, as well as the biliary excretion of penicillin G itself. In order to study the effect of penicillin G on the hepatic excretory pathway, the effect of colchicine and genipin on the increase of biliary glutathione excretion induced by penicillin G was studied in rats. The effect of penicillin G on the biliary excretion of sulfobromophthalein and erythromycin was also studied, together with the effect of penicillin G on cholestasis induced by estradiol-17??-glucuronide.

Methods

After bile duct cannulation, penicillin G was administered to rats at the rate of 0.5???mol/min/100?g. The effect was examined of colchicine pretreatment (0.2?mg/100?g) and genipin administration (0.5???mol/min/100?g) on biliary glutathione excretion increased by penicillin G infused at the rate of 0.5???mol/min/100?g. The effect of penicillin G on the biliary excretion of sulfobromophthalein and erythromycin (0.2 and 0.1???mol/min/100?g for 90?min, respectively) was studied, together with the effect of penicillin G on cholestasis induced by estradiol-17??-glucuronide (0.075???mol/min/100?g for 20?min).

Results

Penicillin G increased bile flow and biliary glutathione excretion, which were not inhibited by colchicine or genipin. Biliary penicillin G excretion was markedly reduced in Eisai hyperbilirubinemic rats (EHBR) and Mrp2-deficient rats. Biliary sulfobromophthalein and erythromycin excretion was unchanged by penicillin G. Cholestasis induced by estradiol-17??-glucuronide was not relieved by penicillin G.

Conclusions

It was shown that colchicine-sensitive vesicular transport has no role on the penicillin G-induced insertion of Mrp2 into the canalicular membrane, as has been observed with genipin. Although the choleresis of penicillin G is thought to be due to the increased biliary excretion of glutathione and penicillin G itself by Mrp2, the mechanism of Mrp2 insertion by penicillin G is thought to be partly different from that by genipin.     

Effect of genipin on the biliary excretion of cholephilic compounds in rats.

Aim: Genipin, a metabolite of geniposide, is reported to stimulate the insertion of multidrug resistance protein 2 (Mrp2) in the bile canalicular membrane, and to cause choleresis by increasing the biliary excretion of glutathione, which has been considered to be a substrate of Mrp2. In the present study, the effect of colchicine on the choleretic effect of genipin was investigated. The effect of genipin on the biliary excretion of the substrates of bile salt export pump and Mrp2 was also studied. Methods: After bile duct cannulation into rats, genipin was administered at the rate of 0.2 mumol/min/100 g, and the effect of colchicine pretreatment (0.2 mg/100 g) was examined. Metabolites of genipin in the bile were examined by a thin layer chromatography. Taurocholate (TC), sulfobromophthalein (BSP), and pravastatin were infused at the rate of 1.0, 0.2 and 0.3 mumol/min/100 g, respectively, and the effect of genipin co-administration was examined. Results: Genipin increased bile flow and the biliary glutathione excretion, and those increases were not inhibited by colchicine. The biliary excretion of genipin glucuronide was less than 10% of the genipin excreted into bile. The biliary excretion of TC, BSP, and pravastatin was unchanged by genipin co-administration. Conclusion: It was indicated that colchicine-sensitive vesicular transport has no role on the genipin-induced insertion of Mrp2 to the canalicular membrane. Choleresis of genipin is considered to be mainly due to the increased biliary glutathione excretion by genipin, not by the biliary excretion of glucuronide. TC had no effect on the biliary glutathione excretion.     

Effect of cyclosporin A on the biliary excretion of cholephilic compounds in rats.

Cyclosporin A (CsA) is known to cause cholestasis. CsA is reported to competitively inhibit the transport of the substrates of the bile salt export pump (Bsep), multidrug resistance protein 2 (Mrp2) and P-glycoprotein (P-gp) in the canalicular membrane vesicles. However, the inhibitory effect of CsA on various substrates of the canalicular ATP-dependent transporters in vivo is unknown. Therefore, in the present study, the acute effect of CsA on the biliary excretion of the substrates of Bsep, Mrp2 and P-gp was examined under the same condition. Ten minutes after the intravenous administration of CsA (25mg/kg), the biliary excretion of various bile acids and organic anions and cations was studied. CsA decreased the biliary excretion of tracer amounts of taurocholate, leukotriene C(4), estradiol-17beta-glucuronide, pravastatin, vinblastine and erythromycin. In contrast, the biliary excretion of high doses of taurocholate and sulfobromophthalein was only slightly or not inhibited by CsA. In conclusion, CsA may competitively inhibit biliary excretion of substrates of Bsep, Mrp2 and P-gp also in vivo, and CsA is considered to inhibit bile acid-dependent bile flow by the competitive inhibition of the canalicular transport of bile acids by Bsep.     

Altered localization and activity of canalicular Mrp2 in estradiol-17beta-D-glucuronide-induced cholestasis

Estradiol-17beta-D-glucuronide (E(2)17G), an endogenous metabolite of estradiol, induces a potent dose-dependent and reversible inhibition of bile flow in the rat. We analyzed the effect of a single dose of E(2)17G (15 micromol/kg, intravenously) to female rats on bile flow and the endocytic retrieval and function of the canalicular multidrug resistance-associated protein 2 (Mrp2) and the effect of pretreatment with dibutyryl-cyclic AMP (DBcAMP; 20 micromol/kg) on these measures. Bile flow was maximally inhibited by 85% within 10 minutes of E(2)17G and returned to 50% and 100% of control levels within 75 and 120 minutes, respectively. Western analysis of total homogenates and mixed plasma and intracellular membranes suggested partial internalization of Mrp2 during the acute phase of cholestasis at 20 minutes and during the period of recovery from cholestasis at 75 minutes, which returned to control levels by 180 minutes after E(2)17G. Confocal analysis confirmed Western studies and demonstrated endocytic retrieval of Mrp2 from the canalicular membrane into pericanalicular and intracellular domains. The biliary concentration and excretion of the model Mrp2 substrate, dinitrophenyl-S-glutathione (DNP-SG), was impaired in parallel with the extent of Mrp2 retrieval. Pretreatment with DBcAMP partially protected against maximal E(2)17G cholestasis and the endocytic retrieval and decreased function of Mrp2 at 20 minutes and significantly accelerated the exocytic insertion of Mrp2 into the canalicular membrane and the recovery of bile flow and biliary excretion of DNP-SG. In conclusion, these data indicate that E(2)17G induces endocytic internalization of Mrp2, which occurs in parallel with decreased bile flow and Mrp2 transport activity.     

Lithocholate-3-O-glucuronide-induced cholestasis

The mechanism of lithocholate-3-O-glucuronide-induced cholestasis is unknown. In this study, we investigated the cholestatic effects of this agent in a congenital hyperbilirubinemic rat, EHBR. We also studied the effects of ursodeoxycholate-3-O-glucuronide and tauroursodeoxycholate on lithocholate-3-O-glucuronide-induced cholestasis in rats. Lithocholate-3-O-glucuronide, administered at the rate of 0.1 mol/min/100 g for 40 min, a cholestatic dose in control rats, failed to cause cholestasis in EHBR, and biliary lithocholate-3-O-glucuronide excretion was delayed. Biliary concentrations of this agent did not correlate with the severity of cholestasis. Both tauroursodeoxycholate and ursodeoxycholate-3-O-glucuronide, infused at the rate of 0.2 mol/min/100 g for 120 min, completely inhibited cholestasis induced by lithocholate-3-O-glucuronide administered at the rate of 0.1 mol/min/100 g for 40 min. Only tauroursodeoxycholate enhanced biliary lithocholate-3-O-glucuronide excretion. These findings indicate that lithocholate-3-O-glucuronide-induced cholestasis is induced by damage at the level of the bile canalicular membrane. Ursodeoxycholate-3-O-glucuronide inhibits this cholestasis, possibly by inhibiting the access of lithocholate-3-O-glucuronide to the bile canalicular membrane.     

Biliary excretion of bile acid conjugates in a hyperbilirubinemic mutant Sprague-Dawley rat

The hepatic transport of bile acid conjugates was studied in the Eisai hyperbilirubinuria rat, a Sprague-Dawley mutant rat with conjugated hyperbilirubinemia. Serum bile acid levels were increased, bile acid-independent bile flow was decreased and biliary glutathione concentrations were markedly decreased in the Eisai hyperbilirubinuria rat. Biliary excretion of sulfobromophthalein was markedly impaired and almost no glutathione conjugate was excreted in the bile of the Eisai hyperbilirubinuria rat. Biliary excretion of lithocholate-3-O-glucuronide and lithocholate-3-sulfate in the Eisai hyperbilirubinuria rat was markedly delayed, whereas that of lithocholate was only slightly delayed. After [14C]chenodeoxycholate infusion (1 mumol/min/100 gm for 60 min), the increases in bile flow and biliary excretion of isotope in the Eisai hyperbilirubinuria rat were not so prominent as those observed in control rats, and the glucuronide of chenodeoxycholate, which constituted about 15% of biliary chenodeoxycholate in control rats, was not observed in the Eisai hyperbilirubinuria rat. Initial uptake of lithocholate and its glucuronide and sulfate by isolated hepatocytes was not impaired in the Eisai hyperbilirubinuria rat; the profiles of cytosolic bile acid binding proteins in Eisai hyperbilirubinuria rat liver were identical to those in control liver. These data indicate that the Eisai hyperbilirubinuria rat has excretory impairment of organic anions, bile acid glucuronide and sulfate and that it has characteristics very similar to those of the hyperbilirubinemic mutant Wistar rats TR- and GY.     

Steroid hormones strongly support bovine articular cartilage integration in the absence of interleukin‐1β

Objective

Posttraumatic integration of articular cartilage at fracture sites is essential for mechanical stability of cartilage, and ruptured cartilage is a prerequisite for early osteoarthritis. This study was undertaken to investigate effects on articular cartilage integration mediated by steroid hormones, interleukin‐1β (IL‐1β), and combinations thereof.

Methods

Articular cartilage blocks were cultured in partial apposition for 2 weeks with ascorbic acid, testosterone, 17β‐estradiol, and dehydroepiandrosterone (DHEA), with or without IL‐1β. Mechanical integration was measured as adhesive strength, i.e., the maximum force at rupture of integrated cartilage blocks divided by the overlap area. Glycosaminoglycan content was used to study synthesized extracellular matrix.

Results

Culture in medium without supplements did not lead to integration (adhesive strength 0 kPa). With administration of ascorbic acid (100 μg/ml), the median adhesive strength was 49 kPa. In comparison with ascorbic acid alone, all steroid hormones induced a strong, concentration‐dependent stimulation of integration (with maximum values observed with DHEA at 3 × 10⁻⁵M, testosterone at 10⁻⁸M, and 17β‐estradiol at 10⁻¹¹M). For testosterone and 17β‐estradiol, this was also reflected by an increase of glycosaminoglycan content. Adhesive strength was increased with IL‐1β at 10 pg/ml, but not at 1 pg/ml or 100 pg/ml. In the presence of both IL‐1β and sex hormones, integration of articular cartilage was reduced.

Conclusion

This is the first study to demonstrate that steroid hormones such as 17β‐estradiol, DHEA, and testosterone stimulate articular cartilage integration. This effect is abrogated by low concentrations of IL‐1β. In the absence of IL‐1β or after neutralization of IL‐1β, steroid hormones might be favorable adjuvant compounds to optimize cartilage integration.

     

Amyloid‐β neurotoxicity in organotypic culture is attenuated by melatonin: involvement of GSK‐3β, tau and neuroinflammation

Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder marked by accumulation of extracellular deposits of amyloid‐β (Aβ) peptide in brain regions that are important for memory and cognition. The buildup of Aβ aggregates in the AD is followed by the formation of intracellular neurofibrillary tangles and activation of neuroinflammatory reactions. The present study investigated whether melatonin possesses a neuroprotective effect against Aβ‐induced toxicity. For this purpose, organotypic hippocampal slices were cultured and exposed to 25 μm of Aβ_25–35 in the absence or in the presence of melatonin (25, 50, or 100 μm ). In addition, the authors have investigated the involvement of GSK‐3β, tau protein, astroglial, and microglial activation, and cytokine levels in the melatonin protection against Aβ‐induced neurotoxicity. Melatonin prevented the cell damage in hippocampus induced by the exposure to Aβ_25–35. In addition, melatonin significantly reduced the activation of GSK‐3β, the phosphorylation of tau protein, the glial activation and the Aβ‐induced increase of TNF‐α and IL‐6 levels. On the basis of these findings, we speculate that melatonin may provide an effective therapeutic strategy for AD, by attenuating Aβ‐induced phosphorylation of tau protein, and preventing GSK‐3β activation and neuroinflammation.     

Matrine improves 17α-ethinyl estradiol-induced acute cholestasis in rats

Aim:  To explore the effects of matrine (MT) on acute intrahepatic cholestasis induced by 17α-ethinyl estradiol (EE) in rats.
Methods:  Acute intrahepatic cholestasis in rats were induced by EE, and the effects of MT on acute intrahepatic cholestasis were explored and compared with ursodeoxycholic acid (UDCA) by serum biochemical determination and bile excretion experiments.
Results:  The serum biochemical and bile biochemical results indicated that MT and UDCA had notable hepatoprotective effects by counteracting cholestasis induced by EE. The bile flow and the bile excretion of glycocholic acid (GC, a substrate of bile salt export pump [Bsep]), ketoprofen glucuronide (KPG) and rhodamine 123 (Rh123, a substrate of multidrug resistance protein 1 [MDR1]) decreased by EE, were significantly improved after administration of MT.
Conclusion:  MT exhibited potential protection against EE-induced acute intrahepatic cholestasis.     

17β‐estradiol induces CD40 expression in dendritic cells via MAPK signaling pathways in a minichromosome maintenance protein 6–dependent manner

Objective

The human immune system exhibits sexual dimorphism in autoimmune diseases such as systemic lupus erythematosus (SLE). Female sex hormones, including 17β‐estradiol, are strongly implicated in the gender bias in SLE. CD40 is a costimulatory molecule and plays a crucial role in modulating the immune response of effector cells. We have previously shown that 17β‐estradiol up‐regulated CD40 expression and altered minichromosome maintenance protein 6 (MCM6) gene expression in dendritic cells (DCs). The mechanism of the correlation between CD40 and MCM6 in the presence of 17β‐estradiol remains unknown. This study was undertaken to elucidate this mechanism and to explain the role of MCM6 in the gender bias in SLE.

Methods

Bone marrow–derived DCs transfected with small interfering RNA (siRNA) for MCM6 were treated with 17β‐estradiol in the absence or presence of CpG. The expression levels of costimulatory molecules, activity of MAPKs, and levels of MCM6 protein were measured. Moreover, the functions of DCs, including proliferation, apoptosis, endocytosis, and cytokine production, were analyzed. In addition, levels of messenger RNA for MCM6 were detected in DCs purified from SLE patients.

Results

Regardless of the presence or absence of CpG, 17β‐estradiol induced CD40 expression via the activation of p38 and JNK, but not ERK. The activation of p38 and JNK enhanced MCM6 expression, which then induced CD40 expression. Suppression of MCM6 in DCs abolished the up‐regulation of 17β‐estradiol–induced CD40 expression. Importantly, MCM6 expression was significantly increased in SLE patients compared with healthy controls.

Conclusion

Our findings indicate that 17β‐estradiol induces CD40 expression in DCs via p38 and JNK MAPKs in an MCM6‐dependent manner. MCM6 may be a critical mediator of sex‐based differences in autoimmune disease.

     

Biliary excretion of azelnidipine, a calcium antagonist, in rats

BACKGROUND AND AIMS: Azelnidipine (CS-905) is a novel dihydropyridine calcium antagonist that is known to be excreted in feces. To examine the mechanism of biliary excretion of azelnidipine, the authors studied its biliary excretion in Eisai hyperbilirubinuria rats (EHBR), multidrug resistance protein (Mrp)2-deficient rats, and the effect of cholephilic compounds on the biliary excretion of azelnidipine in rats. METHODS: Radiolabeled azelnidipine was intravenously administered to EHBR and control rats, and the biliary excretion of radiolabeled metabolites was studied. Furthermore, the effect of sulfobromophthalein, taurocholate and vinblastine on the biliary excretion of azelnidipine metabolites was also studied in control rats. RESULTS: The biliary excretion of azelnidipine metabolites was delayed in EHBR. The biliary excretion of azelnidipine metabolites was inhibited by sulfobromophthalein and vinblastine, but was not inhibited by taurocholate or phenothiazine pretreatment. CONCLUSION: These findings suggest that the metabolites of azelnidipine are excreted into the bile partly by Mrp2 and P-glycoprotein.     

Changes in biliary excretory mechanisms in rats with ethinyloestradiol-induced cholestasis

Several excretory pathways for cholephilic compounds have been known. To examine the changes in excretory pathways in cholestasis induced by ethinyloestradiol, various bile acids, organic anions and organic cations were intravenously administered to ethinyloestradiol-treated rats and their biliary excretion was studied. Biliary excretion of taurocholate was slightly delayed, but its excretory maximum was markedly decreased. Biliary excretion of lithocholate-3-O-glucuronide, leukotriene C4, sulphobromophthalein and pravastatin was markedly impaired to a similar extent. Biliary excretion of vinblastine, a P-glycoprotein substrate, was increased, suggesting increased expression of P-glycoprotein. In contrast, biliary excretion of erythromycin, a cationic antibiotic, was markedly impaired. In conclusion, ethinyloestradiol treatment altered the biliary excretion of organic compounds, which may partly be related to changes of the canalicular transporters.     

Effects of Japanese herbal medicine inchin-ko-to on endotoxin-induced cholestasis in the rat

Background/Objective. Inchin-ko-to (ICKT) is an herbal medicine used in Japan to treat jaundice and liver fibrosis. We investigated the effect of oral ICKT supplementation on endotoxin-induced cholestasis in the rat.Material and methods.Lipopolysaccharide (LPS) injection (1 mg/kg body weight i.p.) was used as a model of sepsis-induced cholestasis. Bile flow, biliary bile salt secretion, biliary glutathione secretion and protein expression of the main hepatobiliary transporters Na(+)-taurocholate-cotransporting peptide (Ntcp), multidrug resistance protein 2 (Mrp2) and bile salt export pump (Bsep) were analyzed by conventional techniques in ICKT treated and non-treated animals.Results.Injection of LPS induced a significant decrease of bile flow (-24%), biliary bile salts (-40%) and glutathione excretion (-70%) as well as a significant decrease in Ntcp (-90%) and Mrp2 (-80%) protein levels. ICKT supplementation partially prevented the effects of LPS determining a less intense reduction in bile flow (-10%), a normalization of glutathione excretion as well as a significant increase in Mrp2 protein levels to 60% of the levels observed in control animals. ICKT administration did not modify the effects of LPS on BS secretion or Ntcp protein levels.Conclusion.Our data show that oral supplementation of ICKT partially prevents LPS-induced cholestasis by increasing Mrp2 protein levels and biliary glutathione excretion thus increasing bile salt-independent flow.     

Effect of tauro-α-muricholate and tauro-β-muricholate on oestradiol-17β-glucuronide-induced cholestasis in rats

The effect of tauro-β-muricholate (βMC-tau) and tauro-α-muricholate (αMC-tau) on oestradiol-17β-glucuronide (E₂17G)-induced cholestasis was compared with that of taurourso-deoxycholate (UDC-tau) in rats. Like UDC-tau, αMC-tau and βMC-tau infused at the rate of 0.2 μmol/min per 100 g bodyweight (BW) completely inhibited the cholestasis induced by E₂17G infused at the rate of 0.06 μmol/min per 100 g BW for 20 min. These findings indicate that βMC-tau and αMC-tau are useful in protecting against various types of experimental cholestasis, as well as against bile acid-induced cholestasis.     

Effects of colchicine on the maximum biliary excretion of cholephilic compounds in rats

BACKGROUND AND AIM: Colchicine, an inhibitor of intracellular vesicular transport, has been reported to inhibit the biliary excretion of bile acids and organic anions, but the previous findings are controversial. In order to systematically evaluate the effect of colchicine on the biliary excretion of cholephilic compounds, we studied the effect of colchicine on the biliary excretion of substrates of various canalicular transporters, which were administered at or above the excretory maximum in rats. METHODS: Substrates of various canalicular adenosine triphosphate-binding-cassette transporters were infused at or above the rate of maximum excretion into rats, and the effect of colchicine (0.2 mg/100 g), which was intraperitoneally injected 3 h before, on the biliary excretion was studied. Furthermore, the effect of tauroursodeoxycholate (TUDC) co-infusion on the biliary excretion of taurocholate (TC) after colchicine treatment was also studied. RESULTS: The biliary excretion of TC and cholate administered at the rate of 1 micro mol/min/100 g was markedly inhibited by colchicine, whereas that of TUDC was not inhibited even with the infusion rate of 2 micro mol/min/100 g. TUDC co-infusion at the rate of 1 micro mol/min/100 g increased the biliary excretion of TC (1 micro mol/min/100 g), which was decreased by the colchicine pretreatment. The biliary excretory maximum of taurolithocholate-sulfate and sulfobromophthalein, substrates of the multidrug resistance protein 2, of erythromycin, a substrate of the P-glycoprotein, and of indocyanine green were not affected by colchicine. CONCLUSIONS: The different excretory maximums of TC and TUDC and the different effect of colchicine on the excretion of these bile acids are considered to be a result of different regulatory mechanisms of vesicular targeting of the bile salt export pump to the canalicular membrane by these bile acid conjugates. The vesicular targeting of the multidrug resistance protein 2 and the P-glycoprotein to the canalicular membrane is considered to be colchicine insensitive in the absence of bile acid coadministration.     

Effects of ursodeoxycholate and its conjugates on biliary glutathione excretion in rats

The effects of ursodeoxycholate and its conjugates on biliary glutathione excretion were studied in rats. Ursodeoxycholate had no effect on glutathione excretion, but tauroursode-oxycholate (10µmol/100 g body wt) transitionally increased biliary glutathione excretion. Ursodeoxycholate-3-O-glucuronide (2 and 10µmol/100 g body wt) markedly inhibited biliary glutathione excretion, but ursodeoxycholate-3-sulfate (2µmol/100 g body wt) and ursode-oxycholate-3,7-disulfate (10µmol/100 g body wt) did not. These findings indicate the existence of several biliary excretion pathways for bile acid glucuronides and sulfates and that one of them for the glucuronides is shared by biliary glutathione excretion.     

Improvement of estradiol 17 beta-D-glucuronide cholestasis by intravenous administration of dimethylethanolamine in the rat.

The intravenous administration of dimethylethanolamine in the rat promotes a selective enrichment of liver membranes with polyunsaturated phosphatidylcholines. The effect of dimethylethanolamine pretreatment on cholestasis induced by estradiol 17 beta-D-glucuronide, a potent cholestatic agent, was assessed in this study. Dimethylethanolamine, dissolved in sodium-taurocholate was infused intravenously (0.3 mg/kg/min) for 15 hr. One group of control rats (estradiol 17 beta-D-glucuronide controls) received the bile salt only. An estradiol 17 beta-D-glucuronide bolus was then injected intravenously (10.4 mg/kg) into dimethylethanolamine-pretreated and estradiol 17 beta-D-control rats, and its effect on bile flow and biliary lipid secretion was compared for 3 hr. The estradiol 17 beta-D-glucuronide inhibitory effect on bile flow and biliary lipid secretion was significantly antagonized by dimethylethanolamine pretreatment. The maximum inhibition of bile flow was found 30 min after estradiol 17 beta-D-glucuronide administration, when it decreased from 3.5 +/- 0.4 microliters/min/100 gm (basal) to 0.9 +/- 0.3 microliters/min/100 gm in estradiol 17 beta-D-glucuronide controls, whereas in dimethylethanolamine-pretreated rats this decreased only from 3.2 +/- 0.4 (basal) to 2.3 +/- 0.4 microliters/min/100 gm. Bile flow and the biliary secretion of cholesterol, phosphatidylcholine and bile salts were significantly higher in the dimethylethanolamine-pretreated rats than in estradiol 17 beta-D-glucuronide controls (p less than 0.02) during the cholestatic phase. The inhibitory effect of estradiol 17 beta-D-glucuronide on bile flow was associated with a marked decrease of membrane fluidity (p less than 0.001) assessed by 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and with a cholesterol enrichment of microsomes, sinusoidal and canalicular liver plasma membranes and inhibition of sinusoidal Na+,K(+)-ATPase activity (p less than 0.05). These membrane alterations persisted 180 min after estradiol 17 beta-D-glucuronide administration despite complete normalization of bile flow. Dimethylethanolamine pretreatment significantly counteracted the reduction of membrane fluidity (p less than 0.001), the cholesterol enrichment and the inhibition of Na+,K(+)-ATPase (p less than 0.05) promoted by estradiol 17 beta-D-glucuronide administration in all membrane subfractions 30 and 180 min after administration. In addition, dimethylethanolamine-pretreated rats had more polyunsaturated fatty acids in membrane phosphatidylcholine with respect to the control groups. Dilatation of canaliculi and loss of microvilli were evident in estradiol 17 beta-D-glucuronide controls 180 min after estradiol 17 beta-D-glucuronide administration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Estrone/17β‐estradiol conversion to,and tumor necrosis factor inhibition by,estrogen metabolites in synovial cells of patients with rheumatoid arthritis and patients with osteoarthritis

Objective

The role of estrogens in rheumatoid arthritis (RA) is debated since both proinflammatory and antiinflammatory effects have been reported. Important evidence of the dual role of estrogens is conversion to various proinflammatory or antiinflammatory metabolites. This study was undertaken to examine the downstream conversion of estrogens in synovial cells from patients with RA or osteoarthritis (OA).

Methods

We studied serum levels of estrone, estrone sulfate, and estrone sulfate membrane transporters, intracellular interconversion of estrone and 17β‐estradiol, and conversion of estrone/17β‐estradiol to various estrogen metabolites in RA and OA synovial cells. The effect of estrogen metabolites on tumor necrosis factor (TNF) secretion was also studied in RA and OA synovial cells.

Results

Serum levels of estrone sulfate were similar in healthy controls and RA patients. Estrone sulfate transporters were present in synovial tissue. Interconversion of estrone and 17β‐estradiol and the expression of converting enzymes of the cytochrome P450 family were similar in RA and OA cells. Using estrone and 17β‐estradiol as substrates, RA and OA synovial cells produced 16α‐, 4‐, and 2‐hydroxylated estrogens and their 4‐ and 2‐methylation products. The levels of 16α‐hydroxylated estrone/17β‐estradiol (16αOH‐estrone/16αOH‐17β‐estradiol) were higher than the levels of all other estrogen metabolites. RA synovial cells produced more 16αOH‐estrone than did OA synovial cells. Importantly, the 16αOH estrogens did not inhibit TNF secretion, whereas all other estrogen metabolites had marked inhibitory effects.

Conclusion

Our findings indicate that precursor estrogens are converted to proinflammatory metabolites, particularly in RA synovial cells. RA synovial cells mainly produce the proproliferative 16αOH‐estrone, which, in addition to 16αOH‐17β‐estradiol, is one of the only 2 estrogens studied that does not inhibit TNF secretion. A preponderance of 16α‐hydroxylated estrogens is an unfavorable sign in synovial inflammation.